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Fish & Shellfish Immunology 39 (2014) 206e214

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Fish & Shellfish Immunology

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Investigation of immunoglobulins in skin of the Antarctic teleostTrematomus bernacchii

Maria Rosaria Coscia a,*, Palma Simoniello b,c, Stefano Giacomelli a, Umberto Oreste a,Chiara Maria Motta b

a Institute of Protein Biochemistry, CNR, via P. Castellino 111, 80131 Naples, ItalybDepartment of Biological Science, University of Naples Federico II, via Mezzocannone 8, 80134 Naples, ItalycGSI Helmholtzzentrum für Schwerionenforschung, Planckstrasse 1, Darmstadt, Germany

a r t i c l e i n f o

Article history:Received 6 December 2013Received in revised form1 April 2014Accepted 23 April 2014Available online 10 May 2014

Keywords:Skin immunityCutaneous mucusTeleost IgMImmunohistochemistryIn situ hybridization

* Corresponding author. Tel.: þ39 0816132556; faxE-mail address: mr.coscia@ibp.cnr.it (M.R. Coscia).

http://dx.doi.org/10.1016/j.fsi.2014.04.0191050-4648/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

The presence and production of IgM in the skin of the Antarctic teleost Trematomus bernacchii wereinvestigated in this study. Immunoglobulins purified from cutaneous mucus and analysed by SDS-PAGErun under non-reducing and reducing conditions, were composed of heavy and light chains of 78 kDaand 25 kDa respectively, with a relative molecular mass of 830 kDa indicating that mucus IgM are tet-ramers as the serum IgM. Mature transcripts encoding the constant domains of both the secretory andmembrane-bound Igm chain were seen in T. bernacchii skin using a PCR strategy and the expression of thesecretory Igm chain in the skin was compared with that in other tissues by Real-time PCR. Cytologicalinvestigations revealed the presence of either immunoglobulins or their transcripts in occasional lym-phocytes distributed close to the basal membrane. IgM once produced here, enters the filament-containing cells and is released into the mucus when these cells degenerate and detach from theepidermis. Our findings indicate that a cutaneous defence mechanism, functioning as anatomical andphysiological barrier under subzero conditions, is present in this Antarctic species as an importantcomponent of the immune system.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Fish skin is a complex organ performing a variety of functionsfrom homoeostatic to mechanical and from sensory perception toprotection against pathogens [1]. Composed of a non-keratinizedstratified epithelium of variable thickness, it is responsible forsecreting mucus, a glycopeptide based protective structure. Theseglycopeptides change in amount and composition under varyingstress and environmental conditions [2,3].

The mucous coat, produced mainly by goblet cells, covers theentire animal to facilitate swimming [4] and to reduce pathogenaccess to epithelial cells by constantly moving down stream and offthe trailing edges [5]. Protection against pathogens is also due to itsantimicrobial properties, particularly enzymes, lytic agents [6e8],and various immune components such as lysozyme, complementand immunoglobulins (Igs) [9]. These increase in response to im-munization by both systemic [10] and bath immersion routes [11]

: þ39 0816132277.

and to pathogen attack [12] indicating the existence of a skin-associated lymphoid tissue (SALT) referred to also as the skin im-mune system (SIS) or cutaneous immune system. In fish, the SALT isa mucosal lymphoid tissue that is very different from that ofmammals since having the outermost cells layers alive and activelydividing [13].

Although defensive role of skin Igs in fish is clear, it has beenlong debatedwhether they are secreted or diffuse passively into themucus. In 1971 Di Conza and Halliday [14] suggested that mucus Igswere locally synthesized and were not derived from blood. Somerecent evidence demonstrates the existence of differences in skinand plasma Ig m chains [12,15,16], suggesting the former are pro-duced locally, and independently from systemic Igs [17]. These Igsare of m isotype as are those present in gut mucus, despite the latterbeing in small amounts perhaps due to degradation in this verystrong proteolytic environment [18]. The skin therefore may beconsidered an integral component of the teleost immune system.

