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Toll–IL-1–resistance (TIR) domain–containing adaptor-inducing IFN-β (TRIF)–related adaptor molecule (TRAM) is the fourth TIR domain–containing adaptor protein to be described that participates in Toll receptor signaling. Like TRIF, TRAM activates interferon regulatory factor (IRF)-3, IRF-7, and NF-κB-dependent signaling pathways. Toll-like receptor (TLR)3 and 4 activate these pathways to induce IFN-α/β, regulated on activation, normal T cell expressed and secreted (RANTES), and γ interferon–inducible protein 10 (IP-10) expression independently of the adaptor protein myeloid differentiation factor 88 (MyD88). Dominant negative and siRNA studies performed here demonstrate that TRIF functions downstream of both the TLR3 (dsRNA) and TLR4 (LPS) signaling pathways, whereas the function of TRAM is restricted to the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor–like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These studies suggest that TRIF and TRAM both function in LPS-TLR4 signaling to regulate the MyD88-independent pathway during the innate immune response to LPS.

Certain C8-substituted and N7, C8-disubstituted guanine ribonucleosides comprise a class of small molecules with immunostimulatory activity. In a variety of animal models, these agents stimulate both humoral and cellular immune responses. The antiviral actions of these guanosine analogs have been attributed to their ability to induce type I IFNs. However, the molecular mechanisms by which the guanosine analogs potentiate immune responses are not known. Here, we report that several guanosine analogs activate Toll-like receptor 7 (TLR7). 7-Thia-8-oxoguanosine, 7-deazaguanosine, and related guanosine analogs ac- trihydrochloride)} were not inhibited by chloroquine, whereas TLR9 activation by CpG oligodeoxynucleotides was abolished. In summary, we present evidence that guanosine analogs activate immune cells via TLR7 by a pathway that requires endosomal maturation. Thus, the B cell-stimulating and antiviral activities of the guanosine analogs may be explained by their TLR7-activating capacity.

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Rat astrocytes, immunologically competent glial cells of the central nervous system (CNS), released a variety of cytokines after activation. Lipopolysaccharidestimulated astrocytes produced tumor necrosis factor (TNF) as demonstrated by Northern blot analysis using a mouse TNF probe and by functional assay. Biological activity of rat astrocyte-derived TNF was neutralized by rabbit antiserum against recombinant murine TNF. Stimulation of astrocytes by lipopolysaccharide also activated the interleukin 1 and interleukin 6 genes. We have also investigated whether a neurotropic paramyxovirus, Newcastle disease virus, triggers cytokine production by astrocytes. This virus induced astrocytes to produce TNF, lymphotoxin, interleukin 6, and a-and 13-interferons.Thus, stimulation by endotoxin and virus activated distinct, yet overlapping, sets of cytokine genes. We propose that astrocytes and the cytokines they produce may play a significant role in the pathogenesis of immunologically and/or virally mediated CNS disease, in CNS intercellular communication, and in the interactions between the nervous and immune systems.Astrocytes are macroglial cells of the central nervous system (CNS) that express a variety of immunological characteristics. Astrocytes stimulated with y-interferon (IFN-y) express class I and class II major histocompatibility complex (MHC) antigens in rodents (1-3). Astrocytes expressing class II antigens can present foreign antigen to T cells in a MHCrestricted fashion (3,4). Lipopolysaccharide (LPS) stimulates astrocytes to produce prostaglandins (5), complement components C3 and factor B (6), and cytokines with biological activities similar to interleukin 1 (IL-1) (5) and IL-3 (7). These observations indicate that astrocytes are immunologically competent cells that share many important functional characteristics with macrophages.Accumulating evidence has revealed that astrocytes, like macrophages (8) MATERIALS AND METHODSCell Cultures. Primary cultures of rat astrocytes were established as described (13) (13). WEHI 164 clone 13, a murine fibrosarcoma line (14), was used for TNF functional assays.RNA Preparation and Analysis. Total RNA was isolated from cells by the guanidinium isothiocyanate method (15). RNA was denatured by formaldehyde treatment, electrophoresed through a 0.8% agarose gel, and transferred to nitrocellulose as described (16,17). The transferred RNA blots were hybridized with probes of high specific activity. Membranes probed with 32P-labeled DNA fragments were hybridized for 2 days at 370C and washed at 550C, twice in 2X SSC (ix SSC = 0.15 M NaC1/0.015 M sodium citrate)/0.1% SDS and twice in 0.5x SSC/0.1% SDS. Membranes probed with 32P-labeled RNA were hybridized overnight at 650C and washed at 650C as described above.Probes. Mouse cytokine probes were used in all experiments. DNA probes for IL-la, IL-1f3, IL-3, and lymphotoxin were constructed by using an oligolabeling reaction kit (Pharmacia), and RNA probes for TNF and the type I IFN were prepared using an RNA probe vector s...

