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The Journal of Immunology, 2006, 176: 390-394.
Copyright © 2006 by The American Association of Immunologists

Altered Self-Erythrocyte Recognition and Destruction in an Inbred Line of Tilapia (Oreochromis aureus)

Andrey Shirak*,{dagger}, Anna Bendersky*, Gideon Hulata{dagger}, Micha Ron{dagger} and Ramy R. Avtalion1,*

* Laboratory of Fish Immunology and Genetics, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel; and {dagger} Institute of Animal Science, Agricultural Research Organization, Bet-Dagan, Israel


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Carboxyfluorescein diacetate (cFDA)-stained autologous and syngeneic tilapia (Oreochromis aureus) erythrocytes are recognized by effector peripheral blood leukocytes and lysed after a short culture period of 4 h. The hemolysis level was evaluated by measuring the fluorescence of the released cFDA. The degree of lysis of stained target erythrocytes of 60 individuals revealed a trimodal distribution statistically stratified into three groups of low (LR), intermediate (IR), and high (HR) responders. Depletion of the majority of phagocytes from leukocytes lowered the lysis level of HR to that of LR. A highly significant increase of LR cytotoxicity was obtained after the addition of conditioned medium from HR but only in the presence of phagocytes. Genetic analysis of offspring from four crosses (IR x HR, IR x LR, HR x LR, and LR x LR) revealed a quantitative trait locus (QTL) segregating for the level of response linked to markers UNH207 and UNH231 on linkage group 6 of tilapia. Based on segregation analysis of 58 gynogenetic BIU-1 offspring, the distances from the centromere were estimated as 21.5, 11.5, and 9.0 cM for UNH207, UNH231, and the QTL, respectively. It is suggested that 1) self-target recognition and destruction requires both cFDA-altered self-erythrocyte membrane and membrane structures normally present in autologous, syngeneic, and xenogeneic targets; 2) natural cytotoxic cells and/or macrophages are involved in erythrocyte lysis; and 3) the lysis level is codominantly inherited by a QTL segregating on tilapia linkage group 6.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Early in vivo and in vitro studies have shown that teleosts exhibit a wide variety of adaptive and innate immune responses, which, for the most part, are similar to those found in higher vertebrates. These include specific humoral and cell-mediated immune responses against Ags, natural cytotoxic cells (NCCs)2 equivalent to mammalian NK (1), and different phagocytic cells (2, 3). Identifying the immune-relevant genes orthologous to those of mammals confirmed the notion that the basic aspects of immunity have been highly conserved during evolution, including self/nonself recognition (4, 5). NK cells (6, 7, 8) and macrophages (9) express several families of cell surface receptors that play a central role in target cell recognition; in contrast to T and B cell response to Ag, which typically requires a proliferation phase, these two cell types have the ability to respond immediately. More specifically, these receptors bind to MHC class I proteins that are able to discriminate among different MHC alleles. Both types of cells express inhibitory receptors and can secrete potent levels of cytokines (10). Serological and biochemical studies revealed the presence of class I MHC Ags in erythrocytes of different vertebrate species including fish (11, 12). The most extensively studied cytotoxic cells in teleosts are the nonspecific cytotoxic cells, which are able to spontaneously kill a variety of targets (13, 14, 15). The inheritance of phagocytosis and lysozyme activity levels in tilapia was reported by Sarder et al. (16). We studied a marked variability for innate nonspecific cytotoxic lysis of both autologous and syngeneic erythrocytes using an inbred line of Oreochromis aureus. Genetic analysis showed a quantitative trait locus (QTL) that segregated on tilapia linkage group 6 (17), which was previously shown to include the residual polymorphic region with deleterious alleles (18, 19).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Fish

The O. aureus meiogynogenetic line (BIU-1) was produced in the Laboratory of Fish Immunology and Genetics, Bar Ilan University, by breeding four successive generations by meiogynogenesis, each followed by full-sib mating (20, 21). Artificial meiogynogenesis is a method in which the female chromosomal complement is duplicated after second meiosis while the genetic contribution of the male is eliminated. The heterogametic O. aureus female (WZ) segregates into both females and males after meiogynogenesis (19).

