Growth and reproduction complex in the rat. Genes linked to the major histocompatibility complex that affect development.

The linkage of the major histocompatibility complex (MHC) and the growth and reproduction complex (Grc) in the rat was studied in an F2 hybrid population generated from female BIL/1 (RT1l-Grc) and male YO (RT1u-Grc+) animals: 1.722 offspring were born, and 1,568 were weaned and studied. The body weights of the offspring segregated with the RT1 haplotype of the MHC, and the RT1l homozygotes were significantly smaller than their RT1l/u and RT1u/u littermates. The growth rate of the RT1l/l animals was approximately the same as that of the BIL/1 animals, and both were significantly less than the growth rates of the RT1l/u, RT1u/u, and YO (RT1u) animals. The testes of the RT1l animals showed an arrest of spermatogenesis at the early pachytene stage of the primary spermatocytes, and they were approximately 1/10 as heavy as the testes of the RT1l/u and RT1u/u animals. The ovaries in females of all three haplotypes had the same weight, but there was a decrease in the number of ova released per cycle in the RT1 l/l animals. The major loss of the RT1l homozygotes, which caused distortion of the phenotypic ratios among the offspring, did not occur in utero but in the early postnatal period before weaning. There were 7/1568 recombinants between the MHC, using the RT1.A antigen as the marker, and the Grc, using small body size (dw-3) as the marker, and 1/1568 recombinant between the loci influencing body size (dw-3) and fertility (ft) of the Grc. These data gave the following map distances (95% confidence levels): RT1.A to dw-3, 0.45 (0.25-0.96) centimorgans and dw-3 to ft, 0.07 (0.04-0.40) centimorgans. A female recombinant was used develop an inbred line carrying the RT1.Al-Grc+ chromosome.

Differential expression of MHC class I antigens on the placenta of the rat. A mechanism for the survival of the fetal allograft.

In some mating combinations in rats, there is a maternal antibody response to the maternal antigenic components of the placenta without any previous immunization of the mother. The highest response occurs in the WF (u) female mated to the DA (a) male, and it is against a unique MHC-encoded class I antigen, the Pa antigen, and not against the major allele-specific transplantation antigen of the DA strain, RT1.Aa. The development of mAbs to the Pa and Aa antigens allowed us to localize these antigens on the placenta and to explore the reason for the differential antibody response to them using immunohistochemical and biochemical techniques. Both antibodies reacted with the WF X DA placenta and stained the endovascular and interstitial trophoblast of the decidua, the basal trophoblast, Reichert's membrane, and the yolk sac epithelium, but they did not stain the labyrinthine trophoblast. Blocking studies showed that each antibody reacted with a separate molecule in the placenta. Anti-class II mAbs reactive with the a or u haplotype did not stain the WF X DA, DA X DA, or WF X WF placenta; hence, there are no class II antigens in the placenta. Electron microscopic studies of the semiallogeneic WF X DA placenta using the immunogold technique with both single- and double-labeling showed that only the Pa antigen was expressed on the surface of the basal trophoblast, but that both the Pa and Aa antigens were in the cytoplasm of these cells; neither antigen was found in the labyrinthine trophoblast. By contrast, the placenta from the syngeneic DA X DA mating expressed both the Pa and Aa antigens on the surface of the basal trophoblast as well as in the cytoplasm; neither antigen was found in the labyrinthine trophoblast. These observations were quantified morphometrically using electron photomicrographs of single-labeled tissues. Both the Pa and Aa antigens isolated from the plasma membrane of lymphocytes have heavy chains of 46 kD, but those antigens isolated from the plasma membrane of basal trophoblast cells have heavy chains of 43 kD. Based on densitometric measurements of autoradiographs, the Pa/Aa ratio in the basal trophoblast membrane is 23.5, whereas it is 0.46 in lymphocyte membranes. These studies show that there is differential regulation of the expression of class I antigens on basal trophoblast cells in semiallogeneic pregnancies, but not in syngeneic pregnancies, such that the major allele-specific transplantation antigen is scarcely expressed on the surface of the basal trophoblast.(ABSTRACT TRUNCATED AT 400 WORDS)

Maternal-fetal interaction and immunological memory.

