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Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii
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Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii Q3
Maria Rosaria Coscia a, *, Palma Simoniello b, c, Stefano Giacomelli a, Umberto Oreste a, Chiara Maria Motta b a b c
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Institute of Protein Biochemistry, CNR, via P. Castellino 111, 80131 Naples, Italy Department of Biological Science, University of Naples Federico II, via Mezzocannone 8, 80134 Naples, Italy GSI Helmholtzzentrum für Schwerionenforschung, Planckstrasse 1, Darmstadt, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2013 Received in revised form 1 April 2014 Accepted 23 April 2014 Available online xxx
The presence and production of IgM in the skin of the Antarctic teleost Trematomus bernacchii were investigated in this study. Immunoglobulins purified from cutaneous mucus and analysed by SDS-PAGE run under non-reducing and reducing conditions, were composed of heavy and light chains of 78 kDa and 25 kDa respectively, with a relative molecular mass of 830 kDa indicating that mucus IgM are tetramers as the serum IgM. Mature transcripts encoding the constant domains of both the secretory and membrane-bound Igm chain were seen in T. bernacchii skin using a PCR strategy and the expression of the secretory Igm chain in the skin was compared with that in other tissues by Real-time PCR. Cytological investigations revealed the presence of either immunoglobulins or their transcripts in occasional lymphocytes distributed close to the basal membrane. IgM once produced here, enters the filamentcontaining cells and is released into the mucus when these cells degenerate and detach from the epidermis. Our findings indicate that a cutaneous defence mechanism, functioning as anatomical and physiological barrier under subzero conditions, is present in this Antarctic species as an important component of the immune system. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Skin immunity Cutaneous mucus Teleost IgM Skin morphology Immunohistochemistry In situ hybridization
1. Introduction Fish skin is a complex organ performing a variety of functions from homoeostatic to mechanical and from sensory perception to protection against pathogens [1]. Composed of a non-keratinized stratified epithelium of variable thickness, it is responsible for secreting mucus, a glycopeptide based protective structure. These glycopeptides change in amount and composition under varying stress and environmental conditions [2,3]. The mucous coat, produced mainly by goblet cells, covers the entire animal to facilitate swimming [4] and to reduce pathogen access to epithelial cells by constantly moving down stream and off the trailing edges [5]. Protection against pathogens is also due to its antimicrobial properties, particularly enzymes, lytic agents [6e8], and various immune components such as lysozyme, complement and immunoglobulins (Igs) [9]. These increase in response to immunization by both systemic [10] and bath immersion routes [11]
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* Corresponding author. Tel.: þ39 0816132556; fax: þ39 0816132629. E-mail address: [email protected] (M.R. Coscia).
and to pathogen attack [12] indicating the existence of a skinassociated lymphoid tissue (SALT) referred to also as the skin immune system (SIS) or cutaneous immune system. In fish, the SALT is a mucosal lymphoid tissue that is very different from that of mammals since having the outermost cells layers alive and actively dividing [13]. Although defensive role of skin Igs in fish is clear, it has been long debated whether they are secreted or diffuse passively into the mucus. In 1971 Di Conza and Halliday [14] suggested that mucus Igs were locally synthesized and were not derived from blood. Some recent evidence demonstrates the existence of differences in skin and plasma Ig m chains [12,15,16], suggesting the former are produced locally, and independently from systemic Igs [17]. These Igs are of m isotype as are those present in gut mucus, despite the latter being in small amounts perhaps due to degradation in this very strong proteolytic environment [18]. The skin therefore may be considered an integral component of the teleost immune system. The Nototheniids are teleosts species adapted to the extreme environmental conditions of the Antarctic Ocean [19]. We have previously highlighted in the secretory and membrane-bound form of IgM of serum and lymphoid tissues some peculiar features that
http://dx.doi.org/10.1016/j.fsi.2014.04.019 1050-4648/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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have evolved as compensatory adjustments in these species [20,21]. In this work we demonstrate the existence of a skin mucosal immunity in a red-blooded Antarctic Nototheniid, the emerald rockcod Trematomus bernacchii. The IgM from the skin and cutaneous mucus were isolated and characterised and the question of a possible local production was addressed by localising the IgM and their mRNA by immunohistochemistry and in situ hybridization, using antibodies and probes produced in our laboratory [22]. Our results indicate that in this species skin production of IgM occurs and that a mucosal immunity system is present. 2. Materials and methods 2.1. Species and sampling Adult specimens of T. bernacchii (Boulenger, 1902) were collected by nets at a depth ranging between 15 and 75 m in front of the Italian Station “Mario Zucchelli” at Terra Nova Bay during the XXV expedition of the Italian National Program for Antarctic Research (PNRA, 2009e2010). Fish were anaesthetised in aqueous MS222 (tricaine methane sulphonate) at a concentration of 1:15000 (w/v). Blood was collected by caudal venipuncture and serum, obtained by centrifugation at 2000 g for 10 min, was stored at 20 C. Mucus was carefully collected by gently wiping the body surface of ten T. bernacchii specimens with tissue paper to avoid damage of the skin and contamination with blood. Saturated tissues were placed in sterile 50-ml conical tubes containing approximately 10 ml of TBS buffer (Tris-buffered saline; 50 mM TriseHCl, pH 8.0, containing 150 mM NaCl) per tube. The content of each tube was homogenized and centrifuged at maximum speed for 15 min. The collected supernatants were pooled, filtered, and the absorbance (A280) was measured. Aliquots were frozen at 20 C. This pooled mucus sample was then processed as described below. Samples of skin, spleen, head kidney, intestine, heart, muscle, and liver were collected and processed for histology or, if required for molecular analyses, frozen in liquid nitrogen, and brought back to Italy at 80 C. The experiments were carried out in Italy in compliance with ethical provisions established by the European Union and authorized by the National Committee of the Italian Ministry of Health for in vivo experimentation (Department for Veterinary Public Health, Nutrition and Food Safety). All the experiments were organised to minimise pain and the number of animals used. 2.2. Purification and biochemical analysis of mucus IgM The mucus sample was thawed, vortexed and then centrifuged at 13,000 rpm for 30 min at 4 C. The supernatant was recovered and dialysed with 125 mM ammonium bicarbonate using 3.5 kDa cut off membrane tubing (SpectrumLabs, CA, USA). Protein concentration was determined by the Bio-Rad kit using bovine serum albumin as standard. IgM was purified from the dialysed mucus proteins as previously described [23]. T. bernacchii IgM concentration was determined in cutaneous mucus by ELISA from a standard titration curve constructed using purified serum T. bernacchii IgM as described in Ref. [23]. Purified IgM was analysed by SDS-PAGE under reducing and non-reducing conditions using a 10% or 5% polyacrylamide gel respectively. The gels were stained using 0.1% Coomassie Brilliant Blue R-250 or the Silver Stain kit (Bio-Rad). Proteins separated by SDS-PAGE were electrophoretically transferred onto nitrocellulose at 24 mA for 18 h; the nitrocellulose sheets were incubated with rabbit anti-T. bernacchii Ig m and/or L chain antisera raised as previously described in Ref. [22] for 1 h and following incubation with peroxidase labelled anti-rabbit antibodies for 1 h. Finally, the
specific binding was revealed by the ECL kit (GE Healthcare Lifescience) following manufacturer’s instructions. The preimmune serum, collected from the same animal in which the antiserum was raised, was used in control experiments. Analysis of the carbohydrate moiety of the mucus IgM was done using the DIG-Glycan Detection kit (Roche Molecular Biochemicals). 2.3. Purification and biochemical analysis of skin proteins and IgM Protein extraction from skin was carried out by placing a piece of frozen tissue in a cryogenic tube with 50 mM PBS, pH 7.5, containing 50 ml/ml protease inhibitor cocktail (Sigma); the sample was shaken for 2 min at 2000 Hz and centrifuged at 13,000 rpm for 90 min. The supernatant was collected and the protein content estimated using a kit from Bio-Rad. The extracted proteins were analysed by 10% SDS-PAGE and immunoblot as described in Section 2.2. 2.4. Preparation of skin samples for cytological analyses Skin samples for light microscopy were fixed in Bouin’s (6e12 h) and processed for wax embedding according to standard protocols. Samples for transmission electron microscopy were treated as previously described [24]. Briefly, they were fixed in Karnowsky solution [25] and post fixed for 2 h in 1% osmium tetroxide or in 2% glutaraldehyde buffered to pH 7.2 with 0.025 M PIPES [26] at room temperature for 6 h if intended for immunogold. All samples were then dehydrated in a graded series of saline alcohols and embedded in epoxy resin. Ultrathin sections were stained with lead citrate and uranyl acetate and observed with a Philips electron microscope. 2.5. Immunohistochemistry (IHC) Sections were washed in TBS (10 mM, pH 7.3) for 15 min, microwaved in citrate buffer (0.01 M, pH 6.0) for 6 min to unmask the antigens, washed in buffer (TBS, BSA 1%, Tween20 0.05%) for 15 min, in 3% H2O2 for 20 min to block endogenous peroxidases and in serum, for 15 min, to reduce non-specific binding. After that, they were incubated for 2 h, at 4 C, with the primary antibodies raised in rabbit against the T. bernacchii Igm and L chains [22], diluted 1:50 in PBS. Antibody binding was detected by a secondary goat antirabbit Ig antibody (1:200 in PBS, for 2 h), conjugated to peroxidase, intensified by incubation with a tertiary anti-PAP antibody (1:100 in PBS, 90 min) and revealed with DAB-urea. Negative controls were prepared by omitting the primary antibodies. The immunogold procedure was as described in Ref. [27]. Briefly, ultrathin sections were treated with sodium metaperiodate and incubated with the anti-T. bernacchii Igm antibody (diluted 1:200). Binding was detected using a secondary antibody linked to 10 nm colloidal gold particles. Sections were examined after mild staining with lead citrate and uranyl acetate. 2.6. In situ hybridisation (ISH) Sections post-fixed in paraformaldehyde (4% in PBS, pH 7.4) for 30 min were washed in PBS-H2ODEPC, then digested for 5 min in PK buffer (TriseHCl, 20 mM, pH 7.0, EDTA 1 mM, pH 8, proteinase K, 0.09 U/ml, H2ODEPC) at 37 C. After washing once in PBS, the sections were fixed again in paraformaldehyde then washed twice in PBS-H2ODEPC and in SSC (Saline tri-Sodium Citrate) and in Tris/ glycine buffer. The sections were incubated at 55 C for 1 h in a prehybridization mix containing formamide 40%, SSC 5, Denhart’s solution 1, 100 mg/ml Salmon testis DNA, 100 mg/ml tRNA in H2ODEPC. Hybridization was carried out at 55 C, overnight, using a dig-labelled riboprobe (see Section 2.5).
