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 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. 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.

Epithelial interfaces with the external environment rely on innate immune defense mechanisms for rapid recognition and defense against infection. The skin is an important example, acting as a physical and chemical barrier against external pathogens in a variety of extreme situations such as wound repair, where infection becomes a major cause of morbidity and mortality. Individual cells comprising the skin are actively involved in this process (1). One way these individual cells are involved is through production and release of antimicrobial peptides (2, 3, 4).

In the skin, antimicrobial peptides can be released by resident cells such as keratinocytes, mast cells, and eccrine gland epithelia or are deposited by recruited cells such as neutrophils (5). Cathelicidins comprise one such family of antimicrobial peptides and exert their effects on a broad range of microbes, including Gram-positive and -negative bacteria as well as fungi and enveloped viruses (6, 7). Humans have a single cathelicidin gene encoding hCAP18/LL-37 (8, 9). In addition to being expressed by several cells of the skin, hCAP18/LL-37 is abundantly expressed in neutrophils and in multiple mucosal epithelia, such as airways (10), buccal mucosa, tongue, esophagus, cervix, vagina (11), and salivary glands (12). In certain cell types, such as those found in eccrine sweat glands, cathelicidin is constitutively expressed (12). In other cells, such as keratinocytes, cathelicidin expression is inducible (3) and is rapidly up-regulated in the epidermis after injury (13). The role that cathelicidins play in host defense extends beyond their ability to directly kill pathogens. The hCAP18 precursor protein is processed upon neutrophil granule fusion to a 37-aa peptide form, LL-37, by cleavage with a serine protease (14). This form of the human cathelicidin has been shown to be involved in chemotaxis of mast cells, neutrophils, and CD4 T cells (15, 16) and can modify dendritic cell and keratinocyte function (17, 18). The chemotactic property of LL-37 has been associated with binding via formyl peptide receptor-like 1 (FPRL-1)2 (19), which belongs to the Gi protein-coupled receptor family. Other host cell activities appear to be independent of FPRL-1 and can involve upstream activation of P2X7 or epidermal growth factor receptors (EGFR) (20, 21).

Human cathelicidin peptides can exist naturally in forms other than LL-37. In sweat, three additional forms have been found: 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 (22). The importance of processing human cathelicidin from LL-37 as the form first released from neutrophils to smaller peptide fragments is unclear.

To investigate the functional implications of LL-37 in the skin, the current study explored the gene expression profile of keratinocytes after exposure to LL-37 and found profound changes in cytokine and chemokine expression and release. The structural basis of this response was studied by generating a library of LL-37-derived synthetic peptide fragments. Observations from these experiments defined specific domains within LL-37 that are important for antibacterial, antifungal, and antiviral activity as well as host immunostimulatory activity. Peptides with the ability to stimulate keratinocytes did not completely correlate with those that were antimicrobial, suggesting that processing of cathelicidin peptides can be an important modifier of host cell immune responsiveness. Additional mechanistic studies suggested that membrane-active LL-37 peptides may indirectly stimulate keratinocyte cell surface receptors and activate downstream signaling cascades, leading to changes in gene expression by host cells.

Neonatal human epidermal keratinocytes (Cascade Biologics) were grown in EpiLife medium (Cascade Biologics) supplemented with 0.06 mM Ca2+, 1% EpiLife defined growth supplement, and 1% penicillin/streptomycin (Invitrogen Life Technologies). Cells were grown at 37°C in a humidified atmosphere of 5% CO2 and 95% air. The Wyeth strain Vaccinia was obtained from the Centers of Disease Control and Prevention.

Normal human keratinocytes were cultured to confluence in 100-mm dishes and treated with 0 or 10 μM LL-37 for 8 h. Total RNA was extracted using the Qiagen RNeasy kit. The Veterans Administration Medical Center GeneChip Core Facility processed five HuGeneFL GeneChips (Affymetrix) using 250–300 ng of total RNA/chip. Two chips were used for control keratinocytes, and three chips were used for the 10 μM LL-37-treated keratinocytes. Data were analyzed using GeneChip operating software and GeneSpring (Silicon Genetics). Data were filtered by genes showing >3-fold change in peptide-treated vs medium alone, and by statistical significance with p < 0.05 by two-tailed Student’s t test.