Female rats of the poorly responding, inbred F344 strain were immunized with poly(Glu(52)Lys(33)Tyr(15)) aggregated with methylated bovine serum albumin, and then they were mated. The first and second litters in the F(1) generation and in the F(2) generation showed an enhanced immune response. When poly-lysine was used as the aggregating agent, enhancement occurred in only the first litter of the F(1) generation and in the F(2) generation. In both cases, antigen was transmitted from the immunized female to her offspring, where it localized in the bone marrow and, in a few cases, in the thymus and spleen also. The transplacental passage of antigen is probably the basis for the enhanced antibody response, which is a manifestation of immunological memory.

The rat as an experimental animal.

The development and characterization of many inbred, congenic, and recombinant strains of rats in recent years has led to the detailed genetic description of this species, especially in regard to its major histocompatibility complex. This information has contributed substantially to the study of comparative genetics and has greatly enhanced the utility of the rat in a variety of areas of biomedical research. This article focuses on the use of the rat in immunogenetics, transplantation, cancer-risk assessment, cardiovascular diseases, and behavior.

Cell lines from grc congenic strains of rats having different susceptibilities to chemical carcinogens.

The growth and reproduction complex (grc-) strains of rats have a 70-kilobase deletion in the major histocompatibility complex (MHC)-linked grc-G/C region that is associated with embryonic death, developmental defects, and an increased susceptibility to chemical carcinogens. To study further the effects associated with the deletion, fibroblastic cell lines from grc-, grc+, and grc+/- rat embryos were developed: BIL-derived cell lines are congenic for the MHC and grc, whereas R16-derived cell lines are congenic for the grc alone. In early passages, all cell lines expressed the MHC class I antigen RT1.A, had a diploid chromosome number, and did not display anchorage-independent growth or in vivo tumorigenicity. The grc- cells [median population doubling time (PDT), 47 h] grew more slowly than the grc+ (PDT, 30.5 h) and grc+/- (PDT, 33 h) cells. All cells underwent crisis, but the crisis stage began earlier and lasted longer in the grc- cells. The established grc- cell lines (PDT, 32.5 h) grew faster than the grc+ (PDT, 48.5 h) and grc+/- (PDT, 54 h) cell lines. Two of the three BIL-derived grc- lines that survived crisis became anchorage independent in tissue culture and tumorigenic in histocompatible F1 rats (highly malignant fibrosarcomas) at passages 33 and 48, respectively; by contrast, none of the R16-derived grc- cell lines transformed. None of 8 grc+ or 8 grc+/- cell lines that survived crisis displayed anchorage-independent growth or tumorigenicity under the same conditions up to passage 50. All of the established cell lines, including the two tumorigenic ones, expressed MHC class I antigens. Southern and Northern blot analyses of BIL-derived cell lines before and after crisis showed that they all constitutively expressed H-ras and Rb and that no cell line showed rearrangement, amplification, or overexpression of c-myc, H-ras, Rb, and p53 either before or after crisis. These observations indicate that: (a) the homozygous grc- deletion is necessary but not sufficient for in vitro transformation; (b) another genetic factor(s) required for transformation is linked to, or possibly in, the MHC; and (c) passage through crisis, spontaneous transformation, or carcinogen treatment does not alter the cellular expression of MHC class I antigens or of several oncogenes and tumor suppressor genes.

Genetic control of susceptibility to diethylnitrosamine carcinogenesis in inbred ACP (grc+) and R16 (grc) rats.

