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Structure-Function Relationships among Human Cathelicidin Peptides: Dissociation of Antimicrobial Properties from Host Immunostimulatory Activities This information is current as of October 1, 2021. Marissa H. Braff, Mi'i A. Hawkins, Anna Di Nardo, Belen Lopez-Garcia, Michael D. Howell, Cathy Wong, Kenneth Lin, Joanne E. Streib, Robert Dorschner, Donald Y. M. Leung and Richard L. Gallo

J Immunol 2005; 174:4271-4278; ; Downloaded from doi: 10.4049/jimmunol.174.7.4271 http://www.jimmunol.org/content/174/7/4271

References This article cites 54 articles, 21 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/174/7/4271.full#ref-list-1

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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 © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Structure-Function Relationships among Human Cathelicidin Peptides: Dissociation of Antimicrobial Properties from Host Immunostimulatory Activities

Marissa H. Braff,* Mi‘i A. Hawkins,* Anna Di Nardo,*‡ Belen Lopez-Garcia,* Michael D. Howell,† Cathy Wong,* Kenneth Lin,* Joanne E. Streib,† Robert Dorschner,* Donald Y. M. Leung,† and Richard L. Gallo1*

Cathelicidins and other antimicrobial peptides are deployed at epithelial surfaces to defend against infection. These molecules have broad-spectrum killing activity against microbes and can have effects on specific mammalian cell types, potentially stimulating additional immune defense through direct chemotactic activity or induction of cytokine release. In humans, the cathelicidin hCAP18/LL-37 is processed to LL-37 in neutrophils, but on skin it can be further proteolytically processed to shorter forms. The Downloaded from influence of these cathelicidin peptides on keratinocyte function is not known. In the current study, DNA microarray analysis and confirmatory protein analysis showed that LL-37 affects the expression of several chemokines and cytokines by keratinocytes. Analysis of a synthetic peptide library derived from LL-37 showed that antimicrobial activity against bacterial, fungal, and viral skin pathogens resides within specific domains of the parent peptide, but antimicrobial activity does not directly correlate with the ability to stimulate IL-8 production in keratinocytes. IL-8 release was induced by D- and L-amino acid forms of cathelicidin and

correlated with membrane permeability, suggesting that highly structure-speciﬁc binding to a cell surface receptor is not likely. http://www.jimmunol.org/ However, this effect was inhibited by either pertussis toxin or AG1478, an epidermal growth factor receptor tyrosine kinase inhibitor, suggesting that cathelicidin may indirectly stimulate multiple signaling pathways associated with cell surface receptors. Taken together, these observations suggest that proteolytic processing may alter the balance between cathelicidin antimicrobial and host immunostimulatory functions. The Journal of Immunology, 2005, 174: 4271–4278.

pithelial interfaces with the external environment rely on vary glands (12). In certain cell types, such as those found in ec- innate immune defense mechanisms for rapid recognition crine sweat glands, cathelicidin is constitutively expressed (12). In and defense against infection. The skin is an important other cells, such as keratinocytes, cathelicidin expression is induc-

E by guest on October 1, 2021 example, acting as a physical and chemical barrier against external ible (3) and is rapidly up-regulated in the epidermis after injury pathogens in a variety of extreme situations such as wound repair, (13). The role that cathelicidins play in host defense extends be- where infection becomes a major cause of morbidity and mortality. yond their ability to directly kill pathogens. The hCAP18 precursor Individual cells comprising the skin are actively involved in this protein is processed upon neutrophil granule fusion to a 37-aa process (1). One way these individual cells are involved is through peptide form, LL-37, by cleavage with a serine protease (14). This production and release of antimicrobial peptides (2–4). form of the human cathelicidin has been shown to be involved in In the skin, antimicrobial peptides can be released by resident chemotaxis of mast cells, neutrophils, and CD4 T cells (15, 16) and cells such as keratinocytes, mast cells, and eccrine gland epithelia can modify dendritic cell and keratinocyte function (17, 18). The or are deposited by recruited cells such as neutrophils (5). Cathe- chemotactic property of LL-37 has been associated with binding licidins comprise one such family of antimicrobial peptides and via formyl peptide receptor-like 1 (FPRL-1)2 (19), which belongs exert their effects on a broad range of microbes, including Gram- to the Gi protein-coupled receptor family. Other host cell activities positive and -negative bacteria as well as fungi and enveloped appear to be independent of FPRL-1 and can involve upstream viruses (6, 7). Humans have a single cathelicidin gene encoding activation of P2X7 or epidermal growth factor receptors (EGFR) hCAP18/LL-37 (8, 9). In addition to being expressed by several (20, 21). cells of the skin, hCAP18/LL-37 is abundantly expressed in neu- Human cathelicidin peptides can exist naturally in forms other trophils and in multiple mucosal epithelia, such as airways (10), than LL-37. In sweat, three additional forms have been found: buccal mucosa, tongue, esophagus, cervix, vagina (11), and sali- KR-20, a 20-aa derivative; RK-31, a 31-aa derivative; and KS-30, a 30-aa derivative. All three processed peptides exhibit an overall gain in antimicrobial activity compared with full-length LL-37 *Division of Dermatology, University of California, and Veterans Affairs San Diego (22). The importance of processing human cathelicidin from Healthcare Center, San Diego, CA 92161; †Division of Allergy and Immunology, Department of Pediatrics, National Jewish Medical and Research Center, Denver, CO LL-37 as the form ﬁrst released from neutrophils to smaller pep- 80206; and ‡San Gallicano Dermatological Institute, Rome, Italy tide fragments is unclear. Received for publication August 12, 2004. Accepted for publication January 12, 2005. 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 with 18 U.S.C. Section 1734 solely to indicate this fact. 2 Abbreviations used in this paper: FPRL-1, formyl peptide receptor-like 1; EGFR, 1 Address correspondence and reprint requests to Dr. Richard L. Gallo, Division of epidermal growth factor receptor; GPCR, G protein-coupled receptor; LDH, lactate Dermatology, University of California, and Veterans Affairs San Diego Healthcare dehydrogenase; PI, propidium iodide; PTX, pertussis toxin; RTK, receptor tyrosine Center, San Diego, CA 92161. E-mail address: [email protected] kinase.

