- Email: [email protected]
Transgenic mouse as a tool for the study of autoimmune disease: Insulin-dependent diabetes mellitus
0192-0561/92 $5.00 + .00 Pergamon Press plc. International Society for lmmunopharmacology.
Int. J, lmmunopharmac., Vol. 14, No. 3, pp. 451-455, 1992. Printed in Great Britain.
T R A N S G E N I C M O U S E AS A TOOL FOR THE S T U D Y OF A U T O I M M U N E DISEASE: I N S U L I N - D E P E N D E N T D I A B E T E S MELLITUS KEN-ICHI YAMAMURA,* TOHRU MIYAZAKI, MASASHI UNO a n d JUN-ICHI MIYAZAKI + Institute for Medical Genetics, Kumamoto University Medical School, Kuhonji, Kumamoto 862, Japan
Abstract - - Transgenic mice have been used for analyses of cis-acting elements which are involved in the tissue-specific and developmental-specific expression, for analyses of physiological function of genes, or for the production of a human disease model. This approach is especially successful in the fields of immunology and oncology. Several years ago it was shown that the major histocompatibility complex (MHC) class II gene is identical to the immune response gene by demonstrating that the immune response can be restored by the new expression of class II molecules on immunocompetent cells. Recent evidence suggests that the class II molecule is involved in the generation of autoimmune disease, such as insulin-dependent diabetes mellitus (IDDM). The NOD (non-obese diabetic) mouse is shown to be a mouse model for human IDDM. Concerning the class II genes, the NOD mouse has two characteristic features, the lack of I-E and the presence of unique I-A. It is discussed how the role of class II molecules in the development of IDDM in the NOD mouse can be analyzed. In addition, the transgenic technique can be applied to the study of differentiation and oncogenesis of lymphoid cells. Factors or molecules that affect these processes will also be discussed.
It is n o t surprising t h a t the i m m u n e response can only be achieved by the interactions of m a n y molecules o f m a n y types o f cell, because m a n y molecules are also involved in other biological reactions. H o w e v e r , one i m p o r t a n t aspect in the i m m u n e response is t h a t each molecule m a y f u n c t i o n at a different stage d u r i n g d e v e l o p m e n t a n d growth. F r o m this point o f view, analyses o n not only the spatial b u t also the t e m p o r a l expression of each molecule are quite i m p o r t a n t . F o r this purpose we need a n experimental system in which we can analyze the f u n c t i o n of each molecule t h r o u g h the whole animal. O n e o f the p r o m i s i n g a p p r o a c h e s is the transgenic m o u s e system. W e will discuss h o w we can utilize this system for the analysis of the i m m u n e response a n d the a u t o i m m u n e disease, insulind e p e n d e n t diabetes mellitus.
B-lymphocytes a n d monocytes. These molecules are s h o w n to have at least two i m m u n o l o g i c a l functions. First, they are expressed in the t h y m u s a n d are involved in the negative or positive selection of i m m a t u r e T-cells. Second, they are expressed o n the cell surface o f certain i m m u n o c o m p e t e n t cells, including B-lymphocytes a n d monocytes, a n d are involved in the antigen p r e s e n t a t i o n to self-educated T-cells. E a c h class II molecule consists o f two subunits, a a n d /3 chains, a n d the genes e n c o d i n g these subunits have been well characterized at the molecular level. In 1985, we a n d others showed t h a t the class II gene is identical to the i m m u n e response gene ( Y a m a m u r a et al., 1985; Le M e u r et al., 1985; P i n k e r t et al., 1985). In this experiment, we p r o d u c e d transgenic mice by i n t r o d u c i n g the m u r i n e E a d gene into the fertilized eggs of C 5 7 B L / 6 mice. W e showed t h a t E a d genes were expressed to f o r m the I-E d'b molecule o n the surface of B-lymphocytes a n d m o n o c y t e s a n d t h a t these molecules are f u n c t i o n a l in terms of the i n d u c t i o n of a mixed lymphocyte reaction a n d the r e s t o r a t i o n o f i m m u n e responsiveness to poly(L-glutamic acid-L-lysine-cphenylalanine) (GL-Phe).
F U N C T I O N OF T H E MAJOR H I S T O C O M P A T I B I L 1 T Y C O M P L E X CLASS II G E N E
The m u r i n e class II m a j o r h i s t o c o m p a t i b i l i t y antigens, I-A a n d l-E, have been detected o n the cell surface o f certain i m m u n o c o m p e t e n t cells, including
*Author to whom correspondence should be addressed. +Present address: Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan. 451
452
K.-I. YAMAMURA e/al.
ROLE OF THE CLASS ii GENE IN A U T O I M M U N E DISEASES
There is accumulating evidence that the class II gene is involved in the generation of a u t o i m m u n e diseases (Todd et al., 1988). Insulin-dependent diabetes mellitus (IDDM) is a typical a u t o i m m u n e disease and is well studied at both the cellular and molecular level. This is chiefly because we have a good mouse model for human I D D M . This mouse model, the non-obese diabetic (NOD) mouse, was established by Makino et al. (1980). The disease is characterized by the infiltration of lymphocytes into Langerhans islets of the pancreas (insulitis) at around 4 weeks of age. By 20 weeks of age more than 80% of male and female mice develop insulitis. This is followed by the complete destruction of islets leading to overt DM. However, the incidence of DM is at most 80% in female and 20% in male. Many immunological studies demonstrated that an autoimmune mechanism is involved in the generation of insulitis (Bendelac et al., 1987; Miller et al., 1988; Serreze et al., 1988; Charlton & Mandel, 1988; Haskins & McDuffie, 1990), In addition, breeding studies between N O D and other strains indicate that two or three genetic loci contribute to disease susceptibility (Makino et al., 1985; Hattori et al., 1986; Prochazka et al., 1987). One of these loci is known to be linked to the M H C on chromosone 17 (Hattori et aL, 1986; Prochazka et al., 1987). In fact, there are two characteristic features in M H C of N O D mouse. First, they do not express I-E molecules because of a defect of the Ea gene (Makino et al., 1985). Second is the uniqueness of the I-A molecule. Acha-Orbea & McDevitt (1987) demonstrated that the 3' half of A/3 chain including the second external domain, the transmembrane domain, and the intracellular domain, is identical to that of Ap chain of the 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. However, one region has five consecutive nucleotide changes that are unique to the N O D mouse. These substitutions result in two amino acid changes at positions 56 and 57 from P r o - A s p to H i s - S e r . These results suggest that the abnormal expression of class II molecules might be involved in the development of a u t o i m m u n e insulitis. To test this hypothesis, we produced N O D transgenic mice by introducing various class II genes. I-E N O D T R A N S G E N I C MOUSE
Previously we (Nishimoto el al., 1987) demonstrated that the development of a u t o i m m u n e insulitis
NOD
E C o
Log fluorescence intensity Fig. 1. Expression of I-E on the surface of peripheral blod lymphocytes in I-E NOD lransgenic mice.