Keratinocytes were cultured to preconfluence, confluence, or 2 days postconfluence and treated with 0, 3, or 10 μM LL-37 for 24 h. Cytokine release was measured using the LINCOplex Human Cytokine Multiplex kit according to the manufacturer’s instructions (Linco Research). Briefly, the filter plate was blocked for 10 min, followed by the addition of standards, samples, and beads. The plate was sealed, covered with aluminum foil, and incubated with shaking for 1 h. Wells were then washed twice, and detection Ab was added. The plate was again sealed and covered with aluminum foil, then incubated with shaking for 30 min. Streptavidin-PE was added to each well, and the plate was incubated an additional 30 min. Wells were washed twice, and sheath fluid was added. The plate was shaken for 5 min, then read using Luminex100.

Normal human epidermal keratinocytes were cultured to confluence and treated with 0 or 10 μM LL-37 in keratinocyte medium for 1 or 24 h at 37°C. IL-1α production was determined by IL-1α ELISA (R&D Systems) according to the manufacturer’s instructions. Briefly, 96-well plates were coated overnight with capture Ab. Wells were blocked for 1 h, after which standards and samples (1/10 dilution) were added, and the plate was incubated for 2 h. Wells were incubated with detection Ab for an additional 2 h, followed by streptavidin-HRP for 20 min in the dark. The plate was incubated with substrate solution for 20 min in the dark. Without washing, stop solution was added to each well, and the plate was immediately read at 450 nm with correction at 570 nm.

LL-37, LL-25, LL-20, LL-15, LL-12, GD-23, RK-19, EK-20, KR-20, VQ-17, EK-15, GK-17, EF-15, RK-31, and KS-30 peptides were commercially prepared by Synpep. These sequences were selected based on predicted α-helical domains of LL-37 from alignments of α-helical cathelicidin family members from various species. These peptides represent N- and C-terminal LL-37 peptide fragments as well as those central domains conserved among species and include sequences from both α-helical domains and disrupted helices. Peptide amino acid sequences are given in Fig. 2. All synthetic peptides were purified by HPLC to >95% purity, and identity was confirmed by mass spectrometry. Peptides were tested for LPS contamination by Limulus assay according to the manufacturer’s instructions.

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.

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.

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Bacteria were grown overnight in sterile tryptic soy broth. Bacteria in log phase were suspended to 1 × 106 CFU/ml in 10% tryptic soy broth (30 g/l stock; Sigma-Aldrich) in 10 mM phosphate buffer (27.6 g/l NaH2PO4-H2O and 53.65 g/l Na2HPO4-7H2O, pH 7.4). Peptide was added and incubated overnight at 37°C. The OD was then measured at 600 nm. Peptide activity against Candida albicans was determined in Dixon medium (0.6% peptone, 4% malt extract, 1% glucose, 0.1% ox bile, and 1% Tween 80) in sterile 96-well plates (Corning Glass) at a final volume of 50 μl. The assay mixtures contained 1–2.5 × 104 CFU/ml C. albicans, 20% Dixon medium, 0.6 mM phosphate buffer (pH 7.0), and 16 μg/ml chloramphenicol. Plates were incubated at 25°C for 24 h with peptides, and absorbance was read at 600 nm.

BS-C-1 (American Type Culture Collection; CCL-26) African green monkey kidney cells (2 × 105 cells/well) were seeded in 24-well tissue culture plates in MEM-10% FCS and penicillin/streptomycin and allowed to grow overnight before the supernatant was removed and replaced with MEM-2.5% FCS for virus incubation. Peptides were diluted to the proper concentrations in 0.01× tryptic soy broth containing 10 mM sodium phosphate 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 mixture was added to the cells in 0.5 ml of MEM-2.5% FCS and allowed to infect for 48 h for plaque development. The medium was removed, and 0.5 ml of 0.1% crystal violet in PBS was added to the wells for 5 min at room temperature. Wells were then aspirated and air-dried for visualization of plaques.

Keratinocytes were cultured to confluence and treated with the peptide of interest in keratinocyte medium at 37°C. For the cathelicidin peptide fragment panel and dose-response assays, peptides were incubated with keratinocytes for 6 h. For the time-course assay, cathelicidin peptides were used at a concentration of 3 μM. Supernatants were removed and placed in a sterile 96-well plate for ELISA. IL-8 production was determined by ELISA (BD Biosciences) according to the manufacturer’s instructions. Briefly, 96-well plates were coated with capture Ab and incubated overnight at 4°C. Wells were washed three times, blocked for 1 h at room temperature, and again washed three times. Standards and 1/20 dilutions of samples were added to each well and incubated for 2 h at room temperature, followed by five washes. Detection Ab was added and incubated for 1 h at room temperature. Wells were washed seven times. Substrate solution was added to each well, and plate was incubated for 30 min at room temperature in the dark. Stop solution was added to each well, and the OD was observed at 450 nm with correction at 570 nm. IL-8 secretion from keratinocytes treated with pertussis toxin (Sigma-Aldrich), the FPRL-1 agonist WKYMVM-NH2 (Phoenix Pharmaceuticals), or the EGFR inhibitor AG1478 (Calbiochem) was assessed by IL-8 ELISA as described.

