The rnu (Rowett Nude) and rnuN (nznu, New Zealand Nude) Rat: An Update

Dr. Schuurman is from the Laboratory for Pathology, National Institute for Public Health and Environmental Protection, in Bilthoven, The Netherlands; and the Division of Histochemistry and Electron Microscopy, Departments of Pathology and Internal Medicine University Hospital in Utrecht, The Netherlands. He is currently located at the Department of Preclinical Research/Immunology, Sandoz Pharma A.G. in Basel, Switzerland. Dr. Hougen is from the Bartholin Institut and University Institute of Forensic Pathology, in Copenhagen, Denmark. Dr. van Loveren is from the Laboratory for Pathology, National Institute for Public Health and Environmental Protection, in Bilthoven, The Netherlands. Dr. Schuurman is from the Laboratory for Pathology, National Institute for Public Health and Environmental Protection, in Bilthoven, The Netherlands; and the Division of Histochemistry and Electron Microscopy, Departments of Pathology and Internal Medicine University Hospital in Utrecht, The Netherlands. He is currently located at the Department of Preclinical Research/Immunology, Sandoz Pharma A.G. in Basel, Switzerland. Dr. Hougen is from the Bartholin Institut and University Institute of Forensic Pathology, in Copenhagen, Denmark. Dr. van Loveren is from the Laboratory for Pathology, National Institute for Public Health and Environmental Protection, in Bilthoven, The Netherlands. Dr. Schuurman is from the Laboratory for Pathology, National Institute for Public Health and Environmental Protection, in Bilthoven, The Netherlands; and the Division of Histochemistry and Electron Microscopy, Departments of Pathology and Internal Medicine University Hospital in Utrecht, The Netherlands. He is currently located at the Department of Preclinical Research/Immunology, Sandoz Pharma A.G. in Basel, Switzerland. Dr. Hougen is from the Bartholin Institut and University Institute of Forensic Pathology, in Copenhagen, Denmark. Dr. van Loveren is from the Laboratory for Pathology, National Institute for Public Health and Environmental Protection, in Bilthoven, The Netherlands. In 1953 rats with nude characteristics appeared for the first time in a rat colony at the Rowett Research Institute, Aberdeen, Scotland. At that time nothing was known about the function of the thymus in the immune system, that is, in generation of T (thymus-derived) lymphocytes in the cell-mediated branch of immunological reactions. Therefore the severe cellular immunodeficiency was not recognized, and no precautions were undertaken to isolate the mutation. Because the mutation reappeared in the 1970s in the same outbred colony ( May et al., 1977 ; Festing et al., 1978 ), one may conclude that the mutation was retained within the colony at a rather low gene frequency. The autosomal recessive mutation is designated rnu . Independently, a second athymic mutant was observed in a colony of random-bred albino rats maintained at Victoria University, Wellington, New Zealand, designated rnuN , nznu , or rnunz ( Berridge et al., 1979 ). The congenitally athymic rat is characterized by hairlessness, which is almost complete in the rnuN/rnuN animal. The rnu / rnu rat usually shows some short and thin hairs ( Figure 1 ). Immunologically, the main feature of the nude mutation is congenital plasia of the thymus, which is only present in a rudimentary form without any lymphocyte population. ( Fossum et al., 1980 ; Vos et al., 1980a ). At day 14 of fetal life, a thymus anlage is seen in both euthymic and athymic animals, but in rats with the nude mutation, this anlage does not become populated by leukocytes (lymphocytes, macrophages, interdigitating cells) later on in life. In rnu / rnu animals the thymus anlage shows undifferentiated strands of epithelium and small cysts or ducts, with differentiation into serous and mucous acini ( Fossum et al., 1980 ; Hougen and Klausen, 1984 ; Vos et al., 1980a ). Normal euthymic rat (a), and congenitally athymic rnu / rnu rat (b), from the breeding colony at the National Institute of Public Health and Environmental Protection, Bilthoven, The Netherlands. Note the hairlessness (that is not complete) in the rat with the rnu mutation. (Photograph courtesy of the authors.) Neither the rnu nor the rnuN mutant genes have been localized for the genome. Breeding data indicate that the mutations are allelic ( Festing et al., 