Immunocytochemical analysis of a monoclonal antibody specific for rainbow trout (Oncorhynchus mykiss) granulocytes and thrombocytes

Veterinary Immunology and lmmunopathology 46 (1995) 349-360

Veterinary immunology arKi immunopathology

Immunocytochemical analysis of a monoclonal antibody specific for rainbow trout (Oncorhynchus mykiss) granulocytes and thrombocytes W.J. Slierendrecht?“,

N. Lorenzenb, J. Glamannc, C. Koch’, J.H.W.M. Romboutd

“Danish Trout Culture Research Station. Br#ns M@lle\aej7. DK-6780 Skm-blek. Denmark “National Veterinary Laboratory, Hangevej 2. DK-8200 Aarhus. Denmark ‘Stutens Senaninstitrrt, Artillerivej 5, DK-2300 Copenhagen, Denmark “Depurtment of Experimental Animal Morphology and Cell Biology. Agricultural University. P.0. Box- 338, 6700 AH Wageningen. Netherlands

Accepted 14 July 1994

Abstract A monoclonal antibody against rainbow trout peripheral blood leucocytes was selected for its lack of reactivity with rainbow trout immunoglobulin. Its reactivity with leucocytes from peripheral blood, head kidney and spleen was analysed by flow cytometry and electron microscopy, and compared with that of monoclonal antibodies directed against rainbow trout immunoglobulin, which reacted with B cells. B lymphoblasts and plasma cells. The antibody reacted with S-20% of the peripheral blood leucocytes, 8-9% of head kidney leucocytes and 5-7% of spleen leucocytes. Electron microscopical immunocytochemistry revealed that the antibody reacted strongly with granulocytes and weakly with thrombocytes, and not with erythrocytes, lymphocytes, monocytes or macrophages. The antibody has possible applications in the identification and isolation of rainbow trout leucocytes, either alone or in combination with other monoclonal antibodies.

1. Introduction

The study of different leucocyte subpopulations in fish is complicated by the fact that differentiation primarily relies on morphological criteria (Ellis, 1977; Rowley et al.. 1988). For mammals, and especially for mice and humans, many monoclonal antibodies (MAbs) against specific membrane antigens are available, which permit the identification of leuco* Corresponding author. 01652427/95/$09.50

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cyte subpopulations (reviewed by Roitt et al., 1989). A similar panel of MAbs, which could identify different subpopulations of fish leucocytes, would considerably facilitate the study of the fish immune system. As in mammals, B cells in fish can be identified by membrane-associated immunoglobulin (Ig) molecules, as has been shown for channel catfish (Lobb and Clem, 1982), carp (Secombes et al., 1983) and rainbow trout (DeLuca et al., 1983; Thuvander et al., 1990). MAbs reacting with other (sub)populations of leucocytes have only been reported for channel catfish, carp and Atlantic salmon. Miller et al. (1987) described a MAb which reacted with T cells, thymocytes, neutrophilic granulocytes and thrombocytes of channel catfish. A MAb reacting with presumed Tcells in channel catfish was reported by Ainsworth et al. (1990), although this MAb needs further characterisation to determine its possible reactivity with other cell types. The reactivity of a MAb with channel catfish neutrophilic granulocytes was demonstrated by Bly et al. (1990) and Ainsworth et al. (1990). Nonspecific cytotoxic cells (NCC) in channel catfish, reacted with a MAb produced by Evans et al. ( 1988). A MAb against carp thrombocytes was produced by Koumans-van Diepen (1993). For Atlantic salmon, a MAb reacting only with granulocytes and monocytes in peripheral blood was reported (Devold Maaseide et al., 1993)) and this MAb seems to cross-react with rainbow trout leucocytes (Hamdani et al., 1993). So far, other MAbs against rainbow trout Ig-negative leucocytes have not been reported. The present report describes the production and characterisation of a MAb against Ignegative leucocytes from peripheral blood, head kidney and spleen of the rainbow trout. The proportion of reactive leucocytes was determined by flow cytometry, whereas electron microscopy was used for the morphological characterisation of immunoreactive peripheral blood and head kidney leucocytes. Different leucocyte types were identified by the criteria defined by Ellis (1977) and Rowley et al. (1988) for fish in general, and by Chiller et al. (1969), Lehmann and Stiirenberg (1975) and Thuvander et al. (1987) for the rainbow trout.

