- Email: [email protected]
Immunodeficiency in IL-2-knockout mice
CLINICAL IMMUNOLOGYAND IMMUNOPATHOLOGY Vol. 76, No. 3, September, pp. $172-$173, 1995
Immunodeficiency in IL-2-Knockout Mice IVAN HORAK Institute of Virology and Immunobiology, University of Wiirzburg, Versbacher Strasse 7, D 97078 Wiirzburg, Germany
Interleukin-2-deficient m i c e (IL-2 -/-) g e n e r a t e d b y t a r g e t e d m u t a g e n e s i s p r o v e d to be an e x c e l l e n t s y s t e m to s t u d y the f u n c t i o n of this l y m p h o k i n e in the c o n t e x t of w h o l e i m m u n e system. In addition, t h e y p r o v i d e an a n i m a l m o d e l o f a p r i m a r y i m m u n o d e f i c i e n c y c a u s e d by a single-gene defect. Our results i n d i c a t e that the e s s e n t i a l d e g e c t in IL-2 -/- mice is the lack of a n e g a t i v e r e g u l a t o r y function, w h i c h leads to breakd o w n of s e l f t o l e r a n c e . T h e d e v e l o p m e n t o f autoi m m u n e d i s e a s e s in IL-2°deficient mice s t r e s s e s the link b e t w e e n a u t o i m m u n i t y and p r i m a r y i m m u n o d e f i ciencies. ,~ 1995 Academic Press, Inc.
tern. To address the role of IL-2 in in vivo, we have introduced a null mutation in the third exon of the IL-2 gene by inserting the neomycine-resistance gene and generated mice deficient in IL-2 activity (IL-2 -/-) (12). N O R M A L D E V E L O P M E N T O F I M M U N E SYSTEM
We have reported t h a t IL-2 -/- mice bred on a mixed genetic background, 129/O1a x C57BL6, develop normally during the first 3 to 4 weeks of age. Also, the ontogeny of the immune system seems not to be affected by the IL-2 deficiency, as seen from normal development of lymphoid organs and the presence of all major lymphocyte subsets (12).
INTRODUCTION
P R O L I F E R A T I O N O F T AND B C E L L S
IL-2 is a pivotal cytokine regulating the growth, differentiation, and function of lymphocytes (for review see 1,2). It is a glycoprotein of about 15 kDa which mediates cell communications via a complex receptor system composed of three subunits, a13~/(for review see 3). Only the a-chain is specific for IL-2, the 6-subunit is also used by IL-15/IL-T (4, 5), and the V-subunit is used by several other ligands (6-8). Since IL-2 is considered an indispensable factor for cytotoxic responses, it is used as an immunotherapeutic agent for the t r e a t m e n t of patients with disseminated cancers and with infectious diseases (9, 10). The exact role of IL-2 in vivo is not known. Its elucidation was hampered by a lack of suitable experimental systems t h a t would allow evaluation of the pleiotropic activites of this interleukin in the context of the whole immune system. IL-2-deficient mice proved to be an important tool in resolving these obstacles. This article summarizes the results obtained with this animal model.
Since IL-2 was first described as a T cell growth factor (TCGF) and is widely used to maintain the growth of T cells in in vitro cultures, it was important to asses directly the effect of lack of IL-2 on T cell proliferation. Paradoxically, proliferation of both T and B cells in IL-2 -/- mice increased, as revealed by lymphocyte counts and by BrdU incorporation (13). The observed high levels of IgG1 and IgE isotypes in sera of IL-2 -/a n i m a l s indicated a p r e f e r e n t i a l use of the IL-4directed swith and suggested t h a t the lack of IL-2 might be compensated by IL-4. However, mice made deficient for both IL-2 and IL-4 possess normal subsets oflymphocytes and also show increased proliferation of lymphocytes (14).
IL-2-DEFICIENT MICE
Generation of mouse m u t a n t s by targeted recombination (11) proved to be an excellent tool in particular to investigate functions of genes in the immune sys'Presented as part of the fifth Jeffrey Modell I m m u n o d e f i c i e n c y Symposium titled "Advances in Primary Immunodeficiency Disease," October 10-11, 1994, Paris, F r a n c e .
