Synovial fluids from 6 of 12 patients with rheumatoid arthritis (RA) and from <em>3</em> of 11 patients with reactive arthritis contained measurable levels of tumor necrosis factor <em>alpha</em> (TNF <em>alpha</em>). Seven of 12 sera from RA patients contained TNF <em>alpha</em>, while only 1 of those from reactive arthritis patients was positive. Gamma-<em>interferon</em> was detected in the synovial fluids and sera of only the RA patients. Tumor necrosis factor beta was not detected in any sera or synovial fluids. RA patients with detectable TNF <em>alpha</em> had higher erythrocyte sedimentation rates and synovial fluid leukocyte counts.

Human cytomegalovirus (HCMV) is a ubiquitous human herpesvirus that can cause life-threatening disease in the fetus and the immunocompromised host. Upon attachment to the cell, the virus induces robust inflammatory, <em>interferon</em>- and growth-factor-like signalling. The mechanisms facilitating viral entry and gene expression are not clearly understood. Here we show that platelet-derived growth factor-<em>alpha</em> receptor (PDGFR-<em>alpha</em>) is specifically phosphorylated by both laboratory and clinical isolates of HCMV in various human cell types, resulting in activation of the phosphoinositide-<em>3</em>-kinase (PI(<em>3</em>)K) signalling pathway. Upon stimulation by HCMV, tyrosine-phosphorylated PDGFR-<em>alpha</em> associated with the p85 regulatory subunit of PI(<em>3</em>)K and induced protein kinase B (also known as Akt) phosphorylation, similar to the genuine ligand, PDGF-AA. Cells in which PDGFR-<em>alpha</em> was genetically deleted or functionally blocked were non-permissive to HCMV entry, viral gene expression or infectious virus production. Re-introducing human PDGFRA gene into knockout cells restored susceptibility to viral entry and essential viral gene expression. Blockade of receptor function with a humanized PDGFR-<em>alpha</em> blocking antibody (IMC-<em>3</em>G<em>3</em>) or targeted inhibition of its kinase activity with a small molecule (Gleevec) completely inhibited HCMV viral internalization and gene expression in human epithelial, endothelial and fibroblast cells. Viral entry in cells harbouring endogenous PDGFR-<em>alpha</em> was competitively inhibited by pretreatment with PDGF-AA. We further demonstrate that HCMV glycoprotein B directly interacts with PDGFR-<em>alpha</em>, resulting in receptor tyrosine phosphorylation, and that glycoprotein B neutralizing antibodies inhibit HCMV-induced PDGFR-<em>alpha</em> phosphorylation. Taken together, these data indicate that PDGFR-<em>alpha</em> is a critical receptor required for HCMV infection, and thus a target for novel anti-viral therapies.

CD40 is a member of the tumor necrosis factor (TNF) receptor family of cell surface proteins and was originally described as a B cell restricted antigen. Treatment of primary human monocytes with granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin <em>3</em> (IL-<em>3</em>), or <em>interferon</em> gamma (IFN-gamma) resulted in the induction of CD40 mRNA and enhancement of cell surface protein expression. CD40 was found to mediate monocyte adhesion to cells expressing recombinant CD40 ligand. CD40 ligand-transfected cells provided a potent costimulus for monocyte TNF-<em>alpha</em> and IL-6 production in the presence of GM-CSF, IL-<em>3</em>, or IFN-gamma, and enhanced IL-8 production stimulated by GM-CSF or IL-<em>3</em>. In addition, CD40 ligand-transfected cells acting in the absence of a costimulus induced monocytes to become tumoricidal against a human melanoma cell target. Collectively, these data indicate that CD40 ligand is pleiotropic with potent biological activity on monocytes.

Macrophages (Mphi) comprise a heterogeneous population of cells with various immune and homeostatic functions. Recently, we have described type-1 and type-2 human monocyte-derived Mphi subsets. Although both support outgrowth of intracellular mycobacteria, Mphi-1 secretes interleukin (IL)-2<em>3</em>/IL-12 and supports T helper cell type 1 (Th1) responses, whereas Mphi-2 fails to produce IL-2<em>3</em>/IL-12, predominantly secretes IL-10, and inhibits Th1 function. Here, we further describe the phenotypic and functional profiles of Mphi-1 and Mphi-2 in response to microbial antigens and <em>interferon</em>-gamma (IFN-gamma) and CD40L as costimulatory T cell back-talk signals. Activated IL-2<em>3</em>(+)/IL-12(+) Mphi-1 secreted IL-1beta, IL-18, IL-6, and tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>), as well as IL-8, monocyte chemoattractant protein-1 (MCP-1), IFN-inducible protein 10 (IP-10), Mphi inflammatory protein-1beta (MIP-1beta), regulated on activation, normal T expressed and secreted (RANTES), Mphi-derived chemokine (MDC), and (low levels of) pulmonary and activation-regulated chemokine and thymus and activation-regulated chemokine (TARC), corroborating their proinflammatory function. Regardless of the stimulus, Mphi-2 maintained their IL-10(+) signature cytokine profile and produced no or relatively low levels of IL-12p40, IL-1beta, IL-6, TNF-<em>alpha</em>, MDC, or TARC. It is remarkable that Mphi-2 secreted high levels of IL-8, MCP-1, IP-10, MIP-1beta, and RANTES, suggesting an active role for these cells in regulating cellular immunity and homeostasis. Mphi-1 and Mphi-2 expressed similar levels of Toll-like receptor and dendritic cell-specific intercellular adhesion molecule-<em>3</em>-grabbing nonintegrin as microbial pattern recognition receptors. Mphi-2, unlike Mphi-1 but like other nonclassical Mphi described previously, expressed CD16<em>3</em> and down-modulated human leukocyte antigen and costimulatory molecules specifically upon activation. These findings demonstrate how Mphi-1/Mphi-2 polarization can differentially skew the host response toward pro- or anti-inflammatory immune responses, respectively. This is likely to be relevant for host-pathogen interactions in chronic bacterial infections and provides a model for dissecting pro- and anti-inflammatory cascades.

