Interferon-␥ (IFN␥) and tumor necrosis factor-␣ (TNF␣) cooperate to induce the expression of many gene products during inflammation. The present report demonstrates that a portion of this cooperativity is mediated by synergism between two distinct transcription factors: signal transducer and activator of transcription 1 (STAT1) and nuclear factor B (NF-B). IFN␥ and TNF␣ synergistically induce expression of mRNAs encoding interferon regulatory factor-1 (IRF-1), intercellular adhesion molecule-1, Mig (monokine induced by ␥-interferon), and RANTES (regulated on activation normal T cell expressed and secreted) in normal but not STAT1-deficient mouse fibroblasts, indicating a requirement for STAT1. Transient transfection assays in fibroblasts using site-directed mutants of a 1.3-kilobase pair sequence of the IRF-1 gene promoter revealed that the synergy was dependent upon two sequence elements; a STAT binding element and a B motif. Artificial constructs containing a single copy of both a STAT binding element and a B motif linked to the herpes virus thymidine kinase promoter were able to mediate synergistic response to IFN␥ and TNF␣; such response varied with both the relative spacing and the specific sequence of the regions between these two sites. Cooperatively responsive sequence constructs bound both STAT1␣ and NF-B in nuclear extracts prepared from IFN␥-and/or TNF␣-stimulated fibroblasts, although binding of individual factors was not cooperative. Thus, the frequently observed synergy between IFN␥ and TNF␣ in promoting inflammatory response depends in part upon cooperation between STAT1␣ and NF-B, which is most likely mediated by their independent interaction with one or more components of the basal transcription complex.Intercellular communication by cytokines during an inflammatory reaction is integral to the subsequent orchestration and resolution of the response. IFN␥ 1 and TNF␣ are pleiotrophic cytokines that play often critical roles in this process (1, 2). Although both cytokines independently exert a number of biological activities in a cell type-specific fashion, they have been shown in many circumstances to function cooperatively or antagonistically in controlling expression of a variety of cytokines and cell surface molecules (3-7). Much recent work on cytokine-mediated intracellular signaling pathways has provided a general paradigm for the molecular mechanisms by which extracellular signals induce transcription of target genes (8 -11). A variety of cytokines, growth factors, and hormones trigger phosphorylation of latent cytoplasmic transcription factors termed signal transducers and activators of transcription (STATs) via one or more members of the Janus (Jak) family of protein tyrosine kinases. Tyrosinephosphorylated STATs assemble in dimeric or oligomeric form, translocate to the nucleus, and bind to specific DNA sequence motifs or STAT binding elements (SBEs) (12). IFN␥ has been shown to induce tyrosine phosphorylation of STAT1␣, and a homodimeric form of STAT1␣ binds to the IFN␥-activation seque...

mRNA transcribed from the mouse KC chemokine gene accumulated to significantly higher levels in multiple cell types after treatment with interleukin 1␣ (IL-1␣) as compared with tumor necrosis factor-␣ (TNF␣). Although TNF␣ and IL-1␣ both signaled the activation of nuclear factor B and enhanced transcription of the KC gene with equal potency, only IL-1␣ treatment resulted in stabilization of KC mRNA. Nucleotide sequences that confer sensitivity for IL-1␣-mediated mRNA stabilization were identified within the 5-and 3-untranslated regions (UTRs) of KC mRNA using transient transfection of chimeric plasmids containing specific portions of KC mRNA linked to the chloramphenicol acetyltransferase (CAT) gene. When plasmids containing either the 3-or 5-UTR of KC mRNA were used, the half-life of CAT mRNA was unaltered either in untreated or IL-1␣-stimulated cells. In contrast, CAT mRNA transcribed from plasmids that contained both the 5-and 3-UTRs of the KC mRNA decayed more rapidly than control CAT mRNA, and this enhanced decay was prevented in cells treated with IL-1␣. A cluster of four overlapping AUUUA motifs within the 3-UTR was required, whereas the 5-UTR region exhibited orientation dependence. These findings indicate that cooperative function of the two nucleotide sequences involves a distinct signaling pathway used by IL-1␣ but not TNF␣.

Interleukin-4 (IL-4 Regulation of inflammatory and immune responses involves intercellular communication through a network of secreted cytokines (1). Cytokines derived from T helper (Th)1 1 cells (prototypically IFN␥) promote the development of cellular immunity, while Th2-derived cytokines such as IL-4 promote humoral immunity and antagonize Th1-dependent activities (2-5). At least a portion of the anti-inflammatory function of IL-4 is targeted to mononuclear phagocytes where expression of inducible genes encoding cytokines (e.g. TNF␣, IL-1␣, IL-1␤, various chemokines) (6 -16) and cell surface molecules (FcR␥ and ICAM-1) (17, 18) is suppressed, while expression of other anti-inflammatory products such as the IL-1 receptor antagonist is amplified (9 -11, 19, 20).The molecular mechanisms mediating cytokine-induced gene transcription have been extensively characterized in recent studies on the Janus kinase (JAK) family of protein tyrosine kinases and signal transducers and activators of transcription (STATs) (21-25). IFN␥ has been shown to induce phosphorylation of STAT1, which, in homodimeric form, binds to the IFN␥ activation sequence (GAS) found in many IFN␥-inducible genes (26 -29). In similar fashion, IL-4 stimulates tyrosine phosphorylation of STAT6 (30 -32), which can bind to GAS motifs as well as IL-4-responsive STAT binding elements (SBEs) found in IL-4-inducible genes (20,(33)(34)(35)(36)(37). Interestingly, STAT6 exhibits transactivating function only in the context of a subset of SBE sequence motifs (20,34,(37)(38)(39)(40).IL-4 and IFN␥ exhibit antagonistic effects on macrophage gene expression (14 -18). We have previously observed that IFN␥-mediated induction and IL-4-mediated suppression utilize the same regulatory sequences (the ISRE in the IP-10 gene and the GAS motif in the IRF-1 gene) (16, 40). Furthermore, IL-4 does not inhibit the activation of STAT1 by IFN␥ and IL-4-induced STAT6 is able to bind the IFN␥-responsive site without transactivation (40). Recent studies have shown that STAT1-dependent transcription requires transcriptional coactivators p300 and CREB-binding protein (CBP), which may be present in limiting amounts (41,42). On the basis of these lines of evidence, we postulated that the inhibitory effect of IL-4 may result, at least in part, from the direct action of IL-4-induced STAT6 on IFN␥-induced STAT1-dependent transcription. The present study was undertaken to determine if IL-4-induced STAT6 is required for the suppressive action of IL-4 on IFN␥-induced gene transcription and to extend our analysis to include the IFN␥-responsive element (␥RE), which controls expression of the MIG chemokine gene (43, 44), a third form of IFN␥-sensitive nucleotide regulatory motif. The results demonstrate that IL-4-dependent suppression of IFN␥-induced MIG gene expression is abolished in peritoneal macrophages from mice in which the STAT6 gene has been deleted (45). Both the MIG gene promoter and the ␥RE from the MIG promoter are sensitive to the stimulatory action of IFN␥ and the inhibitory effec...

Immun. 55:947-954, 1987), we showed that human gingival fibroblasts spontaneously produce thymocyteactivating factor (FTAF), which stimulates mitogen-induced thymocyte proliferation. In the present study, we examined the effect of Bacteroides gingivalis fimbriae on FTAF production by the cells, because the fimbriae may be involved in attachment of the organism to periodontal tissues. We show here that the fimbriae bind to the cells, which may subsequently lead to the stimulation of FTAF production by the cells.