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ILAR Journal V34(1/2) 1992 [FORMERLY ILAR NEWS]
Immunodeficient Rodents

GENETICS

The nude (nu) mutation was discovered in 1962, was found to be autosomal recessive in 1966, and was found to cause thymic dysgenesis in 1968 (Rygaard, 1973). The gene is located on chromosome 11 and has pleiotropic effects. Until recently, heterozygotes (nu/+) were thought to be phenotypically normal (i.e., they have thymuses and hair); however, it is now known that they also have reduced numbers of bone marrow stem cells, lower thymus weights, and increased variability of antibody responses to thymus-dependent antigens (Holub, 1989). Consequently, the mutation might be referred to as semi-dominant, and heterozygotes (nu/+) can no longer be considered phenotypically normal.

An independent remutation occurred spontaneously in the AKR/J strain and was named streaker, with the gene symbol nu^str (Shultz et al., 1978). Both the nude and the streaker mutation were transferred into many defined laboratory strains, thus also assessing the effects of background modifying genes. (Hansen, 1978).

PATHOPHYSIOLOGY

Mice homozygous for the nu mutation have a basic defect that is thought to involve all three embryonic germ layers. The thymic dysgenesis appears to be caused by an ectodermal and endodermal metabolic error and inadequate mesenchymal mass in the mediastinal area. This abnormality results in a failure in the homing of prothymocytes to the developing thymic rudiment, accompanied by a deficiency of Ia⁺ stromal cells and hyperplasia of mucopolysaccharide-producing epithelial cells, which form peculiar structures (Holub, 1989). In the skin, there is defective keratinization of hair and degeneration of hair follicles, resulting in hairlessness despite an increased frequency of hair growth cycles (Eaton, 1976).

Recent studies of nude mice have focused on the significant numbers of T cells that develop with age in an extrathymic pathway. However, the nude mouse cannot be regarded as a model of extrathymic differentiation of T cells because there is in vivo evidence that the dysgenic thymus is not completely bypassed by these cells. That evidence includes some epithelial cell-lymphocyte associations in the area of the thymus, and the secretion by the thymus of some immunoregulatory factors (e.g., CSF-GM) (Holub, 1989; Jiraskova et al., 1990). The only thing missing in the dysgenic thymus is the macrophage deletion of forbidden lymphocytic clones, and it has been shown that there is no negative selection for maturing T cells (Yuuki et al., 1990).

T cells in nude mice slowly develop the surface markers Thy-1, CD3, CD4, CD8 (MacDonald et al., 1986), and CD5 with increasing age (in most background strains, only after 3-4 months of life). T-cell antigen receptor (TCR) expression is 2 to 3 times lower in the lymph nodes of aging nude mice than in euthymic controls (MacDonald et al., 1987), and a high percentage of CD3⁺ cells express g/d TCR (about 50 percent compared with 2 to 4 percent in euthymic mice) (Lake et al., 1991). Older nude mice produce IL-2 and express most IL-2R chains, but their ability to respond to mitogens is limited (Kung and Thomas, 1988). Major histocompatibility complex class II-specific Th 1 cells with a/b TCRs do develop (Sydora and Kronenberg, 1991).

The first line of defense of the mutant, in addition to g/d cells, is an increased population of NK cells (Holub et al., 1989), especially in exposed areas such as superficial lymph nodes. Also, macrophages appear to be more efficient in nude mice byond the fetal stage (see Holub, 1989). Both IL-1 and tumor necrosis factors play more important roles in nude than euthymic mice. Thermoregulatory problems caused by the lower insulating capacity of the hairless skin at ambient temperatures under 30ºC are solved by social and nonshivering thermogenesis (Holub, 1989). The reaction to cold is evident both in terms of brown adipose tissue hyperplasia and an incrase of paraimmunologic functions that are more pronounced in nude mice than in euthymic hairless mice (Holub, in press). A small subpopulation of lymph node L3T4⁺ T cells produce large amounts of IL-3 and CSF-GM in nude mice (Kimoto et al., 1989). Such production may be related to one of the peculiarities of the otherwise normal B-cell population in which precursors lacking IL-3R receptors fail to undergo terminal differentiation (Palacios and Leu, 1986). Antibody formation in nude mice is limited mainly to the IgM isotype and to primary responses. IgG levels fluctuate considerably among nude mice in individual colonies; however, a general defect in IgA and a decreased heterogeneity of Ig have been found consistently (Mink et al., 1980). A recent study of C57BL/6J-nu/nu mice between 10 and 40 weeks of age has established a significant decrease only in IgG2b (Klein-Schneegans et al., 1990). Like antibody formation, absolute Ig levels depend on background strain and microbiological status, and there is often considerable variation among colonies.

The discovery that the deficiencies in immunologic and paraimmunologic defenses of nude mice are not as great as was thought in the early seventies and the introduction of mice with a mutation called severe combined immune deficiency (scid) have decreased the value of the nude mouse as a host for human tissue. However, the nude mutant continues to be a key model for studying T-cell differentiation, specific cellular reactions in the absence of the fully developed thymus, and thermogenesis in immunodeficient states.

HUSBANDRY

Under germ-free conditions, the maximum survival time of nude mice is almost identical to that found in wild type mice (Holland et al., 1978). However, the mean total survival time of nude mice is shorter than that of euthymic littermates because they more frequently develop lymphomas. Under SPF conditions the mean survival time of streaker mice has been found to be 34 weeks compared to 44 weeks for euthymic controls (Shultz et al., 1978).