The R16 strain, which carries the major histocompatibility complex-linked growth and reproduction complex (grc), and its normal counterpart, the ACP strain, were initiated at 8 wk of age with a single i.p. dose of diethylnitrosamine (DEN), and 2 wk later they were fed either a choline-deficient (CD) or a choline-supplemented (CS) diet. The rats were sacrificed 2, 4, 6, 10, and 12 mo later; complete autopsies were performed, and all of the tissues were examined histologically. Sections of the liver were also examined histochemically for gamma-glutamyl transpeptidase activity. Shortly after the administration of DEN, the R16 strain showed a significant increase in the number and size of gamma-glutamyl transpeptidase-positive foci and more severe histological changes (disruption of the lobular architecture, bile duct and oval cell proliferation, cellular atypia, and accumulation of fat) compared with the ACP strain. These changes occurred in animals fed either CD or CS diet, but they were much more extensive and severe in the animals on the CD diet. They did not occur in rats of either strain fed the diets alone. The first hepatocellular carcinoma appeared in the R16 rats on the CD diet at 4 mo after administration of the DEN and on the CS diet, at 10 mo. The only hepatocellular carcinoma that occurred in the ACP rats did so at 12 mo in one animal on the CD diet. Combining the data at 10 and 12 mo for the rats on the CD diet, 50% (20 of 40) of the R16 rats had hepatocellular carcinomas, whereas only 3% (one of 30) of ACP rats did. The R16 strain (22%, 9 of 40), but not the ACP strain (0 of 30), also had a variety of other malignancies: squamous cell carcinomas (8%); renal cell carcinomas (8%); lymphomas (5%); and pheochromocytoma (3%). A similar pattern of malignancies also occurred in the R16 rats on the CS diet, and there were no malignancies in the ACP rats. These observations indicate that the grc confers unusual susceptibility to the induction of cancer by the chemical carcinogen DEN and that this genetic susceptibility to cancer of the R16 strain extends beyond the primary target organ of the carcinogen used.

Genic interaction causing embryonic mortality in the rat: epistasis between the Tal and grc genes.

The autosomal dominant mutant gene, tail anomaly lethal (Tal), of the rat is lethal when homozygous but affects tail morphology (kinks and reduced length) and body weight when heterozygous. There is no apparent sex effect on the expression of Tal. It is incompletely penetrant; has variable expressivity, which is influenced partly by its genetic background; and is not linked to the major histocompatibility complex (MHC). The heterozygous Tal gene and the homozygous grc genes, which are linked to the MHC and affect body size and fertility, interact to cause intrauterine death at a time between implantation (five to seven days post-fertilization) and 15 days of gestation. This interaction shifts the time of death from the immediate postnatal period when the homozygous grc genes act to the time during gestation when the homozygous Tal gene would cause death. This description of lethal epistatic interaction in the rat appears to be the first report of this phenomenon in mammals.

The Ah phenotype. Survey of forty-eight rat strains and twenty inbred mouse strains.

Forty-four inbred and four randombred rat strains and 20 inbred mouse strains were examined for their Ah phenotype by determining the induction of liver microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity (EC 1.14.14.1) by intraperitoneal treatment with either beta-naphthoflavone or 3-methylcholanthrene. All 48 rat strains were found to be Ah-responsive. The maximally induced hydroxylase specific activities of the ALB/Pit, MNR/Pit, MR/Pit, SHR/Pit, and Sprague-Dawley strains were of the same order of magnitude as the basal hydroxylase specific activities of the ACI/Pit, F344/Pit, OKA/Pit, and MNR/N strains. Six of the 20 mouse strains were Ah-nonresponsive (i.e. lacking the normal induction response and presumably lacking detectable amounts of the Ah receptor). The basal hydroxylase specific activities of the BDL/N, NFS/N, STAR/N, and ST/JN mouse strains were more than twice as high as the maximally induced hydroxylase specific activity of the CBA/HT strain.--To date, 24 Ah-nonresponsive mouse strains have been identified, out of a total of 68 known to have been characterized. The reasons for not finding a single Ah-nonresponsive inbred rat strain--as compared with about one Ah-nonresponsive inbred mouse strain found for every three examined--remain unknown.

Genetic analysis of liver neuraminidase isozymes in Rattus norvegicus: independent control of NEU-1 and NEU-2 phenotypes.

