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ILAR Journal V35(1) 1993 [FORMERLY ILAR NEWS]
Models of Type I Diabetes - Part One

The NOD Mouse: A Model for Analyzing the Interplay Between Heredity and Environment in Development of Autoimmune Disease
Edward H. Leiter
Edward H. Leiter, Ph.D., is a senior staff scientist at The Jackson Laboratory in Bar Harbor, Maine.

INTRODUCTION

Inbred colonies of NOD (nonobese diabetic) mice have become widely established around the world since the publication of the initial report (Makino et al., 1980) describing this strain's susceptibility to spontaneous development of autoimmune (type 1) insulin-dependent diabetes mellitus (IDDM). The availability of NOD mice has provided important new immunogenetic and pathophysiologic insights into autoimmune disease and its prevention. Because of their genetic predisposition to develop IDDM, NOD mice are in great demand by investigators wishing to test compounds or devices that will either prevent the development of diabetes or provide therapy after the disease has developed. These mice present a special challenge to laboratory animal health specialists and colony managers. One of the most important lessons "taught" by the NOD mouse is that the quality of the breeding and maintenance environment is not a trivial matter. Whereas there is only circumstantial evidence supporting environmental triggers for IDDM in humans (e.g., dietary ingredients and pathogens), studies using the NOD mouse clearly demonstrate that the penetrance of a diabetes-susceptible genotype is strongly modified by both the microbial and the dietary environment. The immunopathogenic features associated with IDDM in this mouse have recently been reviewed (Kikutani and Makino, 1992). Because of the large numbers of publications reporting on research with the NOD mouse, particularly research on immunotherapy, this review cannot be comprehensive. Our purpose will be to provide an overview of the NOD mouse as a model for analysis of autoimmune diabetes, to identify those characteristics most closely associated with development of autoimmune disease, and to provide a basis for understanding why stringent control of the environment is essential for research in which this model is used.

DEVELOPMENT OF THE NOD STRAIN

The NOD and related inbred strains were developed at the Shionogi Research Laboratories in Aburahi, Japan, by Dr. S. Makino (Makino et al., 1980, 1985). A combination of inbreeding and selective breeding began in 1966 with progeny from an outbred JcI:ICR female mouse that exhibited cataracts and microphthalmia. This breeding program led to the development of one strain in which all mice develop cataracts (now designated CTS, for cataract Shionogi) and a second, control strain that is cata-ract-free (designated NCT). Both are now beyond 100 generations (Fl00) of brother x sister matings.

At F6, mice free of cataracts but exhibiting anomalously high fasting blood glucose, were separated from the cataract-prone line; breeders were selected on the basis of the high fasting blood glucose trait. At Fl3, mice exhibiting normal fasting blood glucose were separated from the hyperglycemic line as a potential control strain (Kikutani and Makino, 1992). In 1974, at F20, a female spontaneously developed overt IDDM. Paradoxically, this female was found not in the line with fasting hyperglycemia, in which diabetes development was expected, but instead in the line being inbred as a diabetes-free control line. The NOD/Shi strain originated from subsequent selective breeding of glycosuric mice from this control line. Based upon a recent report, the diabetes incidence in NOD/Shi mice beyond F20 in the specific-pathogen-free (SPF) source colony at Aburahi Laboratories has remained relatively constant, with a 70-80 percent incidence in females versus a 20 percent incidence in males (Kikutani and Makino, 1992). This gender dimorphism is controlled, in part, by gonadal sex steroids. Gonadectomy at 5 weeks of age markedly increases diabetes development in NOD/Shi males, while it depresses diabetes incidence in females (Makino et al., 1981).

The inbred strain that was developed from the subline with fasting hyperglycemia has been designated NON (nonobese nondiabetic). Because of its similarity to the NOD strain, NON mice are extremely useful for genetic analyses; they apparently share with NOD some, but not all, of the diabetogenic loci predisposing them to diabetes (Leiter, 1989). Although NON mice do not develop autoimmune diabetes, NON/Lt males at The Jackson Laboratory develop marked obesity by 20 weeks of age (NRC, 1989, pp. 103-4). Moreover, NON mice of both sexes are intolerant to glucose loading and develop severe glomerulosclerotic kidney lesions (Tochino et al., 1983). As NON/Lt mice age, immunoregulatory anomalies become apparent; for example, by 20 weeks of age, T-lymphocytopenia and functional T-cell anergy develop in NON/Lt splenocytes (Leiter et al., 1986). Thus, NON mice are not necessarily the best controls for establishing certain "normative" physiologic or immunologic baselines. The issue of potential controls to match with the NOD strain will be discussed later.

PATHOPHYSIOLOGY OF DIABETES IN NOD MICE

Prior to 1986, the availability of NOD mice was limited to a group in Japan called the NOD Mouse Study Group. A book reporting the results of these early studies has been published and contains a wealth of pathophysiologic information (Tarui et al., 1986).

