Immunology 1979 37 777

Comparative patterns of serum immunoglobulin levels in specific-pathogen-free congenitally athymic (nude), hereditarily asplenic (Dh/ +), congenitally athymic-asplenic (lasat) and splenectomized athymic mice

D. D. ECKELS, M. E. GERSHWIN, J. DRAGO* & L. FAULKINt Department of Internal Medicine, Section of Rheumatology-Clinical Immunology, * Department of Surgery, School of Medicine, and t Department of Veterinary Anatomy, School of Veterinary Medicine, University of California, Davis, California, U.S.A.

Acceptedfor publication 26 January 1979

Summary. Serial determinations of serum immunoglobulin levels were assessed in congenitally athymic (nude), hereditarily asplenic (Dh/ +) and congenitally athymic-asplenic (lasat) mice and the results compared to normal intact littermate controls (nu/ +), neonatally splenectomized nu/ + and neonatally splenectomized nude mice. Quantification of Ig levels was accomplished by radial immunodiffusion, for IgM, IgGI, IgG2a, IgG2b and IgA antibody isotypes. Intact spleen and/or thymus function was shown to have marked effects on the age-dependent development of serum IgM, IgG2b and IgA production. Furthermore, because of higher levels of IgA in congenitally athymic-asplenic mice and neonatally splenectomized nude mice v. sham splenectomized nude mice, it is suggested that an IgA-specific suppressor population resides in the spleen. Finally, because of frequent problems in the literature in interpretation of immunoglobulin values, the criteria for the statistical evaluation of such data in establishing normal serum Ig values and ascertaining real differences between treatment groups are emphasized.

INTRODUCTION There has been significant information recently gathered in our understanding of the role(s) of the thymus and spleen in the ontogenesis of immunological responsiveness (Machado, Lozzio & Lair, 1976; Gershwin, Ahmed, Ikeda, Shifrine & Wilson, 1978; Fletcher, Ikeda & Gershwin, 1977; Battisto, Borek & Busci, 1971). Much of this data has been generated by study of two major murine mutants, the congenitally athymic (nude) and the hereditarily asplenic (Dh/ +) mouse. Both of these mutants were discovered in Great Britain and have been reviewed recently in the literature (Fletcher et al., 1977; Gershwin, Merchant, Gelfand, Vickers, Steinberg & Hansen, 1975). Indeed, numerous investigators have quantified serum immunoglobulins in nude (Gershwin et al., 1975), asplenic (Lozzio & Wargon, 1974; Fiocchi, 1976), splenectomized (Busci, Borek & Battisto, 1972) and/or thymectomized (Parrott & East, 1964) mice. Moreover, immunoglobulin (Ig) levels of crosses of nude and Dh/ + mice (congenitally athymic-asplenic or lasat) have likewise been studied (Machado et al., 1976; Gershwin et al., 1978). Nonetheless, several controversies exist in the literature regarding levels of immunoglobulins in such mice and this problem has recently been cited as a major discrepancy in comparing data from several laboratories. In view of these

Correspondence: Dr M. Eric Gershwin, Department of Internal Medicine, Section of Rheumatology-Clinical Immunology, School of Medicine, University of California, Davis, California 95616, U.S.A. 00 19-2805/79/0800-0777$02.00 Cc 1979 Blackwell Scientific Publications

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issues and the availability of nude, Dh/ + and lasat mice in this laboratory, we have assessed serial serum immunoglobulin levels in mice with both congenital asplenia and thymic agenesis (nu/nu Dh/ +) and compared the data with levels obtained in nude (nu/nu), nu/ +, Dh/ +, splenectomized nu/nu (Spx nu/nu) and splenectomized nu/ + (Spx nu/ +) mice.

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MATERIALS AND METHODS Mice Congenitally athymic mice (nu/nu) and hereditarily asplenic mice (Dh/ +) both on an outbred N: NIH(S) background were maintained under specific-pathogen-free (SPF) conditions at the Animal Resource Services of the University of California at Davis. A colony of congenitally athymic-asplenic mice on an N: NIH(S) background was produced from the above colonies by the crossing of Dh/ + females with nu/nu males and subsequent backcrossing of nu/ + Dh/ + offspring with nu/nu animals. Neonatal or sham splenectomy was performed on nu/nu and nu/ + mice using dry ice anaesthesia. The completeness of splenectomy was assessed grossly and when questionable, by histology at the termination of the experiment. Only female mice were studied. Serum immunoglobulin quantification Three to five mice of each group were bled at 1, 3, 6, 9 and 12 months from the orbital venous plexus. Each serum was handled individually. Levels of IgM, IgG1, IgG2a, IgG2b and IgA were quantified by radial immunodiffusion using monospecific antisera. Statistical analysis was conducted: (i) to assess whether or not in fact, the values obtained conformed to a normal (Gaussian) distribution by use of a x2 test for goodness-of-fit, and (ii) to detect the statistical significance of differing group means computed by way of a Student's t test modified to correct for the possibility of unequal variances being present in compared groups (Bailey, 1975). RESULTS

Total serum Ig Total serum immunoglobulin levels were strikingly different from group to group (Fig. 1). In particular, nude mice had significant reductions compared to intact littermate controls throughout the course of the

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12 9 6 3 Age (months) Figure 1. Total serum immunoglobulin levels in mg% (mean + SE) of nu/nu (o) nu/ + (-), Dh/ + (A) and nu/nu Dh/ + (A) mice.

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+), congenitally athymic-asplenic (lasat) and splenectomized athymic mice.

Immunology 1979 37 777 Comparative patterns of serum immunoglobulin levels in specific-pathogen-free congenitally athymic (nude), hereditarily asplen...
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