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Killing of isogeneic erythrocytes by neutrophils in ginbuna crucian carp (Carassius auratus langsdorfii)
Fish & Shellfish Immunology (1998) 8, 531–544 Article No. fi980156
Killing of isogeneic erythrocytes by neutrophils in ginbuna crucian carp (Carassius auratus langsdorfii) UWE FISCHER1*, MITSURO OTOTAKE2
AND
TERUYUKI NAKANISHI2
1
Institute of Applied Virology, Federal Research Centre for Virus Diseases of Animals, Friedrich-Loeffler-Institutes, Insel Riems, D-17498, Germany, and 2 National Research Institute of Aquaculture, Inland Station, Immunology Section, Tamaki, Mie, 519-04 Japan (Received 8 January 1998, accepted after revision 18 May 1998) Cytotoxic activity of fish neutrophils against isogeneic erythrocytes is described. Unsensitised neutrophil-rich leucocytes from clonal ginbuna crucian carp, separated by density gradient centrifugation, were cultured with isogeneic erythrocytes. Lysis of isogeneic erythrocytes was determined by measuring the release of haemoglobin into the supernatant using tetramethylbenzidine (TMB). The level of cytotoxicity obtained was proportional to both the content of granulocytes in e#ector cell fractions and the e#ector to target cell ratio. Lysis of isogeneic erythrocytes occurred only after direct contact of neutrophils with target erythrocytes. No erythrolysis was observed when granulocytes and erythrocytes were cultured in the same medium separated by a polycarbonate membrane, thus allowing exchange of only supernatant but not cells. Erythrolysis was therefore not due to a soluble factor released into the supernatant by the granulocytes. When erythrocytes and neutrophils were cultured together by immobilising them in agarose, erythrolysis was only observed microscopically in cases where erythrocytes were in contact with neutrophils. Thus, neutrophils appear able to cause lysis of histocompatible cells. 1998 Academic Press Key words:
ginbuna crucian carp, in vitro cell-mediated cytotoxicity, neutrophils, erythrocytes, autologous killing, Carassius auratus langsdorfii.
I. Introduction Vertebrate neutrophils are mobilised in response to chemotaxins (e.g. to the site of inflammation), are able to phagocytose foreign material or cells and kill various cells including bacteria. After infiltrating tissues by diapedesis, neutrophils release granules and generate toxic oxygen products. Mammalian neutrophils release cytotoxic substances, for example tumour necrosis factor, reactive oxygen intermediates or defensins (Lehrer, 1993). At sites of inflammation they secrete proteases and other enzymes which contribute to localised breakdown of the foreign material. These functions, with the exception of cytotoxicity have been extensively studied in fish (reviewed by MacArthur & Fletcher, 1985; Ainsworth, 1992; Secombes, 1996). *Author to whom correspondence should be addressed. Email [email protected] 1050–4648/98/070531+14 $30.00/0
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Several reports deal with cytotoxicity mediated by granulocytes in man. Granulocytes are capable of antibody-dependent cell-mediated cytotoxicity (Jahr et al., 1986). Bacterial endotoxins contribute to cytotoxicity of neutrophils against bronchial epithelial and endothelial cells in man (Koyama et al., 1991; Barnett et al., 1995). Little information is available on cell-mediated cytotoxicity of granulocytes in lower vertebrates. In leopard frogs (Rana pipiens), NK-like cells showing morphological similarities to human large granular lymphocytes, are capable of killing allogeneic erythrocytes and xenogeneic tumour cells (Ghoneum & Cooper, 1987). Weissmann et al. (1978) showed cytotoxicity of dogfish granulocytes against sea urchin eggs. Cytotoxic activity of neutrophils against xenogeneic cells has also been described in carp (Kurata et al., 1995; Nakayasu et al., 1997) and ginbuna crucian carp (Kurata et al., 1996). However, it is unclear whether cyprinid neutrophils are capable of killing autologous cells. In man, a natural killer-like cell exhibiting cytotoxicity against autologous erythrocytes shows phenotypic similarities to T-lymphocyte precursors (Pukhova et al., 1988). It is well known that human neutrophils exhibit cytotoxicity against autologous cells in the presence of bacterial endotoxins (Koyama et al., 1991). Cytolytic activity of immune cells against autologous hapten-modified (Verlhac et al., 1990) or virus-infected (Hogan et al., 1996) cells has been shown in channel catfish. However, the e#ector cells are not granulocytes. During studies on cell-mediated cytotoxicity of ginbuna crucian carp leucocytes against allogeneic erythrocytes (Fischer et al., 1998) we found a killing activity of neutrophils against autologous erythrocyte controls. The phenomenon of autologous erythrocyte killing has not yet been described before in fish. In the present paper we describe the killing activity of neutrophilic granulocytes against isogeneic erythrocytes and show its dependence on cell to cell contact. II. Materials and Methods FISH
Clonal triploid ginbuna crucian carp (Carassius auratus langsdorfii) is a naturally occurring gynogenetic species, widely distributed throughout Japan. Two clones from di#erent locations: Lake Suwa in Nagano Prefecture (S3n, 40–60 g) and Okushiri Island in Hokkaido (OB1, 200–250 g; Nakanishi, 1987a–c) were used. Tetraploid hybrids (S4n) were produced by insemination of S3n fish with goldfish (Carassius auratus) sperm as described previously (Nakanishi, 1987a). S4n fish possess a triploid set of chromosomes from S3n clone and a haploid set of chromosomes from goldfish. Progenies weighing between 30 and 50 g were reproduced gynogenetically by artificial insemination of sperm. Fish were maintained at 25 C in 60 l tanks supplied with aerated running spring water and were fed commercial dry pellets. Clonality of the three clones was determined by DNA fingerprinting (unpublished data) and transplantation experiments, where isogeneic scale grafting did not result in graft rejection (Nakanishi, 1987a).
