Cytokine treatment of NK cells results in alterations in multiple cellular responses that include cytotoxicity, cytokine production, proliferation, and chemotaxis. To understand the molecular mechanisms underlying these responses, microarray analysis was performed and the resulting gene expression patterns were compared between unstimulated, IL-2, IL-2 plus IL-12, and IL-2 plus IL-18-stimulated NK92 cells. RNase protection assays and RT-PCR confirmed microarray predictions for changes in mRNA expression for nine genes involved in cell cycle progression, signal transduction, transcriptional activation, and chemotaxis. Multiprobe RNase protection assay also detected changes in the expression of CCR2 mRNA, a gene that was not imprinted on the microarray. We subsequently expanded our search for other chemokine receptor genes absent from the microarray and found an IL-2- and IL-12-dependent decrease in CXCR3 receptor mRNA expression in NK92 cells. A detailed analysis of CXCR3 expression in primary NK cells revealed that an IL-2 and an IL-12 together significantly decreased the CXCR3 receptor mRNA and receptor surface expression by 6 and 24 h of treatment, respectively. This decrease in receptor expression was associated with a significant reduction in chemotaxis in the presence of IFN-γ-inducible protein-10. The decline in CXCR3 mRNA was due to transcriptional and posttranscriptional mechanisms as the addition of actinomycin D to IL-2- and IL-12-treated NK92 slightly altered the half-life of the CXCR3 mRNA. Collectively, these data suggest that IL-2 and IL-12 directly affect NK cell migratory ability by rapid and direct down-regulation of chemokine receptor mRNA expression.
Natural killer (NK) cells are large granular lymphocytes that play an important role in the defense against virally infected or malignant cells (1). Their activity can be characterized as nonadaptive and independent of MHC restriction (1, 2). A variety of NK cell functions including cytotoxicity, proliferation, chemotaxis, and cytokine production are modulated by regulatory cytokines including IFN-αβ, IL-2, IL-12, IL-18, IL-10, and TNF (reviewed in Refs. 3 and 4). Because cytokines induce such a broad range of effects in NK cells, the potential for alterations in gene expression in stimulated cells is very great. To determine which genes are regulated in response to cytokine stimulation, our laboratory has used cDNA microarray technology to examine gene expression in NK cells. Microarray technology is very useful because it allows for large-scale examination of gene expression. Additionally, this technology has proved useful in identifying physiologically relevant gene expression patterns in eukaryotic systems such as yeast (5) and fibroblasts (6) as well as predicting patterns of gene expression in tumor cells (7, 8). To examine gene expression in response to cytokine stimulation, a human NK cell line, NK92, was stimulated with IL-2 alone or in combination with IL-12 or IL-18. These cytokines were chosen because of their ability to induce NK cell responses; however, little is known about the repertoire of genes that are activated by these cytokines. Microarray analysis of gene expression in NK92 cells identified a variety of genes whose mRNA expression patterns change in response to cytokine stimulation. The genes encoding the mRNAs are not specific to any one pathway; however, changes in cytokine, chemokine, and chemokine receptor gene mRNAs were prevalent. Our mRNA studies on chemokine receptor gene expression were extended to cell surface analysis of receptor densities in cytokine-treated primary NK cells. Using FACS analysis, we observed a significant decrease in CXCR3 receptor expression in NK cells treated for 24 h with IL-2 and IL-12 alone or in combination. Recently, alterations in chemokine receptor expression were reported in IL-2-stimulated NK cells (9); however, the cells were cultured in IL-2 for 8–10 days. In contrast, our data demonstrate that cytokines can modify chemokine receptor function within hours, thus supporting a model whereby cytokines, in particular IL-2 and IL-12, regulate chemokine receptor expression in a direct, rapid, and novel manner.
|Title||IL-2 and IL-12 alter NK cell responsiveness to IFN-γ-inducible protein 10 by down-regulating CXCR3 expression|
|Authors||D. L. Hodge, William B. Schill, Ji Ming Wang, I. Blanca, D. A. Reynolds, J. R. Ortaldo, H. A. Young|
|Publication Subtype||Journal Article|
|Series Title||Journal of Immunology|
|Record Source||USGS Publications Warehouse|
|USGS Organization||Leetown Science Center|