Characterisation of a monoclonal antibody detecting Atlantic salmon endothelial and red blood cells, and its association with the infectious salmon anaemia virus cell receptor

Authors


Correspondence

Knut Falk, Norwegian Veterinary Institute, Ullevålsveien 68, PO Box 750 Sentrum, 0106 Oslo, Norway. T: + 47 2 3216132; F: + 47 2 3216001; E: Knut.Falk@vetinst.no

Abstract

Endothelial cells (ECs) line the luminal surfaces of the cardiovascular system and play an important role in cardiovascular functions such as regulation of haemostasis and vasomotor tone. A number of fish and mammalian viruses target these cells in the course of their infection. Infectious salmon anaemia virus (ISAV) attacks ECs and red blood cells (RBCs) of farmed Atlantic salmon (Salmo salar L.), producing the severe disease of infectious salmon anaemia (ISA). The investigation of ISA has up to now been hampered by the lack of a functional marker for ECs in Atlantic salmon in situ. In this study, we report the characterisation and use of a novel monoclonal antibody (MAb) detecting Atlantic salmon ECs (e.g. vessel endothelium, endocardial cells and scavenger ECs) and RBCs. The antibody can be used with immunohistochemistry, IFAT and on Western blots. It appears that the epitope recognised by the antibody is associated with the ISAV cellular receptor. Besides being a tool to identify ECs in situ, it could be useful in further studies of the pathogenicity of ISA. Finally, the detection of an epitope shared by ECs and RBCs agrees with recent findings that these cells share a common origin, thus the MAb can potentially be used to study the ontogeny of these cells in Atlantic salmon.

Introduction

Endothelial cells (ECs) are mesoderm-derived cells that form the luminal surface of the cardiovascular system. The structure and physiological roles of ECs vary widely in accordance with the type of vessel and local, functional requirements. However, ECs are always polarised with a specialised apical, luminal surface covered by a glycocalyx (Reitsma et al. 2007) with junctions laterally, and they are basally anchored to the basement membrane (Sumpio et al. 2002). Vessel endothelia range from continuous layers with no paracellular permeability in the blood–brain barrier to fenestrated or discontinuous layers in the intestine that aid absorption (Braet & Wisse, 2002; Engelhardt, 2003). Morphologically, the ECs can be difficult to distinguish from other cell types. They are equally heterogeneous, ranging from thin and flat squamous cells to highly active cuboidal cells such as the scavenger ECs that perform endocytosis and catabolise waste macromolecules (Seternes et al. 2002). There even appear to be circulating progenitor ECs that can participate in repair of damaged endothelium and neovascularisation in adults (Steinmetz et al. 2010). The ECs are responsive and play an active role in cardiovascular functions, such as regulation of haemostasis and vasomotor tone (Cines et al. 1998). ECs participate in adaptive as well as innate immune responses including the cardinal signs of inflammation (tumor, rubor, calor, dolor) and the migration of white blood cells (WBCs) into surrounding tissue. The functionality of the ECs relies on numerous surface receptors and endocrine and paracrine activities. Given such heterogeneity it is not surprising that a unique, simple marker identifying ECs removed from their characteristic anatomical position is lacking. In human ECs, the presence of von Willebrand factor is the most unique feature of ECs (Sumpio et al. 2002).

Endothelial cells receive much attention in studies of cardiovascular diseases and in atherosclerosis, diabetes, cancer angiogenesis and malignancy, where EC dysfunction or inappropriate response contributes significantly to the pathogenesis (Cines et al. 1998; Lampugnani, 2012). However, the vital importance and disseminated structure of the circulatory system means that virtually any disease involves the EC to some extent. When an infectious disease involves haemogenic dissemination, especially in septicaemia, disruption of endothelial control of permeability and vascular leakiness are the central causes of hypovolemic shock. More specific involvement of the ECs has been found for a number of viral infections. These include highly pathogenic influenza viruses in poultry (Feldmann et al. 2000; Klenk, 2005) and Nipah virus (Maisner et al. 2009), Dengue virus (Dalrymple & Mackow, 2011) and Marburg virus (Schnittler et al. 1993) in humans, and several pathogenic viruses of fishes such as infectious haematopoietic necrosis virus (Ludwig et al. 2011), endothelial cell necrosis virus of eel (Mizutani et al. 2011) and, to an extent, viral haemorrhagic septicaemia (Brudeseth et al. 2005). However, only a few agents, such as the rickettsiae (spotted fever- and typhus groups), human herpes virus 8 and hantaviruses are well documented to have ECs as their main target (Valbuena & Walker, 2006). In addition, infectious salmon anaemia virus (ISAV) replicates mainly in Atlantic salmon (Salmo salar L.) ECs and attaches to RBC surfaces, causing severe anaemia, ascites, haemorrhages and high cumulative mortality in farmed Atlantic salmon (Aamelfot et al. 2012). ISAV is a segmented ssRNA virus in the Orthomyxoviridae family (Kawaoka et al. 2005) with two glycoproteins embedded in the virus envelope, the haemagglutinin esterase (HE) protein and the fusion (F) protein, both important for virus uptake and cell tropism. HE is essential for receptor binding (Falk et al. 2004) and recognises and attaches to specific receptors, 4-O-acetylated sialic acids (Hellebø et al. 2004).

