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Immunoglobulin producing cells in the spotted wolffish (Anarhichas minor Olafsen): localization in adults and during juvenile development
Developmental and Comparative Immunology 27 (2003) 569–578 www.elsevier.com/locate/devcompimm
Immunoglobulin producing cells in the spotted wolffish (Anarhichas minor Olafsen): localization in adults and during juvenile development Randi Nygaard Grøntvedt*, Sigrun Espelid Norwegian Institute of Fisheries and Aquaculture, Tromsø 9291, Norway Received 25 March 2002; revised 24 June 2002; accepted 31 December 2002
Abstract The presence of immunocompetent cells was studied in the larval and adult stages of the spotted wolffish, Anarhichas minor. In situ hybridization with a probe complementary to the secretory Igm-chain was used to localize immunoglobulin producing cells or plasma cells in organs from adult fish and the appearance of these cells in lymphoid tissues during juvenile development. Plasma cells were located in pronephros, spleen, gut, gills and skin of adult wolffish. In juveniles, the first plasma cells were detected in the kidney 1 week post-hatching and the appearance in other lymphoid organs was in the order spleen, gut and thymus. No plasma cells were detected in skin and gills during the sampling period of juveniles (, 10 cm). Our study confirmed that plasma cells are present in both the systemic and mucosal compartments of adult fish but during ontogeny there is an earlier appearance of plasma cells in the gut compared to gill and skin compartments. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Anarhichas minor; Spotted wolffish; Ig producing cells; Lymphoid organs; In situ hybridization
1. Introduction Knowledge about cells and organs involved in the fish immune system is important to improve fish immunprophylaxis. Teleosts lack bone marrow and lymph nodes and the major lymphoid organs are thymus, kidney, spleen and gut-associated lymphoid tissue (GALT) [1]. The thymus in teleosts is a paired organ present in the dorsolateral chamber of the gill cavity. The major role of thymus in higher vertebrates * Corresponding author. Tel.: þ47-776-29000; fax: þ 47-77629100. E-mail address: [email protected] (R.N. Grøntvedt).
is T-cell maturation and this presumably also occurs in fish thymus [2]. The kidney in fish consists of an anterior segment (pronephros or head kidney) and a middle and posterior segment (trunk or body kidney). The pronephros contains predominantly hemopoietic tissue while the trunk kidney is dominated by renal tissue [3]. The spleen is a dark red to black organ located usually ventral and caudal to the stomach. The red pulp area mainly consists of reticular cell network supporting blood-filled sinusoids, while the white pulp is known to contain melanomacrophage centres and ellipsoids. The GALT in fish lacks Peyer’s patches associated to the gut, but fish possess leukocytes in lamina propria and epithelium of
0145-305X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0145-305X(03)00028-4
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the hindgut [1]. Skin and gills have significant protective functions in fish as an outer barrier to the environment. Reports of antigen uptake in gills and/or skin of several fish species [4] indicate that these organs also have immunologically important functions. Extensive studies have been performed on the ontogeny of lymphoid organs in commercially important freshwater species like salmon (Salmo salar) [5], rainbow trout (Oncorhynchus mykiss) [6], carp (Cyprinus carpio) [7] and in marine species like cod (Gadus morhua) [8], turbot (Scophthalmus maximus) [9], yellowtail (Seriola quinqueradiata), red sea bream (Pagrus major), Japanese flounder (Paralichthys olivaceus) [10], spiny plunder fish (Harpagifer antarcticus) [11] and rock fish (Sebastiscus marmoratus) [12]. To study immunocompetence during ontogeny immunodetection has been used to observe surface immunoglobulin (Ig) on Bcells or cytoplasmic Ig in plasma cells in salmon [5], rainbow trout [13], carp [14], sea bass [15,16] and Atlantic cod [17]. The spotted wolffish, Anarhichas minor Olafsen, a species of the Perciformes, is now produced in the aquaculture industry in northern Norway. The eggs have a long incubation period for 4– 5 months and the larvae are on average 22 mm long and well developed when hatched [18]. Nothing is yet known about the presence of immunocompetent cells in the wolffish larvae/juvenile. In this paper, we have used cloned cDNA sequences of the immunoglobulin heavy chain transcript [19] and in situ hybridization to describe both the appearance of immunoglobulin producing cells in the spotted wolffish juveniles and the localization of these cells in various tissues of adult fish.
2. Materials and methods 2.1. Fish sampling and preparation of tissues Eggs, larvae and juveniles of the spotted wolffish were bred and provided from a production plant in northern Norway (Troms Steinbit AS). Three to five individuals were sampled and fixed once a week over a period of 10 months. When sampling prior to hatching, the larvae were dissected out of the eggs.
