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* Division of Dermatology, University of California, San Diego, and VA San Diego Health Care System, San Diego, California, USA;
Department of Dermatology, University of Pennsylvania, Philadelphia, Pennsylvania, USA;
Inserm, U563, Toulouse, F-31300, France; Université Paul-Sabatier, Toulouse, France, and
CHU Purpan, Department of Genetics, France
1Correspondence: MC 9111B, 3350 La Jolla Village Dr., San Diego, CA 92161, USA. E-mail: rgallo{at}ucsd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: antimicrobial peptide • stratum corneum tryptic enzyme • stratum corneum chymotryptic protease • lympho-epithelial Kazal-type related inhibitor • SELDI-TOF-MS
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, birds (2)
, and fish (3)
. They are best known for their action as innate antimicrobials that protect the host against infection by Gram-positive bacteria (4)
, Gram-negative bacteria (5
, 6)
, and some viruses (7)
. Analysis of several animal models (4)
, and human clinical conditions (8)
, have demonstrated that the presence of cathelicidin correlates with the ability of the host to effectively mount a defense against infection. Cathelicidin deficiency has also been reported in Kostmann syndrome and is associated with severe congenial neutropenia and frequent oral infection (9)
. Although antimicrobial peptides such as the cathelicidins represent an evolutionarily ancient form of immune response, current evidence strongly supports the conclusion that these molecules are an integral element of human immunity (10)
. Antimicrobial peptides such as cathelicidins act both to kill microbes and to initiate or modify other cellular immune events. As such, these molecules have been alternatively referred to as "alarmins" (11)
. Due to this activity on the host, strict control of cathelicidin expression and function is necessary to restrict activity of the peptide to conditions where maximal defense against microbial invasion is required. The nascent cathelicidin protein is inactive and consists of an N-terminal cathelin domain that is conserved among mammalian species and a C-terminal domain encoding the mature cathelicidin peptide. A comparison of cathelicidin peptides purified from various species or predicted by their respective cDNA sequences has revealed that these potent antimicrobial molecules are variable sequences of 20 to 40 amino acids in the C-terminal domain. In humans and mice the cathelicidin peptides are alpha-helical, cationic, and amphipathic. These properties enable association and integration with negatively charged cell membranes.
Expression of cathelicidins, such as the human LL-37 released from neutrophils, is regulated by transcriptional and post-transcriptional processing. Cathelicidin transcript and total protein abundance in vivo is induced by infection (12)
, inflammation (13)
, wounding (14)
, and differentiation (15
, 16)
. In humans, 1,25-dihydroxyvitamin D3 is a direct inducer of cathelicidin expression (17)
whereas in mice the cathelicidin mCRAMP (1)
(mouse cathelin-related antimicrobial peptide) expression is dependent on HIF-1a (hypoxia-inducible factor 1, alpha) (18)
. Although these stimuli modify cathelicidin mRNA abundance, factors that regulate the translation and final activation of cathelicidin are unclear. Recent observations of cathelicidins in vivo suggest that the final proteolytic processing of cathelicidin dictates antimicrobial or alarmin activity (19)
.
The nascent human cathelicidin gene product is designated hCAP18 (human 18-kDa cationic antimicrobial protein) (20)
. hCAP18 has been shown to be processed to the antimicrobial peptide LL-37 by proteinase-3 in neutrophils (21)
. The prostate-derived protease gastricsin can also process hCAP18 to ALL-38 (22)
. At the skin surface, unidentified proteases present in human sweat can cleave LL-37 to smaller peptides such as RK-31, KS-30, and KR-20 (23)
. The native cathelicidin peptides at the skin surface are unknown, but these smaller peptides generated in vitro have more potent antimicrobial activity than LL-37. Analysis of alternatively processed human cathelicidin peptides has further shown that processing alters the immunostimulatory capacity of cathelicidins, eliminating their ability to stimulate interleukin (IL)-8 release (19)
. These observations demonstrate the importance of proteolytic processing to the activity of cathelicidins and, by extension, their importance to immune defense in general.