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PREPARATION OF EFFECTOR AND TARGET CELLS
Fish were anaesthetised with ethyl 4-amino-benzoate (EAB; WAKO) at a concentration of 0·1 mg/ml. Approximately 0·3 ml of blood was drawn from the caudal blood vessel using a syringe rinsed with heparin (WAKO) at 1000 IU/ml in phosphate bu#ered saline without magnesium and calcium salts (PBS ). Blood was immediately diluted in a five-fold volume of cold ERDF (enriched RPMI 1640, Dulbecco’s MEM and Ham’s F12 with 2:1:1 ratio; Murakami et al., 1982) medium (Kyokuto) supplemented with RD-1 (insulin, transferrin, sodium selenite, ethanolamine; Kyokuto; 16·53 g/vial/l) and 0·2% bovine serum albumin (initial fraction, Sigma). In the case of smaller fish, blood from several clonal fish was pooled, then diluted with medium (e.g. 21 ml blood plus 8 ml medium). The blood cells were pelleted, supernatant was removed and the pellet was resuspended in 3 ml of Percoll (Sigma)-in-Hanks’ balanced salt solution (Nissui) with a density of 1·098 g/cm3. Onto this suspension, 3 ml each of Percoll-in-Hanks’ balanced salt solutions with densities of 1·080, 1·070, 1·040 g/cm3 and medium were layered sequentially. After centrifugation for 40 min at 650g at 4 C peripheral blood leucocytes (PBL) were collected from the interfaces with a Pasteur pipette, washed twice with ERDF (200g, 4 C, 10 min), stained with trypan blue (Sigma) then counted using a Thoma haemocytometer and a phase contrast microscope. Cells within fractions isolated from di#erent interfaces (1·0951/0·080, 1·080/1·070, 1·070/1·040 g/cm3) were morphologically identified as lymphocytes, monocytes, granulocytes, thrombocytes, erythrocytes and dead cells using trypan blue staining and a phase contrast microscope. Nephros and pronephros were cut into pieces of about 4 mm3 and immediately transferred into a cold Potter-Elvehjem homogeniser previously filled with 7 ml of supplemented ERDF as above containing 30 units/ml of heparin. Kidneys were gently homogenised and the cell suspensions passed through a steel mesh (size 150 gauge) to remove coarse aggregates. The cells were pelleted and kidney leucocyte (KL) subpopulations were isolated and counted as described above for PBL. For preparation of target cells 300 ìl of blood was collected, diluted in supplemented ERDF described above, and centrifuged for 10 min at 200g at 4 C. Bu#y coat was removed with Pasteur pipette. The remaining erythrocytes were washed once with supplemented ERDF (200g and 4 C for 10 min) and adjusted to a final concentration of 2105 cells per ml (2104 erythrocytes/100 ìl/well). NON-RADIOACTIVE CYTOTOXICITY ASSAY
A non-radioactive cell-mediated cytotoxicity assay was used according to the technique described by Rüegg & Jungi (1988) with modifications. The assay had originally been designed to examine antibody-mediated erythrolysis of antigen-coated human and sheep erythrocytes by human macrophages and monocytes and uses 3,3 ,5,5 -tetramethylbenzidine (TMB) dihydrochloride (Sigma) to detect haemoglobin released from lysed target erythrocytes. Target cell control (TSR=target cell spontaneous release and TMR= target cell maximum release) and e#ector cell control (ESR=e#ector cell
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spontaneous release) wells of a 96-well U-bottom tissue culture plate (Falcon, U.S.A.) were filled with 100 ìl of ERDF. A volume of 200 ìl of medium was added to the medium background (Me) and volume correction control (VCC) wells. ESR and experimental (EXP) wells were filled with PBL or KL corresponding to a ratio of 20 e#ector cells to one target erythrocyte. The target cell number added to TSR, TMR and EXP wells was constant and set to 2104 erythrocytes per well. In each experiment triplicate wells were used. After adding medium, target and e#ector cells to the wells, plates were centrifuged for 5 min at 200g, then incubated in a CO2 incubator in a humidified atmosphere at 25 C for 5 h. An aqueous solution of Triton X100 (Sigma) (10 ìl of 0·01%) was added to the TMR and VCC wells 45 min before harvesting the supernatants. Prior to harvesting the supernatants, cells were gently mixed by pipetting and plates were again centrifuged for 5 min at 200g. Fifty ìl of supernatant was removed from each well and transferred to 96-well flat-bottom E.I.A. microtitration plates (ICN). A solution of 3,3 ,5,5 -tetramethylbenzidine (TMB) dihydrochloride (Sigma) substrate was freshly prepared by dissolving 50 ìg of TMB in 10 ml of 90% acetic acid to which was added 10 ml of distilled water containing 20 ìl of 30% H2O2 solution. Undiluted particles were removed from the substrate solution by centrifuging at 1700g for 5 min at 20 C. Fifty ìl of the substrate solution was added to the wells containing the supernatants. Plates were incubated for 30 min at room temperature in the dark, then read in an ELISA reader (BIO-RAD, model 3550) at 405 nm. The percentage of specific cytotoxicity was calculated using the following formula. % Specific cytotoxicity=((EXPESR)TSR)/(TMRVCC)TSR) 100%. Firstly e#ector leucocytes from OB1 or S3n fish were tested against isogeneic target erythrocytes, then S3n leucocytes were retested against allogeneic erythrocytes from S4n fish. TMB assay showed clear correlation to a 51Cr release assay (Fischer et al., 1998). CULTURE OF NEUTROPHIL ENRICHED KL WITH TARGET ERYTHROCYTES IN INSERT PLATES
Twenty-four well cell culture plates (Falcon) and 9 mm cell culture polycarbonate membrane inserts with either a 1·0 or 3·0 ìm pore size (Falcon) were used to co-culture isogeneic neutrophils and erythrocytes from OB1 clonal fish separately. For separate co-culture, 1 ml of medium containing 2·8106 e#ector kidney neutrophils was added to each well, and 800 ìl of medium containing 1·6105 target erythrocytes was then added to the inserts. For direct co-culture control, target and e#ector cells were resuspended in 1 ml of medium, then added to the inserts, and 800 ìl of cell-free medium was added to the inserts. Cell concentrations corresponded to an e#ector to target cell ratio of 17·5 to 1. E#ector and target cells were cultured for 6·5 or 30 h. Supernatants were taken from wells or inserts and investigated in TMB assay.
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% cytotoxicity
100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0%
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2.5:1
5:1 10:1 20:1 Effector : target cell ratio
40:1
Fig. 1. Cell-mediated cytotoxicity of neutrophil enriched head kidney leucocytes (Percoll fraction 1·095/1·080 g/cm3) from OB1 clone against isogeneic erythrocytes. The content of neutrophils in the e#ector cell population is 44·4%. Cytotoxicity is directly dependent from e#ector to target cell ratio. Vertical bars represent the standard deviation obtained from triplicate wells.
AGAROSE ASSAY
Glass slides for histology were pre-coated by dipping the slides into 0·3% agarose (gel point 36 C; FMC Bio products) followed by drying in horizontal position at 60 C in a convection oven. Slides were stored at 4 C. Kidney neutrophil enriched fraction (Percoll 1·095/1·070 g/cm3) and erythrocytes were prepared from OB1 clonal fish as described above. Erythrocytes (107) and neutrophils (105) were resuspended in 2 ml of 0·4% agarose (type VII-A, gel point 26 C; Sigma) in supplemented ERDF at 30 C. Immediately after resuspending the cells, 1 ml of the cell–agarose mixture was spread onto the pre-coated glass slides. The slides were incubated at 25 C for 30 min in a humidified chamber, then examined under a light microscope. STATISTICS
Results were compared statistically by an f-test followed by a two-tailed Student’s t-test using MS Excel 5.0 (Microsoft Corp.) and were considered to be significantly di#erent at Pc0·05. III. Results KILLING OF ISOGENEIC ERYTHROCYTES BY NEUTROPHILS
Cytotoxicity of neutrophils against isogeneic erythrocytes was evident, and cytotoxicity increased as the e#ector to target cell ratio increased (Fig. 1). The level of cytotoxicity was directly dependent on the number of neutrophils in e#ector cell fractions. Neutrophils with distinct granulation lysed erythrocytes more e$ciently than granulocytes with weak granulation. PBL and KL from the highest Percoll density (between 1·095 and 1·080 g/cm3) which
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100%
58%
90% 23%
% cytotoxicity
80% 70%
58%
60% 44%
50% 40% 31%
30% 20%
30%
10% 0%
n.t. 0% OB1 KL versus OB1 erythrocytes
OB1 PBL versus OB1 erythrocytes
18% 31% S3n KL versus S3n erythrocytes
18%
31%
S3n KL versus S4n erythrocytes
Fig. 2. Cell-mediated cytotoxicity of di#erent KL and PBL fractions separated by Percoll gradient centrifugation against isogeneic and allogeneic erythrocytes. E#ector:target cell ratios 20:1; culturing time 5 h. Lysis results are presented as mean of triplicate wells. Percentages on columns indicate contents of neutrophils in e#ector cell fraction.
contained the highest level of neutrophils (up to 58%) showed highest cytotoxicities (Fig. 2). Furthermore, the KL from this Percoll fraction contained the highest density of well granulated neutrophils. In contrast, fractions of lower Percoll density contained fewer neutrophils with weaker granulation. These cells showed lower or almost no cytotoxicity toward isogeneic erythrocytes. PBL neutrophils were only found in the 1·095/1·080 g/ cm3 Percoll fraction and showed a well developed granulation. OB1 KL from the low density fraction (1·070/1·040 g/cm3) exhibited a cytotoxicity against isogeneic erythrocytes of more than 10%, whereas corresponding S3n KL showed almost no cytotoxicity against isogeneic erythrocytes. Furthermore, S3n neutrophil enriched KL, isolated from the interface between 1·095 and 1·080 g/cm3 killed allogeneic S4n erythrocytes more e$ciently than isogeneic S3n erythrocytes (Fig. 2). KL from S3n fish isolated from less dense Percoll fractions, on the other hand, contained few neutrophils. These cells exhibited weaker granulation and were unable to lyse isogeneic or allogeneic erythrocytes. REQUIREMENT OF EFFECTOR TO TARGET CELL CONTACT FOR KILLING OF ERYTHROCYTES
E#ector cells and erythrocytes were co-cultured by either separating them with a membrane of 3·0 ìm pore size or in direct contact, to assay whether erythrocytes are killed by neutrophils after direct cell contact, or if cytotoxic substances released by neutrophils into the supernatant result in erythrocyte destruction. High levels of cytotoxicity were observed when neutrophil enriched KLs were in direct contact with isogeneic erythrocytes (Fig. 3, targets and e#ectors both in well). However, the percentage of cytotoxicity
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KILLING OF ISOGENEIC ERYTHROCYTES BY NEUTROPHILS 80% 70%
d b
% cytotoxicity
60%
c 50% 40% 30% a
20% 10% 0%
b a
c
6.5 h p.incub.