The length of all individuals was measured before fixation. Adult fish were bred at the Aquaculture Research Station, Ka˚rvika, Tromsø. Liver, pronephros, spleen, heart, gut, skin and gills were cut into smaller parts suitable for fixation. All samples were fixed over night in 4% paraformaldehyde in phosphate-buffered saline (PBS) and transferred to 70% ethanol before paraffin embedding. All tissues examined were cut into 5 mm sections and attached to poly-L -lysine (Sigma) treated slides. 2.2. In situ hybridization A plasmid containing cDNA of the complete immunoglobulin heavy chain (IgH) from the spotted wolffish has been described [19]. Two sets of primers were designed, one to pick out the Cm1, Cm2, Cm3 and Cm4 domains of the Igm-chain (1200 bp) and an other to pick out specifically the Cm4 domain and untranslated region of secretory Ig (600 bp) by polymerase chain reaction (PCR). The two PCR products were cloned into a pCR-Scripte AMP SK(þ ) transcription vector (Stratagene). The DIG RNA labeled probes were synthesized by in vitro transcription of DNA using digoxigenin-labeled uridine-triphosphate as substrate (DIG RNA Labeling Kit; Roche Molecular Biochemicals). After linearizing the vectors with Kpn I (USB) the inserts were transcribed with T3 RNA polymerase giving RNAprobes complementary to mRNA Igm in the tissue. Linearizing the vector with Sac I (Stratagene), and transcribing with T7 RNA polymerase gave probes homologous to mRNA in tissues. These probes were used as hybridization controls. Unamplified controls were also checked according to the manual, where complementary probes were included and TSA reagents excluded (see below). No staining of cells was observed in any of these controls. When analyzing the spotted wolffish juveniles, pronephros or spleen tissues from adult fish were always included as positive controls. The Cm1 –4 probe was used throughout the study and the Cm4 probe was included to confirm that the cells produced mRNA specific for secretory IgM. Tissue sections were dewaxed, rehydrated and treated with 1% H2O2 in methanol for 30 min to block endogenous peroxidase activity. The slides were
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pretreated for 15 min with 0.3% Triton X-100 in PBS at room temperature, and then with 20 mg ml21 proteinase K (Promega) for 7 min at 37 8C. Between the two steps, the slides were washed twice at room temperature with PBS and 0.1 M Tris –HCl pH 7.5, respectively. Tissue sections were post-fixed in 4% paraformaldehyde in PBS for 5 min at 4 8C. Each section was hybridized either with a complementary DIG RNA probe or a control DIG RNA probe diluted in 10 ml hybridization mixture (5 £ Denhardt’s, 50% formamide, 2 £ SSC and 50 mg ml21 calf thymus DNA). Slides with coverslips pretreated with Repelsilane (Pharmacia Biotech) were wrapped into Parafilm and incubated in a moisted chamber overnight at 42 8C. After removal of Parafilm and coverslips, the slides were washed twice in 2 £ SSC for 15 min in room temperature, followed by a bath in 0.1 M Tris – HCl/0.15 M NaCl pH 7.5 for 30 min. Hybridization was visualized by using a tyramide signal amplification kit (TSAe-Indirect (ISH); NENe Life Science Products) and peroxidase-conjugated anti-DIG Fab fragments (Roche Molecular Biochemicals). Color reaction was developed by the AEC Chromogen Kit (Sigma). Finally, sections were counterstained with Mayer’s haematoxyline (Fluka) and mounted with Aquamount (BDH Laboratory).
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tissue with a network of blood vascular sinusoids and a few renal tubules. Immunoglobulin producing cells were detected in the pronephros surrounding the blood vessels (Fig. 1a) or as scattered cells in the hemopoietic tissue (Fig. 1b). The spleen in spotted wolffish is a small circular organ with dark red color. As in the pronephros the spleen consists of hemopoietic tissue, and blood vessels are easily recognizable. There is no clear distinction between the red and white pulp. The localization of Ig producing cells in the spleen was similar to the findings in pronephros; as evenly distributed cells, as assemblage surrounding the blood vessels (Fig. 1c) and as circulating cells in the blood (Fig. 1d). Ig producing cells were also detected in gut, skin and gills. In the gut, single cells were located in the lamina propria (Fig. 2a) and in the submucosa (Fig. 2b). In the skin, stained cells were found both in the epithelium near the basal membrane (Fig. 2c) and further out in the epidermis in vicinity with smaller cells that could resemble lymphocytes (Fig. 2d). Ig producing cells were also detected in the primary gill lamellae (Fig. 2e) and in gill filaments along blood vessels (Fig. 2f). Finally, positive cells were occasionally seen in heart and liver sections, but only located in the blood vessels of these tissues (not shown). 3.2. Ig producing cells in juveniles