Human respiratory syncytial virus (HRSV) is the leading cause of serious pediatric acute respiratory tract infections, and a better understanding is needed of the host response to HRSV and its attenuated vaccine derivatives. It has been shown previously that HRSV nonstructural proteins 1 and 2 (NS1 and NS2) inhibit the induction of <em>alpha</em>/beta <em>interferon</em> (IFN-<em>alpha</em>/beta) in A549 cells and human macrophages. Two principal transcription factors for the early IFN-beta and -<em>alpha</em>1 response are <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) and nuclear factor kappaB (NF-kappaB). At early times postinfection, wild-type HRSV and the NS1/NS2 deletion mutants were very similar in the ability to activate IRF-<em>3</em>. However, once NS1 and NS2 were expressed significantly, they acted cooperatively to suppress activation and nuclear translocation of IRF-<em>3</em>. Since these viruses differed greatly in the induction of IFN-<em>alpha</em>/beta, NF-kappaB activation was evaluated in Vero cells, which lack the structural genes for IFN-<em>alpha</em>/beta and would preclude confounding effects of IFN-<em>alpha</em>/beta. This showed that deletion of the NS2 gene sharply reduced the ability of HRSV to induce activation of NF-kappaB. Since recombinant HRSVs from which the NS1 or NS2 genes have been deleted are being developed as vaccine candidates, we investigated whether the changes in activation of host transcription factors and increased IFN-<em>alpha</em>/beta production had an effect on the epithelial production of proinflammatory factors. Viruses lacking NS1 and/or NS2 stimulated modestly lower production of RANTES (Regulated on Activation Normal T-cell Expressed and Secreted), interleukin 8, and tumor necrosis factor <em>alpha</em> compared to wild-type recombinant RSV, supporting their use as attenuated vaccine candidates.

<em>Interferons</em> (IFN)s are involved in numerous immune interactions during viral infections and contribute to both induction and regulation of innate and adaptive antiviral mechanisms. IFNs play a pivotal rule in the outcome of a viral infection, as demonstrated by the impaired resistance against different viruses in mice deficient for the receptors IFNAR-2 and IFNGR. During viral infections, IFNs are involved in numerous immune interactions as inducers, regulators, and effectors of both innate and adaptive antiviral mechanisms. IFN-<em>alpha</em>/beta is produced rapidly when viral factors, such as envelope glycoproteins, CpG DNA, or dsRNA, interact with cellular pattern-recognition receptors (PRRs), such as mannose receptors, toll-like receptors (TLRs), and cytosolic receptors. These host-virus interactions signal downstream to activate transcription factors needed to achieve expression from IFN-<em>alpha</em>/beta genes. These include IFN regulatory factor-<em>3</em> (IRF-<em>3</em>), IRF-5, IRF-7, c-Jun/ATF-2, and NF-kappaB. In contrast, IFN-gamma is induced by receptor-mediated stimulation or in response to early produced cytokines, including interleukin-2 (IL-12), IL-18, and IFN-<em>alpha</em>/beta, or by stimulation through T cell receptors (TCRs) or natural killer (NK) cell receptors. IFNs signal through transmembrane receptors, activating mainly Jak-Stat pathways but also other signal transduction pathways. Cytokine and TCR-induced IFN-gamma expression uses distinct signal transduction pathways involving such transcription factors as NFAT, Stats and NF-kappaB. This results in induction and activation of numerous intrinsic antiviral factors, such as RNA-activated protein kinase (PKR), the 2-5A system, Mx proteins, and several apoptotic pathways. In addition, IFNs modulate distinct aspects of both innate and adaptive immunity. Thus, IFN-<em>alpha</em>/beta and IFN-gamma affect activities of macrophages, NK cells, dendritic cells (DC), and T cells by enhancing antigen presentation, cell trafficking, and cell differentiation and expression profiles, ultimately resulting in enhanced antiviral effector functions. This review focuses on the latest findings regarding induction and regulation of IFNs, primarily during the early phase of an antiviral immune response. Both cellular and molecular aspects are discussed from the perspective of host-virus interactions.