The Nototheniids are teleosts species adapted to the extremeenvironmental conditions of the Antarctic Ocean [19]. We havepreviously highlighted in the secretory andmembrane-bound formof IgM of serum and lymphoid tissues some peculiar features that

M.R. Coscia et al. / Fish & Shellfish Immunology 39 (2014) 206e214 207

have evolved as compensatory adjustments in these species[20,21]. In this work we demonstrate the existence of a skinmucosal immunity in a red-blooded Antarctic Nototheniid, theemerald rockcod Trematomus bernacchii. The IgM from the skin andcutaneous mucus were isolated and characterised and the questionof a possible local production was addressed by localising the IgMand their mRNA by immunohistochemistry and in situ hybridiza-tion, using antibodies and probes produced in our laboratory [22].Our results indicate that in this species skin production of IgMoccurs and that a mucosal immunity system is present.

2. Materials and methods

2.1. Species and sampling

Adult specimens of T. bernacchii (Boulenger, 1902) werecollected by nets at a depth ranging between 15 and 75m in front ofthe Italian Station “Mario Zucchelli” at Terra Nova Bay during theXXV expedition of the Italian National Program for AntarcticResearch (PNRA, 2009e2010).

Fish were anaesthetised in aqueous MS222 (tricaine methanesulphonate) at a concentration of 1:15000 (w/v). Blood wascollected by caudal venipuncture and serum, obtained by centri-fugation at 2000 g for 10 min, was stored at �20 �C. Mucus wascarefully collected by gently wiping the body surface of tenT. bernacchii specimens with tissue paper to avoid damage of theskin and contamination with blood. Saturated tissues were placedin sterile 50-ml conical tubes containing approximately 10 ml ofTBS buffer (Tris-buffered saline; 50 mM TriseHCl, pH 8.0, con-taining 150 mM NaCl) per tube. The content of each tube washomogenised and centrifuged at maximum speed for 15 min. Thecollected supernatants were pooled, filtered, and the absorbance(A280) was measured. Aliquots were frozen at �20 �C. This pooledmucus sample was then processed as described below. Samples ofskin, spleen, head kidney, intestine, heart, muscle, and liver werecollected and processed for histology or, if required for molecularanalyses, frozen in liquid nitrogen, and brought back to Italyat �80 �C. The experiments were carried out in Italy in compliancewith ethical provisions established by the European Union andauthorized by the National Committee of the Italian Ministry ofHealth for in vivo experimentation (Department for VeterinaryPublic Health, Nutrition and Food Safety). All the experiments wereorganised 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 centrifugedat 13,000 rpm for 30 min at 4 �C. The supernatant was recoveredand dialysed with 125 mM ammonium bicarbonate using 3.5 kDacut off membrane tubing (SpectrumLabs, CA, USA). Protein con-centration was determined by the Bio-Rad kit using bovine serumalbumin as standard. IgM was purified from the dialysed mucusproteins as previously described [23].

T. bernacchii IgM concentration was determined in cutaneousmucus by ELISA from a standard titration curve constructed usingpurified serum T. bernacchii IgM as described in Ref. [23].

Purified IgM was analysed by SDS-PAGE under reducing andnon-reducing conditions using a 10% or 5% polyacrylamide gelrespectively. The gels were stained using 0.1% Coomassie BrilliantBlue R-250 or the Silver Stain kit (Bio-Rad). Proteins separated bySDS-PAGE were electrophoretically transferred onto nitrocelluloseat 24 mA for 18 h; the nitrocellulose sheets were incubated withrabbit anti-T. bernacchii Ig m and/or L chain antisera raised as pre-viously described in Ref. [22] for 1 h and following incubation withperoxidase labelled anti-rabbit antibodies for 1 h. Finally, the

specific binding was revealed by the ECL kit (GE Healthcare Life-science) following manufacturer’s instructions. The preimmuneserum, collected from the same animal in which the antiserumwasraised, was used in control experiments.

