Regulation of nuclear gamma interferon gene expression by interleukin 12 (IL-12) and IL-2 represents a novel form of posttranscriptional control.

Posttranscriptional control of gamma interferon (IFN-gamma) gene expression has not been extensively studied and is poorly understood. Our work describes a posttranscriptional mechanism that modulates IFN-gamma mRNA expression in stimulated natural killer (NK) cells through nuclear retention of the IFN-gamma mRNA. This is evidenced by the elevated and sustained nuclear accumulation of both precursor and processed IFN-gamma mRNAs in NK cells stimulated with interleukin-12 (IL-12). The elevated nuclear mRNA accumulation persists long after transcriptional activity has subsided and the rate of cytoplasmic IFN-gamma mRNA accumulation has dropped. The IL-12-induced nuclear retention of the IFN-gamma mRNA prevails until a secondary cytokine stimulus is received. The secondary stimulus, which is initiated by IL-2, mediates transcription-independent movement of the nuclear IFN-gamma mRNA. Concurrent with the nucleocytoplasmic movement of the IFN-gamma mRNA, we have observed increases in the amount of processed nuclear IFN-gamma mRNA that are greater than that seen for the unprocessed IFN-gamma mRNA. The increase in processed IFN-gamma mRNA appears to be due to increased mRNA stability which then promotes increased nucleocytoplasmic shuttling of the mature IFN-gamma mRNA. These data support a model whereby mobilization of nuclear IFN-gamma mRNA stores allows NK cells to rapidly and robustly respond to secondary cytokine activators in a transcription-independent manner, thus shortening the time for overall cellular response to inflammatory signals.

Complex regulation of the Csk homologous kinase (Chk) by IL-4 family cytokines and IFN-gamma in human peripheral blood monocytes.

Csk homologous kinase (Chk) is a tyrosine kinase that shares homology with Csk and, like Csk, has the potential to inhibit src-family kinase function through phosphorylation. In myeloid lineage cells, Chk expression is dependent on monocytic differentiation. IL-4 and IL-13 are cytokines involved in monocytic differentiation that have recently been shown to induce Chk expression in peripheral blood monocytes (PBMs). In this study, we show that two other members of the IL-4 family, IL-3 and GM-CSF, can also induce Chk expression at RNA and protein levels. Interestingly, Chk induction is both blocked and reversed by IFN-gamma treatment. Additionally, a short pretreatment with IFN-gamma is sufficient to prevent Chk induction, and the effects of IFN-gamma are dependent on protein synthesis. Collectively, these results suggest that activation of Chk expression and signaling may have a role in the IL-4 family-mediated differentiation of myeloid cells, and inhibition of Chk activation may be one mechanism by which IFN-gamma alters IL-4-mediated affects.

Regulation of Ly49D/DAP12 signal transduction by Src-family kinases and CD45.

Activating, DAP12-coupled members of the Ly-49 family of NK cell receptors help control viral infections in mice. However, the kinases and/or phosphatases mediating tyrosine phosphorylation of Ly-49D-associated DAP12 have not been elucidated. In this study, we show for the first time that Src family tyrosine kinases are physically and functionally associated with Ly-49D/DAP12 signaling in murine NK cells. Specifically, we demonstrate the following: 1) inhibition of Src family kinases suppresses DAP12 phosphorylation and downstream DAP12 signals; 2) both Fyn and Lck are capable of phosphorylating DAP12; and 3) both kinases coimmunoprecipitate with the Ly-49D/DAP12 complex in NK cells. Although we detect enhanced phosphorylation of Fyn upon Ly-49D cross-linking in NK cells, Ly-49D-mediated events in both Fyn-/- and Fyn/Lck-/- mice appear normal, reinforcing the theme of redundancy in the ability of Src family kinases to initiate activation events. In contrast to disruption of specific Src family enzymes, Ly-49D/DAP12-mediated calcium mobilization and cytokine production by CD45 null NK cells are defective. Although others have ascribed the effects of CD45 mutation solely on the suppression of Src family activity, we demonstrate in this study that DAP12 is hyperphosphorylated in CD45 null NK cells, resulting in uncoordinated tyrosine-mediated signaling upon Ly-49D ligation. Therefore, although our data are consistent with a Src kinase activity proximally within DAP12 signaling, DAP12 also appears to be a substrate of CD45, suggesting a more complex role for this phosphatase than has been reported previously.

Aberrant DAP12 signaling in the 129 strain of mice: implications for the analysis of gene-targeted mice.

NK cells are implicated in antiviral responses, bone marrow transplantation and tumor immunosurveillance. Their function is controlled, in part, through the Ly49 family of class I binding receptors. Inhibitory Ly49s suppress signaling, while activating Ly49s (i.e., Ly49D) activate NK cells via the DAP12 signaling chain. Activating Ly49 signaling has been studied primarily in C57BL/6 mice, however, 129 substrains are commonly used in gene-targeting experiments. In this study, we show that in contrast to C57BL/6 NK cells, cross-linking of DAP12-coupled receptors in 129/J mice induces phosphorylation of DAP12 but not calcium mobilization or cytokine production. Consistent with poor-activating Ly49 function, 129/J mice reject bone marrow less efficiently than C57BL/6 mice. Sequence analysis of receptors and DAP12 suggests no structural basis for inactivity, and both the 129/J and C57BL/6 receptors demonstrate normal function in a reconstituted receptor system. Most importantly, reconstitution of Ly49D in 129/J NK cells demonstrated that the signaling deficit is within the NK cells themselves. These unexpected findings bring into question any NK analysis of 129/J, 129Sv, or gene-targeted mice derived from these strains before complete backcrossing, and provide a possible explanation for the differences observed in the immune response of 129 mice in a variety of models.