3. Results In situ hybridization with a DIG-labeled probe complementary to secretory Igm mRNA (1200 bp), revealed staining of cells with high content of cytoplasm in the tissue sections as seen in Figs. 1a – d and 2a– f. The strongly stained cells in the various organs had similar morphology. The 600 bp probe complementary to the Cm4 domain and the untranslated region of secretory Igm detected cells with the same morphology as the complete Cm1 – 4 probe verifying that the stained cells produced mRNA for secretory Ig. 3.1. Ig producing cells in adult wolffish In spotted wolffish, the pronephros is located as two outlets branching from the trunk kidney. The pronephros consists predominantly of hemopoietic
Fig. 3 shows age and the average length (mm) of the sampled larvae/juveniles. The specimens increased from 17 mm as unhatched larvae to 86 mm juveniles during the 20 week period. The time of hatching was defined as week 0 and in situ hybridization was performed at the time-points indicated. The results of the in situ hybridization are summarized in Table 1. No positive cells were found in the larvae prior to hatching (results not shown). The first appearance of immunoglobulin producing cells was in kidney from one out of three larvae sampled 1 week after hatching (, 25 mm) although the number of stained cells were few (Fig. 4a). The number of positive cells in kidney increased during the first 4 weeks posthatching (Fig. 4b). The cells were located not only in the head kidney but also along the renal part of the kidney. Two weeks after hatching Ig producing
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Fig. 1. Plasma cells detected by hybridizing with Cm1–4 probe in kidney (a,b) and spleen (c,d) from adult wolffish: (a) plasma cells surrounding the blood vessels and (b) as scattered cells in the hemapoietic tissue of the kidney, (c) plasma cells surrounding a blood vessel and (d) as circulating cells in venous blood of spleen. bv, blood vessel; T, tubule and vb, venous blood (phase contrast microscopy £ 1200).
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Fig. 3. Age and average growth rate in length (mm) of the weekly sampled spotted wolffish larvae/juveniles. The time of hatching is defined as week 0. In situ hybridization was done at the time-points indicated.
cells were distinguishable in the spleen and the appearance of stained cells gradually increased with time comparable to findings in the kidney. Immunoglobulin producing cells were detected in spleen from all sampled juveniles at week 18. The first positive cells in gut and thymus appeared somewhat later compared to kidney and spleen. At week 18, the juveniles measured on average 68 mm and showed consistently stained cells in kidney, spleen and gut, while two of three juveniles had positive cells in thymus. The thymus was located in the gill cavity overlaying the gill arches. Haematoxylin staining separated the thymus into a pale and a dark colored zone, and Ig producing cells were detected in both zones. Structures like epithelial cysts were observed in several of the largest juveniles. By week 21, positive cells were detected in thymus of all the sampled juveniles and in higher numbers compared to week 18 (Fig. 5). No positive cells were found associated to the tissues of skin and gills during the sampling period, although a few positive cells were occasionally seen among the red blood cells in the capillaries of the secondary lamellae of the gills.
4. Discussion In situ hybridization with a DIG-labelled probe covering the Cm1 –4 domains of the immunoglobulin heavy chain strongly stained the cytoplasm of large cells in the lymphoid tissues of spotted wolffish. The Cm1 –4 probe has three domains in common with membrane bound Ig and could detect B-cells as well. However, hybridization with a probe complementary to the Cm4-domain and UTR specific for secretory Ig, showed staining of cells with similar morphology and of equal numbers in serial tissue sections. We therefore conclude that the cells detected with the Cm1 –4 and Cm4– probes are plasma cells with high expression of secretory Igm transcripts. The spotted wolffish hatches 4– 5 months after fertilization as 20– 25 mm active juveniles [18] and our study shows that the first plasma cells emerge between week 1 and 4 post-hatching. For comparison, the Atlantic cod hatches only 9 days after fertilization as 3– 4 mm long passive larvae [20], and plasma cells do not appear until 2 months later at the size of approximately 30 mm [17], which is also the size of 4 weeks old wolffish juveniles.
Fig. 2. Plasma cells detected by hybridizing with Cm1–4 probe in gut (a,b), skin (c,d) and gills (e,f) from adult wolffish: (a) plasma cells in lamina propria and (b) submucosa in gut, (c) plasma cells located near the basal membrane and (d) further out in the epidermis of skin, (e) plasma cells located in gill tissue in the primary lamellae and (f) in gill filaments along blood vessels. lp, lamina propria; sm, submucosa; mc, mucus cells; bm, basal membrane and bv, blood vessel (phase contrast microscopy £ 900).
