Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, interleukin-11, and ciliary neurotrophic factor bind to receptor complexes that share the signal transducer gp130. Upon binding, the ligands rapidly activate DNA binding of acute-phase response factor (APRF), a protein antigenically related to the p91 subunit of the interferon-stimulated gene factor-3 alpha (ISGF-3 alpha). These cytokines caused tyrosine phosphorylation of APRF and ISGF-3 alpha p91. Protein kinases of the Jak family were also rapidly tyrosine phosphorylated, and both APRF and Jak1 associated with gp130. These data indicate that Jak family protein kinases may participate in IL-6 signaling and that APRF may be activated in a complex with gp130.

Interleukin-6 (IL-6) is known to be a major mediator of the acute-phase response in liver. We show here that IL-6 triggers the rapid activation of a nuclear factor, termed acute-phase response factor (APRF), both in rat liver in vivo and in human hepatoma (HepG2) cells in vitro. APRF bound to IL-6 response elements in the 5'-flanking regions of various acute-phase protein genes (e.g., the %-macroglobulin, fibrinogen, and al-acid glycoprotein genes). These elements contain a characteristic hexanucleotide motif, CTGGGA, known to be required for the IL-6 responsiveness of these genes. Analysis of the binding specificity of APRF revealed that it is different from NF-IL6 and NF-KB, transcription factors known to be regulated by cytokines and involved in the transcriptional regulation of acute-phase protein genes. In HepG2 cells, activation of APRF was observed within minutes after stimulation with IL-6 or leukemia-inhibitory factor and did not require ongoing protein synthesis. Therefore, a preexisting inactive form of APRF is activated by a posttranslational mechanism. We present evidence that this activation occurs in the cytoplasm and that a phosphorylation is involved. These results lead to the conclusions that APRF is an immediate target of the IL-6 signalling cascade and is likely to play a central role in the transcriptional regulation of many IL-6-induced genes.During an acute inflammation, cytokines released by different cell types, including monocytes, fibroblasts, and endothelial cells, stimulate the synthesis and secretion of a set of plasma proteins, the so-called acute-phase proteins, by the liver. These proteins play a protective role during the acute-phase reaction, e.g., by inactivating proteases, supporting the wound-healing process, or scavenging free oxygen radicals (for a review, see reference 26). According to their regulation by different cytokines, acute-phase proteins have been divided into two subclasses (8). The synthesis of class 1 acute-phase proteins (e.g., a1-acid glycoprotein, C-reactive protein, haptoglobin, and serum amyloid A) is induced by interleukin-1 (IL-1) or combinations of IL-1 and IL-6, whereas the genes for class 2 acute-phase proteins (e.g., a2-macroglobulin, cxl-antichymotrypsin, and fibrinogen) are regulated mainly by IL-6 and glucocorticoids. The 5'-flanking regions of many acute-phase protein genes have been studied in detail, with the goal of identifying regulatory elements required for the cytokine induction of these genes. One type of cytokine response elements found in the promoters of several class 1 acute-phase protein genes represents binding sites for members of the C/EBP family of transcription factors (15,21,49,51,62 (36). In addition, NF-iB or NF-KB-like factors were found to be involved in the regulation of the angiotensinogen, serum amyloid A, and complement factor B genes by 48,54).Less is known about transcription factors regulating the promoters of class 2 acute-phase protein genes. On the basis of a sequence comparison of the 5'-flanking regions of the...

