SummaryPicolinic acid, a catabolite of L-tryptophan, activates the transcription of the inducible nitric oxide synthase gene (iNOS) in IFN-y-treated murine macrophages . We performed functional studies on the 5' flanking region of the iNOS gene linked to a CAT reporter gene to identify the cis-acting element(s) responsible for the activation ofiNOS transcription by picolinic acid. Transient transfection assays showed that the full-length iNOS promoter in the murine macrophage cell line ANA-1 was activated by the synergistic interaction between IFN -'Y and picolinic acid. Deletion or mutation of the iNOS promoter region from -227 to -209, containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE), decreased picolinic acid-but not LPS-induced CAT activity by more than 70% . Functional studies using a tk promoter-CAT reporter gene plasmid demonstrated that the iNOS-HRE was sufficient to confer inducibility by picolinic acid but not by IFN-y or LPS. Electrophoretic mobility shift assays confirmed that picolinic acid alone induced a specific binding activity to the iNOS-HRB . Furthermore, we found that the iNOS-HRE activity was inducible by hypoxia and that hypoxia in combination with IFN-y activated the iNOS promoter in transient transfection assays and induced iNOS transcription and mRNA expression . These data establish that the iNOS-HRE is a novel regulatory element of the iNOS promoter activity in murine macrophages and provide the first evidence that iNOS is a hypoxia-inducible gene.

Interferon-␥ (IFN-␥) is critical for defense against pathogens, but the molecules that mediate its antimicrobial responses are largely unknown. IGTP is the prototype for a family of IFN-␥-regulated genes that encode 48-kDa GTP-binding proteins that localize to the endoplasmic reticulum. We have generated IGTP-deficient mice and found that, despite normal immune cell development and normal clearance of Listeria monocytogenes and cytomegalovirus infections, the mice displayed a profound loss of host resistance to acute infections of the protozoan parasite Toxoplasma gondii. By contrast, IFN-␥ receptor-deficient mice have increased susceptibility to all three pathogens. Thus, IGTP defines an IFN-␥-regulated pathway with a specialized role in antimicrobial resistance.

We have previously reported that a 19-base pair element of the 5-flanking region of the inducible nitric oxide synthase (iNOS) gene containing a sequence homology to a hypoxia-responsive enhancer (iNOS-HRE) mediates picolinic acid (PA)-or hypoxia-induced activation of the iNOS promoter in interferon-␥ (IFN-␥)-treated murine macrophages. The iron chelator desferrioxamine (DFX) induces the activity of the human erythropoietin enhancer in Hep3B cells. We have investigated the influence of DFX on the activation of the iNOS promoter and iNOS gene expression in ANA-1 macrophages. We have found that DFX induced DNAbinding activity to the hypoxia-inducible factor 1 (HIF-1) consensus sequence of the iNOS promoter and activated the iNOS-HRE in murine macrophages. These activities of DFX were associated with a synergistic induction of iNOS mRNA expression and iNOS transcription in IFN-␥-treated ANA-1 macrophages. Functional analysis of the 5-flanking region of the iNOS gene demonstrated that IFN-␥ plus DFX activated the full-length iNOS promoter and that the iNOS-HRE was required for DFX-induced iNOS transcriptional activity. We also investigated the role of iron metabolism in the DFX-or PA-dependent induction of HIF-1 activity and iNOS expression. We demonstrate that addition of iron sulfate completely abolished DFX or PA induction of HIF-1 binding and iNOS-HRE activation and abrogated IFN-␥ plus either DFX-or PA-induced iNOS expression. These data establish that DFX is a co-stimulus for the transcriptional activation of the iNOS gene in IFN-␥-treated macrophages, and they provide evidence that the iNOS-HRE is required for the DFX-dependent activation of the iNOS promoter. Furthermore, our results indicate that the iNOS-HRE is a regulatory element of the iNOS promoter responsive to iron chelation.

