The Journal of Immunology Inactivation of Human ␤-Defensins 2 and 3 by Elastolytic Cathepsins1 Clifford C. Taggart,2* Catherine M. Greene,* Stephen G. Smith,† Rodney L. Levine,‡ Paul B. McCray, Jr.,§ Shane O’Neill,* and Noel G. McElvaney* ␤-Defensins are antimicrobial peptides that contribute to the innate immune responses of eukaryotes. At least three defensins, human ␤-defensins 1, 2, and 3 (HBD-1, -2, and -3), are produced by epithelial cells lining the respiratory tract and are active toward Gram-positive (HBD-3) and Gram-negative (HBD-1, -2, and -3) bacteria. It has been postulated that the antimicrobial activity of defensins is compromised by changes in airway surface liquid composition in lungs of patients with cystic fibrosis (CF), therefore contributing to the bacterial colonization of the lung by Pseudomonas and other bacteria in CF. In this report we demonstrate that HBD-2 and HBD-3 are susceptible to degradation and inactivation by the cysteine proteases cathepsins B, L, and S. In addition, we show that all three cathepsins are present and active in CF bronchoalveolar lavage. Incubation of HBD-2 and -3 with CF bronchoalveolar lavage leads to their degradation, which can be completely (HBD-2) or partially (HBD-3) inhibited by a cathepsin inhibitor. These results suggest that ␤-defensins are susceptible to degradation and inactivation by host proteases, which may be important in the regulation of ␤-defensin activity. In chronic lung diseases associated with infection, overexpression of cathepsins may lead to increased degradation of HBD-2 and -3, thereby favoring bacterial infection and colonization. The Journal of Immunology, 2003, 170: 931–937. efensins are antimicrobial peptides that possess a high HBD-1 is constitutively expressed and is not up-regulated by positive charge and three intramolecular disulfide bacterial or cytokine stimuli (10, 11). However, it has been shown D bridges, the main subfamilies of which are the ␣- and that HBD-1 can be up-regulated by IFN-␥ and LPS in monocyte- ␤-defensins (1). Defensins possess a wide range of microbicidal derived-macrophages (12). In contrast, HBD-2 and -3 expression activity toward Gram-positive and Gram-negative bacteria as well is induced by a variety of proinflammatory stimuli, such as LPS, as against some fungi and enveloped viruses (2). The ␣-defensins IL-1␤, and TNF-␣ (13–15), with HBD-3 being particularly strongly are produced by intestinal Paneth cells and neutrophils; in the latter up-regulated by IFN-␥ (16). Although all four HBDs share some sim- they are stored as mature peptides in the azurophilic granules (3). ilar bactericidal activities, differences exist. HBD-3 has microbicidal Four have been identified in neutrophils, human neutrophil pep- activity toward Staphylococcus aureus, whereas HBD-1 and -2 have by guest on October 1, 2021. Copyright 2003 Pageant Media Ltd. tides-1, -2, -3, and -4 (HNP-1 to -4),3 and two, HD-5 and HD-6, little or no activity toward this microorganism (7). have been identified in the secretory granules of Paneth cells. (4). Defensins are part of the innate immune system, which keeps ␤-Defensins are expressed by epithelial cells of the kidneys, uro- the airways sterile and free of bacteria. However, in cystic fibrosis genital tract, respiratory epithelium, and other mucosal sites. To (CF) the pulmonary mucosal defenses are impaired, leading to date four ␤-defensins of epithelial origin have been identified, hu- bacterial colonization, initially by Haemophilus influenzae and S. man ␤-defensins 1 (HBD-1), -2, -3, and -4 (5–8). In addition, aureus and later on by Pseudomonas aeruginosa (17). This bac- recent evidence indicates that there is a much larger family of terial colonization of the lung is chronic and is associated with ␤-defensin genes in humans (9). increased morbidity and mortality in CF. Changes in ␤-defensin http://classic.jimmunol.org activity, specifically HBD-1, have been linked to the lung patho- genesis of CF. It has been proposed that the high salt concentration present in the airway surface liquid (ASL) of the lung in CF in- *Pulmonary Research Division, Department of Medicine, Royal College of Surgeons hibits the microbicidal activity of HBD-1 (18). Indeed, CF airway in Ireland, Education and Research Center, Beaumont Hospital; and †Department of Microbiology, Moyne Institute, Trinity College Dublin, Ireland; ‡Laboratory of Bio- epithelia fail to kill apically applied P. aeruginosa, contrasting chemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, with the bacterial properties of normal airway epithelia (19). It has Bethesda, MD 20892; and §Department of Pediatrics, University of Iowa College of Downloaded from Medicine, Iowa City, IA 52242 since been demonstrated that a number of antimicrobial proteins Received for publication October 7, 2001. Accepted for publication April 7, 2003. and peptides present in respiratory tract secretions are salt-sensi- tive, and it has been theorized that a high salt concentration in the The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance CF airways may compromise the microbicidal activity of many with 18 U.S.C. Section 1734 solely to indicate this fact. innate immune factors (20). 