Targets in insert - effectors in well; supernatant from insert Targets and effectors both in well; supernatant from insert Targets in insert - effectors in well; supernatant from well Targets and effectors both in well; supernatant from well
d 30 h p.incub. insert well plate scheme
Fig. 3. Direct and separated co-culture of neutrophil enriched KL e#ectors and isogeneic erythrocyte targets. The content of neutrophils in the e#ector cell population was 50%. Targets and e#ectors were co-cultured either under direct cell to cell contact (target and e#ector cells are both in the well) or separately (target erythrocytes in an insert with a pore size of 3 ìm and e#ector cells in the well). Considerable amounts of cytotoxicity could be detected if targets and e#ectors were co-cultured so as to allow direct cell to cell contact. Vertical bars represent the standard deviation obtained from triplicate wells. The letters a, b, c and d denote groups with significantly di#erent means (Pc0·05).
was less than 4% when targets and e#ectors were co-cultured in the same medium separated by a membrane (Fig. 3, targets in insert – e#ectors in well). Levels of cytotoxicity obtained after co-culturing the cells in direct contact with each other did not increase with incubation time (up to 30 h). However, the amount of haemoglobin present in the supernatants collected from the inserts after 30 h was higher than after 6 h, probably because it took time to allow the haemoglobin to pass through the membrane. Similar results were obtained using inserts with 1 ìm pore size (data not shown). MORPHOLOGICAL EVIDENCE OF KILLING OF ISOGENEIC ERYTHROCYTES BY NEUTROPHILS
To confirm the finding that erythrocytes were killed by neutrophils during direct cell to cell contact, we cultured neutrophil enriched KL with isogeneic erythrocytes partially immobilised in agarose. The content of neutrophils in KL was 54·3% on average (data obtained from three separate experiments). Erythrocytes were only lysed when they were in direct contact with neutrophils (Figs 4, 5). Active movement of neutrophils toward erythrocytes was not observed with distances greater than a cell diameter. If erythrocytes and neutrophils came into contact, neutrophilic granules were
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Fig. 4. Killing of ginbuna crucian carp erythrocytes by isogeneic neutrophils. (a) before killing, two erythrocytes are in contact with a neutrophil; (b) after killing, one of the erythrocytes has collapsed and only the nucleus remains visible. Note: the lymphocyte (arrowed) was not able to lyse two neighbouring erythrocytes.
observed moving to the pole of the cell in contact with the erythrocyte. In some cases neutrophils also appeared to move along the membrane of the erythrocyte. Several minutes after contact, erythrocytes collapsed, but their nuclei remained visible (Fig. 4b). Other cell types such as lymphocytes were not able to kill erythrocytes (Fig. 4a, b).
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Fig. 5. Killing of ginbuna crucian carp erythrocytes by isogeneic neutrophils. A neutrophil killing an erythrocyte.
IV. Discussion In this report cytotoxicity of neutrophils against isogeneic erythrocytes by direct contact of e#ector neutrophils with target erythrocytes was demonstrated. This finding seems to be uncommon and it has to be questioned why neutrophils do not cause lysis of autologous erythrocytes in vivo. The possibility of killing due to genetic di#erences between e#ector and target cells can be excluded in our studies as clonality of fish was confirmed by DNA fingerprinting (unpublished data), scale grafting and cell transfer studies (Nakanishi et al., 1987). Isogeneic as well as autologous erythrocytes stained with a fluorescent cell linker survived for more than 2 weeks in the recipient, while allogeneic erythrocytes were killed and disappeared within a few days (unpublished data). We cannot deny the possibility that erythrocytes may change their surface antigens when removed from their physiological in vivo conditions and treated ex vivo. Altered surface antigens may act as receptors for neutrophils and induce killing of the altered erythrocytes. Under physiological conditions aged mammalian erythrocytes are phagocytosed by specialised macrophages in the spleen, liver and bone marrow (Schiebler & Schneider, 1991). The mechanism by which aged erythrocytes are removed from circulation in fish remains unclear. Human natural killer cells with the phenotype of T-lymphocyte precursors show cytotoxicity toward autologous erythrocytes (Pukhova et al., 1988). It has been suggested that these cells may play a role in the induction of erythroid di#erentiation of haematopoietic stem cells and in the killing of aged erythrocytes (Pukhova et al., 1984). It has been shown that
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mammalian neutrophils may exhibit a cytotoxic activity against autologous erythrocytes. However, in these cases the neutrophils had been stimulated by zymosan (Claster et al., 1984) or phorbol myristate acetate (Weiss, 1980, 1982; Vercellotti et al., 1985). Human erythrocytes express significant levels of the integrin-associated protein (IAP) on their surface. IAP is involved in signal transduction during neutrophil activation (Rosales et al., 1992; Lindberg et al., 1994). If fish erythrocytes, like those of man, express IAP on their surface, this may explain the activation of neutrophils in our experiments. Another possible mechanism of enhanced cytotoxicity by neutrophils is the up-regulation of the intercellular adhesion molecule (ICAM). In mice, upregulation of ICAM on target cells promotes cytotoxicity by neutrophils (Barnett et al., 1995). It can be concluded that the culture conditions of the erythrocytes in our experiments were close to physiological, since the spontaneous haemoglobin release of erythrocytes was negligible after 30 h of culture. However, the possibility of surface antigen alterations of erythrocytes cannot be excluded. Ex vivo handling of neutrophils may also have had an influence on their activity. Glasser & Fiederlein (1990) established that the conditions used to separate neutrophils influenced their function, such as chemotaxis and chemiluminescence. Youssef et al. (1995) reported that isolation of human neutrophils at temperatures lower than 37 C causes a significant increase in the expression of â-2 integrin (CD11b) and other surface adhesion molecules. Integrin-like molecules similar to LFA-1 have been reported on catfish leucocytes (Yoshida et al., 1995). L-selectin (CD62L), on the surface of human neutrophils, is decreased after separation procedures involving sedimentation and hypotonic lysis of erythrocytes. Physiological disorders resulting from hypoxaemia in humans leads to an up-regulation of phagocytosis by polymorphonuclear leucocytes (Knowles et al., 1995). Cross-linking of L-selectin induces adhesion of neutrophils to human endothelial cells (Simon et al., 1995). The up-regulation of adhesion molecules may be one reason for isogeneic killing by neutrophils in vitro. In our experiments lysis of erythrocytes occurred only after e#ector to target cell contact and was not caused by substances released by neutrophils into the supernatant. This was shown by co-culturing of neutrophils and erythrocytes separated by polycarbonate membranes and in the agarose assay. It can therefore be suggested that e#ector cells do not release cytotoxic substances into the supernatant, or that the amount of cytotoxic factors if released into the medium is not su$cient to kill the erythrocytes. Spontaneous killer cells from frog also require contact to allogeneic erythrocytes for e$cient killing (Ghoneum & Cooper, 1987). Malorni et al. (1993) designated the contact region between cytotoxic e#ectors and targets as ‘ closed chamber ’ which appears to contribute to killing e$ciency by creating a contact region in which cytotoxic factors can induce target cell lysis. Requirement of cell contact for killing was further confirmed from observations of neutrophils attaching to the membranes of erythrocytes. In the agarose experiments we observed a movement of intracellular granules toward the pole of the neutrophil, which was in contact with the erythrocyte. It has also been shown in neutrophil mediated ADCC against antigen-coated mammalian
