The Journal of Heredity 2001:92(4)
© 2001 The American Genetic Association 92:305-308
MHC Variation and Tissue Transplantation in Fish
From the Department of Biology, Arizona State University, Tempe, AZ 85287-1501.
Address correspondence to Philip Hedrick at the address above or e-mail: philip.hedrick{at}asu.edu.
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Abstract |
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Major histocompatibility complex (MHC) genes were originally discovered because of their role in tissue rejection in mammals and have subsequently been implicated in the incidence of autoimmune diseases and resistance to infectious diseases. Here we present the first demonstration that a gene defined by molecular sequence in the fish MHC, specifically a class II locus, plays an important role in tissue rejection. This effect in the endangered Gila topminnows appears to be additive and depends on the number of MHC alleles shared between the host and the recipient fish of the scale transplants. In addition, there was lower success of scale transplants in MHC-matched individuals in a population with high microsatellite variation than in a population with low variation. This suggests that other loci, presumably other MHC loci, play a significant role in transplantation success in fishes, as they do in mammals.
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Introduction |
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The major histocompatibility complex (MHC) was discovered because of its role in the acceptance or rejection of tissue transplants in mammals. When transplanting organs in humans, the likelihood of acceptance is increased by matching donors with potential recipients at three MHC loci (HLA-A, HLA-B, and HLA-DR). When relatives are matched at these loci, even higher success occurs than for matched nonrelatives. The difference in transplant success between similarly matched relatives and nonrelatives suggests that loci other than these three have significant effects in determining transplant success.
There has been extensive research on tissue transplantation in teleost fishes (Kallman 1970), including grafting a variety of tissues such as fins, scales, spleens, and liver. From studies of crosses and their progeny, Kallman (1960, 1964) concluded that there are a large number of histocompatibility loci and that these act in an additive fashion. In addition, it has been shown that tissue transplants can be used to identify fish clones and transplant success is related to the level of homozygosity in inbred laboratory stocks and natural populations (Eisenbrey and Moore 1981; Moore and Eisenbrey 1979). Much of this early tissue transplant research was in live-bearing fishes (Poeciliidae), closely related to the Gila topminnows that we utilized in our research here. For citations and discussion of recent fish transplant studies, see Ristow et al. (1996) and Nakanishi and Ototake (1999).
Although the MHC is best characterized in mammals, particularly in humans and mice, the MHC has been found in nearly all vertebrates in recent years (Edwards and Hedrick 1998). In mammals, the MHC appears to be on a single chromosome in one linkage group, but the structure of the MHC appears to be quite different in fishes. For example, in zebrafish (Bingulac-Popovic et al. 1997), trout (Hansen et al. 1999), and probably in other fishes, class I and class II genes appear to be unlinked. Further, in zebrafish the class II genes are divided into two linkage groups (Bingulac-Popovic et al. 1997), in cichlids the class II genes are spread over more than 10 map units (Malaga-Trillo et al. 1998), and two unlinked class II genes have been identified in platyfish (McConnell et al. 1998) and sticklebacks (Sato et al. 2000). In other words, the mammalian paradigm of a single linkage group containing all the important expressed major histocompatibility genes does not appear to be true in any fish that has been closely examined.
From the studies summarized above, there is compelling evidence that histocompatibility loci influence tissue acceptance or rejection in fish. However, there has been no demonstration that MHC genes (and alleles) defined by molecular sequence data are in fact responsible for transplantation acceptance or rejection. Here we show that a classical class II locus (and/or loci in linkage disequilibrium with it) is a major determinant of tissue transplantation success in fish and that other loci, unlinked or not in strong linkage disequilibrium with it, also appear to be a factor in transplant success.
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Materials and Methods |
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Background on Gila Topminnows
The Gila topminnow (Poeciliopsis o. occidentalis), a small, live-bearing fish, was once the most common fish in the Gila River, which drains nearly all of southern Arizona. However, it is now an endangered species found naturally in only a few locations in the United States, primarily because of water developments and the introduction of nonnative mosquito fish (Sheffer et al. 1997). We have maintained stocks with a thousand or more breeding adults captured from four locations for the past 5 years. For this study we used randomly caught fish from our raceways that originated primarily from two of these sites, Bylas Spring and Cienega Creek.