Table 1. Incidence of insulitis in NOD - El! transgenic mice
Negative littermate Transgenic offspring
Sex
No. of mice
I-E
lnsulitis
F M F M
16 14 11 14
(- ) (-) (+ ) (+ )
14 (88%) 12 (86%) 0 0
Pancreata of mice were examined at 19 weeks of age.
can be prevented with the expression of I-E molecules by backcrossing I-E expressing C57BL/6 transgenic mice ( B 6 - E a d) (Yamamura et al., 1985) to N O D mice. However, we could not exclude the possible involvement of other genes adjacent to the Ea J transgene from B6 - Ea d mice. Thus, we directly introduced the Ecra gene into the fertilized eggs of N O D mice to produce N O D transgenic mice ( N O D - E a d) (Uehira et al., 1989). Offspring from the founder transgenic mouse were used in the following analyses. The expression of Ea d was analyzed by staining peripheral blood lymphocytes with anti-I-E (13/4). As shown in Fig. 1, one-quarter of the lymphocytes from transgenic mice could be stained by the anti-I-E antibody and the staining intensities of lymphocytes were equal or stronger than those of control B A L B / c mice. To examine the effect of I-E expression, partial pancreatectomy was performed at 19 weeks of age,
Insulin-dependent Diabetes Mellitus
NOD i C3H i
Ak
E o
m
I
~ e r : !
i
k
k
:
r
Logfluorescence intensity Fig. 2. Expression of 1-A~ on the surface of peripheral blood lymphocytes in I-A NOD transgenic mice. and the presence of insulitis was examined histochemically. As expected, more than 80°7o of the negative littermates displayed insulitis. However, none of the transgenic offspring developed insulitis (Table 1). This result confirms our previous report and suggest that the expression of I-E alone can prevent the development of insulitis.
I-A NOD TRANSGENIC MICE
About one-quarter of backcross progeny between NOD and C3H (Hattori et al., 1986), or C57BL/6 (Makino et al., 1985) was shown to develop insulitis. This suggests that the lack of I-E in M H C region is not the only cause for insulitis, because a C57BL/6 mouse also does not express I-E. In addition, Todd et al. (1987) demonstrated that the HLA-DQ/3 alleles, having non-Asp at position 57 were enriched in the Caucasian IDDM population and that the homozygosity of the 57Asp negative gene is necessary for
453
the development of IDDM in most Caucasians. These data clearly suggest that the presence of unique I-A is also involved in the development of autoimmune insulitis. To address this question we (Miyazaki et al., 1990) produced NOD transgenic mice carrying either Aa k ( N O D - A a k) or A/3k ( N O D - A / 3 k) genes. Then, two lines of transgenic mice were mated to produce a double transgenic mouse carrying both Aa k and A/~k genes ( N O D - AakAflk). The expression of Ao:k or Ap k was examined by staining peripheral blood lymphocytes with anti-Aa k (116/32) or anti-Aft k (40F). As shown in Fig. 2, in single transgenic mice carrying either Aa k or Aftk the level of expression of AakA/3N°D or A a d A f l t was less than 50% of that of AatA/3k (I-A k) in a control C3H mouse. The low level expression of AakA/3N°Dor AadA/3k in single transgenic mice may be due to the low pairing efficiency between Aa k and A/3N°D, or Aa J and A/3k, respectively (Germain et al., 1985). In fact, in almost all N O D - A a k A f l k transgenic mice, the level of I-A t expression was more than 100% of that of a C3H mouse. The presence of insulitis was examined histochemically at 20 weeks of age. As shown in Table 2, the incidence of insulitis was the same as that of nontransgenic NOD mice. I t - i s of interest that in N O D - A a k A / 3 t transgenic mice the incidence decreased to about one-third of that in nontransgenic NOD mice. This partial prevention of insulitis was neither due to the decreased level of endogenous I-A N°b expression, nor due to the nonspecific suppression of immune response. This partial prevention of insulitis is not directly related to the level of I-A k expression, because insulitis could occur in such a transgenic mouse expressing three times as much I-A t as a C3H mouse. But the number of Langerhans islets with lymphocytic infiltration was small, suggesting that diabetes will not occur in these transgenic mice. Actually, Slattery et al. (1990) showed that none of the transgenic mice developed diabetes up to 5 - 8 months of age. In order to analyze the role of the amino acid at position 57, we carried out in vitro mutagenesis using a mutagenic primer to replace the coding-sequence for Asp (GAC) with that o f S e r (TCC). Then, we obtained NOD mice tra nsgenic with a mutant A/3k (NOD-Aflk: 57Ser), and with both a Aa k and a A/3k ( N O D - AakA/3k: 57Set) by breeding Aa k transgenic mice with Apk:57Ser transgenic mice. As expected, the level of expression of AadAflk:57Ser in N O D A/3k:57Ser mice and AakAflk:57Ser in N O D AakA/3k:57Ser mice was less than 20% and around 100% of that of I-A t in a control C3H mouse, respectively.