1978 ), and that the rnu mutation is dominant to rnuN ( Hedrich, 1990 ). The detection of heterozygotes is hampered because heterozygous (+/ rnu ) animals are similar in almost every aspect to homozygous wild-type animals. The only difference may be a lower lymphocyte density of the paracortical areas in lymph nodes of +/ rnu animals ( Hougen et al., 1987 ). The original rnu mutant has been backcrossed onto a number of inbred backgrounds to produce congenic strains. Table 1 represents a list of rnu and rnuN strains that have been studied in an international comparative analysis that focussed on immunological characteristics ( Schuurman et al., 1992 ). Some institutes where congenitally athymic rat strains are held The LEW congenic strains have been independently derived: it is not known whether all LEW backgrounds are authentic. However, it is known that LEW/Mol differs from standard LEW rats. The strains listed have been subjected to a comparative analysis ( Schuurman et al., 1992 ). These strains showed major differences in weight of body and spleen (matched for age, only male animals were analysed), and morphometric data of cells bearing αβ-T-cell receptors in spleen and of splenic red pulp identified by an anti-macrophage antibody. In all strains the number of cells bearing αβ-T-cell receptors increased with age (analysed were animals at 1 ½-2 months of age and at ½ year of age). None of the animals tested showed a response to ovalbumin immunization (antibody formation and delayed-type hypersensitivity ear challenge). Some institutes where congenitally athymic rat strains are held The LEW congenic strains have been independently derived: it is not known whether all LEW backgrounds are authentic. However, it is known that LEW/Mol differs from standard LEW rats. The strains listed have been subjected to a comparative analysis ( Schuurman et al., 1992 ). These strains showed major differences in weight of body and spleen (matched for age, only male animals were analysed), and morphometric data of cells bearing αβ-T-cell receptors in spleen and of splenic red pulp identified by an anti-macrophage antibody. In all strains the number of cells bearing αβ-T-cell receptors increased with age (analysed were animals at 1 ½-2 months of age and at ½ year of age). None of the animals tested showed a response to ovalbumin immunization (antibody formation and delayed-type hypersensitivity ear challenge). The rnu mutant has been studied more extensively than the rnuN mutant. The following is a review of published data on the mutant with respect to life span and reproduction, immunological characteristics, and host resistance to infections. The descriptions refer to the rnu mutant unless stated otherwise. Depending on the background strain, the body weight of nude rats is approximately 20 percent lower than that of heterozygous animals ( Schuurman et al., 1992 ). Under conventional conditions, rnu / rnu rats have a lifespan of about 9 months, while rnuN/rnuN rats rarely live longer than 4 months ( Hedrich, 1990 ). When maintained under specific pathogen-free (SPF) conditions, wasting and infections do not occur as frequently as in the nude mouse counterpart. SPF rnu / rnu animals have a maximum life span of 1.5 to 2 years or more ( Salomon and Fogh, 1982 ; Vaessen et al., 1986 ; Hedrich, 1990 ; authors' unpublished observations). The maximum life span of rnuN/rnuN rats is also almost 2 years when maintained under SPF conditions ( Deerberg, 1991 ). A number of endocrinological parameters have been found to be unaffected by the rnu mutation ( Festing et al., 1978 ; Vos et al., 1980a ). For instance, female rnu / rnu rats have lower body weights than do males. They normally are fertile and produce the usual number of offspring; but they can rear only a few newborns ( Hedrich, 1990 ). Therefore, breeding is usually performed by mating +/ rnu or +/+ females with rnu / rnu males. In some animal facilities, +/ rnu females and +/ rnu males are mated. The offspring of heterozygous females and homozygous males usually contains less than 50 percent rnu / rnu animals, indicating a loss in utero ( Festing, 1981 ). Heterozygous matings yield approximately 25 percent rnu / rnu animals, as expected. The breeding of rnuN rats is more difficult than that of rnu rats ( Berridge et al., 1979 ; Festing 1981 ; Hedrich, 1990 ). The congenital absence of the thymus results in a severely