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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Sections were washed in SSC 5, SSC 0.5 and 20% formamide (1 h) and in NTE (NaCl 0.5 M, Tris/HCl 10 mM, EDTA 5 mM, 15 min). Free riboprobe was digested in NTE containing RNase A 0.3 U/ml for 30 min and washed away in NTE and SSC 0.5, 20% formamide. Finally, the sections were washed in SSC 2 for 30 min. Slides were then immerged for 30 min in a Blocking solution prepared with 2% BM blocking reagent (Roche), maleic acid 100 mM and H2Odist. Digoxigenin was revealed by incubating sections overnight with an AP-conjugated anti-dig antibody, diluted 1:400 in Blocking solution with 10% sheep serum (Sigma). The antibody was revealed with a staining mix (1 ml BM Purple, Roche, 10 ml levamisoleTween20 100). Reaction was stopped in PBS with 1 mM EDTA. 2.7. Amplification of secretory and membrane Igm chain transcripts Total RNA was isolated from skin using the SV total RNA isolation system (Promega), and subjected to reverse transcription using MMLV Reverse Transcriptase (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Double-stranded cDNA PCR amplification for secretory and membrane-bound Igm chain was carried out by using the following sense primer: hinge3, 50 TGTCTGTTTGAGGGGAAA-30 ; anti-sense primers: anti-CH4endTb, 50 -CGGGGATGTTCATGTTGAG-30 (for the secretory form); TManti, 50 -TCTACCTCTGTGCTTCCC-30 (for the membrane-bound form). The primers used were all designed on the T. bernacchii Igm chain sequence (AF094531), previously determined. The amplification was performed as follows: 95 C for 3 min, 30 cycles of 95 C (30 s), annealing 56 C (30 s), and 72 C (1 min) with a final extension at 72 C for 10 min. PCR products were visualised on 1% agarose gels using ethidium bromide staining and UV light transillumination. Products were then cloned into pSC-A by using the StrataClone PCR cloning kit (Stratagene) and positive clones were identified by blue/white selection. All sequences were determined on an ABI PRISM 3730 DNA Analyzer sequencer at Primm (Milan, Italy). For the IgL chain probe, the clone L180 (EF421439.1) in p-GEM-T Easy Vector (Promega) was used. Transcription of antisense and sense RNA probes was carried out with DIG RNA labeling kit (Roche).
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in triplicate each time. Data were normalised according to the Pfaffl method [28], by calculating the relative expression ratio as follows:
ðEAct ÞCt sample
.
EIgM
Ct sample
OðEAct ÞCt calibrator
.
EIgM
Ct calibrator
where b-actin was used as reference gene and the skin as a calibrator that all other samples were compared to. The PCR amplification conditions were: 95 C for 5 min, followed by 45 cycles consisting of 10 s denaturation at 95 C, 20 s annealing at 60 C, and 10 s extension at 72 C. In order to verify amplification specificity and the absence of primer dimers, a final dissociation step was run to generate a melting curve. In all melting curve analyses, single specific peaks were observed. The Real-time PCR products were subjected to gel electrophoresis using a 2% (w/v) agarose gel, further confirming that in all cases, the amplifications were specific and no amplification was observed in negative controls. All the results are reported as mean value of triplicates obtained in three independent experiment standard deviation. 3. Results 3.1. Biochemical analysis of skin and mucus IgM Total proteins in skin extracts showed an average concentration of 8.4 mg/ml. The presence of Igm and IgL chains was assessed by immunoblotting (Fig. 1) using a mixture of equal volumes of anti-m and anti-L rabbit antisera. Mean cutaneous mucus protein content was 1.2 mg/ml. Protein composition, analysed by SDS-PAGE (Fig. 2A), indicated the presence of several bands ranging between 78 and 25 kDa. The digoxigenin labelling method for carbohydrates (Fig. 2B) gave a positive reaction on the immobilized 78 kDa band, corresponding to the Igm chain, whereas the L chain band was clearly negative. An additional positive band (50 kDa), probably corresponding to a galectin family component, was observed. IgMs were purified from the dialysed mucus proteins by following a method previously used [23]. High relative molecular
2.8. Real-time PCR Total RNA was extracted from seven tissues (spleen, head kidney, intestine, skin, liver, heart and muscle) as described above, each tissue type pooled from five individuals. cDNA was then synthesised from 5 mg of total RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). Real-time PCR experiments were performed using the LightCycler 480 (Roche). The reaction consisted of 2 ml of the first strand cDNA mixed with 10 ml of LightCycler 480 SYBR Green I Master (Roche) in a final volume of 20 ml with a final concentration of 0.2 mM of each primer, according to the manufacturer’s instructions. Specific PCR primers were designed for the amplification of about 150 bp products from T. bernacchii Igm chain (AF094531): rtFR4.2 (sense primer specific for VH region), 50 TGGGGAAAAGGAACAATGGTG-30 ; rtCH1M1 (anti-sense primer specific for Cm region), 50 -AGGTCAGCGGAGACGGTGC-30 ; b-actin (GQ229124.1) was used as housekeeping gene: BACTfw (sense primer), 50 -CCCAGATCATGTTCGAGACC-30 ; BACTrev (anti-sense primer), 50 -CAGCGACGGACAGGAAG-30 . The reaction conditions were established through a series of preliminary optimization experiments. Firstly the efficiency for each primer couple (EAct for bactin, and EIgM for Igm chain) was tested using serial diluitions of spleen cDNA: EAct was 1.92, while EIgM was 1.88. Real-time PCR experiments were performed three times and all tissue samples, including water as non-template control, were run
Fig. 1. Immunoblotting analysis of skin IgM. A mixture of equal volumes of antiT. bernacchii Ig m and L chain antisera was used to reveal the chains whose relative molecular mass is reported on the left.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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Fig. 2. Biochemical analysis of total cutaneous mucus proteins. A) SDS-PAGE on 10% acrylamide gel run under reducing conditions and stained with Coomassie Brilliant Blue R-250. M: Broad range markers (Bio-Rad). B) Analysis of carbohydrate moiety in the Ig m and L chains by the digoxigenin labelling method.