A N T I V I R A L R E S P O N S E S I N VIVO
Contrary to some previous expectation, young IL-2deficient mice generated only partially reduced in vivo cytotoxic T cell responses and helper-dependent and -independent T cell responses when challenged with various viruses (15). ULCERATIVE COLITIS-LIKE DISEASE
The relative normal development and immune responsponsiveness observed in young IL-2 -/- mice on 129/O1a xC57BL6 genetic background is in contrast to a rapid onset of disease symptoms with mortality of about 50% within 9 weeks of age. All IL-2 -/- mice which survive past 10 weeks of age develop an inflam-
S172 0090-1229/95 $12.00 Copyright © 1995 by Academic Press, Inc.
All rights of reproduction in any form reserved.
IMMUNODEFICIENCY IN IL-2-KNOCKOUT MICE
matory bowel disease (IBD) with striking clinical and histological similarity to ulcerative colitis in humans (13). An antigenic stimulation of the mucosal immune system probably by nonpathogenic intestinal flora is required for triggering of IBD in IL-2 -/- mice, since mice kept under germ-free conditions do not develop the disease. Ulcerative colitis together with morbus Crohn belongs to chronic inflammatory bowel diseases of unknown etiology. A defective immune system and enviromental factors have been implicated in the pathogenesis of ulcerative colitis (16). Interestingly, similar IBD-like syndromes described for IL-2 -/- mice (13) have also been reported in IL-10 -/(17) and TCR -/- (18) mice. It should be stressed that in all three murine IBD models, the disease is dependent on the presence of normal, nonpathogenic intestinal microflora. Collectively, these results reflect the tight regulation of mucosal immune responses and indicate that several different gene defects can disturb the mucosal homeostasis, resulting in similar IBD syndromes. Similar pathomechanisms might, also be involved in h u m a n IBD. GENERALIZED AUTOIMMUNE DISEASE
To study a possible contribution of genetic factors in the pathogenesis of ulcerative colitis, we have bred the IL-2-deficient mice to various genetic backgrounds. IL2 -/- mice on BALB/c genetic background develop a generalized autoimmune disease, which manifests preferentially as autoimmune hemolytic anemia (Sadlack et al., submitted). The primary alteration of the immune system seen in IL-2-deficient BALB/c mice is an uncontrolled polyclonal activation and proliferation of T and B cells, which is apparently associated with an increased production of antibodies of various self specificities. CONCLUSIONS
IL-2-deficient mice generated by gene targeting provide an invaluable tool to elucidate the exact role of this lymphokine. The insights obtained from this single-gene-defect animal model proved to be instrumental in dissecting the pathomechanisms of h u m a n primary immunodeficiencies. Furthermore, the development of autoimmune diseases in IL-2-deficient mice stresses the link between autoimmunity and primary immunodeficiencies. The common feature of diseases seen in IL-2 -/- mice is an uncontrolled activation and proliferation of lymphocytes. It indicates that the essential defect in IL-2 -/- mice is the lack of a negative regulatory function'which leads to breakdown of self tolerance. Received March 16, 1995; accepted May 12, 1995
$173
ACKNOWLEDGMENTS Experimental work from the author's laboratory was supported by the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie. REFERENCES 1. Smith, K. A., Interleukin-2: Inception, impact, and implications. Science 240, 1169-1176, 1988. 2. Paul, W. E., Pleiotropy and redundancy: T cell-derived lymphokines in the immune response. Cell 57, 521-524, 1989. 3. Taniguchi, T., and Minami, Y., The IL-2/IL-2 receptor system: A current overview. Cell 73, 5-8, 1993. 4. Girl, J. G., Aheieh, M., Eisenman, J., Shanebeck, K., Grabstein, K., Kumaki, S., Namen, A., Park, L. S., Cosman, D., and Anderson, D., Utilization of the/3 and 3' chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13, 2822-2830, 1994. 