Analysis of the carbohydrate moiety of the mucus IgMwas doneusing the DIG-Glycan Detection kit (Roche MolecularBiochemicals).

2.3. Purification and biochemical analysis of skin proteins and IgM

Protein extraction from skinwas carried out by placing a piece offrozen tissue in a cryogenic tubewith 50mMPBS, pH 7.5, containing50 ml/ml protease inhibitor cocktail (Sigma); the samplewas shakenfor 2 min at 2000 Hz and centrifuged at 13,000 rpm for 90 min. Thesupernatant was collected and the protein content estimated usinga 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 aspreviously described [24]. Briefly, they were fixed in Karnowskysolution [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 roomtemperature for 6 h if intended for immunogold. All samples werethen dehydrated in a graded series of saline alcohols and embeddedin epoxy resin. Ultrathin sections were stainedwith lead citrate anduranyl 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 unmaskthe antigens, washed in buffer (TBS, BSA 1%, Tween20 0.05%) for15min, in 3% H2O2 for 20min to block endogenous peroxidases andin serum, for 15min, to reduce non-specific binding. After that, theywere incubated for 2 h, at 4 �C, with the primary antibodies raisedin rabbit against the T. bernacchii Igm and L chains [22], diluted 1:50in PBS. Antibody binding was detected by a secondary goat anti-rabbit Ig antibody (1:200 in PBS, for 2 h), conjugated to peroxi-dase, intensified by incubation with a tertiary anti-PAP antibody(1:100 in PBS, 90 min) and revealed with DAB-urea. Negativecontrols were prepared by omitting the primary antibodies.

The immunogold procedure was as described in Ref. [27].Briefly, ultrathin sections were treated with sodium metaperiodateand incubated with the anti-T. bernacchii Igm antibody (diluted1:200). Binding was detected using a secondary antibody linked to10 nm colloidal gold particles. Sections were examined after mildstaining with lead citrate and uranyl acetate.

2.6. In situ hybridisation (ISH)

Sections post-fixed in paraformaldehyde (4% in PBS, pH 7.4) for30 min were washed in PBS-H2ODEPC, then digested for 5 min in PKbuffer (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 sec-tions were fixed again in paraformaldehyde then washed twice inPBS-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 pre-hybridization mix containing formamide 40%, SSC 5�, Denhart’ssolution 1�, 100 mg/ml Salmon testis DNA, 100 mg/ml tRNA inH2ODEPC. Hybridization was carried out at 55 �C, overnight, using adig-labelled riboprobe (see Section 2.5).

Fig. 1. Immunoblotting analysis of skin IgM. A mixture of equal volumes of anti-T. bernacchii Ig m and L chain antisera was used to reveal the chains whose relativemolecular mass is reported on the left.

M.R. Coscia et al. / Fish & Shellfish Immunology 39 (2014) 206e214208

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 riboprobewas digested in NTE containing RNase A 0.3 U/ml for30 min and washed away in NTE and SSC 0.5�, 20% formamide.Finally, the sections were washed in SSC 2� for 30 min. Slides werethen immerged for 30 min in a Blocking solution prepared with 2%BM blocking reagent (Roche), maleic acid 100 mM and H2Odist.

Digoxigeninwas revealed by incubating sections overnight withan AP-conjugated anti-dig antibody, diluted 1:400 in Blocking so-lution with 10% sheep serum (Sigma). The antibody was revealedwith a staining mix (1 ml BM Purple, Roche, 10 ml levamisole-Tween20 100�). Reaction was stopped in PBS with 1 mM EDTA.

2.7. Amplification of secretory and membrane Igm chain transcripts

Total RNAwas isolated from skin using the SV total RNA isolationsystem (Promega), and subjected to reverse transcription using M-MLV Reverse Transcriptase (Ambion, Austin, TX, USA) according tothe manufacturer’s instructions. Double-stranded cDNA PCRamplification for secretory and membrane-bound Igm chain wascarried 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). Theprimers used were all designed on the T. bernacchii Igm chainsequence (AF094531), previously determined. The amplificationwas 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 at72 �C for 10 min. PCR products were visualised on 1% agarose gelsusing ethidium bromide staining and UV light transillumination.