454
K.-I. YAMAMURA et al.
Table 2. Incidence of insulitis in I-A NOD transgenic mice Transgenic mice NOD N O D - A~ N O D _ A~° N O D - A~ : 57Ser N O D - A~ A~ N O D - A ~ A~ : 57Set
Sex
No. of mice
Level of I-A k
F M F M
16 14 24 21
(-)
F M F M F M F M
4 4 7 10 14 15 5 7
",, 20% ~
5070
~, 50% 43 ,-o 872% 48 ~ 134%
lnsulitis 13/16 12/14 20/24 18/21
(81%) (85%) (83°70) (86°7o)
3/4 ,*/4 7/7 10/10 5/14 4/15 1/5 1/7
(75%) (100%) (I00%) (100%) (36%) (27%) (2O%) (14%)
Pancreata of mice were examined at 20 weeks of age. Interestingly e n o u g h , insulitis has occurred w i t h o u t exception in N O D - A / / k : 5 7 S e r mice. M o r e o v e r , the degree o f insulitis of each m o u s e was m o r e severe a n d one male a n d two females out of 17 mice developed diabetes at the age o f 4, 7 or 10 weeks. So far, such an early onset o f diabetes has never been observed in n o n - t r a n s g e n i c N O D mice (Makino et al., 1985). By contrast, in N O D - A a k A / / k : 5 7 S e r mice, the d e v e l o p m e n t of insulitis was prevented a n d the incidence was lower t h a n t h a t in N O D - AakA//k mice. It is of interest that the prevention o f insulitis is complete in I-E transgenic mice, but not in I-A k transgenic mice. All these results suggest that the presence o f a unique I-A in the N O D m o u s e is also involved in the d e v e l o p m e n t o f a u t o i m m u n e insulitis, a n d that the m e c h a n i s m o f p r e v e n t i o n in I-A k transgenic mice might be different f r o m that in I-E transgenic mice. CONCLUSIONS Several i m p o r t a n t conclusions can be d r a w n f r o m these experiments a n d are as follows. First, not only the lack of I-E but also the presence o f u n i q u e I-A are involved in the d e v e l o p m e n t o f a u t o i m m u n e insulitis in a N O D mouse. Second, the m e c h a n i s m o f prevention in I-A k transgenic mice might be different f r o m that in I-E transgenic mice a l t h o u g h the negative selection o f autoreactive T-cells in the t h y m u s has been suggested in I-E transgenic mice. Third, the single a m i n o acid s u b s t i t u t i o n f r o m aspartic acid to serine at position 57 o f the A / / c h a i n is not sufficient for the d e v e l o p m e n t o f insulitis. Actually, L u n d et al. (1990) d e m o n s t r a t e d that residue 56 in the N O D Aft molecule is i m p o r t a n t in the d e v e l o p m e n t of insulitis a n d disease. However,
the a m i n o acid at position 57 m a y be involved in the c o n f o r m a t i o n a l change o f the I-A molecule in such a way as to alter the interaction with the T-cell receptor leading to the positive or negative selection of autoreactive T-cells. F o u r t h , the difference in incidence of insulitis between N O D - A e k A / / k : 5 7 S e r a n d N O D - A / / k : 5 7 S e r suggests that the A a chain is also involved in the d e v e l o p m e n t of insulitis p r o b a b l y t h r o u g h the d e t e r m i n a t i o n of the c o n f o r m a t i o n of the I-A molecule. This is consistent with earlier findings that a specific c o m b i n a t i o n between D Q A 1 and D Q B 1 genes c o n t r i b u t e s to susceptibility to h u m a n I D D M (Todd et al., 1989). Fifth, the ectopic expression of class II molecules in B-cells m a y not be the primary event for the generation of insulitis because we could not detect I-E molecules in//-cells of I-E transgenic mice. Besides the identification of the responsible gene(s) in the M H C region, a n o t h e r i m p o r t a n t question to address is the n o m i n a l a u t o a n t i g e n which is expected to be expressed by/3 cells. In a n a t t e m p t to identify the n o m i n a l a u t o a n t i g e n , we established a // cell line f r o m i n s u l i n o m a o b t a i n e d by targeted expression of the simian virus 40 T antigen gene in transgenic mice (Miyazaki et al., 1990). A l t h o u g h the study is still in progress, the transgenic mice shown here should be a powerful tool for the analysis of IDDM. Acknowledgements - - We thank Drs M. Steinmetz, N. Hozumi and M. Watanabe for the gift of the Aa ~ and A/3~ genes. This work required the joint efforts of several individuals besides the authors: Drs K. Kishimoto, H. Kikutani, H. Nishimoto M. Uehira and M. Kimoto. This work was supported by the Japanese Ministry of Education, Science and Culture, and by the Science and Technology Agency of the Japanese Government.