Keratinocytes were cultured to confluence and treated with the peptide of interest in keratinocyte medium for 6 h at 37°C. Total RNA was isolated from treated cells using an RNeasy Mini kit (Qiagen). Isolated RNA was retrotranscribed by reverse transcriptase using the Retroscript kit (Ambion). Briefly, first-strand synthesis combined RNA with 0.4 mM dNTPs and 4 μM random decamers in 16 μl at 80°C for 3 min. The reaction was iced, and 2 μl of 10× RT-PCR buffer, 1 μl of RNase inhibitor, and 1 μl of Moloney murine leukemia virus RT were added and incubated at 42°C for 1 h, then denatured at 92°C for 10 min. Real-time quantitative PCR was performed using an Applied Biosystems 7000 Sequence Detection System. TaqMan predeveloped assay reagents were used to detect human IL-8 (Applied Biosystems), and GAPDH primers and probe were used as an endogenous control. Briefly, 2 μl of the RT reaction was added to 25 μl of TaqMan Universal PCR Master Mix (Roche), 2.5 μl of target primers and probe, and 20.5 μl of RNase/DNase-free water. The thermal profile was 50°C for 2 min, 95°C for 10 min, and 40 cycles of 94°C for 15 s, followed by 60°C for 1 min. Results were analyzed using the comparative cycle threshold method (Applied Biosystems). Briefly, cycle threshold values were normalized to GAPDH, which was used as an endogenous control, then normalized to the sample chosen as a calibrator. Product specificity was confirmed by generating dissociation curves for the reactions.

Keratinocytes were incubated for 6 h at 37°C with 0, 3, or 10 μM cathelicidin peptide. Supernatants were collected into clean 96-well plates. Cells were washed three times. Basal medium (100 μl) was added to each well. Viability was assessed using the CellTiter96 Non-Radioactive Cell Proliferation Assay (MTT; Promega) according to the manufacturer’s instructions. Briefly, dye solution (15 μl) was added to each well, and the plate was incubated 4 h at 37°C. Solubilization/stop solution (100 μl) was added to each well, and the plate was incubated in a dark, moist chamber overnight at room temperature. OD was determined at 570 nm, with correction at 660 nm. Alternatively, cells were incubated briefly with 1.3 μg/ml propidium iodide (PI) after removal of supernatants, and fluorescence intensity was measured using a fluorescence microplate reader at an excitation of 530 nm and an emission of 620 nm. For evaluation of toxicity by membrane permeability to lactate dehydrogenase (LDH), LDH release from cells was measured using the Cytotoxicity Detection kit (Roche). Briefly, 50 μl of catalyst/dye solution was added to 50 μl 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 at 492 nm, with correction at 620 nm.

The human cathelicidin antimicrobial peptide LL-37 has been implicated in a variety of effects on the host immune system, including chemotaxis (16, 19), dendritic cell differentiation (17), and cytokine release (22). Epithelial surfaces such as the skin are exposed to abundant levels of LL-37 during the response to injury or infection. It was of interest to evaluate the response of epidermal keratinocytes to LL-37 at concentrations similar to those found in vivo (13). DNA microarray analysis of cultured keratinocytes exposed to LL-37 for 8 h revealed effects on the transcripts of various genes (Table I). Many genes involved in inflammation, including those encoding IL-8, cyclooxygenase 2, pro-IL-1β, TNF-α-induced protein 3, and IL-6, were significantly up-regulated.

Table I.

Change in transcript expression in LL-37-treated human keratinocytesa

Transcripts Up-RegulatedFold IncreaseTranscripts Down-RegulatedFold 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-1β (IL1β) 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, α2 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-α-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   
Transcripts Up-RegulatedFold IncreaseTranscripts Down-RegulatedFold 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-1β (IL1β) 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, α2 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-α-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 μM 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 μM 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 < 0.05.

To confirm the microarray analysis data showing that keratinocytes stimulated with LL-37 up-regulate the expression of cytokines and chemokines, quantitative immunodetection assays were performed on culture supernatants from treated cells. Consistent with the results of the microarray, IL-6 and IL-8 protein levels increased. The production of TNF-α and GM-CSF also increased. No changes were seen in the abundance of IL-1β, IL-2, IL-4, or IFN-γ (Fig. 1,A). For analysis of IL-1α, ELISA of cultured supernatant from LL-37-treated keratinocytes was performed and demonstrated a large increase in IL-1α release compared with cells treated with medium only (Fig. 1 B). Assays were performed on preconfluent, confluent, and postconfluent cells in the presence of low or high calcium, and no significant differences were found (data not shown).

