Production of a monoclonal antibody for carp (Cyprinus carpioL.) phagocytic cells and separation of the cells

Fish & Shellfish Immunology (1998) 8, 91–100

Production of a monoclonal antibody for carp (Cyprinus carpio L.) phagocytic cells and separation of the cells CHIHAYA NAKAYASU, MIYUKI OMORI, SATOSHI HASEGAWA, OSAMU KURATA NOBUAKI OKAMOTO*

AND

Department of Aquatic Biosciences, Tokyo University of Fisheries, Konan 4, Minato-ku, Tokyo 108, Japan (Received 10 June 1997, accepted 16 June 1997) A monoclonal antibody (MAb; TCL-BE8) for carp peripheral blood leucocytes (PBL) was produced, and its reactivity was analysed by electron microscopy and flow cytometry. Electron microscopy using immunogold labelling showed that this MAb reacted with neutrophils and monocytes. The antibody reacted with 98% of the cells in a fraction of granulocytes separated by flow cytometry. The antigen detected by this was MAb defined as a membrane protein of Mr of 112 kDa. Using a magnetic separator, cells reactive with this antibody were separated from leucocytes with a density of 1·08 g ml "1 or 1·08–1·09 g ml "1. MAb-positive cells in the PBL with a density of 1·08 g ml "1 consisted of a mixture of neutrophils and monocytes, whilst in PBL with a density of 1·09 g ml "1 they consisted of only neutrophils. The MAb-negative fractions contained mainly lymphocytes and thrombocytes. The MAb-positive cells showed strong phagocytosis, which is a characteristic function of neutrophils and monocytes, but this was absent from the MAb-negative fraction. Purification of monocytes and neutrophils, or isolation of neutrophils, can be achieved by using this MAb allowing more e#ective analysis of the carp leucocyte functions. ? 1998 Academic Press Limited Key words:

monoclonal antibody, carp, monocytes, cell separation, phagocytosis.

I. Introduction The existence of various leucocyte subpopulations in teleost fish was reported on the basis of the morphology of the cells (Ellis, 1977). Recently, leucocyte subpopulations have been characterised using monoclonal antibodies (MAbs) against specific membrane antigens. In fish, production of MAbs against serum immunoglobulin of carp (Secombes et al., 1983), rainbow trout (DeLuca et al., 1983), channel catfish (Lobb & Clem, 1982) and sea bream (Navarro et al., 1993) enabled B lymphocytes to be identified. MAbs against other subpopulations of leucocytes have also been reported for channel catfish T cells (Ainsworth et al., 1990), channel catfish nonspecific cytotoxic cells (Evans et al., 1988), carp thymocytes or T lymphocytes (Secombes et al., 1983; Rombout et al., 1997), sea bass thymocytes (Scapigliati et al., 1995), rainbow trout thrombocytes and granulocytes (Slierendrecht et al., 1995) and carp *Author to whom correspondence should be addressed. 1050–4648/98/020091+10 $25.00/0/fi970125

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thrombocytes (Nakayasu et al., 1997). A MAb that reacted with neutrophils was described in channel catfish by Bly et al. (1990). Separation of leucocyte subpopulations is important in order to investigate cell function. Although leucocyte subpopulations have been separated mainly by density gradient centrifugation with Percoll, using MAbs for separation of the subpopulations is more e#ective than ordinary methods. In fish, separation of lymphocyte subpopulations by MAbs has been accomplished using the immune a$nity adherence technique of panning (DeLuca et al., 1983; Miller et al., 1987) or magnetic cell separation system (Koumans-van Diepen et al., 1994). The present study examined the production of a MAb against phagocytic cells of carp, and the characterisation of the cell populations separated with the MAb by a combination of a magnetic cell separation system and density gradient centrifugation. II. Materials and Methods FISH AND PREPARATION OF CELLS FOR PRODUCTION OF MAbS

Carp (Cyprinus carpio L.) weighing approximately 300 g were obtained from Yoshida Research and Training Station, Tokyo University of Fisheries. The fish were maintained in the laboratory in a 400-l tank with running water at a temperature of 25&1) C and fed commercial pellets. Heparinised blood samples were taken from the caudal blood vessel. The blood cells were washed by centrifugation (800#g, 5 min, 4) C) in RPMI-1640 (Nissui Pharmaceutical Co.) and the bu#y-coat cells were collected. The collected cells were then overlaid onto Histopaque 1077 (Sigma Chemical Co.) and centrifuged (350#g, 20 min, 4) C) to separate peripheral blood leucocytes (PBL). PBL with density of 1·077 g ml "1 were collected and washed three times with RPMI-1640.