To investigate the functional implications of LL-37 in the skin, terminal LL-37 peptide fragments as well as those central domains con- the current study explored the gene expression profile of keratin- served among species and include sequences from both ␣-helical domains ocytes after exposure to LL-37 and found profound changes in and disrupted helices. Peptide amino acid sequences are given in Fig. 2. All synthetic peptides were purified by HPLC to Ͼ95% purity, and identity cytokine and chemokine expression and release. The structural ba- was confirmed by mass spectrometry. Peptides were tested for LPS con- sis of this response was studied by generating a library of LL-37- tamination by Limulus assay according to the manufacturer’s instructions. derived synthetic peptide fragments. Observations from these ex- Determination of minimal inhibitory concentration periments defined specific domains within LL-37 that are important for antibacterial, antifungal, and antiviral activity as well Bacteria were grown overnight in sterile tryptic soy broth. Bacteria in log 6 as host immunostimulatory activity. Peptides with the ability to phase were suspended to 1 ϫ 10 CFU/ml in 10% tryptic soy broth (30 g/l stock; Sigma-Aldrich) in 10 mM phosphate buffer (27.6 g/l NaH PO -H O stimulate keratinocytes did not completely correlate with those that 2 4 2 and 53.65 g/l Na2HPO4-7H2O, pH 7.4). Peptide was added and incubated were antimicrobial, suggesting that processing of cathelicidin pep- overnight at 37°C. The OD was then measured at 600 nm. Peptide activity tides can be an important modifier of host cell immune respon- against Candida albicans was determined in Dixon medium (0.6% pep- siveness. Additional mechanistic studies suggested that mem- tone, 4% malt extract, 1% glucose, 0.1% ox bile, and 1% Tween 80) in ␮ brane-active LL-37 peptides may indirectly stimulate keratinocyte sterile 96-well plates (Corning Glass) at a final volume of 50 l. The assay mixtures contained 1–2.5 ϫ 104 CFU/ml C. albicans, 20% Dixon medium, cell surface receptors and activate downstream signaling cascades, 0.6 mM phosphate buffer (pH 7.0), and 16 ␮g/ml chloramphenicol. Plates leading to changes in gene expression by host cells. were incubated at 25°C for 24 h with peptides, and absorbance was read at 600 nm. Materials and Methods Antiviral assay Cell culture and virus source BS-C-1 (American Type Culture Collection; CCL-26) African green mon- Downloaded from Neonatal human epidermal keratinocytes (Cascade Biologics) were grown key kidney cells (2 ϫ 105 cells/well) were seeded in 24-well tissue culture in EpiLife medium (Cascade Biologics) supplemented with 0.06 mM plates in MEM-10% FCS and penicillin/streptomycin and allowed to grow 2ϩ Ca , 1% EpiLife defined growth supplement, and 1% penicillin/strepto- overnight before the supernatant was removed and replaced with MEM- mycin (Invitrogen Life Technologies). Cells were grown at 37°C in a hu- 2.5% FCS for virus incubation. Peptides were diluted to the proper con- midified atmosphere of 5% CO2 and 95% air. The Wyeth strain Vaccinia centrations in 0.01ϫ tryptic soy broth containing 10 mM sodium phosphate was obtained from the Centers of Disease Control and Prevention. buffer (pH 7.4). Virus diluted in the same buffer was added to the peptides