mass components were fractionated by FPLC (Fig. 3A); the main peak was analysed by SDS-PAGE run without reducing agents, and its relative molecular mass was estimated to be 830 kDa (Fig. 3B). The 830-kDa fraction was further purified by affinity chromatography using a column coupled with rabbit anti-T. bernacchii Ig m chain antibodies. To determine the mean concentration of IgM, skin mucus was collected from ten adult specimens and analysed by ELISA as described in Ref. [23]. IgM concentrations were calculated from the standard titration curve obtained by using purified T. bernacchii serum IgM and found to be 135 mg/ml. SDS-PAGE analysis of purified mucus IgM performed under reducing conditions (Fig. 4A) gave two bands with a relative molecular mass of 78 kDa and 25 kDa, corresponding to Ig m and L chains respectively as found for serum IgM [23]. These results were confirmed by immunoblotting performed using rabbit antiserum specific for T. bernacchii Ig m or L chains (Fig. 4B) and preimmune serum, as negative control, collected from the same animal in which the antiserum was raised (Fig. 4B).
Fig. 3. Purification of T. bernacchii cutaneous mucus Ig. A) FPLC elution profile of mucus Ig (upper), using a Bio-Prep SE-1000/17 column (Bio-Rad) precalibrated with marker proteins (Gel filtration standard, Bio-Rad): Thyroglobulin (670,000), g-globulin (158,000), Ovalbumin (44,000), Myoglobin (17,000), Vitamin B12 (1350). Black arrows indicate the elution position of the molecular weight markers. Immunoblotting analysis (lower) of the three main peaks (indicated with solid horizontal lines with the number on the top) performed with rabbit anti-T. bernacchii Ig m antiserum. B) SDSPAGE on 5% polyacrylamide gel under non-reducing conditions of the FPLC major peak (indicated by red arrow in A) containing most IgM, whose relative molecular mass was estimated to be 830 kDa. The gel was stained using the Silver Stain kit (BioRad). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
cytoplasm of the filament-containing cells (Fig. 6AeD) but not in the goblet cells (Fig. 6A, C) that remained unlabelled as well as all cells in negative controls (Fig. 6EeF) prepared by omitting the primary antibodies. The distribution of the labelled filament-containing cells in the skin varied significantly in the different samples obtained from different animals. IgMs were uniformly dispersed in all cells of the basal and intermediate strata which were mildly positive to the IHC reaction (Fig. 6A, C, D). In several cases however, labelling appeared very intense (Fig. 6G, H) or localised on isolated cells of
3.2. Skin morphology The integument in T. bernacchii is particularly thick and characterised by a multilayered epidermis (Fig. 5AeB) composed mostly of filament-containing cells. Their shape is significantly varied with those located close to the basal membrane typically columnar; those present in the intermediate stratum were approximately spherical and those located in the superficial stratum (pavement cells) tended to be more flattened (Fig. 5B). In the epidermis, large mucous (goblet) cells could also be recognized (Fig. 5C). The dermis was thin and contained scales (Fig. 5A) and occasional melanophores (Fig. 9D). 3.3. Immunolocalisation of IgM in T. bernacchii skin Immunocytochemistry indicated that Ig m and L chains were abundant in the mucus (Fig. 6A, C) and in the perinuclear cytoplasm of occasional lymphocytes located close to the basal membrane (Fig. 6AeD). A pale but significant labelling was also present in the
Fig. 4. Biochemical analysis of T. bernacchii cutaneous mucus purified IgM. A) SDSPAGE on 10% acrylamide gel run under reducing conditions using broad range markers (Bio-Rad). Staining with Coomassie Brilliant Blue R-250. B) Immunoblotting analysis with rabbit anti-T. bernacchii Ig m or L chain antisera. Purified serum IgM are reported for comparison. NC: the preimmune serum, collected from the same animal in which the antiserum was raised, was used as negative control.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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3.4. Localisation of the IgM mRNA in T. bernacchii skin In situ hybridization with an anti-sense mRNA demonstrated the expression of both Ig L (Fig. 9AeB) and m (Fig. 9C) chains in occasional lymphocytes located close to the basal membrane. Filamentcontaining cells and goblets cells were always unstained. No signal was detected in negative controls when hybridization was carried out using a sense probe (Fig. 9D). Fig. 5. Skin morphology in T. bernacchii. A) Multi-layered epidermis (e) and scales (arrows) in a thin dermis. B) Detail of the epidermis showing the filament-containing cells changing their shape from columnar (*), close to the basal membrane (arrow), to flattened (**), close to the surface. Notice the absence in this area of goblet cells. Arrow: basal membrane. C) Epidermis with several goblet cells (arrow) among the filamentcontaining cells (*).
the intermediate layer (Fig. 6I) and on cells forming a monolayer between the intermediate and superficial stratum (Fig. 6J). In all samples, Igs were absent from cells of the superficial stratum (Fig. 6A, C, GeJ) and electron microscopy using immunogold was carried out to verify this observation. Transmission electron microscopy revealed that the filament-containing cells (Fig. 7A) had a cytoplasm rich in mitochondria, endoplasmic reticulum and vesicles containing a filamentous material. The cortical cytoplasm was characterised by a thick layer of regularly arranged microfilaments. Adjacent cells were interconnected by several desmosomes and showed extended membrane interdigitations. Pavement cells, the filament-containing cells in contact with the sea water also showed typical short microridges protruding in the mucus (Fig. 7CeD). Immunogold labelling demonstrated the presence of IgM in all these cells (Fig. 7B, D). In contrast, no IgM could be observed in either the cytoplasm or the secretory granules of goblet cells (Fig. 7EeG). Several of the pavement cells showed clear signs of degeneration including disorganised cytoplasm, pale microfilaments and loss of microridges (Fig. 8A). In these cells the cytoplasm was still rich in Igs as demonstrated by the presence of gold particles (Fig. 8B). Many cells were undergoing shedding/desquamation (Fig. 8C).
3.5. Gene expression analysis Mature transcripts encoding the constant domains of the secretory and membrane-bound Igm chain were observed in T. bernacchii skin using a PCR strategy (Fig. 10). A 450-bp fragment of the membrane-bound form and an 800bp fragment of the secretory form were amplified by using respective specific primers. The two PCR products were cloned and sequenced, confirming that both were the expected ones. Furthermore, the expression levels for the secretory Igm chain were analysed in T. bernacchii skin and compared to other tissues (spleen, head kidney, intestine, skin, liver, heart, and muscle) by Real-time PCR. The Igm chain expression levels were normalised to b-actin levels and are shown in Fig. 11. Igm transcripts were found to be expressed at the highest levels in the head kidney, followed by the spleen. The overall expression levels of the Igm chain in the skin were found to be significantly lower than the levels measured in the head kidney. 4. Discussion It is well known that IgM are the predominant immunoglobulin in the serum of teleost fishes. In this work we have investigated the presence of IgM in the skin of the Antarctic teleost T. bernacchii and have shown that IgMs are present in the cutaneous mucus at higher concentration, 135 mg/ml vs 8e90 mg/ml reported in other species [13], therefore likely play also an important role as defence molecules. These IgMs are very similar to those present in the serum
Fig. 6. Immunolocalisation of Ig L (AeB) and m (C,D, GeJ) chains in the skin of T. bernacchii. A, C) Epidermis with labelled mucus (*) and lymphocytes (arrows). Filament-containing cells (**) show a diffuse labelling while goblet cells (white arrow) are always completely unstained. B, D) Details showing the labelled lymphocytes. E, F) negative controls for anti-L and anti-m chains antibodies respectively. All cells (*) are completely unstained; nuclei are counterstained with haematoxylin. GeJ) distribution of Ig m in the dorsal epidermis of different animals. Labelling may be diffused (G) or very intense (H) on basal filament-containing cells (**); it may be located on occasional filament-containing cell (I, arrow) of the intermediate stratum or on cells of the intermediate stratum organised in a monolayer (J, arrow). Mucus (*) is always stained, goblet cells (white arrow) always unstained.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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Fig. 7. Immunogold localisation of Ig m chain in T. bernacchii skin. A) Filament-containing cells connected by desmosomes (arrows) and interdigitations (white arrow). The cytoplasm, rich in mitochondria (m) and vesicles (v), is surrounded by a thick layer of microfilaments (*). B) Ig m chains (gold particles, small black dots) in the cytoplasm of filamentcontaining cells. Note the desmosomes (arrows), the endoplasmic reticulum (r), the microfilament layer (*) and the dense nucleus (n). C) Pavement cell with microridges (m). Nucleus (n) and cytoplasm (c). D) Ig chains (small black dots) in the cytoplasm of a pavement cell; microridge (m), nucleus (n), cytoplasm filled with organelles (c) and microfilaments (*). E) Large goblet cell (*) among filament-containing cells (**). Note the basal nucleus (n) and the cytoplasm engulfed with secretory vesicles (*). F) Detail of the unlabelled secretory vesicles (*). Note the presence of Igs (small black dots, arrow) in the cytoplasm of an adjacent filament-containing cell (**). G) Further detail of the unlabelled secretory vesicles (*) of a goblet cell. Unlabelled endoplasmic reticulum (r).