Antibodies are widely used as markers in biological disciplines including in situ studies of disease pathogenesis. For mammals, especially mice and humans, a vast variety of antibodies are available, and in a few cases these can also be applied to fish, such as anti-PCNA (Haugarvoll et al. 2008; Gjessing et al. 2009), anti-cytokeratin (Bunton, 1993; Haugarvoll et al. 2008; Weli et al. 2013) and anti-VEGF (Khatib et al. 2010). However, knowledge acquired in mammals cannot always be transferred to ectothermic vertebrates, such as fish. Thus, still there is a paucity of working antibodies in fish, including in Atlantic salmon. In this study we characterise a novel monoclonal antibody (MAb), 10E4, and its endothelial reactivity pattern on species, cell and molecular levels and explore its relation to the ISAV receptor.

Materials and methods

Cells and virus

The Norwegian ISAV isolate Glesvaer/2/90 (Dannevig et al. 1995) was used throughout the study. Cultures of ASK-II cells (Devold et al. 2000) were used for virus propagation. Cells were incubated at 15 °C after inoculation with virus. Other cell cultures used for characterisation of the MAb included SHK-1 (Dannevig et al. 1995), TO (Wergeland & Jakobsen, 2001), CHSE-214 (Fryer et al. 1965), BF-2 (Hay, 1992), and EPC cells (Fijan et al. 1983). All of these cell cultures were grown at 20 °C in Leibovitz L-15 medium (L-15) supplemented with 10% fetal bovine serum, glutamine (4 mm), and gentamicin (50 μg mL–1).

Production of hybridomas

Antigen for immunisation of female BALB/c mice was prepared from ISAV-infected ASK-II cell culture supernatant, and purified by sucrose gradient centrifugation as described previously (Falk et al. 1997). Hybridoma cultures were produced as previously described (Falk et al. 1998) by fusing spleen cells and SP2/0-Ag-14 cells at a ratio of 4 : 1 in polyethylene glycol 1500 (Boehringer Mannheim). ISAV-infected ASK-II cells in 96-well plates were used for primary screening of hybridoma supernatants using indirect immunofluorescent (IFAT) labelling. Cells were given a dose resulting in approximately 20% infected cells. These cells were subsequentally fixed in 80% acetone in ddH2O. See below for the IFAT procedure. Positive clones were immediately subcloned twice by the limited dilution method in 96-well culture plates. Secondary screening of IFAT-positive hybridomas was performed by immunohistochemistry (IHC) on formalin-fixed, paraffin-embedded kidney tissue sections from ISAV-infected Atlantic salmon. See below for the IHC procedure. Monoclonal antibody class and subclass were determined by an ELISA-based mouse Ig Isotyping Kit (Southern Biotechnology Associates Inc.) according to the manufacturer's instructions.

Haemagglutination inhibition (HI)

The HI test was performed as previously described (Falk et al. 1998), using standard amount of virus (4 HAU) and RBCs (0.6%) for agglutination, and various dilutions of MAb.