FIGURE 1.

LL-37 influences cytokine production by human keratinocytes. LL-37 was diluted in keratinocyte medium to a final concentration of 3 or 10 μM. A, Keratinocytes were incubated with medium only or with LL-37 for 24 h at 37°C. Supernatants were analyzed for the indicated cytokines by Luminex assay. B, Keratinocytes were incubated with medium only or with 10 μM LL-37 for 1 or 24 h. Supernatants were analyzed for IL-1α by ELISA. Results are expressed as the mean ± SEM of one representative experiment of three, each performed in triplicate.

FIGURE 1.

LL-37 influences cytokine production by human keratinocytes. LL-37 was diluted in keratinocyte medium to a final concentration of 3 or 10 μM. A, Keratinocytes were incubated with medium only or with LL-37 for 24 h at 37°C. Supernatants were analyzed for the indicated cytokines by Luminex assay. B, Keratinocytes were incubated with medium only or with 10 μM LL-37 for 1 or 24 h. Supernatants were analyzed for IL-1α by ELISA. Results are expressed as the mean ± SEM of one representative experiment of three, each performed in triplicate.

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To compare the mechanisms used in antimicrobial activity to those responsible for activating keratinocytes, a library of LL-37-derived peptides was synthesized. The sequences of these peptides are shown in Fig. 2. Select peptides exhibited a broad range of antimicrobial activities against test organisms, which included Gram-negative and -positive bacteria, Vaccinia virus, and C. albicans (Table II). The peptide fragments RK-31 and KS-30 showed the highest overall antimicrobial properties, with minimal inhibitory concentrations ranging from 2 to 16 μM. Peptide fragment GD-23 exhibited the lowest minimal inhibitory concentration against Staphylococcus aureus mprF at 2 μM. LL-25, EK-20, KR-20, GK-17, RK-31, and KS-30 also inhibited the growth of S. aureus mprF better than LL-37. Under these culture conditions, several fragments showed higher activity than LL-37 against Escherichia coli, including LL-25, GD-23, RK-19, EK-20, EK-15, GK-17, EF-15, RK-31, and KS-30.

Table II.

MIC (μM) of LL-37 and LL-37 synthetic peptide fragmentsa

MIC (μM)
E. coli (O29)GAS (NZ131)S. aureus (ATCC 25932)S. aureus mprFVaccinia (Wyeth strain)C. albicans (ATCC 14053)
LL-37 64 8–16 >64 32 32 16 
LL-25 16 >64 >64 16 
LL-20 >64 >32 >64 >32 >64 >32 
LL-15 >64 >32 >64 >32 >64 >32 
LL-12 >64 >32 >64 >32 >64 >32 
GD-23 16 >32 >64 >64 32 
RK-19 32 >32 >64 >32 >64 16 
EK-20 32 >64 8–16 64 32 
KR-20 >64 >64 >64 16 
VQ-17 >64 >32 >64 >32 >64 >32 
EK-15 >32 >32 >320 >32 >64 >32 
GK-17 32 16 >320 16 32 16 
EF-15 >32 32 >320 32 >64 32 
RK-31 16 16 
KS-30 16 
MIC (μM)
E. coli (O29)GAS (NZ131)S. aureus (ATCC 25932)S. aureus mprFVaccinia (Wyeth strain)C. albicans (ATCC 14053)
LL-37 64 8–16 >64 32 32 16 
LL-25 16 >64 >64 16 
LL-20 >64 >32 >64 >32 >64 >32 
LL-15 >64 >32 >64 >32 >64 >32 
LL-12 >64 >32 >64 >32 >64 >32 
GD-23 16 >32 >64 >64 32 
RK-19 32 >32 >64 >32 >64 16 
EK-20 32 >64 8–16 64 32 
KR-20 >64 >64 >64 16 
VQ-17 >64 >32 >64 >32 >64 >32 
EK-15 >32 >32 >320 >32 >64 >32 
GK-17 32 16 >320 16 32 16 
EF-15 >32 32 >320 32 >64 32 
RK-31 16 16 
KS-30 16 
a

Bacterial and fungal minimal inhibitory concentration (MIC) values were determined by solution killing assay. Peptides ranging in concentration from 2 to 320 μM 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.