The Gila topminnow has been the subject of extensive genetic investigation, including recent examination of variation in a class II gene (Hedrick and Parker 1998) and variation at polymorphic microsatellite loci (Parker et al. 1999). (Previously we called this MHC gene a DRB gene, but because it may not be homologous to the human DRB gene, we call it the DAB gene here.) In the study of DAB variation by Hedrick and Parker (1998), nine different alleles were found over the four populations. Most of the sequence variation was nonsynonymous, resulting in alleles that differed in from 1 to 12 amino acids in exon 2. The Bylas Spring population was monomorphic for allele Pooc-1 and the Cienega Creek population was polymorphic for two alleles, Pooc-1 and Pooc-5, in equal frequencies. Alleles Pooc-1 and Pooc-5 differ by five amino acids with no synonymous differences. Bylas Spring also had very low genetic variation for microsatellite loci, being polymorphic at only one of the five loci examined, with an average heterozygosity of 0.075, while Cienega Creek was polymorphic at three of the five loci, with an average heterozygosity of 0.264.
Transplant Procedure
To examine the effect of MHC variation on tissue transplant success, we carried out three experiments, all of which had paired observations. That is, in all experiments, we transplanted two highly pigmented dorsal scales, each containing several melanophores, to a site between lightly pigmented ventral scales. In the autograft (transplant within fish) experiment, for a given fish a scale was taken from the left side and transplanted to the right side and a scale was taken from the right side and transplanted to the left side. In the allograft experiments (transplants between fish from the same population), fish were randomly paired so that they were both recipients and donors. Two scales were taken from opposite dorsal sides of the first fish and inserted between ventral scales of the second fish. Then two scales were taken from opposite dorsal sides of the second fish and inserted between ventral scales on opposite sides of the first fish. All of the fish were kept in a controlled temperature (27°C) and the transplants and the subsequent observations were carried out between 9 A.M. and 12 A.M.
In determining the success of transplantation, fish were observed three times a week for 65 days. There were three categories of response: (1) complete acceptance, in which there was no sign of rejection; (2) delayed acceptance, in which the rejection process was initiated, the melanophores were lost, the scales started to discolor, but the scales were not sloughed off and remained on the fish for the duration of the experiment; and (3) rejection, in which the melanophores in the transplanted scales began to disappear, the scale became opaque, and it was eventually sloughed off (the number of days until rejection was monitored in this case). It is possible that the scales, which we classified as showing delayed acceptance, may have eventually been rejected. However, we observed no change in these individuals over the last 5 weeks of the experiment (between day 28 and day 65) so we feel that this category is different from either complete acceptance or rejection. Scales that were missing or did not appear to be properly transplanted after 2 days were not included.
Experiment 1 involved transplanting scales between the left and right sides of individuals (autografts) from Cienega Creek and a third population, Monkey Spring. We carried out two experiments in which we transplanted scales between fish from the same population (allografts). In experiment 2 we transplanted scales between different fish from Bylas Spring, all of which were homozygous for the DAB allele Pooc-1. In experiment 3 we transplanted scales between random fish from Cienega Creek. After the scale transplant experiment was concluded, we determined the three DAB genotypes of these fish, Pooc-1/Pooc-1, Pooc-1/Pooc-5, or Pooc-5/Pooc-5, using SSCP (single-strand conformation polymorphism) (for experimental details, see Hedrick and Parker 1998). The donor and recipient fish could share either 2, 1, or 0 alleles. Because the scale transplant study was concluded before the DAB typing was carried out, there was no way for us to know the extent of DAB sharing for individual fish during the period of scale transplant monitoring.
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Results |
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The transplant success of the two replicate scales per individual were very similar (not statistically different in all experiments using a t test for paired samples), so we averaged the right and left side values in our analyses below. In the 27 individuals used in the autograft experiment, all scales were completely accepted and there was no initiation of rejection (Table 1). In the allografts of the 42 individuals from Bylas Spring (all homozygous for Pooc-1), there were two categories of response: delayed acceptance, which was observed in 60% of the individuals, and rejection, observed in 40% of the individuals (the mean time to initiation of rejection was 14.3 ± 0.8 days).
