Postsecretory Processing Generates Multiple Cathelicidins for Enhanced Topical Antimicrobial Defense This information is current as Masamoto Murakami, Belen Lopez-Garcia, Marissa Braff, of October 1, 2021. Robert A. Dorschner and Richard L. Gallo J Immunol 2004; 172:3070-3077; ; doi: 10.4049/jimmunol.172.5.3070 http://www.jimmunol.org/content/172/5/3070 Downloaded from References This article cites 35 articles, 15 of which you can access for free at: http://www.jimmunol.org/content/172/5/3070.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Postsecretory Processing Generates Multiple Cathelicidins for Enhanced Topical Antimicrobial Defense1 Masamoto Murakami, Belen Lopez-Garcia, Marissa Braff, Robert A. Dorschner, and Richard L. Gallo2 The production of antimicrobial peptides and proteins is essential for defense against infection. Many of the known human antimicrobial peptides are multifunctional, with stimulatory activities such as chemotaxis while simultaneously acting as natural antibiotics. In humans, eccrine appendages express DCD and CAMP, genes encoding proteins processed into the antimicrobial peptides dermcidin and LL-37. In this study we show that after secretion onto the skin surface, the CAMP gene product is processed by a serine protease-dependent mechanism into multiple novel antimicrobial peptides distinct from the cathelicidin LL-37. These peptides show enhanced antimicrobial action, acquiring the ability to kill skin pathogens such as Staphylococcus aureus and Candida albicans. Furthermore, although LL-37 may influence the host inflammatory response by stimulating IL-8 Downloaded from release from keratinocytes, this activity is lost in subsequently processed peptides. Thus, a single gene product encoding an important defense molecule alters structure and function in the topical environment to shift the balance of activity toward direct inhibition of microbial colonization. The Journal of Immunology, 2004, 172: 3070–3077. eptides with antimicrobial activity are found throughout activity becomes available at the most external interface, providing nature and are known to be important for the immune de- an inhibitory barrier to infection. P fense of plants, insects, and animals (1). Many of these The clinical consequences of antimicrobial peptide expression http://www.jimmunol.org/ peptides exhibit a broad spectrum of antimicrobial activity, inhib- are demonstrated by observations that patients with atopic derma- iting or killing Gram-positive and Gram-negative bacteria, fungi, titis lack the ability to increase cathelicidins and defensins in re- and viruses. In mammals, antimicrobial peptides belonging to the sponse to inflammatory stimuli (15). As patients with atopic der- cathelicidin family have been shown to be important for the anti- matitis are uniquely susceptible to infection compared with normal microbial efficacy of neutrophils, macrophages, and mast cells (2– individuals or those with elevated expression, as seen in psoriasis, 6). In addition, epithelia of lung, gut, urinary bladder, oral mucosa, this first-line immune defense mechanism appears to have an es- and skin produce antimicrobial peptides of the defensin and cathe- sential function for resistance against skin infections. Similarly, licidin families constitutively or in response to injury (7–9). At the patients with Kostmann syndrome, a rare inherited disorder char- by guest on October 1, 2021 epithelial interface with the external environment, these molecules acterized by frequent infections and neutrophil dysfunction, have a are thought to serve as a rapid first line defense for inhibition of deficiency in production and processing of cathelicidin (16). Such microbial proliferation and invasion. emerging clinical associations support the need to further explore In mammalian skin, cathelicidins have been directly shown to be the function and regulation of this evolutionarily ancient aspect of essential for defense against invasive bacterial infection by group the human immune system. A streptococcus (10). Found in abundance in neutrophils and mast Most antimicrobial peptides are synthesized as inactive prepro- cells, the cathelicidins are expressed at relatively low levels in proteins that require enzymatic processing for release of