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ILAR Journal V35(2) 1993 [FORMERLY ILAR NEWS]
Models of Type I Diabetes - Part Two

Transgenic Models of Insulin-Dependent Diabetes Mellitus
Jun-ichi Miyazaki and Fumi Tashiro

Jun-ichi Miyazaki, M.D., Ph.D., is a professor and Fumi Tashiro is an assistant professor in the Department of Disease-related Gene Regulation Research (Sandoz) at the University of Tokyo, Faculty of Medicine, in Tokyo, Japan.

INTRODUCTION

Transgenic mice carry a foreign gene that usually is introduced by direct microinjection into fertilized mouse eggs (Hogan et al., 1986). This technique allows us to study the effects of a single gene during the development and growth of an animal, which cannot be done in cell-culture systems. It has been used to study complex biological processes, such as developmental gene regulation, the role of oncogenes in tumorigenesis, and the function of the immune system (Hanahan, 1989). Thus, transgenic technology provides a powerful tool to explore the mechanisms of human genetic disorders and autoimmune diseases using animal models. Recently, transgenic mouse technology has been used to study type I or insulin-dependent diabetes mellitus (IDDM).

The literature on the pathogenesis of IDDM in mice and rats suggests that there is an autoimmune response directed against insulin-secreting pancreatic b cells (see reviews by Eisenbarth, 1986; Guberski, 1993; Leiter, 1993). Overt diabetes develops during the final stages of the disease. Anti-islet T cells appear to have a dominant role in the progression of the disease (see Guberski, 1993; Leiter, 1993). This disease is strongly correlated with some types of HLA molecules (Todd et al., 1987, 1989). Other susceptibility genes may also be present in humans and in mice (Todd and Bain, 1992). Some environmental factors are believed to trigger the disease, including b-cell toxic agents and b-cell tropic viruses (see Guberski, 1993; Leiter, 1993).

A number of transgenic mice have been produced to investigate the exact sequence of events in IDDM. One line was designed to examine the effects of introducing candidate disease-susceptibility genes into IDDM mouse models, and another to establish mouse models for IDDM that reproduce at least part of the autoimmune process (Table 1). These transgenic mice have provided valuable information on molecular mechanisms of autoimmunity against b cells, roles of major histocompatibility complex (MHC) antigen expression on b cells, and disease susceptibility genes. This review will discuss the current studies.

TRANSGENIC MOUSE MODELS FOR IDDM

The first transgenic mouse model of IDDM was produced by introducing into one-cell mouse embryos a hybrid gene combining regulatory sequences of the rat insulin II gene with simian virus 40 (SV40) large-T antigen protein coding information (Adams et al., 1987). In the transgenic lineages, large-T antigen was expressed exclusively in the b cells of the islets of Langerhans. In some lineages, transgene expression began before birth, and the progeny were naturally self-tolerant to the T antigen. However, in other lineages, transgene expression was delayed until the age of approximately 2-3 months. Progeny of those lineages failed to become self-tolerant to T antigen; they gradually developed an autoimmune response against b cells and insulitis. This b-cell response was shown to correlate with the MHC haplo-type of the mice, although diabetes did not develop in any of the strain backgrounds they tested (Skowronski et al., 1990). This study suggests that b-cell dysfunction damages b-cells and leads to exposure of b-cell-specific antigens to the immune system, which triggers anti-b-cell autoimmunity.

To investigate the potential association between virus and IDDM, Roman et al. (1990) generated three lines of transgenic mice (called RIP-HA) that express the hemagglutinin (HA) of a strain of influenza virus specifically on their b cells. All three expressed HA in the islets by day 12 of embryonic life; therefore, these mice should have been immunologically tolerant to HA. However, they developed insulitis, accompanied by a humoral response against b-cell-surface antigens, including HA. Furthermore, they exhibited hyperglycemia, although at a low frequency (approximately 13-27 percent). Interestingly, the incidence of hyperglycemia is affected by the haplotype of the MHC locus, reminiscent of the heritable susceptibility of humans to IDDM (see Analysis of IDDM Susceptibility Genes). RIP-HA mice may provide a useful system in which to study cellular interactions involved with the induction of self-toler-ance and autoimmunity.