Diabetes development in NOD mice is characterized by insulitis, which is a leukocytic infiltrate of the pancreatic islets. In the NOD/Lt colony at the Jackson Laboratory, a pervasive leukocytic infiltrate emanating from the pancreatic vasculature and secretory ducts is first observed at a time when the islets are free of lesions. Pancreatic islets are concentrated in the perivascular/periductular areas; consequently, large numbers of leukocytes aggregate at the periphery of islets (peri-insulitis). The aggregates usually start at one pole but eventually surround the entire islet perimeter. Widespread insulitis, entailing the erosion of b-cell mass as leukocytes penetrate into the islet core, develops between 5 and 7 weeks of age in females and several weeks later in males. Interestingly, a period of islet growth partially compensates for this early insulitis. NOD/Lt islets in mice between 5 and 12 weeks of age are quite large in comparison with those of the closely-related NON/Lt strain, despite the heavy leukocytic aggregations surrounding many of them.

Marked decreases in pancreatic insulin content are demonstrable in NOD/Lt females at around 12 weeks of age (Gaskins et al., 1992a) and several weeks later in males (E. Leiter, unpublished observation). Onset of diabetes is marked by the appearance of moderate glycosuria (1+ reading on Lilly Tes-TapeTM) and by a non-fasting plasma glucose higher than 250 mg/dl. Both glycosuria and hyperglycemia become progressively more severe over a 34 week period during which weight loss, polydipsia, and polyuria occur. Diabetic mice are hypo-insulinemic and hyperglucagonemic, a finding that correlates with the histologic profile of selective destruction of b, but not non-b, islet cells. Without insulin treatment, diabetic mice become severely hyperglycemic and ketonemic, but they do not become ketoacidotic (Harano et al., 1986).

In most specific-pathogen-free colonies, untreated diabetic mice will survive for 3-4 weeks after the first detection of glycosuria. Most investigators monitor NOD mice for development of glycosuria at weekly intervals beginning after 10 weeks of age. Weight loss and the appearance of polydipsia and polyuria indicate the onset of hyperglycemia. Documentation of increasing levels of glycosuria over 2 consecutive weeks, coupled with a serum or plasma glucose measurement in excess of 300 mg/dl, are acceptable measures for a diagnosis of IDDM. It is difficult in diabetic NOD mice to maintain serum glucose within a normal range by insulin treatment, although body weight can be maintained and lifespan prolonged (Ohneda et al., 1984). A morning and evening injection of a 1:1 mixture of regular and NPH insulin at a dose between 1 and 3 units per mouse, depending upon the level of glycosuria as measured using Tes-TapeTM, has been reported (Doi et al., 1990).

IMMUNOPATHOGENESIS

Although B lymphocytes and macrophages are present in the early insulitic infiltrates (Jarpe et al., 1991; Signore et al., 1989), T lymphocytes predominate (Miyazaki et al., 1985). Diabetogenesis is T-lymphocyte dependent and to a large extent T-cell mediated, with both CD4+ and CD8+ subsets required for the initiation of destructive insulitis (Christianson et al., 1993; Miller et al., 1988; Yagi et al., 1992). Unlike the diabetes-prone BBDP/ Wor rat, which is T-lymphopenic and exhibits strong natural killer cell activity, NOD/Lt mice exhibit elevated percentages of T lymphocytes in peripheral lymphoid organs (T-lymphoaccumulation), whereas NK cells are functionally defective (Serreze and Leiter, 1988). Table 1 presents a partial listing of some of the aberrant immu-nophenotypes described in the NOD mouse. NOD/Lt mice homozygous for the severe combined immune deficiency (scid) mutation fail to develop functional T and B lymphocytes and consequently, are insulitis- and diabe-tes-free (Christianson et al., 1993). Purified splenic CD4+, but not CD8+, T lymphocytes from diabetic NOD/Lt donors (already primed in vivo to the b-cell antigens released as a consequence of destructive insulitis) adoptively transfer insulitis and diabetes into unirradiated NOD/ Lt-scid/scid recipients (Christianson et al., 1993). However, purified splenic CD4+ T lymphocytes from prediabetic NOD/Lt mice are incapable of transferring disease in the absence of CD8+ T lymphocytes, confirming that CD8+ T lymphocytes are required for the initiation of b-cell destruction during the natural course of the disease in euthymic NOD mice (Christianson et al., 1993). Macrophage infiltration into the islets is required to recruit or activate diabetogenic T lymphocytes (Hutchings et al., 1990; Ihm and Yoon, 1990). A longitudinal flow cytometric analysis of islet-infiltrating leukocytes in NOD/Uf (detected at 5 weeks of age in females and 7 weeks of age in males) indicates that class II+, Ig- monocytes and CD8+ T lymphocytes are the first cell types to infiltrate pancreatic islets (Jarpe et al., 1991). The failure to detect CD4+ T lymphocytes in the earliest infiltrates by flow cytometry apparently reflects an activation-associated down-regulation of the CD4 molecule because CD4 transcripts are detected by polymerase chain reaction amplification of reverse transcriptase products (Dr. A. B. Peck, University of Florida, Department of Pathology, Gainesville, personal communication). A second influx of CD8+, as well as a major influx of CD4+ cells has been observed between 10 and 12 weeks of age, quickly followed by the development of overt diabetes (Jarpe et al., 1991). Of further interest is the finding that the cytokine profile of CD4+ T lymphocytes from the islets of 14-week-old females is that of a T-helper 2 cell (A. B. Peck, personal communication). Interestingly, pancreatic b cells from 3-week-old mice of both sexes are coated with autoantibody, suggesting that the initial lesion may entail antibody-dependent cell-mediated cytotoxicity. NOD b cells are distinguished at the ultrastructural level from non-b islet cells by the presence in the former of an aberrant intracisternal type C virus particle (Nakagawa et al., 1992), which is apparently encoded by an endogenous xenotropic (Xmv) proviral gene (Gaskins et al., 1992a). Since autoimmune-prone mouse strains commonly develop high titers of antibodies to retroviral gene products, it is conceivable that maternally-transmitted antibodies may "target" b cells in juvenile NOD mice for T-lymphocyte-mediated destruction. Adoptive transfer of autoantibodies from mother to offspring would explain the finding that diabetogenic T-effector cells will adoptively transfer disease into neonates whose endogenous B-lymphocyte functions have been suppressed by treatment with anti-m chain monoclonal antibody (Bendelac et al., 1988).

