Role of cathepsin S in ozone-induced airway hyperresponsiveness and inflammation

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Abstract

Ambient ozone has been linked to the worsening of symptoms of patients with obstructive diseases such as chronic obstructive pulmonary disease (COPD) and asthma. We investigated the role of cathepsin S on ozone-induced airway hyperresponsiveness (AHR) and inflammation, using the selective cathepsin S inhibitor, Compound A. Balb/c mice were exposed to ozone at a concentration of 3 ppm or air for 3 h, following administration by gavage of Compound A or vehicle. Bronchoalveolar lavage (BAL) was performed 3 h and 20–24 h following exposure, AHR was measured at 20–24 h only. Ozone exposure, compared to air exposure increased BAL cathepsin S levels, AHR and BAL inflammatory cells. Compound A (30 mg kg−1 p.o.) dosing compared to vehicle dosing inhibited ozone-induced AHR (−log PC100 vehicle: −0.70 ± 0.12, n = 8 vs. cathepsin S inhibitor: −1.30 ± 0.06, P < 0.001, n = 8) at 20–24 h and BAL neutrophilia at 3 h and 20–24 h (P < 0.05, n = 6). Ozone exposure increased levels of BAL cytokines IL-6, TNF-α and IFN-γ. Compound A reduced IL-6 at 3 h and 20–24 h (P < 0.05, n = 5) and TNF-α, at 20–24 h (P < 0.05, n = 6). These data indicate an important role for cathepsin S in the regulation of ozone-induced AHR and neutrophil cell recruitment and suggest that cathepsin S may be a target in the treatment of oxidative stress-induced AHR and inflammation.

Introduction

Ozone is a potent oxidising pollutant. Experimental ozone exposure induces oxidative stress, airways hyperresponsiveness (AHR) and lung neutrophilia [1], [2], [3], [4], [5], [6]. The mechanisms underlying ozone-induced AHR and inflammation are unclear, although the influx of neutrophils may be due to their recruitment by ozone-induced release of pro-inflammatory cytokines and chemokines. These include cytokine-induced neutrophil chemoattractant (CINC), MIP-2, TNF-α and IL-1β [3], [7], [8], [9], [10], [11].

Cathepsin S is a papain-like cysteine protease that is highly homologous to other cysteine protease family members, with 57% homology to cathepsin L, 49% to cathepsin K and 16% to cathepsin B. In contrast to cathepsins B and L, the tissue distribution of cathepsin S is restricted to spleen, heart and lung. Therefore, its localisation in the lung may pinpoint to an important function for cathepsin S. Thus, cathepsin S is also expressed in antigen-presenting cells (APC), such as macrophages and dendritic cells, and in MHC Class II cells, and could mediate CD4+ T-cell mediated immune responses. In addition, it is expressed in non-professional APC, such as epithelial cells [12], [13], [14]. Such distribution strongly suggests a role for cathepsin S in immune and inflammatory processes such that it is provoked by ozone exposure. While other cathepsin family members are limited to operating within the alkaline conditions of the lysosome, cathepsin S is less restricted and is bioactive in the acidic conditions of the extracellular matrix. This property of cathepsin S enables it to be active in the acidic sites of inflamed tissues, potentially exacerbating inflammatory processes and causing tissue destruction.

Previous studies indicate that cathepsin S may have an important role in lung remodelling [15], [16], [17]. It has been proposed that the protease–antiprotease imbalance may contribute to the development of emphysema [18]. Increases in cathepsin S may be IFN-γ dependent, and IFN-γ stimulates cathepsin S release from smooth muscle cells and inhibits the antiprotease secretory leukocyte protease inhibitor (SLPI) [19]. Cathepsin S knockout mice bred with IFN-γ transgenic mice are protected against IFN-γ mediated epithelial cell apoptosis and emphysema [16]. Interestingly, macrophages from cathepsin E−/− mice exhibit decreased surface receptors for TLR2 and TLR4 indicating a role for cathepsins in innate immune recognition [20]. We have recently highlighted the importance of TLRs and Myeloid Differentiation Factor (MyD)-88 in ozone-induced AHR and neutrophilia [21]. Overall, these data indicate that cathepsin S may be important in the process of lung tissue destruction in disease and may have a role in the response to oxidative stress. We, therefore, investigated the role of cathepsin S in ozone-induced AHR, neutrophilia and inflammation, using a selective cathepsin S inhibitor, Compound A.

Section snippets

Ozone exposure

Pathogen-free, 6–8-week-old male BALB/c mice (Harlan, UK) were housed within ‘maximiser’ filter-topped cages (Maximiser, Theseus caging system Inc., Hazelton, PA, USA) and were used throughout all experiments. Mice were exposed to ozone produced by an Ozoniser (Model 500 Sander Ozoniser, Germany), mixed with medical air (BOC, UK) for 3 h at a concentration of 3 parts per million (ppm) in a sealed Perspex container. Ozone concentration was continually monitored with an ozone probe (ATi

Effect of ozone on AHR, inflammatory cell recruitment and cathepsin S activity

Compared to air exposure, mice exposed to ozone required a lesser concentration of ACh to cause a 100% increase in baseline RL (−log PC100 Ozone: −1.47 ± 0.06 vs. Air: −2.12 ± 0.11; P < 0.001; Fig. 1B). Ozone increased total cells recovered from the bronchoalveolar lavage (BAL) fluid, greatest at 20–24 h following exposure (P < 0.05), as compared to air exposure. These numbers were reflected by increases in macrophages, maximal at 48 h (P < 0.05) (Fig. 1D), while neutrophil numbers were increased at 3 h,

Discussion

We have demonstrated that inhibition of cathepsin S using Compound A reduces ozone-induced airway hyperresponsiveness, BAL fluid neutrophil and macrophage numbers and reduces the inflammatory mediators, IL-6 and TNF-α, in BAL fluid. These results indicate the important contribution of cathepsin S in mediating ozone-induced effects in the airways including AHR and inflammation.

The demonstration of cathepsin S involvement was achieved by using a relatively selective inhibitor of cathepsin S that

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