The aim of this study was to explore further the hypothesis that early stages of normal human hematopoiesis might be coregulated by autocrine/paracrine regulatory loops and by cross-talk among early hematopoietic cells. Highly purified normal human CD<em>3</em>4(+) cells and ex vivo expanded early colony-forming unit-granulocyte-macrophage (CFU-GM)-derived, burst forming unit-erythroid (BFU-E)-derived, and CFU-megakaryocyte (CFU-Meg)-derived cells were phenotyped for messenger RNA expression and protein secretion of various growth factors, cytokines, and chemokines to determine the biological significance of this secretion. Transcripts were found for numerous growth factors (kit ligand [KL], FLT<em>3</em> ligand, fibroblast growth factor-2 [FGF-2], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], insulinlike growth factor-1 [IGF-1], and thrombopoietin [TPO]); cytokines (tumor necrosis factor-<em>alpha</em>, Fas ligand, <em>interferon</em> <em>alpha</em>, interleukin 1 [IL-1], and IL-16); and chemokines (macrophage inflammatory protein-1<em>alpha</em> [MIP-1<em>alpha</em>], MIP-1beta, regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein-<em>3</em> [MCP-<em>3</em>], MCP-4, IL-8, <em>interferon</em>-inducible protein-10, macrophage-derived chemokine [MDC], and platelet factor-4 [PF-4]) to be expressed by CD<em>3</em>4(+) cells. More importantly, the regulatory proteins VEGF, HGF, FGF-2, KL, FLT<em>3</em> ligand, TPO, IL-16, IGF-1, transforming growth factor-beta1 (TGF-beta1), TGF-beta2, RANTES, MIP-1<em>alpha</em>, MIP-1beta, IL-8, and PF-4 were identified in media conditioned by these cells. Moreover, media conditioned by CD<em>3</em>4(+) cells were found to inhibit apoptosis and slightly stimulate the proliferation of other freshly isolated CD<em>3</em>4(+) cells; chemo-attract CFU-GM- and CFU-Meg-derived cells as well as other CD<em>3</em>4(+) cells; and, finally, stimulate the proliferation of human endothelial cells. It was also demonstrated that these various hematopoietic growth factors, cytokines, and chemokines are expressed and secreted by CFU-GM-, CFU-Meg-, and BFU-E-derived cells. It is concluded that normal human CD<em>3</em>4(+) cells and hematopoietic precursors secrete numerous regulatory molecules that form the basis of intercellular cross-talk networks and regulate in an autocrine and/or a paracrine manner the various stages of normal human hematopoiesis.

Several cytokines, in particular tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) and <em>interferon</em>-gamma (IFN-gamma), have been shown to be responsible for pathological reactions which may lead to shock and death observed in infection with Gram-negative bacteria and in response to endotoxins (lipopolysaccharides, LPS). Priming of mice with the avirulent Bacille Calmette Guérin (BCG) vaccine strain of Mycobacterium bovis increases the sensitivity of mice to the lethal effect of LPS and results in an efficient priming for cytokine production. In response to low doses (1 microgram/mouse) of LPS, BCG-primed mice produce interleukin-12 (IL-12) which controls IFN-gamma production, as demonstrated by the ability of neutralizing anti-IL-12 antibodies to suppress IFN-gamma production. However, the concentration of the biologically active IL-12 p70 heterodimer is similar in the serum of both BCG-primed or unprimed mice, reaching levels of 1-<em>3</em> ng/ml at <em>3</em>-6 h after LPS injection, whereas IFN-gamma production was observed only in BCG-primed mice. The priming effect of BCG on IFN-gamma production appears to be mostly due to its ability to increase TNF-<em>alpha</em> production, which acts as cofactor with LPS-induced IL-12 in inducing IFN-gamma production, as shown by the ability of injection of TNF-<em>alpha</em> and LPS (1 microgram/mouse), but not LPS alone, to induce IFN-gamma production. However, in addition to TNF-<em>alpha</em>, other LPS-induced cofactor(s) are required in cooperation with IL-12 to induce optimal IFN-gamma production, because co-injection of TNF-<em>alpha</em> and IL-12, sufficient to induce serum concentrations of both cytokines higher and more persistent than those obtained by injection of LPS, was not sufficient to induce IFN-gamma production in vivo. Neutralizing anti-IL-12 antibodies, in addition to inhibiting the in vivo LPS-induced IFN-gamma production, also completely protect BCG-primed mice injected with up to 10 micrograms of LPS from shock-induced death. Thus, IL-12 is required for IFN-gamma production and lethality in an endotoxic shock model in mice.