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erythrocytes that granules accumulate near the target–e#ector interface before erythrocytes undergo lysis (Petty et al., 1992). Previous studies on cell-mediated cytotoxicity of ginbuna crucian carp leucocytes against allogeneic erythrocytes showed that the TMB assay clearly correlates to a 51Cr release assay and shows even higher sensitivity (Fischer et al., 1998). In these studies, leucocytes exhibited lysis of autologous erythrocyte controls only when the e#ector cells contained neutrophils whereas unsensitised leucocytes containing no neutrophils but high percentages of lymphocytes did not kill either isogeneic or allogeneic erythrocytes. Therefore, in these studies neutrophils were excluded from the e#ector cell fraction by density gradient centrifugation. Kurata et al. (1995, 1996) and Nakayasu et al. (1997) reported that neutrophil enriched head kidney leucocytes of common carp and ginbuna crucian carp killed xenogeneic cells derived from humans. Cytotoxicity of neutrophils to xenogeneic cells was attributed to a natural killer cell-like activity. It cannot be excluded that neutrophils from carp also show cytotoxic activity against autologous cells, e.g. erythrocytes, although this has not yet been examined. In our experiments we found that neutrophil enriched fractions of KL exhibited a stronger cytotoxicity toward allogeneic erythrocytes than to isogeneic erythrocytes, suggesting that alloantigens enhance the cytotoxic reaction of neutrophils. On the other hand, leucocyte fractions containing low density neutrophils exhibited lower or no cytotoxicity against erythrocytes. Those granulocytes were less granulated. The weaker killing activity can most likely be attributed to the low maturation stage of the granulocytes. Several reports in mammals support the suggestion, that low density cells of the myeloid lineage are functionally immature in fish too (Ross et al., 1978; Bol & Williams, 1980; Olo#son et al., 1980; Berkow & Dodson, 1986; Glasser & Fiederlein, 1987). In preliminary experiments, we used cytocentrifugation of Percoll fractions from KL and PBL followed by Giemsa staining and flow cytometry where granulocytes were identified as large granulated cells (data not shown). Ginbuna crucian carp PBL separated into three fractions by Percoll according to the protocol described in this paper are positive for Sudan black B and peroxidase staining in the lowest fraction suggesting that the corresponding cells are granulocytes. Esterase positive cells can also be isolated from lighter Percoll fractions and seemed to be monocytes because this reaction was inhibited under the presence of NaF. However, there are medium-sized negative cells for both Sudan black B and Peroxidase in the lowest fraction (Ainsworth, 1992). Numbers of e#ector cells used in our studies were 2·5 to 40 times higher than those of target erythrocytes, and consequently the probability of neutrophils coming into contact with erythrocytes was very high. However, in blood the situation is di#erent since the number of erythrocytes exceeds that of neutrophils more than 1000 times (Fänge, 1992). If neutrophils are able to recognise damaged erythrocytes and lyse them, activated neutrophils may also kill neighbouring intact erythrocytes. This mechanism would markedly enhance the killing activity under conditions of high e#ector to target cell ratios. However, it seems that this does not happen in vivo. It is well known that neutrophils contain a wide range of endogenous enzymes including peroxidase. The peroxidase activity is measurable in
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supernatants from lysed ginbuna crucian carp neutrophil rich leucocytes using TMB (unpublished data). To exclude the spontaneous release of peroxidase into the supernatant we subtracted the e#ector cell spontaneous release (ESR) when calculating the % cytotoxicity. Using monocytes as e#ector cells and erythrocytes as target cells, Rüegg & Jungi (1988) systematically calculated higher cytotoxicities in TMB assay than in the chromium release assay. However, the authors did not subtract ESR from the experimental release. It is strongly recommended to subtract the ESR whenever detection of endogenous enzyme activity is the principle of a test. The lactate dehydrogenase release assay which is used as a cytotoxicity detection kit also requires subtraction of ESR (Boehringer Mannheim or Promega). In conclusion, neutrophils of ginbuna crucian carp are able to kill isogeneic erythrocytes in vitro by cell to cell contact. Whenever e#ector cells which contain neutrophils are used, cytotoxicity of the neutrophils has to be taken into consideration. We are grateful to Dr Th. C. Mettenleiter, Federal Research Centre for Virus Diseases of Animals, Insel Riems, Germany, and Dr Kimberley D. Thompson, Institute of Aquaculture, University of Stirling, Scotland, for critical reading of the manuscript. This study was supported by a grant from the Science and Technology Agency of Japan and in part by a grant-in-aid (Bio Media Program) from the Ministry of Agriculture, Forestry and Fisheries of Japan (BMP 97-V-2-1-4).
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Jahr, H., Kiowski, S. & Baehr, R. (1986). Bestimmung der antikörperabhängigen Zytotoxizität von Granulozyten, Monozyten und Lymphozyten unter Verwendung humaner Erythrozyten als Targetzellen. Allerg. Immunol. 32, 277–268. Klempau, A. E. & Cooper, E. L. (1984). T-lymphocyte and B-lymphocyte dichotomy in anuran amphibians: III. Assessment and identification of inducible killer T-lymphocytes (IKTL) and spontaneous killer T-lymphocytes (SKTL). Developmental and Comparative Immunology 8, 649–661. Knowles, R., Keeping, H., Nguyen, K., Garner, C., D’Amico, R. & Simms, H. H. (1995). Hypoxaemia up-regulates interleukin-8 stimulated phagocytosis of polymorphonuclear leukocytes by di#erential regulation of CD32w and CD35 messenger RNA expression. Surgery 118, 177–184. Koyama, S., Rennard, S. I., Leikauf, G. D., Shoji, S., Von Essen, S., Claassen, L. & Robbins, R. A. (1991). Endotoxin stimulates bronchial epithelial cells to release chemotactic factors for neutrophils. A potential mechanism for neutrophil recruitment, cytotoxicity, and inhibition of proliferation in bronchial inflammation. Journal of Immunology 147, 4293–4301. Kurata, O., Okamoto, N. & Ikeda, Y. (1995). Neutrophilic granulocytes in carp, Cyprinus carpio, possess a spontaneous cytotoxic activity. Developmental and Comparative Immunology 19, 315–325. Kurata, O., Hasegawa, S., Okamoto, N., Nakanishi, T. & Ikeda, Y. (1996). Spontaneous cytotoxic activity of neutrophilic granulocytes in ginbuna crucian carp and channel catfish. Fish Pathology 31, 51–52. Lehrer, R. I., Lichtenstein, A. K. & Ganz, T. (1993). Defensins: antimicrobial and cytotoxic peptides of mammalian cells. Annual Review of Immunology 11, 105–128. Lindberg, F. P., Lublin, D. M., Telen, M. J., Veile, R. A., Miller, Y. E., Donis-Keller, H. & Brown, E. J. (1994). Rh-related antigen CD47 is the signal-transducer integrinassociated protein. Journal of Biological Chemistry 269, 1567–1570. MacArthur, J. I. & Fletcher, T. C. (1985). Phagocytosis in fish. In: Fish Immunology (M. J. Manning & M. F. Tatner, eds) pp. 29–46. London: Academic Press. Malorni, W., Iosi, F., Zarcone, D., Grossi, C. E. & Arancia, G. (1993). Role of adhesion molecules in the mechanism of non-MHC (major histocompatibility complex) restricted cell-mediated cytotoxicity. Scanning Microscopy 7, 323–332. Nakanishi, T. (1987a). Histocompatibility analyses in tetraploid induced from clonal triploid crucian carp and in gynogenetic diploid goldfish. Journal of Fish Biology 31 (Suppl. A), 35–40. Nakanishi, T. (1987b). Kinetics of transfer of immunity by immune leukocytes and PFC response to HRBC in isogenic ginbuna crucian carp. Journal of Fish Biology 30, 723–729. Nakanishi, T. (1987c). Transferability of immune plasma and pronephric cells in isogeneic, allogeneic and xenogeneic transfer systems in crucian carp. Developmental and Comparative Immunology 11, 521–528. Nakayasu, C., Hasegawa, S., Yoshitomi, T. & Okamoto, N. (1997). Granulocytes in carp (Cyprinus carpio L.) peripheral blood are responsible for natural cytotoxicity. Developmental and Comparative Immunology 21, 222. Nakayasu, C., Omori, M., Hasegawa, S., Kurata, O. & Okamoto, N. (1997). Production of a monoclonal antibody for carp (Cyprinus carpio L.) phagocytic cells and separation of the cells. Fish & Shellfish Immunology 8, 91–100. Olofsson, T., Gartner, I. & Olsson, I. (1980). Separation of human bone marrow cells in density gradients of polyvinylpyrrolidone coated silica gel (Percoll). Scandinavian Journal of Haematology 24(3), 254–262. Petty, H. R., Liang, B. & Maher, R. J. (1992). Mapping the entry of reactive oxygen metabolites into target erythrocytes during neutrophil-mediated antibodydependent cellular cytotoxicity. Journal of Cellular Physiology 150, 447–450. Pukhova, Y. I., Terskov, I. A. & Ponomarenko, L. D. (1984). Normal lymphocyte killers in the mechanism of the selective physiological recognition and destruction of old erythrocytes. Doklady Akademii Nauk SSSR 277, 509–512.
