Submitted on April 15, 2009
Revised on May 28, 2009
Accepted on June 15, 2009
TLR3 activation stimulates cytokine secretion without altering agonist-induced human small airway contraction or relaxation
Philip R Cooper1, Roberta Lamb2, Nicole D Day2, Patrick J Branigan2, Radhika Kajekar2, Lani San Mateo3, Pamela J Hornby2, and Reynold A Panettieri, Jr.1*
1 University of Pennsylvania
2 Centocor
3 Centocor, Inc.
* To whom correspondence should be addressed. E-mail: rap{at}mail.med.upenn.edu.
Background: Respiratory infections exacerbate chronic lung diseases promoting airway inflammation and hyper-reactivity. Toll-like receptor 3 (TLR3), recognizes viral double-stranded (ds)RNA such as polyinosinic-polycytidylic acid (poly(I:C)) and stimulates innate immune responses. Objective: To test the hypothesis that dsRNA promotes lung inflammation and alters airway responsiveness to cholinergic and 
-adrenergic receptor agonists in human lung slices. Methods: Human ASM was incubated for 24 hours in poly(I:C) +/- TNF
and a TLR3 monoclonal antibody. Precision-cut lung slices (PCLS; 250 µm thickness), from healthy human lungs, containing a small airway were incubated in 0, 10 or 100 µg/ml poly(I:C) for 24 hours. 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 multi-plex bead assay. Results: In human ASM, poly(I:C) (0.5 µg/ml) increased MIP1 and RANTES that was prevented by a TLR3 monoclonal receptor antibody. Incubation of human PCLS with poly(I:C) (10 & 100 µg/ml) had little effect on the log EC50 or Emax for contraction and relaxation in response to carbachol and isoproterenol, respectively. The responsiveness of the same human PCLS to poly(I:C) incubation was confirmed by the robust increase in chemokines and cytokines. In separate experiments incubation of PCLS with IL-13 or TNF (100 ng/ml) increased airway sensitivity to carbachol. Conclusions: Poly(I:C) promotes inflammatory mediator release that was not associated with enhanced bronchoconstriction or attenuated bronchodilation in normal healthy human lung slices. Transduction at the TLR3 initiated by dsRNA stimulates downstream innate immune responses.