deficient cell-mediated branch of the immune system. The data reported in the literature are summarized in Table 2 . Histology of secondary lymphoid organs ( Figure 2 ) shows a severe lymphodepletion of the periarteriolar lymphocyte sheath in the spleen ( Figure 2d ), interfollicular areas (paracortex) in lymph nodes ( Figure 2b ) and Peyer's patches along the intestinal tract. Similarly, interfollicular areas in the bronchial-associated lymphoid tissue and nasal-associated lymphoid tissue are lymphodepleted (Kuper, 1991). It should be noted that the anlage of the thymus-dependent areas in secondary lymphoid organs is present in nude rats in similar or somewhat smaller size than in euthymic rats ( Figures 2b, d ). The bone marrow in nude rats has not been studied in much detail, but it does not appear to be different from that of euthymic rats. The absence of T lymphocytes can be shown by using immunohistochemistry for T-cell differentiation antigens ( Figure 3 ). The absence of cell-mediated immunity in nude rats is evident in functional in vitro assessments such as lymphocyte stimulation with T-cell mitogens and alloreactivity in mixed leukocyte reactions with subsequent cell-mediated lympholysis. In vivo assessments show that thymus-dependent immunity, including the response to thymus-dependent antigens, alloreactivity in graft rejection, and graft-versus-host reactivity, is lower than normal or absent in nude rats. Overview of a mesenteric lymph node (a and b, magnification 30x) and a more detailed view on the white pulp in spleen (c and d, magnification 125x) from a normal euthymic rat (a,c) and a congenitally athymic rnu/rnu rat (b,d). The lymph node from the euthymic rat (a) shows a normal architecture comprising follicles with germinal centers (F), paracortex (P), and medulla (M). Note the dense population of follicles and paracortex by lymphocytes. In the lymph nde of the athymic rat (b,d), follicles (F) are present but do not manifest a germinal center; the paracortex (P) is almost completely lymphocyte-depleted; and the medulla (M) shows a normal architecture. The athymic situation is reflected by the absence of T lymphocytes in the paracortex. Germinal centers are not present since germinal center formation is a T-cell dependent process. The white pulp of spleen shows the periarteriolar lymphocyte sheath (P), follicles (F), and the marginal zone (M), and is surrounded by the red pulp. Euthymic and athymic rat spleen show a dense population by B lymphocytes in follicles and marginal zone (being B-lymphocyte areas); the periarteriolar lymphocyte sheath, being a T-cell area, is well populated in spleen from the euthymic rat (c), but shows a severe lymphodepletion in spleen from the athymic rat (d). Formalin-fixed material, embedded in paraffin, secion stained with hematoxylin and cosin. (Photograph courtesy of the authors. Immunohistochemistry on sections of spleen from 7 week-old congenitally athymic rnu/rnu rat. The white pulp with the arteriole in the middle is shown, surrounded by red pulp. The method applied was an indirect immunoperoxidase reaction on frozen tissue section, with hematoxylin counterstain (magnification 125x). Immunolabeling was done with the following monoclonal antibodies: (a) R73 to αβ-T-cell receptor (provided by Dr. Th. Hünig, Würzburg, Germany); (b) ER2 (CD4) to T cells of helper-inducer phenotype, in adition to a subset of macrophages (provided by Dr. J. Rozing, Leiden, The Netherlands); (c) OX8 (CD8) to T cells of suppressor-cytotoxic phenotype, in addition to natural-killer cells (provided by Dr. A.F. Williams, Oxford, England); (d) ED1 to all macrophages (provided by Dr. C. Dijkstra, Amsterdam, The Netherlands). (a) There is no specific immunolabeling for αβ-T-cell receptor; the black dots, mainly in the red pulp, represent endogenous peroxidase activity. (b) Around the arteriole some cells are labeled by ER2 (indicated by arrows), and in addition a diffuse immunolabeling for ER2 is observed in the red pulp; this immunolabeling presumably represents macrophage staining. (c) Some cells in the white pulp, and at a higher density cells in the red pulp show immunolabeling by OX8 (some indicated by arrows), presumably representing labeling of natural-killer cells. (d) Macrophages in the white pulp, especially around the arteriole, and in the red pulp are labeled by ED1. (Photographs courtesy of the authors). Immune