Animal and organ samples

Atlantic salmon of approximately 400 g were obtained at Solbergstrand research station, Norway. Wild Atlantic salmon were captured in Drammenselven, Norway. Peripheral blood was collected in heparin, and tissues from heart, liver, gills, kidney, anterior kidney, spleen, skin, muscle, pyloric caeca, hind gut and brain were collected in 10% buffered formalin or snap-frozen in liquid nitrogen and stored at −80 °C. Leucocytes were isolated from blood using a discontinuous Percoll gradient (GE Healthcare) as described previously (Braun-Nesje et al. 1982) followed by centrifugation of 105 cells per slide at 200 g for 5 min in a cytocentrifuge (Shandon Cytospin 2, Pittsburgh, PA, USA). The slides were air-dried for 2 h at room temperature (RT) and stored at −80 °C until assayed. All fish were anaesthetised with methane tricaine sulphonate (MS222, Sigma, 0.1 mg mL−1) before handling. Archival, formalin-fixed, paraffin-embedded tissues collected from ISAV-infected Atlantic salmon, and tissues from 71 other species including mammals, birds, reptiles, amphibians and several different fish species (Table S1) from diagnostic and research samples at the Norwegian Veterinary Institute were included in the study.

RBC membrane isolates

RBC membranes were isolated as described by Michel & Rudloff (1989). Briefly, heparinised blood from Atlantic salmon, rainbow trout (Oncorhynchus mykiss) and cow (Bos taurus) was centrifuged at 1400 g for 10 min. Buffy coat was removed, and the blood washed four times with Fish Ringer (145 mm NaCl, 5 mm CaCl, 1 mm MgSO4, 4 mm KCl, 10 mm Hepes, 5 mm glucose, pH 7.9) 1 : 1 at 800 g for 10 min each time. Haemolysis was performed by making a 1 : 15 dilution of the packed cells in ice-cold ddH2O. During haemolysis and all subsequent homogenisation steps, PMSF (phenylmethylsulphonyl fluoride) was added to a final concentration of 0.15 mm (15 μL mL−1). The solution was kept on ice for 5 min, and then homogenised with a tight-fitting dounce (10 strokes). MET buffer (12.5 mm MgCl, 15 mm EDTA, 75 mm Tris, pH 7.5) was added 1 : 1 and the solution was homogenised again. The solution was centrifuged at 1400 g for 2 min and the supernatant saved. Homogenisation was repeated four times and supernatant fractions were pooled and pellet by centrifugation at 40 000 g for 30 min. The pellet was dissolved in a small volume of MET buffer and frozen at −80 °C.

Immunohistochemistry

Immunohistochemical labelling was performed on formalin-fixed tissue sections to identify MAb 10E4-positive cells in situ. Formalin-fixed, paraffin-embedded sections were de-waxed in xylene and hydrated in graded alcohols, followed by treatment with peroxidase block (EnVision, DAKO) for 5 min. Non-specific blocking was performed by incubation with 5% bovine serum albumin (BSA) in Tris-buffered saline (TBS; pH 7.6) for 20 min, followed by incubation with MAb and an HRP-conjugated, anti-mouse Ig-amplified detection system (EnVision, DAKO) with 3,3′-diaminobenzidine (DAB) as substrate.

To test for the binding specificity of the MAb, the following controls were included: (i) The sections were treated with 0.1 n NaOH for 30 min at room temperature for saponification. This results in de-O-acetylation of sialic acids on glycoproteins and glycolipids (Reuter & Schauer, 1994). (ii) The sections were treated with sialidase from Vibrio cholera (Sigma) for 24 h at 37 °C. This results in the removal of sialic acids on glycoproteins and glycolipids, except for sialic acids that are O-acetylated in the C-4 position (Schauer, 1982; Klausegger et al. 1999). (iii) The sections were subjected to combined treatment starting with saponification followed by sialidase, which will enable the sialidase to remove O-acetylated sialic acids. (iv) The MAb was pre-incubated with ASK-II cell culture suspension (dilution 1 : 50) for 30 min on a rotator at room temperature prior to incubation on tissue sections. The preparation was centrifuged prior to being overlaid the tissue sections. (v) Tissue sections were pre-incubated with ISAV HE antigen (100 HAU mL−1) for 30 min.