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Recognition of microbial components by APCs and their activation through Toll-like receptors (TLR) leads to the induction of adaptive immune responses. In this study, we show that activation of TLR2 by its synthetic ligand Pam3Cys, in contrast to activation of TLR9 by immunostimulatory DNA (ISS-ODN), induces a prominent Th2-biased immune response. Activation of APCs by Pam3Cys resulted in the induction of Th2-associated effector molecules like IL-13, and IL-1β, GM-CSF and up-regulation of B7RP-1, but low levels of Th1-associated cytokines (IL-12, IFNα, IL-18, IL-27). Accordingly, TLR2 ligands aggravated experimental asthma. These data indicate that the type of TLR stimulation during the initial phase of immune activation determines the polarization of the adaptive immune response and may play a role in the initiation of Th2-mediated immune disorders, such as asthma.

Interferon regulatory factors (IRFs) are critical components of virus-induced immune activation and type I interferon regulation. IRF3 and IRF7 are activated in response to a variety of viruses or after engagement of Toll-like receptor (TLR) 3 and TLR4 by double-stranded RNA and lipopolysaccharide, respectively. The activation of IRF5, is much more restricted. Here we show that in contrast to IRF3 and IRF7, IRF5 is not a target of the TLR3 signaling pathway but is activated by TLR7 or TLR8 signaling. We also demonstrate that MyD88, interleukin 1 receptor-associated kinase 1, and tumor necrosis factor receptor-associated factor 6 are required for the activation of IRF5 and IRF7 in the TLR7 signaling pathway. Moreover, ectopic expression of IRF5 enabled type I interferon production in response to TLR7 signaling, whereas knockdown of IRF5 by small interfering RNA reduced type I interferon induction in response to the TLR7 ligand, R-848. IRF5 and IRF7, therefore, emerge from these studies as critical mediators of TLR7 signaling.Members of the Toll-like receptor family are essential recognition and signaling components of mammalian anti-viral host defense (1). TLR3, 1 TLR7, TLR8, and TLR9 recognize viral nucleic acids and induce type I IFNs. TLR7 and TLR8 are similar in sequence and together with TLR9 form an evolutionarily related subgroup within the TLR superfamily (2, 3).Whereas unmethylated CpG DNA (4), herpes simplex virus (HSV) type 1 (5), and HSV type 2 genomic DNA (6) specifically stimulate TLR9 (7, 8), TLR7 is activated by infections with single-stranded RNA viruses, including influenza virus and vesicular stomatitis virus (VSV) (7, 9). Consequently, plasmacytoid dendritic cells (pDCs) from TLR7-deficient mice fail to produce type I IFNs upon infection with influenza virus or VSV (7, 10). In addition to single-stranded RNA, the synthetic imidazoquinoline, imiquimod, a low molecular weight immune response modifier, activates TLR7 in both humans and mice, whereas its derivative resiquimod (R-848) activates TLR7 and TLR8 in humans but only TLR7 in mice (10, 11). Both imiquimod and R-848 elicit robust anti-viral and anti-tumor immune responses in vivo, which correlate with a strong induction of type I IFNs (12-14). As a consequence of this activity, imiquimod is used for the treatment of external genital warts caused by human Papillomavirus (15).Interferon regulatory factors (IRFs) coordinate the expression of type I IFNs (16 -19) as well as chemokines such as IP-10 and RANTES (regulated on activation normal T cell expressed and secreted) (20 -22). Viral infections, dsRNA, or LPS signaling can activate . In contrast, the activation of IRF5, another member of the IRF family, is much more restricted. Only certain viruses, including Newcastle disease virus (NDV), VSV, and herpes simplex virus type 1, have been shown to activate IRF5 (22), whereas Sendai virus (SeV) and dsRNA poly(I) poly(C) (pI:C), which activate IRF3 and IRF7, do not activate IRF5 (22). These observations suggest that IRF5 is activated by distinct...