status of congenitally athymic (nude) rats a References to studies on the mun mutant are underlined. Bell et al., 1987 Berridge et al., 1979 Brooks et al., 1980 De Jong et al., 1980 Douglas-Jones et al., 1981 Festing et al., 1978 Fossum et al., 1980 Fossum, 1983 Fossum et al., 1983a Fossum et al., 1983b Gilhar et al., 1986 Hedrich, 1990 Hedrich et al., 1987 Hougen and Klausen, 1984 Hougen et al., 1987 Hougen and Klausen, 1987 Hougen et al., 1989 Hougen et al., 1990 Klausen et al., 1982 Klausen and Hougen, 1987 Kumararatne et al., 1981 Lotzova et al., 1984 Marshall and Miller, 1981 Pritchard and Eady, 1980 Reynolds et al., 1982 Rolstad and Ford, 1983 Rolstad et al., 1983 Rolstad and Fossum, 1987 Salomon and Fogh, 1982 Schuurman et al., 1985 Schuurman et al., 1986 Schuurman et al., 1989 Schwinzer et al., 1987 a Schwinzer et al., 1987 b Sfaksi et al., 1985 Svendsen et al., 1981 Taubman et al., 1983 Terada et al., 1980 Tønnesen and Rolstad, 1983 Vaessen et al., 1986 Van Loveren et al., 1987 Van Loveren et al., 1988 Vos et al., 1979 Vos et al., 1980a Vos et al., 1980b Wonigeit et al., 1987 Yoshie et al., 1985 Immune status of congenitally athymic (nude) rats a References to studies on the mun mutant are underlined. Bell et al., 1987 Berridge et al., 1979 Brooks et al., 1980 De Jong et al., 1980 Douglas-Jones et al., 1981 Festing et al., 1978 Fossum et al., 1980 Fossum, 1983 Fossum et al., 1983a Fossum et al., 1983b Gilhar et al., 1986 Hedrich, 1990 Hedrich et al., 1987 Hougen and Klausen, 1984 Hougen et al., 1987 Hougen and Klausen, 1987 Hougen et al., 1989 Hougen et al., 1990 Klausen et al., 1982 Klausen and Hougen, 1987 Kumararatne et al., 1981 Lotzova et al., 1984 Marshall and Miller, 1981 Pritchard and Eady, 1980 Reynolds et al., 1982 Rolstad and Ford, 1983 Rolstad et al., 1983 Rolstad and Fossum, 1987 Salomon and Fogh, 1982 Schuurman et al., 1985 Schuurman et al., 1986 Schuurman et al., 1989 Schwinzer et al., 1987 a Schwinzer et al., 1987 b Sfaksi et al., 1985 Svendsen et al., 1981 Taubman et al., 1983 Terada et al., 1980 Tønnesen and Rolstad, 1983 Vaessen et al., 1986 Van Loveren et al., 1987 Van Loveren et al., 1988 Vos et al., 1979 Vos et al., 1980a Vos et al., 1980b Wonigeit et al., 1987 Yoshie et al., 1985 Non-thymus-dependent cytotoxicity, including natural killer cell activity and macrophage cytotoxicity, is increased, which may be a compensatory mechanism for the animal to cope with the deficient T-cell-mediated defense. Also allogeneic cells can be rejected by natural cytotoxic cells, which kill in a non-MHC-restricted manner ( Rolstad and Fossum, 1987 ; Tünnesen and Rolstad, 1983 ), different from cytotoxic T cells. The B-lymphocyte branch of the immune system is intact, insofar as it can function without T-cell assistance. Thus, peripheral lymphoid organs show primary follicles ( Figures 2b, d ), but secondary follicles are almost absent because the formation of germinal centers in such follicles requires T-cell assistance. The same applies to in vivo or in vitro antibody formation, which occurs after stimulation with thymus-independent antigens but is absent after stimulation with thymus-dependent antigens. Like other rodent mutants with a severely defective cell-mediated immune system (for instance, mice with the nu or the scid mutation), nude rats have lymphocytes with T-cell characteristics. This has been demonstrated by flow cytometry using cell suspensions from the thoracic duct, spleen, and lymph nodes. Early studies in this area used monoclonal antibodies that were not truly T-cell specific, that is, they would react with more than one type of cell. Examples are antibodies in the CD4 (T-helper-inducer phenotype, also reactive to some macrophages) and CD8 clusters (T-suppressor-cytotoxic phenotype, also reactive to natural killer cells). This phenomenon is illustrated in Figures 3b and c for immunohistochemistry on splenic sections. However, after introduction of truly T-cell specific reagents, including antibodies to the -heterodimeric T-cell receptor structure, cells expressing T-cell characteristics could still be detected. These cells are designated as T-like cells because they have not been ‘educated’ in a thymic microenvironment, that is, they have not been subjected to positive and negative selection in the thymus. The proportions of these cells vary between different background strains and increase with age ( Schuurman