Ligation of Toll-like receptor 3 differentially regulates M2 and M3 muscarinic receptor expression and function in human airway smooth muscle cells.
BACKGROUND
Viral infection causes asthma exacerbations and airway hyperreactivity. Toll-like receptor 3 (TLR3) recognizes double-stranded RNA (dsRNA) of viral or synthetic origin in a fashion different from protein kinase R (PKR). The aim of this study was to examine the expression and function of TLR3 in human airway smooth muscle (ASM) cells.


METHODS
Expression of TLR3 and muscarinic receptor (MR), histamine receptor (HR), and cysteinyl leukotriene receptor (CysLTR) subtypes was analyzed by quantitative real-time PCR, flow cytometry, or Western blotting. It was assessed whether ASM cells respond to polyinosinic-polycytidylic acid (poly I:C), a synthetic analog of dsRNA, with alterations in M2R, M3R, H1R, and CysLT1R expression. The function of these subtypes was evaluated by cholinergic regulation of forskolin-stimulated cyclic AMP accumulation or by mobilization of intracellular calcium upon stimulation.


RESULTS
ASM cells expressed TLR3 and PKR, and intracellular TLR3 expression was demonstrated. Poly I:C caused decreased M2R and increased M3R expression, without affecting H1R and CysLT1R expression. Poly I:C-treated cells showed decreased cholinergic inhibition of forskolin-stimulated cyclic AMP accumulation and enhanced calcium flux in response to acetylcholine, but not to histamine and LTD4. These modulating effects of poly I:C were reversed by chloroquine, but not by 2-aminopurine.


CONCLUSIONS
The data indicate that poly I:C internalized by ASM cells differentially regulates M2R and M3R expression and function by interacting with TLR3 rather than with PKR, suggesting that these changes may contribute to airway hyperreactivity.
Morishima
Hirotaka
H
Clinical Research Center for Allergy and Rheumatology, National Hospital Organization, Sagamihara Hospital, Sagamihara, Japan.
Kajiwara
Keiichi
K
Akiyama
Kazuo
K
Yanagihara
Yukiyoshi
Y
D015075:2-Aminopurine / Q000494:pharmacology; D016535:Bronchial Hyperreactivity / Q000503:physiopathology*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured / Q000187:drug effects / Q000378:metabolism; D002738:Chloroquine / Q000494:pharmacology; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects; D006632:Histamine / Q000494:pharmacology; D006801:Humans; D017998:Leukotriene D4 / Q000494:pharmacology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D018727:Muscarinic Antagonists / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D011070:Poly I-C / Q000494:pharmacology*; D043585:Receptor, Muscarinic M2 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D043587:Receptor, Muscarinic M3 / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D011969:Receptors, Histamine H1 / Q000096:biosynthesis / Q000235:genetics; D018077:Receptors, Leukotriene / Q000096:biosynthesis / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects; D051196:Toll-Like Receptor 3 / Q000502:physiology*; D019892:eIF-2 Kinase / Q000502:physiology
D008565:Membrane Proteins; D018727:Muscarinic Antagonists; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D011969:Receptors, Histamine H1; D018077:Receptors, Leukotriene; C495344:TLR3 protein, human; D051196:Toll-Like Receptor 3; D005576:Colforsin; D015075:2-Aminopurine; D017998:Leukotriene D4; D006632:Histamine; D002738:Chloroquine; D000242:Cyclic AMP; D019892:eIF-2 Kinase; C083169:leukotriene D4 receptor; D011070:Poly I-C