Toll-like receptor 9 (TLR9) is present in murine liver sinusoidal endothelial cells (LSECs) and mediates the effect of CpG-oligonucleotides
Introduction
The liver is the primary site of blood clearance of microbial products, including LPS [1] and bacterial DNA [2]. Thus liver sinusoidal endothelial cells (LSECs) may represent an important interface between the host and the pathogens.
The immunostimulatory activity of bacterial DNA is attributed to the presence of unmethylated CpG motifs, that are abundant in bacterial DNA. Synthetic oligonucleotides containing unmethylated CpG motifs (CpGs) can mimic the ability of bacterial DNA to activate immune cells such as macrophages, B-cells, NK-cells and dendritic cells to produce cytokines [3], [4], [5]. The administration of CpG DNA induces potent Th1-like immune responses that are protective against several immune disorders in animal models [6]. Therefore CpG DNA may represent a clinically useful adjuvant for vaccines against infectious diseases and cancer [7].
Toll-like receptors (TLRs) are a recently discovered family of transmembrane proteins that play a principal role in distinct pathogen recognition and the initiation of innate immune responses [5]. To date, ten different human TLRs have been cloned, each of which are involved in the recognition of pathogen-derived material [8], [9], [10].
TLR9, a member of the TLR family, mediates signaling of CpGs. Recent studies have demonstrated that mice deficient in TLR9 were unresponsive to CpGs despite normal activation by LPS [11]. Internalization of CpGs by receptor-mediated endocytosis and subsequent endosomal maturation seems to be required for activity since inhibitors of endosomal maturation abolish CpG-mediated cell activation [12], [13]. Upon recognizing CpGs, intracellular TLR9 recruits adaptor protein, myeloid differentiation marker 88 (MyD88), and activates MAPKs and NF-κB through the MyD88/IRAK/TRAF6/TAK1 kinase cascade [14], [15].
Activation of the NF-κB pathway leads to production of inflammatory mediators such as IL-1, IL-6, IL-8, IL-12, TNF-α, chemokines and induction of costimulatory molecules [16].
Previous studies on distribution of intravenously administered DNA oligonucleotides revealed that the majority of the injected material was taken up by LSECs [17], [18]. In the present study we demonstrate for the first time that functional TLR9 is present in LSECs. We also present evidence that CpGs are first taken up by LSECs scavenger receptor(s) (SR) and then delivered to endo/lysosomally located TLR9, leading to signal transduction and subsequent production of IL-1β and IL-6.
Section snippets
Reagents
Percoll and Na 125I were from Amersham-Pharmacia Biotech (Uppsala, Sweden); BSA, chloroquine, monensin, LPS and Limulus-test kit from Sigma-Aldrich (Oslo, Norway); Collagenase P from Roche Diagnostics Gmbh (Mannheim, Germany), and Iodogen from Pierce Biotechnology Inc. (Rockford, IL, USA). Vitrogen® 100 was from Cohesion Technologies (Palo Alto, CA, USA) and RPMI 1640 from Gibco BRL (Roskilde, Denmark). CpGs: Flu 1668 (5′-T*C*C*-A*TG-ACG-TTC-CTG-A*T*G*-C*T-3′), 5′FITC labeled Flu 1668
In vivo distribution and serum clearance
Intravenously injected 125I-CpGs were cleared from the blood, with 50% remaining in the blood after 4 min (Fig. 1A). This rapid clearance is also observed upon intravenous injections of hyaluronan [21], FITC-collagen-α-chains [22] and AGE-BSA [23] that are normally cleared from circulation by LSECs. By 10 min liver and kidney contained most ligand, (>50 and 37% of recovered radioactivity), with minor amounts in other organs (Fig. 1B).
In vivo and in vitro uptake of FITC-CpGs
Intraveneous injection of FITC-CpGs was performed to determine
Discussion
The present study confirms that the liver is the main organ of uptake of circulating CpGs in mice [2], [17]. The kidneys also take up large amounts of CpGs. Butler et al. [2] showed that the main site of accumulation of oligonucleotides in the kidney is the epithelia of the glomerular capsule and proximal tubule, where most reabsorption occurs. We here show that 50% of the injected 125I-CpGs are eliminated from the blood 4 min after injection. This clearance is similar to the rate of other
Acknowledgements
This project was supported by the Norwegian Research Council (grant no. 146804/120 and grant no. 149091) and the Norwegian Cancer Society.
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