PRODUCTION OF MAbS

The collected PBL were washed twice with Ringer’s solution (0·75% NaCl, 0·02% KCl, 0·75% CaCl2 and 0·002% NaHCO3), resuspended in 100 ìl of the same solution. The cell suspension was injected intraperitoneally into a 4-week-old Balb/c mouse, and similar injections were given 21 and 42 days later. Three days after the final injection, the spleen was removed from the mouse, and splenocytes were prepared by pressing the tissue through a stainless steel sieve in RPMI-1640. Hybridomas were produced by the fusion method using polyethylene glycol as described by Gefter et al. (1977), with P3U1 myeloma cells. After the fusion, the cells were suspended in Git medium (Wako) containing HAT and plated on a thymocyte feeder layer in 96-well microculture plates. After incubation at 37) C for 14 days, 100 ìl aliquots of the hybridoma supernatants were put on acetone-fixed carp PBL preparations on slides and were screened by immuno glucose oxidase-staining using Vectastain (ABC-GO kit, Vector Laboratories). A hybridoma could be selected by light microscopy because the supernatant reacted with only a part of the

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cell populations. The positive hybridoma was cloned twice and cultured in GIT, and a hybridoma cell line (TCL-BE8) was established. The isotype of this MAb was the IgG2b subclass. IMMUNOELECTRON MICROSCOPY

Carp PBL suspensions (1#107 cells 100 ìl "1) were incubated with 100 ìl of a MAb (TCL-BE8) for 45 min on ice. The cells were washed twice with RPMI-1640 and incubated with 100 ìl of a 1:25 dilution of 15 nm goldconjugated goat anti-mouse Ig antibody (British Biocell International) for 45 min on ice. After washing twice in RPMI-1640, the cell pellets were fixed with 2·5% glutaraldehyde in 0·1 M phosphate bu#er on ice. After 60 min, the cells were washed six times with 7% sucrose and postfixed with 1% OsO4 in phosphate-bu#ered saline for 90 min on ice. The cells were then dehydrated in graduated ethanol concentrations and embedded in Spurr resin (Taab Laboratories). Ultrathin sections were cut and stained with uranyl acetate and lead acetate. As a negative control, cells were incubated with isotype matched irrelevant primary antibody. The samples were observed with a Hitachi H-7000 transmission electron microscope (TEM; Hitachi). FLOW CYTOMETRIC ANALYSIS

Carp PBL were adjusted to 1#106 cells 100 ìl "1 of RPMI-1640 supplemented with 10% heat-inactivated foetal bovine serum (FBS: Commonwealth Serum Laboratories) and 0·1% sodium azide (NaN3), and incubated with 100 ìl of hybridoma supernatants for 45 min on ice. After washing the cells twice in Ringer’s solution, 50 ìl of fluorescein isothiocyanate (FITC)-labelled goat anti-mouse Ig antibody (Bio Source International) diluted 1:100 with RPMI-1640 containing 10% FBS was added to the cells and incubated for 30 min on ice. After washing twice in Ringer’s solution, the cells were screened on a flow cytometer (EPICS XL, Coulter). Cells in a negative control were incubated with isotype matched irrelevant primary antibody. IMMUNOPRECIPITATION

Leucocytes suspension (1#107 cells) in 1 ml phosphate bu#ered saline (PBS) was centrifuged (400#g, 4) C, 5 min), and the pellets resuspended in 1 ml lysis bu#er (250 mM NaCl, 25 mMTris-HCl, pH 7·5, 5 mM EDTA, pH 8·0, 1% NP-40, 2 ìg ml "1 aprotinin, 2 ìg ml"1 leupeptin, 1 mM phenylmethylsulfonyl fluoride, 0·5% sodium deoxycholate) and incubated for 20 min on ice. The mixture was then centrifuged (12 000#g, 4) C, 10 min), the supernatant collected, labelled with D-biotinoyl-å-aminocaproic acid-N-hydroxysuccinimide ester (biotin-7-NHS; Cellular Labelling and Immunoprecipitation Kit, Boehringer Mannheim) and stored at "80) C. After absorption for 2 h at 4) C by Protein G-Agarose (Boehringer Mannheim), the cell lysate was centrifuged (12 000#g, 4) C, 10 min) and the supernatant incubated with MAb TCL-BE8 for 2 h at 4) C. Next, Protein G-Agarose was added and the mixture incubated overnight at 4) C. After washing to remove unbound proteins, the Protein G-Agarose complex was centrifuged (12 000#g, 4) C, 20 sec) and resuspended