Autologous and syngeneic cytotoxic reactions were performed using 60 16-mo-old full-sibs of the fourth generation (F4 x F4) as blood donors. The fish were randomly divided into groups of 10 fish each and reciprocally examined using all possible effector-target combinations (90 syngeneic and 10 autologous reactions in each group). Cytotoxicity tests of the effectors from these donors against erythrocytes of xenogeneic fish (Tilapia zillii and Oreochromis niloticus from local Israeli stocks) and targets of O. aureus (F4 x F4) x O. niloticus hybrids were performed. Inheritance of the cytotoxicity level was examined by testing 24 randomly selected offspring of four crosses between two males and four females from the F4 x F4 generation.

The fifth gynogenetic generation of the BIU-1 line was produced using UV-irradiated milt from T. zillii males (local Israeli stock) and eggs from a selected F4 x F4 female (tagged as no. 16) as described by Shirak et al. (21).

Genotyping and linkage analysis

The associations between cytotoxicity and 11 UNH microsatellite markers (101, 103, 107, 148, 159, 187, 190, 207, 216, 231, and 232) that were found heterozygous in the fifth gynogenetic generation of the BIU-1 line were investigated. Three of the markers (UNH159, 216, and 231) were previously found to be linked to a residual polymorphic region that includes deleterious alleles. DNA was extracted from fin tissue by the salting-out procedure (18, 19). PCR was performed using dye-labeled dUTPs and 30 ng of genomic DNA; the amplified products were separated on an ABI-377 DNA sequencer (Applied Biosystems) and automatically sized by comparison with the internal standard, as described by Palti et al. (18). Fifty-eight meiogynogenetic individuals obtained in two successive spawns of a selected F4 x F4 female (no. 16) were genotyped for 11 UNH markers. Two of these markers, which were found to be associated with the cytotoxicity level, were used for mapping the QTL for the cytotoxic reaction. The proportion of heterozygous individuals in the meiogynogenetic progeny (K) is a function of the distance between the locus and the centromere. A coefficient of the interference value (k) of 0.2 was used to convert K to the corrected map distances (22, 23).

Preparation of effector and target cells

Fish were bled by puncture of the caudal vein with a heparinized syringe. Blood was immediately diluted 6-fold by volume with cold DMEM, and peripheral blood leukocytes (PBLks) were separated on a Lymphoprep (Nycomed) bed by centrifugation (350 x g, 15 min). PBLks were collected from the interphase and RBCs from the sediment. Next, the cells were washed twice in Ca2+- and Mg2+-enriched PBS. Finally, the PBLks were resuspended in DMEM plus 2% FCS at a concentration of 2 x 106 cells/ml and used as effector cells. The RBCs were then labeled with carboxyfluorescein diacetate (cFDA) (Sigma-Aldrich) and used as target cells.

RBC labeling with cFDA

One milliliter of cFDA (5-CFDA; Sigma-Aldrich) working solution (75 µg/ml in PBS; prepared from a cFDA stock solution of 20 mg/ml in acetone, stored at –20°C) was added to a pellet of 1–2 x 106 RBCs and incubated at 28°C in a humidified 5% CO2 incubator for 25 min. The RBCs were then washed three times in PBS containing 1% BSA and resuspended in DMEM containing 2% FCS at a concentration of 1 x 105 cells/ml.

Cytotoxic assay

The assay is a modification of the technique used by Provinciali et al. (24) for determining NK activity. Briefly, 100 µl of target cells (1 x 104) were incubated with 100 µl of effector cells, at a ratio of 1:20, in 96-well U-bottom microtiter plates (Nunc). The test was performed in triplicate. First, the plates were incubated at 28°C in a humidified 5% CO2 incubator for 4 h and then centrifuged at 700 x g for 5 min. Next, the supernatant was separated from the cellular fraction by rapidly inverting the plate and flicking the supernatant out. Finally, 100 µl of a 1% solution of Triton X-100 in 0.05 M borate buffer (pH 9.0) was added to each well.

After incubation for 10 min at room temperature (~23°C), or for 20 h to 3 days at 4°C, the plates were measured for fluorescence with SPECTRA FluorPlus (TECAN). The percentage of specific lysis was calculated using the following formula: % cytotoxic lysis = ((Fmed – Fexp) x 100)/Fmed, where Fmed represents the fluorescence of the resuspended cell pellet from the target incubated in medium alone, and Fexp represents the fluorescence of the resuspended cell pellet from the target incubated with effector cells.