Insulin-dependent Diabetes Mellitus
455
REFERENCES
ACHA-ORBEA,H. & McDEVITT, H. O. (1987). The first external domain of the nonobese diabetic mouse class II I-A 13chain is unique. Proc. natn. Acad. Sei. U.S.A., 84, 2435-2439. BENDELAC,A., CARNAUD,C., BOITARD,C. & BACH, J. F. (1987). Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates - - requirement for both L3T4 + and Lyt-2 + T cells. J. exp. Med., 166, 823-832. CHARLTON, B. & MANDEL, T. E. (1988). Progression from insulitis to B-cell destruction in NOD mouse requires L3T4 + T-lymphocytes. Diabetes, 37, 1108 - 1112. GERMAIN, R., BENTLY, D. M. & QUILL, H. (1985). Influence of allelic polymorphism on the assembly and surface expression of class II MHC (Ia) molecules. Cell, 43, 233 -242. HASK1NS, K. & McDUEFIE, M. (1990). Acceleration of diabetes in young NOD mice with a CD4 + islet-specific T cell clone. Science, 249, 1433 - 1436. HATTORI, M., BUSE, J. B., JACKSON, R. A., GLIMCHER, L., DORF, M. E., MINAMI, M., MAKINO, S., MORIWAKI, K., KUZUYA, H., IMURA, H., STRAUSS,W. M., SEIDMAN, J. G. & EISENBARTH,G. S. (1986). The NOD mouse: recessive diabetogenic gene in the major histocompatibility complex. Science, 2 3 1 , 7 3 3 - 735. LE MEUR, M., GERLINGER, P., BENOIST, C. & MATH1S, D. (1985). Correcting an immune-response deficiency by creating Ea gene transgenic mice. Nature, 316, 3 8 - 42. LUND, T., O'REILLY, L., HUTCHINGS, P., KANAGAWA,O., SIMPSON,E., GRAVELY,R., CHANDLER,P., DYSON, J., PICARD, J. K., EDWARDS, A., KIOUSSIS, D. & COOKE, A. (1990). Prevention of insulin-dependent diabetes mellitus in nonobese diabetic mice by transgenes encoding modified I-A O-chain or normal I-E a-chain. Nature, 345, 727- 729. MAKINO, S., KUN1MOTO,K., MURAOKA,Y., KATAGIRI,K. & TOCHINO, Y. (1980). Breeding of a non-obese diabetic strain of mice. Expl Anim., 29, 1 - 13. MAKINO, S., MURAOKA,Y., KISHIMOTO,Y. & HAYASHI,Y. (1985). Genetic analysis for insulitis in NOD mice. ExplAnirn., 34, 425 - 432. MILLER, B. J., APPEL, M. C., O'NEIL, J. J. & WICKER, L. S. (1988). Both the Lyt-2 ~ and L3T4 + T cell subsets are required for the transfer of diabetes in nonobese diabetic mice. J. lmmun., 140, 52-58. MIYAZAKI, J., ARAKI, K., YAMATO, E., IKEGAMI, H., ASANO, T., SHIBASAKI, Y., OKA, Y. & YAMAMURA,K. (1990). Establishment of a pancreatic/3 cell line that retains glucose-inducible insulin secretion: special reference to glucose transporter isoforms. Endocrinology, 127, 126- 132. MIYAZAK1, T., UNO, M., UEHIRA, M., KIKUTANI, H., KISHIMOTO, T., KIMOTO, M., N1SHIMOTO, H., MIYAZAK1, J. & YAMAMURA, K. (1990). Direct evidence for the contribution of the unique I-A N°D to the development of insulitis in non-obese diabetic mice. Nature, 345, 722-724. NISHIMOTO, H., KIKUTANI,H., YAMAMURA,K. & KISH1MOTO,T. (1987). Prevention of autoimmune insulitis by expression of I-E molecules in NOD mice. Nature, 328, 432- 434. PINKERT, C. A., WIDERA, G., COWING, C., HEBER-KATZ,E., PALMITER,R. D., FLAVELL,R. A. & BRINSTER, R. L. (1985). Tissue-specific, inducible and functional expression of the Ea a MHC class 11 gene in transgenic mice. EMBO J., 4, 2225 - 2230. PROCHAZKA, M., LEITER, E. H., SERREZE, D. V. & COLEMAN, D. L. (1987). Three recessive loci required for insulindependent diabetes in nonobese diabetic mice. Science, 237, 286-289. SERREZE, D. V., LEITER, E. H., WORTHEN, S. M. & SHULTZ, L. D. (1988). NOD marrow stem cells adoptively transfer diabetes to resistant (NOD x NON)F1 mice. Diabetes, 37, 252-255. SLATTERY, R. M., KJER-NIELSEN, L., ALLISON, J., CHARLTON, B., MANDEL, T. E. & MILLER, J. F. A. P. (1990). Prevention of diabetes in non-obese diabetic I-A k transgenic mice. Nature, 345, 724-726. TODD, J. A., ACHA-ORBEA,H., BELL, J. I., CHAO, N., FRONECK, Z., JACOB, C. O., MCDERMOTT, M., SINHA, A. A., TIMMERMAN, K., STEIMAN, L. & MCDEVlTT, H. O. (1988). A molecular basis for MHC class lI-associated autoimmunity. Science, 240, 1003- 1009. TODD, J. A., BELL, J. I. & McDEVlTT, H. O. (1987). HLA-DQ/3 gene contributes to susceptibility and resistance to insulindependent diabetes. Nature, 329, 599- 604. TODD, J. A., MIJOVIC, C., FLETCHER, J., JENKINS, D., BRADWELL, A. R. & BARNETT, A. H. (1989). Identification of susceptibility loci for insulin-dependent diabetes mellitus by trans-racial gene mapping. Nature, 338, 587-589. UEHIRA, M., UNO, M., KURNER, T., K1KUTAN1,H., MORI, K., INOMOTO, T., UEDE, T., MIYAZAKI,J., NISHIMOTO, H., KISHIMOTO, T. & YAMAMURA,K. (1989). Development of autoimmune insulitis is prevented in Ea but not in A/3~NOD transgenic mice. Int. Immun., 1, 209-213. YAMAMURA,K., KIKUTANI, H., FOLSOM, V., CLAYTON, L., KIMOTO, M., AK1RA, S., KASHIWAMURA,S., TONEGAWA,S. & KISHIMOTO, T. (1985). Functional expression of a microinjected Ead gene in C57BL/6 transgenic mice. Nature, 316, 67 - 69.