Two recently identified isozymes of neuraminidase in rat liver were examined for transmission patterns and linkage relationships, and for variation among inbred strains. The isozymes, designated neuraminidase-1 (NEU-1) and neuraminidase-2 (NEU-2), exhibited no electrophoretic mobility variants among the 22 inbred strains examined, but did possess striking interstrain variation in activity phenotypes on electrophoretic gels. The results of a backcross analysis involving the KGH and ACP strains revealed that NEU-1 and NEU-2 phenotypes are independently controlled, each by a single autosomal locus with additively acting alleles. The two loci are unlinked to one another, but the gene controlling NEU-1 is tightly linked to RT1, the rat major histocompatibility complex. This gene is almost certainly identical to Neu-1, a gene identified previously through its effect on "total" activity levels of liver neuraminidase as determined by fluorometric assay of tissue homogenates. NEU-2 and the gene controlling its phenotype were not detected by the fluorometric technique. We designate the genes controlling the NEU-1 and NEU-2 phenotypes as Neu-1 and Neu-2, respectively. Data from this and other studies place Neu-1 between Glo-1 and dw-3. The location of Neu-2 is unknown.

Cellular events associated with peripherally induced rejection of mature neural xenografts placed into neonatal rat brains.

Various circumstances have brought about a dispute concerning the immunologically priviledged status of the central nervous system (CNS). Using a transplantation paradigm, we have examined the cellular events associated with an experimentally induced focal assault on the CNS by the immune system. Chunks of embryonic mouse cortex were transplanted into neonatal rat brains and allowed to survive for 4 weeks. The adult rats then received a skin graft of donor origin to induce rejection of the transplanted tissue. Animals were sacrificed at various time points and examined histologically and immunocytochemically. Under these circumstances, the transplant is rejected via a first-set rejection response, and astrocytes of donor origin appear to be the primary target of the host immune system. Expression of class I and class II major histocompatibility antigens is noted to correlate with lymphocytic invasion of the transplant.

Growth of rat-mouse hybridomas in nude mice and nude rats.

Athymic (nude) mice and rats were inoculated intraperitoneally with rat-mouse hybridoma cells secreting monoclonal antibodies to rat MHC class I antigens in order to improve the yield of antibodies. Pristane priming and subsequent intraperitoneal injection of the hybridoma cells in to nude mice yielded ascites which contained antibody in high concentration (10-15 mg/ml). Complete Freund's adjuvant, mineral oil, pristane or antibody-antigen complexes were used to induce ascites in nude rats, but only pristane priming did so consistently. The hybridoma cells in the ascitic fluid failed to secrete antibody, although they contained intracellular antibody. However, when the pristane-primed nude rats received 250-500 rads of total body radiation prior to injection with the hybridoma cells, they produced large amounts of antibody. When the nude rats were splenectomized and injected with the hybridoma cells, they also produced antibody in high titers. There was no in vitro inhibition of antibody formation by the hybridoma cells cultured in medium containing 15% serum from nude rats, but co-culture of the hybridoma cells with splenic lymphocytes from normal or nude rats markedly inhibited antibody production. These results indicate that the defect in antibody secretion by the hybridoma cells in the ascites of nude rats is due to the presence of radiation-sensitive suppressor cells in the spleen.

The genetic control of the antibody response to the Pa antigen in the rat.

The antibody response to the pregnancy-associated (Pa) antigen in the pregnant female segregated with the major histocompatibility complex (MHC) when the female progeny in the (PVG x WF)F1 x PVG testcross, in which the PVG strain is a low responder to Pa and the WF strain is a high responder, were mated to DA male rats. These results show that non-MHC immune response genes do not play a role, or at least any significant role, in the immune response to the paternal component of the placental antigens: the response approximates a unigenic trait with simple Mendelian segregation. Serological analysis showed that the antibodies formed by both the high responder (GMT 15, titer 1:32,768) and low responder (GMT 3, titer 1:8) female rats were directed against the Pa antigen. These findings show that the genetic control mechanism and the specificity of the antibody elicited by the placental antigen Pa are different from those in the responses to organ grafts and to soluble antigens.

Pregnancy-induced monoclonal antibody to a unique fetal antigen.