Since the presence of cathelicidin in skin is critical for normal microbial defense and innate immunity, we sought in the present study to identify the cathelicidins in human skin and define the proteases responsible for cathelicidin activation. We show that novel cathelicidin peptide forms are present at the skin surface and that kallikreins, a family of serine proteases known for their influence on the development of the epidermis, are responsible for their generation. Moreover, we show that cathelicidin processing is altered in vivo in the absence of the serine protease inhibitor LEKTI. These data suggest that the function of kallikreins in the skin is in part to regulate the immune barrier, both by modifying the physical structure of the epidermis and by determining innate antimicrobial peptide function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Protein chips (RS100 protein chip array, Ciphergen Biosystems, Fremont, CA, USA) were coated with 4 µl of rabbit anti-LL-37 antibody (Ab) (0.73 mg/ml) for 2 h at room temperature, followed by blocking with 0.5 M etahnolamine in PBS (pH 8.0). After washing three times with PBS/0.5% Triton X, protein chips were assembled in the BioprocessorTM reservoir, and samples (50 µl) were applied and incubated for 2 h at room temperature. Protein chips were washed three times with 1 x RIPA buffer, twice with PBS/0.5% Triton X-100, and three times with PBS, followed by soaking in 10 mM HEPES buffer, then air dried. A half microliter of energy absorbance molecule (50%-saturated alpha-cyano-4-hydroxy cinnamic acid in 50% acetonitrile, 0.5% trifluoric acid) was applied twice, and all spots were completely dried up. Samples were analyzed on a SELDI mass analyzer PBS II with a linear time-of-flight mass spectrometer (Ciphergen Biosystems) using time-lag focusing. Specificity and accuracy in this system were confirmed by several synthetic cathelicidin peptides as standards (19
, 23)
. Synthetic LL-37 and KR-20 peptides were used as an internal reference. Skin extracts from three individuals were analyzed and showed similar patterns of cathelicidin peptides.
Collection and assay of skin proteases
Protease activity at the skin surface was evaluated in sweat collected as described for collection of human sweat (24)
. Activity was monitored by EnzCheck® Protease Assay Kit green fluorescence (Molecular Probes, Inc., Eugene, OR, USA) according to the manufacturer’s instructions. Briefly, 100 µl of the aqueous solution collected from the skin surface was mixed with 100 µl of BODIPY FL casein substrate and incubated at 37°C for designated periods. Protease activity was monitored as increased fluorescence with SpectraMax GEMINI EM (Molecular Devices Corp., Sunnyvale, CA, USA). In some experiments protease inhibitors were added, including mixed protease inhibitor mixture (complete EDTA-free, 1 tablet/50 ml; Roche, Indianapolis, IN, USA), 200 µg/ml bestatin, 20 µg/ml E-64, and 20 µg/ml aprotinin (Sigma-Aldrich, St. Louis, MO, USA), 200 µM 4-(2-aminoethyl)-benzenesulfonylfluoride (AEBSF), 200 µM human neutrophil elastase inhibitor (methoxysuccinyl-Ala-Ala-Pro-Ala-chloromethyl ketone, Calbiochem, San Diego, CA, USA), 200 µM human leukocyte elastase inhibitor (methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone, Calbiochem), 100 µM chymostatin (Roche), or 10 µM leupeptin (Roche).
Purification of protease activity
Sweat (240 ml) collected from the skin surface was concentrated using MacrosepTM Centrifugal Devices (3 kDa cutoff; Pall Life Science, Ann Arbor, MI, USA). Separation of enzyme activity was performed using an 
KTA purification system (Amersham Pharmacia Biotech, Piscataway, NJ, USA) on a reverse phase HPLC (µRPC C2/C18 ST 4.6/100 column; Amersham Pharmacia Biotech). Columns were equilibrated in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min and eluted using gradients of 0–60% acetonitrile for 91 min. Column effluent was monitored at 214, 230, and 280 nm. All fractions collected (1 ml) were lyophilized and suspended in 50 µl of distilled water for protease assay and immunoassay.
Skin surface fractions purified by HPLC were evaluated by quantitative dot blot. Five microliters of each fraction was compared with a standard curve of recombinant SCTE (25)
applied onto NitroBond nitrocellulose membrane (GE Osmonics Labstore, Minnetonka, MN, USA). For immunoblot, membranes were blocked with 5% nonfat milk and 3% BSA in 0.1% TTBS (0.1% Tween 20/TBS (150 mM NaCl and 10 mM Tris base, pH 7.4) for 60 min at room temperature, then incubated with mouse anti-human SCTE monoclonal antibody (mAb) (1/1000 in the blocking solution; R&D Systems Inc., Minneapolis, MN, USA) overnight at 4°C. After washing three times with 0.1% TTBS, the membrane was incubated with HRP-conjugated goat antimouse Ab (1/5000 in the blocking solution; DAKO, Carpinteria, CA, USA) for 60 min at room temperature. After washing the membrane again with 0.1% TTBS, the membrane was immersed in enhanced chemiluminescence (ECL) solution (Western LightningTM Chemiluminescence Reagents Plus; Perkin-Elmer Life Sciences, Boston, MA, USA) for 60 s, then exposed to X-ray film (X-OmatTM; Eastman Kodak, Rochester, NY, USA).