and incubated for 24 h at 37°C. Twenty microliters of the peptide/virus http://www.jimmunol.org/ DNA microarray analysis mixture was added to the cells in 0.5 ml of MEM-2.5% FCS and allowed Normal human keratinocytes were cultured to confluence in 100-mm to infect for 48 h for plaque development. The medium was removed, and dishes and treated with 0 or 10 ␮M LL-37 for 8 h. Total RNA was ex- 0.5 ml of 0.1% crystal violet in PBS was added to the wells for 5 min at tracted using the Qiagen RNeasy kit. The Veterans Administration Medical room temperature. Wells were then aspirated and air-dried for visualization Center GeneChip Core Facility processed five HuGeneFL GeneChips (Af- of plaques. fymetrix) using 250–300 ng of total RNA/chip. Two chips were used for Measurement of IL-8 release from keratinocytes control keratinocytes, and three chips were used for the 10 ␮M LL-37- treated keratinocytes. Data were analyzed using GeneChip operating soft- Keratinocytes were cultured to confluence and treated with the peptide of ware and GeneSpring (Silicon Genetics). Data were filtered by genes show- interest in keratinocyte medium at 37°C. For the cathelicidin peptide frag- ing Ͼ3-fold change in peptide-treated vs medium alone, and by statistical ment panel and dose-response assays, peptides were incubated with kera- by guest on October 1, 2021 significance with p Ͻ 0.05 by two-tailed Student’s t test. tinocytes for 6 h. For the time-course assay, cathelicidin peptides were used at a concentration of 3 ␮M. Supernatants were removed and placed in a Cytokine expression analysis sterile 96-well plate for ELISA. IL-8 production was determined by ELISA Keratinocytes were cultured to preconfluence, confluence, or 2 days post- (BD Biosciences) according to the manufacturer’s instructions. Briefly, confluence and treated with 0, 3, or 10 ␮M LL-37 for 24 h. Cytokine 96-well plates were coated with capture Ab and incubated overnight at 4°C. release was measured using the LINCOplex Human Cytokine Multiplex kit Wells were washed three times, blocked for1hatroom temperature, and according to the manufacturer’s instructions (Linco Research). Briefly, the again washed three times. Standards and 1/20 dilutions of samples were filter plate was blocked for 10 min, followed by the addition of standards, added to each well and incubated for2hatroom temperature, followed by samples, and beads. The plate was sealed, covered with aluminum foil, and five washes. Detection Ab was added and incubated for1hatroom tem- incubated with shaking for 1 h. Wells were then washed twice, and detec- perature. Wells were washed seven times. Substrate solution was added to tion Ab was added. The plate was again sealed and covered with aluminum each well, and plate was incubated for 30 min at room temperature in the foil, then incubated with shaking for 30 min. Streptavidin-PE was added to dark. Stop solution was added to each well, and the OD was observed at each well, and the plate was incubated an additional 30 min. Wells were 450 nm with correction at 570 nm. IL-8 secretion from keratinocytes washed twice, and sheath fluid was added. The plate was shaken for 5 min, treated with pertussis toxin (Sigma-Aldrich), the FPRL-1 agonist then read using Luminex100. WKYMVM-NH2 (Phoenix Pharmaceuticals), or the EGFR inhibitor AG1478 (Calbiochem) was assessed by IL-8 ELISA as described. Measurement of IL-1␣ release from keratinocytes Quantitative real-time PCR Normal human epidermal keratinocytes were cultured to confluence and treated with 0 or 10 ␮M LL-37 in keratinocyte medium for 1 or 24 h at Keratinocytes were cultured to confluence and treated with the peptide of 37°C. IL-1␣ production was determined by IL-1␣ ELISA (R&D Systems) interest in keratinocyte medium for6hat37°C. Total RNA was isolated according to the manufacturer’s instructions. Briefly, 96-well plates were from treated cells using an RNeasy Mini kit (Qiagen). Isolated RNA was coated overnight with capture Ab. Wells were blocked for 1 h, after which retrotranscribed by reverse transcriptase using the Retroscript kit (Am- standards and samples (1/10 dilution) were added, and the plate was in- bion). Briefly, first-strand synthesis combined RNA with 0.4 mM dNTPs and 4 ␮M random decamers in 16 ␮l at 80°C for 3 min. The reaction was cubated for 2 h. Wells were incubated with detection Ab for an additional ␮ ϫ ␮ ␮ 2 h, followed by streptavidin-HRP for 20 min in the dark. The plate was iced, and 2 lof10 RT-PCR buffer, 1 l of RNase inhibitor, and 1 l incubated with substrate solution for 20 min in the dark. Without washing, of Moloney murine leukemia virus RT were added and incubated at 42°C stop solution was added to each well, and the plate was immediately read for 1 h, then denatured at 92°C for 10 min. Real-time quantitative PCR was at 450 nm with correction at 570 nm. performed using an Applied Biosystems 7000 Sequence Detection System. TaqMan predeveloped assay reagents were used to detect human IL-8 (Ap- LL-37 and LL-37 peptide fragment synthesis plied Biosystems), and GAPDH primers and probe were used as an endogenous control. Briefly, 2 ␮l of the RT reaction was added to 25 ␮lof LL-37, LL-25, LL-20, LL-15, LL-12, GD-23, RK-19, EK-20, KR-20, VQ- TaqMan Universal PCR Master Mix (Roche), 2.5 ␮l of target primers and 17, EK-15, GK-17, EF-15, RK-31, and KS-30 peptides were commercially probe, and 20.5 ␮l of RNase/DNase-free water. The thermal profile was prepared by Synpep. These sequences were selected based on predicted 50°C for 2 min, 95°C for 10 min, and 40 cycles of 94°C for 15 s, followed ␣-helical domains of LL-37 from alignments of ␣-helical cathelicidin fam- by 60°C for 1 min. Results were analyzed using the comparative cycle ily members from various species. These peptides represent N- and C- threshold method (Applied Biosystems). Briefly, cycle threshold values The Journal of Immunology 4273 were normalized to GAPDH, which was used as an endogenous control, inflammation, including those encoding IL-8, cyclooxygenase then normalized to the sample chosen as a calibrator. Product specificity 2, pro-IL-1␤, TNF-␣-induced protein 3, and IL-6, were signif- was confirmed by generating dissociation curves for the reactions. icantly up-regulated. Measurement of cell viability To confirm the microarray analysis data showing that keratinocytes stimulated with LL-37 up-regulate the expression of cyto- Keratinocytes were incubated for6hat37°C with 0, 3, or 10 ␮M cathelicidin peptide. Supernatants were collected into clean 96-well plates. Cells kines and chemokines, quantitative immunodetection assays were were washed three times. Basal medium (100 ␮l) was added to each well. performed on culture supernatants from treated cells. Consistent Viability was assessed using the CellTiter96 Non-Radioactive Cell Prolif- with the results of the microarray, IL-6 and IL-8 protein levels eration Assay (MTT; Promega) according to the manufacturer’s instruc- ␣ ␮ increased. The production of TNF- and GM-CSF also increased. tions. Briefly, dye solution (15 l) was added to each well, and the plate ␤ was incubated4hat37°C. Solubilization/stop solution (100 ␮l) was added No changes were seen in the abundance of IL-1 , IL-2, IL-4, or to each well, and the plate was incubated in a dark, moist chamber over- IFN-␥ (Fig. 1A). For analysis of IL-1␣, ELISA of cultured super- night at room temperature. OD was determined at 570 nm, with correction natant from LL-37-treated keratinocytes was performed and dem- at 660 nm. Alternatively, cells were incubated briefly with 1.3 ␮g/ml pro- onstrated a large increase in IL-1␣ release compared with cells pidium iodide (PI) after removal of supernatants, and fluorescence intensity treated with medium only (Fig. 1B). Assays were performed on was measured using a fluorescence microplate reader at an excitation of 530 nm and an emission of 620 nm. For evaluation of toxicity by mem- preconfluent, confluent, and postconfluent cells in the presence of brane permeability to lactate dehydrogenase (LDH), LDH release from low or high calcium, and no significant differences were found cells was measured using the Cytotoxicity Detection kit (Roche). Briefly, (data not shown). 50 ␮l of catalyst/dye solution was added to 50 ␮l of cultured supernatant removed from the cells used for the MTT or PI assays described above.