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Table 1 Appearance of plasma cells in the different organs of the wolffish juveniles as detected by in situ hybridization Time (weeks)
Length (mm)
Kidney
Spleen
Gut
Thymus
Gill
Skin
0 1 2 3 4 8 13 18 21
24 25 26 28 30 32 53 68 86
2222 2 þ2 2 þþ 2 þþ 2 þ þþ þ þþ þ þþ þ þþ þ þþ
222222 22 2 þ2 2 þ2 2 þ2 2 þ2 2 þþ 2 þþ þ þþ þ
2222 2 22 2 22 2 22 2 22 2 þ2 2 þþ 2 þ þþ þ þþ
2222 2 22 2 22 2 22 2 þ2 2 22 2 þ2 2 þþ 2 þþ þ
2222 2 22 2 22 2 22 2 22 2 22 2 22 2 22 2 22 2
2222 2 22 2 22 2 22 2 22 2 22 2 22 2 22 2 22 2
Three individuals were analyzed at each sampling, except at week 0, where five larvae were analyzed. þ or 2 indicate positive or negative staining of individuals (i.e. þ22 ¼ one of three fish show positive staining).
Although the ontogeny of various fish species is different, it is suggested that the maturation of the adaptive immune system is more dependent on size than age after hatching [21].
In our study, the first plasma cells were detected in cells of the head kidney of wolffish larvae 1 week post-hatching. The head kidney is a major lymphoid organ with differentiation of B-cells into plasma cells
Fig. 4. Plasma cells in kidney from spotted wolffish juveniles by hybridizing with a probe complementary to the Cm4 and UTR region: (a) 1 week post-hatching a few positive cells (arrows) are detected in kidney tissue among tubules (light microscopy £ 720) and (b) 4 weeks after hatching several cells are present in hemapoietic tissue in pronephros (phase contrast microscopy £ 1440). pn ¼ pronephros.
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Fig. 5. Plasma cells (arrows) detected in thymus by hybridizing with Cm1–4 probe in juvenile 18 weeks post-hatching (light microscopy £ 530). Magnified section shows one plasma cell (phase contrast microscopy £ 1440).
which is in accordance with results from carp [14,22], rainbow trout [23], sea bass [15] and cod [17]. The appearance of plasma cells in the spleen a week later in the wolffish is also similar to previous reports from trout [23], carp [14] and sea bass [24], suggesting the spleen too as a major lymphoid organ in fish. The kidney and spleen are the major sites for trapping of antigens and the production of antibodies, and the predominant source of serum Ig is suggested to be plasma cells in the head kidney [25]. The presence of plasma cells in kidney and spleen of spotted wolffish a few weeks after hatching indicates an important role of adaptive and protective immune responses in the early free-living stages of this species. The stained sections of kidney and spleen from adult wolffish revealed plasma cells both as singly distributed cells and more densely packed around blood vessels. Clusters of immunoglobulin producing cells surrounding blood vessels and melanomacrophage centres have been observed in other fish species
[8,26], but melanomacrophages were not distinctive in the wolffish tissues. Plasma cells were detected in both the outer and inner zones of the thymus in wolffish juveniles. Thymus was localized in the juveniles only, and the difficulty in finding thymus in adult fish could be due to age related involution, which is reported from other fish species [27]. The thymus is important for maturation of T-cells, at least in higher vertebrates. The appearance of cells of B-cell origin in this organ is disputed since the number of cells is low and it is also suggested that these cells may originate from blood. Some reports, however, conclude that plasma cells are present in the thymus of fish [8,28]. In wolffish thymus the stained cells were clearly associated to the lymphoid tissue and not to blood vessels. Plasma cells in the gut tissue of wolffish juveniles appeared later compared to kidney and spleen which is in the same order as detection of Igþ cells in sea
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bass [29]. Positively stained cells were located in the lamina propria of both the anterior and posterior gut which is in accordance with previous findings from carp [30], sea bass [24,31], cod [8] and turbot [26]. The existence of a common mucosal immune system in fish is suggested since cells involved in immune responses are present in the tissues of gut, gills and skin [25,32,33]. Mucosal Ig is qualitatively and quantitatively described in several fish species [34 – 38] and these molecules could either be produced locally or enter the epithelium from blood. Several authors report the presence of serological variants of IgM in mucus compared to serum, in addition to varying peak antibody responses in the two compartments after different antigen administration routes [35,39 – 42], and these antigenic variations are probably due to post-translational processes. Our study confirmed that plasma cells with secretory Cm transcripts are present in the mucosal compartments of adult wolffish but during ontogeny there is an obvious distinction in the appearance of plasma cells in the gut compared to the gill and skin compartments. The relatively early localization of these cells in the gut could be explained by a massive exposure to antigens through the gut lumen, since the wolffish larvae immediately after hatch feed on external nutrition, but one should expect gills and skin to be exposed to external antigens from the environment as well. An alternative explanation is that the ontogeny of immune cells is different in the various mucosal tissues. The protective role of gills and skin could therefore be of limited importance in the early life stages of spotted wolffish. However, results from sea bass, another perciform species [43], show that antibody production is induced in the gills of 0.1 g juveniles after immersion in bacterin, indicative of an early presence of immunocompetent cells in mucosal tissue. Further work on the spotted wolffish will elucidate if antibody production can be induced earlier in the gills and skin of juveniles exposed to bath vaccination and challenge experiments.