Cells respond to viral infection or double-stranded RNA with the transcriptional induction of a subset of <em>alpha</em>/beta <em>interferon</em>-stimulated genes by a pathway distinct from the <em>interferon</em> signal pathway. The transcriptional induction is mediated through a DNA sequence containing the <em>alpha</em>/beta <em>interferon</em>-stimulated response element (ISRE). We previously identified a novel transcription factor, designated double-stranded RNA-activated factor 1 (DRAF1), that recognizes this response element. The DNA-binding specificity of DRAF1 correlates with transcriptional induction, thereby distinguishing it as a positive regulator of <em>alpha</em>/beta <em>interferon</em>-stimulated genes. Two of the components of DRAF1 have now been identified as <em>interferon</em> regulatory factor <em>3</em> (IRF-<em>3</em>) and the transcriptional coactivator CREB-binding protein (CBP)/p<em>3</em>00. We demonstrate that IRF-<em>3</em> preexists in the cytoplasm of uninfected cells and translocates to the nucleus following viral infection. Translocation of IRF-<em>3</em> is accompanied by an increase in serine and threonine phosphorylation. Coimmunoprecipitation analyses of endogenous proteins demonstrate an association of IRF-<em>3</em> with the transcriptional coactivators CBP and p<em>3</em>00 only subsequent to infection. In addition, antibodies to the IRF-<em>3</em>, CBP, and p<em>3</em>00 molecules react with DRAF1 bound to the ISRE target site of induced genes. The cellular response that leads to DRAF1 activation and specific gene expression may serve to increase host survival during viral infection.

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for <em>3</em>-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with <em>interferon</em>-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in <em>alpha</em>-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular <em>alpha</em>-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.

Poxviruses encode a large number of proteins that attenuate the inflammatory and immune responses to infection. In this report we demonstrate that a number of orthopoxviruses express a type I <em>interferon</em> (IFN)-binding protein, which is encoded by the B18R open reading frame in the WR strain of vaccinia virus. The B18R protein has significant regions of homology with the <em>alpha</em> subunits of the mouse, human, and bovine type I IFN receptors, bound human IFN <em>alpha</em> 2 with high affinity, and inhibited transmembrane signaling as demonstrated by inhibition of Fc receptor factor gamma 1/gamma 2 and <em>interferon</em>-stimulated gene factor-<em>3</em> formation as well as inhibition of the IFN <em>alpha</em> antiviral response. Among viral host response modifiers, the B18R protein is unique inasmuch as it exists as a soluble extracellular as well as a cell surface protein and thus should effectively block both autocrine and paracrine functions of IFN.

TANK-binding kinase 1 (TBK1) and IkappaB kinase epsilon (IKKepsilon) regulate the production of Type 1 <em>interferons</em> during bacterial and viral infection, but the lack of useful pharmacological inhibitors has hampered progress in identifying additional physiological roles of these protein kinases and how they are regulated. Here we demonstrate that BX795, a potent and relatively specific inhibitor of TBK1 and IKKepsilon, blocked the phosphorylation, nuclear translocation, and transcriptional activity of <em>interferon</em> regulatory factor <em>3</em> and, hence, the production of <em>interferon</em>-beta in macrophages stimulated with poly(I:C) or lipopolysaccharide (LPS). In contrast, BX795 had no effect on the canonical NFkappaB signaling pathway. Although BX795 blocked the autophosphorylation of overexpressed TBK1 and IKKepsilon at Ser-172 and, hence, the autoactivation of these protein kinases, it did not inhibit the phosphorylation of endogenous TBK1 and IKKepsilon at Ser-172 in response to LPS, poly(I:C), interleukin-1<em>alpha</em> (IL-1<em>alpha</em>), or tumor necrosis factor <em>alpha</em> and actually enhanced the LPS, poly(I:C), and IL-1<em>alpha</em>-stimulated phosphorylation of this residue. These results demonstrate that the phosphorylation of Ser-172 and the activation of TBK1 and IKKepsilon are catalyzed by a distinct protein kinase(s) in vivo and that TBK1 and IKKepsilon control a feedback loop that limits their activation by LPS, poly(I:C) and IL-1<em>alpha</em> (but not tumor necrosis factor <em>alpha</em>) to prevent the hyperactivation of these enzymes.