Plates were incubated for 25 min at room temperature. OD was determined Downloaded from at 492 nm, with correction at 620 nm. Derivatives of LL-37 have diverse antimicrobial activity To compare the mechanisms used in antimicrobial activity to those Results responsible for activating keratinocytes, a library of LL-37-derived LL-37 induces cytokine and chemokine production by normal peptides was synthesized. The sequences of these peptides are human keratinocytes shown in Fig. 2. Select peptides exhibited a broad range of anti-

The human cathelicidin antimicrobial peptide LL-37 has been im- microbial activities against test organisms, which included Gram- http://www.jimmunol.org/ plicated in a variety of effects on the host immune system, includ- negative and -positive bacteria, Vaccinia virus, and C. albicans ing chemotaxis (16, 19), dendritic cell differentiation (17), and (Table II). The peptide fragments RK-31 and KS-30 showed the cytokine release (22). Epithelial surfaces such as the skin are ex- highest overall antimicrobial properties, with minimal inhibitory posed to abundant levels of LL-37 during the response to injury concentrations ranging from 2 to 16 ␮M. Peptide fragment GD-23 or infection. It was of interest to evaluate the response of epi- exhibited the lowest minimal inhibitory concentration against dermal keratinocytes to LL-37 at concentrations similar to those Staphylococcus aureus mprF at 2 ␮M. LL-25, EK-20, KR-20, GK- found in vivo (13). DNA microarray analysis of cultured ker- 17, RK-31, and KS-30 also inhibited the growth of S. aureus mprF atinocytes exposed to LL-37 for 8 h revealed effects on the better than LL-37. Under these culture conditions, several frag- transcripts of various genes (Table I). Many genes involved in ments showed higher activity than LL-37 against Escherichia coli, by guest on October 1, 2021

Table I. Change in transcript expression in LL-37-treated human keratinocytesa

Transcripts Up-Regulated Fold Increase Transcripts Down-Regulated Fold Increase

IL-8 11.32 Thioredoxin interacting protein 9.14 A kinase (PRKA) anchor protein (gravin) 12 9.26 Estrogen receptor 1 7.35 PG endoperoxide synthase 2 (PTGS2, COX2) 9.18 Ataxia-telangiectasia locus protein 4.84 Cyclooxygenase 2 gene 6.75 18S rRNA gene 4.69 Chemokine (CXC motif) ligand 3 (CXCL3) 5.94 Proﬁlaggrin gene 4.49 Pro-IL-1␤ (IL1␤) gene 5.91 BRCA1 4.31 GOS2 gene 5.09 Primase (PRIM1) 3.17 Urokinase-type plasminogen receptor (UPAR, URKR) 5.08 BRCA2 gene 3.00 IL-13 receptor, ␣2 4.76 Pim-1 oncogene 4.74 Putative chemokine receptor 4.68 Decay accelerating factor for complement 4.55 Chemokine (CXC motif) ligand 2 (CXCL2) 4.54 Melanoma growth stimulatory activity (MGSA) gene (CXCL1, GRO1) 4.40 Solute carrier family 2 (facilitated glucose transporter) 4.25 Thioredoxin reductase 1 4.11 Human tumor Ag (L6) 3.99 Baculoviral IAP repeat-containing 1 3.83 Aldehyde dehydrogenase 1 3.60 Dual speciﬁcity phosphatase 1 3.60 TNF-␣-induced protein 3 3.45 IL-6 3.45 Urokinase inhibitor (PAI2) gene 3.33 B-cell translocation gene 1, antiproliferative 3.09 Bone morphogenetic protein 2 3.06

a Keratinocytes grown to confluence were treated with 0 or 10 ␮M LL-37 for 8 h. Total RNA was extracted and analyzed for transcript change by HuGeneFL GeneChips. Approximately 250–300 ng of RNA was used for each chip. Fold change represents an average of the difference among five GeneChips: two for the control compared with three for the 10 ␮M LL-37-treated cells. The data were analyzed by GeneChip Operating Software (Affymetrix) and GeneSpring (Silicon Genetics). Statistical analysis was performed by the paired Student t test. All values shown are significant to p Ͻ 0.05. 4274 RELATIONSHIPS AMONG HUMAN CATHELICIDIN PEPTIDES