Products were then cloned into pSC-A by using the StrataClonePCR cloning kit (Stratagene) and positive clones were identified byblue/white selection. All sequences were determined on an ABIPRISM 3730 DNA Analyzer sequencer at Primm (Milan, Italy).

For the IgL chain probe, the clone L180 (EF421439.1) in p-GEM-TEasy Vector (Promega) was used. Transcription of antisense andsense RNA probes was carried out with DIG RNA labeling kit(Roche).

2.8. Real-time PCR

Total RNA was extracted from seven tissues (spleen, head kid-ney, intestine, skin, liver, heart and muscle) as described above,each tissue type pooled from five individuals. cDNA was thensynthesised from 5 mg of total RNA using Transcriptor First StrandcDNA Synthesis Kit (Roche).

Real-time PCR experiments were performed using the Light-Cycler 480 (Roche). The reaction consisted of 2 ml of the first strandcDNA mixed with 10 ml of LightCycler 480 SYBR Green I Master(Roche) in a final volume of 20 ml with a final concentration of0.2 mM of each primer, according to the manufacturer’s in-structions. Specific PCR primers were designed for the amplifica-tion 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 primerspecific for Cm region), 50-AGGTCAGCGGAGACGGTGC-30; b-actin(GQ229124.1) was used as housekeeping gene: BACTfw (senseprimer), 50-CCCAGATCATGTTCGAGACC-30; BACTrev (anti-senseprimer), 50-CAGCGACGGACAGGAAG-30. The reaction conditionswere established through a series of preliminary optimization ex-periments. Firstly the efficiency for each primer couple (EAct for b-actin, and EIgM for Igm chain) was tested using serial diluitions ofspleen cDNA: EAct was 1.92, while EIgM was 1.88.

Real-time PCR experiments were performed three times and alltissue samples, including water as non-template control, were run

in triplicate each time. Data were normalised according to the Pfafflmethod [28], by calculating the relative expression ratio as follows:

ðEActÞCt sample.�

EIgM�Ct sample

OðEActÞCt calibrator.�

EIgM�Ct calibrator

where b-actin was used as reference gene and the skin as a cali-brator that all other samples were compared to.

The PCR amplification conditions were: 95 �C for 5 min, fol-lowed by 45 cycles consisting of 10 s denaturation at 95 �C, 20 sannealing at 60 �C, and 10 s extension at 72 �C. In order to verifyamplification specificity and the absence of primer dimers, a finaldissociation step was run to generate a melting curve. In all meltingcurve analyses, single specific peaks were observed. The Real-timePCR products were subjected to gel electrophoresis using a 2% (w/v)agarose gel, further confirming that in all cases, the amplificationswere specific and no amplification was observed in negative con-trols. All the results are reported as mean value of triplicates ob-tained 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 concentrationof 8.4 mg/ml. The presence of Igm and IgL chains was assessed byimmunoblotting (Fig. 1) using a mixture of equal volumes of anti-mand anti-L rabbit antisera.

Mean cutaneous mucus protein content was 1.2 mg/ml. Proteincomposition, analysed by SDS-PAGE (Fig. 2A), indicated the pres-ence of several bands ranging between 78 and 25 kDa. The digox-igenin labelling method for carbohydrates (Fig. 2B) gave a positivereaction on the immobilized 78 kDa band, corresponding to the Igmchain, whereas the L chain band was clearly negative. An additionalpositive band (50 kDa), probably corresponding to a galectin familycomponent, was observed.