The effects of LL-37 synthetic peptide fragments as well as the structurally divergent d-amino acid form of LL-37 were next compared with full-length LL-37 in assays for keratinocyte IL-8 production. For initial screening, keratinocytes were cultured for 6 h in the presence of peptides at a final concentration of 3 μM. d-LL-37 stimulated the highest production of IL-8 by keratinocytes, followed by LL-37, KS-30, and RK-31, respectively (Fig. 3).

FIGURE 3.

Specific LL-37 peptides influence IL-8 production by human keratinocytes. Peptides were diluted to a concentration of 3 μM in keratinocyte medium. Cells were incubated with peptide for 6 h at 37°C. Supernatants were removed and analyzed for IL-8 by ELISA. Results are expressed as the mean ± SEM of one representative experiment of three, each performed in triplicate. Statistical analysis was performed using the paired Student’s t test: ∗, p < 0.01.

FIGURE 3.

Specific LL-37 peptides influence IL-8 production by human keratinocytes. Peptides were diluted to a concentration of 3 μM in keratinocyte medium. Cells were incubated with peptide for 6 h at 37°C. Supernatants were removed and analyzed for IL-8 by ELISA. Results are expressed as the mean ± SEM of one representative experiment of three, each performed in triplicate. Statistical analysis was performed using the paired Student’s t test: ∗, p < 0.01.

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Because EK-20, RK-31, and KS-30 were found to have good broad-spectrum antimicrobial activity, but little or no apparent ability to stimulate keratinocyte IL-8 release, dose and time responses were evaluated (Fig. 4). Treatment of keratinocytes with LL-37 resulted in the highest dose-dependent release of IL-8 from keratinocytes, although KS-30 also induced an increase in keratinocyte IL-8 production at higher concentrations. Peptide fragments RK-31 and EK-20 did not stimulate IL-8 above the medium background (Fig. 4,A). To observe the time-dependent release of IL-8 by keratinocytes, peptides were diluted to 3 μM. IL-8 accumulated with time in both control and peptide-treated keratinocytes. Full-length LL-37 again stimulated the highest levels of IL-8 release from keratinocytes, demonstrating a 6-fold increase at 24 h 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. 4 B). The increasing accumulation of IL-8 over time in response to these peptides was similar to constitutive release from keratinocytes treated with medium alone.

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, Peptides were diluted to 2, 4, 8, or 10 μM in keratinocyte medium. Cells were incubated with peptides for 6 h at 37°C. Supernatants were removed and analyzed for IL-8 by ELISA. B, Peptides were diluted to 3 μM in keratinocyte medium. Cells were incubated with peptides for 2, 6, 12, or 24 h at 37°C. Supernatants were removed and analyzed for IL-8 by ELISA. Experiments were performed three times in triplicate, with a representative outcome shown as the mean ± SEM.

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, Peptides were diluted to 2, 4, 8, or 10 μM in keratinocyte medium. Cells were incubated with peptides for 6 h at 37°C. Supernatants were removed and analyzed for IL-8 by ELISA. B, Peptides were diluted to 3 μM in keratinocyte medium. Cells were incubated with peptides for 2, 6, 12, or 24 h at 37°C. Supernatants were removed and analyzed for IL-8 by ELISA. Experiments were performed three times in triplicate, with a representative outcome shown as the mean ± SEM.

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To evaluate the effects of LL-37, EK-20, RK-31, and KS-30 on the membrane permeability of keratinocytes, LDH release and reduction of tetrazolium to formazan were studied by LDH and MTT assays, respectively. Minimal membrane disruption was observed by LDH assay for all peptides and concentrations tested (Fig. 5,A). Keratinocyte viability, as evaluated by MTT assay, was clearly reduced upon exposure to 10 μM LL-37 and, to a lesser extent, RK-31 and KS-30 (Fig. 5 B).

FIGURE 5.

Assessment of cytotoxicity to keratinocytes by LL-37 and LL-37 peptide fragments. A, Peptides were diluted to 3 or 10 μM in keratinocyte medium. Cells were incubated for 6 h at 37°C, and 50 μl of supernatants were removed and assessed for LDH release. B, Peptides were diluted to 3 or 10 μM in keratinocyte medium. Cells were incubated for 6 h at 37°C. Supernatants were removed and replaced with 100 μl of medium. Cells were assessed by MTT assay according to the manufacturer’s instructions. Experiments were performed three times in triplicate, with a representative outcome shown as the mean ± SEM.

FIGURE 5.

