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Interleukin-4-deficient mice
THE IMMUNOBIOLOGY Schreiber, H. (1989), in “Fundamental immunology, 2” (W.E. Paul) (p. 927). Raven Press, New York. Taylor, C.W., Grogan, T.M. & Salmon, S.E. (1990), Effects of interleukin-4 on the in vitro growth of human lymphoid and plasma cell neoplasms. Blood, 75, 1114-1118. Tepper, R.I., Pattengale, P.K. & Leder, P. (1989), Murine interleukin-4 displays potent antitumor activity in vivo. Cell, 57, 503-512. Tepper, R.I., Levinson, D.A., Stanger, B.Z., CamposTorres, J., Abbas, A.K. & Leder, P. (1990), IL-4 induces allergic-like inflammatory disease and alters T cell development in transgenic mice. Cell, 62, 457-467. Tepper, R.I. (1992). The tumor-cytokine transplantation assay and the antitumor activity of interleukin-4. Bone Marrow Transplant., 9 (suppl. I), 177-181. Tepper, R.I., Coffman, R.L. & Leder, P. (1992), An eosinophil-dependent mechanism of the antitumor effect of IL-4. Science, 257, 548-551. Thornhill, M.H. & Haskard, D.O. (1990), IL-4 regulates endothelial cell activation by IL-I, tumor necrosis factors, or IFN-y. J. Inlrnunol., 145, 865-872. Thornhill, M.H., Wellicome, S.M., Mahiouz, D.L., Lanchbury, J.S.S., Kyan-Aung, U. & Haskard, D.O. (1991). Tumor necrosis factor combines with IL-4 or IFN-y to selectively enhance endothelial cell adhesiveness for T cells. The contribution of vascular cell adhesion molecule-l-dependent and -independent binding mechanisms. J. Immunol., 146, 592-598. Totpal, K. & Aggarwal, B.B. (1991), Interleukin 4 potentiates the antiproliferative effects of tumor necrosis factor on various tumor cell lines. Cancer Res., 5 I, 42664270. Tsu, C.L., Duckett, T., de Kernion, J.B. & Belldegrun, A.S. (1992), Modulation of tumor-infiltrating lymphocytes derived from human renal cell carcinoma by interleukin-4. J. Immunother., 12, 82-89.
OF INTERLEUKIN
K. Rajewsky
Insritufe for Generics, University
Cytokines such as interleukin 4 (IL4) are pleiotropic molecules active on many cell types, inducing or modifying multiple cell proliferation and differentiation processes. At least two methods are available to assess the biological functions of cytokines in vivo. The most common strategy utilizes injections of large quantities of anti-cytokine and/or
637
Van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., De Plaen, E., Van den Eynde, B., Knuth, A. & Boon, T. (1991). A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science, 254, 1643-1647. Van Pel, A., Vessiere, F. & Boon, T. (1983), Protection against two spontaneous mouse leukemias conferred by immunogenic variants obtained by mutagenesis. J. Exp. Med., 157, 1992-1001. Vitetta, E.S., Fulton, R.J., May, R.D., Till, M. & Ujr, J.W. (1987), Redesigning nature’s poisons to create antitumor reagents. Science, 238, 1098-l 104. Walsh, G.M., Mermod, J.J., Hartnell, A., Kay, A.B. & Wardlaw, A.J. (1991), Human eosinophil, but not neutrophil, adherence to IL-l-stimulated human umbilical vascular endothelial cells is a,,p, (very late antigen-4) dependent. J. Immunol., 146, 3419-3423. Wang, J .M., Rambaldi. A., Biondi, A., Chen, Z.G., Sanderson, C.J. & Mantovani, A. (1989), Recombinant human interleukin 5 is a selective eosinophil chemoattractant. Europ. J. Itntuunol., 19, 701-705. Watanabe, Y., Kuribayashi, K., Miyatake, S., Nishihara, K., Nakayama, E., Taniyama, T. & Sakata, T. (1989). Exogenous expression of mouse interferon ycDNA in mouse neuroblastoma Cl300 cells results in induced tumorigenicity by augmented anti-tumor immunity. Proc. Nutl. Acud. Sci. (Wash.), 86, 9456-9460. Yamaguchi, Y., Hayashi, Y., Sugama, Y., Miura, Y., Kasahara, T., Kitamura, S., Torisu, M., Mita, S., Tominaga, A. & Takatsu, K. (1988), Highly purified murine interleukin 5 (IL-5) stimulates eosinophil function and prolongs in vitro survival. IL-5 as an eosinophil chemotactic factor. J. Exp. Med., 167, 1737-1742. Yu, J.S., Wei, M.X., Chiocca. E.A., Martuza, R.L.&Tepper, R.I. (1993). Treatment of glioma by engineered interleukin-4 secreting cells. Cancer Res., 53, 3125-3128.