being exclusively tetrameric, with m and L chains having the same relative molecular mass, and composition of the carbohydrate moieties, despite dimeric Igs being also observed in the skin mucus of sheephead [29]. No redox forms of tetrameric IgM that had been previously found in T. bernacchii serum [23] or the unique redox form consisting of halfmeric components H1L1 of relative molecular mass of 100 kDa, seen in rainbow trout skin mucus [30], have been observed in the skin mucus of T. bernacchii. Thus, mucus IgMs do not show significantly different protein assembly or antigenicity in comparison to the serum IgM. This conclusion is also supported by the fact that skin mucus IgMs were detected with rabbit antisera raised against m or L chains purified from serum IgM.
As described for the majority of teleost species, the PCR results confirmed those previously obtained that mRNA splicing, leading to the exclusion of the CH4 exon and thus to a shorter transcript for the membrane-bound form, occurs in T. bernacchii [20]. Detection of the IgM membrane-bound form in the skin, besides the secretory form, suggests that B cells are present in the skin and in turn the complexity of the adaptive immune response is already present in teleost fish skin. The overall expression levels of the Igm chain were analysed by Real-time PCR: the values obtained for skin and intestine were significantly lower than those collected for head kidney and spleen, the former being well known sites of the mucosal immunity in which other Ig isotypes have a predominant role [31,32]. The value relative to muscle is surprising and needs to be
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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Fig. 8. Immunogold localisation of Ig m chains in degenerating pavement cells of T. bernacchii skin. A) Cell with pale microfilaments (*) and clearly disorganised cytoplasm (c). Healthy cells (**). B) Detail of the cytoplasm (c) of a degenerating pavement cell showing the presence of Igs (small black dots, arrow). Microfilaments (*), cytoplasm (c). C) Pavement cell (arrow) detaching from the skin (*).
low levels in the skin and gill [33]. More importantly, the higher expression of IgM observed by Real-time PCR experiments in the head kidney compared to the spleen and gut might be attributed to the first appearance of Ig-producing cells in the head kidney, which mature here and then migrate to sites of activation, either the spleen or the mucosal compartment [34]. Recent gene expression studies in zebrafish have shown IgM as more predominant in both lymphoid and non-lymphoid organs when compared to IgZ/T [35], a new isotype found in several teleosts [36,37] and shown to be the prevalent isotype in the gut [31] and skin mucus [32] of rainbow trout. This indicates that fish IgT play a specialised role in mucosal immunity. It remains to be verified whether in T. bernacchii other components of the cutaneous immune system are also present, although IgT has been recently identified in the skin of two Antarctic fish species (Coscia et al., manuscript in preparation).
Fig. 9. Localisation of Ig L (AeB) and m (C) chain mRNAs in the skin of T. bernacchii. A, B) labelling is on occasional lymphocytes (arrow) located in the basal epidermis. Filament-containing cells (*) and goblet cells (g) are unstained. C) labelled lymphocytes (arrows) among negative filament-containing cells (*). Messenger is concentrated in the cytoplasm. D) negative control hybridised with a sense probe with no labelling observed (*). Melanophores (arrowheads) and scales (s) in the dermis.
further investigated taking into account that Antarctic fish are heavily parasitised [22], and thus immune response against pathogens may take also place in tissues other than lymphoid ones. Moreover, these results may vary according to different bacterial or parasitic infections. In adult fish, IgM is constitutively expressed in renal haematopoietic tissue, spleen and thymus, but expressed at
Fig. 10. RT-PCR amplification for detection of the membrane-bound (lane 1) and secretory (lane 2) IgM forms. M: GeneRuler 100 bp DNA ladder (Fermentas).
Fig. 11. Analysis by Real-time PCR of relative expression levels of Igm transcripts in different tissues. The results are reported as folds of difference relative to skin. Data were normalised to b-actin using the Pfaffl method, considering efficiency value for each primer couple. The results are reported as mean value of triplicates obtained in three independent experiment standard deviation.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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The cytological investigations revealed that T. bernacchii skin has a thick, multilayered epidermis which provides a multifunctional barrier and may represent an adaptation to the hostile, cold environment in protection against freezing [38,39]. Thick skin, typical in relatively cold waters is also present in other species such as the trout [40]. It is evident from the ISH analyses that skin IgMs are produced in situ by lymphocytes localised in the basal epidermis as Igm and L chains and their respective mRNAs were present thereby confirming the local production of functional IgM. Ig diffusion from lymphocytes would spread throughout the epidermis to reach the apical mucus. This movement is similar to that seen in other fish species for example, in the skin of fugu (Takifugu rubripes), common carp (Cyprinus carpio), and orangespotted grouper (Epinephelus coioides) [15,41,42]. Ig secreted into bile from the liver of T. bernacchii [43] and other teleosts [44,45], also diffuses in this manner. Diffusion through the extracellular spaces would be discouraged by the presence of desmosomes and interdigitations among adjacent cells. Goblet cells would not be involved as they are always negative to the anti-Ig antibodies and probes. Detailed Ig transport mechanisms are yet to be clarified. Polymeric immunoglobulin receptor (pIgR) is probably involved and would be responsible for transporting and protecting the secretory Ig from proteolytic degradation [46]. A gene encoding a pIgR has been isolated from several teleost fish species [15,42,46,47]. Teleost skin thus adopts the same Ig transport system as mammalian intestine or other mucosal sites, via a unique pIgR, expressed on the surface of epithelial cells, whose secretory component size seems to be species-specific (w60 kDa in fugu, w95 kDa in sheepshead, w35 kDa in trout) [15,29,32]. The release of Ig from the skin into the mucous coat may occur in two different ways. The first is that it simply diffuses, released by the skin pIgR. With this respect, the efficiency of the secretion would be increased by the flattening of the pavement cells and the presence of microridges. The second possibility is that the release occurs when the degenerating pavement cells undergo sloughing. Such cells, in fact, are still rich in Ig. Their asynchronous dismission would ensure a continuous supply to the skin mucus of new Ig and, probably, also of enzymes and various other substances. An interesting aspect emerging from our observations is the Ig distribution pattern in the different samples examined. Since they all come from a dorsal area of the trunk, one possible explanation is the existence of significant variation among different individuals. Such differences could represent a sort of fingerprint left by a previous pathogen attack or perhaps, one in progress. It should be mentioned that in several animals the liver appeared infested by nematode parasites, and a clear immune response against them in Antarctic fish has been previously well documented [22,48]. The differences observed would not depend on sex even though the majority of animals examined were females. As such, we currently have only a partial picture of the skin secretory immune system in these fish. How Igs interact with antigens at the mucosal surface constantly exposed to water and swimming forces remains completely unknown but an adaptation in antigeneantibody interactions might be expected in such a particular mucosal environment. This work demonstrates the existence of a local immunity based on the production of IgM in the skin of an Antarctic teleost species. The protection is highly efficient due to the high concentration of IgM. The Antarctic environment is very hostile with competition for resources particularly severe and with a high parasite infection load. Thus, efficient protection against pathogens may represent a significant advantage to survive under extreme conditions.