Int. J, lmmunopharmac., Vol. 14, No. 3, pp. 451-455, 1992. Printed in Great Britain.
T R A N S G E N I C M O U S E AS A TOOL FOR THE S T U D Y OF A U T O I M M U N E DISEASE: I N S U L I N - D E P E N D E N T D I A B E T E S MELLITUS KEN-ICHI YAMAMURA,* TOHRU MIYAZAKI, MASASHI UNO a n d JUN-ICHI MIYAZAKI + Institute for Medical Genetics, Kumamoto University Medical School, Kuhonji, Kumamoto 862, Japan
Abstract - - Transgenic mice have been used for analyses of cis-acting elements which are involved in the tissue-specific and developmental-specific expression, for analyses of physiological function of genes, or for the production of a human disease model. This approach is especially successful in the fields of immunology and oncology. Several years ago it was shown that the major histocompatibility complex (MHC) class II gene is identical to the immune response gene by demonstrating that the immune response can be restored by the new expression of class II molecules on immunocompetent cells. Recent evidence suggests that the class II molecule is involved in the generation of autoimmune disease, such as insulin-dependent diabetes mellitus (IDDM). The NOD (non-obese diabetic) mouse is shown to be a mouse model for human IDDM. Concerning the class II genes, the NOD mouse has two characteristic features, the lack of I-E and the presence of unique I-A. It is discussed how the role of class II molecules in the development of IDDM in the NOD mouse can be analyzed. In addition, the transgenic technique can be applied to the study of differentiation and oncogenesis of lymphoid cells. Factors or molecules that affect these processes will also be discussed.
It is n o t surprising t h a t the i m m u n e response can only be achieved by the interactions of m a n y molecules o f m a n y types o f cell, because m a n y molecules are also involved in other biological reactions. H o w e v e r , one i m p o r t a n t aspect in the i m m u n e response is t h a t each molecule m a y f u n c t i o n at a different stage d u r i n g d e v e l o p m e n t a n d growth. F r o m this point o f view, analyses o n not only the spatial b u t also the t e m p o r a l expression of each molecule are quite i m p o r t a n t . F o r this purpose we need a n experimental system in which we can analyze the f u n c t i o n of each molecule t h r o u g h the whole animal. O n e o f the p r o m i s i n g a p p r o a c h e s is the transgenic m o u s e system. W e will discuss h o w we can utilize this system for the analysis of the i m m u n e response a n d the a u t o i m m u n e disease, insulind e p e n d e n t diabetes mellitus.
B-lymphocytes a n d monocytes. These molecules are s h o w n to have at least two i m m u n o l o g i c a l functions. First, they are expressed in the t h y m u s a n d are involved in the negative or positive selection of i m m a t u r e T-cells. Second, they are expressed o n the cell surface o f certain i m m u n o c o m p e t e n t cells, including B-lymphocytes a n d monocytes, a n d are involved in the antigen p r e s e n t a t i o n to self-educated T-cells. E a c h class II molecule consists o f two subunits, a a n d /3 chains, a n d the genes e n c o d i n g these subunits have been well characterized at the molecular level. In 1985, we a n d others showed t h a t the class II gene is identical to the i m m u n e response gene ( Y a m a m u r a et al., 1985; Le M e u r et al., 1985; P i n k e r t et al., 1985). In this experiment, we p r o d u c e d transgenic mice by i n t r o d u c i n g the m u r i n e E a d gene into the fertilized eggs of C 5 7 B L / 6 mice. W e showed t h a t E a d genes were expressed to f o r m the I-E d'b molecule o n the surface of B-lymphocytes a n d m o n o c y t e s a n d t h a t these molecules are f u n c t i o n a l in terms of the i n d u c t i o n of a mixed lymphocyte reaction a n d the r e s t o r a t i o n o f i m m u n e responsiveness to poly(L-glutamic acid-L-lysine-cphenylalanine) (GL-Phe).
F U N C T I O N OF T H E MAJOR H I S T O C O M P A T I B I L 1 T Y C O M P L E X CLASS II G E N E
The m u r i n e class II m a j o r h i s t o c o m p a t i b i l i t y antigens, I-A a n d l-E, have been detected o n the cell surface o f certain i m m u n o c o m p e t e n t cells, including
*Author to whom correspondence should be addressed. +Present address: Department of Disease-related Gene Regulation Research (Sandoz), Faculty of Medicine, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113, Japan. 451
452
K.-I. YAMAMURA e/al.
ROLE OF THE CLASS ii GENE IN A U T O I M M U N E DISEASES
There is accumulating evidence that the class II gene is involved in the generation of a u t o i m m u n e diseases (Todd et al., 1988). Insulin-dependent diabetes mellitus (IDDM) is a typical a u t o i m m u n e disease and is well studied at both the cellular and molecular level. This is chiefly because we have a good mouse model for human I D D M . This mouse model, the non-obese diabetic (NOD) mouse, was established by Makino et al. (1980). The disease is characterized by the infiltration of lymphocytes into Langerhans islets of the pancreas (insulitis) at around 4 weeks of age. By 20 weeks of age more than 80% of male and female mice develop insulitis. This is followed by the complete destruction of islets leading to overt DM. However, the incidence of DM is at most 80% in female and 20% in male. Many immunological studies demonstrated that an autoimmune mechanism is involved in the generation of insulitis (Bendelac et al., 1987; Miller et al., 1988; Serreze et al., 1988; Charlton & Mandel, 1988; Haskins & McDuffie, 1990), In addition, breeding studies between N O D and other strains indicate that two or three genetic loci contribute to disease susceptibility (Makino et al., 1985; Hattori et al., 1986; Prochazka et al., 1987). One of these loci is known to be linked to the M H C on chromosone 17 (Hattori et aL, 1986; Prochazka et al., 1987). In fact, there are two characteristic features in M H C of N O D mouse. First, they do not express I-E molecules because of a defect of the Ea gene (Makino et al., 1985). Second is the uniqueness of the I-A molecule. Acha-Orbea & McDevitt (1987) demonstrated that the 3' half of A/3 chain including the second external domain, the transmembrane domain, and the intracellular domain, is identical to that of Ap chain of the 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. However, one region has five consecutive nucleotide changes that are unique to the N O D mouse. These substitutions result in two amino acid changes at positions 56 and 57 from P r o - A s p to H i s - S e r . These results suggest that the abnormal expression of class II molecules might be involved in the development of a u t o i m m u n e insulitis. To test this hypothesis, we produced N O D transgenic mice by introducing various class II genes. I-E N O D T R A N S G E N I C MOUSE
Previously we (Nishimoto el al., 1987) demonstrated that the development of a u t o i m m u n e insulitis
NOD
E C o
Log fluorescence intensity Fig. 1. Expression of I-E on the surface of peripheral blod lymphocytes in I-E NOD lransgenic mice.