1 This work was supported by the Higher Education Authority of Ireland, the Health Proteases present in CF ASL have previously been demon- Research Board of Ireland, and the Royal College of Surgeons in Ireland. strated to cleave and inactivate antimicrobial proteins. Lactoferrin 2 Address correspondence and reprint requests to Dr. Clifford Taggart, Pulmonary and transferrin are degraded by neutrophil elastase and Pseudo- Research Division, Department of Medicine, Royal College of Surgeons in Ireland, Education and Research Center, Beaumont Hospital, Dublin 9, Ireland. E-mail ad- monas elastase, and Pseudomonas elastase cleaves lysozyme (21, dress: [email protected] 22). However, due to the small size of members of the defensin 3 Abbreviations used in this paper: HNP, human neutrophil peptide; ASL, airway family as well as their positive charge and the constrained structure surface liquid; BAL, bronchoalveolar lavage; CF, cystic fibrosis; HBD, human ␤-de- fensin; ILD, interstitial lung disease; MALDI-TOF, matrix-assisted laser desorption adopted as a result of disulfide bridging, it has been assumed that ionization time-of-flight; SLPI, secretory leukoprotease inhibitor. these peptides are resistant to proteolysis. We have previously Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 932 CATHEPSIN CLEAVAGE OF HBD-2 AND -3 demonstrated that another antiprotease/antimicrobial protein, se- bridges. Twenty-three microliters of 10% trifluoroacetic acid was added to cretory leukoprotease inhibitor (SLPI), a small m.w. protein, with each sample to bring the pH to 2–3, and the samples were analyzed by a similar charge and function as the defensins, is susceptible to matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) spectrometry using a Hewlett-Packard (model G2025A) instrument with cleavage and antiprotease inactivation by members of the elasto- ␣-cyano-4-hydroxycinnamic acid (Hewlett-Packard, Palo Alto, CA) as the lytic cathepsin family of proteases (cathepsins B, L, and S) (23). matrix. The sample and matrix (0.4 ␮l each) were mixed on the target and Cathepsins are cysteine proteases produced predominantly by dried under vacuum. The instrument was calibrated with an external stan- macrophages and are involved in matrix remodeling and Ag pro- dard mixture of peptides (Agilent Technologies, Palo Alto, CA). cessing (24). Elevated levels of active cathepsin L have been Radial diffusion assay shown in the bronchoalveolar lavage (BAL) of smokers and indi- Various concentrations of cathepsins B, L, and S (50–1000 ng) were in- viduals with emphysema due to the increased numbers of macro- cubated with a fixed amount of HBD-2 and -3 (1 ␮g) as outlined above (24 phages present, and they are thought to play an important role in hat37°C), and the reactions were stopped with the cathepsin inhibitor, ␮ the lung destruction observed in the emphysematous lung (23, 25). Z-Phe-Ala-CHN2 (50 M). The samples were then tested for microbicidal The observation of SLPI proteolysis by cathepsins B, L, and S has activity according to the method of Lehrer et al. (26). Following incuba- tion, the samples were lyophilized and reconstituted in 5 ␮l of 0.01% acetic raised the question of whether members of the defensin family are acid/0.1% human serum albumin. An exponential phase culture of PAO1 also susceptible to proteolytic cleavage by the cathepsins. We was prepared by diluting an overnight culture 1/100 in trypticase soy broth present evidence that HBD-2 and -3 are degraded by cathepsins B, until an A600 of between 0.1 and 0.4 was reached. The cells were washed L, and S, which affect the microbicidal activity of both ␤-defensins in cold sterile 10 mM sodium phosphate and resuspended at 4 ϫ 106 toward P. aeruginosa. In addition, we show that members of the CFU/ml of molten agar (10 mM sodium phosphate, 1% trypticase soy broth, and 1% agarose), poured onto sterile petri dishes, and allowed to set. elastolytic cathepsin family are present and active in CF BAL. Three-micrometer plugs were punched out of the gel and removed with a Incubation of recombinant HBD-2 and -3 with CF BAL resulted in sterile tip, into which the 5-␮l sample incubations and HBD-2 standard their degradation, which was completely (HBD-2) or partially were pipetted. The plates were incubated at 37°C for 3 h, after which each (HBD-3) inhibited using a cathepsin inhibitor. These observations plate was overlayed with more agar (8 ml of molten agar plus 2 ml of 5% sterile agarose gel). As soon as the gel solidified, the plates were in- provide evidence that small antimicrobial peptides are susceptible verted and placed in an incubator at 37°C overnight.