5. Burton, J. D., Bamford, R. N., Peters, C., Grant, A. J., Kurys, G., Goldman, C. K., Brennan, J., Roessler, E., and Waldmann, T. A., New lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphonkine-activated killer cells. Proc. Natl. Acad. Sci. USA 91, 4935--4939, 1994. 6. Noguchi, M., Nakamura, Y., Russell, S. M., Ziegler, S. F., Tsang, M., Cao, X., and Leonard, W. J., Interleukin-2 receptor 3' chain: A functional component of the interleukin-7 receptor. Science 262, 1877-1880, 1994. 7. Russell, S. M., Keegan, A. D., Harada, N., Nakamura, Y., Noguchi, M., Leland, P., Friedman, M. C., Miyajima, A., Purl, R. J., Paul, W. E., and Leonard, W. J., Interleukin-2 receptor v chain: A functional component of the interleukin-4 receptor. Science 262, 1880-1883, 1994. 8. Kondo, M., Takeshita, T., Ishii, N., Nakamura, M., Watanabe, S., Arai, K., and Sugamura, K., Sharing of the interleukin-2 (IL-2) receptor 3' chain between receptors for IL-2 and IL-4. Science 262, 1874-1877, 1994. 9. Rosenberg, S. A., Immunotherapy of cancer using interleukin-2. Immunol. Today 9, 58-62, 1988. 10. Fauci, A. S., Multifacorial nature of human immunodeficiency virus disease: Implications for therapy. Science 262, 1101-1018, 1993. 11. Thomas, K. R., and Capecchi, M. R., Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503-512, 1987 12. Schorle, H., Holtschke, T., Htinig, T., Schimpl, A., and Horak, I., Development and function of T cells in mie rendered interleukin-2 deficient by gene targeting. Nature 352, 621-624, 1991. 13. Sadlack, B., Merz, H., Schorle, H., Schimpl, A., Feller, A., and Horak, I., Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75, 253-261, 1993 14. Sadlack, B., Ktihn, R., Schorle, H., Rajewsky, K., l~Iuller, W., and Horak, I., Development and function oflymphocytes in mice deficient for both interleukins 2 and 4. Eur. J. Immunol. 24, 281284, 1994. 15. Kiindig, T. M., Schorle, H., Bachmann, M., Hengartner, H., Zinkernagel, R. M., and Horak, I., Immune responses in interleukin-2 deficient mice. Science 262, 1959-1061, 1993. 16. Podolsky, D. K., Inflammatory bowel disease. N. Engl. J. Med. 325, 928-937, 1991. 17. Ktihn, R., LShler, J., Rennick, D., Rajewsky, K., and Mtiller, W., Interleukin-10-deficientmice develop chronic enterocolitis. Cell 75, 263-274, 1993. 18. Mombaerts, P., Mizoguchi, E., Grusby, M.J., Glimcher, L. H., Bhan, A. K., and Tonegawa, S., Spontaneous development of inflammatory bowel disease in T-cell receptor mutant mouse. C~ll 75, 275-282, 1993.
Immunodeficiency in IL-2-Knockout Mice IVAN HORAK Institute of Virology and Immunobiology, University of Wiirzburg, Versbacher Strasse 7, D 97078 Wiirzburg, Germany
Interleukin-2-deficient m i c e (IL-2 -/-) g e n e r a t e d b y t a r g e t e d m u t a g e n e s i s p r o v e d to be an e x c e l l e n t s y s t e m to s t u d y the f u n c t i o n of this l y m p h o k i n e in the c o n t e x t of w h o l e i m m u n e system. In addition, t h e y p r o v i d e an a n i m a l m o d e l o f a p r i m a r y i m m u n o d e f i c i e n c y c a u s e d by a single-gene defect. Our results i n d i c a t e that the e s s e n t i a l d e g e c t in IL-2 -/- mice is the lack of a n e g a t i v e r e g u l a t o r y function, w h i c h leads to breakd o w n of s e l f t o l e r a n c e . T h e d e v e l o p m e n t o f autoi m m u n e d i s e a s e s in IL-2°deficient mice s t r e s s e s the link b e t w e e n a u t o i m m u n i t y and p r i m a r y i m m u n o d e f i ciencies. ,~ 1995 Academic Press, Inc.
tern. To address the role of IL-2 in in vivo, we have introduced a null mutation in the third exon of the IL-2 gene by inserting the neomycine-resistance gene and generated mice deficient in IL-2 activity (IL-2 -/-) (12). N O R M A L D E V E L O P M E N T O F I M M U N E SYSTEM
We have reported t h a t IL-2 -/- mice bred on a mixed genetic background, 129/O1a x C57BL6, develop normally during the first 3 to 4 weeks of age. Also, the ontogeny of the immune system seems not to be affected by the IL-2 deficiency, as seen from normal development of lymphoid organs and the presence of all major lymphocyte subsets (12).