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Pukhova, Y. I., Terskov, I. A. & Kriger, N. V. (1988). Heterogeneity of e#ector cells of normal killer activity against autologous erythrocytes. Transplanted from Doklady Akademii Nauk SSSR, Vol. 297, No. 2, 477–479, 1987. Cytology, 603–605. Rosales, C., Gresham, H. D. & Brown, E. J. (1992). Expression of the 50-kDa integrinassociated protein on myeloid cells and erythrocytes. Journal of Immunology 149, 2759–2764. Ross, G. D., Jarowski, C. I., Rabellino, E. M. & Winchester, R. J. (1978). The sequential appearance of Ia-like antigens and two di#erent complement receptors during the maturation of human neutrophils. Journal of Experimental Medicine 147(3), 730–744. Rüegg, S. J. & Jungi, T. W. (1988). Antibody-mediated erythrolysis and erythrophagocytosis by human monocytes, macrophages and activated macrophages. Evidence for distinction between involvement of high-a$nity and low-a$nity receptors for IgG by using di#erent erythroid target cells. Immunology 63, 513–520. Secombes, C. J. (1996). The nonspecific immune system: cellular defenses. In: Fish Physiology, XV (G. K. Iwama & T. Nakanishi, Eds) pp. 63–103. Academic Press. Schiebler, T. H. & Schneider, F. (1991). Histologie: Zytologie, Histologie und mikroskopische Anatomie des Menschen. Springer, 313. Simon, S. I., Burns, A. R., Taylor, A. D., Gopalan, P. K., Lynam, E. B., Sklar, L. A. & Smith, C. W. (1995). L-selectin (CD62L) cross-linking signals neutrophil adhesive functions via the Mac-1 (CD11b/CD18) beta 2-integrin. Journal of Immunology 155, 1502–1514. Vercellotti, G. M., van Asbeck, B. S. & Jacob, H. S. (1985). Oxygen radical-induced erythrocyte hemolysis by neutrophils. Critical role of iron and lactoferrin. Journal of Clinical Investigation 76(3), 956–962. Verlhac, V., Sage, M. & Deschaux, P. (1990). Cytotoxicity of carp (Cyprinus carpio) leukocytes induced against TNP-modified autologous spleen cells and influence of acclimatization temperature. Developmental and Comparative Immunology 14, 475–480. Weiss, S. J. (1980). The role of superoxide in the destruction of erythrocyte targets by human neutrophils. Journal of Biological Chemistry 255(20), 9912–9917. Weiss, S. J. (1982). Neutrophil-mediated methemoglobin formation in the erythrocyte. The role of superoxide and hydrogen peroxide. Journal of Biological Chemistry 256(6), 2947–2953. Weissmann, G., Finkelstein, M. C., Csernansky, J., Quigley, J. P., Quinn, R. S., Techner, L., Troll, W. & Dunham, P. B. (1978). Attack of sea urchin eggs by dogfish phagocytes: a model of phagocyte-mediated cellular cytotoxicity. Proceedings of the National Academy of Sciences, USA 75, 1825–1829. Yoshida, S. H., Stuge, T. B., Miller, N. M. & Clem, L. W. (1995). Phylogeny of lymphocyte heterogeneity: cytotoxic activity of channel catfish peripheral blood leukocytes directed against allogeneic targets. Developmental and Comparative Immunology 19, 71–77. Youssef, P. P., Mantzioris, B. X., Roberts-Thomson, P. J., Ahern, M. J. & Smith, M. D. (1995). E#ects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils. Journal of Immunological Methods 186, 217–224.
Killing of isogeneic erythrocytes by neutrophils in ginbuna crucian carp (Carassius auratus langsdorfii) UWE FISCHER1*, MITSURO OTOTAKE2
AND
TERUYUKI NAKANISHI2
1
Institute of Applied Virology, Federal Research Centre for Virus Diseases of Animals, Friedrich-Loeffler-Institutes, Insel Riems, D-17498, Germany, and 2 National Research Institute of Aquaculture, Inland Station, Immunology Section, Tamaki, Mie, 519-04 Japan (Received 8 January 1998, accepted after revision 18 May 1998) Cytotoxic activity of fish neutrophils against isogeneic erythrocytes is described. Unsensitised neutrophil-rich leucocytes from clonal ginbuna crucian carp, separated by density gradient centrifugation, were cultured with isogeneic erythrocytes. Lysis of isogeneic erythrocytes was determined by measuring the release of haemoglobin into the supernatant using tetramethylbenzidine (TMB). The level of cytotoxicity obtained was proportional to both the content of granulocytes in e#ector cell fractions and the e#ector to target cell ratio. Lysis of isogeneic erythrocytes occurred only after direct contact of neutrophils with target erythrocytes. No erythrolysis was observed when granulocytes and erythrocytes were cultured in the same medium separated by a polycarbonate membrane, thus allowing exchange of only supernatant but not cells. Erythrolysis was therefore not due to a soluble factor released into the supernatant by the granulocytes. When erythrocytes and neutrophils were cultured together by immobilising them in agarose, erythrolysis was only observed microscopically in cases where erythrocytes were in contact with neutrophils. Thus, neutrophils appear able to cause lysis of histocompatible cells. 1998 Academic Press Key words:
ginbuna crucian carp, in vitro cell-mediated cytotoxicity, neutrophils, erythrocytes, autologous killing, Carassius auratus langsdorfii.
I. Introduction Vertebrate neutrophils are mobilised in response to chemotaxins (e.g. to the site of inflammation), are able to phagocytose foreign material or cells and kill various cells including bacteria. After infiltrating tissues by diapedesis, neutrophils release granules and generate toxic oxygen products. Mammalian neutrophils release cytotoxic substances, for example tumour necrosis factor, reactive oxygen intermediates or defensins (Lehrer, 1993). At sites of inflammation they secrete proteases and other enzymes which contribute to localised breakdown of the foreign material. These functions, with the exception of cytotoxicity have been extensively studied in fish (reviewed by MacArthur & Fletcher, 1985; Ainsworth, 1992; Secombes, 1996). *Author to whom correspondence should be addressed. Email [email protected] 1050–4648/98/070531+14 $30.00/0
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Several reports deal with cytotoxicity mediated by granulocytes in man. Granulocytes are capable of antibody-dependent cell-mediated cytotoxicity (Jahr et al., 1986). Bacterial endotoxins contribute to cytotoxicity of neutrophils against bronchial epithelial and endothelial cells in man (Koyama et al., 1991; Barnett et al., 1995). Little information is available on cell-mediated cytotoxicity of granulocytes in lower vertebrates. In leopard frogs (Rana pipiens), NK-like cells showing morphological similarities to human large granular lymphocytes, are capable of killing allogeneic erythrocytes and xenogeneic tumour cells (Ghoneum & Cooper, 1987). Weissmann et al. (1978) showed cytotoxicity of dogfish granulocytes against sea urchin eggs. Cytotoxic activity of neutrophils against xenogeneic cells has also been described in carp (Kurata et al., 1995; Nakayasu et al., 1997) and ginbuna crucian carp (Kurata et al., 1996). However, it is unclear whether cyprinid neutrophils are capable of killing autologous cells. In man, a natural killer-like cell exhibiting cytotoxicity against autologous erythrocytes shows phenotypic similarities to T-lymphocyte precursors (Pukhova et al., 1988). It is well known that human neutrophils exhibit cytotoxicity against autologous cells in the presence of bacterial endotoxins (Koyama et al., 1991). Cytolytic activity of immune cells against autologous hapten-modified (Verlhac et al., 1990) or virus-infected (Hogan et al., 1996) cells has been shown in channel catfish. However, the e#ector cells are not granulocytes. During studies on cell-mediated cytotoxicity of ginbuna crucian carp leucocytes against allogeneic erythrocytes (Fischer et al., 1998) we found a killing activity of neutrophils against autologous erythrocyte controls. The phenomenon of autologous erythrocyte killing has not yet been described before in fish. In the present paper we describe the killing activity of neutrophilic granulocytes against isogeneic erythrocytes and show its dependence on cell to cell contact. II. Materials and Methods FISH
Clonal triploid ginbuna crucian carp (Carassius auratus langsdorfii) is a naturally occurring gynogenetic species, widely distributed throughout Japan. Two clones from di#erent locations: Lake Suwa in Nagano Prefecture (S3n, 40–60 g) and Okushiri Island in Hokkaido (OB1, 200–250 g; Nakanishi, 1987a–c) were used. Tetraploid hybrids (S4n) were produced by insemination of S3n fish with goldfish (Carassius auratus) sperm as described previously (Nakanishi, 1987a). S4n fish possess a triploid set of chromosomes from S3n clone and a haploid set of chromosomes from goldfish. Progenies weighing between 30 and 50 g were reproduced gynogenetically by artificial insemination of sperm. Fish were maintained at 25 C in 60 l tanks supplied with aerated running spring water and were fed commercial dry pellets. Clonality of the three clones was determined by DNA fingerprinting (unpublished data) and transplantation experiments, where isogeneic scale grafting did not result in graft rejection (Nakanishi, 1987a).