IgMs were purified from the dialysed mucus proteins byfollowing a method previously used [23]. High relative molecular

Fig. 2. Biochemical analysis of total cutaneous mucus proteins. A) SDS-PAGE on 10%acrylamide gel run under reducing conditions and stained with Coomassie BrilliantBlue R-250. M: Broad range markers (Bio-Rad). B) Analysis of carbohydrate moiety inthe Ig m and L chains by the digoxigenin labelling method.

Fig. 3. Purification of T. bernacchii cutaneous mucus Ig. A) FPLC elution profile ofmucus Ig (upper), using a Bio-Prep SE-1000/17 column (Bio-Rad) precalibrated withmarker proteins (Gel filtration standard, Bio-Rad): Thyroglobulin (670,000), g-globulin(158,000), Ovalbumin (44,000), Myoglobin (17,000), Vitamin B12 (1350). Black arrowsindicate the elution position of the molecular weight markers. Immunoblotting anal-ysis (lower) of the three main peaks (indicated by solid horizontal lines with thenumber on the top) performed with rabbit anti-T. bernacchii Ig m antiserum. B) SDS-PAGE on 5% polyacrylamide gel under non-reducing conditions of the FPLC majorpeak (indicated by red arrow in A) containing most IgM, whose relative molecularmass was estimated to be 830 kDa. The gel was stained using the Silver Stain kit (Bio-Rad). (For interpretation of the references to colour in this figure legend, the reader isreferred to the web version of this article.)

Fig. 4. Biochemical analysis of T. bernacchii cutaneous mucus purified IgM. A) SDS-PAGE on 10% acrylamide gel run under reducing conditions using broad rangemarkers (Bio-Rad). Staining with Coomassie Brilliant Blue R-250. B) Immunoblottinganalysis with rabbit anti-T. bernacchii Ig m or L chain antisera. Purified serum IgM arereported for comparison. NC: the preimmune serum, collected from the same animalin which the antiserum was raised, was used as negative control.

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mass components were fractionated by FPLC (Fig. 3A); the mainpeak was analysed by SDS-PAGE run without reducing agents, andits relative molecular mass was estimated to be 830 kDa (Fig. 3B).The 830-kDa fraction was further purified by affinity chromatog-raphy using a column coupled with rabbit anti-T. bernacchii Ig mchain antibodies. To determine themean concentration of IgM, skinmucus was collected from ten adult specimens and analysed byELISA as described in Ref. [23]. IgM concentrations were calculatedfrom the standard titration curve obtained by using purifiedT. bernacchii serum IgM and found to be 135 mg/ml.

SDS-PAGE analysis of purified mucus IgM performed underreducing conditions (Fig. 4A) gave two bands with a relative mo-lecular mass of 78 kDa and 25 kDa, corresponding to Ig m and Lchains respectively as found for serum IgM [23]. These results wereconfirmed by immunoblotting performed using rabbit antiserumspecific for T. bernacchii Ig m or L chains (Fig. 4B) and preimmuneserum, as negative control, collected from the same animal inwhich the antiserum was raised (Fig. 4B).

3.2. Skin morphology

The integument in T. bernacchii is particularly thick and char-acterised by amultilayered epidermis (Fig. 5AeB) composedmostlyof filament-containing cells. Their shape is significantly varied withthose located close to the basal membrane typically columnar;those present in the intermediate stratum were approximatelyspherical and those located in the superficial stratum (pavementcells) tended to be more flattened (Fig. 5B). In the epidermis, largemucous (goblet) cells could also be recognized (Fig. 5C). The dermiswas thin and contained scales (Fig. 5A) and occasional melano-phores (Fig. 9D).

3.3. Immunolocalisation of IgM in T. bernacchii skin

Immunocytochemistry indicated that Ig m and L chains wereabundant in themucus (Fig. 6A, C) and in the perinuclear cytoplasmof occasional lymphocytes located close to the basal membrane(Fig. 6AeD). A pale but significant labelling was also present in the

cytoplasm of the filament-containing cells (Fig. 6AeD) but not inthe goblet cells (Fig. 6A, C) that remained unlabelled as well as allcells in negative controls (Fig. 6EeF) prepared by omitting theprimary antibodies.