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Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii Q3
Maria Rosaria Coscia a, *, Palma Simoniello b, c, Stefano Giacomelli a, Umberto Oreste a, Chiara Maria Motta b a b c
Q2
Institute of Protein Biochemistry, CNR, via P. Castellino 111, 80131 Naples, Italy Department of Biological Science, University of Naples Federico II, via Mezzocannone 8, 80134 Naples, Italy GSI Helmholtzzentrum für Schwerionenforschung, Planckstrasse 1, Darmstadt, Germany
a r t i c l e i n f o
a b s t r a c t
Article history: Received 6 December 2013 Received in revised form 1 April 2014 Accepted 23 April 2014 Available online xxx
The presence and production of IgM in the skin of the Antarctic teleost Trematomus bernacchii were investigated in this study. Immunoglobulins purified from cutaneous mucus and analysed by SDS-PAGE run under non-reducing and reducing conditions, were composed of heavy and light chains of 78 kDa and 25 kDa respectively, with a relative molecular mass of 830 kDa indicating that mucus IgM are tetramers as the serum IgM. Mature transcripts encoding the constant domains of both the secretory and membrane-bound Igm chain were seen in T. bernacchii skin using a PCR strategy and the expression of the secretory Igm chain in the skin was compared with that in other tissues by Real-time PCR. Cytological investigations revealed the presence of either immunoglobulins or their transcripts in occasional lymphocytes distributed close to the basal membrane. IgM once produced here, enters the filamentcontaining cells and is released into the mucus when these cells degenerate and detach from the epidermis. Our findings indicate that a cutaneous defence mechanism, functioning as anatomical and physiological barrier under subzero conditions, is present in this Antarctic species as an important component of the immune system. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Skin immunity Cutaneous mucus Teleost IgM Skin morphology Immunohistochemistry In situ hybridization
1. Introduction Fish skin is a complex organ performing a variety of functions from homoeostatic to mechanical and from sensory perception to protection against pathogens [1]. Composed of a non-keratinized stratified epithelium of variable thickness, it is responsible for secreting mucus, a glycopeptide based protective structure. These glycopeptides change in amount and composition under varying stress and environmental conditions [2,3]. The mucous coat, produced mainly by goblet cells, covers the entire animal to facilitate swimming [4] and to reduce pathogen access to epithelial cells by constantly moving down stream and off the trailing edges [5]. Protection against pathogens is also due to its antimicrobial properties, particularly enzymes, lytic agents [6e8], and various immune components such as lysozyme, complement and immunoglobulins (Igs) [9]. These increase in response to immunization by both systemic [10] and bath immersion routes [11]
Q1
* Corresponding author. Tel.: þ39 0816132556; fax: þ39 0816132629. E-mail address: [email protected] (M.R. Coscia).
and to pathogen attack [12] indicating the existence of a skinassociated lymphoid tissue (SALT) referred to also as the skin immune system (SIS) or cutaneous immune system. In fish, the SALT is a mucosal lymphoid tissue that is very different from that of mammals since having the outermost cells layers alive and actively dividing [13]. Although defensive role of skin Igs in fish is clear, it has been long debated whether they are secreted or diffuse passively into the mucus. In 1971 Di Conza and Halliday [14] suggested that mucus Igs were locally synthesized and were not derived from blood. Some recent evidence demonstrates the existence of differences in skin and plasma Ig m chains [12,15,16], suggesting the former are produced locally, and independently from systemic Igs [17]. These Igs are of m isotype as are those present in gut mucus, despite the latter being in small amounts perhaps due to degradation in this very strong proteolytic environment [18]. The skin therefore may be considered an integral component of the teleost immune system. The Nototheniids are teleosts species adapted to the extreme environmental conditions of the Antarctic Ocean [19]. We have previously highlighted in the secretory and membrane-bound form of IgM of serum and lymphoid tissues some peculiar features that
http://dx.doi.org/10.1016/j.fsi.2014.04.019 1050-4648/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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have evolved as compensatory adjustments in these species [20,21]. In this work we demonstrate the existence of a skin mucosal immunity in a red-blooded Antarctic Nototheniid, the emerald rockcod Trematomus bernacchii. The IgM from the skin and cutaneous mucus were isolated and characterised and the question of a possible local production was addressed by localising the IgM and their mRNA by immunohistochemistry and in situ hybridization, using antibodies and probes produced in our laboratory [22]. Our results indicate that in this species skin production of IgM occurs and that a mucosal immunity system is present. 2. Materials and methods 2.1. Species and sampling Adult specimens of T. bernacchii (Boulenger, 1902) were collected by nets at a depth ranging between 15 and 75 m in front of the Italian Station “Mario Zucchelli” at Terra Nova Bay during the XXV expedition of the Italian National Program for Antarctic Research (PNRA, 2009e2010). Fish were anaesthetised in aqueous MS222 (tricaine methane sulphonate) at a concentration of 1:15000 (w/v). Blood was collected by caudal venipuncture and serum, obtained by centrifugation at 2000 g for 10 min, was stored at 20 C. Mucus was carefully collected by gently wiping the body surface of ten T. bernacchii specimens with tissue paper to avoid damage of the skin and contamination with blood. Saturated tissues were placed in sterile 50-ml conical tubes containing approximately 10 ml of TBS buffer (Tris-buffered saline; 50 mM TriseHCl, pH 8.0, containing 150 mM NaCl) per tube. The content of each tube was homogenized and centrifuged at maximum speed for 15 min. The collected supernatants were pooled, filtered, and the absorbance (A280) was measured. Aliquots were frozen at 20 C. This pooled mucus sample was then processed as described below. Samples of skin, spleen, head kidney, intestine, heart, muscle, and liver were collected and processed for histology or, if required for molecular analyses, frozen in liquid nitrogen, and brought back to Italy at 80 C. The experiments were carried out in Italy in compliance with ethical provisions established by the European Union and authorized by the National Committee of the Italian Ministry of Health for in vivo experimentation (Department for Veterinary Public Health, Nutrition and Food Safety). All the experiments were organised to minimise pain and the number of animals used. 2.2. Purification and biochemical analysis of mucus IgM The mucus sample was thawed, vortexed and then centrifuged at 13,000 rpm for 30 min at 4 C. The supernatant was recovered and dialysed with 125 mM ammonium bicarbonate using 3.5 kDa cut off membrane tubing (SpectrumLabs, CA, USA). Protein concentration was determined by the Bio-Rad kit using bovine serum albumin as standard. IgM was purified from the dialysed mucus proteins as previously described [23]. T. bernacchii IgM concentration was determined in cutaneous mucus by ELISA from a standard titration curve constructed using purified serum T. bernacchii IgM as described in Ref. [23]. Purified IgM was analysed by SDS-PAGE under reducing and non-reducing conditions using a 10% or 5% polyacrylamide gel respectively. The gels were stained using 0.1% Coomassie Brilliant Blue R-250 or the Silver Stain kit (Bio-Rad). Proteins separated by SDS-PAGE were electrophoretically transferred onto nitrocellulose at 24 mA for 18 h; the nitrocellulose sheets were incubated with rabbit anti-T. bernacchii Ig m and/or L chain antisera raised as previously described in Ref. [22] for 1 h and following incubation with peroxidase labelled anti-rabbit antibodies for 1 h. Finally, the
specific binding was revealed by the ECL kit (GE Healthcare Lifescience) following manufacturer’s instructions. The preimmune serum, collected from the same animal in which the antiserum was raised, was used in control experiments. Analysis of the carbohydrate moiety of the mucus IgM was done using the DIG-Glycan Detection kit (Roche Molecular Biochemicals). 2.3. Purification and biochemical analysis of skin proteins and IgM Protein extraction from skin was carried out by placing a piece of frozen tissue in a cryogenic tube with 50 mM PBS, pH 7.5, containing 50 ml/ml protease inhibitor cocktail (Sigma); the sample was shaken for 2 min at 2000 Hz and centrifuged at 13,000 rpm for 90 min. The supernatant was collected and the protein content estimated using a kit from Bio-Rad. The extracted proteins were analysed by 10% SDS-PAGE and immunoblot as described in Section 2.2. 2.4. Preparation of skin samples for cytological analyses Skin samples for light microscopy were fixed in Bouin’s (6e12 h) and processed for wax embedding according to standard protocols. Samples for transmission electron microscopy were treated as previously described [24]. Briefly, they were fixed in Karnowsky solution [25] and post fixed for 2 h in 1% osmium tetroxide or in 2% glutaraldehyde buffered to pH 7.2 with 0.025 M PIPES [26] at room temperature for 6 h if intended for immunogold. All samples were then dehydrated in a graded series of saline alcohols and embedded in epoxy resin. Ultrathin sections were stained with lead citrate and uranyl acetate and observed with a Philips electron microscope. 2.5. Immunohistochemistry (IHC) Sections were washed in TBS (10 mM, pH 7.3) for 15 min, microwaved in citrate buffer (0.01 M, pH 6.0) for 6 min to unmask the antigens, washed in buffer (TBS, BSA 1%, Tween20 0.05%) for 15 min, in 3% H2O2 for 20 min to block endogenous peroxidases and in serum, for 15 min, to reduce non-specific binding. After that, they were incubated for 2 h, at 4 C, with the primary antibodies raised in rabbit against the T. bernacchii Igm and L chains [22], diluted 1:50 in PBS. Antibody binding was detected by a secondary goat antirabbit Ig antibody (1:200 in PBS, for 2 h), conjugated to peroxidase, intensified by incubation with a tertiary anti-PAP antibody (1:100 in PBS, 90 min) and revealed with DAB-urea. Negative controls were prepared by omitting the primary antibodies. The immunogold procedure was as described in Ref. [27]. Briefly, ultrathin sections were treated with sodium metaperiodate and incubated with the anti-T. bernacchii Igm antibody (diluted 1:200). Binding was detected using a secondary antibody linked to 10 nm colloidal gold particles. Sections were examined after mild staining with lead citrate and uranyl acetate. 2.6. In situ hybridisation (ISH) Sections post-fixed in paraformaldehyde (4% in PBS, pH 7.4) for 30 min were washed in PBS-H2ODEPC, then digested for 5 min in PK buffer (TriseHCl, 20 mM, pH 7.0, EDTA 1 mM, pH 8, proteinase K, 0.09 U/ml, H2ODEPC) at 37 C. After washing once in PBS, the sections were fixed again in paraformaldehyde then washed twice in PBS-H2ODEPC and in SSC (Saline tri-Sodium Citrate) and in Tris/ glycine buffer. The sections were incubated at 55 C for 1 h in a prehybridization mix containing formamide 40%, SSC 5, Denhart’s solution 1, 100 mg/ml Salmon testis DNA, 100 mg/ml tRNA in H2ODEPC. Hybridization was carried out at 55 C, overnight, using a dig-labelled riboprobe (see Section 2.5).
