Abstract

BackgroundNeutrophils play an important role in the pathophysiology of RSV, though RSV does not appear to directly activate neutrophils in the lower airways. Therefore locally produced cytokines or other molecules released by virally-infected airway epithelial cells are likely responsible for recruiting and activating neutrophils. Heat shock proteins (HSPs) are generally regarded as intracellular proteins acting as molecular chaperones; however, HSP72 can also be released from cells, and the implications of this release are not fully understood.MethodsHuman bronchial epithelial cells (16HBE14o-) were infected with RSV and Hsp72 levels were measured by Western blot and ELISA. Tracheal aspirates were obtained from critically ill children infected with RSV and analyzed for Hsp72 levels by ELISA. Primary human neutrophils and differentiated HL-60 cells were cultured with Hsp72 and supernatants analyzed for cytokine production. In some cases, cells were pretreated with polymyxin B prior to treatment with Hsp72. IκBα was assessed by Western blot and EMSA's were performed to determine NF-κB activation. HL-60 cells were pretreated with neutralizing antibody against TLR4 prior to Hsp72 treatment. Neutrophils were harvested from the bone marrow of wild type or TLR4-deficient mice prior to treatment with Hsp72.ResultsInfection of 16HBE14o- with RSV showed an induction of intracellular Hsp72 levels as well as extracellular release of Hsp72. Primary human neutrophils from normal donors and differentiated HL-60 cells treated with increasing concentrations of Hsp72 resulted in increased cytokine (IL-8 and TNFα) production. This effect was independent of the low levels of endotoxin in the Hsp72 preparation. Hsp72 mediated cytokine production via activation of NF-κB translocation and DNA binding. Using bone marrow-derived neutrophils from wild type and TLR4-mutant mice, we showed that Hsp72 directly activates neutrophil-derived cytokine production via the activation of TLR4.ConclusionCollectively these data suggest that extracellular Hsp72 is released from virally infected airway epithelial cells resulting in the recruitment and activation of neutrophils.

Highlights

  • The heat shock response is an ancient, highly conserved, endogenous cellular defense mechanism characterized by the rapid upregulation of a specific group of proteins called heat shock proteins [1]

  • We found that the preparation of respiratory syncytial virus (RSV) does contain a negligible amount of Hsp72 (0.32 ng/μl)

  • Similar to what we have previously shown in human airway epithelial cells and mouse tracheal epithelial cells [13], Hsp72-induced IL-8 gene expression in neutrophils is dependent upon activation of both Toll-like receptor (TLR)-4 and NF-κB

Read more

Summary

Introduction

The heat shock response is an ancient, highly conserved, endogenous cellular defense mechanism characterized by the rapid upregulation of a specific group of proteins called heat shock proteins [1]. Increased expression of the 72 kDa heat shock protein (Hsp72) appears to abrogate proinflammatory gene expression through inhibition of the transcription factor, NF-κB [3] Heat shock proteins, such as Hsp and Hsp appear to be intimately involved in the recognition of so-called pathogen-associated molecular patterns (PAMPs), such as lipopolysaccharide (LPS). The release of Hsp into the extracellular environment may serve to signal an impending danger signal to neighboring cells [8] In this context, Hsp is included in a growing list of so-called alarmins, a group of endogenous proteins that are released by necrotic cells or secreted via non-classical pathways in order to convey a danger signal to surrounding cells [9]. Heat shock proteins (HSPs) are generally regarded as intracellular proteins acting as molecular chaperones; HSP72 can be released from cells, and the implications of this release are not fully understood

Methods
Results
Discussion
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call