The distribution of the labelled filament-containing cells in theskin varied significantly in the different samples obtained fromdifferent animals. IgM were uniformly dispersed in all cells of thebasal and intermediate strata which were mildly positive to theIHC reaction (Fig. 6A, C, D). In several cases however, labellingappeared very intense (Fig. 6G, H) or localised on isolated cells of

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-containingcells changing their shape from columnar (*), close to the basal membrane (arrow), toflattened (**), 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 filament-containing cells (*).

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the intermediate layer (Fig. 6I) and on cells forming a monolayerbetween the intermediate and superficial stratum (Fig. 6J).

In all samples, Igs were absent from cells of the superficialstratum (Fig. 6A, C, GeJ) and electron microscopy using immuno-gold was carried out to verify this observation. Transmission elec-tron microscopy revealed that the filament-containing cells(Fig. 7A) had a cytoplasm rich in mitochondria, endoplasmic re-ticulum and vesicles containing a filamentous material. The corticalcytoplasm was characterised by a thick layer of regularly arrangedmicrofilaments. Adjacent cells were interconnected by severaldesmosomes and showed extended membrane interdigitations.Pavement cells, the filament-containing cells in contact with thesea water also showed typical short microridges protruding in themucus (Fig. 7CeD). Immunogold labelling demonstrated the pres-ence of IgM in all these cells (Fig. 7B, D). In contrast, no IgM could beobserved in either the cytoplasm or the secretory granules of gobletcells (Fig. 7EeG). Several of the pavement cells showed clear signsof degeneration including disorganised cytoplasm, pale microfila-ments and loss of microridges (Fig. 8A). In these cells the cytoplasmwas still rich in Igs as demonstrated by the presence of gold par-ticles (Fig. 8B). Many cells were undergoing shedding/desquama-tion (Fig. 8C).

Fig. 6. Immunolocalisation of Ig L (AeB) and m (C,D, GeJ) chains in the skin of T. bernacchii. Acells (**) show a diffuse labelling while goblet cells (white arrow) are always completely unsand anti-m chains antibodies respectively. All cells (*) are completely unstained; nuclei aredifferent animals. Labelling may be diffused (G) or very intense (H) on basal filament-contaiintermediate stratum or on cells of the intermediate stratum organised in a monolayer (J,

3.4. Localisation of the IgM mRNA in T. bernacchii skin

In situ hybridizationwith an anti-sensemRNA demonstrated theexpression of both Ig L (Fig. 9AeB) and m (Fig. 9C) chains in occa-sional lymphocytes located close to the basal membrane. Filament-containing cells and goblets cells were always unstained. No signalwas detected in negative controls when hybridization was carriedout using a sense probe (Fig. 9D).

3.5. Gene expression analysis

Mature transcripts encoding the constant domains of thesecretory and membrane-bound Igm chain were observed inT. bernacchii skin using a PCR strategy (Fig. 10).

A 450-bp fragment of the membrane-bound form and an 800-bp fragment of the secretory form were amplified by usingrespective specific primers. The two PCR products were cloned andsequenced, confirming that both were the expected ones.Furthermore, the expression levels for the secretory Igm chain wereanalysed in T. bernacchii skin and compared to other tissues (spleen,head kidney, intestine, skin, liver, heart, and muscle) by Real-timePCR. The Igm chain expression levels were normalised to b-actinlevels and are shown in Fig. 11. Igm transcripts were found to beexpressed at the highest levels in the head kidney, followed by thespleen. The overall expression levels of the Igm chain in the skinwere found to be significantly lower than the levels measured inthe head kidney.