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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Sections were washed in SSC 5, SSC 0.5 and 20% formamide (1 h) and in NTE (NaCl 0.5 M, Tris/HCl 10 mM, EDTA 5 mM, 15 min). Free riboprobe was digested in NTE containing RNase A 0.3 U/ml for 30 min and washed away in NTE and SSC 0.5, 20% formamide. Finally, the sections were washed in SSC 2 for 30 min. Slides were then immerged for 30 min in a Blocking solution prepared with 2% BM blocking reagent (Roche), maleic acid 100 mM and H2Odist. Digoxigenin was revealed by incubating sections overnight with an AP-conjugated anti-dig antibody, diluted 1:400 in Blocking solution with 10% sheep serum (Sigma). The antibody was revealed with a staining mix (1 ml BM Purple, Roche, 10 ml levamisoleTween20 100). Reaction was stopped in PBS with 1 mM EDTA. 2.7. Amplification of secretory and membrane Igm chain transcripts Total RNA was isolated from skin using the SV total RNA isolation system (Promega), and subjected to reverse transcription using MMLV Reverse Transcriptase (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Double-stranded cDNA PCR amplification for secretory and membrane-bound Igm chain was carried out by using the following sense primer: hinge3, 50 TGTCTGTTTGAGGGGAAA-30 ; anti-sense primers: anti-CH4endTb, 50 -CGGGGATGTTCATGTTGAG-30 (for the secretory form); TManti, 50 -TCTACCTCTGTGCTTCCC-30 (for the membrane-bound form). The primers used were all designed on the T. bernacchii Igm chain sequence (AF094531), previously determined. The amplification was performed as follows: 95 C for 3 min, 30 cycles of 95 C (30 s), annealing 56 C (30 s), and 72 C (1 min) with a final extension at 72 C for 10 min. PCR products were visualised on 1% agarose gels using ethidium bromide staining and UV light transillumination. Products were then cloned into pSC-A by using the StrataClone PCR cloning kit (Stratagene) and positive clones were identified by blue/white selection. All sequences were determined on an ABI PRISM 3730 DNA Analyzer sequencer at Primm (Milan, Italy). For the IgL chain probe, the clone L180 (EF421439.1) in p-GEM-T Easy Vector (Promega) was used. Transcription of antisense and sense RNA probes was carried out with DIG RNA labeling kit (Roche).
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in triplicate each time. Data were normalised according to the Pfaffl method [28], by calculating the relative expression ratio as follows:
ðEAct ÞCt sample
.
EIgM
Ct sample
OðEAct ÞCt calibrator
.
EIgM
Ct calibrator
where b-actin was used as reference gene and the skin as a calibrator that all other samples were compared to. The PCR amplification conditions were: 95 C for 5 min, followed by 45 cycles consisting of 10 s denaturation at 95 C, 20 s annealing at 60 C, and 10 s extension at 72 C. In order to verify amplification specificity and the absence of primer dimers, a final dissociation step was run to generate a melting curve. In all melting curve analyses, single specific peaks were observed. The Real-time PCR products were subjected to gel electrophoresis using a 2% (w/v) agarose gel, further confirming that in all cases, the amplifications were specific and no amplification was observed in negative controls. All the results are reported as mean value of triplicates obtained in three independent experiment standard deviation. 3. Results 3.1. Biochemical analysis of skin and mucus IgM Total proteins in skin extracts showed an average concentration of 8.4 mg/ml. The presence of Igm and IgL chains was assessed by immunoblotting (Fig. 1) using a mixture of equal volumes of anti-m and anti-L rabbit antisera. Mean cutaneous mucus protein content was 1.2 mg/ml. Protein composition, analysed by SDS-PAGE (Fig. 2A), indicated the presence of several bands ranging between 78 and 25 kDa. The digoxigenin labelling method for carbohydrates (Fig. 2B) gave a positive reaction on the immobilized 78 kDa band, corresponding to the Igm chain, whereas the L chain band was clearly negative. An additional positive band (50 kDa), probably corresponding to a galectin family component, was observed. IgMs were purified from the dialysed mucus proteins by following a method previously used [23]. High relative molecular
2.8. Real-time PCR Total RNA was extracted from seven tissues (spleen, head kidney, intestine, skin, liver, heart and muscle) as described above, each tissue type pooled from five individuals. cDNA was then synthesised from 5 mg of total RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche). Real-time PCR experiments were performed using the LightCycler 480 (Roche). The reaction consisted of 2 ml of the first strand cDNA mixed with 10 ml of LightCycler 480 SYBR Green I Master (Roche) in a final volume of 20 ml with a final concentration of 0.2 mM of each primer, according to the manufacturer’s instructions. Specific PCR primers were designed for the amplification of about 150 bp products from T. bernacchii Igm chain (AF094531): rtFR4.2 (sense primer specific for VH region), 50 TGGGGAAAAGGAACAATGGTG-30 ; rtCH1M1 (anti-sense primer specific for Cm region), 50 -AGGTCAGCGGAGACGGTGC-30 ; b-actin (GQ229124.1) was used as housekeeping gene: BACTfw (sense primer), 50 -CCCAGATCATGTTCGAGACC-30 ; BACTrev (anti-sense primer), 50 -CAGCGACGGACAGGAAG-30 . The reaction conditions were established through a series of preliminary optimization experiments. Firstly the efficiency for each primer couple (EAct for bactin, and EIgM for Igm chain) was tested using serial diluitions of spleen cDNA: EAct was 1.92, while EIgM was 1.88. Real-time PCR experiments were performed three times and all tissue samples, including water as non-template control, were run
Fig. 1. Immunoblotting analysis of skin IgM. A mixture of equal volumes of antiT. bernacchii Ig m and L chain antisera was used to reveal the chains whose relative molecular mass is reported on the left.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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Fig. 2. Biochemical analysis of total cutaneous mucus proteins. A) SDS-PAGE on 10% acrylamide gel run under reducing conditions and stained with Coomassie Brilliant Blue R-250. M: Broad range markers (Bio-Rad). B) Analysis of carbohydrate moiety in the Ig m and L chains by the digoxigenin labelling method.
mass components were fractionated by FPLC (Fig. 3A); the main peak was analysed by SDS-PAGE run without reducing agents, and its relative molecular mass was estimated to be 830 kDa (Fig. 3B). The 830-kDa fraction was further purified by affinity chromatography using a column coupled with rabbit anti-T. bernacchii Ig m chain antibodies. To determine the mean concentration of IgM, skin mucus was collected from ten adult specimens and analysed by ELISA as described in Ref. [23]. IgM concentrations were calculated from the standard titration curve obtained by using purified T. bernacchii serum IgM and found to be 135 mg/ml. SDS-PAGE analysis of purified mucus IgM performed under reducing conditions (Fig. 4A) gave two bands with a relative molecular mass of 78 kDa and 25 kDa, corresponding to Ig m and L chains respectively as found for serum IgM [23]. These results were confirmed by immunoblotting performed using rabbit antiserum specific for T. bernacchii Ig m or L chains (Fig. 4B) and preimmune serum, as negative control, collected from the same animal in which the antiserum was raised (Fig. 4B).