Table 1. Incidence of insulitis in NOD - El! transgenic mice
Negative littermate Transgenic offspring
Sex
No. of mice
I-E
lnsulitis
F M F M
16 14 11 14
(- ) (-) (+ ) (+ )
14 (88%) 12 (86%) 0 0
Pancreata of mice were examined at 19 weeks of age.
can be prevented with the expression of I-E molecules by backcrossing I-E expressing C57BL/6 transgenic mice ( B 6 - E a d) (Yamamura et al., 1985) to N O D mice. However, we could not exclude the possible involvement of other genes adjacent to the Ea J transgene from B6 - Ea d mice. Thus, we directly introduced the Ecra gene into the fertilized eggs of N O D mice to produce N O D transgenic mice ( N O D - E a d) (Uehira et al., 1989). Offspring from the founder transgenic mouse were used in the following analyses. The expression of Ea d was analyzed by staining peripheral blood lymphocytes with anti-I-E (13/4). As shown in Fig. 1, one-quarter of the lymphocytes from transgenic mice could be stained by the anti-I-E antibody and the staining intensities of lymphocytes were equal or stronger than those of control B A L B / c mice. To examine the effect of I-E expression, partial pancreatectomy was performed at 19 weeks of age,
Insulin-dependent Diabetes Mellitus
NOD i C3H i
Ak
E o
m
I
~ e r : !
i
k
k
:
r
Logfluorescence intensity Fig. 2. Expression of 1-A~ on the surface of peripheral blood lymphocytes in I-A NOD transgenic mice. and the presence of insulitis was examined histochemically. As expected, more than 80°7o of the negative littermates displayed insulitis. However, none of the transgenic offspring developed insulitis (Table 1). This result confirms our previous report and suggest that the expression of I-E alone can prevent the development of insulitis.
I-A NOD TRANSGENIC MICE
About one-quarter of backcross progeny between NOD and C3H (Hattori et al., 1986), or C57BL/6 (Makino et al., 1985) was shown to develop insulitis. This suggests that the lack of I-E in M H C region is not the only cause for insulitis, because a C57BL/6 mouse also does not express I-E. In addition, Todd et al. (1987) demonstrated that the HLA-DQ/3 alleles, having non-Asp at position 57 were enriched in the Caucasian IDDM population and that the homozygosity of the 57Asp negative gene is necessary for
453
the development of IDDM in most Caucasians. These data clearly suggest that the presence of unique I-A is also involved in the development of autoimmune insulitis. To address this question we (Miyazaki et al., 1990) produced NOD transgenic mice carrying either Aa k ( N O D - A a k) or A/3k ( N O D - A / 3 k) genes. Then, two lines of transgenic mice were mated to produce a double transgenic mouse carrying both Aa k and A/~k genes ( N O D - AakAflk). The expression of Ao:k or Ap k was examined by staining peripheral blood lymphocytes with anti-Aa k (116/32) or anti-Aft k (40F). As shown in Fig. 2, in single transgenic mice carrying either Aa k or Aftk the level of expression of AakA/3N°D or A a d A f l t was less than 50% of that of AatA/3k (I-A k) in a control C3H mouse. The low level expression of AakA/3N°Dor AadA/3k in single transgenic mice may be due to the low pairing efficiency between Aa k and A/3N°D, or Aa J and A/3k, respectively (Germain et al., 1985). In fact, in almost all N O D - A a k A f l k transgenic mice, the level of I-A t expression was more than 100% of that of a C3H mouse. The presence of insulitis was examined histochemically at 20 weeks of age. As shown in Table 2, the incidence of insulitis was the same as that of nontransgenic NOD mice. I t - i s of interest that in N O D - A a k A / 3 t transgenic mice the incidence decreased to about one-third of that in nontransgenic NOD mice. This partial prevention of insulitis was neither due to the decreased level of endogenous I-A N°b expression, nor due to the nonspecific suppression of immune response. This partial prevention of insulitis is not directly related to the level of I-A k expression, because insulitis could occur in such a transgenic mouse expressing three times as much I-A t as a C3H mouse. But the number of Langerhans islets with lymphocytic infiltration was small, suggesting that diabetes will not occur in these transgenic mice. Actually, Slattery et al. (1990) showed that none of the transgenic mice developed diabetes up to 5 - 8 months of age. In order to analyze the role of the amino acid at position 57, we carried out in vitro mutagenesis using a mutagenic primer to replace the coding-sequence for Asp (GAC) with that o f S e r (TCC). Then, we obtained NOD mice tra nsgenic with a mutant A/3k (NOD-Aflk: 57Ser), and with both a Aa k and a A/3k ( N O D - AakA/3k: 57Set) by breeding Aa k transgenic mice with Apk:57Ser transgenic mice. As expected, the level of expression of AadAflk:57Ser in N O D A/3k:57Ser mice and AakAflk:57Ser in N O D AakA/3k:57Ser mice was less than 20% and around 100% of that of I-A t in a control C3H mouse, respectively.