FIGURE 2. Sequences of LL-37 and synthetic peptide fragments of LL- 37. Peptides were commercially prepared, endotoxin free by Limulus assay, and puriﬁed by HPLC. Identity was conﬁrmed by mass spectrometry.

at a dose near the minimal IL-8 stimulatory concentration. EK-20, RK-31, and KS-30 displayed minimal effects on keratinocyte IL-8 production at the time points examined (Fig. 4B). The increasing Downloaded from accumulation of IL-8 over time in response to these peptides was similar to constitutive release from keratinocytes treated with medium alone. To evaluate the effects of LL-37, EK-20, RK-31, and KS-30 on the membrane permeability of keratinocytes, LDH release and re-

duction of tetrazolium to formazan were studied by LDH and MTT http://www.jimmunol.org/ assays, respectively. Minimal membrane disruption was observed by LDH assay for all peptides and concentrations tested (Fig. 5A). Keratinocyte viability, as evaluated by MTT assay, was clearly FIGURE 1. LL-37 influences cytokine production by human keratino- reduced upon exposure to 10 ␮M LL-37 and, to a lesser extent, cytes. LL-37 was diluted in keratinocyte medium to a final concentration of 3or10␮M. A, Keratinocytes were incubated with medium only or with RK-31 and KS-30 (Fig. 5B). LL-37 for 24 h at 37°C. Supernatants were analyzed for the indicated Stimulation of IL-8 production by LL-37 and D-LL-37 directly cytokines by Luminex assay. B, Keratinocytes were incubated with medium only or with 10 ␮M LL-37 for 1 or 24 h. Supernatants were analyzed correlates with membrane permeability by guest on October 1, 2021 for IL-1␣ by ELISA. Results are expressed as the mean Ϯ SEM of one Because 3 ␮M D-LL-37 was shown to exhibit potent IL-8 immu- representative experiment of three, each performed in triplicate. nostimulatory ability in Fig. 3, dose-response experiments were performed to compare the activities of D-LL-37 and LL-37 (Fig. 6). Both LL-37 and D-LL-37, but not EK-20, increased IL-8 protein including LL-25, GD-23, RK-19, EK-20, EK-15, GK-17, EF-15, production in a dose-dependent manner (Fig. 6A). Maximal IL-8 RK-31, and KS-30. induction occurred at a lower concentration of D-LL-37 (4 ␮M), but both peptides showed equivalent immunostimulatory activity Ability of cathelicidin peptides to stimulate IL-8 production at the highest dose of 10 ␮M. Therefore, although D-LL-37 appears correlates with reduction in keratinocyte viability, but is to be more potent than LL-37, both peptides have equivalent max- independent of peptide antimicrobial activity imal IL-8 stimulatory capacity. Quantitative real-time PCR anal- The effects of LL-37 synthetic peptide fragments as well as the ysis showed induction of IL-8 transcript by keratinocytes in re- structurally divergent D-amino acid form of LL-37 were next com- sponse to LL-37 and D-LL-37, but not EK-20 (Fig. 7). It was pared with full-length LL-37 in assays for keratinocyte IL-8 pro- interesting to note that IL-8 production in response to LL-37 and duction. For initial screening, keratinocytes were cultured for 6 h D-LL-37 correlated directly with membrane permeability, as mea- in the presence of peptides at a final concentration of 3 ␮M. D- sured by PI incorporation, suggesting that these events may be LL-37 stimulated the highest production of IL-8 by keratinocytes, connected (Fig. 6B). followed by LL-37, KS-30, and RK-31, respectively (Fig. 3). Because EK-20, RK-31, and KS-30 were found to have good Cathelicidin peptide-induced IL-8 production in keratinocytes broad-spectrum antimicrobial activity, but little or no apparent involves EGFR and Gi protein signaling ability to stimulate keratinocyte IL-8 release, dose and time re- The strict sequence requirement for keratinocyte immunostimula- sponses were evaluated (Fig. 4). Treatment of keratinocytes with tory activity contrasted with the less stringent domain require- LL-37 resulted in the highest dose-dependent release of IL-8 from ments for broad-spectrum antimicrobial activity. However, the po- keratinocytes, although KS-30 also induced an increase in kera- tent effects of LL-37 synthesized with D-amino acids suggested tinocyte IL-8 production at higher concentrations. Peptide frag- that a traditional ligand-receptor binding mechanism for keratino- ments RK-31 and EK-20 did not stimulate IL-8 above the medium cyte recognition of LL-37 was unlikely. Previous investigations background (Fig. 4A). To observe the time-dependent release of have implicated FPRL-1, a Gi-coupled receptor, in the chemotaxis IL-8 by keratinocytes, peptides were diluted to 3 ␮M. IL-8 accu- of neutrophils, monocytes, and T cells in response to LL-37 (19). mulated with time in both control and peptide-treated keratino- However, under the culture conditions used in this study, keratin- cytes. Full-length LL-37 again stimulated the highest levels of IL-8 ocytes were not responsive to the FPRL-1 ligand WKYMVM-NH2 release from keratinocytes, demonstrating a 6-fold increase at 24 h at concentrations up to 100 times greater than those reported to be The Journal of Immunology 4275

Table II. MIC (␮M) of LL-37 and LL-37 synthetic peptide fragmentsa

MIC (␮M)