active normal keratinocytes, but are rapidly induced during inflammation peptides. Lack of processing, such as seen in ␣-defensins from (11). The recruitment of cathelicidin-rich cells and the increase in Panath cells of matrilysin-deficient mice (17), leads to impaired expression by the epithelial keratinocytes lead to accumulation of clearance of infection in the gut. Similarly, inhibition of processing cathelicidins in wound fluid and in the overlying crust (12). Re- of porcine cathelicidins by elastase impairs bacterial clearance in cently, cathelicidins and dermcidin have also been found consti- wounds (18). The enzymatic processing of cathelicidins from a tutively produced by the eccrine apparatus and secreted constitu- pro-protein to a two-component solution consisting of the cathelin tively into human sweat (13, 14). In this scenario, antimicrobial prodomain and the C-terminal cationic antimicrobial peptide, LL- 37, can have multiple consequences for immune defense. The Division of Dermatology, University of California, and Veterans Affairs San Diego cathelin domain is itself antimicrobial and functions to inhibit cys- Healthcare Center, San Diego, CA 92161 teine proteases such as cathepsin L (19). LL-37, although func- Received for publication August 27, 2003. Accepted for publication December tional as a broad spectrum antimicrobial, can also stimulate che- 15, 2003. motaxis and angiogenesis by binding formyl peptide receptor- The costs of publication of this article were defrayed in part by the payment of page like-1 (20, 21) and improve re-epithelialization to enhance wound charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. repair (22). In this study we determined whether additional antimicrobial 1 This work was supported by a Veterans Affairs merit award and National Institutes of Health Grants RO1AR45676 and AI52453 (to R.L.G.), the Japan-North America peptides are present in the most superficial barrier topically pro- Medical Exchange Foundation (to M.M.), and the Ramon Areces Foundation (to vided by human sweat. Our findings suggest that human catheli- B.L.-G.). cidin is further processed and enables a shift in biological activity 2 Address correspondence and reprint requests to Dr. Richard L. Gallo, MC 911B, 3350 LaJolla Village Drive, San Diego, CA 92161. E-mail address: rgallo@vapop. toward antimicrobial function and away from the ability to stim- ucsd.edu ulate a host response. Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00 The Journal of Immunology 3071 Materials and Methods Mass spectrometry and protein sequence analysis Sweat collection and processing Mass spectrometry was performed by Center for Mass Spectrometry, The Scripps Research Institute (La Jolla, CA). MALDI-MS spectra were ob- Sweat was collected on paper tissues (Kimwipes; Kimberly-Clark, Neenah, tained with a Voyager DE-RP MALDI-TOF mass spectrometer (PerSep- WI) from healthy volunteers after exercise as previously described (13). tive Biosystems, Framingham, MA) equipped with a nitrogen laser (337 After collection, 20 ml of sweat was centrifuged at 2000 ϫ g for 15 min nm, 3-ns pulse). Spectra were collected in the reflector mode. The accel- at 4°C, filtered through a 0.20-␮l filter (Acrodisc syringe filter, 0.2 ␮m, low erating voltage in the ion source was 20 kV. Data were acquired with a protein binding; Fisher Scientific, Tustin, CA), and frozen at Ϫ80°C. For transient recorder with 2-ns resolution. The matrix used in this work was some experiments sweat was lyophilized to dryness, then suspended in 400 a-cyano-4-hydroxycinnamic acid dissolved in water/acetonitrile (1/1, v/v) ␮l of distilled water (DW3; cell culture grade, endotoxin free; Life Tech- to give a saturated solution at room temperature. To prepare the sample for nologies, Grand Island, NY). For analysis of LL37 processing by sweat, 1.6 ␮ analysis, 1 ␮l of the peptide solution (containing 1–10 pmol of protein in nmol of LL-37 synthetic peptide was incubated in 50 l of sweat for 0, 1, ␮ 6,and24hat37or4°C. In some experiments proteinase inhibitors in- 0.1% trifluoroacetic acid) was added to 1 l of the matrix solution and cluding mixed protease inhibitor mixture (1 tablet/10 ml; Roche, Indianap- applied to a stainless steel sample plate. The mixture was then allowed