Interleukin-4-deficient W. Miiller,
4
mice and R. Kuhn
of Cologne, 50931 Cologne (Germany)
anti-cytokine receptor antibodies into mice. The advantages of this method are that it can be used as soon as suitable antibodies are developed and that it can be applied to all available inbred strains and mutant mice (Finkelman, 1992). The major disadvantage is that one cannot be absolutely sure that the antibody treatment completely blocks cytokine
638
53rd FORUM
action in the animal. In addition, cytokine-mediated processes early in development cannot be studied by this method. As an alternative strategy, we decided to inactivate the IL4 gene in the mouse germline by gene targeting (Kiihn et al., 1991). In mice homozygous for the inactivating mutation (IL4T), one can evaluate in which respect, if any, IL4 is essential for the development and function of the immune system. IL4 turned out to be required neither for the generation of T-cell subsets in the thymus, as defined by antiCD4 and anti-CD8 antibodies, nor for the generation and function of B cells (Ki.ihn et al., 1991). In addition, IL4T mice develop normal germinal centres during primary immune responses towards a T-celldependent antigen and, furthermore, they are able to mount a normal secondary immune response, as characterized by somatically mutated, affinity matured antibodies (unpublished observations). On the other hand, IL4 seems essential for the production of antibodies of the IgE subclass (as was already strongly suggested by experiments in which antiILCantibody-treated mice were used) and controls IgGl dominance of certain T-cell-dependent immune responses (Kiihn et a/., 1991). Finally, we could demonstrate the existence of an IL4-independent pathway of Ig class switching to IgG1 (Kiihn et al., 1991), probably induced via a yet unidentified T-cellmembrane-bound molecule (Schulz et al., 1992). We would like to point out that one has to be careful not to overinterpret “negative” results in cytokine-deficient mice. For example, “normal” T-cell-development in IL4T mice does not mean that IL4 does not participate in normal T-cell development. Rather, it means that the function of IL4, if used during normal T-cell development, can be replaced by other signal-transmitting molecules. This argument holds true for all other compartments and functions of the immune system in IL4T mice which showed no alterations compared to controls. Recently, a second ILCdeficient mutant strain has been reported (Kopf et al., 1993). The analysis of this strain confirmed our results. In addition, Kopf et al. claim that the “disruption of the murine IL4 gene
IN IMMUNOI~OG
Y
blocks Th2 cytokine responses”. For this, they analysed the in vivo generation of Th2 cells during the primary infection of mice by the nematode Nipposlrongylus brasiliensis (Nb). They showed that the levels of IL5 and IL10 produced upon activation of CD4+ cells by anti-CD3 antibodies are reduced in their ILCdeficient mice compared to controls. In contrast, in our own experiments (in collaboration with B. Coffman, DNAX), now including the analysis of secondary Nb infection, we arrive at a somewhat different conclusion concerning the role of IL4 in the generation of the Th2 subset. Under appropriate experimental conditions, IL5 and IL10 levels produced upon anti-CD3 stimulation by CD4+ cells from IL4T mice were indistinguishable from controls. We therefore conclude that IL4 is not essential for the generation of the Th2 response to infection with a helminth parasite, although it may enhance that response (Kiihn er al., in preparation). IL4T mice may be useful to assess the role of IL4 in diseases such as allergy, asthma, parasite infections and autoimmunity. The IL4T strain developed in our laboratory is available from B&K Universal, Great Britain, FAX 44-964-527006. This work is supported by the Bundesminsterium fiir schung und Technologie through the Genzentrum Kiiln.