E. coli (O29) GAS (NZ131) S. aureus (ATCC 25932) S. aureus mprF Vaccinia (Wyeth strain) C. albicans (ATCC 14053)

LL-37 64 8–16 Ͼ64 32 32 16 LL-25 16 8 Ͼ64 8 Ͼ64 16 LL-20 Ͼ64 Ͼ32 Ͼ64 Ͼ32 Ͼ64 Ͼ32 LL-15 Ͼ64 Ͼ32 Ͼ64 Ͼ32 Ͼ64 Ͼ32 LL-12 Ͼ64 Ͼ32 Ͼ64 Ͼ32 Ͼ64 Ͼ32 GD-23 16 Ͼ32 Ͼ64 2 Ͼ64 32 RK-19 32 Ͼ32 Ͼ64 Ͼ32 Ͼ64 16 EK-20 32 8 Ͼ64 8–16 64 32 KR-20 Ͼ64 8 Ͼ64 8 Ͼ64 16 VQ-17 Ͼ64 Ͼ32 Ͼ64 Ͼ32 Ͼ64 Ͼ32 EK-15 Ͼ32 Ͼ32 Ͼ320 Ͼ32 Ͼ64 Ͼ32 GK-17 32 16 Ͼ320 16 32 16 EF-15 Ͼ32 32 Ͼ320 32 Ͼ64 32 RK-31 8 4 16 8 16 4 KS-30 8 4 16 8 8 2

a Bacterial and fungal minimal inhibitory concentration (MIC) values were determined by solution killing assay. Peptides ranging in concentration from 2 to 320 ␮M were added to bacteria in tryptic soy broth or to C. albicans in Dixon medium. Results indicate minimum concentration required for killing. Viral MIC was determined by the plaque method. Each assay was performed three times in triplicate, with representative data shown. Downloaded from effective in other cell types (100 nM; data not shown). Similarly, has also been shown to stimulate host inflammatory events (21, FPRL-1 expression was not seen in cultured keratinocytes or in 24). The skin has demonstrated dependence on cathelicidin ex- whole skin by immunostaining. This result agrees with recent stud- pression for resistance to infection by Streptococcus pyogenes in ies demonstrating that FPRL-1 is not involved in LL-37-induced mice (25), and cathelicidin expression correlates with resistance to activation of epithelial cells (21) or monocytes (23). human skin infections (26). Therefore, this study analyzed the im- http://www.jimmunol.org/ Previous investigations showed that cell activation and signaling munomodulatory effects of LL-37 peptides on keratinocytes for by LL-37 can be Gi protein dependent (19) or independent (23). comparison with antimicrobial activity. LL-37 had significant ef- Therefore, the IL-8 response was evaluated after treatment of ker- fects on the expression of a wide range of transcripts from kera- atinocytes with the Gi inhibitor, pertussis toxin (PTX), in the pres- tinocytes, with IL-8 showing the most abundant increase in steady ence or the absence of LL-37 or D-LL-37. Because LL-37 and D-LL-37 have different potencies, peptide concentrations that induce equivalent levels of IL-8, 10 ␮M LL-37 and 4 ␮M D-LL-37, were chosen for inhibitor experiments. PTX significantly inhibited by guest on October 1, 2021 IL-8 release by keratinocytes in response to either LL-37 or D- LL-37 (Fig. 8). Because LL-37 has also previously been shown to transactivate the EGFR (21), IL-8 production was also assessed in keratinocytes treated with equivalent stimulatory doses of LL-37 and D-LL-37 in the presence of the EGFR tyrosine kinase inhibitor, AG1478. Similar to PTX, treatment with AG1478 significantly inhibited IL-8 release from keratinocytes in response to either LL-37 or D-LL-37 (Fig. 8). Taken together, these results suggest that LL-37 and D-LL-37 activate keratinocyte production of IL-8 through pathways involving both Gi protein signaling and EGFR activation.

Discussion Cathelicidins, such as LL-37, possess an inherent ability to kill Gram-negative and -positive bacteria, fungi, and viruses. LL-37

FIGURE 4. Dose response and time course of IL-8 production by human keratinocytes in response to LL-37 and LL-37 peptide fragments. A, FIGURE 3. Speciﬁc LL-37 peptides inﬂuence IL-8 production by hu- Peptides were diluted to 2, 4, 8, or 10 ␮M in keratinocyte medium. Cells man keratinocytes. Peptides were diluted to a concentration of 3 ␮Min were incubated with peptides for6hat37°C. Supernatants were removed keratinocyte medium. Cells were incubated with peptide for6hat37°C. and analyzed for IL-8 by ELISA. B, Peptides were diluted to 3 ␮Min Supernatants were removed and analyzed for IL-8 by ELISA. Results are keratinocyte medium. Cells were incubated with peptides for 2, 6, 12, or expressed as the mean Ϯ SEM of one representative experiment of three, 24 h at 37°C. Supernatants were removed and analyzed for IL-8 by ELISA. each performed in triplicate. Statistical analysis was performed using the Experiments were performed three times in triplicate, with a representative .p Ͻ 0.01. outcome shown as the mean Ϯ SEM ,ء :paired Student’s t test 4276 RELATIONSHIPS AMONG HUMAN CATHELICIDIN PEPTIDES

FIGURE 7. Treatment of keratinocytes with LL-37 and D-LL-37 peptides induces IL-8 mRNA expression. Cells were incubated with medium alone, LL-37, D-LL-37, or EK-20 for6hat37C. RNA was extracted using the RNeasy kit, and cDNA was made using the Retroscript kit. Quantitative real-time PCR was performed using TaqMan primers and probes for IL-8 and GAPDH. Experiments were performed three times in triplicate, with a representative outcome shown as the mean Ϯ SEM.