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in gel-loading bu#er (0·125 M Tris-HCl, pH 6·8, 20% glycerol, 10% 2-mercaptoethanol, 4·6% SDS, 0·0025% bromophenol blue). This suspension was boiled for 3 min to denature proteins and then the Protein G-Agarose was removed by centrifugation (12 000#g, room temperature, 20 sec). The supernatant was transferred to a fresh tube. Samples were analysed by sodium dodecyl sulphate-polyacrylamide (10%) gel electrophoresis (SDS-PAGE). After electrophoretic transfer onto a PVDF membrane (PVDF-PLUS; Micron Separation Inc.), the labelled proteins were detected on a film (X-Omat AR; KODAK) by the BM Chemiluminescence Blotting Kit (Boehringer Mannheim). As a negative control, immunoprecipitation with an isotypematched control antibody was performed. PHAGOCYTIC CELL SEPARATION BY THE MAb

By density gradient centrifugation using Percoll (Sigma Chemical Co.), cells with densities of 1·08 g ml "1 and 1·08–1·09 g ml "1 were separately collected into tubes. The collected cells were adjusted to a concentration of 1#107 cells 100 ìl "1 with RPMI-1640 containing 10% FBS and 0·1% sodium azide. These cells were incubated with the MAb TCL-BE8 on ice for 60 min, and washed twice in RPMI-1640 without FBS. Aliquots (5#107) of washed cells were then incubated for 20 min at 4) C with 500 ìl of a 1:5 dilution of magnetic bead-conjugated goat anti-mouse Ig antibody (Miltenyi Biotec GmbH). Cell separation was carried out by using a magnetic separation system (Mini Macs; Miltenyi Biotec GmbH). The complex was applied to a plastic column to which an external magnet was attached. MAb-positive cells remained in the column by magnetic attraction, and MAb-negative cells flowed through the column. Smears of the collected MAb-positive and MAb-negative cells were stained with either Giemsa (Merck) and/or Sudan black B (Sigma Chemical Co.), the latter to identify neutrophilic granulocytes before observation under the light microscope. Phagocytosis of the separated cell populations was investigated with the bacterial thin-layer method of Seki et al. (1989) as modified by Nakayasu et al. (1995). The pathogenic fish bacteria Edwardsiella tarda, was suspended in distilled water, heated at 60) C for 10 min and incubated in plastic dishes for 1 h. The fluid of each dish was then removed and the dishes were dried rapidly. The MAb-positive and MAb-negative cells collected by the magnetic separation system and unfractionated PBL (2#106 cells each) were laid on the bacterial thin-layer on a dish. The dishes were incubated at 20) C for 30 min. Samples were carefully removed and the dishes were gently washed using RPMI-1640 without FBS. These dishes were then stained with Pfei#er solution for a bacterial stain. Plaques on the bacterial thin-layer, due to the phagocytic activity of leucocytes, were counted in 10 microscopic fields (magnification: #100) for each dish. III. Results IMMUNOELECTRON MICROSCOPY AND FLOW CYTOMETRIC ANALYSIS

Examination of the immunoreaction of MAb TCL-BE8 with cell suspensions confirmed the binding of the gold particles on the membranes of neutrophils

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Fig. 1. Transmission electon micrograph of a neutrophil immunoreacted with MAb TCL-BE8. Bar=1 ìm. Gold particles (arrows) were present at the surface of the neutrophil, which possesses numerous granules containing a dense rod-shaped core.

that had an eccentric nucleus and granules containing a dense rod-shaped core (Fig. 1), and of monocytes that were rich in cytoplasmic organelles and had reniform nucleus and granules in the cytoplasm (Fig. 2). Such binding was not observed on other leucocytes. The control, which was incubated with an isotype matched irrelevant primary antibody, did not show any binding to the carp PBL. The forward (FS) and sideward (SS) scatter patterns of the flow cytometer divided carp PBL into two fractions (A and B) (Fig. 3a). Fraction A consisted of granulocytes and fraction B consisted of a mixture of lymphocytes and thrombocytes. Monocytes could not be detected as a fraction because of their low number in PBL. The cells reacting with MAb TCL-BE8 were granulocytes (Fig. 3b). The MAb TCL-BE8 reacted positively with approximately 98% of the cells in fraction A (Fig. 3c) and 1–4% in fraction B (Fig. 3d). No reaction was found in the negative control. IMMUNOPRECIPITATION

SDS-PAGE analysis of leucocyte membranes absorbed with Protein G-Agarose indicated the presence of many protein bands (Fig. 4 lane C). Immunoprecipitation of PBL membrane protein with TCL-BE8 resulted in the isolation of a protein membrane molecule of Mr of approximately 112 kDa (Fig. 4, lane A). Immunoprecipitation with an isotype matched control antibody did not leave any membrane proteins in the lysate (Fig. 4, lane B). PHAGOCYTIC CELL SEPARATION BY THE MAb

Carp PBL with density of 1·08 g ml "1 mainly consisted of lymphocytes, thrombocytes, granulocytes and monocytes (Fig. 5a): approximately 1–3%

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Fig. 2. Transmission electron micrograph of a monocyte immunoreacted with MAb TCL-BE8. Bar=1 ìm. Gold particles (arrows) were present at the surface of the monocyte, which showed pseudopodia, and contained many vesicles of variable size and a reniform, largely euchromatic nucleus.