We have generated primary effector populations from naive CD8 T cells in response to antigen and determined their patterns of cytokine secretion upon restimulation. The effect of exogenous factors on the effector generation was examined and compared with responses of antigen-specific CD4 effectors generated under comparable conditions. CD8 cells from bm1 mice were stimulated with C57BL/6 (B6) antigen presenting cells (APCs) bearing allogeneic class I and CD8 cells from female severe combined immunodeficiency (SCID) B6 mice, transgenic for a T cell receptor <em>alpha</em>/beta (TCR-<em>alpha</em>/beta) that recognizes H-Y on Db, were stimulated with APCs from male mice. In parallel, CD4 cells from bm12 mice were stimulated with alloantigen and CD4 cells from V beta <em>3</em>/V <em>alpha</em> 11 TCR transgenics were stimulated with a peptide of pigeon cytochrome c on IEk. T cells from both transgenic mice were of naive phenotype whereas normal mice contained 10-20% memory cells. Effector CD8 populations generated were L-selectin low, CD45RB high, and CD44 high. Naive CD8 cells from SCID anti-H-Y mice made little or no cytokine immediately upon stimulation in contrast to naive CD4 which produced large amounts of interleukin 2 (IL-2). Both populations, however, generated primary effectors over 4-5 d that made substantial quantities of many cytokines upon restimulation. Both CD8 and CD4 effectors produced similar patterns of cytokines with alloantigen or specific antigen. Cytokines present during naive CD8 stimulation influenced the cytokine secretion profile of the effectors, as previously shown for CD4 cells, although secretion by CD8 effectors was generally lower than that of CD4 effectors. CD8 cells cultured with IL-2 alone made predominantly <em>interferon</em> gamma (IFN-gamma) and no IL-4 or IL-5, similar to CD4 cells. Priming with IFN-gamma increased IFN-gamma secretion from CD4 effectors, but had little if any effect on CD8 cells. In contrast, priming with IL-12 generated CD8 effectors, as well as CD4 effectors, producing elevated quantities of IFN-gamma, with similar levels from both the CD4 and CD8 populations. The presence of IL-4 during effector cell generation promoted synthesis of IL-4 and IL-5 from both CD8 and CD4 cells while downregulating IFN-gamma secretion. CD8 cells made only small amounts of IL-4, more than 100-fold less than CD4 cells, whereas significant levels of IL-5 were induced, only <em>3</em>-10-fold lower than from CD4.(ABSTRACT TRUNCATED AT 400 WORDS)

Rip1 is required for IkappaB kinase activation in response to tumor necrosis factor <em>alpha</em> (TNF-<em>alpha</em>) and has been implicated in the Toll-like receptor <em>3</em> (TLR<em>3</em>) response to double-stranded RNA. Cytokine production is impaired when rip1-/- cells are treated with TNF-<em>alpha</em>, poly(I-C), or lipopolysaccharide, implicating Rip1 in the Trif-dependent TLR<em>3</em> and TLR4 pathways. To examine the role of Rip1 in the Trif-dependent TLR4 pathway, we generated rip1-/- MyD88-/- cells. Lipopolysaccharide failed to stimulate NF-kappaB activation in rip1-/-MyD88-/- cells, revealing that Rip1 is also required for the Trif-dependent TLR4-induced NF-kappaB pathway. In addition to activating NF-kappaB, TLR<em>3</em>/4 pathways also stimulate <em>interferon</em> regulatory factor <em>3</em> activation. However, we find that Rip1 expression stimulates NF-kappaB but not <em>interferon</em> regulatory factor <em>3</em> activity. In the TNF-<em>alpha</em> pathway, Rip1 interacts with the E<em>3</em> ubiquitin ligase Traf2 and is modified by polyubiquitin chains. Upon TLR<em>3</em> activation, Rip1 is also modified by polyubiquitin chains and is recruited to TLR<em>3</em> along with Traf6 and the ubiquitin-activated kinase Tak1. These studies suggest that Rip1 uses a similar, ubiquitin-dependent mechanism to activate IkappaB kinase-beta in response to TNF-<em>alpha</em> and TLR<em>3</em> ligands.

Human peripheral blood mononuclear cells (PBMC) were induced by recombinant interleukin 2 and mitogens to secrete two distinct cytotoxic polypeptides, tumor necrosis factor-<em>alpha</em> (TNF-<em>alpha</em>) and tumor necrosis factor-beta (TNF-beta), previously called lymphotoxin. Treatment of PBMC with recombinant human interleukin 2 (rIL 2) or mitogens in combination with recombinant human <em>interferon</em>-gamma (rIFN-gamma) resulted in augmented production of both TNF-<em>alpha</em> and TNF-beta. rIFN-gamma alone had no effect on production of either cytotoxic polypeptide. TNF-<em>alpha</em> was produced within 2 to <em>3</em> hr after induction and was the major cytotoxin produced by PBMC during the first 48 hr of culture, after which time TNF-beta became the predominant species. TNF-beta was first secreted into the media after 8 hr of induction. Enhanced levels of both TNF-<em>alpha</em> and TNF-beta were seen when the PBMC were separated into adherent and nonadherent cells. Both TNF-<em>alpha</em> and TNF-beta were induced in different tumor cell lines of hematopoietic origin. The results demonstrate that the production of TNF-<em>alpha</em> and TNF-beta can be enhanced by two lymphokines, IL 2 and IFN-gamma.

Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) has been shown to antagonize numerous cellular pathways, including the antiviral <em>interferon</em>-<em>alpha</em> response. However, the capacity of this protein to interact with the viral polymerase suggests a more direct role for NS5A in genome replication. In this study, we employed two bacterially expressed, soluble derivatives of NS5A to probe for novel functions of this protein. We find that NS5A has the capacity to bind to the <em>3</em>'-ends of HCV plus and minus strand RNAs. The high affinity binding site for NS5A in the <em>3</em>'-end of plus strand RNA maps to the polypyrimidine tract, an element known to be essential for genome replication and infectivity. NS5A has a preference for single-stranded RNA containing stretches of uridine or guanosine. Values for the equilibrium dissociation constants for high affinity binding sites were in the 10 nM range. Two-dimensional gel electrophoresis followed by Western blotting revealed the presence of unphosphorylated NS5A in Huh-7 cells stably expressing the subgenomic replicon. Moreover, RNA immunoprecipitation and NS5A pull-down experiments showed the capacity of replicon-derived NS5A to bind to synthetic RNA and the HCV genome, respectively. Deletion of all of the casein kinase II phosphorylation sites in NS5A supported stable replication of a subgenomic replicon in Huh-7. However, this derivative could not be labeled with inorganic phosphate, suggesting that extensive phosphorylation of NS5A is not required for the replication functions of NS5A. The discovery that NS5A is an RNA-binding protein defines a new functional target for development of agents to treat HCV infection and a new structural class of RNA-binding proteins.

1046 base-pairs (bp) of genomic DNA spanning the first exon of the human <em>alpha</em>/beta-<em>interferon</em> (IFN)-inducible gene 6-16 have been analysed for their role in induction. The whole gene or 5'-flanking deletion derivatives of it were assayed for inducibility in populations of stably transfected mouse cells. 5'-Flanking DNA fragments were assayed for their ability to confer inducibility on a reporter gene in stably and transiently transfected mouse and human cells. The data suggest that a <em>3</em>9 bp sequence is sufficient to confer transcriptional inducibility and can account in large part for the response of 6-16. Two copies of this sequence, one of which contains a dinucleotide insert, are located in tandem 88 bp upstream of the 6-16 transcriptional initiation site. For at least one of the repeat units the 5' limit of a subregion required for induction lies in the sequence GGGAAAAT. The motif GGAAA occurs in several well characterized enhancers. Furthermore, one residue <em>3</em>' of the GGAAA there is a second motif, TGAAACT, which is conserved in the regulatory regions of other IFN-induced genes. In gel retardation assays the oligonucleotide GGGAAAATGAAACT competes with the repeat element for binding to IFN-modulated protein(s) but a mutated oligonucleotide, GGGAAAATGACACT does not. These results identify an <em>alpha</em>/beta IFN response element partially homologous to those described previously for the genes of the MHC complexes.

The interaction of <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) with a specific cell-surface receptor elicits physiological changes that rely on rapid transcriptional activation of a group of IFN-<em>alpha</em>-stimulated genes (ISGs). The IFN-stimulated response element (ISRE), a conserved regulatory element of all ISGs, is the target for transcriptional activation by the positive regulator IFN-stimulated gene factor-<em>3</em> (ISGF<em>3</em>). We reported previously that post-translational activation of ISGF<em>3</em> in the cytoplasm of IFN-<em>alpha</em>-treated cells requires two cytoplasmic activities (ISGF<em>3</em> <em>alpha</em> and ISGF<em>3</em> gamma) to produce an ISRE-binding complex that accumulates in the nucleus. In this study, we show that these activities are actually distinct subunits of the ISGF<em>3</em> complex, which associate through noncovalent interaction. Sedimentation analysis, protein renaturation, and photoaffinity cross-linking of enriched preparations of cytoplasmic ISGF<em>3</em> <em>alpha</em> and ISGF<em>3</em> gamma and of nuclear ISGF<em>3</em> demonstrated that ISGF<em>3</em> gamma was a 48-kD polypeptide with intrinsic, low-affinity DNA-binding activity. Four polypeptides of 48, 84, 91, and 11<em>3</em> kD bound to the ISRE in vitro; the larger three polypeptides most likely compose the ISGF<em>3</em> <em>alpha</em> component. These ISGF<em>3</em> <em>alpha</em> polypeptides were unable to bind DNA alone but formed a DNA-binding complex in conjunction with ISGF<em>3</em> gamma. The resulting heteromeric complex had the same ISRE-binding specificity as the individual ISGF<em>3</em> gamma polypeptide but approximately 25-fold higher affinity. Whereas ISGF<em>3</em> gamma partitioned between the cytoplasm and nucleus in unstimulated cells, ISGF<em>3</em> <em>alpha</em> was stimulated to translocate to the nucleus only following IFN-<em>alpha</em> treatment, resulting in preferential nuclear accumulation of both ISGF<em>3</em> <em>alpha</em> and ISGF<em>3</em> gamma as a stable ISGF<em>3</em>-ISRE complex. This regulated nuclear translocation of an activated transcription factor subunit maintained the specificity and rapidity of the IFN-<em>alpha</em> signaling pathway.