Immunofluorescent antibody test (IFAT)

Immunofluorescent antibody test was performed as previously described (Falk et al. 1997). Briefly, cell cultures on 13-mm coverslips, cytospot preparations, cryostat sections and RBC-preparations were fixed in either 80% acetone in ddH2O or 10% buffered formalin for 10 min. Formalin-fixed samples were either stained directly or permeabilised with 0.15% NP-40 in phosphate-buffered saline (PBS) before staining. Labelling was performed by incubation with MAb 10E4 and Alexa Fluor® 488 conjugated anti-mouse IgM (Molecular Probes). Co-labelling was performed on ISAV-infected cell cultures or cryostat sections from ISAV-infected Atlantic salmon. The MAb and antibody to either ISAV nucleoprotein (Aspehaug et al. 2004) (cell culture) or antibody to ISAV haemagglutinin esterase (HE) (Falk et al. 1998) (cryostat sections) were used. Alexa Fluor® 488 conjugated anti-mouse IgM and Alexa Fluor® 596 anti-mouse IgG or anti-rabbit Ig were used for detection. Sections were mounted in SlowFade® Gold (Molecular Probes).

Virus histochemistry

To identify the cells expressing the ISAV receptor, virus histochemistry was performed as previously described (Aamelfot et al. 2012). Labelling was performed with ISAV HE antigen (100 HAU mL−1), MAb to ISAV HE (Falk et al. 1998) and an HRP-conjugated, anti-mouse, Ig-amplified detection system (EnVision) with DAB substrate. To test for binding specificity, tissue sections were pre-incubated with MAb 10E4 for 30 min, followed by modified virus histochemistry using MAb to ISAV HE and Alexa Fluor (R) 488 anti-mouse IgG (Molecular probes) for the detection of bound ISAV antigen.

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot

A series of samples to be tested against MAb 10E4 (SHK-1, ASK-II and EPC cell cultures, and isolated RBC membranes from Atlantic salmon, rainbow trout and cow) were treated with lysis buffer (50 mm Tris–HCl pH 7.5, 150 mm NaCl, 2 mm EDTA, 1% NP-40, 0,5% deoxcholate) for 30 min on ice, then boiled for 5 min in sample buffer with reducing agent (Bio-Rad), prior to loading in a 12% Mini-Protean II gel (Bio-Rad). Separation of proteins was carried out as described elsewhere (Laemmli, 1970). Gels were stained with Blue Safe Protein Stain (Thermo Scientific) or they were electroblotted onto polyvinylidene fluoride (PVDF) membrane using the iBlot® Dry Blotting system (Invitrogen). PVDF membranes were immunolabelled immediately after blotting. Non-specific binding sites were blocked with 5% non-fat dry milk (Nestlé) in PBS containing 0.1% Tween 20 (pH 7.6) for 30 min. MAb was added and incubated for 60 min, followed by incubation with rabbit anti-mouse Ig/HPR (DAKO) diluted 1/1000 for 60 min and DAB substrate (Sigma), resulting in brown-coloured precipitate at the binding site.

Results

Generation of MAb 10E4

This MAb was established during attempts to produce anti ISAV-MAb that could be used for IHC on formalin-fixed, paraffin-embedded tissue sections. During initial screening of hybridoma cultures we identified a culture with a labelling pattern that resembled what we expected for an ISAV-positive MAb. However, when this MAb was used for IHC labelling of tissues sections from uninfected control salmon, a labelling pattern suggesting specificity for ECs appeared. Further preliminary testing confirmed this binding reactivity and also indicated binding to RBCs. The isotype of the MAb was determined to be IgM and κ (kappa) chain-positive.

MAb 10E4 binds to Atlantic salmon ECs, RBCs and a small fraction of Percoll gradient isolated cells

Strong and consistent labelling of ECs of arteries, arterioles, capillaries, venules and veins, as well as RBCs, was seen in all organs of Atlantic salmon investigated with the MAb by IHC. Figure 1 shows this labelling and a detailed description of selected organs. Epithelial cells and parenchymal cells were negative. Heart showed labelling of the endocardial cells in all compartments (sinus venosus, atrium, ventricle, and bulbus arteriosus). The endothelium of the central vein, the hepatic artery and the portal veins in the liver were positive, as were the sinusoids, whereas the bile duct epithelium and the hepatocytes were negative. Diffuse labelling of red pulp was seen in the spleen. In the kidney, the glomerular and peritubular ECs were strongly positive, whereas the tubular epithelium was negative. The haematopoietic tissue in the anterior kidney (analogue to the bone marrow of mammals) and the scavenger ECs labelled positive with a reticular labelling pattern. In sections from all parts of the digestive system, including oesophagus, stomach, pyloric caeca and mid- and hindgut, labelling of ECs in the capillaries, arteries, and veins was observed. Muscle and epithelial cells in the digestive system were not labelled. The specialised pillar ECs in the gills and the secondary vessels in gill and skin were positive. The interbranchial lymphoid tissue (ILT), previously described as an avascular structure (Haugarvoll et al. 2008; Koppang et al. 2010), labelled negatively. Other organs tested displayed the same endothelial and RBC labelling pattern. These included gonads, brain, skin, muscle, pseudo branch and eye.