Elimination of phagocytes

Phagocytes were eliminated from peripheral blood as previously described by Eisenthal et al. (25). Briefly, iron-carbonyl powder (Sigma-Aldrich) was added to the blood samples in a proportion of 0.01 g to 1 ml of blood. The samples were incubated with gentle shaking for 30 min at 28°C. Magnetic removal of the iron-absorbing phagocytes was performed using a magnetic device adapted to a test tube (Dynal Biotech), for 10 min at 4°C. Removal of iron-absorbing cells was repeated twice.

Conditioned medium (CM) preparation

Supernatant from the cytotoxic test was used as the CM. Supernatant collected by centrifugation from a cytotoxic reaction with unlabeled target cells and effector cells from high-responding individuals was diluted 1/2 with DMEM containing 2% FCS before being added to the effector target reaction.

Statistical analysis

The cytotoxic reaction was analyzed by one-way ANOVA with effectors or donors as classes. Based on the trimodal distribution, groups of high (HR), intermediate (IR), and low (LR) responders were stratified. Relationships between genotypic segregation of microsatellite markers and cytotoxicity levels were assessed by the {chi}2 test. This test was also used for analysis of distortion from the expected 1:1 segregation between the two classes of homozygotes. The cytotoxic reaction response using the three types of effectors against xenogeneic and allogeneic targets was compared using Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Syngeneic and autologous cytotoxic reactions

One-way ANOVA revealed that the effector donors had a marked effect on the extent of the cytotoxic reaction. Based on the trimodal distribution (Fig. 1), the three groups of effectors were stratified according to low, intermediate, and high response. The IR group boundaries were formed by effectors with erythrocytes lysed from 9 to 27%, and the HR and LR groups were characterized by >27 and <9% erythrocyte lysis, respectively. Significant differences were found between the HR and IR groups (p < 0.0001) but not between the IR and LR groups.



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  		FIGURE 1. Trimodal distribution of cytotoxic reaction.

 
Kinetics of the cytotoxic reaction

The kinetics of the cytotoxic reaction revealed different curves of cytotoxicity specific for the activities of the LR, IR, and HR groups of effectors against the pooled syngeneic and autologous erythrocytes. All curves peaked on the fourth hour after effector-target incubation (Fig. 2).



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  		FIGURE 2. Kinetics of the cytotoxic reaction. Each point represents mean lysis percentage ± SE of three or four individuals, each tested in triplicate.

 
Inheritance of cytotoxicity levels in the BIU-1 line

Twenty-four offspring, randomly selected from the progeny of four different spawns from the BIU-1 line, were subjected to genetic analysis (Fig. 3) as follows. Two parental males were assessed as HR (H-3) or LR (L-11). Four parental females were assessed as LR (L-15 and L-18) or IR (I-16 and I-17). A sample of seven progeny of the cross L-15 x H-3 were all found to be IR. A sample of six progeny of the cross L-18 x L-11 were all found to be LR. Two other crosses (I-16 x L-11 and I-17 x H-3) showed offspring segregated into IR and LR, and IR and HR, respectively. These results indicate that the QTL has codominant inheritance of cytotoxicity levels.



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  		FIGURE 3. Inheritance of cytotoxicity levels of individuals of the BIU-1 line of O. aureus. Circle, Female; square, male; black, HR; gray, IR; white, LR.

 
Mapping of the QTL for the cytotoxic reaction

Eleven offspring from the segregated crosses (I-16 x L-11 and I-17 x H-3) and their four parents were analyzed for linkage with polymorphic UNH microsatellite markers for cytotoxicity. UNH207 and UNH231 markers, mapped to LG6 in the latest tilapia linkage map (17), showed complete cosegregation according to the level of cytotoxicity. In low-responding individuals, only the 144-bp and 170-bp alleles of UNH207 and UNH231, respectively, were amplified, whereas in high-responding individuals, only the 148- and 174-bp alleles of UNH207 and UNH231, respectively, were amplified. Heterozygous individuals for both markers showed an intermediate level of cytotoxicity. To estimate the distance of the QTL, UNH207, and UNH231 marker loci from the centromere, we used 58 gynogenetic progeny of female I-16. This individual was assigned to the IR group and thus was assumed to be heterozygous for the QTL. This female was also heterozygous for the UNH207 and UNH231 markers. Allele segregation analysis of these progeny supported the association of the UNH207 and UNH231 markers with a QTL for cytotoxicity at significance levels of p < 0.00005 and p < 0.005, respectively (Table I). The proportions of heterozygotes for these markers and of individuals with intermediate levels of cytotoxicity enabled us to estimate the distances of UNH231, UNH207, and the QTL from the centromere. These distances were 21.5, 11.5, and 9.0 cM, as calculated from their respective initial values (K) 0.397, 0.224, and 0.172 (Table II). A linkage map that includes UNH231, UNH207, and the QTL was constructed, assuming (see Discussion) that all three loci are located on the same chromosome arm. The between-loci distances (UNH231-QTL, UNH231-UNH207, and UNH207-QTL) were calculated as 12.5, 10, and 2.5 cM, respectively, by subtracting the locus-centromere distances (Fig. 4).