The spontaneously occurring autoimmune disease in NOD mice differs significantly from experimentally induced models of organ-specific autoimmunity. Although myelin basic protein-autoreactive T lymphocytes elicited in experimentally induced allergic encephalomyelitis (EAE) in susceptible strains of mice show very restricted T-cell receptor (TCR) Vb gene utilization, analysis of TCR Vb gene utilization by islet-infiltrating T lymphocytes has indicated an extensive polyclonal expression of the repertoire (Maeda et al., 1991; Waters et al., 1992). Similarly, the immune system of NOD mice responds to a plethora of different candidate b-cell antigens, including insulin, glutamic acid decarboxylase (formerly a 64 kilo-dalton [kd] autoantigen) and a 52 kd autoantigen, as well as to endogenous retroviral gene products (Gaskins et al., 1992a).

Although most analyses of diabetogenesis in NOD mice focus on T lymphocytes because of their role as disease effectors, functional defects in bone marrow-derived antigen-presenting cells (APCs), which are summarized in Table 1, very likely underlie the generation of b-cell-autoreactive T lymphocytes. F1 hybrids between NOD and other inbred strains are diabetes-resistant; however, diabetogenic T lymphocytes develop when those F1 hybrids are lethally-irradiated and reconstituted with NOD bone marrow (Serreze et al., 1988; Wicker et al., 1988). These defects have been attributed to APCs, such as macrophages and dendritic cells, that develop from myelocytic precursors in the marrow. APCs play an important role in shaping the T-lymphocyte repertoire by presenting self-antigens intrathymically, and they stimulate T-lymphocyte responses in the periphery by presenting processed peptides in association with major histocom-patibility complex (MHC) molecules to antigen-specific T lymphocytes. Two immunoregulatory defects that are probably central to the diabetes susceptibility of NOD/Lt mice have been ascribed to defective APC functions, including an inability to activate functional immunoregulatory T (suppressor) cells in the periphery, as measured in a syngeneic mixed lymphocyte reaction (SMLR) (Serreze and Leiter, 1988; Serreze et al., 1990), and an inability to block the development of b-cell-autoreactive T lymphocytes in the thymus (Serreze and Leiter, 1991). The genetic basis for the expression of these defects at the APC level entails a complex trans-interaction between diabetogenic MHC and non-MHC-linked genes (Leiter and Serreze, 1992).

It should be noted that aging NOD mice exhibit a spectrum of autoimmune pathologies distributed through a variety of organs (Leiter, 1990a). These multi-organ lesions are a reflection of the T-lymphoaccumulation peculiar to this strain. The thymus gland is very slow to involute in NOD/Lt mice when compared with NON/Lt mice, and NOD thymocytes do not proliferate in response to mitogen (Ziptis et al., 1991), potentially reflecting an underlying defect in normal intrathymic apoptosis. Morphologic anomalies in the NOD thymus have also been reported (Savino et al., 1991). As in other inbred strains with autoimmune susceptibilities, aging NOD mice develop a wide spectrum of neoplasms, the most common of which are lymphomas (Leiret, 1990a).

GENETICS

The susceptibility of NOD mice to type I autoimmune IDDM is under complex polygenic control, with environmental factors also exerting strong effects on gene pen-etrances. However, it is quite clear from genetic analyses (reviewed in Kikutani and Makino, 1992; Leiter, 1990a; and Leiter and Serreze, 1992) that the major component of this susceptibility is the unique MHC haplotype (H-2g7, on Chr 17). The MHC-encoded susceptibility entails both a lack of expression of I-E (homologous to DR in humans) and expression of a unique I-Ab locus (histidine at residue 56, serine at residue 57; homologous to "diabetogenic" HLA-DQb non-aspartic acid57-containing alleles) (Todd et al., 1988). In genetic outcross/ backcross analyses, heterozygous expression of the diabetogenic H-2g7 haplotype is permissive for insulitis development, but insulitis sufficiently widespread to produce the clinical phenotype of diabetes is rarely observed in segregants that are not homozygous for the diabetogenic class II MHC alleles. Thus, if only insulitis induction is considered, the haplotype functions in a codominant fashion with other genetic factors in the NOD strain background. However, when development of overt IDDM is considered, recessive components within this haplotype are clearly recognizable. An example of a recessive gene effect is the inability of the H-2g7 haplotype to express cell surface I-E molecules on APCs due to mutations in the Ea locus. NOD mice expressing a functional Ead transgene have been reported to be both insulitis- and diabetes-resistant when compared with standard (I-E null) NOD mice (Lund et al., 1990; Uehira et al., 1989).