Ohashi et al. (1991) have produced transgenic mice (called GP mice) that express the glycoprotein (GP) of lymphocytic choriomeningitis virus (LCMV) and have bred them with transgenic mice whose peripheral T cells predominantly express the T-cell receptor (TCR) derived from an anti-GP cytotoxic T-cell line. In the resulting double transgenic mice (called TCR-GP mice), most T cells express GP-specific TCR transgenes in the periphery, although their b cells express GP on the cell surface. Therefore, these T cells seem to be positively selected in the thymus. They do not exhibit an immune attack on their GP-expressing b cells but on macrophages infected with LCMV in vitro. GP transgenic mice develop diabetes approximately 9-11 days after infection with LCMV and die in 15-20 days. The onset of diabetes is accelerated in TCR-GP mice (3-4 days after LCMV infection). The islets of Langerhans in GP mice were infiltrated with cytotoxic T lymphocytes (CTLs) and with CD4⁺ T cells. Ohashi et al. (1991) suggest that self-reactive CTLs may remain functionally unresponsive due to a lack of appropriate T-cell activation, which may require CD4⁺ T cells. Oldstone et al. (1991) have reported similar observations.

TRANSGENIC MICE EXPRESSING CYTOKINE

The central role of CD4⁺ T cells in the activation of anti-islet autoimmunity has also been suggested from studies of NOD mice and diabetes-prone and diabetes-resistant rats (Guberski, 1993; Leiter, 1993). However, it is not clear whether b cells are damaged by CD4⁺ T cells, CD4+-activated CD8⁺ T cells, or cytokines (such as interferon, tumor necrosis factor, interleukin-1, and interleukin-2) secreted by CD4⁺ or other inflammatory cells. To address this question, transgenic mice were produced by microinjection with a hybrid gene comprised of the human insulin gene promoter and the mouse interferon-g (IFN_g) gene (Sarvetnick et al., 1988). Some of those transgenic mice exhibited inflammatory destruction of the islets and overt diabetes. However, it is still unclear whether islet destruction was caused by infiltrating lymphocytes or by deleterious effects of IFN_g. Further analysis showed that engrafted MHC-compatible islets are destroyed in these transgenic mice and that lymphocytes from the transgenic mice are cytotoxic to normal islets in vitro, which indicates that the pancreatic expression of IFN~ can result in a loss of tolerance to normal islets (Sarvetnick et al., 1990).

TRANSGENIC MICE EXPRESSING MHC ANTIGENS

Enhanced expression of MHC class I molecules and ectopic expression of class II molecules have been observed on [3 cells with insulitis in humans (Bottazzo et al., 1985) and in animal models (see Guberski, 1993; Leiter, 1993). Such aberrant expression of MHC molecules might be induced by local cytokine production caused by viral infection and might enable the b-cell to function as an antigen-presenting cell, leading to autoimmunity. To examine this possibility, several lines of transgenic mice were produced in which class I H-2K^b (Allison et al., 1988), class II I-E^b (Lo et al., 1988), and class II I-A^d (Sarvetnick et al., 1988) proteins were expressed at high levels exclusively on b cells of MHC-compatible mice. All of these transgenic mice developed early-onset diabetes without evidence of lymphocytic infiltration into the islets. Clonal anergy could be shown in these mice expressing extrathymic antigens (Markmann et al., 1988). It was also reported that overexpression of b₂-micro-globulin in transgenic mouse b cells causes hyperglycemia (Allison et al., 1991). Similarly, transgenic mice with an elevated b-cell expression of calmodulin, which is implicated as a regulator of insulin secretion, (Epstein et al., 1989) and those expressing an activated form of Ha-ras (Efrat et al., 1990) exhibit b-cell destruction and diabetes. These results suggest that various transgene molecules that are over-expressed in b cells can cause islet dysfunction, which leads to diabetes. Consistent with this, Böhme et al. (1989) showed that expression of I-A^k cDNA on islet cells at levels comparable with those on resting B lymphocytes does not perturb the function of b cells. It is interesting to note that these transgenic mice were not tolerant to the I-A^k antigen but did not develop diabetes. Therefore, over-expression of MHC class I molecules or aberrant expression of class II molecules does not necessarily result in an autoimmune reaction.