of microorganisms. However, not all peptide fragments of LL-37 that demonstrated antimicrobial activity induced cytokine release by keratinocytes. Furthermore, LL-37 synthesized with amino ac-

FIGURE 5. Assessment of cytotoxicity to keratinocytes by LL-37 and Downloaded from ids in the D configuration, D-LL-37, was a potent stimulator of IL-8 LL-37 peptide fragments. A, Peptides were diluted to 3 or 10 ␮M in ker- release by keratinocytes, an effect that was significantly inhibited atinocyte medium. Cells were incubated for6hat37°C, and 50 ␮lof supernatants were removed and assessed for LDH release. B, Peptides were either by a specific EGFR tyrosine kinase inhibitor, AG1478, or by diluted to 3 or 10 ␮M in keratinocyte medium. Cells were incubated for 6 h PTX. Overall, these data suggest that the native processing of at 37°C. Supernatants were removed and replaced with 100 ␮l of medium. cathelicidins to alternate peptide forms can have important conse- Cells were assessed by MTT assay according to the manufacturer’s in- quences for host defense responses. structions. Experiments were performed three times in triplicate, with a The findings presented in this study show that LL-37 induces the http://www.jimmunol.org/ Ϯ representative outcome shown as the mean SEM. expression of numerous inflammatory mediators by keratinocytes. The transcripts for IL-8, cyclooxygenase 2, pro-IL-1␤, and IL-6 state transcript abundance by DNA microarray analysis. Several were among those significantly up-regulated. Confirmation of pro- peptides derived from LL-37 and sharing the sequence defined by tein expression levels by Luminex assay also showed an increase ␣ EK-20 showed potent antimicrobial activity against a wide range in the proinflammatory mediators TNF- and GM-CSF. The Lu- minex assay did not show an increase in IL-1␤ synthesis despite the DNA microarray findings and the prior report of IL-1␤ induction via upstream activation of the P2X7 receptor (20). This find- ing was not unexpected; although keratinocytes are able to make by guest on October 1, 2021 both IL-1␣ and IL-1␤, they lack the protease required to cleave pro-IL-1␤ to its active form (27). It is interesting to note that IL-1␣ is induced after only1hofkeratinocyte stimulation with LL-37. This immediate response is probably due to the release of stored IL- 1␣, rather than increased synthesis. The additional increase in IL-1␣ observed at 24 h probably results from increased synthesis by keratinocytes. The ability of LL-37 to increase proinflammatory mediator release from keratinocytes may have a significant impact on the initial phase of cutaneous inflammation. In fact, epidermal resident cells, including keratinocytes, comprise a major independent component of the acute inflammatory response, which is dictated by chemokine and

FIGURE 6. Dose response of keratinocyte IL-8 production and membrane permeability in response to LL-37 and D-LL-37 peptides. A, Peptides were diluted to 2, 4, 6, 8, or 10 ␮M in keratinocyte medium. Cells were FIGURE 8. PTX and AG1478 inhibit IL-8 induction in response to LL-37 incubated with peptides for6hat37°C. Supernatants were removed and and D-LL-37 peptides. Keratinocytes were pretreated for 1 h with medium analyzed for IL-8 by ELISA. B, Cells were treated with various peptide alone, 100 ng/ml PTX, or 2 ␮M AG1478, then stimulated for6hat37°C in doses for6hat37°C. Supernatants were removed and replaced with 1.3 the presence or the absence of LL-37 or D-LL-37. Supernatants were removed ␮g/ml PI in PBS. Fluorescence was measured at an excitation of 530 nm and analyzed for IL-8 by ELISA. Experiments were performed three times in and an emission of 620 nm. Experiments were performed three times in triplicate, with representative outcome shown as the mean Ϯ SEM. Statistical triplicate, with a representative outcome shown as the mean Ϯ SEM. analysis was performed using the paired Student’s t test. The Journal of Immunology 4277 cytokine expression (28, 29). Before additional cleavage to immuno- viously implicated in the host stimulatory effects of LL-37, includ- logically inactive forms, early deposition of LL-37 in the epidermis ing FPRL-1, EGFR, and P2X7 (19–21, 23). The current data could serve as an additional inflammatory mediator through its prop- suggest that specific binding to one of these receptors is an un- erties as a chemoattractant or its ability to stimulate expression of likely explanation for the response observed in keratinocytes, be- other potent chemotactic agents, such as IL-8. cause both LL-37 and D-LL-37 induced a maximal IL-8 response. The results of this study suggest that cathelicidin antimicrobial The potent activity of D-LL-37 argues against a highly structure- properties have domain specificity. Specifically, the sequences within specific association of cathelicidin peptides with a cell surface re- EK-20 appeared to be necessary, but not sufficient, for optimal anti- ceptor, although potential binding to a promiscuous pattern recog- microbial activity. LL-20, LL-15, and LL-12, which lack the sequence nition receptor could account for activation by peptides in both the VQRIKDFLRN, exhibited the lowest overall antimicrobial activity. L and D forms, but not by the shorter peptides. Interestingly, treat- The first six or seven amino acids of LL-37 did not appear to be ment with AG1478 and PTX, either independently or in combina- essential for antimicrobial activity, because RK-31 and KS-30 lack tion, blocked keratinocyte IL-8 induction in response to LL-37 and these amino acids. Generation of an additional peptide library with D-LL-37. Reduced IL-8 production by keratinocytes treated with site-specific amino acid modifications would serve to better define the these inhibitors suggests that the signaling mechanism generated at domain responsible for cathelicidin antimicrobial activity. the membrane by LL-37 and D-LL-37 involves both the EGF re- Of principal interest in the current study was the lack of direct ceptor and membrane-associated Gi proteins. Gi proteins, which correlation between antimicrobial activity and host stimulatory ef- are classically described as being associated with G protein-cou- fects, which was indicated by measuring IL-8 production. IL-8 was pled receptors (GPCRs), are recruited to the intracellular side of chosen as the indicator for host immunomodulatory activity due to

