In these studies, IFN gamma-inducing factor (IGIF), unlike IL-12, did not drive Th1 development in BALB/c or C57BL/6 mice, but like IL-1alpha, potentiated IL-12-driven Th1 development in BALB/c mice. IGIF and IL-12 synergized for IFN gamma production from Th1 cells. Unlike IL-1alpha, IGIF had no effect on Th2 cells. IGIF signaled through IRAK, IL-1 receptor-associated kinase, to induce nuclear translocation of p65/p50 NFkappaB in Th1 cells. IL-1alpha had no effect on proliferation, cytokine production, or NFkappaB activation in Th1 cells but activated NFkappaB and proliferation in Th2 cells. Thus, Th1 and Th2 cells may differ in responsiveness and receptor expression for IL-1 family molecules. IGIF and IL-1alpha may differentially amplify Th1 and Th2 effector responses, respectively.

Interleukin‐3 (IL‐3) is an important regulator of hemopoiesis and considerable effort has been directed towards the study of its mechanism of signal transduction. In this paper, we describe the first molecular identification of a STAT transcription factor that is activated by IL‐3. STATs exist in a cytoplasmic, transcriptionally inactive form which, in response to extracellular signals, become tyrosine phosphorylated and translocate to the nucleus where they bind to specific DNA elements. Several of these DNA elements were found which bind proteins in an IL‐3‐responsive manner. Analysis of these bandshift complexes with available antibodies to the known STATs suggests that IL‐3 activates the DNA‐binding ability of STAT5, a protein which was originally characterized as a prolactin‐responsive transcription factor in sheep. IL‐5 and granulocyte‐macrophage colony stimulating factor (GM‐CSF), which share a common signaling receptor subunit with IL‐3, also activate STAT5. Unexpectedly, two murine STAT5 homologs, 96% identical to each other at the amino acid level, were isolated and IL‐3‐dependent GAS binding could be reconstituted in COS cells transfected with IL‐3 receptor and either STAT5 cDNA. In IL‐3‐dependent hemopoietic cells, both forms of STAT5 are expressed and activated in response to IL‐3.

STAT (signal transducers and activators of transcription) proteins are transcription factors which are activated by phosphorylation on tyrosine residues upon stimulation by cytokines. Seven members of the STAT family are known, including the closely related STAT5A and STAT5B, which are activated by various cytokines. Except for prolactin-dependent ␤-casein production in mammary gland cells, the biological consequences of STAT5 activation in various systems are not clear. We applied PCR-driven random mutagenesis and a retrovirus-mediated expression screening system to identify constitutively active forms of STAT5. By this strategy, we have identified a constitutively active STAT5 mutant which has two amino acid substitutions; one is located upstream of the putative DNA binding domain (H299R), and the other is located in the transactivation domain (S711F). The mutant STAT5 was constitutively phosphorylated on tyrosine residues, localized in the nucleus, and was transcriptionally active. Expression of the mutant STAT5 partially dispenses with interleukin 3 (IL-3) as a growth stimulant of IL-3-dependent cell lines. Further analyses of the mutant STAT5 have demonstrated that both of the mutations are required for nuclear localization, efficient transcriptional activation, and induction of IL-3-independent growth of an IL-3-dependent cell line, Ba/F3, and have indicated that a molecular basis for the constitutive activation is the stability of the phosphorylated form of the mutant STAT5.Stimulation of cytokine receptors leads to activation of multiple signal transduction pathways, including the Ras-Raf-MEK-mitogen-activated protein kinase (MAPK) and the JAK-STAT pathways (14,28,34,42,44). The latter signaling pathway was originally found downstream of the interferon receptors and is now recognized as a common pathway downstream of most cytokine receptors. Upon stimulation with cytokines, receptor-associated JAKs are activated and phosphorylate STAT factors on tyrosine residues. The phosphorylated STAT molecules then form homo-or heterodimers through SH2-mediated interactions and translocate into nuclei to activate transcription of various target genes. Seven members of the STAT family (STAT1 through 4, -5A, -5B, and -6) are known; STAT5A and STAT5B are closely related. With the exception of STAT4 and STAT6, which were shown to be specifically activated by only one or two cytokines, interleukin 12 (IL-12) or both IL-4 and IL-13, respectively (13, 15), most of the other STATs are activated by multiple cytokines. In particular, both STAT5A and STAT5B are activated by numerous cytokines, including prolactin, IL-2, IL-3, IL-5, IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, erythropoietin (Epo), thrombopoietin, and growth hormone (GH).Using the receptor for the human GM-CSF as a model system, members of our group previously showed that activation of the Ras-Raf-MEK-MAPK pathway inhibits apoptosis while the region of the GM-CSF receptor, which is responsible for activation of JAK2 and STAT5 an...

Interleukin‐3 (IL3) was shown recently to utilize the transcription factor Stat5, but the genes regulated by this pathway and the biological consequence of Stat5 activation remained to be determined. In order to study the role of Stat5 in IL3 signalling, we constructed a dominant‐negative Stat5 protein by C‐terminal truncation, and inducibly expressed it in an IL3‐dependent cell line. The effect of dominant‐negative Stat5 induction on expression of IL3 early response genes was examined, and expression of several genes, including cis, osm and pim‐1 was inhibited profoundly. The expression of c‐fos was also reduced, but to a lesser extent. While activated Ras alone (though not Stat5 alone) could induce c‐fos, maximal expression required the action of both Ras and Stat5. Interestingly, although the membrane‐proximal region of the IL3 receptor beta‐chain is responsible for both Jak2‐Stat5 activation and c‐myc induction, c‐myc levels were not affected by the dominant‐negative Stat5. Thus, the signals directed by this membrane‐proximal domain, which is essential for transducing a DNA synthesis signal, can be separated further into Stat5 or c‐myc pathways. The net effect of dominant‐negative Stat5 expression was partial inhibition of IL3‐dependent growth. This provides the first direct evidence that Stat5 is involved in regulation of cell proliferation.

We recently reported that SHIP restrains LPS-induced classical (M1) activation of in vitro differentiated, bone marrow-derived macrophages (BMMPhis) and that SHIP upregulation is essential for endotoxin tolerance. Herein, we show that in vivo differentiated SHIP-/- peritoneal (PMPhis) and alveolar (AMPhis) macrophages, unlike their wild-type counterparts, are profoundly M2 skewed (alternatively activated), possessing constitutively high arginase I (ArgI) and Ym1 levels and impaired LPS-induced NO production. Consistent with this, SHIP-/- mice display M2-mediated lung pathology and enhanced tumor implant growth. Interestingly, BMMPhis from SHIP-/- mice do not display this M2 phenotype unless exposed to TGFbeta within normal mouse plasma (MP) during in vitro differentiation. Our results suggest that SHIP functions in vivo to repress M2 skewing and that macrophage polarization can occur during differentiation in response to TGFbeta if progenitors have elevated PIP3.