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In the 48 random fish from Cienega Creek used in the second allograft experiment, there were 18 that shared two alleles (both were Pooc-1/Pooc-1, Pooc-1/Pooc-5, or Pooc-5/Pooc-5), 24 that shared one allele (Pooc-1/Pooc-5 with Pooc-1/Pooc-1 or Pooc-5/Pooc-5), and 6 that shared zero alleles (Pooc-1/Pooc-1 with Pooc-5/Pooc-5). None of these fish had either complete or delayed acceptance. All three categories rejected the scale transplants, and initiation of rejection was statistically significantly faster than the allografts from the Bylas Spring fish. The initiation of rejection was slowest for the fish that shared two DAB alleles (10.5 days), faster for fish that shared only one DAB allele (8.7 days), and fastest for fish that shared no DAB alleles (6.2 days) (Table 1, Figure 1). Initiation of rejection was statistically significantly associated with the number of alleles shared (P = .013), suggesting a dosage effect for DAB alleles determining tissue acceptance. Initiation of rejection is about 70% slower when the recipient and donor are matched for the locus than when they differ. It addition, the intermediate value for pairs that share one allele is consistent with the notion that the histocompatibility effect is additive, that is, it acts cumulatively.
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Discussion |
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Although MHC genes were originally discovered because of their role in tissue rejection in mammals, this is the first demonstration that MHC genes (and alleles) defined by molecular sequence play a similar role in fishes. Of course, we cannot distinguish the effect of alleles at other loci in linkage disequilibrium with the DAB locus and the intrinsic effects of the DAB locus. It appears that the effects on transplantation success are additive within the DAB locus so that success is highest when there is complete sharing, lower when only one allele is shared, and lowest when both alleles differ.
In addition, because Bylas Spring fish are monomorphic for the DAB locus and yet 40% of the fish rejected transplants, it appears that other genes are involved. Cienega Creek fish that also shared two alleles showed a faster rejection rate than Bylas Spring fish, and none of Cienega Creek fish exhibited delayed acceptance, possibly reflecting the higher genetic variation for other loci in this population. There is threefold higher microsatellite heterozygosity for Cienega Creek fish than for Bylas Spring fish, assumed to a random sample of genetic variation at other loci in the two groups, suggesting that individuals identical for DAB in Cienega Creek would be much less similar for other loci than would random individuals (also identical at DAB) from Bylas Spring. In other words, the lower success of DAB-matched Cienega Creek individuals than Bylas Spring individuals is consistent with the lower genetic similarity at other loci for random individuals from Cienega Creek than from Bylas Spring. The demonstrations that class I and class II genes appear to be unlinked in fish (Bingulac-Popovic et al. 1997; Hansen et al. 1999) and that class II genes may be in at least two linkage groups (Bingulac-Popovic et al. 1997; McConnell et al. 1998; Sato et al. 2000) or not tightly linked (Malaga-Trillo et al. 1998) are therefore relevant here. Our findings that genes unlinked to the DAB loci, or linked but in linkage equilibrium with this class II locus, may influence tissue transplantation suggest these other genes, either class I, class II, or even minor histocompatibility genes, may be important in determining transplantation rejection or acceptance.
In addition to their role in tissue rejection in mammals, the MHC has been shown to influence the incidence of autoimmune diseases in humans and resistance to infectious diseases in mammals and birds, and secondarily, the MHC may also influence maternal-fetal interaction and mate choice in mammals (e.g., Edwards and Hedrick 1998). However, demonstrating the importance of the MHC in resistance to infectious diseases, the most widely accepted effect of the MHC except that for the autoimmune diseases in humans, has been difficult (Hedrick and Kim 2000), although several recent studies have shown a relationship of MHC variation in humans and resistance to hepatitis (Thurz et al. 1997) and HIV (Carrington et al. 1999). Hopefully with greater knowledge of the MHC in other organisms besides humans, details of its important effects will be understood in a number of organisms and the functional role of MHC will become known.
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Acknowledgments |
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We appreciate the support of the Hughes Program (to T.N.C.), the support of the Ullman Professorship (to P.W.H.), and the assistance of K. Parker in determining the MHC alleles. The stocks used were collected under U.S. Fish and Wildlife Service permits and the research was carried out using a protocol approved by the Arizona State University Animal Care Committee.
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Footnotes |
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Corresponding Editor: Lisa Seeb
Received April 21, 2000
Accepted January 15, 2001
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References |
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