Differential screening of a cDNA library constructed from human umbilical vein endothelial cells exposed for 1 h to interleukin-1 beta (IL-1 beta) has led to the identification of a novel gene (PTX<em>3</em>) related to pentaxins (C-reactive protein and serum amyloid P component in man), a subclass of acute phase proteins. Sequencing of the full-length cDNA clone and RNase mapping revealed that the PTX<em>3</em> transcript is 1861 base pairs long and has a unique transcription start site. The predicted protein sequence of <em>3</em>81 amino acids is highly similar to pentaxins in its COOH-terminal half where it also contains a typical 8-amino acid "pentaxin signature" sequence. The NH2-terminal half of PTX<em>3</em> shows no similarity to any known protein sequence and initiates with a putative signal peptide indicating that PTX<em>3</em> is secreted. The genome of PTX<em>3</em> is organized into three exons. Interestingly, the region of homology between PTX<em>3</em> and pentaxins corresponds to the third PTX<em>3</em> exon. The PTX<em>3</em> gene has been localized on human chromosome <em>3</em> band q25 by Southern blots of somatic cell hybrids and by in situ hybridization. The PTX<em>3</em> mRNA is induced in endothelial, hepatic, and fibroblastic cells by IL-1 beta and tumor necrosis factor <em>alpha</em> but not by IL-6 and <em>interferon</em>-gamma. PTX<em>3</em> may represent a novel marker of inflammatory reactions, particularly those involving the vessel wall.

The expression of specific tumor necrosis factor (TNF) membrane receptors and biological effects of recombinant TNF (rTNF)-<em>alpha</em> on normal human T lymphocytes were studied. Although resting T cells lacked specific binding capacity for rTNF-<em>alpha</em>, high affinity (Kd 70 pM) TNF receptors were de novo induced upon primary activation of T cells. Comparison of TNF receptor expression with that of high affinity interleukin 2 (IL-2) and <em>interferon</em>-gamma (IFN-gamma) receptors, respectively, revealed similarities to IL 2-receptor expression with respect to kinetics of induction. However, maximum expression of TNF receptors (approximately equal to 5000/cell at day 6) and subsequent decline occurred approximately <em>3</em> days after the peak of IL 2-receptor expression. In contrast, no change in the expression of IFN-gamma receptors (Kd 10 pM, <em>3</em>00 to 400 receptors/cell) was found in the course of T cell activation. On activated TNF receptor positive T cells, TNF-<em>alpha</em> exerted multiple stimulatory activities. Thus TNF increased the expression of HLA-DR antigens and high affinity IL 2 receptors. As a consequence, TNF-treated T cells showed an enhanced proliferative response to IL 2. Moreover, TNF-<em>alpha</em> was effective as a co-stimulator of IL 2-dependent IFN-gamma production. These data indicate that TNF-<em>alpha</em> may regulate growth and functional activities of normal T cells.

<em>Interferon</em> regulatory factor (IRF)-<em>3</em> is a master transcription factor that activates host antiviral defense programs. Although cell culture studies suggest that IRF-<em>3</em> promotes antiviral control by inducing <em>interferon</em> (IFN)-beta, near normal levels of IFN-<em>alpha</em> and IFN-beta were observed in IRF-<em>3</em>(-/-) mice after infection by several RNA and DNA viruses. Thus, the specific mechanisms by which IRF-<em>3</em> modulates viral infection remain controversial. Some of this disparity could reflect direct IRF-<em>3</em>-dependent antiviral responses in specific cell types to control infection. To address this and determine how IRF-<em>3</em> coordinates an antiviral response, we infected IRF-<em>3</em>(-/-) mice and two primary cells relevant for West Nile virus (WNV) pathogenesis, macrophages and cortical neurons. IRF-<em>3</em>(-/-) mice were uniformly vulnerable to infection and developed elevated WNV burdens in peripheral and central nervous system tissues, though peripheral IFN responses were largely normal. Whereas wild-type macrophages basally expressed key host defense molecules, including RIG-I, MDA5, ISG54, and ISG56, and restricted WNV infection, IRF-<em>3</em>(-/-) macrophages lacked basal expression of these host defense genes and supported increased WNV infection and IFN-<em>alpha</em> and IFN-beta production. In contrast, wild-type cortical neurons were highly permissive to WNV and did not basally express RIG-I, MDA5, ISG54, and ISG56. IRF-<em>3</em>(-/-) neurons lacked induction of host defense genes and had blunted IFN-<em>alpha</em> and IFN-beta production, yet exhibited only modestly increased viral titers. Collectively, our data suggest that cell-specific IRF-<em>3</em> responses protect against WNV infection through both IFN-dependent and -independent programs.