2. Material and methods 2.1. Fish Thirty rainbow trout (Oncorhynchus mykiss (Walbaum) ) of 300400 g body weight were kept in a UV-filtered recirculation system at a water temperature of 15-16°C. Before the sampling of blood, spleen or head kidney, trout were anaesthetised or killed in a solution of 75 mg l- ’ tricaine methane sulphonate (TMS; Crescent Research Chemicals, Phoenix, USA), buffered with 150 mg I- ’ Na,C03. 2.2. Collection of leucocytes Heparinised blood samples were taken from the caudal blood vessel. The heparin concentration in the final sample was 50 IU ml-‘. Peripheral blood leucocytes (PBL) were collected after centrifuging the samples over a discontinuous density gradient (Lymphoprep@; Nycomed, Oslo, Norway), according to the supplier’s instructions. Head kidney

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leucocytes (HKL) or spleen leucocytes (SL) were obtained by squeezing tissue pieces of head kidney (pronephros) or spleen through a nylon gauze filter. A continuous density gradient was prepared by centrifuging 60% Percoll@ (Pharmacia, Uppsala, Sweden) for 45 min at 15 000 X g; HKL and SL were collected after centrifuging for 30 min at 840 X g over this gradient. 2.3. MAbs Production of MAbs followed the principles given by Kohler and Milstein ( 1975). MAb Hyb106-9 was obtained by immunising mice with Ig-negative PBL. Ig-positive cells were depleted by immuno-absorption. In brief, lo8 PBL in Tris-buffered Eagle’s minimum essential medium (C&co, Paisley, UK) were incubated for 30 min at 4°C with a 1:5 dilution of MAb 3D7 against rainbow trout Ig (Thuvander et al., 1990)) followed by washing and incubation for 15 min at 4°C with a rabbit anti-mouse Ig antiserum (dilution 1:25; Dakopatts, Glostrup, Denmark). After washing twice, cells were incubated for 20 min with Protein-A sepharose 6MB beads (Pharmacia) and unbound cells were collected for immunisation. Less than 1% of these cells appeared to be Ig-positive in immunofluorescence microscopy. MAbs were selected by means of an enzyme-linked immunosorbent assay (ELISA), in which 30 ~1 per well of 10’ PBL ml- ’ were used for coating. After drying at 37”C, cells were fixed with methanol:acetone 1: 1. Bound antibody was demonstrated with horseradish peroxidase-conjugated rabbit anti-mouse Ig (dilution 1: 1000; Dakopatts, Glostrup, Denmark) followed by the H,O,-orthophenylenediamine (OPD) staining method. Absorbance was read at 492 nm in a TIM10 ELISA reader (Life Technologies, Roskilde, Denmark). Of seven MAbs obtained, the IgM MAb Hyb106-9 was selected because it did not react with rainbow trout Ig, as demonstrated by a catching ELISA for Ig and immunoblotting of purified Ig from this species. MAb 2E9 was obtained by immunising mice with rainbow trout buffy coat cells. This MAb appeared to react with the heavy chain of rainbow trout Ig and hence with rainbow trout B cells. The reactivity of this MAb (of IgG 1 class) was identical with that of an earlier described MAb against rainbow trout Ig, 3D7 (Thuvander et al., 1990). Both MAbs (2E9 and 3D7) were used in this study to detect or isolate Ig-positive cells. 2.4. Flow cytometry Approximately lo6 leucocytes were incubated with 250 ~1 of a 1:50dilution of hybridoma supernatant for 30 min on ice. After washing in RPM1 + buffer (RPM1 1640; Gibco, UK) with 1% bovine serum albumin (Sigma, St. Louis, MO, USA), 0.1% sodium azide (Merck, Darmstadt, Germany) and 20 IU heparin ml-’ (Leo, Denmark), the leucocytes were incubated with 250 ~1 of a 1:200 dilution of fluorescein isothiocyanate (FITC) labelled rabbit anti-mouse Ig antibody (Dakopatts, Glostrup, Denmark) for 15 min on ice. After washing twice in RPMI+ buffer, 104 cells per sample were screened on a FACStar or FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA, USA). The Consort 30 or FACScan software program was used for analysis. The MAbs Hyb106-9 and 2E9 were tested individually on PBL, HKL and SL, or simultaneously on PBL. In addition, a double staining was carried out using tetramethylrhodamine