Analysis of cathelicidin processing
LL-37 was synthesized and prepared as described previously (23)
. For analysis of LL-37 processing, 32 nmol of LL-37 synthetic peptide was incubated with proteases in 100 µl for 0, 1, 6, and 24 h at 37°C. The buffer used was 0.2 M NaCl, 0.1 M MOPS, pH 7.0 for SCTE and 2.0 M NaCl, 0.045 M Tris-HCl, pH 8.0 for SCCE. After incubation, peptides were separated by reverse phase HPLC (Sephasil peptide C18 12 µm, ST 4.6/250 column; Amersham Pharmacia Biotech). Column was equilibrated in 10% acetonitrile with 0.1% trifluoroacetic acid at a flow rate of 2 ml/min and cleaved peptides were eluted using gradients of 10–20 and 20–70% acetonitrile for 2 and 31 min, respectively. Column effluent was monitored at 214, 230, and 280 nm. All collected fractions (1 ml) were lyophilized and suspended in 10 µl of distilled water for antimicrobial radial diffusion assay or directly analyzed by mass spectrometry.
For Western blot, recombinant hCAP18 (26)
was incubated with proteases and separated by 16% Tris-tricine gel (GeneMate® Express gels; ISC BioExpress, Kaysville, UT, USA), then transferred onto a polyvinylidene difluoride membrane (Immobilon-P; Millipore, Billerica, MA, USA). For the positive control, 5 pmol of LL-37 synthetic peptide was applied. Membranes were blocked with 5% nonfat milk and 3% BSA in 0.1% TTBS, incubated with anticathelin domain chicken Ab (1/5000 in the blocking solution) or anti-LL37 rabbit Ab (1/5000 in the blocking solution), and developed as described above.
Mass spectrometry and protein sequence analysis
Mass spectrometry was performed by the Mass Spectrometry Facility in the Department of Chemistry and Biochemistry at University of California, San Diego. The matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) spectra were obtained with a Finnigan LCQ DECA mass spectrometer (Thermo Electron Corporation, Waltham, MA, USA). The instrument has unit mass resolution over a mass range of 50–2000 m/z with a typical mass accuracy of 100 ppm (0.01%). Protein sequence analysis for target HPLC fractions was performed by the Division of Biology Protein Sequencer Facility, University of California, San Diego. Amino acid sequencing was performed on Applied Biosystems Procise model 494 sequencer (Foster City, CA, USA) using the pulsed liquid program supplied by the manufacturer.
Antimicrobial assays
For screening of antimicrobial activity, radial diffusion assay was used as described previously (24)
. Lyophilized HPLC fractions were dissolved in 10 µl of MOPS buffer (pH 7.0) and tested against Staphylococcus aureus mprF (gift from A. Peschel, Microbial Genetics, University of Tubingen, Tubingen, Germany). This strain of S. aureus was selected for screening due to its increased sensitivity to cationic peptides. Thin plates (1 mm) of 1% SeaKem® GTG® Agarose (Cambrex Corp., East Rutherford, NJ, USA) and 1% LB Broth (EM Science, Gibbstown, NJ, USA) in 10 mM phosphate buffer (pH 7.2) containing 5 x 106 cells/ml of S. aureus mprF were used. One-millimeter wells were punched in the plates, and 2 µl of samples were loaded in each well. As a positive control, synthetic LL-37 was applied to separate wells. After incubation at 37°C overnight, the inhibition zone diameters were measured.
Fluorescence immunohistochemistry
Frozen sections of normal skin (6 µm) were fixed with paraformaldehyde, sections were blocked with 5% goat serum, then incubated simultaneously with polyclonal rabbit anti-LL-37 and monoclonal mouse anti-SCTE primary antibodies. Goat anti-rabbit IgG conjugated to AlexaFluor488 (Molecular Probes) and goat antimouse IgG conjugated to tetramethylrhodamine isothiocyanate (TRITC) were used as secondary antibodies, respectively. Nuclei were stained with 4',6'-diam idino-2-phenylidole (DAPI) and sections were mounted in ProLong Anti-Fade reagent (Molecular Probes). Images were obtained using an Olympus BX41 fluorescent microscope (Scientific Instrument Company, Temecula, CA, USA).