Assessment of cytotoxicity to keratinocytes by LL-37 and LL-37 peptide fragments. A, Peptides were diluted to 3 or 10 μM in keratinocyte medium. Cells were incubated for 6 h at 37°C, and 50 μl of supernatants were removed and assessed for LDH release. B, Peptides were diluted to 3 or 10 μM in keratinocyte medium. Cells were incubated for 6 h at 37°C. Supernatants were removed and replaced with 100 μl of medium. Cells were assessed by MTT assay according to the manufacturer’s instructions. Experiments were performed three times in triplicate, with a representative outcome shown as the mean ± SEM.

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Because 3 μM d-LL-37 was shown to exhibit potent IL-8 immunostimulatory 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 production in a dose-dependent manner (Fig. 6,A). Maximal IL-8 induction occurred at a lower concentration of d-LL-37 (4 μM), but both peptides showed equivalent immunostimulatory activity at the highest dose of 10 μM. Therefore, although d-LL-37 appears to be more potent than LL-37, both peptides have equivalent maximal IL-8 stimulatory capacity. Quantitative real-time PCR analysis showed induction of IL-8 transcript by keratinocytes in response to LL-37 and d-LL-37, but not EK-20 (Fig. 7). It was interesting to note that IL-8 production in response to LL-37 and d-LL-37 correlated directly with membrane permeability, as measured by PI incorporation, suggesting that these events may be connected (Fig. 6 B).

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 μM in keratinocyte medium. Cells were incubated with peptides for 6 h at 37°C. Supernatants were removed and analyzed for IL-8 by ELISA. B, Cells were treated with various peptide doses for 6 h at 37°C. Supernatants were removed and replaced with 1.3 μg/ml PI in PBS. Fluorescence was measured at an excitation of 530 nm and an emission of 620 nm. Experiments were performed three times in triplicate, with a representative outcome shown as the mean ± SEM.

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 μM in keratinocyte medium. Cells were incubated with peptides for 6 h at 37°C. Supernatants were removed and analyzed for IL-8 by ELISA. B, Cells were treated with various peptide doses for 6 h at 37°C. Supernatants were removed and replaced with 1.3 μg/ml PI in PBS. Fluorescence was measured at an excitation of 530 nm and an emission of 620 nm. Experiments were performed three times in triplicate, with a representative outcome shown as the mean ± SEM.

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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 for 6 h at 37C. 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 ± SEM.

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 for 6 h at 37C. 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 ± SEM.

Close modal

The strict sequence requirement for keratinocyte immunostimulatory activity contrasted with the less stringent domain requirements for broad-spectrum antimicrobial activity. However, the potent effects of LL-37 synthesized with d-amino acids suggested that a traditional ligand-receptor binding mechanism for keratinocyte recognition of LL-37 was unlikely. Previous investigations have implicated FPRL-1, a Gi-coupled receptor, in the chemotaxis of neutrophils, monocytes, and T cells in response to LL-37 (19). However, under the culture conditions used in this study, keratinocytes were not responsive to the FPRL-1 ligand WKYMVM-NH2 at concentrations up to 100 times greater than those reported to be effective in other cell types (100 nM; data not shown). Similarly, FPRL-1 expression was not seen in cultured keratinocytes or in whole skin by immunostaining. This result agrees with recent studies demonstrating that FPRL-1 is not involved in LL-37-induced activation of epithelial cells (21) or monocytes (23).

Previous investigations showed that cell activation and signaling by LL-37 can be Gi protein dependent (19) or independent (23). Therefore, the IL-8 response was evaluated after treatment of keratinocytes with the Gi inhibitor, pertussis toxin (PTX), in the presence 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 μM LL-37 and 4 μM d-LL-37, were chosen for inhibitor experiments. PTX significantly inhibited 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.

FIGURE 8.

PTX and AG1478 inhibit IL-8 induction in response to LL-37 and d-LL-37 peptides. Keratinocytes were pretreated for 1 h with medium alone, 100 ng/ml PTX, or 2 μM AG1478, then stimulated for 6 h at 37°C in the presence or the absence of LL-37 or d-LL-37. Supernatants were removed and analyzed for IL-8 by ELISA. Experiments were performed three times in triplicate, with representative outcome shown as the mean ± SEM. Statistical analysis was performed using the paired Student’s t test.

FIGURE 8.

PTX and AG1478 inhibit IL-8 induction in response to LL-37 and d-LL-37 peptides. Keratinocytes were pretreated for 1 h with medium alone, 100 ng/ml PTX, or 2 μM AG1478, then stimulated for 6 h at 37°C in the presence or the absence of LL-37 or d-LL-37. Supernatants were removed and analyzed for IL-8 by ELISA. Experiments were performed three times in triplicate, with representative outcome shown as the mean ± SEM. Statistical analysis was performed using the paired Student’s t test.

