IFN-α Enhances TLR3-Mediated Antiviral Cytokine Expression in Human Endothelial and Epithelial Cells by Up-Regulating TLR3 Expression This information is current as of September 30, 2021. Jorma Tissari, Jukka Sirén, Seppo Meri, Ilkka Julkunen and Sampsa Matikainen J Immunol 2005; 174:4289-4294; ; doi: 10.4049/jimmunol.174.7.4289 http://www.jimmunol.org/content/174/7/4289 Downloaded from References This article cites 32 articles, 13 of which you can access for free at: http://www.jimmunol.org/content/174/7/4289.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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The Journal of Immunology IFN-␣ Enhances TLR3-Mediated Antiviral Cytokine Expression in Human Endothelial and Epithelial Cells by Up-Regulating TLR3 Expression1 Jorma Tissari,† Jukka Sire´n,* Seppo Meri,† Ilkka Julkunen,* and Sampsa Matikainen2* TLRs play a critical role in early innate immune response to virus infection. TLR3 together with TLR7 and TLR8 constitute a powerful system to detect genetic material of RNA viruses. TLR3 has been shown to bind viral dsRNA whereas TLR7 and TLR8 are receptors for viral single-stranded RNA. In this report we show that TLR7 or TLR8 are not expressed in human epithelial A549 cells or in HUVECs. Accordingly, A549 cells and HUVECs were unresponsive to TLR7/8 ligand R848. TLR3 was expressed at a higher level in HUVECs than in A549 cells. The TLR3 ligand poly(I:C) up-regulated IFN-␤, IL-28, IL-29, STAT1, and TLR3 expression in HUVECs but not in A549 cells. An enhanced TLR3 expression by transfection or by IFN-␣ stimulation conferred Downloaded from poly(I:C) responsiveness in A549 cells. Similarly, IFN-␣ pretreatment strongly enhanced poly(I:C)-induced activation of IFN-␤, IL-28, and IL-29 genes also in HUVECs. In conclusion, our results suggest that IFN-␣-induced up-regulation of TLR3 expression is involved in dsRNA activated antiviral response in human epithelial and endothelial cells. The Journal of Immunology, 2005, 174: 4289–4294. oll-like receptors play a crucial role in regulating the ac- (6–8). At least ssRNA from HIV and influenza virus is recognized http://www.jimmunol.org/ tivation of innate immune response to invading microbes by TLR7/8. Therefore, TLR7/8 together with TLR3 constitute a T (1). These evolutionarily well-conserved receptors recog- powerful system to detect the genetic material of RNA viruses. nize structural motifs of microbes, called pathogen-associated mi- The key cytokine that regulates innate immune responses crobial patterns (2). Pathogen-associated microbial patterns in- against viruses is IFN-␣ (9). IFN-␣ has antiviral and immunoregu- clude bacterial cell wall components of both Gram-positive and latory functions. IFN-␣ serves as an important link between innate Gram-negative bacteria, as well as flagellin, bacterial and viral and adaptive immunity (10). It activates dendritic cells (DCs),3 NK DNA and viral dsRNA and ssRNA (1). Ten human TLRs have cells, and macrophages. In synergy with IL-18, IFN-␣ enhances been characterized so far. They are predominantly expressed by IFN-␥ production in NK and T cells (11–13). IFN-␥ in turn stim- by guest on September 30, 2021 cells involved in immune functions, like in splenic macrophages ulates Ag presentation in DCs and macrophages. This results in the and peripheral blood leukocytes, as well as in tissues exposed to activation of adaptive immunity and the elimination of virus-in- the external environment such as lungs and the gastrointestinal fected cells by CTLs. Recently, a new family of type I IFN-like tract (1). TLRs are essential elements in host defense against most cytokines has been characterized (14, 15). These novel cytokines ␭ microbes by activating innate immune response which is a prereq- include IL-28A and B and IL-29 (also known as IFN- 2/3 and ␭ ␣ ␤ uisite for the induction of the adaptive immunity (3). IFN- 1, respectively). Like IFN- and IFN- , IL-28 and IL-29 Many RNA viruses express dsRNA at some point during their have antiviral activities (14, 15) but their immunoregulatory func- replication cycle, and dsRNA is involved in the activation of innate tions have not been characterized so far. immunity during virus infection. TLR3 is a receptor for viral In addition to immune cells, epithelial and endothelial cells are dsRNA and binding of dsRNA to its receptor activates IFN-␣ pro- important primary targets of virus infection. Both epithelial and duction (4). However, viruses have evolved strategies to sequester vascular endothelial cells are highly susceptible to