INTRODUCTION
P R O L I F E R A T I O N O F T AND B C E L L S
IL-2 is a pivotal cytokine regulating the growth, differentiation, and function of lymphocytes (for review see 1,2). It is a glycoprotein of about 15 kDa which mediates cell communications via a complex receptor system composed of three subunits, a13~/(for review see 3). Only the a-chain is specific for IL-2, the 6-subunit is also used by IL-15/IL-T (4, 5), and the V-subunit is used by several other ligands (6-8). Since IL-2 is considered an indispensable factor for cytotoxic responses, it is used as an immunotherapeutic agent for the t r e a t m e n t of patients with disseminated cancers and with infectious diseases (9, 10). The exact role of IL-2 in vivo is not known. Its elucidation was hampered by a lack of suitable experimental systems t h a t would allow evaluation of the pleiotropic activites of this interleukin in the context of the whole immune system. IL-2-deficient mice proved to be an important tool in resolving these obstacles. This article summarizes the results obtained with this animal model.
Since IL-2 was first described as a T cell growth factor (TCGF) and is widely used to maintain the growth of T cells in in vitro cultures, it was important to asses directly the effect of lack of IL-2 on T cell proliferation. Paradoxically, proliferation of both T and B cells in IL-2 -/- mice increased, as revealed by lymphocyte counts and by BrdU incorporation (13). The observed high levels of IgG1 and IgE isotypes in sera of IL-2 -/a n i m a l s indicated a p r e f e r e n t i a l use of the IL-4directed swith and suggested t h a t the lack of IL-2 might be compensated by IL-4. However, mice made deficient for both IL-2 and IL-4 possess normal subsets oflymphocytes and also show increased proliferation of lymphocytes (14).
IL-2-DEFICIENT MICE
Generation of mouse m u t a n t s by targeted recombination (11) proved to be an excellent tool in particular to investigate functions of genes in the immune sys'Presented as part of the fifth Jeffrey Modell I m m u n o d e f i c i e n c y Symposium titled "Advances in Primary Immunodeficiency Disease," October 10-11, 1994, Paris, F r a n c e .
A N T I V I R A L R E S P O N S E S I N VIVO
Contrary to some previous expectation, young IL-2deficient mice generated only partially reduced in vivo cytotoxic T cell responses and helper-dependent and -independent T cell responses when challenged with various viruses (15). ULCERATIVE COLITIS-LIKE DISEASE
The relative normal development and immune responsponsiveness observed in young IL-2 -/- mice on 129/O1a xC57BL6 genetic background is in contrast to a rapid onset of disease symptoms with mortality of about 50% within 9 weeks of age. All IL-2 -/- mice which survive past 10 weeks of age develop an inflam-
S172 0090-1229/95 $12.00 Copyright © 1995 by Academic Press, Inc.
All rights of reproduction in any form reserved.
IMMUNODEFICIENCY IN IL-2-KNOCKOUT MICE
matory bowel disease (IBD) with striking clinical and histological similarity to ulcerative colitis in humans (13). An antigenic stimulation of the mucosal immune system probably by nonpathogenic intestinal flora is required for triggering of IBD in IL-2 -/- mice, since mice kept under germ-free conditions do not develop the disease. Ulcerative colitis together with morbus Crohn belongs to chronic inflammatory bowel diseases of unknown etiology. A defective immune system and enviromental factors have been implicated in the pathogenesis of ulcerative colitis (16). Interestingly, similar IBD-like syndromes described for IL-2 -/- mice (13) have also been reported in IL-10 -/(17) and TCR -/- (18) mice. It should be stressed that in all three murine IBD models, the disease is dependent on the presence of normal, nonpathogenic intestinal microflora. Collectively, these results reflect the tight regulation of mucosal immune responses and indicate that several different gene defects can disturb the mucosal homeostasis, resulting in similar IBD syndromes. Similar pathomechanisms might, also be involved in h u m a n IBD. GENERALIZED AUTOIMMUNE DISEASE
To study a possible contribution of genetic factors in the pathogenesis of ulcerative colitis, we have bred the IL-2-deficient mice to various genetic backgrounds. IL2 -/- mice on BALB/c genetic background develop a generalized autoimmune disease, which manifests preferentially as autoimmune hemolytic anemia (Sadlack et al., submitted). The primary alteration of the immune system seen in IL-2-deficient BALB/c mice is an uncontrolled polyclonal activation and proliferation of T and B cells, which is apparently associated with an increased production of antibodies of various self specificities. CONCLUSIONS
