The liver, lungs and kidneys serve as unique model organs to understand the mechanisms by which parenchymal cells and leukocytes communicate in different microenvironments. These studies have relevance to understanding the regulation and optimization of development of innate immune responses and their relationship to metastasis formation and disease-induced inflammation. IL-12 and IL-18 are potent immunoregulatory cytokines for natural killer (NK), NKT, and T cells, and they induce beneficial antitumor activities in numerous experimental models. Our results have shown that although both IL-12 and IL-18 induced potent IFN-gamma-dependent antimetastatic effects, they have very distinctive effects on the NKT/NK subsets in different organs. Specifically, we have found that while IL-12 administration results in an overall increase in total liver-associated leukocytes, there is a rapid reduction in the total number of detectable NKT cells in the liver, and a later reduction in NK cells. The ability of IL-12 to recruit T cells to the liver is dependent on MIP1alpha. Conversely, IL-18 increases the number of NKT and NK cells, and this increase in liver leukocytes is dependent on CXCR3 binding chemokines. In addition to studying the different immunoregulatory effects of cytokines on NKT and NK cells, we are also studying the interactions between these two subsets and the relative roles each plays in antitumor defenses. At present the only efficient means to selectively stimulate NKT cells in vivo is alpha-galactosyl ceramide (alpha-GalCer). However, alpha-GalCer effectively stimulates and then diminishes the number of detectable NKT cells. It also exhibits a potent, indirect ability to activate NK cells. We have now discovered another ceramide compound, ?GalCer (C12), that efficiently diminishes the number of detectable mouse NKT cells in vivo without inducing significant cytokine expression or activation of NK cells. Binding studies employing CD1d tetramers loaded with ?GalCer (C12) demonstrated significant but lower intensity binding to NKT cells when compared to alpha-GalCer, but both ceramides were equally efficient in reducing the number of NKT cells. However, ?GalCer (C12), in contrast to alpha-GalCer failed to increase NK cell size, number and cytolytic activity. Also in contrast to alpha-GalCer, ?GalCer (C12) is a poor inducer of IFN-gamma, TNFalpha GM-CSF, and IL-4 gene expression. These qualitative differences in NKT perturbation/NK activation have important implications for delineating the unique in vivo roles of NKT vs NK cells. Thus, alpha-GalCer (which triggers NKT cells and activates NK cells) efficiently increases the resistance to allogeneic bone marrow transplantation while ?GalCer (C12) (which triggers NKT cells but does not activate NK cells) fails to enhance bone marrow graft rejection. Our results show ?GalCer (C12) can effectively discriminate between NKT and NK-mediated responses in vivo. These results indicate the use of different TcR-binding ceramides can provide a unique approach for understanding the intricate immunoregulatory contributions of these two cell types.