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  		Table I. Segregation of the UNH207 and UNH231 markers, and their relationship to the cytotoxicity levels in the meiogynogenetic offspring of I-16 femalea

 

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  		Table II. Segregation of the UNH231 and UNH207 genotypes in relation to the cytotoxicity levels in the meiogynogenetic offspring of I-16 females

 


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  		FIGURE 4. Mapping of the QTL for cytotoxicity relative to UNH207, UNH231, and the centromere.

 
Xenogeneic cytotoxic reaction of O. aureus (line BIU-1) against erythrocytes of two different tilapia species (O. niloticus and T. zillii)

The cytotoxic reaction of effector cells from HR and LR against xenogeneic target erythrocytes resulted in low levels of cytotoxicity, which was significantly higher against T. zillii (a distant species) than against O. niloticus (a closely related species). However, no significant differences could be seen between the HR and LR effector cells (Table III).


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  		Table III. Cytotoxic reaction against xenogeneic target erythrocytesa

 
Cytotoxic reaction of O. aureus (line BIU-1) against erythrocytes of hybrids with O. niloticus

PBLks from HR, IR, and LR of the BIU-1 line were incubated with erythrocyte targets from four of their hybrids with O. niloticus. The difference between the levels of cytotoxic reaction of the three effector groups was significant (p < 0.05) over all targets. No significant differences (>0.05) were discerned between these results and those obtained in the syngeneic and autologous reactions with the same responders (Table IV).


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  		Table IV. Cytotoxic activity of O. aureus effector leukocytes against erythrocytes of four of their hybrids with O. niloticus

 
Effect of phagocytes on the cytotoxicity of effector cells

The cytotoxicity of leukocytes from HR and LR was tested before and after magnetic removal of iron-treated phagocytes. Removing most of the phagocytes from leukocytes caused a significant decrease in the percentage of target lysis in both LR (p = 0.04) and HR (p = 0.001) (Fig. 5).



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  		FIGURE 5. Cytotoxic reaction with (+M{varphi}) and without (–M{varphi}) phagocytic cells. Each point represents mean ± SE of four individuals, each tested in triplicate.

 
Effect of CM on the cytotoxic reaction

Adding HR CM to the cytotoxic reaction of HR and LR significantly increased (p < 0.05) the percentage of target cells lysed in the presence of phagocytes only. Interestingly, when phagocytes were removed, the CM had no effect on the cytotoxic reaction. However, the addition of CM to LR, in the presence of phagocytes, significantly elevated (p < 0.05) their cytotoxic lysis to the level of IR (Fig. 6).



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  		FIGURE 6. Effect of CM on the cytotoxic reaction. Each point represents mean ± SE of four individuals, each tested in triplicate. {cjs2108} and {blacksquare}, Without and with the addition of CM.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Syngeneic tests between individuals of the BIU-1 inbred line showed three levels of cytotoxic activity stratified into three groups: LR, IR, and HR. Because of the low level of cytotoxicity obtained in xenogeneic erythrocytes, the different levels of cytotoxicity (LR, IR, and HR) that characterized syngeneic and autologous effector cells are thought to be the result of altered self membrane molecules, provided by the cFDA dye, and/or of missing self recognition resulting from this alteration. This is in line with the self/nonself strategies of the innate immune response described by Medzhitov and Janeway (4, 5) and by others in both mammalian NK cells (7, 8) and macrophages (9). However, the lower cytotoxicity level obtained with xenogeneic erythrocytes, which was contrary to what was expected, is difficult to explain. In this respect, it was reasonable to assume that high activities of both NCCs and macrophages are simultaneously involved in this kind of cytotoxicity. Moreover, we assumed that macrophage activity genetically controls the cytotoxicity level against self and syngeneic targets against their hybrid offspring with O. niloticus, but not the cytotoxicity of NCCs, which are equivalently active in all responding cell groups, as shown against xenogeneic target cells. This assumption is strengthened by the fact that addition of CM from the HR reaction to the LR effectors resulted in a marked elevation of cytotoxicity, probably by supplying missing stimulatory molecules. Previous results reported in channel catfish lymphoid cell lines lG8 and 64M2 with virus-infected or uninfected, but 51Cr-labeled, allogeneic targets (26) are also in agreement with this assumption.