Poxviruses encode proteins that suppress host immune responses, including secreted decoy receptors for pro-inflammatory cytokines such as interleukin-1 (IL-1) and the vaccinia virus proteins A46R and A52R that inhibit intracellular signaling by members of the IL-1 receptor (IL-1R) and Toll-like receptor (TLR) family. In vivo, the TLRs mediate the innate immune response by serving as pathogen recognition receptors, whose oligomerized intracellular Toll/IL-1 receptor (TIR) domains can initiate innate immune signaling. A family of TIR domain-containing adapter molecules transduces signals from engaged receptors that ultimately activate NF-kappaB and/or <em>interferon</em> regulatory factor <em>3</em> (IRF<em>3</em>) to induce pro-inflammatory cytokines. Data base searches detected a significant similarity between the N1L protein of vaccinia virus and A52R, a poxvirus inhibitor of TIR signaling. Compared with other poxvirus virulence factors, the poxvirus N1L protein strongly affects virulence in vivo; however, the precise target of N1L was previously unknown. Here we show that N1L suppresses NF-kappaB activation following engagement of Toll/IL-1 receptors, tumor necrosis factor receptors, and lymphotoxin receptors. N1L inhibited receptor-, adapter-, TRAF-, and IKK-<em>alpha</em> and IKK-beta-dependent signaling to NF-kappaB. N1L associated with several components of the multisubunit I-kappaB kinase complex, most strongly associating with the kinase, TANK-binding kinase 1 (TBK1). Together these findings are consistent with the hypothesis that N1L disrupts signaling to NF-kappaB by Toll/IL-1Rs and TNF superfamily receptors by targeting the IKK complex for inhibition. Furthermore, N1L inhibited IRF<em>3</em> signaling, which is also regulated by TBK1. These studies define a role for N1L as an immunomodulator of innate immunity by targeting components of NF-kappaB and IRF<em>3</em> signaling pathways.

Viral infections trigger innate immune responses, including the production of type I <em>interferons</em> (IFN-<em>alpha</em> and -beta) and other proinflammatory cytokines. Novel antiviral cytokines IFN-lambda1, IFN-lambda2, and IFN-lambda<em>3</em> are classified as type III IFNs and have evolved independently of type I IFNs. Type III IFN genes are regulated at the level of transcription and induced by viral infection. Although the regulatory mechanism of type I IFNs is well elucidated, the expression mechanism of IFN-lambdas is not well understood. Here, we analyzed the mechanism by which IFN-lambda gene expression is induced by viral infections. Loss- and gain-of-function experiments revealed the involvement of RIG-I (retinoic acid-inducible gene I), IPS-1, TBK1, and <em>interferon</em> regulatory factor-<em>3</em>, key regulators of the virus-induced activation of type I IFN genes. Consistent with this, a search for the cis-regulatory element of the human ifnlambda1 revealed a cluster of <em>interferon</em> regulatory factor-binding sites and a NF-kappaB-binding site. Functional analysis demonstrated that all of these sites are essential for gene activation by the virus. These results strongly suggest that types I and III IFN genes are regulated by a common mechanism.

There is now evidence that repeated administration of <em>interferon</em>-<em>alpha</em> (IFN-<em>alpha</em>) to patients with chronic active hepatitis and cancers induces depressive symptoms. There is also evidence that induction of the cytokine network modulates the serotonergic system and that major depression is related to activation of the cytokine network and disturbances in the serotonergic metabolism. The aims of this study were to examine the effects of IFN-<em>alpha</em>-based immunotherapy on the development of depressive symptoms in relation to its effects on plasma tryptophan and kynurenine and serum serotonin (5-HT). Eighteen patients affected by chronic active hepatitis C were treated with IFN-<em>alpha</em> (<em>3</em>-6 million units subcutaneously three to six times a week for 6 months) and had measurements of the previous parameters before starting immunotherapy and 2, 4, 16, and 24 weeks later. Severity of depression and anxiety were measured with the Montgomery-Asberg Depression Rating Scale (MADRS) and the Hamilton Rating Scale for Anxiety (HAM-A) scale, respectively. Immunochemotherapy with IFN-<em>alpha</em> (1) significantly increased the MADRS and HAM-A scores and serum kynurenine concentrations and (2) significantly reduced plasma tryptophan and serum 5-HT concentrations. IFN-<em>alpha</em>-based immunotherapy significantly increased the kynurenine per tryptophan quotient, which estimates the activity of indoleamine 2,<em>3</em>-dioxygenase, the major tryptophan-catabolizing enzyme, which is induced by IFNs. There are significant relationships between the IFN-<em>alpha</em>-induced changes in the MADRS score and serum kynurenine (positive) and 5-HT (negative) concentrations. Immunotherapy with IFN-<em>alpha</em> significantly increases the severity of depressive symptoms. The latter is related to changes in the serotonergic system, such as depletion of serum 5-HT and induction of the catabolism of tryptophan to kynurenine. It is suggested that the IFN-<em>alpha</em>-induced changes in the serotonergic turnover could play a role in the development of IFN-<em>alpha</em>-induced depressive symptoms.