ANALYSIS OF IDDM SUSCEPTIBILITY GENES

The NOD (non-obese diabetic) mouse, which was established from ICR mice (Makino et al., 1980), is a good model of IDDM. Typically, NOD mice develop insulitis at approximately 4 weeks, and by 20 weeks more than 80 percent of the mice have developed insulitis (Figure 1) (Makino et al., 1980). The development of insulitis is followed by the complete destruction of islets and leads to overt diabetes. However, the incidence of diabetes is at most 80 percent in females and 20 percent in males (Makino et al., 1985). Many immunological studies have demonstrated that an autoimmune mechanism is involved in the generation of insulitis (Bendelac et al., 1987; Miller et al., 1988). In addition, breeding studies between NOD and other strains indicate that two or three genetic loci contribute to disease susceptibility (Hattori et al., 1986; Makino et al., 1985; Prochazka et al., 1987; Todd et al., 1991), one of which is linked to the MHC on chromosome 17 (Hattori et al., 1986; Prochazka et al., 1987). There are two characteristic features of the MHC of NOD mice. First, it does not express I-E molecules because of a deletion in the promoter region and the first exon of the E_a gene (Hattori et al., 1986). This defect is not unique to NOD mice, but is shared by other mice of H-2^b and H-2^s haplotypes. Second, the I-A molecule of the NOD mouse is unique. Acha-Orbea and McDevitt (1987) demonstrated that the 3' half of the I-A_b chain, including the second external domain, the transmembrane domain, and the intracellular domain, is identical to that of the I-Al3 chain in mice of the H-2^d haplotype. The first external domain carries several amino acid changes and deletions, but most of these differences are shared by at least one other haplotype (Acha-Orbea and McDevitt, 1987). However, one region has five consecutive nucleotide changes that are unique to the NOD mouse and cause substitutions in the amino acids at position 56 (histidine replaces proline) and position 57 (serine replaces aspartic acid) (Acha-Orbea and McDevitt, 1987). In humans, Todd et al. (1987) have demonstrated that HLA-DQ_b alleles in which alanine, valine, or serine are substituted for aspartic acid at position 57 are positively associated with IDDM in Caucasians. These and other studies suggest that the expression of abnormal class II molecules might be involved in the development of autoimmune insulitis (Table 2) (Lund et al., 1990; Miyazaki et al., 1990a, Slattery et al., 1990; Uno et al. 1991).

I-E TRANSGENIC NOD MOUSE

Nishimoto et al. (1987) have demonstrated by backcr0ssing C57BL/6 transgenic mice (B6-Ea^d), which express I-E (Yamamura et al., 1985), with NOD mice, which do not, that the expression of I-E molecules in NOD mice can prevent the development of autoimmune insulitis. Although this is an extremely intriguing finding, it is difficult to exclude the possible involvement of other genes adjacent to the Ea^d transgene from B6-Ea^d mice. Therefore, Uehira et al. (1989) generated NOD mice that carry the Ea^d transgene and confirmed that I-E expression completely prevents the development of insulitis (Figure 1) and cyclophosphamide-induced diabetes (Uno et al., 1991 ). Lund et al. (1990) obtained similar results. The mechanism by which I-E molecules protect NOD mice from diabetes is not known, although some speculate that this protection is mediated through the deletion of specific Vb families of T-cell receptors (Lund et al., 1990).

I-A TRANSGENIC NOD MICE

Breeding studies between NOD and C57BL/6 mice, both of which lack I-E expression, suggest the presence of an MHC-linked IDDM susceptibility locus other than I-E. To investigate whether the unique I-A molecule of NOD mice (I-A^g7)is related to IDDM susceptibility, several lines of transgenic NOD mice were produced with the Aa^k gene and the Ab^k gene (Miyazaki et al., 1990a; Slattery et al., 1990). As summarized in Table 2, when either Aa^k or Ab^k were expressed, there was no effect on insulitis, but when Aa^kAb^k was expressed (i.e., I-A^k), the incidence of insulitis was decreased to one-third that in normal NOD mice. Further analysis showed that l-A^k expression almost completely prevents diabetes (F. Tashiro, Department of Disease-related Gene Regulation Research (Sandoz), University of Tokyo, Faculty of Medicine, Tokyo, Japan, unpublished).

Using site-directed mutagenesis, Miyazaki et al. (1990a) have shown that the protective effect of transgenic I-A^k molecules in which aspartic acid was replaced by serine at position 57 is enhanced (Table 2). Lurid et al. (1990) took a complementary approach, making transgenic NOD mice that harbored a mutated Ab^g7 gene in which codon 56 is changed from histidine to proline. Interestingly, this transgene also conferred protection against diabetes.