4. Discussion

It is well known that IgM are the predominant immunoglobulinin the serum of teleost fishes. In this work we have investigated thepresence of IgM in the skin of the Antarctic teleost T. bernacchii andhave shown that IgM are present in the cutaneous mucus at higherconcentration, 135 mg/ml vs 8e90 mg/ml reported in other species[13], therefore likely play also an important role as defence mole-cules. These IgM are very similar to those present in the serum

, C) Epidermis with labelled mucus (*) and lymphocytes (arrows). Filament-containingtained. B, D) Details showing the labelled lymphocytes. E, F) negative controls for anti-Lcounterstained with haematoxylin. GeJ) distribution of Ig m in the dorsal epidermis ofning cells (**); it may be located on occasional filament-containing cell (I, arrow) of thearrow). Mucus (*) is always stained, goblet cells (white arrow) always unstained.

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 cyto-plasm, 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 filament-containing 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 micro-filaments (*). E) Large goblet cell (*) among filament-containing cells (**). Note the basal nucleus (n) and the cytoplasm engulfed with secretory vesicles (*). F) Detail of theunlabelled 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 unlabelledsecretory vesicles (*) of a goblet cell. Unlabelled endoplasmic reticulum (r).

M.R. Coscia et al. / Fish & Shellfish Immunology 39 (2014) 206e214 211

being exclusively tetrameric, with m and L chains having the samerelative molecular mass, and composition of the carbohydratemoieties, despite dimeric Igs being also observed in the skin mucusof sheephead [29].

No redox forms of tetrameric IgM that had been previouslyfound in T. bernacchii serum [23] or the unique redox form con-sisting of halfmeric components H1L1 of relative molecular mass of100 kDa, seen in rainbow trout skin mucus [30], have beenobserved in the skin mucus of T. bernacchii. Thus, mucus IgM do notshow significantly different protein assembly or antigenicity incomparison to the serum IgM. This conclusion is also supported bythe fact that skin mucus IgM were detected with rabbit antiseraraised against m or L chains purified from serum IgM.

As described for the majority of teleost species, the PCR resultsconfirmed those previously obtained thatmRNA splicing, leading tothe exclusion of the CH4 exon and thus to a shorter transcript forthe membrane-bound form, occurs in T. bernacchii [20]. Detectionof the IgMmembrane-bound form in the skin, besides the secretoryform, suggests that B cells are present in the skin and in turn thecomplexity of the adaptive immune response is already present inteleost fish skin. The overall expression levels of the Igm chain wereanalysed by Real-time PCR: the values obtained for skin and in-testine were significantly lower than those collected for head kid-ney and spleen, the former being well known sites of the mucosalimmunity in which other Ig isotypes have a predominant role[31,32]. The value relative to muscle is surprising and needs to be

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 lympho-cytes (arrows) among negative filament-containing cells (*). Messenger is concen-trated in the cytoplasm. D) negative control hybridised with a sense probe with nolabelling observed (*). Melanophores (arrowheads) and scales (s) in the dermis.

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) Pavementcell (arrow) detaching from the skin (*).

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further investigated taking into account that Antarctic fish areheavily parasitised [22], and thus immune response against path-ogens may take also place in tissues other than lymphoid ones.Moreover, these results may vary according to different bacterial orparasitic infections. In adult fish, IgM is constitutively expressed inrenal haematopoietic tissue, spleen and thymus, but expressed at

Fig. 10. RT-PCR amplification for detection of the membrane-bound (lane 1) andsecretory (lane 2) forms of the Igm chain. M: GeneRuler 100 bp DNA ladder(Fermentas).

low levels in the skin and gill [33]. More importantly, the higherexpression of IgM observed by Real-time PCR experiments in thehead kidney compared to the spleen and gut might be attributed tothe first appearance of Ig-producing cells in the head kidney, whichmature here and then move to the spleen and the mucosalcompartment as sites of activation [34].

Recent gene expression studies in zebrafish have shown IgM asmore predominant in both lymphoid and non-lymphoid organswhen compared to IgZ/T [35], a new isotype found in several tel-eosts [36,37] and shown to be the prevalent isotype in the gut [31]and skin mucus [32] of rainbow trout. This indicates that fish IgTplay a specialised role in mucosal immunity. It remains to beverifiedwhether inT. bernacchii other components of the cutaneousimmune system are also present, although IgT has been recentlyidentified in the skin of two Antarctic fish species (Coscia et al.,manuscript in preparation).