Interleukin-6 (IL-6), leukemia inhibitory factor, oncostatin M, IL-11, and ciliary neurotropic factor are a family of cytokines and neuronal differentiation factors which bind to composite plasma membrane receptors sharing the signal transducing subunit gpl30. We have shown recently that IL-6 and leukemia inhibitory factor rapidly activate a latent cytoplasmic transcription factor, acute-phase response factor (APRF), by tyrosine phosphorylation, which then binds to IL-6 response elements of various IL-6 target genes. Here we demonstrate that APRF is activated by all cytokines acting through gpl30 and is detected in a wide variety of cell types, indicating a central role of this transcription factor in gpl30-mediated signaling. APRF activation is also observed in vitro upon addition of IL-6 to cell homogenates. Protein tyrosine kinase inhibitors block both the tyrosine phosphorylation and DNA binding of APRF. The factor was purified to homogeneity from rat liver and shown to consist of a single 87-kDa polypeptide, while two forms (89 and 87 kDa) are isolated from human hepatoma cells. As reported earlier, the binding sequence specificity of APRF is shared by gamma interferon (IFN-y) activation factor, which is formed by the Stat9l protein. Partial amino acid sequence obtained from purified rat APRF demonstrated that it is likely to be related to Stat9l. In fact, an antiserum raised against the amino-terminal portion of Stat9l cross-reacted with APRF, suggesting the relatedness of APRF and Stat9l. Altogether, these data indicate that APRF belongs to a growing family of Stat-related proteins and that IFN-y and IL-6 use similar signaling pathways to activate IFN-y activation factor and APRF, respectively.Communication between cells interacting in the immune and hematopoietic systems is mediated by a class of soluble polypeptides generally referred to as cytokines. Most cytokines exert multiple effects on different cell types, a typical example being interleukin-6 (IL-6), which during injuries and infections is released by monocytes, endothelial cells, fibroblasts, and other cells. IL-6 is involved in the differentiation of B and T cells, acts as myeloma growth factor, and is the main mediator of the acute-phase response in the liver (reviewed in references 23 and 33). IL-6 specifically binds to a cell surface receptor which consists of two types of subunits, the ligand-binding glycoprotein gp8O and the signal transducer gpl3O (24,67). Binding of IL-6 to gp8O induces homodimerization and tyrosine phosphorylation of gpl3O (42)

Interleukin-6 (IL-6) and gamma interferon (IFN-y) induce a partially overlapping set of genes, including the genes for interferon regulatory factor 1 (IRF-1), intercellular adhesion molecule 1 (ICAM-1), and the acute-phase protein a2-macroglobulin. We report here that the rat a2-macroglobulin promoter is activated by IFN-y in human hepatoma (HepG2) cells and that the IFN-,y response element maps to the same site previously defined as the acute-phase response element (APRE), which binds the IL-6-activated transcription factor APRF (acute-phase response factor). As was reported for fibroblasts, the IFN--y-regulated transcription factor GAF is phosphorylated at tyrosine after IFN--y treatment of HepG2 cells. IFN-y posttranslationally activates a protein which specifically binds to the a2-macroglobulin APRE. This protein is shown to be identical or closely related to GAF. Although APRF and GAF are shown to represent different proteins, their binding sequence specificities are very similar. APRF and GAF bind equally well to the APRE sequences of various acute-phase protein genes as well as to the IFN--y response elements of the IRF-1, ICAM-1, and other IFN--y-inducible genes. Transient transfection analysis revealed that the IFN--y response elements of the IRF-1 and ICAM-1 promoters are able to confer responsiveness to both IFN--y and IL-6 onto a heterologous promoter. Therefore, APRF and GAF are likely to be involved in the transcriptional induction of these immediate-early genes by IL-6 and IFN-y, respectively. Taken together, these results demonstrate that two functionally distinct hormones, IL-6 and IFN--y, act through common regulatory elements to which different transcription factors sharing almost the same sequence specificity bind.

CD69 is highly expressed by lymphocytes at mucosal surfaces. We aimed to investigate the role of CD69 in mucosal immune responses. The expression of CD69 by CD4 T cells isolated from the spleen, mesenteric lymph nodes, small intestinal lamina propria, and colonic lamina propria was determined in specific pathogen-free B6 and TCR transgenic animals, as well as in germ-free B6 mice. Transfer colitis was induced by transplanting RAG−/− mice with B6 or CD69−/−CD45RBhigh CD4 T cells. CD69 expression by CD4 T cells is induced by the intestinal microflora, oral delivery of specific Ag, and type I IFN (IFN-I) signals. CD4 T cells from CD69−/− animals produce higher amounts of the proinflammatory cytokines IFN-γ, TNF-α, and IL-21, whereas the production of TGF-β1 is decreased. CD69-deficient CD4 T cells showed reduced potential to differentiate into Foxp3+ regulatory T cells in vivo and in vitro. The transfer of CD69−/−CD45RBhigh CD4 T cells into RAG−/− hosts induced an accelerated colitis. Oral tolerance was impaired in CD69−/− and IFN-I receptor 1-deficient mice when compared with B6 and OT-II × RAG−/− animals. Polyinosinic-polycytidylic acid treatment of RAG−/− mice transplanted with B6 but not CD69−/− or IFN-I receptor 1-deficient CD45RBhigh CD4 T cells attenuated transfer colitis. CD69 deficiency led to the increased production of proinflammatory cytokines, reduced Foxp3+ regulatory T cell induction, impaired oral tolerance, and more severe colitis. Hence, the activation Ag CD69 plays an important role in regulating mucosal immune responses.