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B isothiocyanate (TRITC)-conjugated 2E9. As a negative control, cells were incubated with FITC-conjugated rabbit anti-mouse Ig antibody only. 2.5. Electron microscopy Samples were prepared for electron microscopy according to the method of Van Diepen et al. ( 199 1)) with some minor modifications. In short, PBL or HKL (2.5 X lo6 cells per sample) were incubated on ice for 30 min with 250 ~1 RPM1 with MAb diluted 1:25. The cells were washed in RPM1 + buffer and incubated on ice for 45 min with 100 ~1 of 15 nm gold-conjugated goat anti-mouse Ig antibody (Janssen Pharmaceutics, Beerse, Belgium) diluted 1:5. After washing twice in RPM1 + buffer, cell pellets were fixed in 1% K$r,O,, 2% glutaraldehyde and 1% osmium tetroxide in 0.1 M sodium cacodylate buffer, dehydrated and embedded in Epon. Ultrathin sections were cut, stained with uranyl acetate for 5 min and lead citrate for 30 s, and subsequently studied in a Philips 201 electron microscope. Cells incubated with gold-conjugated goat anti-mouse Ig antibody alone were used as a control.

3. Results 3.1. Flow cytometry Fig. 1 shows representative flow cytometry histograms of Hyb106-9 and 2E9-stained PBL. Approximately 5-20% of PBL varying between stained individuals reacted with Hyb106-9, and approximately 40% reacted with 2E9. 2E9-reactive cells appeared to be cells with an intermediate forward scatter (FSC) and a low sideward scatter (SSC). A limited proportion of peripheral blood cells with low FSC/SSC values and almost all cells with higher FSCYSSC values were Hybl06-9 immunoreactive (data not shown), although in head kidney, some of the cells with a high FSC/SSC did not react with Hyb106-9 (Figs. 2(a) and 2(b) ). The usual range of reactive HKL was 8-9%, whereas about 5-7% of the SL were reactive. Double labelling of PBL with TRITC-conjugated 2E9 and Hybl06-9 indirectly labelled with FITC showed that these MAbs reacted with different cells (Fig. 3): around 40% appeared to be Ig-positive and around 7% Hyb106-9 positive. 3.2. Electron microscopical

immunocytochemistry

Control preparations did not show labelling of leucocytes with gold particles. The MAb Hybl06-9 was shown to bind to two leucocyte types: granulocytes and thrombocytes. Gold particles were absent from all observed lymphocytes, monocytes/macrophages and erythrocytes. Granulocytes were characterised by large oval electron-dense granules and an irregularly shaped nucleus. Besides a high local concentration of gold particles on the cell membranes, a considerable number of gold particles was also found in endosomes inside the cells (Fig. 4). Phagocytosed pigment particles from demolished melano-macrophages were occasion-

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-, la)

86%

14%

FL1

FL1

Fig. I. Flow cytometry histogramof peripheral blood leucocytes, incubated with (a) monoclonal antibody Hyb 106. 9 and (b) monoclonal antibody 2E9, followed by FITC-labelled rabbit anti-mouse Ig antibody.