An IgM monoclonal antibody was produced by fusing maternal splenic lymphocytes obtained on the day of delivery of the first litter in a WF X DA mating with P3-653 myeloma cells, and at the time when the lymphocytes were isolated there was no circulating antibody in the pregnant animal. The monoclonal antibody had the same specificity as the antibodies in the postpartum pregnancy serum: it reacted with a unique class I MHC antigen, designated the pregnancy-associated (Pa) antigen, which is not one of the currently known, haplo-type-specific class I antigens. The use of recombinant strains mapped the locus encoding the Pa antigen to the region of RT1.A and to the left of RT1.B.

Prolongation of skin graft survival across different genetic barriers in rats with cyclosporine--and its potentiation by Bordetella pertussis vaccine.

Skin allografting was performed in rats treated with cyclosporine using strain combinations that differed across the RT1.A (class I) or RT1.B (class II) loci of the major histocompatibility complex (MHC) or across non-MHC loci. Injection of cyclosporine (20 mg/kg) for 14 consecutive days was followed by prolonged acceptance (MST = 67 days) of the skin allografts. Bordetella pertussis vaccine potentiated the effect of cyclosporine, and the synergistic effect of the vaccine occurred only when it was given before grafting. The cyclosporine given for 14 days with or without B pertussis failed to prolong second-set skin grafts. Production of hemagglutinating antibodies in cyclosporine-treated animals was completely suppressed when the skin grafts were in place, and it occurred to a much lesser extent following rejection, as compared with untreated animals. The cyclosporine did not, however, influence the antibody response following second set skin graft rejection. A system in which rats were treated for only 6 days with cyclosporine was developed in order to test the effects of disparities at different histocompatibility loci on the response to cyclosporine, because this regimen gave a marginal, but significant, prolongation of skin grafts across a full RT1 (MHC) difference. There was a differential effect of cyclosporine treatment depending upon the genetic differences involved: a skin graft across an RT1.A (class I) difference was indefinitely prolonged, one across an RT1.B (class II) or RT1.AB difference was slightly prolonged (9 to 12 days and 12.5 to 16.5 days, respectively) and a graft across non-MHC differences was not affected. Hence, the immunosuppressive effects of cyclosporine on skin graft rejection appear to depend upon the genetic disparities between donor and recipient. Exploitation of this finding may lead to the design of more effective, multidrug protocols for the treatment of rejection that would have fewer deleterious side-effects.

Instability of neural xenografts placed in neonatal rat brains.

Embryonic mouse retinae placed in neonatal rat brains differentiate normally, form appropriate connections with the host brain, and may survive for longer than 1 year. However, such grafts are susceptible to rejection, either spontaneously or after challenge. Advanced spontaneous rejection of the transplant was identified in about 10% of the animals. In addition, two circumstances have been defined in which mouse retinal grafts can be subsequently induced to undergo rejection. One is following placement of a mouse skin graft on the flank of a rat that has received a retinal transplant in the brain, and the other is following removal of a host eye. After each of these procedures, the neural grafts become infiltrated with lymphocytes and undergo degeneration. It is proposed that this system may provide a useful approach not only for studying the immunology and genetics of neural transplantation, but also for examining the circumstances that precipitate the degenerative events associated with certain autoimmune diseases of the central nervous system.

Regulation of class I antigen expression in the rat.

We have investigated the regulation of expression of class I transplantation antigens encoded by the A and E loci in the rat with regard to resting expression level, induction by interferon, and antigen turnover. Flow cytometric measurements showed that the expression of the A locus products is significantly greater than that of the E locus product. Comparisons among a number of haplotypes indicated that the expression of A locus products varies only marginally (+/- 20%) among strains and that expression of the E locus product is approximately one-fifth that of the A locus products. The influence of nonMHC genes on the expression of class I antigens was evaluated by comparing their expression in inbred and congenic strains: it was not significant in this system. Stimulation of lymphocytes with alpha or gamma interferon resulted in proportional increases in the expression of the antigens encoded by both the A and the E loci, and these increases in cell surface antigen expression were reflected in similar increases in the levels of class I mRNA and in the rate of antigen synthesis. Measurement of turnover kinetics showed that the turnover of the A locus antigens is substantially more rapid than that of the E locus antigen, and the difference in half-lives is 3-4-fold. The addition of a broadly reactive anti-class I monoclonal antibody resulted in significant dose-dependent inhibition of the proliferative response of lymph node lymphocytes stimulated with concanavalin A. The inhibition was maximal when the antibody was added during the early stages of activation.