Close modal

Cathelicidins, such as LL-37, possess an inherent ability to kill Gram-negative and -positive bacteria, fungi, and viruses. LL-37 has also been shown to stimulate host inflammatory events (21, 24). The skin has demonstrated dependence on cathelicidin expression for resistance to infection by Streptococcus pyogenes in mice (25), and cathelicidin expression correlates with resistance to human skin infections (26). Therefore, this study analyzed the immunomodulatory effects of LL-37 peptides on keratinocytes for comparison with antimicrobial activity. LL-37 had significant effects on the expression of a wide range of transcripts from keratinocytes, with IL-8 showing the most abundant increase in steady state transcript abundance by DNA microarray analysis. Several peptides derived from LL-37 and sharing the sequence defined by EK-20 showed potent antimicrobial activity against a wide range 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 acids in the d configuration, d-LL-37, was a potent stimulator of IL-8 release by keratinocytes, an effect that was significantly inhibited either by a specific EGFR tyrosine kinase inhibitor, AG1478, or by PTX. Overall, these data suggest that the native processing of cathelicidins to alternate peptide forms can have important consequences for host defense responses.

The findings presented in this study show that LL-37 induces the expression of numerous inflammatory mediators by keratinocytes. The transcripts for IL-8, cyclooxygenase 2, pro-IL-1β, and IL-6 were among those significantly up-regulated. Confirmation of protein expression levels by Luminex assay also showed an increase in the proinflammatory mediators TNF-α and GM-CSF. The Luminex assay did not show an increase in IL-1β synthesis despite the DNA microarray findings and the prior report of IL-1β induction via upstream activation of the P2X7 receptor (20). This finding was not unexpected; although keratinocytes are able to make both IL-1α and IL-1β, they lack the protease required to cleave pro-IL-1β to its active form (27). It is interesting to note that IL-1α is induced after only 1 h of keratinocyte stimulation with LL-37. This immediate response is probably due to the release of stored IL-1α, rather than increased synthesis. The additional increase in IL-1α 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 cytokine expression (28, 29). Before additional cleavage to immunologically inactive forms, early deposition of LL-37 in the epidermis could serve as an additional inflammatory mediator through its properties as a chemoattractant or its ability to stimulate expression of other potent chemotactic agents, such as IL-8.

The results of this study suggest that cathelicidin antimicrobial properties have domain specificity. Specifically, the sequences within EK-20 appeared to be necessary, but not sufficient, for optimal antimicrobial activity. LL-20, LL-15, and LL-12, which lack the sequence VQRIKDFLRN, exhibited the lowest overall antimicrobial activity. The first six or seven amino acids of LL-37 did not appear to be essential for antimicrobial activity, because RK-31 and KS-30 lack these amino acids. Generation of an additional peptide library with site-specific amino acid modifications would serve to better define the domain responsible for cathelicidin antimicrobial activity.

Of principal interest in the current study was the lack of direct correlation between antimicrobial activity and host stimulatory effects, which was indicated by measuring IL-8 production. IL-8 was chosen as the indicator for host immunomodulatory activity due to the high induction of IL-8 mRNA and protein expression in LL-37-treated keratinocytes. In addition, previous investigations have shown that antimicrobial peptides, including LL-37 and neutrophil defensins, induce IL-8 synthesis and release by epithelial cells (21, 30). For example, LL-37, RK-31, and KS-30 exhibited broad-spectrum antimicrobial properties, but stimulated keratinocytes to increase IL-8 production to varying degrees. EK-20, which also exhibited broad-spectrum antimicrobial activity, did not increase IL-8 release. These peptides did not demonstrate significant cytotoxic effects on keratinocytes at concentrations effective for antimicrobial activity or keratinocyte immune stimulation. However, 10 μM LL-37 did affect keratinocyte reduction of MTT, suggesting potential keratinocyte membrane activity at this concentration in culture. Previous structure-function studies of synthetic antimicrobial peptides indicated a dissociation between antimicrobial and immunomodulatory activities (31, 32, 33). For example, N-terminal arginine residues of PR-39 were shown to be essential for protein binding and syndecan induction in mammalian cells, whereas modification of C-terminal residues specifically eliminated antimicrobial activity (31). Structural changes to PR-39 influenced antimicrobial activity, whereas mammalian effects were mostly dependent on N-terminal charge. The results of the present study endorse the function of cathelicidin peptides as membrane active agents with relative selectivity for microbial membranes (34). Remarkably, the antimicrobial effects of cathelicidin at concentrations <10 μM extended across bacteria, fungi, and viral envelopes, but spared the eukaryotic plasma membrane of keratinocytes.