454
K.-I. YAMAMURA et al.
Table 2. Incidence of insulitis in I-A NOD transgenic mice Transgenic mice NOD N O D - A~ N O D _ A~° N O D - A~ : 57Ser N O D - A~ A~ N O D - A ~ A~ : 57Set
Sex
No. of mice
Level of I-A k
F M F M
16 14 24 21
(-)
F M F M F M F M
4 4 7 10 14 15 5 7
",, 20% ~
5070
~, 50% 43 ,-o 872% 48 ~ 134%
lnsulitis 13/16 12/14 20/24 18/21
(81%) (85%) (83°70) (86°7o)
3/4 ,*/4 7/7 10/10 5/14 4/15 1/5 1/7
(75%) (100%) (I00%) (100%) (36%) (27%) (2O%) (14%)
Pancreata of mice were examined at 20 weeks of age. Interestingly e n o u g h , insulitis has occurred w i t h o u t exception in N O D - A / / k : 5 7 S e r mice. M o r e o v e r , the degree o f insulitis of each m o u s e was m o r e severe a n d one male a n d two females out of 17 mice developed diabetes at the age o f 4, 7 or 10 weeks. So far, such an early onset o f diabetes has never been observed in n o n - t r a n s g e n i c N O D mice (Makino et al., 1985). By contrast, in N O D - A a k A / / k : 5 7 S e r mice, the d e v e l o p m e n t of insulitis was prevented a n d the incidence was lower t h a n t h a t in N O D - AakA//k mice. It is of interest that the prevention o f insulitis is complete in I-E transgenic mice, but not in I-A k transgenic mice. All these results suggest that the presence o f a unique I-A in the N O D m o u s e is also involved in the d e v e l o p m e n t o f a u t o i m m u n e insulitis, a n d that the m e c h a n i s m o f p r e v e n t i o n in I-A k transgenic mice might be different f r o m that in I-E transgenic mice. CONCLUSIONS Several i m p o r t a n t conclusions can be d r a w n f r o m these experiments a n d are as follows. First, not only the lack of I-E but also the presence o f u n i q u e I-A are involved in the d e v e l o p m e n t o f a u t o i m m u n e insulitis in a N O D mouse. Second, the m e c h a n i s m o f prevention in I-A k transgenic mice might be different f r o m that in I-E transgenic mice a l t h o u g h the negative selection o f autoreactive T-cells in the t h y m u s has been suggested in I-E transgenic mice. Third, the single a m i n o acid s u b s t i t u t i o n f r o m aspartic acid to serine at position 57 o f the A / / c h a i n is not sufficient for the d e v e l o p m e n t o f insulitis. Actually, L u n d et al. (1990) d e m o n s t r a t e d that residue 56 in the N O D Aft molecule is i m p o r t a n t in the d e v e l o p m e n t of insulitis a n d disease. However,
the a m i n o acid at position 57 m a y be involved in the c o n f o r m a t i o n a l change o f the I-A molecule in such a way as to alter the interaction with the T-cell receptor leading to the positive or negative selection of autoreactive T-cells. F o u r t h , the difference in incidence of insulitis between N O D - A e k A / / k : 5 7 S e r a n d N O D - A / / k : 5 7 S e r suggests that the A a chain is also involved in the d e v e l o p m e n t of insulitis p r o b a b l y t h r o u g h the d e t e r m i n a t i o n of the c o n f o r m a t i o n of the I-A molecule. This is consistent with earlier findings that a specific c o m b i n a t i o n between D Q A 1 and D Q B 1 genes c o n t r i b u t e s to susceptibility to h u m a n I D D M (Todd et al., 1989). Fifth, the ectopic expression of class II molecules in B-cells m a y not be the primary event for the generation of insulitis because we could not detect I-E molecules in//-cells of I-E transgenic mice. Besides the identification of the responsible gene(s) in the M H C region, a n o t h e r i m p o r t a n t question to address is the n o m i n a l a u t o a n t i g e n which is expected to be expressed by/3 cells. In a n a t t e m p t to identify the n o m i n a l a u t o a n t i g e n , we established a // cell line f r o m i n s u l i n o m a o b t a i n e d by targeted expression of the simian virus 40 T antigen gene in transgenic mice (Miyazaki et al., 1990). A l t h o u g h the study is still in progress, the transgenic mice shown here should be a powerful tool for the analysis of IDDM. Acknowledgements - - We thank Drs M. Steinmetz, N. Hozumi and M. Watanabe for the gift of the Aa ~ and A/3~ genes. This work required the joint efforts of several individuals besides the authors: Drs K. Kishimoto, H. Kikutani, H. Nishimoto M. Uehira and M. Kimoto. This work was supported by the Japanese Ministry of Education, Science and Culture, and by the Science and Technology Agency of the Japanese Government.