viruses that dsRNA to avoid the activation of antiviral pathways (5). In addi- cause a systemic infection such as measles virus, and the spread of infection occurs via endothelial cells. Lung epithelial cells are pri- tion to dsRNA, also ssRNA is a molecular pattern recognized by mary targets of influenza virus infection, and they produce immu- TLRs. It has been shown that viral ssRNA is recognized by murine noregulatory cytokines, including IFN-␤, in response to the infec- TLR7 and human TLR8 which activates innate immune response tion (16). In this report we show that epithelial and endothelial cells do not express TLR7 or TLR8, which are the receptors for viral ssRNA. In contrast, TLR3 which is a receptor for dsRNA is expressed by these cells. Furthermore, TLR3 protein expression is *Department of Microbiology, National Public Health Institute, Helsinki, Finland; and †Haartman Institute, Department of Bacteriology and Immunology, University of strongly up-regulated by IFN-␣ resulting in an enhanced response Helsinki, Helsinki, Finland to viral RNA. Received for publication July 28, 2004. Accepted for publication January 10, 2005. The costs of publication of this article were defrayed in part by the payment of page Materials and Methods charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Cell culture 1 This study was supported by the Medical Research Council of the Academy of A549 human lung carcinoma cells (American Type Culture Collection; Finland, the Sigrid Juselius Foundation, and the Finnish Cancer Foundation. ATCC CCL185) were maintained in Eagle’s MEM supplemented with 0.6 2 Address correspondence and reprint requests to Dr. Sampsa Matikainen, Department of Microbiology, National Public Health Institute, Mannerheimintie 166, FI-00300, Helsinki, Finland. E-mail address: sampsa.matikainen@ktl.fi 3 Abbreviation used in this paper: DC, dendritic cell. Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 4290 IFN-␣ UP-REGULATES TLR3 EXPRESSION ␮g/ml penicillin, 60 ␮g/ml streptomycin, 2 mM L-glutamine, and 10% nucleotides (5Ј-GATCAGTTGAGGGGACTTTCCCAGCC-3Ј) were pur- heat-inactivated FCS (Integro). HUVECs (ATCC CRL1730) and HEK293 chased from DNA Technology A/S. The probes were labeled by Klenow cells (ATCC CRL1573) were maintained in RPMI 1640 medium (Sigma- fill-in reaction and the binding reaction was done at room temperature for Aldrich) with supplements described above. 30 min. The samples were analyzed by electrophoresis in 6% nondenatur- ing low-ionic strength polyacrylamide gels in 0.25ϫ TBE. The gels were Cytokines and TLR ligands dried and visualized by autoradiography. Human leukocyte IFN-␣ was provided by the Finnish Red Cross Blood Transfusion Service and was used at 100 IU/ml. IFN-␤ (Schering-Plough) Results was used at 100 IU/ml. TLR3 ligand poly(I:C) and TLR7/8 ligand R848 TLR3, TLR7, and TLR8 expression and TLR-stimulated response was purchased from Sigma-Aldrich and InVivoGen, respectively. in A549 cells and HUVECs Cloning of TLR3 transfection vector and preparation of anti- TLR3, TLR7, and TLR8 are pattern recognition receptors that de- TLR3 Abs tect viral RNA (4, 6–8). We were interested in seeing whether TLR3 cDNA, kindly provided by Dr. Kastelein (DNAX, Palo Alto, CA), epithelial or endothelial cells express these receptors that would was TAϪ cloned to pcDNA3.1/CT-GFP-TOPO vector (Invitrogen) fol- enable direct sensing of viral RNA. We have previously shown lowed by cloning to pcDNA3.1-FLAG vector (Sigma-Aldrich). The that IFN-␣ can up-regulate TLR3 and TLR7 gene expression in pcDNA3.1-TLR3-FLAG construct was transfected to HEK293 cells and human macrophages (17). Therefore, we also studied the effects of TLR3-Flag expression was confirmed with immunoblotting with anti- IFN-␣ and IFN-␤ on TLR expression in epithelial A549 cells and FLAG mAb M2 (Sigma-Aldrich). Cell lysates prepared from TLR3-Flag- transfected cells were used to analyze rabbit polyclonal anti-TLR3 Abs in HUVECs. A549 cells and HUVECs were left untreated or stim- raised against the TLR3 peptide SIQKIKNNPFVKQKNLIT (anti-TLR3-P ulated with IFN-␣ or IFN-␤ for 6 h, after which total cellular RNA Ab) or guinea pig anti-TLR3 Abs raised against an intracellular part of the was isolated and analyzed by Northern blotting. RNA from IFN- Downloaded from TLR3 (anti-TLR3-IC Ab). Intracellular TLR3 was expressed in Esche- ␣-stimulated macrophages was included as a positive control. richia coli B strain BL21(DE3) as a glutathione S-transferase fusion pro- tein and purified by a preparative SDS-PAGE (Prep-Cell; Bio-Rad).