IL-2-deficient mice generated by gene targeting provide an invaluable tool to elucidate the exact role of this lymphokine. The insights obtained from this single-gene-defect animal model proved to be instrumental in dissecting the pathomechanisms of h u m a n primary immunodeficiencies. Furthermore, the development of autoimmune diseases in IL-2-deficient mice stresses the link between autoimmunity and primary immunodeficiencies. The common feature of diseases seen in IL-2 -/- mice is an uncontrolled activation and proliferation of lymphocytes. It indicates that the essential defect in IL-2 -/- mice is the lack of a negative regulatory function'which leads to breakdown of self tolerance. Received March 16, 1995; accepted May 12, 1995
$173
ACKNOWLEDGMENTS Experimental work from the author's laboratory was supported by the Deutsche Forschungsgemeinschaft and the Fond der Chemischen Industrie. REFERENCES 1. Smith, K. A., Interleukin-2: Inception, impact, and implications. Science 240, 1169-1176, 1988. 2. Paul, W. E., Pleiotropy and redundancy: T cell-derived lymphokines in the immune response. Cell 57, 521-524, 1989. 3. Taniguchi, T., and Minami, Y., The IL-2/IL-2 receptor system: A current overview. Cell 73, 5-8, 1993. 4. Girl, J. G., Aheieh, M., Eisenman, J., Shanebeck, K., Grabstein, K., Kumaki, S., Namen, A., Park, L. S., Cosman, D., and Anderson, D., Utilization of the/3 and 3' chains of the IL-2 receptor by the novel cytokine IL-15. EMBO J. 13, 2822-2830, 1994. 5. Burton, J. D., Bamford, R. N., Peters, C., Grant, A. J., Kurys, G., Goldman, C. K., Brennan, J., Roessler, E., and Waldmann, T. A., New lymphokine, provisionally designated interleukin T and produced by a human adult T-cell leukemia line, stimulates T-cell proliferation and the induction of lymphonkine-activated killer cells. Proc. Natl. Acad. Sci. USA 91, 4935--4939, 1994. 6. Noguchi, M., Nakamura, Y., Russell, S. M., Ziegler, S. F., Tsang, M., Cao, X., and Leonard, W. J., Interleukin-2 receptor 3' chain: A functional component of the interleukin-7 receptor. Science 262, 1877-1880, 1994. 7. Russell, S. M., Keegan, A. D., Harada, N., Nakamura, Y., Noguchi, M., Leland, P., Friedman, M. C., Miyajima, A., Purl, R. J., Paul, W. E., and Leonard, W. J., Interleukin-2 receptor v chain: A functional component of the interleukin-4 receptor. Science 262, 1880-1883, 1994. 8. Kondo, M., Takeshita, T., Ishii, N., Nakamura, M., Watanabe, S., Arai, K., and Sugamura, K., Sharing of the interleukin-2 (IL-2) receptor 3' chain between receptors for IL-2 and IL-4. Science 262, 1874-1877, 1994. 9. Rosenberg, S. A., Immunotherapy of cancer using interleukin-2. Immunol. Today 9, 58-62, 1988. 10. Fauci, A. S., Multifacorial nature of human immunodeficiency virus disease: Implications for therapy. Science 262, 1101-1018, 1993. 11. Thomas, K. R., and Capecchi, M. R., Site-directed mutagenesis by gene targeting in mouse embryo-derived stem cells. Cell 51, 503-512, 1987 12. Schorle, H., Holtschke, T., Htinig, T., Schimpl, A., and Horak, I., Development and function of T cells in mie rendered interleukin-2 deficient by gene targeting. Nature 352, 621-624, 1991. 13. Sadlack, B., Merz, H., Schorle, H., Schimpl, A., Feller, A., and Horak, I., Ulcerative colitis-like disease in mice with a disrupted interleukin-2 gene. Cell 75, 253-261, 1993 14. Sadlack, B., Ktihn, R., Schorle, H., Rajewsky, K., l~Iuller, W., and Horak, I., Development and function oflymphocytes in mice deficient for both interleukins 2 and 4. Eur. J. Immunol. 24, 281284, 1994. 15. Kiindig, T. M., Schorle, H., Bachmann, M., Hengartner, H., Zinkernagel, R. M., and Horak, I., Immune responses in interleukin-2 deficient mice. Science 262, 1959-1061, 1993. 16. Podolsky, D. K., Inflammatory bowel disease. N. Engl. J. Med. 325, 928-937, 1991. 17. Ktihn, R., LShler, J., Rennick, D., Rajewsky, K., and Mtiller, W., Interleukin-10-deficientmice develop chronic enterocolitis. Cell 75, 263-274, 1993. 18. Mombaerts, P., Mizoguchi, E., Grusby, M.J., Glimcher, L. H., Bhan, A. K., and Tonegawa, S., Spontaneous development of inflammatory bowel disease in T-cell receptor mutant mouse. C~ll 75, 275-282, 1993.