Insulin-dependent Diabetes Mellitus
455
REFERENCES
ACHA-ORBEA,H. & McDEVITT, H. O. (1987). The first external domain of the nonobese diabetic mouse class II I-A 13chain is unique. Proc. natn. Acad. Sei. U.S.A., 84, 2435-2439. BENDELAC,A., CARNAUD,C., BOITARD,C. & BACH, J. F. (1987). Syngeneic transfer of autoimmune diabetes from diabetic NOD mice to healthy neonates - - requirement for both L3T4 + and Lyt-2 + T cells. J. exp. Med., 166, 823-832. CHARLTON, B. & MANDEL, T. E. (1988). Progression from insulitis to B-cell destruction in NOD mouse requires L3T4 + T-lymphocytes. Diabetes, 37, 1108 - 1112. GERMAIN, R., BENTLY, D. M. & QUILL, H. (1985). Influence of allelic polymorphism on the assembly and surface expression of class II MHC (Ia) molecules. Cell, 43, 233 -242. HASK1NS, K. & McDUEFIE, M. (1990). Acceleration of diabetes in young NOD mice with a CD4 + islet-specific T cell clone. Science, 249, 1433 - 1436. HATTORI, M., BUSE, J. B., JACKSON, R. A., GLIMCHER, L., DORF, M. E., MINAMI, M., MAKINO, S., MORIWAKI, K., KUZUYA, H., IMURA, H., STRAUSS,W. M., SEIDMAN, J. G. & EISENBARTH,G. S. (1986). The NOD mouse: recessive diabetogenic gene in the major histocompatibility complex. Science, 2 3 1 , 7 3 3 - 735. LE MEUR, M., GERLINGER, P., BENOIST, C. & MATH1S, D. (1985). Correcting an immune-response deficiency by creating Ea gene transgenic mice. Nature, 316, 3 8 - 42. LUND, T., O'REILLY, L., HUTCHINGS, P., KANAGAWA,O., SIMPSON,E., GRAVELY,R., CHANDLER,P., DYSON, J., PICARD, J. K., EDWARDS, A., KIOUSSIS, D. & COOKE, A. (1990). Prevention of insulin-dependent diabetes mellitus in nonobese diabetic mice by transgenes encoding modified I-A O-chain or normal I-E a-chain. Nature, 345, 727- 729. MAKINO, S., KUN1MOTO,K., MURAOKA,Y., KATAGIRI,K. & TOCHINO, Y. (1980). Breeding of a non-obese diabetic strain of mice. Expl Anim., 29, 1 - 13. MAKINO, S., MURAOKA,Y., KISHIMOTO,Y. & HAYASHI,Y. (1985). Genetic analysis for insulitis in NOD mice. ExplAnirn., 34, 425 - 432. MILLER, B. J., APPEL, M. C., O'NEIL, J. J. & WICKER, L. S. (1988). Both the Lyt-2 ~ and L3T4 + T cell subsets are required for the transfer of diabetes in nonobese diabetic mice. J. lmmun., 140, 52-58. MIYAZAKI, J., ARAKI, K., YAMATO, E., IKEGAMI, H., ASANO, T., SHIBASAKI, Y., OKA, Y. & YAMAMURA,K. (1990). Establishment of a pancreatic/3 cell line that retains glucose-inducible insulin secretion: special reference to glucose transporter isoforms. Endocrinology, 127, 126- 132. MIYAZAK1, T., UNO, M., UEHIRA, M., KIKUTANI, H., KISHIMOTO, T., KIMOTO, M., N1SHIMOTO, H., MIYAZAK1, J. & YAMAMURA, K. (1990). Direct evidence for the contribution of the unique I-A N°D to the development of insulitis in non-obese diabetic mice. Nature, 345, 722-724. NISHIMOTO, H., KIKUTANI,H., YAMAMURA,K. & KISH1MOTO,T. (1987). Prevention of autoimmune insulitis by expression of I-E molecules in NOD mice. Nature, 328, 432- 434. PINKERT, C. A., WIDERA, G., COWING, C., HEBER-KATZ,E., PALMITER,R. D., FLAVELL,R. A. & BRINSTER, R. L. (1985). Tissue-specific, inducible and functional expression of the Ea a MHC class 11 gene in transgenic mice. EMBO J., 4, 2225 - 2230. PROCHAZKA, M., LEITER, E. H., SERREZE, D. V. & COLEMAN, D. L. (1987). Three recessive loci required for insulindependent diabetes in nonobese diabetic mice. Science, 237, 286-289. SERREZE, D. V., LEITER, E. H., WORTHEN, S. M. & SHULTZ, L. D. (1988). NOD marrow stem cells adoptively transfer diabetes to resistant (NOD x NON)F1 mice. Diabetes, 37, 252-255. SLATTERY, R. M., KJER-NIELSEN, L., ALLISON, J., CHARLTON, B., MANDEL, T. E. & MILLER, J. F. A. P. (1990). Prevention of diabetes in non-obese diabetic I-A k transgenic mice. Nature, 345, 724-726. TODD, J. A., ACHA-ORBEA,H., BELL, J. I., CHAO, N., FRONECK, Z., JACOB, C. O., MCDERMOTT, M., SINHA, A. A., TIMMERMAN, K., STEIMAN, L. & MCDEVlTT, H. O. (1988). A molecular basis for MHC class lI-associated autoimmunity. Science, 240, 1003- 1009. TODD, J. A., BELL, J. I. & McDEVlTT, H. O. (1987). HLA-DQ/3 gene contributes to susceptibility and resistance to insulindependent diabetes. Nature, 329, 599- 604. TODD, J. A., MIJOVIC, C., FLETCHER, J., JENKINS, D., BRADWELL, A. R. & BARNETT, A. H. (1989). Identification of susceptibility loci for insulin-dependent diabetes mellitus by trans-racial gene mapping. Nature, 338, 587-589. UEHIRA, M., UNO, M., KURNER, T., K1KUTAN1,H., MORI, K., INOMOTO, T., UEDE, T., MIYAZAKI,J., NISHIMOTO, H., KISHIMOTO, T. & YAMAMURA,K. (1989). Development of autoimmune insulitis is prevented in Ea but not in A/3~NOD transgenic mice. Int. Immun., 1, 209-213. YAMAMURA,K., KIKUTANI, H., FOLSOM, V., CLAYTON, L., KIMOTO, M., AK1RA, S., KASHIWAMURA,S., TONEGAWA,S. & KISHIMOTO, T. (1985). Functional expression of a microinjected Ead gene in C57BL/6 transgenic mice. Nature, 316, 67 - 69.