Both autologous and syngeneic individual reactions provided reproducible values of target lysis in repeated experiments at different ages (10 and 16 mo), but all expressed high individual variations that could be stratified into three groups based on the level of the cytotoxic reaction. Interestingly, such stratification was not possible in the original outbred population (data not shown), possibly because of the influence of additional genes on cytotoxicity level due to their allelic variability. In the BIU-1 line used, despite its high level of inbreeding, the current data point to the presence of residual heterozygous gene(s) that control the cytotoxicity level. The residual heterozygosity in this line was reported to be preserved in several loci by sex-specific mortality (12, 14), including one such locus on LG6 linked to a QTL. Analysis of the cytotoxic reaction in the normal offspring of crosses between parents from variably responding groups revealed that the QTL for cytotoxic reactions is codominantly inherited in the BIU-1 inbred line (Fig. 3). This finding is in line with the result of Zimmerman et al. (27) in rainbow trout. An attempt was made to locate this QTL on the UNH tilapia genetic map (13) by using the residual polymorphic UNH markers in our inbred BIU-1 line.

Based on the segregation results obtained in 58 meiogynogenetic cases, we estimated the distances from the centromere as 21.5, 11.5, and 9.0 cM from UNH231, UNH207, and the QTL, respectively. The gradual reduction in deleterious effect occurring from UNH231 through the UNH207 locus and QTL, and the strong correlation of allele segregation among all three loci led us to conclude that these three loci are likely to be mapped on the same arm of the centromere. Hence, the between-loci distances can be calculated by simple subtraction. This procedure for evaluation of the between-loci distances is not highly accurate, mainly because of the possible elimination of specific genotypes (deleterious effect) (12, 14). Nevertheless, the estimated distance between UNH231 and UNH207 (10 cM) approaches the recently updated map distance (8 cM) (13). Thus, we plan to generate crosses between IR x IR and between IR x HR to increase the accuracy of mapping the QTL and lethal gene(s) using recently developed relevant markers (13).

According to Neumann et al. (28), phagocytes can directly act and lyse cells in the case of the primary innate reaction. Additional experiments have pointed to phagocytes as possible participating cytotoxic cells. Elimination of iron-absorbing phagocyte cells from the experimental system markedly inhibited the reaction. The remaining level of cytotoxicity may be 1) because of nonabsolute elimination of phagocytes, and 2) because part of the effector cells were preactivated by soluble mediators (e.g., IL-1) secreted by phagocytes. In fact, by adding CM, especially in the case of LR effector cells, the cytotoxic lysis could be markedly increased. However, it was reported that such soluble mediators could be secreted by one or more of the participating cells (mononuclear cells, macrophages, and even erythrocytes) (28, 29). The effect of increased cytotoxic lysis by adding CM can be obtained only in the system with phagocytes. Accordingly, we suggest that soluble factors can serve in directly activating phagocytes or in inducing cytotoxin secretion by phagocytes and other cells such as NCCs that take part in cytotoxic reactions.


   Acknowledgments
 
We thank Dr. Joel Weller, Dr. Helena Zhevelev, and Dr. Yoram Louzoun for their help.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Address correspondence and reprint requests to Dr. Ramy R. Avtalion, Laboratory of Fish Immunology and Genetics, Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 52900, Israel. E-mail address: avtalr{at}mail.biu.ac.il Back

2 Abbreviations used in this paper: NCC, natural cytotoxic cell; QTL, quantitative trait locus; PBLk, peripheral blood leukocyte; cFDA, carboxyfluorescein diacetate; CM, conditioned medium; HR, high responder; IR, intermediate responder; LR, low responder. Back

Received for publication June 29, 2005. Accepted for publication October 20, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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