Although immunogenetic analysis has concentrated on the diabetogenic contributions of the MHC class II region of the H-2g7 haplotype, current evidence suggests that the haplotype as a whole should be considered as contributing to susceptibility. The most compelling evidence comes from the congeneic transfer of the unique MHC haplotype of the related CTS/Shi strain onto the NOD/Shi genetic background. The MHC of CTS mice apparently contains the same class II alleles as NOD but has distinct class I loci, indicating that loci between these markers may differ as well. When this CTS haplotype (H-2 designation not yet assigned) was transferred onto the NOD inbred background and compared in the ho-mozygous state to segregants homozygous for the H-2g7 haplotype, a lower incidence of diabetes and insulitis was observed in the mice homozygous for the CTS MHC (Kikutani and Makino, 1992; Makino et al., 1991). The reduced diabetogenic potency of the CTS MHC thus provides strong support for the concept that, while the class II region is clearly important to disease development, other loci within the extended H-2g7 haplotype are also contributory. Intra-MHC regions both proximal and distal to the H-2g7 class Il region contain rare or unique alleles that may also contribute to diabetes susceptibility. Among these are a unique heat-shock protein 70 (Hsp70) allele (Gaskins et al., 1990), as well as rare alleles at Tap-1 and Tap-2 (for transporters associated with antigen processing and formerly designated Ham-1 and Ham-2) (Gaskins et al., 1992b) The products of these loci are members of a superfamily of ATP-dependent transport proteins, and they may function to transport processed antigenic peptide fragments generated in endosomal compartments into the lumen of the endoplasmic reticulum for association with MHC class I molecules (Monaco et al., 1990). Mutant mouse and human cell lines lacking the ability to form stable MHC class I peptide complexes all carry mutations that map to regions encoding Tap-1 or homologous genes (Monaco et al., 1990). Although defective Tap gene transcription in the spleens of NOD mice has recently been reported (Faustman et al., 1992), this has not been confirmed in cultured NOD/Lt peritoneal macrophages, in which IFNg-induced upregulation of both Tap-1 and Tap-2 mRNA is normal, as is upregulation of TAP1 content in NOD b cells (Gaskins et al., 1992b). However, the NOD Tap genes have not yet been sequenced, so the possibility remains that TAP functions in NOD macrophages may not be normal. Although the MHC class I H-2K and H-2D loci in the H-2g7 haplotype of NOD are common alleles (e.g., H-2Kd,, H-2Db), intra-cellular and cell surface levels of H-2Kd aberrantly decline in response to IFNg. This aberrant response is cell-specific since it is observed in NOD macrophages but not in b cells (Serreze et al., 1993). This cell-type-specific defect is apparently not due to defective interaction between Tap gene products and MHC class I molecules, because an H-2g7 -identical strain called NOR/Lt, which is related to NOD and is discussed below, exhibits normal MHC class I upregulation in response to IFNg(Serreze et al., 1993).

MHC-unlinked genes also control diabetes development and have been analyzed by outcrossing NOD with other inbred strains; the numbers and locations of these genes is wholly predicated upon the degree of relatedness of the strain chosen for outcross with NOD (Table 3). Analysis of MHC congenic stocks has shown that although the unique H-2g7 haplotype of the NOD strain represents a major component of this strain's genetic susceptibility to IDDM, the diabetogenic H-2g7 haplotype requires interaction with non-MHC linked susceptibility modifiers to mediate diabetogenesis (Leiret and Serreze, 1991; Todd et al., 1991).