ally found in the cytoplasm of the granulocytes, indicating the phagocytic capacity of these cells. Thrombocytes were defined as irregularly shaped cells with large vacuoles in the cytoplasm. Sometimes, labelling was found on the cell membrane, but more often gold particles were situated in vacuoles in the cell (Fig. 5). Only gold particles bound to a MAb were taken up by either cell type, as the negative controls, in which the cells had been incubated with the gold-conjugated second antibody alone, did not show this uptake. Monocytes could be distinguished from mature granulocytes by their more circular shape, the absence of the characteristic oval granules, and the presence of smaller, round, and less electron-dense lysosomes. These monocytes did not bind MAb 106-9 (Fig. 6). In head kidney, some cells were less easy to characterise, as immature granulocytes with fewer or no oval granules strongly resembled monocytes. These intermediate stages were partly positive with Hyb106-9. However, macrophages containing phagocytosed pigment particles were not reactive with Hyb106-9. It could be such macrophages, which are the negative

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Forward

Scatter

‘200 ‘4BB ‘200 ‘Bcm ‘i,,, Forward

Scatter

Fig. 2. Flow cytometry dot plots, showing forward scatter and sideward scatter of cells incubated with Hyb106-9 and FITC-labelled rabbit anti-mouse Ig antibody: (a) all HKL, both reactive and non-reactive with Hyb106-9: (b) only HKL reactive with Hyb106-9.

cells with high FSC-SSC values in the head kidney flow cytometry profiles shown in Fig. 2. Immunogold labelling reactions demonstrated that 2E9 reacted with some of the lymphocytes. In HKL preparations, lymphoblast-like cells and plasma cells were reactive.

Fig. 3. Flow cytometry dot plots of PBL. showing fluorescence I (FITC) and fluorescence 2 (TRITC). The dot plot results differ with respect to the antibodies used in the first incubation step: (a) an incubation with TRITCconjugated 2E9, followed by FITC-labelled rabbit anti-mouse Ig; (b) an incubation with Hyb106-9. followed by FITC-labelled rabbit anti-mouse Ig; (c) an incubation with both TRITC-conjugated 2E9 and unconjugated Hyb106-9, followed by FITC-labelled rabbit anti-mouse Ig.

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Fluorescence

1

Fluorescence

1

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Fig. 4. Electronmicrograph of a Hyb 106-9 immunoreactive granulocyte (G), defined by its lobed, irregular nucleus and its round to oval electron-dense granules. Gold particles are present on the membrane (black arrows), as well as in endosomes (white arrows) inside the cells, whereas gold particles are absent from the lymphoid cells (L)

Fig. 5. Electronmicrograph of a Hyb 106-9 immunoreactive thrombocyte, charactensed by the presence of large vacuoles in its cytoplasm. Note the occurrence of gold particles at the membrane (black arrow) and in the vacuolar structures (white arrow).

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3.57

Fig. 6. Electronmicrograph of a monocyte or macrophage-like cell (M). non-reactive with Hyb106-9. and a granulocyte (G), reactive with Hyb106-9. In the monocyte, the oval granules are absent, whereas some circular lysosomes (L) and a phagosome (P) are present in the cytoplasm.

Lymphoblast-like cells were defined as cells with a lymphoid nucleus, but a higher cytoplasm/nucleus ratio than lymphocytes, and the absence of granular inclusions. Plasma cells were characterised by a well-developed rough endoplasmic reticulum, often with dilated cisternae, a lymphoid nucleus, and a variable number of gold particles. Gold particles were frequently found in clusters of three or more at the surface of B cells and plasma cells.