The observation that d-LL-37 has more potent immunostimulatory activity than LL-37 raises additional questions pertaining to the mechanism by which peptide treatment induces IL-8 production by keratinocytes. d-LL-37 induced maximal IL-8 release at a low concentration (4 μM), whereas a higher concentration of LL-37 (10 μM) was required for equivalent induction at both the mRNA and protein levels. Interestingly, LL-37 caught up with d-LL-37, reaching the same maximum IL-8 level, albeit at a different dose. The more potent activity of d-LL-37, compared with LL-37, may reflect the resistance to proteolytic degradation of proteins composed of amino acids in the synthetic d form. Thus, additional activity may be a consequence of a higher final concentration after incubation with cells producing active proteases. Furthermore, as expected of membrane-active agents, cell permeability and membrane disruption corresponded directly with IL-8 mRNA and protein production in response to these peptides.

The mechanism by which cathelicidin peptides exert their effects on keratinocytes is unclear. Several receptors have been previously implicated in the host stimulatory effects of LL-37, including FPRL-1, EGFR, and P2X7 (19, 20, 21, 23). The current data suggest that specific binding to one of these receptors is an unlikely explanation for the response observed in keratinocytes, because both LL-37 and d-LL-37 induced a maximal IL-8 response. The potent activity of d-LL-37 argues against a highly structure-specific association of cathelicidin peptides with a cell surface receptor, although potential binding to a promiscuous pattern recognition receptor could account for activation by peptides in both the l and d forms, but not by the shorter peptides. Interestingly, treatment with AG1478 and PTX, either independently or in combination, blocked keratinocyte IL-8 induction in response to LL-37 and d-LL-37. Reduced IL-8 production by keratinocytes treated with these inhibitors suggests that the signaling mechanism generated at the membrane by LL-37 and d-LL-37 involves both the EGF receptor and membrane-associated Gi proteins. Gi proteins, which are classically described as being associated with G protein-coupled receptors (GPCRs), are recruited to the intracellular side of the membrane after receptor activation. However, at least three activators of G protein signaling that act independently of these typical receptors have been described (35). Although the data presented in this study suggest that LL-37 peptides permeabilize the cell membrane and are therefore able to enter cells, the ability of these peptides to directly interact with these intracellular activators of G protein signaling was not investigated.

Previous studies have demonstrated ligand-independent transactivation of receptor tyrosine kinases (RTKs), such as EGFR, by GPCRs (36, 37, 38, 39, 40). The occurrence of cross-talk between the GPCR and EGFR signaling pathways, which is regulated by the metalloprotease kuzbanian (ADAM10), may help to explain the inhibitory effects of both PTX and AG1478 on the keratinocyte IL-8 response (41, 42). Other studies have suggested that cellular stresses also perturb the cell surface and alter receptor conformation, thereby activating intracellular signaling pathways (36, 37, 43, 44, 45, 46, 47). In a similar manner, LL-37 may directly interact with the keratinocyte plasma membrane to cause conformational changes that indirectly activate surface receptor domains linked to intracellular signaling molecules. Although the mechanism by which GPCR and RTK signaling pathways converge has not been completely determined, recent work has suggested that these pathways may run parallel and fulfill different signaling functions (48, 49). Therefore, LL-37-mediated activation of transmembrane receptors, including both GPCRs and RTKs, in addition to influences on keratinocyte membrane ionic equilibrium should be further explored to understand the mechanism by which LL-37 peptides activate downstream production of immunostimulatory molecules by keratinocytes.

These observations support previous studies in several cell, animal, and human systems that cathelicidin peptides have properties of both natural antibiotics and host stimulatory molecules (6, 50, 51, 52, 53). The rapid increase in cathelicidin at epithelial interfaces exposed to injury or infection can provide a simultaneous barrier to microbial proliferation and an additional alarm to the host. The term alarmins, which was recently proposed to refer to peptides such as the cathelicidins or defensins, is quite appropriate in this model (54). Proteolytic processing at sites of peptide deposition could enable the epithelia to further modify the spectra of biologic activity of cathelicidin peptides and regulate the balance of activity between host immune modulation and inhibition of microbial growth. Additional studies examining changes in membrane fluidity, ion transport, and receptor aggregation in the presence of LL-37 will offer pertinent information concerning the mechanism by which LL-37 stimulates keratinocytes to produce IL-8 and other proinflammatory molecules.

The authors have no financial conflict of interest.

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, epidermal growth factor receptor; GPCR, G protein-coupled receptor; LDH, lactate dehydrogenase; PI, propidium iodide; PTX, pertussis toxin; RTK, receptor tyrosine kinase.

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