Recognition of double-stranded RNA by Toll-like receptor 3 (TLR3) will increase the production of cytokines and chemokines through transcriptional activation by the NF-B protein.Over 136 single-nucleotide polymorphisms (SNPs) in TLR3 have been identified in the human population. Of these, four alter the sequence of the TLR3 protein. Molecular modeling suggests that two of the SNPs, N284I and L412F, could affect the packing of the leucine-rich repeating units in TLR3. Notably, L412F is reported to be present in 20% of the population and is higher in the asthmatic population. To examine whether the four SNPs affect TLR3 function, each were cloned and tested for their ability to activate the expression of TLR3-dependent reporter constructs. SNP N284I was nearly completely defective for activating reporter activity, and L412F was reduced in activity. These two SNPs did not obviously affect the level of TLR3 expression or their intracellular location in vesicles. However, N284I and L412F were underrepresented on the cell surface, as determined by flow cytometry analysis, and were not efficiently secreted into the culture medium when expressed as the soluble ectodomain. They were also reduced in their ability to act in a dominant negative fashion on the wild type TLR3 allele. These observations suggest that N284I and L412F affect the activities of TLR3 needed for proper signaling.
These findings demonstrate that TLR3 activation by poly(I:C) modulates the local inflammatory response in the lung and suggest a critical role of TLR3 activation in driving lung function impairment. Thus, TLR3 activation may be one mechanism through which viral infections contribute toward exacerbation of respiratory disease.
Dexamethasone is known to have an inhibitory effect on IL-1 production. To determine the mechanism(s) of this inhibition, adherent human blood monocytes were stimulated with Escherichia coli lipopolysaccharide (LPS) (10 sg/ml) in the presence of dexamethasone. Nuclear transcription run-off assays showed that LPS induced IL-1IB gene transcription two-to fourfold and that this induction was unaffected by dexamethasone exposure (10-5 M). The lack of dexamethasone's transcriptional effects was further supported by the absence of any significant change in IL-I1f mRNA accumulation between LPS-stimulated monocytes exposed or unexposed to dexamethasone, as determined by Northern blot analysis.Posttranscriptionally, dexamethasone was found to have multiple effects: slight prolongation of IL-1jf mRNA half-life, moderate inhibition of translation of the IHAlfl precursor, and profound inhibition of the release of IL-igi into the extracellular fluid.The data indicate that IL-1fi is first translated as the 33,000-D pro-IL-1ft protein, the predominant intracellular form, and the processed to a 17,500-D IL-i1f protein before or during extracellular transport. The major inhibitory effects of dexamethasone appear to be directed at the translational and posttranslational steps involved in these events.
Proteins that recognize pathogen-associated molecular patterns are key factors in the cascade of events from the detection to the elimination of an invading organism. This form of innate immunity is conserved in eukaryotes. For example, the Drosophila melanogaster Toll protein is responsible for resistance to fungal and bacterial infections (3, 4), and plants can encode disease-resistance proteins that are important in determining the outcome of infection (5). The vertebrate pathogendetecting proteins called Toll-like receptors (TLRs) 5 are key players in the activation of both the innate and adaptive arms of the immune system (6 -9).The TLRs and related pathogen sensors contain leucine-rich repeat motifs that form docking sites for pathogen ligands or adaptors that bind pathogen ligands, the binding of which will activate signal transduction pathway(s) (10 -12). TLR3 recognizes double-stranded RNA and may be a part of a redundant sensor system to detect viral infections (13-15). Although specific features in the ligands required to interact with TLR3 remain to be identified, TLR3 is activated by polyinosinepolycytidylic acid (poly(I:C)) and has been reported to be activated by RNAs extracted from necrotic cells (16).A number of issues concerning TLR3 structure and function remain to be elucidated. For example, TLR3 can apparently act both on the surface of the plasma membrane, as it does in fibroblasts, and by attaching to the membranes of intracellular vacuoles, where it is proposed to act in immature dendritic cells (17,18). The trafficking of TLRs should be influenced by glycosylation in general, and N-linked glycosylation of TLR2 and TLR4 has been shown to play essential roles in its localization (19,20). A significant recent advance in TLR3 was the elucidation of a 2.1 Å structure of the soluble ectodomain by Choe et al. (1). Bell et al. (2) independently elucidated a highly similar structure. In both studies, the crystallized ectodomains were produced in a baculovirus expression system and formed a horseshoe-shaped solenoid structure that was extensively decorated with glycosyl modifications, some of which were partially resolved in the structure. Whether the glycosylations are important in TLR3 localization and/or function were not directly addressed in these works (1, 2). However, de Bouteiller et al. (21) showed that a change of Asn-247 to an arginine in TLR3 negatively affected TLR3 activity.We expressed the extracellular domain (ECD) of the human TLR3 in human embryonic kidney cells (HEK 293T) and demonstrated that it was modified with N-linked glycosylations. Using the GlcNAc-transferase inhibitor tunicamycin, a concentration-dependent inhibition of TLR3 activity was observed. Systematic mutational analysis of the predicted N-linked glycosylation sites identified two asparagine residues in leucine-rich repeats 8 and 15 that are important for TLR3 activity. The mutant proteins remain expressed at levels similar to wild type. In addition, because our ectodomain was produced in human cells as opp...
The objective of this study was to test the hypothesis that dsRNA promotes lung inflammation and alters airway responsiveness to cholinergic and -adrenergic receptor agonists in human lung slices. Human airway smooth muscle (ASM) was incubated for 24 h in poly(I:C) Ϯ TNF␣ and a TLR3 monoclonal antibody. Precision-cut lung slices (PCLS; 250-m thickness) from healthy human lungs containing a small airway were incubated in 0, 10, or 100 g/ml poly(I:C) for 24 h. Intravital microscopy of lung slices was used to quantify contractile and relaxation responsiveness to carbachol and isoproterenol, respectively. Supernatants of ASM and PCLS were analyzed for cytokine secretion using a 25-multiplex bead assay. In human ASM, poly(I:C) (0.5 g/ml) increased macrophage inflammatory protein-1␣ (MIP-1␣) and RANTES that was prevented by a TLR3 monoclonal receptor antibody. Incubation of human PCLS with poly(I:C) (10 and 100 g/ml) had little effect on the log EC50 or maximum drug effect (Emax) for contraction and relaxation in response to carbachol and isoproterenol, respectively.
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