Fig. 3. Purification of T. bernacchii cutaneous mucus Ig. A) FPLC elution profile of mucus Ig (upper), using a Bio-Prep SE-1000/17 column (Bio-Rad) precalibrated with marker proteins (Gel filtration standard, Bio-Rad): Thyroglobulin (670,000), g-globulin (158,000), Ovalbumin (44,000), Myoglobin (17,000), Vitamin B12 (1350). Black arrows indicate the elution position of the molecular weight markers. Immunoblotting analysis (lower) of the three main peaks (indicated with solid horizontal lines with the number on the top) performed with rabbit anti-T. bernacchii Ig m antiserum. B) SDSPAGE on 5% polyacrylamide gel under non-reducing conditions of the FPLC major peak (indicated by red arrow in A) containing most IgM, whose relative molecular mass was estimated to be 830 kDa. The gel was stained using the Silver Stain kit (BioRad). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
cytoplasm of the filament-containing cells (Fig. 6AeD) but not in the goblet cells (Fig. 6A, C) that remained unlabelled as well as all cells in negative controls (Fig. 6EeF) prepared by omitting the primary antibodies. The distribution of the labelled filament-containing cells in the skin varied significantly in the different samples obtained from different animals. IgMs were uniformly dispersed in all cells of the basal and intermediate strata which were mildly positive to the IHC reaction (Fig. 6A, C, D). In several cases however, labelling appeared very intense (Fig. 6G, H) or localised on isolated cells of
3.2. Skin morphology The integument in T. bernacchii is particularly thick and characterised by a multilayered epidermis (Fig. 5AeB) composed mostly of filament-containing cells. Their shape is significantly varied with those located close to the basal membrane typically columnar; those present in the intermediate stratum were approximately spherical and those located in the superficial stratum (pavement cells) tended to be more flattened (Fig. 5B). In the epidermis, large mucous (goblet) cells could also be recognized (Fig. 5C). The dermis was thin and contained scales (Fig. 5A) and occasional melanophores (Fig. 9D). 3.3. Immunolocalisation of IgM in T. bernacchii skin Immunocytochemistry indicated that Ig m and L chains were abundant in the mucus (Fig. 6A, C) and in the perinuclear cytoplasm of occasional lymphocytes located close to the basal membrane (Fig. 6AeD). A pale but significant labelling was also present in the
Fig. 4. Biochemical analysis of T. bernacchii cutaneous mucus purified IgM. A) SDSPAGE on 10% acrylamide gel run under reducing conditions using broad range markers (Bio-Rad). Staining with Coomassie Brilliant Blue R-250. B) Immunoblotting analysis with rabbit anti-T. bernacchii Ig m or L chain antisera. Purified serum IgM are reported for comparison. NC: the preimmune serum, collected from the same animal in which the antiserum was raised, was used as negative control.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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3.4. Localisation of the IgM mRNA in T. bernacchii skin In situ hybridization with an anti-sense mRNA demonstrated the expression of both Ig L (Fig. 9AeB) and m (Fig. 9C) chains in occasional lymphocytes located close to the basal membrane. Filamentcontaining cells and goblets cells were always unstained. No signal was detected in negative controls when hybridization was carried out using a sense probe (Fig. 9D). Fig. 5. Skin morphology in T. bernacchii. A) Multi-layered epidermis (e) and scales (arrows) in a thin dermis. B) Detail of the epidermis showing the filament-containing cells changing their shape from columnar (*), close to the basal membrane (arrow), to flattened (**), close to the surface. Notice the absence in this area of goblet cells. Arrow: basal membrane. C) Epidermis with several goblet cells (arrow) among the filamentcontaining cells (*).
the intermediate layer (Fig. 6I) and on cells forming a monolayer between the intermediate and superficial stratum (Fig. 6J). In all samples, Igs were absent from cells of the superficial stratum (Fig. 6A, C, GeJ) and electron microscopy using immunogold was carried out to verify this observation. Transmission electron microscopy revealed that the filament-containing cells (Fig. 7A) had a cytoplasm rich in mitochondria, endoplasmic reticulum and vesicles containing a filamentous material. The cortical cytoplasm was characterised by a thick layer of regularly arranged microfilaments. Adjacent cells were interconnected by several desmosomes and showed extended membrane interdigitations. Pavement cells, the filament-containing cells in contact with the sea water also showed typical short microridges protruding in the mucus (Fig. 7CeD). Immunogold labelling demonstrated the presence of IgM in all these cells (Fig. 7B, D). In contrast, no IgM could be observed in either the cytoplasm or the secretory granules of goblet cells (Fig. 7EeG). Several of the pavement cells showed clear signs of degeneration including disorganised cytoplasm, pale microfilaments and loss of microridges (Fig. 8A). In these cells the cytoplasm was still rich in Igs as demonstrated by the presence of gold particles (Fig. 8B). Many cells were undergoing shedding/desquamation (Fig. 8C).
3.5. Gene expression analysis Mature transcripts encoding the constant domains of the secretory and membrane-bound Igm chain were observed in T. bernacchii skin using a PCR strategy (Fig. 10). A 450-bp fragment of the membrane-bound form and an 800bp fragment of the secretory form were amplified by using respective specific primers. The two PCR products were cloned and sequenced, confirming that both were the expected ones. Furthermore, the expression levels for the secretory Igm chain were analysed in T. bernacchii skin and compared to other tissues (spleen, head kidney, intestine, skin, liver, heart, and muscle) by Real-time PCR. The Igm chain expression levels were normalised to b-actin levels and are shown in Fig. 11. Igm transcripts were found to be expressed at the highest levels in the head kidney, followed by the spleen. The overall expression levels of the Igm chain in the skin were found to be significantly lower than the levels measured in the head kidney. 4. Discussion It is well known that IgM are the predominant immunoglobulin in the serum of teleost fishes. In this work we have investigated the presence of IgM in the skin of the Antarctic teleost T. bernacchii and have shown that IgMs are present in the cutaneous mucus at higher concentration, 135 mg/ml vs 8e90 mg/ml reported in other species [13], therefore likely play also an important role as defence molecules. These IgMs are very similar to those present in the serum
Fig. 6. Immunolocalisation of Ig L (AeB) and m (C,D, GeJ) chains in the skin of T. bernacchii. A, C) Epidermis with labelled mucus (*) and lymphocytes (arrows). Filament-containing cells (**) show a diffuse labelling while goblet cells (white arrow) are always completely unstained. B, D) Details showing the labelled lymphocytes. E, F) negative controls for anti-L and anti-m chains antibodies respectively. All cells (*) are completely unstained; nuclei are counterstained with haematoxylin. GeJ) distribution of Ig m in the dorsal epidermis of different animals. Labelling may be diffused (G) or very intense (H) on basal filament-containing cells (**); it may be located on occasional filament-containing cell (I, arrow) of the intermediate stratum or on cells of the intermediate stratum organised in a monolayer (J, arrow). Mucus (*) is always stained, goblet cells (white arrow) always unstained.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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Fig. 7. Immunogold localisation of Ig m chain in T. bernacchii skin. A) Filament-containing cells connected by desmosomes (arrows) and interdigitations (white arrow). The cytoplasm, rich in mitochondria (m) and vesicles (v), is surrounded by a thick layer of microfilaments (*). B) Ig m chains (gold particles, small black dots) in the cytoplasm of filamentcontaining cells. Note the desmosomes (arrows), the endoplasmic reticulum (r), the microfilament layer (*) and the dense nucleus (n). C) Pavement cell with microridges (m). Nucleus (n) and cytoplasm (c). D) Ig chains (small black dots) in the cytoplasm of a pavement cell; microridge (m), nucleus (n), cytoplasm filled with organelles (c) and microfilaments (*). E) Large goblet cell (*) among filament-containing cells (**). Note the basal nucleus (n) and the cytoplasm engulfed with secretory vesicles (*). F) Detail of the unlabelled secretory vesicles (*). Note the presence of Igs (small black dots, arrow) in the cytoplasm of an adjacent filament-containing cell (**). G) Further detail of the unlabelled secretory vesicles (*) of a goblet cell. Unlabelled endoplasmic reticulum (r).
