Hsp72 Induces Inflammation and Regulates Cytokine Production in Airway Epithelium through a TLR4- and NF-κB-Dependent Mechanism

Abstract

Heat shock proteins are generally regarded as intracellular proteins acting as molecular chaperones; however, Hsp72 is also detected in the extracellular compartment. Hsp72 has been identified in the bronchoalveolar lavage fluid (BALF) of patients with acute lung injury. To address whether Hsp72 directly activated airway epithelium, human bronchial epithelial cells (16HBE14o-) were treated with recombinant Hsp72. Hsp72 induced a dose-dependent increase in IL-8 expression, which was inhibited by the NF-κB inhibitor parthenolide. Hsp72 induced activation of NF-κB, as evidenced by NF-κB trans-activation and by p65 RelA and p50 NF-κB1 binding to DNA. Endotoxin contamination of the Hsp72 preparation was not responsible for these effects. Next, BALB/c mice were challenged with a single intratracheal inhalation of Hsp72 and killed 4 h later. Hsp72 induced significant up-regulation of KC, TNF-α, neutrophil recruitment, and myeloperoxidase in the BALF. A similar challenge with Hsp72 in TLR4 mutant mice did not stimulate the inflammatory response, stressing the importance of TLR4 in Hsp72-mediated lung inflammation. Last, cultured mouse tracheal epithelial cells (MTEC) from BALB/c and TLR4 mutant and wild-type mice were treated ex vivo with Hsp72. Hsp72 induced a significant increase in KC expression from BALB/c and wild-type MTEC in an NF-κB-dependent manner; however, TLR4 mutant MTEC had minimal cytokine release. Taken together, these data suggest that Hsp72 is released and biologically active in the BALF and can regulate airway epithelial cell cytokine expression in a TLR4 and NF-κB-dependent mechanism.

Exosomal membrane molecules are potent immune response modulators

“…25 This response was blocked by treatment with either anti-Hsp70 antibody or anti-IL-6 antibody demonstrating that TLR-2 dependent IL-6/Stat3 pathway is involved. Besides being a chaperone, the cytokine properties of Hsp70 are also well documented in studies showing its interaction with TLR4/CD14 complexes 18,23,26 or to TLR2. 23,27 Similarly, studies by Multhoff's group showed that tumor cells that express higher Hsp70 on their surface are killed significantly better by natural-killer (NK) cells compared to tumor cells that express low Hsp70 levels.…”

“…Previous studies have demonstrated that rHsp70 can bind to different TLRs on the cell surface leading to activation of NF-kB and MAP kinase pathways. [17][18][19] Stimulation of TLRs, in general, leads to increased pro-inflammatory response including phagocytosis.20,21 Therefore, it is possible that exosomal Hsp70 interacts with TLRs on the cell surface thereby initiating signaling pathways, such as NF-kB (proposed model depicted in Fig. 2), that ultimately leads to increased phagocytosis and phago-lysosome fusion.…”

“…Increase in phagocytosis of yeast particles with rHsp70 treatment has also been shown to initiate specifically through TLR7 in competitive experiments where phagocytosis induced by rHsp70 was blocked when cells were first exposed to different TLR7 ligands.22 Although this study demonstrated a role for TLR7, it is likely that exosomal Hsp70 may as well bind to other TLRs as reported previously for rHsp70 by some studies. 18,19,23,24 These contradictions might arise due to the possible differences in confirmation of exosomal and purified Hsp70 as well as due to differences in the source and preparation of recombinant Hsp70 used in various studies. miRNA through this route.…”

TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation

“…However, several studies indicate that HSPs are recognized mainly by TLR-4. 37,38 Activation of immune cells by HSPs (and possibly by other DAMPs) via TLR-4 may raise concerns about endotoxin contamination masking the observed immunostimulatory effects. In our model, neutrophil attraction induced by apoptotic cells was independent of TLR-4, which excludes the involvement of HSP60, HSP70 and HSP72.…”

Transgenic Expression of Hsc70 in Pancreatic Islets Enhances Autoimmune Diabetes in Response to β Cell Damage

“…Primers used for hsc70 genotyping and RT-PCR were H1 (3Ј-tggcttagacaaaaaggttgg), and H2 (3Ј-cttggggatacgggttgag) and control ␤-actin primers were Act1 (3Ј-atggatgacgatatcgctgc) and Act2 (3Ј-ctagaagcacttgcggtgcac). Peptide gp [33][34][35][36][37][38][39][40][41] …”