Figure 1.

Immunohistochemical detection using MAb 10E4 on tissues from Atlantic salmon. Labelled cells (brown) are present in capillaries and blood vessels. (A) Heart with labelled endothelial and endocardial cells; Co = compact layer, Sp = spongious layer. (B) Liver with labelled vessel endothelium and sinusoidal (Si) walls (arrow). (C) Gills with labelled endothelial cells (ECs) including pillar cells (arrow); F = filament, L = lamella. (D) Stomach with endothelial cell labelling (arrow); L, lumen. (E) Kidney with labelled vessel endothelium, glomerular (G) (arrow) capillaries and peritubular ECs; T = tubuli. (F) Anterior kidney with labelled endothelium and scavenger ECs (arrow); H = haematopoietic tissue. (G) Spleen with reticular labelling pattern (arrow). (H) Mid-gut with EC labelling (arrow). (I) Skin (S) and muscle (M) with labelling of EC of capillaries and vessels. (J) Midline incision of skin (S), muscle (M) and vessels showing EC labelling (arrow), includes labelling of secondary vessel (SV) system. (K) Brain with labelled EC (arrow). (L) Female gonad with EC labelling (arrow).

Further, Atlantic salmon RBC preparations (blood smears) and cytospots (isolated blood leucocytes) were investigated using immunofluorescent labelling. RBC preparations (blood smears) investigated with confocal microscope showed that the MAb attached to a membrane-bound epitope on RBCs (Fig. 2A). All RBCs tested positive. Approximately 3–5% of the leucocyte-like cells in cytospot preparations tested positive for the MAb.

Figure 2.

IFAT labelling of red blood cell (RBC) preparations and cell cultures with MAb. (A) Labelled RBCs investigated with confocal microscope shows membrane localisation of MAb epitope. (B–D). Labelling of cell cultures investigated with a fluorescent microscope shows the ISAV-permissive cell cultures SHK-1, ASK-II and TO positive to MAb labelling. (B) SHK-1. (C) ASK-II. (D) TO. The labelling appears to be associated with the surface; however, cytoplasmic, nuclear or Golgi labelling cannot be excluded. Other cell cultures tested (i.e. EPC, BF-2 and CHSE) were negative.

RBC membranes and fish cell cultures were separated by SDS-PAGE, Western blotted to PVDF-membrane and probed with MAb. The results are shown in Fig. 3. The Western blot labelling revealed that the MAb functions on Western blot and recognises several proteins in Atlantic salmon RBC membranes and ASK-II and SHK-1 cell cultures. Cow and rainbow trout RBCs and EPC cell culture were negative (data not shown).

Figure 3.

Isolated red blood cell (RBC) membranes and cell cultures were separated by SDS-PAGE, then transferred to PVDF-membrane and probed with MAb. (A) MAb recognises proteins in Atlantic salmon RBCs. (B) MAb recognises proteins in SHK-1 cells. (C) MAb recognises proteins in ASK-II cells.

To investigate the specificity of the MAb, heart tissues from 71 other species, including fish, mammals, birds, reptiles and amphibians (Supporting Information Table S1) were subjected to immunohistochemical labelling with the MAb. Only Grayling (Thymallus thymallus) and Spiny dogfish (Squalus acanthias) tested positive for the MAb on ECs and RBCs. All other species tested negative.

Pre-treatment of the MAb with ASK-II cell culture suspension almost abolished the labelling reaction, indicating that the antigen used for immunisation of the mice indeed came from the cell culture used to cultivate the virus. Pre-incubation of sections with ISAV HE-antigen followed by MAb immunohistochemistry resulted in a normal labelling pattern, as did pre-incubation of sections with MAb followed by virus histochemistry.