The nude mutant is more susceptible to certain natural pathogens than are its euthymic littermates. Such pathogens range from viruses to intestinal parasites. Specific pathogen free conditions, and sterilization of water, chow (preferably with a higher fat and vitamin content), and bedding are advisable. The ambient temperature should not be lower than 28ºC and the humidity should never exceed 60 percent. A consistent light/dark cycle is recommended. Experimental data can be unexpected and not reproducible if nude mice are used immediately after arrival at a facility or are housed among other mice without taking appropriate precautions against infectious diseases.

REPRODUCTION

Homozygous nude females are not efficient breeders, although there may be an occasional female that can lactate and raise a litter. The most effective breeding scheme uses heterozygous females and nude males, with periodic breeding of homozygous normal females to homozygous nude males. Litter size should be reduced by eliminating excess heterozygous males so that the number of heterozygous pups is equal to the number of nude pups. Between fetal day 17 and postnatal day 7, homozygous nude pups can be distinguished from their euthymic littermates by their poorly developed vibrissae. The diminished size of the thymus becomes detectable by fetal day 14. To determine which animals are homozygous for the nude mutation at earlier stages of development, skin must be tested by grafting it onto allogeneic or xenogeneic recipients and demonstrating a lack of hair growth.

REFERENCES

Eaton, G.J. 1976. Hair growth cycles and wave patterns in "nude" mice. Transplantation 22:217-222.

Hansen, C.T. 1978. The nude gene and its effects. Pp. 1-35 in The Nude Mouse in Experimental and Clinical Research, J. Fogh and B. Giovanella, eds. New York:Academic Press.

Holland, J.M., T.J. Mitchell, L.. Gipson, and M.S. Whitaker, 1978. Survival and cause of death in aging germfree athymic nude and normal inbred C3Hf/He mice. J. Natl. Cancer Inst. 61:1357-1361.

Holub, M. 1989. Immunology of Nude Mice. Boca Raton, Fla.:CRC Press.

Holub, M., V. Vêtvicka, J. Houstek, D. Janikoa, Z. Rychter, A. Vrana, and L. Dazdova. 1989. Influence of ambient temperature on nude mouse metabolic and immune status. Pp. 68-77 in Immune-Deficient Animals in Experimental Medicine. Sixth International Workshop on Immune-Deficient Animals, Bing-quan wu and Jie Zheng, eds. Basel:Karger.

Holub, M., J. Houstek, and J. Chalupna. In Press. Effect of ambient temperature on immunocompetent and immunodeficient hairless mice. Folia Biol. (Prague).

Jiraskova, Z., E. Necas, M. Holub, and J. Ludvik. 1990. The dysgenic thymus in nu/nu is a potent source of colongy stimulating factor (CSF). Folia Biol. (Praha) 36:231-235.

Kimoto, M., S. De Kossodo, V. Kindler, M. Detraz, P. Vassalli, and S. Izui. 1989. Presence of a very small population of Thy-1+, L3T4 cells producing large amounts of IL-3 in young athymic nude mice. Immunology 68:557-563.

Klein-Schneegans, A.-S., L. Kuntz, S. Trembleau, P. Foneneau, and F. Loor. 1990. Serum concentrations of IgM, IgG1, IgG2b, IgG3, and IgA in C57BL/6 mice and their congenics at the nu (nude) locus. Thymus 16:45-54.

Kung, J.T., and C.A. Thomas III. 1988. Athymic nude CD4+ 8- cells produce IL-2 but fail to proliferate in response to mitogenic stimuli. J. Immunol. 141:3691-3696.

Lake, J.P., C.W. Pierce, and J.D. Kennedy. 1991. T-cell receptor expression by T cells that mature extrathymically in nude mice. Cell. Immunol. 135:259-265.

MacDonald, H.R., C. Blanc, R.K. Lees, and B. Sordat. 1986. Abnormal distribution of T-cell subsets in athymic mice. J. Immunol. 136:4337-4339.

MacDonald, H.R., R.K. Less, C. Bron, E. Sordat, and G.Miescher. 1987. T-cell antigen receptor expression in athymic (nu/nu) mice. Evidence for an oligoclonal beta chain repertoire. J. Exp. Med. 166:195-209.

Mink, J.G., J. Radl, P. van den Berg, J.J. Haaijman, M.J. van Zwieten, and R. Benner. 1980. Serum immunoglobulins in nude mice and their heterozygous littermates during aging. Immunology 40:539-545.

Palacios, R., and T. Leu. 1986. CCII: A monoclonal antibody specific for interleukin-3-sensitive cells defines two major populations of B-cell precursors in the bone marrow. Immunol. Rev. 93:125-146.

Rygaard, J. 1973. Thymus and Self-immunobiology of the Mouse Mutant Nude. Copenhagen:F.A.D.L.

Shultz, L.D., J.-J. Heiniger, and E.M. Eicher. 1978. Immunopathology of streaker mice: A remutation to the nude in the AKR/J strain. Pp. 211-222 in Animal Models of Comparative and Developmental Aspects of Immunity and Disease, M.E. Gershwin and E.L. Cooper, eds. Elmsford, N.Y.:Pergamon Press.

Sydora, B.C., and M. Kronenberg. 1991. Characterization of a CD4+ T-cell line derived from an athymic (nu/nu) mouse. Cell Immunol. 134:54-64.

Yuuki, H., Y. Yoshihaki, K. Kishihara, A. Iwasaki, G. Matsuzaki, H. Takimoto, and K. Nomoto. 1990. Clonal energy in self-reactive alpha/beta cells is abrogated by heat-shock protein reactive gd T cells in aged athymic nude mice. Eur. J. Immunol. 20:1475-1482.

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