Although there is increasing evidence of the role of extracellular Hsp72 in propagating the inflammatory response to stress in cells of the innate immune system, there are no published data of its direct effects on parenchymal cells. We show here for the first time that extracellular Hsp72, when added to human bronchial epithelial cells, induced the inflammatory cascade, increasing IL-8 gene expression via activation of the NF-κB pathway. Furthermore, we show that this biological effect is operative in vivo, given that direct inhalation of Hsp72 in mice increased cytokine expression and neutrophilia in the airway. The central role of the lung epithelium in this process is supported by the demonstration that isolated MTECs responded to Hsp72 stimulation in the form of cytokine production. In the absence of TLR4, that response is lost both in vivo as well as in MTEC cultured from TLR4 mutants, further supporting the importance of these cells and their TLR4 receptors in initiation of the cytokine response to Hsp72. Thus, lung epithelium can now be classified as a biological target of extracellular Hsp72, along with cells of the innate immune system. The implication of this finding is that other parenchymal cells may respond to the “danger signal” of extracellular Hsp72.
One interesting concept that has arisen through investigations into extracellular Hsp72 is the idea of Hsp72 as an endogenous TLR4 ligand (16
). Given the shared TLR4 and NF-κ
B pathways between LPS and Hsp72, this concept may help to explain the similar pathophysiologic responses of infectious and noninfectious mechanisms of ALI. Any stress in the lung, either ischemia, heat, hypoxia, or others, could lead to the release of extracellular Hsp72 by an as yet unidentified cell type and mechanism, subsequently activating the TLR4 receptor pathway on neighboring cells and modulating the inflammatory cascade in a way that parallels the effect of infection and LPS stimulation. One limitation of this study is that we focused exclusively on the role of TLR4 in Hsp72-induced cytokine expression. It is possible that Hsp72 may interact with other TLRs, including TLR2, TLR7, or TLR9. In fact, studies have shown that Hsp72 binds to both TLR4 and TLR2 (17
). We did not investigate the role of TLR2 in this system and future work is needed to shed light on this important question. In addition, it is possible that other TLR ligands play a role in stabilizing Hsp72/TLR4 interactions. This was also not addressed in the current study but may shed some additional light on the mechanism by which Hsp72 binds TLR4.
Although this rationale might help explain the similar responses between LPS-induced lung injury and other forms of ALI, it also highlights the debate and controversy surrounding the role of LPS contamination in investigations into extracellular Hsp72. As LPS shares the TLR4 and NF-κ
B signaling pathways with Hsp72 and is known to contaminate most laboratory preparations of recombinant proteins, this is a reasonable concern. Because of this, we pursued multiple layers of confirmation as to the validity of our findings. The endotoxin levels of our Hsp72 product were measured independently and that amount added to cells without resultant increases in IL-8 expression. Additionally, we boiled Hsp72 (to denature the protein and leave LPS intact) which eliminated cytokine release in 16HBE14o- cells and BALB/c mice. Furthermore, polymyxin B pretreatment was performed to bind any residual LPS with no measurable decrease in Hsp72 induced IL-8 expression. Importantly, LPS signaling requires cofactors including LPS-binding protein, CD-14, and MD-2. It has been shown in primary cultures of human airway epithelium, little or no MD-2 is expressed (41
). Although we did not measure MD-2 levels, we have previously shown that the 16HBE14o- cells are minimally responsive to LPS (39
). In addition, we treated cells in serum-free conditions thus eliminating a major source of LPS-binding protein (42
). These experiments lend credence to the theory that Hsp72 and not LPS is inducing cytokine expression in airway epithelium.
Other investigators have identified Hsp72 levels of 160 ± 51 ng/ml in the BALF of patients with hydrostatic pulmonary edema vs 603 ± 153 ng/ml in patients with ALI (25
). Our initial mouse inhalation experiments were performed using 100 ng and 1 μ
g of Hsp72 per inhalation. We chose to perform the remaining experiments with 100 ng per inhalation, to keep the levels in a physiologic range. Given previously published Hsp72 levels recovered in BALF these doses seem to be appropriate and biologically relevant.
The source of extracellular Hsp72 in BALF remains somewhat unclear. Some authors have suggested that necrotic or apoptotic cells release Hsp72 into the extracellular environment (43
), but there is also evidence that active release from viable cells also occurs (6
). Furthermore, that release has been shown to involve an exosome-dependent, nonclassical protein secretory pathway in human PBMC and tumor cells (47
). Although both active and necrotic cell release mechanisms may be involved in extracellular Hsp72 signaling, the possibility of active release allows for a markedly inducible response to a variety of insults, providing amplification of a local danger signal to neighboring cells (49
Through this investigation, we have found evidence that supports the presence and biological activity of extracellular Hsp72 in the lung. We have furthermore established that the airway epithelium itself is responsive to this extracellular Hsp72 and that this cytokine response is regulated through the TLR4 and NF-κ
B pathways. The clinical applicability of this finding requires further study. Although our data would suggest that extracellular Hsp72 is responsible for inducing and propagating inflammation, a process at the heart of the pathogenesis of lung injury, Ganter et al. (25
) found extracellular Hsp72 to be a marker of improved alveolar fluid clearance and therefore recovery from lung injury. This and other clinical investigations purporting divergent effects of extracellular Hsp72 would suggest that the mere presence of Hsp72 in the extracellular milieu is not the only factor. Perhaps there exists a threshold of extracellular Hsp72 that is required to maintain adequate signaling, below which the cells are unprepared for the insult, and above which excessive inflammation and therefore increased injury occur. There remain many questions as to the secretion, function, and clinical significance of extracellular Hsp72 in the lung. Although many questions remain, our findings support the importance of extracellular Hsp72 as a functional mediator of inflammation on a parenchymal cell, expanding its breadth of impact, supporting its potential role as an endogenous danger signal, and further increasing the interest in extracellular Hsp72 as a prognostic marker and potential therapeutic target in lung injury as well as other organ system dysfunction.