being exclusively tetrameric, with m and L chains having the same relative molecular mass, and composition of the carbohydrate moieties, despite dimeric Igs being also observed in the skin mucus of sheephead [29]. No redox forms of tetrameric IgM that had been previously found in T. bernacchii serum [23] or the unique redox form consisting of halfmeric components H1L1 of relative molecular mass of 100 kDa, seen in rainbow trout skin mucus [30], have been observed in the skin mucus of T. bernacchii. Thus, mucus IgMs do not show significantly different protein assembly or antigenicity in comparison to the serum IgM. This conclusion is also supported by the fact that skin mucus IgMs were detected with rabbit antisera raised against m or L chains purified from serum IgM.
As described for the majority of teleost species, the PCR results confirmed those previously obtained that mRNA splicing, leading to the exclusion of the CH4 exon and thus to a shorter transcript for the membrane-bound form, occurs in T. bernacchii [20]. Detection of the IgM membrane-bound form in the skin, besides the secretory form, suggests that B cells are present in the skin and in turn the complexity of the adaptive immune response is already present in teleost fish skin. The overall expression levels of the Igm chain were analysed by Real-time PCR: the values obtained for skin and intestine were significantly lower than those collected for head kidney and spleen, the former being well known sites of the mucosal immunity in which other Ig isotypes have a predominant role [31,32]. The value relative to muscle is surprising and needs to be
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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Fig. 8. Immunogold localisation of Ig m chains in degenerating pavement cells of T. bernacchii skin. A) Cell with pale microfilaments (*) and clearly disorganised cytoplasm (c). Healthy cells (**). B) Detail of the cytoplasm (c) of a degenerating pavement cell showing the presence of Igs (small black dots, arrow). Microfilaments (*), cytoplasm (c). C) Pavement cell (arrow) detaching from the skin (*).
low levels in the skin and gill [33]. More importantly, the higher expression of IgM observed by Real-time PCR experiments in the head kidney compared to the spleen and gut might be attributed to the first appearance of Ig-producing cells in the head kidney, which mature here and then migrate to sites of activation, either the spleen or the mucosal compartment [34]. Recent gene expression studies in zebrafish have shown IgM as more predominant in both lymphoid and non-lymphoid organs when compared to IgZ/T [35], a new isotype found in several teleosts [36,37] and shown to be the prevalent isotype in the gut [31] and skin mucus [32] of rainbow trout. This indicates that fish IgT play a specialised role in mucosal immunity. It remains to be verified whether in T. bernacchii other components of the cutaneous immune system are also present, although IgT has been recently identified in the skin of two Antarctic fish species (Coscia et al., manuscript in preparation).
Fig. 9. Localisation of Ig L (AeB) and m (C) chain mRNAs in the skin of T. bernacchii. A, B) labelling is on occasional lymphocytes (arrow) located in the basal epidermis. Filament-containing cells (*) and goblet cells (g) are unstained. C) labelled lymphocytes (arrows) among negative filament-containing cells (*). Messenger is concentrated in the cytoplasm. D) negative control hybridised with a sense probe with no labelling observed (*). Melanophores (arrowheads) and scales (s) in the dermis.
further investigated taking into account that Antarctic fish are heavily parasitised [22], and thus immune response against pathogens may take also place in tissues other than lymphoid ones. Moreover, these results may vary according to different bacterial or parasitic infections. In adult fish, IgM is constitutively expressed in renal haematopoietic tissue, spleen and thymus, but expressed at
Fig. 10. RT-PCR amplification for detection of the membrane-bound (lane 1) and secretory (lane 2) IgM forms. M: GeneRuler 100 bp DNA ladder (Fermentas).
Fig. 11. Analysis by Real-time PCR of relative expression levels of Igm transcripts in different tissues. The results are reported as folds of difference relative to skin. Data were normalised to b-actin using the Pfaffl method, considering efficiency value for each primer couple. The results are reported as mean value of triplicates obtained in three independent experiment standard deviation.
Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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The cytological investigations revealed that T. bernacchii skin has a thick, multilayered epidermis which provides a multifunctional barrier and may represent an adaptation to the hostile, cold environment in protection against freezing [38,39]. Thick skin, typical in relatively cold waters is also present in other species such as the trout [40]. It is evident from the ISH analyses that skin IgMs are produced in situ by lymphocytes localised in the basal epidermis as Igm and L chains and their respective mRNAs were present thereby confirming the local production of functional IgM. Ig diffusion from lymphocytes would spread throughout the epidermis to reach the apical mucus. This movement is similar to that seen in other fish species for example, in the skin of fugu (Takifugu rubripes), common carp (Cyprinus carpio), and orangespotted grouper (Epinephelus coioides) [15,41,42]. Ig secreted into bile from the liver of T. bernacchii [43] and other teleosts [44,45], also diffuses in this manner. Diffusion through the extracellular spaces would be discouraged by the presence of desmosomes and interdigitations among adjacent cells. Goblet cells would not be involved as they are always negative to the anti-Ig antibodies and probes. Detailed Ig transport mechanisms are yet to be clarified. Polymeric immunoglobulin receptor (pIgR) is probably involved and would be responsible for transporting and protecting the secretory Ig from proteolytic degradation [46]. A gene encoding a pIgR has been isolated from several teleost fish species [15,42,46,47]. Teleost skin thus adopts the same Ig transport system as mammalian intestine or other mucosal sites, via a unique pIgR, expressed on the surface of epithelial cells, whose secretory component size seems to be species-specific (w60 kDa in fugu, w95 kDa in sheepshead, w35 kDa in trout) [15,29,32]. The release of Ig from the skin into the mucous coat may occur in two different ways. The first is that it simply diffuses, released by the skin pIgR. With this respect, the efficiency of the secretion would be increased by the flattening of the pavement cells and the presence of microridges. The second possibility is that the release occurs when the degenerating pavement cells undergo sloughing. Such cells, in fact, are still rich in Ig. Their asynchronous dismission would ensure a continuous supply to the skin mucus of new Ig and, probably, also of enzymes and various other substances. An interesting aspect emerging from our observations is the Ig distribution pattern in the different samples examined. Since they all come from a dorsal area of the trunk, one possible explanation is the existence of significant variation among different individuals. Such differences could represent a sort of fingerprint left by a previous pathogen attack or perhaps, one in progress. It should be mentioned that in several animals the liver appeared infested by nematode parasites, and a clear immune response against them in Antarctic fish has been previously well documented [22,48]. The differences observed would not depend on sex even though the majority of animals examined were females. As such, we currently have only a partial picture of the skin secretory immune system in these fish. How Igs interact with antigens at the mucosal surface constantly exposed to water and swimming forces remains completely unknown but an adaptation in antigeneantibody interactions might be expected in such a particular mucosal environment. This work demonstrates the existence of a local immunity based on the production of IgM in the skin of an Antarctic teleost species. The protection is highly efficient due to the high concentration of IgM. The Antarctic environment is very hostile with competition for resources particularly severe and with a high parasite infection load. Thus, efficient protection against pathogens may represent a significant advantage to survive under extreme conditions.
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Please cite this article in press as: Coscia MR, et al., Investigation of immunoglobulins in skin of the Antarctic teleost Trematomus bernacchii, Fish & Shellfish Immunology (2014), http://dx.doi.org/10.1016/j.fsi.2014.04.019
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