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Fig. 3. Flow cytometric analysis of carp peripheral blood leucocytes (PBL). Flow cytometry FS/SS-dot plots: (a) all PBL, granulocytes (gate A) and lymphocytes (gate B); (b) only PBL reacted with the MAb TCL-BE8. Flow cytometry fluorescence histograms of PBL labelled with MAb TCL-BE8 for gate A (c) and for gate B (d).

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Fig. 4. SDS-PAGE analysis of immunoprecipitated leucocytes membranes with MAb TCL-BE8 (lane A), or an isotype-matched control antibody (lane B) and leucocyte membranes absorbed with protein G-agarose (lane C). Arrows and numbers designate Mr values of a set of reference proteins.

neutrophils; 1–2% monocytes. The magnetic cell separation system revealed that neutrophils and monocytes were TCL-BE8 positive in the MAb positive fraction, where they accounted for 75–90% of the cells (Fig. 5b). Neutrophils and monocytes were not seen in the MAb negative fraction (Fig. 5c). On the other hand, PBL with a density of 1·08–1·09 g ml "1 mainly consisted of neutrophils, lymphocytes and thrombocytes: approximately 50–80% neutrophils. Only neutrophils were found in the MAb positive fraction (Fig. 5d). Confirmation that the MAb negative fraction did not contain phagocytic cells was shown in the phagocytosis assay where plaque formation was absent in the MAb negative fraction but present in the positive fraction (Fig. 6 ). IV. Discussion In this study, production of a MAb TCL-BE8, that reacts with carp phagocytic cells (neutrophils and monocytes) was accomplished, and separation of the phagocytic cells or isolation of the neutrophils was completed using this MAb. Flow cytometric analysis of FS/SS profiles of leucocytes in the carp pronephros resulted in three fractions: lymphocytes, monocytes and granulocytes (Verburg-van Kemenade et al., 1994). The results of this study showed that carp PBL were divided into two fractions; granulocytes and a mixture of lymphocytes and thrombocytes. A monocyte fraction was not found because of the low numbers in PBL. The MAb TCL-BE8 reacted with 98% of the cells in the granulocyte fraction, but not with the cells in the lymphocyte/ thrombocyte fraction. After immunogold labelling with the MAb TCL-BE8, membranes of neutrophils and of monocytes were selectively labelled with gold particles. Identification of the cells was based on morphological criteria described by Cenini (1984) and Imagawa et al. (1989). By immunoprecipitation

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Fig. 5. Fractionation of PBL using MAb (TCL-BE8 and a magnetic separator (a) PBL with a density of 1·08 g ml "1; lymphocytes (L), thrombocytes (T) and neutrophils (N) are visible. Monocytes are not shown in this photograph. (b) MAb-positive cells in PBL with a density of 1·08 g ml "1; neutrophils and monocytes were collected with this MAb; (c) MAb-negative cells in PBL with a density of 1·08 g ml "1; neutrophils and monocytes are completely absent, whilst lymphocytes (L) and thrombocytes (T) are visible. (d) MAb-positive cells in PBL with a density of 1·08–1·09 g ml "1; only neutrophils, which were positive for Sudan black B staining, were found. Bar=30 ìm (a, b and c) and 20 ìm (d).

of membrane lysates of carp PBL with TCL-BE8, the antigen recognised by this MAb was defined as a membrane protein of Mr of 112 kDa. The MAb TCL-BE8 positive cells were neutrophils and monocytes using PBL with a density of 1·08 g ml "1 and neutrophils using PBL with a density of 1·08–1·09 g ml "1. Sudan black B staining distinguished between neutrophils and monocytes because only neutrophils were positive. A more precise cell classification in each fraction was carried out with TEM (data not shown). The MAb positive cells had phagocytic activity, which is one of the main functions of neutrophils and monocytes, lacking in the negative cells. Such data show that the MAb TCL-BE8 is specific for carp phagocytic cells and that cells collected with the separation system using this MAb maintain phagocytic activities. The separation and identification of carp phagocytic cells or the isolation of neutrophils that has become possible with this MAb will allow more e#ective analysis of carp leucocyte functions.

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Fig. 6. Plaques (arrowheads) due to phagocytic activity and leucocytes (arrows) were observed on the bacterial thin-layer. (a) phagocytic activity of carp PBL. (b) activity of the MAb positive cells. (c) activity of the MAb negative cells. Bar=100 ìm. The authors wish to thank Ms Hiroko Adachi, National Cancer Research Institution, Japan, for her technical support. This study was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (Nos. 07660239, 08660226) and by a grant from the Japanese Fisheries Agency.

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