For-
References Finkelman, F.D. (1992), Down, but not out for the count. Curr. Biol., 2, 214-215. Kopf, M., Le Gras, G., Bachmann, M., Lamers, M.C., Bluethmann, H. & KGhler, G. (1993). Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nafure (Lond.), 326, 245-247.
Kiihn, R., Rajewsky, K. &Miller,
and analysis of Interleukin-4 254, 707-7 IO. Schulz, C.L., Rothman, P., Kiihn,
W. (1991), Generation
deficient R., Mehry,
mice. M.,
Science, Miiller,
W., Rajewsky, K., Alt, F. & Coffman, R.L. (1992), T helper cell membranes promote IL-4-independent expression of germline cyl transcripts in B cells. J. Jmtnunol.,
149, 60-64.
OF INTERLEUKIN
K. Rajewsky
Insritufe for Generics, University
Cytokines such as interleukin 4 (IL4) are pleiotropic molecules active on many cell types, inducing or modifying multiple cell proliferation and differentiation processes. At least two methods are available to assess the biological functions of cytokines in vivo. The most common strategy utilizes injections of large quantities of anti-cytokine and/or
637
Van der Bruggen, P., Traversari, C., Chomez, P., Lurquin, C., De Plaen, E., Van den Eynde, B., Knuth, A. & Boon, T. (1991). A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma. Science, 254, 1643-1647. Van Pel, A., Vessiere, F. & Boon, T. (1983), Protection against two spontaneous mouse leukemias conferred by immunogenic variants obtained by mutagenesis. J. Exp. Med., 157, 1992-1001. Vitetta, E.S., Fulton, R.J., May, R.D., Till, M. & Ujr, J.W. (1987), Redesigning nature’s poisons to create antitumor reagents. Science, 238, 1098-l 104. Walsh, G.M., Mermod, J.J., Hartnell, A., Kay, A.B. & Wardlaw, A.J. (1991), Human eosinophil, but not neutrophil, adherence to IL-l-stimulated human umbilical vascular endothelial cells is a,,p, (very late antigen-4) dependent. J. Immunol., 146, 3419-3423. Wang, J .M., Rambaldi. A., Biondi, A., Chen, Z.G., Sanderson, C.J. & Mantovani, A. (1989), Recombinant human interleukin 5 is a selective eosinophil chemoattractant. Europ. J. Itntuunol., 19, 701-705. Watanabe, Y., Kuribayashi, K., Miyatake, S., Nishihara, K., Nakayama, E., Taniyama, T. & Sakata, T. (1989). Exogenous expression of mouse interferon ycDNA in mouse neuroblastoma Cl300 cells results in induced tumorigenicity by augmented anti-tumor immunity. Proc. Nutl. Acud. Sci. (Wash.), 86, 9456-9460. Yamaguchi, Y., Hayashi, Y., Sugama, Y., Miura, Y., Kasahara, T., Kitamura, S., Torisu, M., Mita, S., Tominaga, A. & Takatsu, K. (1988), Highly purified murine interleukin 5 (IL-5) stimulates eosinophil function and prolongs in vitro survival. IL-5 as an eosinophil chemotactic factor. J. Exp. Med., 167, 1737-1742. Yu, J.S., Wei, M.X., Chiocca. E.A., Martuza, R.L.&Tepper, R.I. (1993). Treatment of glioma by engineered interleukin-4 secreting cells. Cancer Res., 53, 3125-3128.