MAb 10E4 binds to ISAV permissive cell cultures

To further investigate the binding capacity of the MAb, various fish cell cultures were investigated using IFAT. ISAV permissive cell cultures (i.e. ASK-II SHK-1 and TO) were MAb-positive (Fig. 2B–D), whereas other cell cultures (i.e. CHSE, BF-2 and EPC) were negative (data not shown). In the SHK-1 cells it appears that the MAb binds to the surface of all the cells (Fig. 2B). In the ASK-II cell culture the MAb binds strongly to the surface of a minor population of large, flat cells morphologically resembling cultured endothelial cells, whereas the abundant smaller cells appeared to have weaker labelling (Fig. 2C). In the TO cells the MAb binds to all the cells; however, it appears that the labelling also relates to cytoplasmic structures (e.g. Golgi apparatus), although only after permeabilisation (Fig. 2D). Permeabilisation did not affect the binding pattern of the SHK-1 or ASK-II cells.

MAb 10E4 binds to an epitope putatively related to a 4-O-acetylated sialic acid species

Several controls were included in the IHC to test for the binding specificity of the MAb. Figure 4 gives a simplified model of the controls and the results. First, the sections were subjected to mild alkali treatment. This saponification is known to de-O-acetylate sialic acids (Reuter & Schauer, 1994). This had no effect on the labelling. Secondly, the sections were treated with sialidase from Vibrio cholera, which removes sialic acids from proteins and lipids, except for sialic acids that are O-acetylated in the C-4 position (Schauer, 1982). The sialidase treatment had no effect on the labelling. Thirdly, the sections were subjected to a combined treatment of saponification followed by sialidase treatment. This saponification renders the O-acetylated sialic acids susceptible to sialidase treatment. The combined treatment abolished the labelling, indicating that the MAb binding epitope is associated with a 4-O-acetylated sialic acid species, although not directly involving the acetyl group. Lack of positive ISAV haemagglutination-inhibition test using this antibody further supports the non-involvement of the acetyl group.

Figure 4.

Principle of MAb immunohistochemical labelling after treatment with 0.1 N NaOH (i.e. saponification), sialidase and a combination of 0.1 N NaOH and sialidase.

MAb 10E4-positive epitope co-localise with expression of the ISAV receptor

To document co-localisation, virus histochemical labelling was performed on tissue sections from Atlantic salmon in order to identify expression of ISAV receptor-positive cells. The receptor expression pattern closely mirrored the pattern seen with the MAb, with labelling of ECs and RBCs in all organs. However, some discrepancies were found. The virus receptor was found on epithelial cells in gill (lamellar epithelial cells), skin (basal keratinocytes) and gut (luminal gut-epithelial cells) (Aamelfot et al. 2012) that were negative for the MAb. A comparison of MAb and virus histochemically labelled anterior kidney, gill ILT and thymus tissue sections is shown in Fig. 5. A similar pattern was visible in the sections from heart and anterior kidney. However, the reticular labelling pattern seen with virus histochemistry in the gill ILT and the thymus is not visible with MAb labelling, supporting previous findings that the gill ILT is an avascular tissue (Haugarvoll et al. 2008; Koppang et al. 2010).

Figure 5.

Immunohistochemistry with MAb 10E4 (A–D) and ISA virus histochemistry (E–H) shows co-localisation of MAb marker and ISAV receptor in heart (A,E) and anterior kidney (B,F), but not in gill interbranchial lymphoid tissue (ILT) (C,G) and thymus (D,H). The gill ILT and thymus appear to be devoid of endothelial cells and red blood cells; however, there is abundant expression of the ISAV receptor in both organs.

MAb 10E4-positive cells co-localise with ISAV-infected cells

Co-labelling of MAb-positive cells and ISAV-infected cells on infected ASK-II cell culture and cryostat sections from ISAV-infected Atlantic salmon revealed partial co-localisation of MAb-positive cells and ISAV-infected cells (Fig. 6). In the cryostat sections we found that all infected cells were MAb-positive. In the ISAV-infected ASK-II cell cultures, at 24 h post-infection (p.i.), the large flat cells labelled strongly positive for the MAb. All these large cells became infected by ISAV at this time. The smaller, rounder cells that labelled more weakly with the MAb did not initially become infected by ISAV. However, at 48 h p.i. the smaller, rounder cells had also become infected by ISAV. When investigating labelling of ISAV-infected Atlantic salmon we did not find any changes in the distribution, or signs that the epitope was more or less abundant, indicating that the detected epitope is neither up- or down-regulated as a result of ISAV-infection.