Acknowledgements This work was supported by The Research Council of Norway (project no. 124043/140).
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Immunoglobulin producing cells in the spotted wolffish (Anarhichas minor Olafsen): localization in adults and during juvenile development Randi Nygaard Grøntvedt*, Sigrun Espelid Norwegian Institute of Fisheries and Aquaculture, Tromsø 9291, Norway Received 25 March 2002; revised 24 June 2002; accepted 31 December 2002
Abstract The presence of immunocompetent cells was studied in the larval and adult stages of the spotted wolffish, Anarhichas minor. In situ hybridization with a probe complementary to the secretory Igm-chain was used to localize immunoglobulin producing cells or plasma cells in organs from adult fish and the appearance of these cells in lymphoid tissues during juvenile development. Plasma cells were located in pronephros, spleen, gut, gills and skin of adult wolffish. In juveniles, the first plasma cells were detected in the kidney 1 week post-hatching and the appearance in other lymphoid organs was in the order spleen, gut and thymus. No plasma cells were detected in skin and gills during the sampling period of juveniles (, 10 cm). Our study confirmed that plasma cells are present in both the systemic and mucosal compartments of adult fish but during ontogeny there is an earlier appearance of plasma cells in the gut compared to gill and skin compartments. q 2003 Elsevier Science Ltd. All rights reserved. Keywords: Anarhichas minor; Spotted wolffish; Ig producing cells; Lymphoid organs; In situ hybridization
1. Introduction Knowledge about cells and organs involved in the fish immune system is important to improve fish immunprophylaxis. Teleosts lack bone marrow and lymph nodes and the major lymphoid organs are thymus, kidney, spleen and gut-associated lymphoid tissue (GALT) [1]. The thymus in teleosts is a paired organ present in the dorsolateral chamber of the gill cavity. The major role of thymus in higher vertebrates * Corresponding author. Tel.: þ47-776-29000; fax: þ 47-77629100. E-mail address: [email protected] (R.N. Grøntvedt).
is T-cell maturation and this presumably also occurs in fish thymus [2]. The kidney in fish consists of an anterior segment (pronephros or head kidney) and a middle and posterior segment (trunk or body kidney). The pronephros contains predominantly hemopoietic tissue while the trunk kidney is dominated by renal tissue [3]. The spleen is a dark red to black organ located usually ventral and caudal to the stomach. The red pulp area mainly consists of reticular cell network supporting blood-filled sinusoids, while the white pulp is known to contain melanomacrophage centres and ellipsoids. The GALT in fish lacks Peyer’s patches associated to the gut, but fish possess leukocytes in lamina propria and epithelium of
0145-305X/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0145-305X(03)00028-4
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the hindgut [1]. Skin and gills have significant protective functions in fish as an outer barrier to the environment. Reports of antigen uptake in gills and/or skin of several fish species [4] indicate that these organs also have immunologically important functions. Extensive studies have been performed on the ontogeny of lymphoid organs in commercially important freshwater species like salmon (Salmo salar) [5], rainbow trout (Oncorhynchus mykiss) [6], carp (Cyprinus carpio) [7] and in marine species like cod (Gadus morhua) [8], turbot (Scophthalmus maximus) [9], yellowtail (Seriola quinqueradiata), red sea bream (Pagrus major), Japanese flounder (Paralichthys olivaceus) [10], spiny plunder fish (Harpagifer antarcticus) [11] and rock fish (Sebastiscus marmoratus) [12]. To study immunocompetence during ontogeny immunodetection has been used to observe surface immunoglobulin (Ig) on Bcells or cytoplasmic Ig in plasma cells in salmon [5], rainbow trout [13], carp [14], sea bass [15,16] and Atlantic cod [17]. The spotted wolffish, Anarhichas minor Olafsen, a species of the Perciformes, is now produced in the aquaculture industry in northern Norway. The eggs have a long incubation period for 4– 5 months and the larvae are on average 22 mm long and well developed when hatched [18]. Nothing is yet known about the presence of immunocompetent cells in the wolffish larvae/juvenile. In this paper, we have used cloned cDNA sequences of the immunoglobulin heavy chain transcript [19] and in situ hybridization to describe both the appearance of immunoglobulin producing cells in the spotted wolffish juveniles and the localization of these cells in various tissues of adult fish.
2. Materials and methods 2.1. Fish sampling and preparation of tissues Eggs, larvae and juveniles of the spotted wolffish were bred and provided from a production plant in northern Norway (Troms Steinbit AS). Three to five individuals were sampled and fixed once a week over a period of 10 months. When sampling prior to hatching, the larvae were dissected out of the eggs.