the membrane after receptor activation. However, at least three Downloaded from the high induction of IL-8 mRNA and protein expression in LL- activators of G protein signaling that act independently of these 37-treated keratinocytes. In addition, previous investigations have typical receptors have been described (35). Although the data pre- shown that antimicrobial peptides, including LL-37 and neutrophil sented in this study suggest that LL-37 peptides permeabilize the defensins, induce IL-8 synthesis and release by epithelial cells (21, cell membrane and are therefore able to enter cells, the ability of 30). For example, LL-37, RK-31, and KS-30 exhibited broad-spec- these peptides to directly interact with these intracellular activators trum antimicrobial properties, but stimulated keratinocytes to in-

of G protein signaling was not investigated. http://www.jimmunol.org/ crease IL-8 production to varying degrees. EK-20, which also ex- Previous studies have demonstrated ligand-independent transac- hibited broad-spectrum antimicrobial activity, did not increase tivation of receptor tyrosine kinases (RTKs), such as EGFR, by IL-8 release. These peptides did not demonstrate significant cyto- GPCRs (36–40). The occurrence of cross-talk between the GPCR toxic effects on keratinocytes at concentrations effective for anti- and EGFR signaling pathways, which is regulated by the metal- microbial activity or keratinocyte immune stimulation. However, loprotease kuzbanian (ADAM10), may help to explain the inhib- 10 ␮M LL-37 did affect keratinocyte reduction of MTT, suggest- itory effects of both PTX and AG1478 on the keratinocyte IL-8 ing potential keratinocyte membrane activity at this concentration response (41, 42). Other studies have suggested that cellular in culture. Previous structure-function studies of synthetic antimi- stresses also perturb the cell surface and alter receptor conforma- crobial peptides indicated a dissociation between antimicrobial and tion, thereby activating intracellular signaling pathways (36, 37, by guest on October 1, 2021 immunomodulatory activities (31–33). For example, N-terminal 43–47). In a similar manner, LL-37 may directly interact with the arginine residues of PR-39 were shown to be essential for protein keratinocyte plasma membrane to cause conformational changes binding and syndecan induction in mammalian cells, whereas modification of C-terminal residues specifically eliminated antimi- that indirectly activate surface receptor domains linked to intra- crobial activity (31). Structural changes to PR-39 influenced anti- cellular signaling molecules. Although the mechanism by which microbial activity, whereas mammalian effects were mostly depen- GPCR and RTK signaling pathways converge has not been com- dent on N-terminal charge. The results of the present study endorse pletely determined, recent work has suggested that these pathways the function of cathelicidin peptides as membrane active agents may run parallel and fulfill different signaling functions (48, 49). with relative selectivity for microbial membranes (34). Remark- Therefore, LL-37-mediated activation of transmembrane receptors, ably, the antimicrobial effects of cathelicidin at concentrations including both GPCRs and RTKs, in addition to influences on kera- Ͻ10 ␮M extended across bacteria, fungi, and viral envelopes, but tinocyte membrane ionic equilibrium should be further explored to spared the eukaryotic plasma membrane of keratinocytes. understand the mechanism by which LL-37 peptides activate down- The observation that D-LL-37 has more potent immunostimula- stream production of immunostimulatory molecules by keratinocytes. tory activity than LL-37 raises additional questions pertaining to These observations support previous studies in several cell, an- the mechanism by which peptide treatment induces IL-8 produc- imal, and human systems that cathelicidin peptides have properties tion by keratinocytes. D-LL-37 induced maximal IL-8 release at a of both natural antibiotics and host stimulatory molecules (6, 50– low concentration (4 ␮M), whereas a higher concentration of 53). The rapid increase in cathelicidin at epithelial interfaces ex- LL-37 (10 ␮M) was required for equivalent induction at both the posed to injury or infection can provide a simultaneous barrier to mRNA and protein levels. Interestingly, LL-37 caught up with microbial proliferation and an additional alarm to the host. The D-LL-37, reaching the same maximum IL-8 level, albeit at a dif- term alarmins, which was recently proposed to refer to peptides ferent dose. The more potent activity of D-LL-37, compared with such as the cathelicidins or defensins, is quite appropriate in this LL-37, may reflect the resistance to proteolytic degradation of pro- model (54). Proteolytic processing at sites of peptide deposition teins composed of amino acids in the synthetic D form. Thus, ad- could enable the epithelia to further modify the spectra of biologic ditional activity may be a consequence of a higher final concen- activity of cathelicidin peptides and regulate the balance of activity tration after incubation with cells producing active proteases. between host immune modulation and inhibition of microbial Furthermore, as expected of membrane-active agents, cell perme- growth. Additional studies examining changes in membrane flu- ability and membrane disruption corresponded directly with IL-8 idity, ion transport, and receptor aggregation in the presence of mRNA and protein production in response to these peptides. LL-37 will offer pertinent information concerning the mechanism The mechanism by which cathelicidin peptides exert their ef- by which LL-37 stimulates keratinocytes to produce IL-8 and other fects on keratinocytes is unclear. Several receptors have been pre- proinflammatory molecules. 4278 RELATIONSHIPS AMONG HUMAN CATHELICIDIN PEPTIDES

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