4. Discussion MAb Hyb106-9 reacts with about 520% of the PBL, 8-9% HKL and 5-7% SL. MAb 2E9 reacts specifically with about 40% of the PBL, probably the B lymphocytes and plasma cells. Thuvander et al. (1990) also described almost half of the rainbow trout PBL as Igpositive, using MAb 3D7. Flow cytometric double staining strongly suggests that 2E9 and Hyb106-9 react with two different subpopulations of PBL. After immunogold labelling with MAb Hyb106-9, gold particles were found on granulocytes and thrombocytes, but not on lymphocytes, monocytes and erythrocytes. In head kidney, macrophages characterised by the absence of oval granules and the presence of phagocytosed pigment were also negative. However, it appears to be difficult to distinguish immature granulocytes from monocytes in head kidney, a phenomenon described earlier for light microscopic observations (Lehmann and Stiirenberg, 1975). As some of these cells were Hyb106-9 positive, this MAb may be used to differentiate between developmental stages of granulocytes and monocytes in head kidney. Classification of granulocytes is mainly based on morphological characteristics and rarely on functional studies. In fish, three types of granulocyte are usually distinguished: neutro-

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philic (or heterophilic) , eosinophilic and basophilic granulocytes. Eosinophilic and basophilic granulocytes are reported to be absent or only present in very low numbers in rainbow trout (Ellis, 1977; Thuvander et al., 1987; Rowley et al., 1988), and the commonly observed granulocytes seem to be neutrophilic. Neutrophilic granulocytes are involved in the nonspecific defence mechanisms of the fish, mainly through phagocytosis (Thuvander et al., 1987), and this corresponds well with the frequent presence of phagocytosed pigment particles within the granulocytes of head kidney. In the present study, more gold particles were observed inside thrombocytes than on their cell membrane. The number of gold particles associated with each cell was much lower than for granulocytes. Gold particles were not found in thrombocytes subjected to negative control (incubation without MAb), which indicates that thrombocytes are labelled specifically by the MAb. Apparently the low temperature used (0°C) and the presence of sodium azide do not completely inhibit endocytosis in rainbow trout cells. The function of thrombocytes is still unclear, but seems to be mainly an involvement in blood clotting, and a limited phagocytic activity (Ellis et al., 1977; Rowley et al., 1988). Thuvander et al. ( 1987) classifies rainbow trout thrombocytes as phagocytic cells, because they are able to ingest latex beads. In reviews by Ellis (1977) and Rowley et al. (1988), the phagocytic capacity of thrombocytes is questioned. A possible explanation for the characteristic vacuoles, if they do not reflect phagocytic activity, could be that they are part of a canalicular system of cell membrane invaginations, as described for human platelets (White, 1972). Both lymphocytes and plasma cells labelled with 2E9 showed clustered gold particles on their surface. A similar phenomenon, which does not seem to be related to patching, was also described by Van Diepen et al. (1991) for carp and Navarro et al. (1993) for sea bream. In contrast to mammalian plasma cells (Roitt et al., 1989), most rainbow trout plasma cells appear to have Ig at their surface. This feature has also been described for carp plasma cells (Koumans-van Diepen et al., 1994). It is noteworthy that the number of gold particles bound seems to differ between plasma cells, possibly indicating different stages in plasma cell maturation. The results presented in this report indicate that MAb Hyb106-9 reacts strongly with rainbow trout granulocytes and weakly with thrombocytes. An interesting future application of this antibody will be its use in combination with MAb IE3D9 (Devold Maaseide et al., 1993), which seems to react with both granulocytes and monocytes. A pure monocyte population can be isolated by purifying granulocytes and monocytes from a population of leucocytes by immunoabsorption with IE3D9 as catching antibody, followed by depletion of the granulocytes by means of Hyb106-9. Identification of monocytes will also be possible by double staining with these two antibodies. Another application will be to deplete leucocyte populations of granulocytes and thrombocytes for immunisations to obtain MAbs against other leucocyte subpopulations.

Acknowledgements This work was supported by a grant from the Danish Governmental Biotechnology Programme 1987-1995 and by a grant from the Nordic Council of Ministers. The authors

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wish to thank Nicole Hansen of the Danish Trout Culture Research Station, Denmark, for technical assistance, Ellen Harmsen of the Agricultural University, the Netherlands, for flow cytometry support and Anja Taveme-Thiele of the Agricultural University, the Netherlands, for assisting with the electron microscopy.

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