The length of all individuals was measured before fixation. Adult fish were bred at the Aquaculture Research Station, Ka˚rvika, Tromsø. Liver, pronephros, spleen, heart, gut, skin and gills were cut into smaller parts suitable for fixation. All samples were fixed over night in 4% paraformaldehyde in phosphate-buffered saline (PBS) and transferred to 70% ethanol before paraffin embedding. All tissues examined were cut into 5 mm sections and attached to poly-L -lysine (Sigma) treated slides. 2.2. In situ hybridization A plasmid containing cDNA of the complete immunoglobulin heavy chain (IgH) from the spotted wolffish has been described [19]. Two sets of primers were designed, one to pick out the Cm1, Cm2, Cm3 and Cm4 domains of the Igm-chain (1200 bp) and an other to pick out specifically the Cm4 domain and untranslated region of secretory Ig (600 bp) by polymerase chain reaction (PCR). The two PCR products were cloned into a pCR-Scripte AMP SK(þ ) transcription vector (Stratagene). The DIG RNA labeled probes were synthesized by in vitro transcription of DNA using digoxigenin-labeled uridine-triphosphate as substrate (DIG RNA Labeling Kit; Roche Molecular Biochemicals). After linearizing the vectors with Kpn I (USB) the inserts were transcribed with T3 RNA polymerase giving RNAprobes complementary to mRNA Igm in the tissue. Linearizing the vector with Sac I (Stratagene), and transcribing with T7 RNA polymerase gave probes homologous to mRNA in tissues. These probes were used as hybridization controls. Unamplified controls were also checked according to the manual, where complementary probes were included and TSA reagents excluded (see below). No staining of cells was observed in any of these controls. When analyzing the spotted wolffish juveniles, pronephros or spleen tissues from adult fish were always included as positive controls. The Cm1 –4 probe was used throughout the study and the Cm4 probe was included to confirm that the cells produced mRNA specific for secretory IgM. Tissue sections were dewaxed, rehydrated and treated with 1% H2O2 in methanol for 30 min to block endogenous peroxidase activity. The slides were
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pretreated for 15 min with 0.3% Triton X-100 in PBS at room temperature, and then with 20 mg ml21 proteinase K (Promega) for 7 min at 37 8C. Between the two steps, the slides were washed twice at room temperature with PBS and 0.1 M Tris –HCl pH 7.5, respectively. Tissue sections were post-fixed in 4% paraformaldehyde in PBS for 5 min at 4 8C. Each section was hybridized either with a complementary DIG RNA probe or a control DIG RNA probe diluted in 10 ml hybridization mixture (5 £ Denhardt’s, 50% formamide, 2 £ SSC and 50 mg ml21 calf thymus DNA). Slides with coverslips pretreated with Repelsilane (Pharmacia Biotech) were wrapped into Parafilm and incubated in a moisted chamber overnight at 42 8C. After removal of Parafilm and coverslips, the slides were washed twice in 2 £ SSC for 15 min in room temperature, followed by a bath in 0.1 M Tris – HCl/0.15 M NaCl pH 7.5 for 30 min. Hybridization was visualized by using a tyramide signal amplification kit (TSAe-Indirect (ISH); NENe Life Science Products) and peroxidase-conjugated anti-DIG Fab fragments (Roche Molecular Biochemicals). Color reaction was developed by the AEC Chromogen Kit (Sigma). Finally, sections were counterstained with Mayer’s haematoxyline (Fluka) and mounted with Aquamount (BDH Laboratory).
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tissue with a network of blood vascular sinusoids and a few renal tubules. Immunoglobulin producing cells were detected in the pronephros surrounding the blood vessels (Fig. 1a) or as scattered cells in the hemopoietic tissue (Fig. 1b). The spleen in spotted wolffish is a small circular organ with dark red color. As in the pronephros the spleen consists of hemopoietic tissue, and blood vessels are easily recognizable. There is no clear distinction between the red and white pulp. The localization of Ig producing cells in the spleen was similar to the findings in pronephros; as evenly distributed cells, as assemblage surrounding the blood vessels (Fig. 1c) and as circulating cells in the blood (Fig. 1d). Ig producing cells were also detected in gut, skin and gills. In the gut, single cells were located in the lamina propria (Fig. 2a) and in the submucosa (Fig. 2b). In the skin, stained cells were found both in the epithelium near the basal membrane (Fig. 2c) and further out in the epidermis in vicinity with smaller cells that could resemble lymphocytes (Fig. 2d). Ig producing cells were also detected in the primary gill lamellae (Fig. 2e) and in gill filaments along blood vessels (Fig. 2f). Finally, positive cells were occasionally seen in heart and liver sections, but only located in the blood vessels of these tissues (not shown). 3.2. Ig producing cells in juveniles
3. Results In situ hybridization with a DIG-labeled probe complementary to secretory Igm mRNA (1200 bp), revealed staining of cells with high content of cytoplasm in the tissue sections as seen in Figs. 1a – d and 2a– f. The strongly stained cells in the various organs had similar morphology. The 600 bp probe complementary to the Cm4 domain and the untranslated region of secretory Igm detected cells with the same morphology as the complete Cm1 – 4 probe verifying that the stained cells produced mRNA for secretory Ig. 3.1. Ig producing cells in adult wolffish In spotted wolffish, the pronephros is located as two outlets branching from the trunk kidney. The pronephros consists predominantly of hemopoietic