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PREPARATION OF EFFECTOR AND TARGET CELLS
Fish were anaesthetised with ethyl 4-amino-benzoate (EAB; WAKO) at a concentration of 0·1 mg/ml. Approximately 0·3 ml of blood was drawn from the caudal blood vessel using a syringe rinsed with heparin (WAKO) at 1000 IU/ml in phosphate bu#ered saline without magnesium and calcium salts (PBS ). Blood was immediately diluted in a five-fold volume of cold ERDF (enriched RPMI 1640, Dulbecco’s MEM and Ham’s F12 with 2:1:1 ratio; Murakami et al., 1982) medium (Kyokuto) supplemented with RD-1 (insulin, transferrin, sodium selenite, ethanolamine; Kyokuto; 16·53 g/vial/l) and 0·2% bovine serum albumin (initial fraction, Sigma). In the case of smaller fish, blood from several clonal fish was pooled, then diluted with medium (e.g. 21 ml blood plus 8 ml medium). The blood cells were pelleted, supernatant was removed and the pellet was resuspended in 3 ml of Percoll (Sigma)-in-Hanks’ balanced salt solution (Nissui) with a density of 1·098 g/cm3. Onto this suspension, 3 ml each of Percoll-in-Hanks’ balanced salt solutions with densities of 1·080, 1·070, 1·040 g/cm3 and medium were layered sequentially. After centrifugation for 40 min at 650g at 4 C peripheral blood leucocytes (PBL) were collected from the interfaces with a Pasteur pipette, washed twice with ERDF (200g, 4 C, 10 min), stained with trypan blue (Sigma) then counted using a Thoma haemocytometer and a phase contrast microscope. Cells within fractions isolated from di#erent interfaces (1·0951/0·080, 1·080/1·070, 1·070/1·040 g/cm3) were morphologically identified as lymphocytes, monocytes, granulocytes, thrombocytes, erythrocytes and dead cells using trypan blue staining and a phase contrast microscope. Nephros and pronephros were cut into pieces of about 4 mm3 and immediately transferred into a cold Potter-Elvehjem homogeniser previously filled with 7 ml of supplemented ERDF as above containing 30 units/ml of heparin. Kidneys were gently homogenised and the cell suspensions passed through a steel mesh (size 150 gauge) to remove coarse aggregates. The cells were pelleted and kidney leucocyte (KL) subpopulations were isolated and counted as described above for PBL. For preparation of target cells 300 ìl of blood was collected, diluted in supplemented ERDF described above, and centrifuged for 10 min at 200g at 4 C. Bu#y coat was removed with Pasteur pipette. The remaining erythrocytes were washed once with supplemented ERDF (200g and 4 C for 10 min) and adjusted to a final concentration of 2105 cells per ml (2104 erythrocytes/100 ìl/well). NON-RADIOACTIVE CYTOTOXICITY ASSAY
A non-radioactive cell-mediated cytotoxicity assay was used according to the technique described by Rüegg & Jungi (1988) with modifications. The assay had originally been designed to examine antibody-mediated erythrolysis of antigen-coated human and sheep erythrocytes by human macrophages and monocytes and uses 3,3 ,5,5 -tetramethylbenzidine (TMB) dihydrochloride (Sigma) to detect haemoglobin released from lysed target erythrocytes. Target cell control (TSR=target cell spontaneous release and TMR= target cell maximum release) and e#ector cell control (ESR=e#ector cell
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spontaneous release) wells of a 96-well U-bottom tissue culture plate (Falcon, U.S.A.) were filled with 100 ìl of ERDF. A volume of 200 ìl of medium was added to the medium background (Me) and volume correction control (VCC) wells. ESR and experimental (EXP) wells were filled with PBL or KL corresponding to a ratio of 20 e#ector cells to one target erythrocyte. The target cell number added to TSR, TMR and EXP wells was constant and set to 2104 erythrocytes per well. In each experiment triplicate wells were used. After adding medium, target and e#ector cells to the wells, plates were centrifuged for 5 min at 200g, then incubated in a CO2 incubator in a humidified atmosphere at 25 C for 5 h. An aqueous solution of Triton X100 (Sigma) (10 ìl of 0·01%) was added to the TMR and VCC wells 45 min before harvesting the supernatants. Prior to harvesting the supernatants, cells were gently mixed by pipetting and plates were again centrifuged for 5 min at 200g. Fifty ìl of supernatant was removed from each well and transferred to 96-well flat-bottom E.I.A. microtitration plates (ICN). A solution of 3,3 ,5,5 -tetramethylbenzidine (TMB) dihydrochloride (Sigma) substrate was freshly prepared by dissolving 50 ìg of TMB in 10 ml of 90% acetic acid to which was added 10 ml of distilled water containing 20 ìl of 30% H2O2 solution. Undiluted particles were removed from the substrate solution by centrifuging at 1700g for 5 min at 20 C. Fifty ìl of the substrate solution was added to the wells containing the supernatants. Plates were incubated for 30 min at room temperature in the dark, then read in an ELISA reader (BIO-RAD, model 3550) at 405 nm. The percentage of specific cytotoxicity was calculated using the following formula. % Specific cytotoxicity=((EXPESR)TSR)/(TMRVCC)TSR) 100%. Firstly e#ector leucocytes from OB1 or S3n fish were tested against isogeneic target erythrocytes, then S3n leucocytes were retested against allogeneic erythrocytes from S4n fish. TMB assay showed clear correlation to a 51Cr release assay (Fischer et al., 1998). CULTURE OF NEUTROPHIL ENRICHED KL WITH TARGET ERYTHROCYTES IN INSERT PLATES
Twenty-four well cell culture plates (Falcon) and 9 mm cell culture polycarbonate membrane inserts with either a 1·0 or 3·0 ìm pore size (Falcon) were used to co-culture isogeneic neutrophils and erythrocytes from OB1 clonal fish separately. For separate co-culture, 1 ml of medium containing 2·8106 e#ector kidney neutrophils was added to each well, and 800 ìl of medium containing 1·6105 target erythrocytes was then added to the inserts. For direct co-culture control, target and e#ector cells were resuspended in 1 ml of medium, then added to the inserts, and 800 ìl of cell-free medium was added to the inserts. Cell concentrations corresponded to an e#ector to target cell ratio of 17·5 to 1. E#ector and target cells were cultured for 6·5 or 30 h. Supernatants were taken from wells or inserts and investigated in TMB assay.