Genetic segregation analyses are initiated by an out-cross between NOD and a diabetes-resistant strain, followed by either one backcross (BC1) to NOD or, less frequently, by an FI x F1 intercross to produce an F2 generation. In situations where the diabetes-resistance genes from the second parental strain are incompletely dominant (as usually is the case), the F2 cross will be more informative than a backcross to NOD because the strength of the protective locus can be assessed in the homozygous, as well as in the heterozygous state. The NOD-derived modifiers shown to segregate with disease susceptibility are provisionally designated Idd (Insulin-dependent diabetes) loci. The H-2g7 susceptibility (which encompasses at least two loci within the class II region) has been designated ldd-1, with ldd-1s denoting the susceptibility haplotype and Idd-1r denoting the resistance haplotype (Prochazka et al., 1987). Interval mapping strategies have been employed to define chromosomal locations of the non-MHC susceptibility loci segregating in outcrosses with the NON, C57BL/10J (B 10), and C57BL/ 6J strains. These analyses have shown the presence of genetic factors controlling diabetes susceptibility or resistance on at least eight chromosomes (Leiter and Serreze, 1992). These studies require segregation analysis in diabetic versus non-diabetic progeny using mapped DNA or phenotypic polymorphisms distinguishing the two parental strains. The aim is to type as many polymorphic loci as possible within a discrete region of a chromosome that have been identified by linkage analysis as segregating with diabetes susceptibility. Although phenotypic markers (e.g., polymorphic serum or tissue isoenzymes, immunophenotypic cell surface markers, or physiologic responses known to be under single gene control) are employed, the bulk of the genetic screening for diagnostic polymorphisms is at the DNA level (Prochazka et al., 1987; Todd et al., 1991). Identification and mapping of microsatellite repeat sequences scattered throughout the mouse genome, coupled with the development of the polymerase chain reaction (PCR), have revolutionized this type of genetic analysis (Dietrich et al., 1992; Love et al., 1990; Todd et al., 1991). NOD-derived genes showing the strongest statistical linkage association with the diabetic phenotype are assumed to flank an ldd locus. A linked marker locus that shows no recombination with the diabetic phenotype may be presumed to be a candidate for the Idd locus itself. In practice, only the H-2g7 complex exhibits such tight linkage with diabetes that it can be stated with certainty that this complex contains the ldd-1 susceptibility. This may reflect the fact that although diabetes in NOD mice is clearly under poly-genic control, H-2g7 exerts a stronger diabetogenic influence than many of the non-MHC modifiers. For example, when NOD/Lt is crossed with the related NON/Lt strain, there is no diabetes in the Fl generation. In contrast, in an outcross using an NON/Lt stock congeneic for H-2g7 , a low (6.5 percent) incidence of diabetes develops in F1 females, with a 14 percent incidence in F2 females (Leiter and Serreze, 1992). In a cross between NOD and B10 mice congeneic for H-2g7 , no F1 and less than one percent of F2 progeny develop diabetes, indicating the presence of a larger number of non-MHC genes segregating for susceptibility and resistance when an unrelated inbred strain is employed as the outcross partner (Todd et al., 1991). It should be noted that not all genes derived from a diabetes-resistant strain are disease-protective or disease-neutral. In an outcross between NOD and C57BL/ 10J mice congeneic for H-2g7, B10-derived genes on Chr 7 (ldd-7) and Chr 14 (Idd-8) have been found to be positively associated with diabetogenesis (Todd et al., 1991). Autoimmune lupus and nephritis are similarly accelerated when NZB mice are outcrossed with lupus-prone NZW mice (Babcock et al., 1989).

ENVIRONMENTAL REQUIREMENTS FOR DIABETOGENESIS

The NOD mouse has provided researchers with the most compelling evidence to date that environmental factors are important modulators of genetic susceptibility to IDDM. By the time they are 30 weeks of age, the incidence of diabetes in NOD females is usually 80 percent or higher, whereas in males, incidence is highly variable among colonies, ranging between 100 and 0 percent at different institutions. The environment accounts for a major component of this variation (Leiter, 1990b). Diabetes incidence in NOD males serves as a useful indicator of the presence of environmental factors affecting the penetrance of this strain's genetic susceptibility to IDDM. Transfer of NOD males from a conventional mouse room in Japan into germfree conditions increased the incidence of diabetes in males from 6 to 70 percent (Suzuki et al., 1987). Exposure of NOD mice to a variety of murine viruses (e.g., encephalomyocarditis virus, lymphocytic chorio-meningitis virus, and murine hepatitis virus) prevents diabetes development (Hermite et al., 1990; Oldstone, 1988; Wilberz et al., 1991). These infectious agents apparently protect by providing general immunostimulation, because treatment of prediabetic NOD mice with various types of exogenous immunomodulators, including complete Freund's adjuvant (Sadelain et al., 1990), cytokines (IL-1, TNFa, IL-2, 1L-4), and poly I:C, all circumvent the development of diabetes (Leiter, 1990b; Rapoport et al., 1992). Diabetogenic catalysts are also present in natural ingredient diets, which contain lipoidal moieties that are absent or present in low concentration in semi-purified diets (Coleman et al., 1990).

Certain peripheral immunoregulatory functions appear to be defective in NOD mice maintained in specific-pathogen-free (SPF) environments (see Table 1), as exemplified by defective T-suppressor-cell functions measured in vitro, as well as defects in the differentiation and maturation of APCs developing from bone marrow progenitors (Serreze et al., 1993). Defects in the degree of cytokine-elicited differentiation of APCs from bone marrow have been associated with inefficient presentation of self-antigens (Leiter and Serreze, 1992; Serreze et al., 1993), which may explain not only the defective tolerogenic functions of these cells but also the subnormal secretion of monokines by peripheral macrophages in response to lipopolysaccharide stimulation; subnormal secretion of IL-2 and IL-4 by splenic and thymic T lymphocytes, respectively; depressed NK cell activity; depressed thy-mocyte responses to mitogenic stimulation; and accumulation of T lymphocytes. Presumably, immunomodulatory effects mediated by environmental components serve to regulate some of these defective APC functions, resulting either in more normal thytalc elimination of autoreactive T lymphocytes, more potent activation of immunoregulatory T lymphocytes in the periphery, or both.