Fig. 3 shows age and the average length (mm) of the sampled larvae/juveniles. The specimens increased from 17 mm as unhatched larvae to 86 mm juveniles during the 20 week period. The time of hatching was defined as week 0 and in situ hybridization was performed at the time-points indicated. The results of the in situ hybridization are summarized in Table 1. No positive cells were found in the larvae prior to hatching (results not shown). The first appearance of immunoglobulin producing cells was in kidney from one out of three larvae sampled 1 week after hatching (, 25 mm) although the number of stained cells were few (Fig. 4a). The number of positive cells in kidney increased during the first 4 weeks posthatching (Fig. 4b). The cells were located not only in the head kidney but also along the renal part of the kidney. Two weeks after hatching Ig producing
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Fig. 1. Plasma cells detected by hybridizing with Cm1–4 probe in kidney (a,b) and spleen (c,d) from adult wolffish: (a) plasma cells surrounding the blood vessels and (b) as scattered cells in the hemapoietic tissue of the kidney, (c) plasma cells surrounding a blood vessel and (d) as circulating cells in venous blood of spleen. bv, blood vessel; T, tubule and vb, venous blood (phase contrast microscopy £ 1200).
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Fig. 3. Age and average growth rate in length (mm) of the weekly sampled spotted wolffish larvae/juveniles. The time of hatching is defined as week 0. In situ hybridization was done at the time-points indicated.
cells were distinguishable in the spleen and the appearance of stained cells gradually increased with time comparable to findings in the kidney. Immunoglobulin producing cells were detected in spleen from all sampled juveniles at week 18. The first positive cells in gut and thymus appeared somewhat later compared to kidney and spleen. At week 18, the juveniles measured on average 68 mm and showed consistently stained cells in kidney, spleen and gut, while two of three juveniles had positive cells in thymus. The thymus was located in the gill cavity overlaying the gill arches. Haematoxylin staining separated the thymus into a pale and a dark colored zone, and Ig producing cells were detected in both zones. Structures like epithelial cysts were observed in several of the largest juveniles. By week 21, positive cells were detected in thymus of all the sampled juveniles and in higher numbers compared to week 18 (Fig. 5). No positive cells were found associated to the tissues of skin and gills during the sampling period, although a few positive cells were occasionally seen among the red blood cells in the capillaries of the secondary lamellae of the gills.
4. Discussion In situ hybridization with a DIG-labelled probe covering the Cm1 –4 domains of the immunoglobulin heavy chain strongly stained the cytoplasm of large cells in the lymphoid tissues of spotted wolffish. The Cm1 –4 probe has three domains in common with membrane bound Ig and could detect B-cells as well. However, hybridization with a probe complementary to the Cm4-domain and UTR specific for secretory Ig, showed staining of cells with similar morphology and of equal numbers in serial tissue sections. We therefore conclude that the cells detected with the Cm1 –4 and Cm4– probes are plasma cells with high expression of secretory Igm transcripts. The spotted wolffish hatches 4– 5 months after fertilization as 20– 25 mm active juveniles [18] and our study shows that the first plasma cells emerge between week 1 and 4 post-hatching. For comparison, the Atlantic cod hatches only 9 days after fertilization as 3– 4 mm long passive larvae [20], and plasma cells do not appear until 2 months later at the size of approximately 30 mm [17], which is also the size of 4 weeks old wolffish juveniles.
Fig. 2. Plasma cells detected by hybridizing with Cm1–4 probe in gut (a,b), skin (c,d) and gills (e,f) from adult wolffish: (a) plasma cells in lamina propria and (b) submucosa in gut, (c) plasma cells located near the basal membrane and (d) further out in the epidermis of skin, (e) plasma cells located in gill tissue in the primary lamellae and (f) in gill filaments along blood vessels. lp, lamina propria; sm, submucosa; mc, mucus cells; bm, basal membrane and bv, blood vessel (phase contrast microscopy £ 900).
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Table 1 Appearance of plasma cells in the different organs of the wolffish juveniles as detected by in situ hybridization Time (weeks)
Length (mm)
Kidney
Spleen
Gut
Thymus
Gill
Skin
0 1 2 3 4 8 13 18 21
24 25 26 28 30 32 53 68 86
2222 2 þ2 2 þþ 2 þþ 2 þ þþ þ þþ þ þþ þ þþ þ þþ
222222 22 2 þ2 2 þ2 2 þ2 2 þ2 2 þþ 2 þþ þ þþ þ
2222 2 22 2 22 2 22 2 22 2 þ2 2 þþ 2 þ þþ þ þþ
2222 2 22 2 22 2 22 2 þ2 2 22 2 þ2 2 þþ 2 þþ þ
2222 2 22 2 22 2 22 2 22 2 22 2 22 2 22 2 22 2
2222 2 22 2 22 2 22 2 22 2 22 2 22 2 22 2 22 2
Three individuals were analyzed at each sampling, except at week 0, where five larvae were analyzed. þ or 2 indicate positive or negative staining of individuals (i.e. þ22 ¼ one of three fish show positive staining).
