Research in this laboratory is focused on defining how T cell-derived cytokines such as interferon-gamma (IFN-g), interleukin-4 (IL-4) and IL-10 regulate the expression of specific genes in human monocytes and monocyte-derived macrophages. In this context, we are examining the repertoire of genes that are induced in monocytes by treatment with these cytokines. Particular attention is directed at defining how these cytokines regulate expression of the SOCS genes (Suppressors of Cytokine Signaling) in monocytes and macrophages. The proteins encoded by the SOCS genes play a critical role in regulating cytokine signaling. The second major area of research in this laboratory is focused on characterizing the expression and function of several novel IL-10-related cytokines. These include IL-19, IL-20, IL-22, and IL-24 (MDA-7). At present, very little is known regarding the cell types that produce these cytokines or the stimuli that induce their expression. Even less is known about the cell types that respond to these cytokines or the genes that they induce. Therefore, we are conducting studies to define what cell types express these cytokines and what genes are induced by these ligands in cells that express functional receptors. Although these cytokines exhibit approximately 25% sequence homology with IL-10, the target cells that they act upon and the functional activities that they induce are distinct from those of IL-10. Characterization of the Effects of Recombinant Human Interleukin-12 on Cytokine Gene Expression in Human T Cells. Interleukin-12 (IL-12) is known to upregulate production of interferon-gamma (IFN-g) by activated T cells; however, the effects of IL-12 on expression of other cytokines are less well defined. In this project, we are examining the effects of recombinant human IL-12 (rHuIL-12) on production of multiple cytokines, including IFN-g, IL-2, IL-4, IL-10 and IL-13, by purified normal human CD3+ T cells. Although resting T cells are largely nonresponsive to IL-12, anti-CD3-activated T cell blasts are strongly responsive, as demonstrated by the ability of IL-12 to induce STAT4-mediated DNA-binding activity. We found that activation of purified human T lymphocytes on immobilized anti-CD3 mAb induces rapid expression of TNF-alpha and IL-2 mRNA, and more gradual increases in mRNA levels for IFN-g and IL-10. Changes in mRNA expression levels were measured by RNase protection assay using primer template sets that enable simultaneous analysis of multiple cytokine genes. We found that IL-12 markedly upregulates expression of IFN-g and IL-10, and down-regulates production of IL-2. Inhibition of IL-2 production by IL-12 correlates directly with increased production of IL-10. Moreover, neutralization of IL-10 activity with anti-IL-10 antibodies normalized IL-2 production in IL-12-treated T cells, demonstrating that the inhibitory effects of IL-12 are IL-10-mediated. Thus, we have found that IL-12 simultaneously upregulates production of IFN-g and IL-10, and, by a direct IL-10-dependent pathway, feedback inhibits production of IL-2. The fact that IL-12 differentially regulates synthesis of IFN-g and IL-2 in T cells demonstrates that IL-12 does not globally enhance expression of all Th1-type lymphokines. Furthermore, the ability of IL-12 to upregulate production of IL-10 provides a mechanism for limiting the IL-2-dependent clonal expansion of activated T cells, and defines a novel cytokine regulatory pathway that is inducible by IL-12. In future studies, we plan to further define the effects of IL-12 on production of other cytokines, particularly the Th2-type cytokines IL-4 and IL-13 by activated T cells, and to compare the effects of IL-12 to that of other macrophage-derived immunoregulatory cytokines, including IL-1, IL-18 and TNF. We will also further explore the functional consequences of increased IL-10 production in IL-12-treated T cells. The results of these studies will expand our current understanding of the actions of IL-12 on immune effector cells. This information may be useful in interpreting any therapeutic activity induced by IL-12 in clinical trials that are now underway. It may also provide insight to the physiological basis for the systemic toxicity associated with high dose IL-12 therapy in cancer patients. Furthermore, analysis of the effects of IL-12 on cytokine and cytokine receptor gene expression in vitro may facilitate identification of novel markers of IL-12 bioactivity that could be useful in monitoring the potency of recombinant IL-12 in human recipients. Identification and Characterization of Novel Interleukin-10-Related Genes. Activation of human monocytes by bacterial endotoxin, lipopolysaccharide (LPS), induces expression of many cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1), IL-6 and IL-10. IL-10 expression is delayed relative to that of TNF, IL-1 and IL-6. Furthermore, IL-10 feedback inhibits expression of TNF, IL-1 and IL-6, thus providing an efficient autocrine mechanism for controlling proinflammatory cytokine production in monocytes. In this project, we are examining the mechanism by which IL-10 down-regulates production of cytokines such as TNF and IL-1 in endotoxin-stimulated monocytes. We are also evaluating the effects of IL-10 on signal transduction events that are activated by cytokines such as IFN-gamma (IFN-g) and IL-4. We have found that IL-10 inhibits activation and gene expression induced by IL-4 and IFN-gamma. We have also determined that the ability of IL-10 to inhibit IL-4-inducible gene expression is a consequence of decreased tyrosine phosphorylation and nuclear translocation of the IL-4-inducible transcription factor, STAT6. We are now examining the role of a novel family of JAK/STAT inhibitory genes, the Suppressors of Cytokine Signaling (SOCS) genes, in mediating these IL-10-inducible inhibitory effects. We have found that IL-10 selectively induces expression of at least one member of this newly identified gene family: SOCS-3. Forced expression of SOCS-3 in a macrophage cell line markedly inhibits induction of STAT activity by several cytokines, including IFN-gamma, GM-CSF and IL-4. Therefore, the ability of IL-10 to antagonize cytokine-inducible gene expression is associated with its ability to induce rapid expression of the SOCS-3 gene. In related studies, we are also examining the biological activities of several newly identified IL-10 homologues. One of these cytokines, IL-19, shares approximately 21% amino acid homology with IL-10, but signals through a receptor complex that is distinct from that used by IL-10. We have found that expression of the IL-19 gene is up-regulated by many of the same agents that induce IL-10 gene expression in monocytes, including many Toll-like receptor (TLR) agonists. However, unlike the IL-10 gene, IL-19 is not expressed in activated T cells. Another IL-10 homologue, IL-22, was also recently identified by several groups. In collaboration with scientists at the University of Medicine and Dentistry of New Jersey (UMDNJ), we identified and characterized the gene (CRF2-9) that encodes the ligand-binding chain of the receptor for this novel cytokine. This receptor chain heterodimerizes with the IL-10R-beta chain (IL-10R2) to form the functional IL-22 receptor complex. Therefore, IL-10R2 is an essential component of both the IL-10 and IL-22 receptors. We have found that the IL-22 receptor is expressed at high levels in liver and kidney, but not in hematopoietic tissues such as the spleen and thymus. This suggests a possible functional role for this receptor in regulating gene expression in these tissues. This project incorporates FY2002 projects 1Z01BN002021-07 and 1Z01BN002022-09.