Macrophages and neutrophils represent a mammal's first line of defense against infectious agents and irritants. The knowledge that the genetic defects that predispose NOD mice to IDDM are reflected, in part, as maturational or functional defects in macrophages provides a basis for understanding why exposing prediabetic NOD mice to any of a myriad of environmental pathogens and immunoregulatory cytokines circumvents diabetes (Leiter, 1990b). Many of the therapies that do not entail direct immunosuppression of T cells would be expected to activate macrophage function and stimulate antigen processing and presentation. Even when therapeutic intervention is achieved by a semi-synthetic diet rather than by exposure to a virus or treatment with a cytokine, protection is associated with increased rather than decreased immune responsiveness. For example, when the semi-purified diet AIN-76 is substituted for a natural-ingredient diet (such as Old Guilford 96W, the diet fed to NOD/Lt mice at The Jackson Laboratory), the incidence of diabetes in NOD/Lt females is reduced and the age of onset is delayed (Coleman et al., 1990). This protection is associated with development of high titers of anti-BSA antibodies (D. V. Serreze and E. H. Leiter, The Jackson Laboratory, Bar Harbor, Maine, unpublished data), presumably in response to contaminants in the casein used in the diet. Anti-BSA antibodies found in NOD mice have recently been cited to support the speculation that cow's milk protein is a diabetogenic catalyst for development of IDDM in genetically-predisposed Caucasian children (Karjalainen et al., 1992). The finding in NOD mice that high titers of anti-BSA antibodies do not necessarily correlate with early onset diabetes is not consistent with the hypothesis that BSA or another product of milk in the rodent diet is diabetogenic. Interestingly, digestive hydrolysis of b-casein generates immunomodulatory peptides that stimulate macrophage phagocytosis (Migliore-Samour and Jolles, 1988). The protection afforded by PregestimilTM, a hypoallergenic infant formula, is even more potent than that provided by diet AIN-76 (Coleman et al., 1990). Although PregestimilTM contains casein hydrolysate rather than intact milk protein, small peptides and amino acids are present and may serve as macrophage activators. It is conceivable that either dietary or microbial components are acting as "superantigens" to regulate the T-cell repertoire. However, it now appears that the T-cell response to NOD [3-cell antigens is quite polyclonal. Thus, environmental factors probably decrease the penetrance of diabetes susceptibility genes by upregulating APC functions. More normal APC functions, both intrathymically and in the periphery, should block development of autoreactive, polyclonal cytotoxic T lymphocytes.

Since many investigators who study the NOD model want to perform therapeutic intervention studies that often entail the use of monoclonal antibodies and other biologics (Shizuru et al., 1988; Taki et al., 1991), it is prudent that all biologic reagents to be tested in NOD mice be prescreened for the presence of pathogenic agents. The acute sensitivity of NOD mice to environmental modulators places a special burden on colony managers to strive for the maximum possible degree of environmental control. Ideally, the breeding facility should be separate from rooms in which experiments are performed.

APPROPRIATE CONTROLS FOR NOD MICE

The question of the appropriate control to use for experimentation with NOD mice often arises. Table 5 lists some of the inbred and congenic strains without insulitis and diabetes that have been used to establish experimental "baseline" parameters and are potential controls for the NOD strain. In some of the initial immunologic studies of the NOD mouse in Japan, the outbred ICR strain was used as the standard for comparison, and it was erroneously concluded that NOD mice were T-lymphocytopenic (Kataoka et al., 1983). Considerable size differences distinguish ICR from NOD mice. At the Jackson Laboratory, the SWR/J or the SWR/BmJ substrain has provided a suitable, inbred standard for comparison. SWR mice share with ICR (and NOD) mice a common derivation from the "Swiss" mice brought to the United States by Dr. Clara Lynch in the 1920s. SWR/J mice are approximately the same size as NOD/Lt mice, and they exhibit normal immunoregulatory responses for most parameters that are aberrant in NOD/Lt mice. The H-2q haplotype of SWR, like the H-2g7 haplotype of NOD, does not produce cell surface I-E molecules. The NON/ Lt is not considered a normative control strain for immunologic function because, like CTS mice, NON/Lt mice develop an age-related decline in peripheral T-cell numbers, and like NOD/Lt mice, their SMLR is depressed. Thus, as positive controls for cytokine production assays or SMLR analyses, SWR/J mice are a reasonable choice. The recently developed NOR/Lt incipient congenic stock (presently at F20 and available for distribution from The Jackson Laboratory) shares the diabetogenic H-2g7 with NOD, but carries the genome from the C57BL/KsJ strain on portions of at least five chromosomes (Chr 2, 4, 7, 11, and 12). NOR/Lt mice develop very little insulitis and no diabetes, even after cyclophosphamide treatment (Prochazka, 1992b) In NOD colonies in which the pen-etrance of diabetogenic genes is suppressed (e.g., in colonies with a very low incidence of spontaneous diabetes in males), cyclophosphamide treatment can be used to rapidly elicit the diabetic phenotype (Harada and Makino, 1984). The NOR/Lt stock exhibits a more robust SMLR than NOD/Lt mice, and as discussed above, should provide an excellent control for comparing the nature of diabetogenic interactions between non-MHC-encoded trans-active factors and the diabetogenic H-2g7 MHC haplotype. NOD mice that are rendered diabetes-free because they express an MHC transgene (Ead) or are congeneic for a diabetes-resistant H-2 haplotype are of obvious value for analyzing the role of the unique H-2g7 haplotype in selection and peripheral regulation of a normal T-cell repertoire. Diabetes-resistant NOD congenic stocks carrying chromosomal regions from B10 that are associated with non-MHC-linked resistance are currently under development by Dr. L. Wicker (Merck Research Laboratories, Rahway, New Jersey). These stocks will be essential for associating specific immunodeficiencies in NOD mice with specific ldd genes. To identify and isolate specific Idd genes, one's choice of an outbred partner strain is limited only by the degree of genetic complexity that the investigator is willing to analyze (see Table 3). Although inbred strains derived from wild mice, such as Mus spretus, guarantee widespread genetic polymorphisms at most loci that have been typed, the polygenetics of disease susceptibility are such that very few mice with IDDM will be recovered at the first backcross. In contrast, reasonable numbers of diabetic probands will be obtained when outcrosses with related strains such as NON/Lt or SWR/J are employed, because these related strains share with NOD a certain number of diabetes-permissive genes not shared in unrelated strains, such as B10. However, the disadvantage is that related strains provide reduced numbers of genetic polymorphisms available for segregation analyses.