Although the ontogeny of various fish species is different, it is suggested that the maturation of the adaptive immune system is more dependent on size than age after hatching [21].
In our study, the first plasma cells were detected in cells of the head kidney of wolffish larvae 1 week post-hatching. The head kidney is a major lymphoid organ with differentiation of B-cells into plasma cells
Fig. 4. Plasma cells in kidney from spotted wolffish juveniles by hybridizing with a probe complementary to the Cm4 and UTR region: (a) 1 week post-hatching a few positive cells (arrows) are detected in kidney tissue among tubules (light microscopy £ 720) and (b) 4 weeks after hatching several cells are present in hemapoietic tissue in pronephros (phase contrast microscopy £ 1440). pn ¼ pronephros.
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Fig. 5. Plasma cells (arrows) detected in thymus by hybridizing with Cm1–4 probe in juvenile 18 weeks post-hatching (light microscopy £ 530). Magnified section shows one plasma cell (phase contrast microscopy £ 1440).
which is in accordance with results from carp [14,22], rainbow trout [23], sea bass [15] and cod [17]. The appearance of plasma cells in the spleen a week later in the wolffish is also similar to previous reports from trout [23], carp [14] and sea bass [24], suggesting the spleen too as a major lymphoid organ in fish. The kidney and spleen are the major sites for trapping of antigens and the production of antibodies, and the predominant source of serum Ig is suggested to be plasma cells in the head kidney [25]. The presence of plasma cells in kidney and spleen of spotted wolffish a few weeks after hatching indicates an important role of adaptive and protective immune responses in the early free-living stages of this species. The stained sections of kidney and spleen from adult wolffish revealed plasma cells both as singly distributed cells and more densely packed around blood vessels. Clusters of immunoglobulin producing cells surrounding blood vessels and melanomacrophage centres have been observed in other fish species
[8,26], but melanomacrophages were not distinctive in the wolffish tissues. Plasma cells were detected in both the outer and inner zones of the thymus in wolffish juveniles. Thymus was localized in the juveniles only, and the difficulty in finding thymus in adult fish could be due to age related involution, which is reported from other fish species [27]. The thymus is important for maturation of T-cells, at least in higher vertebrates. The appearance of cells of B-cell origin in this organ is disputed since the number of cells is low and it is also suggested that these cells may originate from blood. Some reports, however, conclude that plasma cells are present in the thymus of fish [8,28]. In wolffish thymus the stained cells were clearly associated to the lymphoid tissue and not to blood vessels. Plasma cells in the gut tissue of wolffish juveniles appeared later compared to kidney and spleen which is in the same order as detection of Igþ cells in sea
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bass [29]. Positively stained cells were located in the lamina propria of both the anterior and posterior gut which is in accordance with previous findings from carp [30], sea bass [24,31], cod [8] and turbot [26]. The existence of a common mucosal immune system in fish is suggested since cells involved in immune responses are present in the tissues of gut, gills and skin [25,32,33]. Mucosal Ig is qualitatively and quantitatively described in several fish species [34 – 38] and these molecules could either be produced locally or enter the epithelium from blood. Several authors report the presence of serological variants of IgM in mucus compared to serum, in addition to varying peak antibody responses in the two compartments after different antigen administration routes [35,39 – 42], and these antigenic variations are probably due to post-translational processes. Our study confirmed that plasma cells with secretory Cm transcripts are present in the mucosal compartments of adult wolffish but during ontogeny there is an obvious distinction in the appearance of plasma cells in the gut compared to the gill and skin compartments. The relatively early localization of these cells in the gut could be explained by a massive exposure to antigens through the gut lumen, since the wolffish larvae immediately after hatch feed on external nutrition, but one should expect gills and skin to be exposed to external antigens from the environment as well. An alternative explanation is that the ontogeny of immune cells is different in the various mucosal tissues. The protective role of gills and skin could therefore be of limited importance in the early life stages of spotted wolffish. However, results from sea bass, another perciform species [43], show that antibody production is induced in the gills of 0.1 g juveniles after immersion in bacterin, indicative of an early presence of immunocompetent cells in mucosal tissue. Further work on the spotted wolffish will elucidate if antibody production can be induced earlier in the gills and skin of juveniles exposed to bath vaccination and challenge experiments.
Acknowledgements This work was supported by The Research Council of Norway (project no. 124043/140).
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