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% cytotoxicity
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2.5:1
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Fig. 1. Cell-mediated cytotoxicity of neutrophil enriched head kidney leucocytes (Percoll fraction 1·095/1·080 g/cm3) from OB1 clone against isogeneic erythrocytes. The content of neutrophils in the e#ector cell population is 44·4%. Cytotoxicity is directly dependent from e#ector to target cell ratio. Vertical bars represent the standard deviation obtained from triplicate wells.
AGAROSE ASSAY
Glass slides for histology were pre-coated by dipping the slides into 0·3% agarose (gel point 36 C; FMC Bio products) followed by drying in horizontal position at 60 C in a convection oven. Slides were stored at 4 C. Kidney neutrophil enriched fraction (Percoll 1·095/1·070 g/cm3) and erythrocytes were prepared from OB1 clonal fish as described above. Erythrocytes (107) and neutrophils (105) were resuspended in 2 ml of 0·4% agarose (type VII-A, gel point 26 C; Sigma) in supplemented ERDF at 30 C. Immediately after resuspending the cells, 1 ml of the cell–agarose mixture was spread onto the pre-coated glass slides. The slides were incubated at 25 C for 30 min in a humidified chamber, then examined under a light microscope. STATISTICS
Results were compared statistically by an f-test followed by a two-tailed Student’s t-test using MS Excel 5.0 (Microsoft Corp.) and were considered to be significantly di#erent at Pc0·05. III. Results KILLING OF ISOGENEIC ERYTHROCYTES BY NEUTROPHILS
Cytotoxicity of neutrophils against isogeneic erythrocytes was evident, and cytotoxicity increased as the e#ector to target cell ratio increased (Fig. 1). The level of cytotoxicity was directly dependent on the number of neutrophils in e#ector cell fractions. Neutrophils with distinct granulation lysed erythrocytes more e$ciently than granulocytes with weak granulation. PBL and KL from the highest Percoll density (between 1·095 and 1·080 g/cm3) which
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100%
58%
90% 23%
% cytotoxicity
80% 70%
58%
60% 44%
50% 40% 31%
30% 20%
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10% 0%
n.t. 0% OB1 KL versus OB1 erythrocytes
OB1 PBL versus OB1 erythrocytes
18% 31% S3n KL versus S3n erythrocytes
18%
31%
S3n KL versus S4n erythrocytes
Fig. 2. Cell-mediated cytotoxicity of di#erent KL and PBL fractions separated by Percoll gradient centrifugation against isogeneic and allogeneic erythrocytes. E#ector:target cell ratios 20:1; culturing time 5 h. Lysis results are presented as mean of triplicate wells. Percentages on columns indicate contents of neutrophils in e#ector cell fraction.
contained the highest level of neutrophils (up to 58%) showed highest cytotoxicities (Fig. 2). Furthermore, the KL from this Percoll fraction contained the highest density of well granulated neutrophils. In contrast, fractions of lower Percoll density contained fewer neutrophils with weaker granulation. These cells showed lower or almost no cytotoxicity toward isogeneic erythrocytes. PBL neutrophils were only found in the 1·095/1·080 g/ cm3 Percoll fraction and showed a well developed granulation. OB1 KL from the low density fraction (1·070/1·040 g/cm3) exhibited a cytotoxicity against isogeneic erythrocytes of more than 10%, whereas corresponding S3n KL showed almost no cytotoxicity against isogeneic erythrocytes. Furthermore, S3n neutrophil enriched KL, isolated from the interface between 1·095 and 1·080 g/cm3 killed allogeneic S4n erythrocytes more e$ciently than isogeneic S3n erythrocytes (Fig. 2). KL from S3n fish isolated from less dense Percoll fractions, on the other hand, contained few neutrophils. These cells exhibited weaker granulation and were unable to lyse isogeneic or allogeneic erythrocytes. REQUIREMENT OF EFFECTOR TO TARGET CELL CONTACT FOR KILLING OF ERYTHROCYTES
E#ector cells and erythrocytes were co-cultured by either separating them with a membrane of 3·0 ìm pore size or in direct contact, to assay whether erythrocytes are killed by neutrophils after direct cell contact, or if cytotoxic substances released by neutrophils into the supernatant result in erythrocyte destruction. High levels of cytotoxicity were observed when neutrophil enriched KLs were in direct contact with isogeneic erythrocytes (Fig. 3, targets and e#ectors both in well). However, the percentage of cytotoxicity
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KILLING OF ISOGENEIC ERYTHROCYTES BY NEUTROPHILS 80% 70%
d b
% cytotoxicity
60%
c 50% 40% 30% a
20% 10% 0%
b a
c
6.5 h p.incub.
Targets in insert - effectors in well; supernatant from insert Targets and effectors both in well; supernatant from insert Targets in insert - effectors in well; supernatant from well Targets and effectors both in well; supernatant from well
d 30 h p.incub. insert well plate scheme
Fig. 3. Direct and separated co-culture of neutrophil enriched KL e#ectors and isogeneic erythrocyte targets. The content of neutrophils in the e#ector cell population was 50%. Targets and e#ectors were co-cultured either under direct cell to cell contact (target and e#ector cells are both in the well) or separately (target erythrocytes in an insert with a pore size of 3 ìm and e#ector cells in the well). Considerable amounts of cytotoxicity could be detected if targets and e#ectors were co-cultured so as to allow direct cell to cell contact. Vertical bars represent the standard deviation obtained from triplicate wells. The letters a, b, c and d denote groups with significantly di#erent means (Pc0·05).
was less than 4% when targets and e#ectors were co-cultured in the same medium separated by a membrane (Fig. 3, targets in insert – e#ectors in well). Levels of cytotoxicity obtained after co-culturing the cells in direct contact with each other did not increase with incubation time (up to 30 h). However, the amount of haemoglobin present in the supernatants collected from the inserts after 30 h was higher than after 6 h, probably because it took time to allow the haemoglobin to pass through the membrane. Similar results were obtained using inserts with 1 ìm pore size (data not shown). MORPHOLOGICAL EVIDENCE OF KILLING OF ISOGENEIC ERYTHROCYTES BY NEUTROPHILS
To confirm the finding that erythrocytes were killed by neutrophils during direct cell to cell contact, we cultured neutrophil enriched KL with isogeneic erythrocytes partially immobilised in agarose. The content of neutrophils in KL was 54·3% on average (data obtained from three separate experiments). Erythrocytes were only lysed when they were in direct contact with neutrophils (Figs 4, 5). Active movement of neutrophils toward erythrocytes was not observed with distances greater than a cell diameter. If erythrocytes and neutrophils came into contact, neutrophilic granules were
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Fig. 4. Killing of ginbuna crucian carp erythrocytes by isogeneic neutrophils. (a) before killing, two erythrocytes are in contact with a neutrophil; (b) after killing, one of the erythrocytes has collapsed and only the nucleus remains visible. Note: the lymphocyte (arrowed) was not able to lyse two neighbouring erythrocytes.
observed moving to the pole of the cell in contact with the erythrocyte. In some cases neutrophils also appeared to move along the membrane of the erythrocyte. Several minutes after contact, erythrocytes collapsed, but their nuclei remained visible (Fig. 4b). Other cell types such as lymphocytes were not able to kill erythrocytes (Fig. 4a, b).
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Fig. 5. Killing of ginbuna crucian carp erythrocytes by isogeneic neutrophils. A neutrophil killing an erythrocyte.