The role of MHC and non-MHC antigens in the rejection of intracerebral allogeneic neural grafts.

Embryonic DA retinal allografts that have survived for prolonged periods after having been transplanted into the brains of neonatal BN rats can be induced to reject following peripheral sensitization with a DA skin graft. The results show that histocompatibility antigens play the major role in the rejection of grafts placed in the CNS and that a disparity between the retinal and skin grafts for MHC antigens induces a more severe rejection response than does a non-MHC antigen disparity.

Immunogenetic aspects of transplantation in the rat brain.

The nature of the brain as an immunologically privileged site is controversial. Using congenic rats, we have demonstrated that grafts that differ from the recipient in the A locus of the MHC produce sensitization following intracerebral transplantation, thus indicating that the brain is not immunologically privileged. The IC grafts show histologic signs of rejection, which are delayed compared with the changes found in the subsequent orthotopic grafts, suggesting that the immune response is weaker in the brain.

Enhancement of induction of intestinal adenocarcinomas by cyclosporine in rats given a single dose of N-methyl N-nitrosourea.

Dietary administration of cyclosporine (CsA) to rats induces lymphoproliferative disorders involving the gut lymphoid plaques with frequent mucosal ulceration. In this study, we investigated whether administration of a known chemical carcinogen prior to CsA treatment modifies the development of the lesions induced by CsA. Male Sprague-Dawley rats were given either a single i.p. injection of N-methyl N-nitrosourea (MNU) (25 mg/kg) or the solvent--and one week thereafter they were fed a diet containing 0.011% CsA. Control rats were fed a basal diet with or without prior MNU treatment. Of the rats that received MNU and CsA, 60% developed adenocarcinomas of the small and large intestine arising in the regions of the intestinal lymphoid plaques. Only 1 of 12 rats given MNU followed by a basal diet developed adenocarcinoma. No tumors developed in rats treated with CsA alone, but there was atypical proliferation of the intestinal mucosa in conjunction with lymphoid hyperplasia of the gut. The mucosal damage secondary to CsA-induced lymphoid lesions probably acted as a promoting stimulus for the development of intestinal carcinomas.

A unique antigen, RT11, in the placenta of the rat.

In the course of exploring the antibody response in the unsensitized WF (u) female pregnant by a DA (a) male, we prepared a hybridoma that secreted an antibody (mAb 213) that was specific to the a haplotype but identified an antigen different from Pa. This antigen was designated RT11. It is present from the twelfth day of gestation on the collagen fibers of the placenta and of all organs in fetal and adult rats. It is particularly prominent on red blood cells; in the yolk sac epithelium; in the walls of the endodermal sinus, blood vessels and bronchioles; and in capsules and trabeculae. A very small amount is present on DA lymphocytes, since 17-20% of them react with mAb 213 by cytofluorimetry. The RT11 antigen is absent from the basal and labyrinthine trophoblast cells, from the parenchymal cells of all organs, and from T and B cells. This distribution pattern is completely different from that of the Aa and Pa antigens. Inhibition and absorption studies showed that RT11 is not an integral part of the collagen molecule. The SDS-PAGE analysis of the immunoprecipitates of RT11 from radioiodinated whole-membrane extracts of red blood cells and from the glycoprotein fraction thereof showed that it is an unglycosylated protein of molecular weight 29,000. The evidence to date suggests that RT11 is a blood group antigen. Studies on the genetic control of the expression of RT11 were undertaken to determine whether a gene linked to the MHC was involved and whether the control mechanism was unigenic or polygenic. Backcrosses generated using inbred strains--(DA x BN)F1 x DA-- and using complementary congenic strains--(DA.1N x BN.1A) F1 x BN.1A--showed that the expression of RT11 was under polygenic control, and that both an MHC-linked gene (1.2 cM from RT1.Aa) and genes not linked to the MHC are involved. By contrast, the expression of the Pa antigen is under the control of an MHC gene only.