Interleukin-4-deficient W. Miiller,
4
mice and R. Kuhn
of Cologne, 50931 Cologne (Germany)
anti-cytokine receptor antibodies into mice. The advantages of this method are that it can be used as soon as suitable antibodies are developed and that it can be applied to all available inbred strains and mutant mice (Finkelman, 1992). The major disadvantage is that one cannot be absolutely sure that the antibody treatment completely blocks cytokine
638
53rd FORUM
action in the animal. In addition, cytokine-mediated processes early in development cannot be studied by this method. As an alternative strategy, we decided to inactivate the IL4 gene in the mouse germline by gene targeting (Kiihn et al., 1991). In mice homozygous for the inactivating mutation (IL4T), one can evaluate in which respect, if any, IL4 is essential for the development and function of the immune system. IL4 turned out to be required neither for the generation of T-cell subsets in the thymus, as defined by antiCD4 and anti-CD8 antibodies, nor for the generation and function of B cells (Ki.ihn et al., 1991). In addition, IL4T mice develop normal germinal centres during primary immune responses towards a T-celldependent antigen and, furthermore, they are able to mount a normal secondary immune response, as characterized by somatically mutated, affinity matured antibodies (unpublished observations). On the other hand, IL4 seems essential for the production of antibodies of the IgE subclass (as was already strongly suggested by experiments in which antiILCantibody-treated mice were used) and controls IgGl dominance of certain T-cell-dependent immune responses (Kiihn et a/., 1991). Finally, we could demonstrate the existence of an IL4-independent pathway of Ig class switching to IgG1 (Kiihn et al., 1991), probably induced via a yet unidentified T-cellmembrane-bound molecule (Schulz et al., 1992). We would like to point out that one has to be careful not to overinterpret “negative” results in cytokine-deficient mice. For example, “normal” T-cell-development in IL4T mice does not mean that IL4 does not participate in normal T-cell development. Rather, it means that the function of IL4, if used during normal T-cell development, can be replaced by other signal-transmitting molecules. This argument holds true for all other compartments and functions of the immune system in IL4T mice which showed no alterations compared to controls. Recently, a second ILCdeficient mutant strain has been reported (Kopf et al., 1993). The analysis of this strain confirmed our results. In addition, Kopf et al. claim that the “disruption of the murine IL4 gene
IN IMMUNOI~OG
Y
blocks Th2 cytokine responses”. For this, they analysed the in vivo generation of Th2 cells during the primary infection of mice by the nematode Nipposlrongylus brasiliensis (Nb). They showed that the levels of IL5 and IL10 produced upon activation of CD4+ cells by anti-CD3 antibodies are reduced in their ILCdeficient mice compared to controls. In contrast, in our own experiments (in collaboration with B. Coffman, DNAX), now including the analysis of secondary Nb infection, we arrive at a somewhat different conclusion concerning the role of IL4 in the generation of the Th2 subset. Under appropriate experimental conditions, IL5 and IL10 levels produced upon anti-CD3 stimulation by CD4+ cells from IL4T mice were indistinguishable from controls. We therefore conclude that IL4 is not essential for the generation of the Th2 response to infection with a helminth parasite, although it may enhance that response (Kiihn er al., in preparation). IL4T mice may be useful to assess the role of IL4 in diseases such as allergy, asthma, parasite infections and autoimmunity. The IL4T strain developed in our laboratory is available from B&K Universal, Great Britain, FAX 44-964-527006. This work is supported by the Bundesminsterium fiir schung und Technologie through the Genzentrum Kiiln.
For-
References Finkelman, F.D. (1992), Down, but not out for the count. Curr. Biol., 2, 214-215. Kopf, M., Le Gras, G., Bachmann, M., Lamers, M.C., Bluethmann, H. & KGhler, G. (1993). Disruption of the murine IL-4 gene blocks Th2 cytokine responses. Nafure (Lond.), 326, 245-247.
Kiihn, R., Rajewsky, K. &Miller,
and analysis of Interleukin-4 254, 707-7 IO. Schulz, C.L., Rothman, P., Kiihn,
W. (1991), Generation
deficient R., Mehry,
mice. M.,
Science, Miiller,
W., Rajewsky, K., Alt, F. & Coffman, R.L. (1992), T helper cell membranes promote IL-4-independent expression of germline cyl transcripts in B cells. J. Jmtnunol.,
149, 60-64.