Figure 6.

Immunofluorescent labelling of ISAV-infected ASK-II cell culture (A–C) and ISAV-infected heart cryostat sections (D–F) analysed by confocal microscopy shows partial co-localisation of MAb and ISAV-infected cells. (A,D) MAb labelling of cells. (B) Antibody to ISAV nucleoprotein (NP). (E) Antibody to ISAV haemagglutinin esterase (HE). (C) Merging of (A) and (B) sections. (F) Merging of (D) and (E) sections.

Discussion

The present study characterises a novel monoclonal antibody (MAb 10E4) that binds to Atlantic salmon ECs (e.g. vessel endothelium, endocardial cells and scavenger ECs) and the surface of RBCs. The epitope recognised by the MAb is associated with the receptor of the Atlantic salmon pathogen ISAV. Our findings suggest that the MAb binds to a carbohydrate epitope related to a 4-O-acetylated sialic acid species.

Our results show a potential for the MAb as a marker for ECs in Atlantic salmon, as the MAb bound to all types of endothelium in the circulatory system including arteries, arterioles, capillaries, venules and veins as well as endocardial cells covering the lumenal surface of the heart muscle. ECs of the secondary vessel system in gills and skin, considered to be an emergency system to secure oxygen supply (Vogel, 2010), were also detected. A number of specialised ECs were labelled, such as the pillar cells of gill lamellae and both kidney glomerular cells and scavenger ECs (Seternes et al. 2002) lining the sinusoids of the kidney. In short, no ECs appeared devoid of labelling, which was somewhat surprising given the heterogenicity of this cell type and the limited availability of general endothelial cell markers in mammals.

The other cell type found to be MAb-positive in Atlantic salmon was the RBCs, where the labelling was confined to the surface. This raises the question of how ECs and RBC are related. Several studies have suggested that ECs and RBCs share a common progenitor cell (Gering et al. 1998; Liao et al. 1998; Vogeli et al. 2006). Recently, a possibly common vertebrate mechanism where adult haematopoietic stem cells develop from haemogenic endothelium of the embryonic dorsal aorta has been described (Lancrin et al. 2009; Bertrand et al. 2010). This could explain why ECs and RBCs share the epitope of the MAb. However, the ontogeny of haematopoietic cells and endothelium has been elusive due to its stepwise and complex nature (Bautch, 2011; Yoder, 2012). It will be interesting to explore Atlantic salmon embryology as well as angiogenesis and neovascularisation using this MAb. Preliminary screenings on yolk-sac fry indicate that vessels growing inside the eye during development are easily visualised with the MAb (data not shown). Interestingly, when the MAb was tested on tissues from other species of fish and selected mammals, birds, reptiles and amphibians, the dual reactivity with only ECs and RBCs was also found in the few other species that reacted with the MAb, Grayling (Thymallus thymallus) and Spiny dogfish (Squalus acanthias).

The MAb did not label epithelial cells and parenchymal cells in general, but some labelling in kidney interstitium (the equivalent of mammalian bone marrow) and spleen stroma was observed and could be explained by their functions in erythropoiesis and RBC degradation, respectively. Leucocytes isolated by gradient centrifugation of blood were generally not labelled by the MAb, except for a small fraction of positive cells. As these cells are selected by physical means, the labelled cells could be erythrocytic cells physically more similar to leucocytes, e.g. erythroblasts (Conroy & Conroy, 2006) or circulating ECs (Steinmetz et al. 2010). The MAb labelling pattern in lymphoid organs such as thymus and gill ILT (Haugarvoll et al. 2008) supports the notion that the labelled cells in the cytospots are not necessarily leucocytes, as both organs were more or less devoid of labelling by the MAb. In contrast, extensive labelling was demonstrated by virus histochemistry detecting 4-O-acetylated sialic acids, as recently demonstrated by Aamelfot et al. (2012) in both thymus and gill ILT. This suggests that the virus histochemistry may detect different 4-O-acetylated species in comparison with the MAb, which attaches to a specific sialic acid directly. O-acetylated sialic acids have been demonstrated in T-cells in humans (Schauer, 1982), and both thymus and gill ILT are dominated by T-cells (Koppang et al. 2010).

