Interleukin 13 (IL-13) is a pleiotropic, anti-inflammatory cytokine produced mainly by activated Th2 T cells. We have recently cloned a naturally occurring mRNA splice variant of IL-13 in which the second exon is omitted (termed IL-13 E2). Our characterization of this splice variant indicates sharp contrasts in the expression and function of IL-13 E2 and IL-13. IL-13 E2 is expressed mainly during inflammatory (Th1 and Th17) responses, while IL-13 is expressed primarily during Th2 immune responses. IL-13 E2 is produced exclusively by activated B cells, while the main cellular source of IL-13 is activated Th2 T cells. Treatment with IL-13 E2, in vitro or in vivo, results in increases in the inflammatory mediators TNF-1 and IL-12. On the other hand, the net effect of IL-13 expression is a decrease in the secretion of these same inflammatory mediators. Lastly, we have also shown that in mice lacking IL-13 E2 expression is highly resistant to induction of experimental autoimmune encephalomyelitis (EAE). Given the expression pattern of IL-13 E2, its ability to modulate the expression of inflammatory cytokines, and the demonstration that lack of IL-13 E2 impairs the development of EAE, we hypothesis that IL-13 E2 is a unique cytokine involved in inflammatory immune responses, including the priming of encephalitogenic T cells. Given the discovery of this novel cytokine, we propose to further demonstrate the indispensable role of IL-13 E2 in the immune responses which cause (EAE). We also propose to investigate the mechanism by which IL-13 E2 promotes EAE in vitro and in vivo, comparing differences in the cytokine milieu and the generation of Th1 and Th17 cells in the presence or absence of IL-13 E2.
The specific aims of this study are:
Specific Aim 1 : Does IL-13 E2 augment the induction of Th1 and Th17 cells in vitro and in vivo? Specific Aim 2: Does IL-13 E2 exert effects during priming and during the effector phase of EAE? Specific Aim 3: Do B cells augment EAE solely by secreting IL-13 E2? Public Health Relevance: Multiple sclerosis is a human autoimmune disease which afflicts more than 400,000 Americans. EAE is commonly accepted as a suitable animal model for studying the immunopathology of multiple sclerosis. These two diseases share many common characteristics such as clinical manifestations and histopathology of CNS lesions. Murine EAE also resembles MS in the development of the chronic relapsing form of the disease. The basic mechanisms of disease initiation in EAE and MS are still unknown. This proposal seeks to characterize a novel cytokine splice variant and its role in the pathogenesis of EAE. Knowledge obtained from this study should provide insight to possible therapeutics for MS. Interleukin 13 is a Th2 type cytokine produced by activated T cells which exhibits anti-inflammatory function on B cells, monocytes, macrophages and dendritic cells. We have recently cloned a naturally occurring mRNA splice variant of IL-13 in which the second exon is omitted (termed IL-13 E2 or simply E2). We have developed specific RT-PCR assays for quantitation of IL-13 and E2 gene expression. We have also cloned and purified each of the molecules as HIS-tagged proteins for use in in vivo and in vitro studies. We find that unlike its full length counterpart, E2 is expressed predominantly under inflammatory conditions. E2 is the predominant form of IL-13 expressed in the lymph nodes of mice immunized using complete Freund's adjuvant (CFA), an adjuvant which induced Th1 responses. We find that addition of exogenous E2 to cultured antigen presenting cells (either macrophages or dendritic cells) induces striking phenotypic changes. These changes include transformation in cell morphology and increases in inflammatory cytokine secretion after activation. Most notably we have shown that mice lacking E2 expression are highly resistant to induction of experimental autoimmune encephalomyelitis (EAE). Given the expression pattern of E2, its ability to modulate the expression of inflammatory cytokines, and the demonstration that lack of E2 impairs the development of EAE, we hypothesis that E2 is a unique cytokine involved in inflammatory immune responses, including the priming of encephalitogenic T cells. We suggest that analysis of cytokine expression by Th1 T cells in other studies may mistakenly identify E2 as IL- 13, since most methods commonly used to measure IL-13 do not discriminate between the splice variants. Given the discovery of this novel cytokine, we propose to further demonstrate the indispensable role of E2 in the immune responses which cause (EAE). We also propose to investigate the mechanism by which E2 promotes EAE in vitro and in vivo, comparing differences in the cytokine milieu and the generation of Th1, Th2 and Th17 cells in the presence or absence of E2. Furthermore, we propose to determine if the development of EAE can be inhibited by selectively blocking the action of E2 in vivo.
Multiple sclerosis is a human autoimmune disease which afflicts more than 400,000 Americans. EAE is commonly accepted as a suitable animal model for studying the immunopathology of multiple sclerosis. These two diseases share many common characteristics such as clinical manifestations and histopathology of CNS lesions. Murine EAE also resembles MS in the development of the chronic relapsing form of the disease. The basic mechanisms of disease initiation in EAE and MS are still unknown. This proposal seeks to characterize a novel cytokine splice variant and its role in the pathogenesis of EAE. Knowledge obtained form this study should provide insight to possible therapeutics for MS. Interleukin 13 is a Th2 type cytokine produced by activated T cells which exhibits anti-inflammatory function on B cells, monocytes, macrophages and dendritic cells. We have recently cloned a naturally occurring mRNA splice variant of IL-13 in which the second exon is omitted (termed IL-13 ?E2 or simply ?E2). We have developed specific RT-PCR assays for quantitation of IL-13 and ?E2 gene expression. We have also cloned and purified each of the molecules as HIS-tagged proteins for use in in vivo and in vitro studies. We find that unlike its full length counterpart, ?E2 is expressed predominantly under inflammatory conditions. ?E2 is the predominant form of IL-13 expressed in the lymph nodes of mice immunized using complete Freund's adjuvant (CFA), an adjuvant which induced Th1 responses. We find that addition of exogenous ?E2 to cultured antigen presenting cells (either macrophages or dendritic cells) induces striking phenotypic changes. These changes include transformation in cell morphology and increases in inflammatory cytokine secretion after activation. Most notably we have shown that mice lacking ?E2 expression are highly resistant to induction of experimental autoimmune encephalomyelitis (EAE). Given the expression pattern of ?E2, its ability to modulate the expression of inflammatory cytokines, and the demonstration that lack of ?E2 impairs the development of EAE, we hypothesis that ?E2 is a unique cytokine involved in inflammatory immune responses, including the priming of encephalitogenic T cells. We suggest that analysis of cytokine expression by Th1 T cells in other studies may mistakenly identify ?E2 as IL- 13, since most methods commonly used to measure IL-13 do not discriminate between the splice variants. Given the discovery of this novel cytokine, we propose to further demonstrate the indispensable role of ?E2 in the immune responses which cause (EAE). We also propose to investigate the mechanism by which ?E2 promotes EAE in vitro and in vivo, comparing differences in the cytokine milieu and the generation of Th1, Th2 and Th17 cells in the presence or absence of ?E2. Furthermore, we propose to determine if the development of EAE can be inhibited by selectively blocking the action of ?E2 in vivo.