IV. Discussion In this report cytotoxicity of neutrophils against isogeneic erythrocytes by direct contact of e#ector neutrophils with target erythrocytes was demonstrated. This finding seems to be uncommon and it has to be questioned why neutrophils do not cause lysis of autologous erythrocytes in vivo. The possibility of killing due to genetic di#erences between e#ector and target cells can be excluded in our studies as clonality of fish was confirmed by DNA fingerprinting (unpublished data), scale grafting and cell transfer studies (Nakanishi et al., 1987). Isogeneic as well as autologous erythrocytes stained with a fluorescent cell linker survived for more than 2 weeks in the recipient, while allogeneic erythrocytes were killed and disappeared within a few days (unpublished data). We cannot deny the possibility that erythrocytes may change their surface antigens when removed from their physiological in vivo conditions and treated ex vivo. Altered surface antigens may act as receptors for neutrophils and induce killing of the altered erythrocytes. Under physiological conditions aged mammalian erythrocytes are phagocytosed by specialised macrophages in the spleen, liver and bone marrow (Schiebler & Schneider, 1991). The mechanism by which aged erythrocytes are removed from circulation in fish remains unclear. Human natural killer cells with the phenotype of T-lymphocyte precursors show cytotoxicity toward autologous erythrocytes (Pukhova et al., 1988). It has been suggested that these cells may play a role in the induction of erythroid di#erentiation of haematopoietic stem cells and in the killing of aged erythrocytes (Pukhova et al., 1984). It has been shown that
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mammalian neutrophils may exhibit a cytotoxic activity against autologous erythrocytes. However, in these cases the neutrophils had been stimulated by zymosan (Claster et al., 1984) or phorbol myristate acetate (Weiss, 1980, 1982; Vercellotti et al., 1985). Human erythrocytes express significant levels of the integrin-associated protein (IAP) on their surface. IAP is involved in signal transduction during neutrophil activation (Rosales et al., 1992; Lindberg et al., 1994). If fish erythrocytes, like those of man, express IAP on their surface, this may explain the activation of neutrophils in our experiments. Another possible mechanism of enhanced cytotoxicity by neutrophils is the up-regulation of the intercellular adhesion molecule (ICAM). In mice, upregulation of ICAM on target cells promotes cytotoxicity by neutrophils (Barnett et al., 1995). It can be concluded that the culture conditions of the erythrocytes in our experiments were close to physiological, since the spontaneous haemoglobin release of erythrocytes was negligible after 30 h of culture. However, the possibility of surface antigen alterations of erythrocytes cannot be excluded. Ex vivo handling of neutrophils may also have had an influence on their activity. Glasser & Fiederlein (1990) established that the conditions used to separate neutrophils influenced their function, such as chemotaxis and chemiluminescence. Youssef et al. (1995) reported that isolation of human neutrophils at temperatures lower than 37 C causes a significant increase in the expression of â-2 integrin (CD11b) and other surface adhesion molecules. Integrin-like molecules similar to LFA-1 have been reported on catfish leucocytes (Yoshida et al., 1995). L-selectin (CD62L), on the surface of human neutrophils, is decreased after separation procedures involving sedimentation and hypotonic lysis of erythrocytes. Physiological disorders resulting from hypoxaemia in humans leads to an up-regulation of phagocytosis by polymorphonuclear leucocytes (Knowles et al., 1995). Cross-linking of L-selectin induces adhesion of neutrophils to human endothelial cells (Simon et al., 1995). The up-regulation of adhesion molecules may be one reason for isogeneic killing by neutrophils in vitro. In our experiments lysis of erythrocytes occurred only after e#ector to target cell contact and was not caused by substances released by neutrophils into the supernatant. This was shown by co-culturing of neutrophils and erythrocytes separated by polycarbonate membranes and in the agarose assay. It can therefore be suggested that e#ector cells do not release cytotoxic substances into the supernatant, or that the amount of cytotoxic factors if released into the medium is not su$cient to kill the erythrocytes. Spontaneous killer cells from frog also require contact to allogeneic erythrocytes for e$cient killing (Ghoneum & Cooper, 1987). Malorni et al. (1993) designated the contact region between cytotoxic e#ectors and targets as ‘ closed chamber ’ which appears to contribute to killing e$ciency by creating a contact region in which cytotoxic factors can induce target cell lysis. Requirement of cell contact for killing was further confirmed from observations of neutrophils attaching to the membranes of erythrocytes. In the agarose experiments we observed a movement of intracellular granules toward the pole of the neutrophil, which was in contact with the erythrocyte. It has also been shown in neutrophil mediated ADCC against antigen-coated mammalian
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erythrocytes that granules accumulate near the target–e#ector interface before erythrocytes undergo lysis (Petty et al., 1992). Previous studies on cell-mediated cytotoxicity of ginbuna crucian carp leucocytes against allogeneic erythrocytes showed that the TMB assay clearly correlates to a 51Cr release assay and shows even higher sensitivity (Fischer et al., 1998). In these studies, leucocytes exhibited lysis of autologous erythrocyte controls only when the e#ector cells contained neutrophils whereas unsensitised leucocytes containing no neutrophils but high percentages of lymphocytes did not kill either isogeneic or allogeneic erythrocytes. Therefore, in these studies neutrophils were excluded from the e#ector cell fraction by density gradient centrifugation. Kurata et al. (1995, 1996) and Nakayasu et al. (1997) reported that neutrophil enriched head kidney leucocytes of common carp and ginbuna crucian carp killed xenogeneic cells derived from humans. Cytotoxicity of neutrophils to xenogeneic cells was attributed to a natural killer cell-like activity. It cannot be excluded that neutrophils from carp also show cytotoxic activity against autologous cells, e.g. erythrocytes, although this has not yet been examined. In our experiments we found that neutrophil enriched fractions of KL exhibited a stronger cytotoxicity toward allogeneic erythrocytes than to isogeneic erythrocytes, suggesting that alloantigens enhance the cytotoxic reaction of neutrophils. On the other hand, leucocyte fractions containing low density neutrophils exhibited lower or no cytotoxicity against erythrocytes. Those granulocytes were less granulated. The weaker killing activity can most likely be attributed to the low maturation stage of the granulocytes. Several reports in mammals support the suggestion, that low density cells of the myeloid lineage are functionally immature in fish too (Ross et al., 1978; Bol & Williams, 1980; Olo#son et al., 1980; Berkow & Dodson, 1986; Glasser & Fiederlein, 1987). In preliminary experiments, we used cytocentrifugation of Percoll fractions from KL and PBL followed by Giemsa staining and flow cytometry where granulocytes were identified as large granulated cells (data not shown). Ginbuna crucian carp PBL separated into three fractions by Percoll according to the protocol described in this paper are positive for Sudan black B and peroxidase staining in the lowest fraction suggesting that the corresponding cells are granulocytes. Esterase positive cells can also be isolated from lighter Percoll fractions and seemed to be monocytes because this reaction was inhibited under the presence of NaF. However, there are medium-sized negative cells for both Sudan black B and Peroxidase in the lowest fraction (Ainsworth, 1992). Numbers of e#ector cells used in our studies were 2·5 to 40 times higher than those of target erythrocytes, and consequently the probability of neutrophils coming into contact with erythrocytes was very high. However, in blood the situation is di#erent since the number of erythrocytes exceeds that of neutrophils more than 1000 times (Fänge, 1992). If neutrophils are able to recognise damaged erythrocytes and lyse them, activated neutrophils may also kill neighbouring intact erythrocytes. This mechanism would markedly enhance the killing activity under conditions of high e#ector to target cell ratios. However, it seems that this does not happen in vivo. It is well known that neutrophils contain a wide range of endogenous enzymes including peroxidase. The peroxidase activity is measurable in
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supernatants from lysed ginbuna crucian carp neutrophil rich leucocytes using TMB (unpublished data). To exclude the spontaneous release of peroxidase into the supernatant we subtracted the e#ector cell spontaneous release (ESR) when calculating the % cytotoxicity. Using monocytes as e#ector cells and erythrocytes as target cells, Rüegg & Jungi (1988) systematically calculated higher cytotoxicities in TMB assay than in the chromium release assay. However, the authors did not subtract ESR from the experimental release. It is strongly recommended to subtract the ESR whenever detection of endogenous enzyme activity is the principle of a test. The lactate dehydrogenase release assay which is used as a cytotoxicity detection kit also requires subtraction of ESR (Boehringer Mannheim or Promega). In conclusion, neutrophils of ginbuna crucian carp are able to kill isogeneic erythrocytes in vitro by cell to cell contact. Whenever e#ector cells which contain neutrophils are used, cytotoxicity of the neutrophils has to be taken into consideration. We are grateful to Dr Th. C. Mettenleiter, Federal Research Centre for Virus Diseases of Animals, Insel Riems, Germany, and Dr Kimberley D. Thompson, Institute of Aquaculture, University of Stirling, Scotland, for critical reading of the manuscript. This study was supported by a grant from the Science and Technology Agency of Japan and in part by a grant-in-aid (Bio Media Program) from the Ministry of Agriculture, Forestry and Fisheries of Japan (BMP 97-V-2-1-4).
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