Congenic stocks of NOD mice rendered genetically T-cell deficient by introduction of the nu (Yagi et al., 1992) or the scid (Christianson et al., 1993) mutations have recently been described. These mice are obviously useful in establishing an endocrinologic or physiologic baseline in the absence of insulitis and impairment of pancreatic b-cell functions. Further, they are useful for analyzing the pathogenic or diabetes-suppressive competence of various cloned T-cell lines established from the NOD mouse (Bradley et al., 1990; Pankewycz et al., 1991; Reich et al., 1989).

A PROBLEM FOR THE FUTURE: GENETIC DIVERGENCE OF NOD SUBSTRAINS

As more and more institutions maintain their own inbred colonies of NOD mice, substrain divergence can be expected and has already occurred to some extent. During the development of the NOD/Shi inbred strain, and apparently before all segregating loci were fixed to ho-mozygosity, a satellite breeding colony was established in Kyoto, Japan. From this colony, breeders were sent to the Walter and Eliza Hall Institute in Australia in 1984, where a new substrain, NOD/Wehi, was developed. The NOD/Lt mice at the Jackson Laboratory were also derived from a breeding colony held by Dr. M. Hattori, Kyoto, who brought the mice to the United States in 1984.

The incidence of spontaneous diabetes in NOD/Wehi mice is significantly lower in both sexes than in NOD/Lt mice imported from the Jackson Laboratory to Australia in 1987 and maintained in the same environment (Baxter et al., 1989, 1991), indicating that there has been significant genetic divergence between the NOD/Lt and NOD/ Wehi substrains. What particularly distinguishes the NOD/Lt substrain from both the NOD/Shi and NOD/Wehi substrains is the higher incidence of diabetes in NOD/Lt males (50-70 percent by 40 weeks of age) when compared with NOD/Shi and NOD/Wehi males (20 percent or less). Because of the gnotobiotic study done in Japan (Suzuki et al., 1987) that showed an environmental influence on the penetrance of diabetes in NOD/Shi males, it has been assumed that the difference between incidences of diabetes in male NOD/Shi and NOD/Lt mice is probably environmentally-mediated. Surprisingly, however, a direct comparison of diabetes incidence in both substrains maintained in a common environment by Dr. L. Herberg (Diabetes Research Institute, Dusseldorf, Germany) indicates that substrain divergence is occurring. By 28 weeks of age, 78 percent of male NOD/LtHI have diabetes (21 out of 27), compared with 14 percent (8 out of 22) of NOD/Shill1 males obtained from CLEA Japan, Inc. Similarly, 96 percent (25 out of 26) of NOD/LtH1 females become diabetic compared with 65 percent (13 out of 20) NOD/ Shill1 females obtained from CLEA Japan, Inc. (Dr. L. Herberg, personal communication). Although castration can increase diabetes incidence in NOD/Shi males, which have a low spontaneous incidence of IDDM (Makino et al., 1981), adolescent castration does not further elevate the relatively high spontaneous incidence observed in NOD/Lt mice (E. H. Leiter, unpublished). Thus, substrain as well as environmental differences must be considered when approaching the scientific literature on NOD mice.

Given the likelihood that inbred NOD colonies worldwide are accumulating independent genetic mutations that will eventually lead to substrain divergence, it is essential that inbred colonies be identified by a laboratory code. For example "HI" is the laboratory code assigned to Dr. L. Herberg. These assignments are available upon request from Dr. Dorothy Greenhouse, Institute of Laboratory Animal Resources, National Research Council, 2101 Constitution Avenue, NW, Washington, DC 20418. Colony managers of inbred NOD mice and related strains are kindly requested to complete a colony registration/data form supplied by the World NOD Registry. Forms are available from Dr. Paolo Pozzilli, Department of Diabetes and Metabolism, St. Bartholomew's Hospital, West Smithfield, London EC1A 7BE, UK. Comparisons of worldwide diabetes incidence in the various NOD colonies will provide important insights into the genetic and environmental factors that, in aggregate, determine the development of diabetes.

ACKNOWLEDGEMENTS

This review was supported by grants from NIH DK 27722 and 36175, The Juvenile Diabetes Foundation International, and the Diabetes Research and Education Foundation. I would like to thank Dr. Lieselotte Herberg for permission to cite her unpublished substrain comparative incidence data.