et al., 1992 ; Schwinzer et al., 1987b ). For instance, the proportions of CD5-positive T cells (pan-T marker, antibody MRC OX19) in spleen cell suspensions from nude rats increases from 5 percent at 2 months of age to 19 percent at 17 months of age. The value in euthymic rats is about 34 percent, without an apparent age-dependency ( Vaessen et al., 1986 . In young nude rats, the T-like cells have the uncommon immunophenotype of immature cells ( Vaessen et al., 1985 ). Schwinzer et al. (1989 ) have shown that the expression of CD45 cells (common leukocyte marker (MRC OX22 antibody) and RT6 antigen expression on CD4-positive or CD8-positive cells in nude rats differs from that in euthymic rats. These observations indicate that the population of cells bearing T-cell markers in nude rats differs from that in euthymic rats. Thus, in nude rats, cells with T-cell characteristics emerge and increase in number during life. It remains to be established whether the generation of these T-like cells with age is influenced by the genotype (background strain) or local environment (housing conditions). The cells do express αβ-T-cell receptors but apparently do not exert functional activity using this receptor. This has been shown by the absence of specific reactivity to antigens requiring interaction with the T-cell receptor on cells that have been appropriately selected in the thymus (e.g., immune response after immunization with ovalbumin, tetanus toxoid, or sheep red blood cells). For nude mice, the oligoclonal character of T cells emerging during life has been documented ( Kung, 1988 ; Maleckar and Sherman, 1987 ; McDonald et al., 1987 ). For nude rats, data on the same aspect is lacking which makes it to whether T-like cells in these animals manifest a complete absence of reactivity or show reactivity to only a small number of antigens. The T-like cells can function in an (e.g., to lymphocyte stimulation with mitogens or and can produce such as following stimulation ( Schwinzer et al., ; Sfaksi et al., 1985 ; Vaessen et al., 1987 ). In these cells may also show some functional reactivity, which can be form the of germinal centers in secondary follicles that requires T-cell assistance. This a of reactivity between that by truly stimulation by receptors other than the T-cell receptor and that by truly reactions. However, data in the literature show in this that the response is but studies on the outbred ( ) rat have including in vitro cytotoxic activity and in vivo ( Table 2 ). When not maintained under SPF conditions, nude rats are to is with and loss of epithelium ( and 1980 ). reported by , and ( Festing, 1981 ). occurs after ( et al., 1984 ). is with especially in animals ( et al., 1985 ). in the literature on host resistance is summarized in Table 3 . These data are for the immune status of the animals There is no reactivity to that requires thymus-dependent For is a response to the and and to rat For these cell-mediated immunity is not or is only For , the that on macrophage activity is increased, but the of the that requires functional cellular immunity is hampered ( Van Loveren et al., 1987 ). The antibody response to is not to or to In this represents an of rnu / rnu rats to d and 1980 et al., 1989 et al., 1987 Hougen et al., 1989 Hougen et al., 1990 et al., 1987 Klausen et al., 1986 et al., 1980 et al., 1983 Rolstad, 1984 et al., 1979 Van Loveren et al., 1987 Van Loveren et al., 1988 Vos et al., 1983 Van Loveren et al., unpublished of rnu / rnu rats to d and 1980 et al., 1989 et al., 1987 Hougen et al., 1989 Hougen et al., 1990 et al., 1987 Klausen et al., 1986 et al., 1980 et al., 1983 Rolstad, 1984 et al., 1979 Van Loveren et al., 1987 Van Loveren et al., 1988 Vos et al., 1983 Van Loveren et al., unpublished The severely deficient branch of cell-mediated immunity, with the size and of the rnu / rnu rat and the absence of major endocrinological observed in some it an for For instance, in the areas of and a of the animal is in The are by the of to the immune status of rats, including monoclonal antibodies to leukocyte by Schuurman et al., 1991 ). for and in vivo host resistance ( Vos et al., 1990 ). The of the rnu mutant has been shown in studies on T-cell differentiation after (e.g., of T cells or thymus ( Schuurman et al., 1988 ; Hougen, 1991 ). studies are to be in the years with the of the functional the expression and function of T-cell receptor