These experiments show that the amino acid sequences in the vicinity of position 57 are related to the diabetogenic propensity of an I-A molecule. A recent study of diabetes in human populations suggests that disease susceptibility is correlated with the presence of DQ molecules formed by particular combinations of a-chains with b-chains (Todd et al., 1989). This conclusion may be supported by Miyazaki et al. (1990a), who showed that the I-A^kb chain protects NOD mice against insulitis only when it associates with the I-A^ka chain, but not when it associates with the endogenous I-A^da chain, as shown in the Ab^k single transgenic NOD mouse (Table 2). These transgenic studies revealed that the roles of MHC class II molecules in the pathogenesis of autoimmune insulitis in NOD mice and IDDM patients closely resemble each other. Recently, it has been shown that transgenic expression of class I-L^d molecules also decrease the incidence of insulitis in NOD mice, suggesting that MHC class I molecules are also involved in the development of insulitis in NOD mice (Miyazaki et al., 1992).

FUTURE PERSPECTIVE

Various transgenic mouse models for IDDM have been produced. Although none of these mice seem to be an exact model of the autoimmune process in humans or in NOD mice, each has provided important information about how the mechanisms of b-cell tolerance are maintained or lost. Studies using transgenic NOD mice clearly demonstrate that MHC class II genes participate in conferring susceptibility or resistance to IDDM. The fine structure or amino acid residues of the class II Ab^g7 chain, which is directly involved in the development of insulitis, will further be identified by introducing mutated Ab genes into NOD mice. The transgenic technique has also been applied to establish pancreatic b-cell lines (Efrat et al., 1988; Hamaguchi et al., 1991; Miyazaki et al., 1990b). These cell lines will make it easier to study and identify autoantigen(s) that trigger IDDM. Techniques developed to target genes via homologous recombination in the embryonic stem cells enable us to directly assess the role of a gene in the disease process (Capecchi, 1989). This technique, together with traditional transgenic techniques, will further be applied to address problems in the pathogenesis of IDDM, including identification of the genes conferring disease susceptibility and the authentic auto-antigen(s).

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TABLE 1 IDDM-related transgenic mice

Transgene producta	Insulitis	Diabetes	Age at onset	References
SV40h large T antigen	+	-		Adams et al., 1987
Influenza virus hemagglutinin	+	+	4-9 weeks	Roman et al., 1990
LCMVc glycoprotein	+	+	Inducible	Ohashi et al., 1991; Oldstone et al., 1991
Mouse IFN-g	+	+	6-10 weeks	Sarvetnick et al., 1988, 1990
MHC class I H-2Kb	-	+	2-3 weeks	Allison et al., 1988
MHC class II I-Eb	-	+	3-5 weeks	Lo et al., 1988
MHC class II I-Ad	-	+	-8 weeks	Sarvetnick et al., 1988
MHC class I1 I-Ak	-	-		Böhme et al., 1989
b2-microglobulin	-	+	-25 days	Allison et al., 1991
Chicken calmodulin	-	+	-3 days	Epstein et al., 1989
Human Ha-ras	-	+	-20 weeks	Efrat et al., 1990

^a promoters used to drive the transgenes were either that of the rat insulin II gene or that of the human insulin gene.
^b simian virus 40
^c lymphocytic choriomeningitis virus

TABLE 2 Effects of MHC transgene expression on the incidence of insulitis in NOD mice^a

Transgene	lnsulitisb	(%)	Diabetes
-	+	(80~90)	+
Ead	-	(0)	-
Aak	®	(80~90)	+
Abk	®	(80~90)	+
Aak Abk	¯	(20~30)	-
Aak Abk(Asp57®Ser)	¯	(10~20)	-
Abg7 (His56®Pro)	¯		-
Ld	¯

® unaffected
¯ decreased incidence

Based on Miyazaki et al., 1990; Slattery et al., 1990; Lund et al., 1990: Uno et al., 1991; Miyazaki et al., 1992.

FIGURE 1 Insulitis was observed in the pancreas of the NOD mouse. Pancrease from an NOD mouse (bottom) and an I-E expressing transgenic NOD mouse (NOD(Ea^d) (top) at 19 weeks of age was sectioned and stained with hematosylin and eosin. Massive lymphocytic infiltration into the islet (insulitis) was observed in the NOD mouse but not in the transgenic mouse.