For further characterisation of the Mab, several fish cell cultures were labelled. Reactivity was confined to cell cultures normally used for ISAV cultivation (i.e. SHK-1, ASK-II and TO). These cells are derived from Atlantic salmon anteriour kidney (mammalian bone marrow analogue) cells and mitogen-stimulated peripheral blood leucocytes. The labelling pattern varied among the positive cell cultures. Specifically, in ASK-II cells the MAb bound strongly to the surface of a minor population of large, flat cells resembling endothelial cells, but more weakly to the dominant population of smaller, rounder cells, indicating a mixed cell population in the ASK-II cells. This suggests a possible potential for the MAb to be used for cell sorting. The SHK-1 and TO cells are described in the literature as macrophage-like (Dannevig et al. 1995; Wergeland & Jakobsen, 2001); however, the results reported here suggest that they may be of endothelial origin, possibly scavenger ECs.

Recently, we showed that ECs and RBCs are the main target cells of ISAV in Atlantic salmon (Aamelfot et al. 2012). The same study showed that ECs, RBCs and some epithelial cells expressed the ISAV receptor molecule, 4-O-acetylated sialic acid, on the cell surface. In this study the MAb reactivity was confined to the ECs and RBCs, ISAV-infected or not. Co-localisation of ISAV-infected cells and MAb epitope on cell culture and cryo-sections points towards an association of the MAb epitope with the ISAV sialic acid virus receptor. Added to this, when investigating the MAb specificity on a molecular level using saponification and sialidase treatment we found that only a combined treatment abolished the reaction, further substantiating the close association between the ISAV cellular receptor and the MAb binding epitope. This, and the fact that the ISAV haemagglutination-inhibition test was negative, suggests that the epitope recognised by the MAb is associated with a 4-O-acetylated sialic acid species, though not directly with the acetyl group where ISAV bind. This could explain why some epithelial cells carry an ISAV receptor but do not react with the MAb. Added to this, the MAb was produced during experiments establishing monoclonal antibodies against ISAV, and it is not unlikely that the cellular receptor was present in the semi-pure virus preparation used for immunisation of the mice.

To conclude, this study provides a detailed analysis and characterisation of a novel cell marker recognising an epitope on Atlantic salmon ECs (e.g. vessel endothelium, endocardial cells, scavenger ECs) and RBCs. The MAb can be used for immunohistochemistry, immunocytochemistry and Western blot analysis, and the epitope of the MAb is associated with the ISAV receptor. The MAb may enable the separation and further analysis of existing cell cultures from Atlantic salmon, as well as the isolation and establishment of endothelial cell cultures, opening new avenues for pathogenesis studies of endotheliotrophic infections. Furthermore, as this is the first report of a monoclonal antibody directed specifically against all types of Atlantic salmon ECs, it opens up new possibilities for studying this highly heterogeneous cell population important for so many aspects of disciplines such as anatomy, ontogeny, physiology and pathophysiology. Due to the broad specificity for ECs and RBCs, the MAb may be an especially good tool for studying salmon haematopoiesis and vasculogenesis.

Acknowledgements

The authors wish to thank the Norwegian Institute for Water Research (NIVA Solbergstrand), T. Poppe, A.K. Jøranlid and J. Schönheit at the Norwegian Veterinary Institute (NVI), K. Zimmer and A. Frøyse at the Norwegian School of Veterinary Science (NSVS), A. Lynghammar at the University of Tromsø, and W.R. Bennett and K. Garver at the Fisheries and Oceans Canada for providing samples from various species, and K. Høybakk, S. Benestad and I. Modahl at NVI and C.M. Olsen at NSVS for technical assistance. The work was funded by the Atlantic Innovation Fund, Canada Inc. and Novartis Animal Health, and the Norwegian Research Council, project no. 186907.

Conflict of interest

The authors declare that they have no conflict of interest.

Authors' contributions

M.A. carried out the experiments and drafted the manuscript. S.W. participated in the WB experiments and the design of the study, and helped to draft the manuscript. K.F. produced the MAb, participated in the design and coordination of the experiment, and aided in drafting of the manuscript. O.B.D. and E.O.K. participated in the design of the study and helped to draft the manuscript. All authors read and approved the final manuscript.

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