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TABLE 1 Aberrant Immunophenotypes in NOD Mice: A Partial Listing

Bone Marrow


Macrophages/APC
T Lymphocytes
NK Cells



TABLE 2 Spectrum of Aging-associated Pathologies Present in NOD/Lt Mice in Addition to Insulitis

Tumors
Lymphomas*
Lymphosarcomas
Osteosarcoma/osteochondrosarcoma
Myoepitheliocarcinoma
Rhabdomyosarcoma
Mammary carcinoma
Heparoma

Infiltrates
Colitis
Sialiris
Harderian/Lacrimal adenitis
Myositis
Neuritis/meningitis
Thyroiditis
Osteomyelitis
Nephritis

*80-100% incidence of thymic lymphomas in NOD-scid/scid mice.



TABLE 3 The Number of Genetic Factors Segregating for Diabetes Susceptibility Varies with the Partner Strain Used in Outcross with NOD

Original outcross# Diabetic/total (both sexes)% DiabeticMinimum estimate of # of chromosomes carrying recessive susceptibility genes
NOD x NON19/2009.53
NOD x BKs1/1150.9>5
NOD x B65/3831.3>5
NOD x SWR15/112 (females)13.03



TABLE 4 Chromosomal Location of Idd Susceptibility Modifiers (designations are provisional)

Locus Symbol (provisional)Chromosome/strongest linkage marker or intervalOutcross in which linkage was establishedComments regarding resistance modifiers from outcross partner strain
ldd-117/AIb , Eaall MHC-disparate outcrosseswhole haplotype may contribute
Idd-2 9/Thy-1NON/Lt(BC l )
NON.NOD-H-2g7 (F2)
B 10.NOD-H-2g7 (BC 1)		incompletely dominant, potential timing gene
ldd-33/lL-2 in NON D3Ndsl- Tshb in B10NON.NOD-H-2g7 (F2)
BIO.NOD-H-2gz (BCI)
C57BL/6J*		stronger than ldd-2, incompletely dominant, probably at least 2 distinct resistance loci contributed by B10
Idd-411/AcrbB 10.NOD-H-2g7 (BC 1)
C57BL/6J*		incompletely dominant,
potential timing gene
ldd-51/1Llrl -Lsh-Bcl-2B 10.NOD-H-2,~7 (BC1)incompletely dominant
ldd-66/Kras-2C57BL/6J*incompletely dominant
ldd-77/CkmmB lO.NOD-H-2g7 (BC 1)**B 10 locus contributes to susceptibility
ldd-814/PlauB 10.NOD-H-2g7 (BC 1)**B 10 locus contributes to susceptibility

*Personal communication, Dr. Edward Wakeland, University of Florida, Gainesville
**Todd et al., 1991; Personal communication, Dr. John Todd, Oxford University



TABLE 5 Potential Diabetes-free Control Strains for NOD Mice

StrainAdvantageDisadvantageBest UseReference
NON/LtClosely related to NOD
Diabetes resistant MHC		Develops obesity
Impaired glucose tolerance
Immunodeficiencies
Difficult to breed		Genetic analysis of hid genes
Potential model for type II diabetes		Leiter et al., 1986
CTS/ShiJosClosely related to NOD
Same MHC class II genes
Different class I genes		Early developing T-lymphocytopenia
Unavailable		Genetic analysis of the contributory role of MHC class II as well as other H-2 alleles in the ldd-I complexIkegami et al., 1988; Kikutani and Makino, 1992
ILl/JicJosClosely related to NOD
MHC-identical		UnavailableGenetic analysis of non-MHC-linked ldd genesHattori et al., 1990
ICR (available as CD-I)Progenitor stock for NOD and related strainsRandomly bredAnalysis of population frequency of rare genetic polymorphisms present in NODlkegami et al., 1990
SWR/JSwiss-derived like ICR and NOD, but inbred and without immunodeficiencies
Available		Genetically very different from NOD, including MHCControl for immune functions that are aberrant in NODSerreze and Leiter, 1988
NOR/LtNOD-derived recombinant congenic stock
Same MHC, differs at relatively few non-MHC loci		Not yet at F20
Exhibits some but not all of NOD's immune dysfunctions		Analysis to establish which ldd genes control aberrant immunophenotypes essential to pathogenesisProchazka et al., 1992a
NOD-Eota transgenicsClosest genetic match to diabetes-developing NOD miceNot widely available-- stock
being developed at The Jackson Laboratory		Analysis of T-cell repertoire development
Presentation of ,B-cell antigens		Uehira et al., 1989; Lund et al., 1990
NOD-NON-H-2.hlDiabetes-resistant MHC from NON/Lt
Available		Exhibits some but not all of NOD's immune dysfunctionsLeiter and Serreze, 1991
Serreze and Leiter, 1991
NON-NOD-H-Diabetogenic MHC from NOD/LtExhibits some but not all of NOD's immune dysfunctionsAll MHC congenic stocks are extremely useful in dissecting the role of MHC versus non-MHC genes in producing aberrant immunophenotypesLeiter and Serreze, 1991;
Serreze and Leiter, 1991
NOD-BIO-H-2bDiabetes-resistant MHC from C57BL/IOJ
Available		Exhibits some but not all of NOD's immune dysfunctionsWicker et al., 1992; Todd et al., 1991
NOD-scidNo endogeneous T- or B-lymphocyte functionsDevelops high incidence of thymoma with ageDelineation of the role of T-cell subsets and autoantibodiesProchazka et al., 1992b; Christianson et al., 1992






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