CD33 is a myeloid specific member of the sialic acid-binding receptor family and is expressed highly on myeloid progenitor cells but at much lower levels in differentiated cells. Human CD33 has two tyrosine residues in its cytoplasmic domain (Y340 and Y358). When phosphorylated, these tyrosines could function as docking sites for the phosphatases, SHP-1 and/or SHP-2, enabling CD33 to function as an inhibitory receptor. Here we demonstrate that CD33 is tyrosine phosphorylated in the presence of the phosphatase inhibitor, pervanadate, and recruits SHP-1 and SHP-2. Co-expression studies suggest that the Src-family kinase Lck is effective at phosphorylating Y340, but not Y358, suggesting that these residues may function in the selective recruitment of adapter molecules and have distinct functions. Further support for overlapping, but nonredundant, roles for Y340 and Y358 comes from peptide-binding studies that revealed the recruitment of both SHP-1 and SHP-2 to Y340 but only SHP-2 to Y358. Analysis using mutants of SHP-1 demonstrated that binding Y340 of CD33 was primarily to the amino Src homology-2 domain of SHP-1. The potential of CD33 to function as an inhibitory receptor was demonstrated by its ability to down-regulate CD64-induced calcium mobilization in U937. The dependence of this inhibition on SHP-1 was demonstrated by blocking CD33-mediated effects with dominant negative SHP-1. This result implies that CD33 is an inhibitory receptor and also that SHP-1 phosphatase has a significant role in mediating CD33 function. Further studies are essential to identify the receptor(s) that CD33 inhibits in vivo and its function in myeloid lineage development.

Posttranscriptional control of gamma interferon (IFN-␥) gene expression has not been extensively studied and is poorly understood. Our work describes a posttranscriptional mechanism that modulates IFN-␥ mRNA expression in stimulated natural killer (NK) cells through nuclear retention of the IFN-␥ mRNA. This is evidenced by the elevated and sustained nuclear accumulation of both precursor and processed IFN-␥ 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-␥ mRNA accumulation has dropped. The IL-12-induced nuclear retention of the IFN-␥ 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-␥ mRNA. Concurrent with the nucleocytoplasmic movement of the IFN-␥ mRNA, we have observed increases in the amount of processed nuclear IFN-␥ mRNA that are greater than that seen for the unprocessed IFN-␥ mRNA. The increase in processed IFN-␥ mRNA appears to be due to increased mRNA stability which then promotes increased nucleocytoplasmic shuttling of the mature IFN-␥ mRNA. These data support a model whereby mobilization of nuclear IFN-␥ 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.

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.

The murine Ly49 family contains nine genes in two subgroups: the inhibitory receptors (Ly49A, B, C, E, F, G2, and I) and the noninhibitory receptors (Ly49D and H). Unlike their inhibitory counterparts, Ly49D and H do not contain immunoreceptor tyrosine-based inhibitory motifs but associate with a recently described coreceptor, DAP12, to transmit positive signals to natural killer (NK) cells. DAP12 is also expressed in myeloid cells, but the receptors coupled to it there are unknown. Here we document the signaling pathways of the Ly49D/ DAP12 complex in NK cells. We show that ligation of Ly49D results in 1) tyrosine phosphorylation of several substrates, including phospholipase C␥1, Cbl, and p44/ p42 mitogen-activated protein kinase, and 2) calcium mobilization. Moreover, we demonstrate that although human DAP12 reportedly binds the SH2 domains of both Syk and Zap-70, ligation of Ly49D leads to activation of Syk but not Zap-70. Consistent with this observation, Ly49D/DAP12-mediated calcium mobilization is blocked by dominant negative Syk but not by catalytically inactive Zap-70. These data demonstrate the dependence of DAP12-coupled receptors on Syk and suggest that the outcome of Ly49D/DAP12 engagement will be regulated by Cbl and culminate in the activation of transcription factors.

Taken together, our results demonstrate that LPS and hypoxia act synergistically to induce HIF-1α-transcriptional activity and they emphasize the existence of a cross talk between hypoxic and non-hypoxic signaling pathways in the regulation of macrophages gene expression.