Fig. 11. Analysis by Real-time PCR of relative expression levels of Igm transcripts indifferent tissues. The results are reported as folds of difference relative to skin. Datawere normalised to b-actin using the Pfaffl method, considering efficiency value foreach primer couple. The results are reported as mean value of triplicates obtained inthree independent experiment � standard deviation.

M.R. Coscia et al. / Fish & Shellfish Immunology 39 (2014) 206e214 213

The cytological investigations revealed that T. bernacchii skin hasa thick, multilayered epidermis which provides a multifunctionalbarrier and may represent an adaptation to the hostile, cold envi-ronment in protection against freezing [38,39]. Thick skin, typical inrelatively cold waters is also present in other species such as thetrout [40].

It is evident from the ISH analyses that skin IgM are produced insitu by lymphocytes localised in the basal epidermis as Igm and Lchains and their respective mRNAs were present thereby con-firming the local production of functional IgM.

Ig diffusion from lymphocytes would spread throughout theepidermis to reach the apical mucus. This movement is similar tothat seen in other fish species for example, in the skin of fugu(Takifugu rubripes), common carp (Cyprinus carpio), and orange-spotted grouper (Epinephelus coioides) [15,41,42]. Igs secreted intobile from the liver of T. bernacchii [43] and other teleosts [44,45],also diffuse in this manner. Diffusion through the extracellularspaces would be discouraged by the presence of desmosomes andinterdigitations among adjacent cells. Goblet cells would not beinvolved as they are always negative to the anti-Ig antibodies andprobes.

Detailed Ig transport mechanisms are yet to be clarified. Poly-meric immunoglobulin receptor (pIgR) is probably involved andwould be responsible for transporting and protecting the secretoryIg from proteolytic degradation [46]. A gene encoding a pIgR hasbeen isolated from several teleost fish species [15,42,46,47]. Teleostskin thus adopts the same Ig transport system as mammalian in-testine or other mucosal sites, via a unique pIgR, expressed on thesurface of epithelial cells, whose secretory component size seems tobe 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 occurin two different ways. The first is that it simply diffuses, released bythe skin pIgR. With this respect, the efficiency of the secretionwould be increased by the flattening of the pavement cells and thepresence of microridges. The second possibility is that the releaseoccurs when the degenerating pavement cells undergo sloughing.Such cells, in fact, are still rich in Ig. Their asynchronous dismissionwould 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 Igdistribution pattern in the different samples examined. Since theyall come from a dorsal area of the trunk, one possible explanation isthe existence of significant variation among different individuals.Such differences could represent a sort of fingerprint left by aprevious pathogen attack or perhaps, one in progress. It should bementioned that in several animals the liver appeared infested bynematode parasites, and a clear immune response against them inAntarctic fish has been previously well documented [22,48]. Thedifferences observed would not depend on sex even though themajority of animals examined were females. As such, we currentlyhave only a partial picture of the skin secretory immune system inthese fish.

How Igs interact with antigens at themucosal surface constantlyexposed to water and swimming forces remains completely un-known but an adaptation in antigeneantibody interactions mightbe expected in such a particular mucosal environment.

This work demonstrates the existence of a local immunitybased on the production of IgM in the skin of an Antarctic teleostspecies. The protection is highly efficient due to the high con-centration of IgM. The Antarctic environment is very hostile withcompetition for resources particularly severe and with a highparasite infection load. Thus, efficient protection against patho-gens may represent a significant advantage to survive underextreme conditions.

Acknowledgements

We acknowledge Prof. Giacomo Zaccone (Università degli Studidi Messina, Italy) for kindly providing us with bibliographic infor-mation. This work was supported by grant PEA2009/A1.12 from theItalian National Program for Antarctic Research.

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