siRNA transfection
siRNA for SCTE and SCCE and transfection reagents were obtained from Darmacon, Inc. (Chicago, IL, USA). The human keratinocyte cell line HaCaT, a generous gift from Dr. Norbert Fusenig (Krebsforschungszentrum, Heidelberg, Germany), was cultured in Dulbecco’s modified Eagle medium (DMEM) plus 10% fetal calf serum and 100 U/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. After cells reached 70% confluence they were treated with 1 µg of siRNA with transfection reagent or with transfection reagent only. Cultured media and cells were collected 48 h after transfection. Buffer with supplements at final concentrations of 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 5 mM EDTA, and 1 µg/ml pepstatin were added and samples were sonicated on ice for 5 min. After centrifuge at 14,000 rpm for 10 min, protease activities of supernatants was monitored by EnzCheck® Protease Assay Kit as described above. The suppression of SCTE and SCCE mRNA were confirmed by the quantitative RT-polymerase chain reaction (RT-PCR) with specific primers and probes (Applied Biosystems). Glyceraldehyde-3-phosphate dehydrogenase primers and probe (Applied Biosystems) were used as an endogenous control. Results of the quantitative polymerase chain reaction (PCR) were plotted as the relative expression to the control using the Comparative Ct Method (User Bulletin #2, Applied Biosystems).
Cathelicidin peptide extraction from mouse skin
Neonatal mouse skin was excised by 8 mm punch biopsy, homogenized in 1 ml of 1 M acetic acid, and incubated at 4°C overnight. After centrifugation for 15 min at 14,000 rpm, soluble fractions were transferred to new tubes and lyophilized. Pellets were dissolved in 50 µl of MOPS buffer (5 mM, pH 7.0). Antimicrobial activity against Staphylococcus aureus mprf was examined by radial diffusion assay as described previously. Antimicrobial activities were determined in solution assay against wild-type (WT) S. aureus [Rosenbach American Type Culture Collection (ATCC) 25923; ATCC, Manassas, VA, USA], methycilin-resistance S. aureus strain (81025 and 82056, and clinical isolate generously provided by Dr. Joshua Fierer, Veterans Affairs Healthcare Systems (San Diego, CA, USA), group A streptococcus (M49 strian NZ131 and M1 strain), and Pseudomonas aeruginosa (clinical isolate generously provided by Dr. Joshua Fierer). Bacteria (1x106-109 CFU) in log-phase growth were suspended in 20 µl of RPMI1640 with 10% FCS, and 5 µl of the skin extracts was added and incubated at 37°C for 3 h. Bacteria were then serially diluted in PBS and plated on TSB agar (tryptic soy broth; 30 g/l, Sigma-Aldrich, BactoTMagar; 10 g/l, BD Bioscience, Sparks, MD, USA) for direct colony count and determination of CFU. Amount of mCRAMP was determined by immuno-dot blot using synthetic CRAMP peptides as references, and mean and SE of three mice were plotted on the graph. Processing of mCRAMP was analyzed by SELDI-TOF-MS as described previously with rabbit anti-CRAMP Ab (14)
(1.55 mg/ml) instead of antihCAP18/LL-37 Ab. To delete cathelicidin activity from skin extracts, skin extracts were incubated with immobilized anti-CRAMP Ab, and supernatants were analyzed as cathelicidin-immunoabsorbed skin extracts. Depletion of cathelicidin was confirmed by a second SELDI-TOF analysis. Serine protease activity with the presence of 5 mM EDTA and 1 µg/ml pepstatin was determined using EnzCheck® as described previously.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The presence of this spectrum of cathelicidin peptides suggested that local proteolysis has a major influence on the identity of cathelicidin. Furthermore, these findings show that prior conclusions were inaccurate when LL-37 was assumed to be the form of cathelicidin in skin.
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Identification of cathelicidin processing enzymes at the skin surface
To identify enzymes at the skin surface that cleave human cathelicidin, protease activity collected from the skin surface of healthy volunteers was first detected by a fluorescence-conjugated casein-based assay. Protease activity was detected by the increase in fluorescence over time compared with that seen when substrate was incubated in a buffer solution of similar ionic composition (Fig. 2
A). Enzyme activity was detectable for up to 4 days after incubation. To determine the nature of these proteases, a panel of inhibitors that distinguish between the various major protease families was included. The only effective inhibitors were aprotinin and AEBSF, which are inhibitors of serine protease (Fig. 2B
). Bestatin (amino peptidase inhibitor), E-64 (cystein protease inhibitor), N.E.I. (neutrophil elastase inhibitor), L.E.I. (leukocyte elastase inhibitor), EDTA (metalloprotease inhibitor), and pepstatin (asparate protease inhibitor) were ineffective. Chymostatin and leupeptin, which specifically inhibit chymotrypsin-like and trypsin-like serine proteases, respectively, were used to characterize this more precisely. Under these conditions, chymostatin suppressed protease activity whereas leupeptin had a minimum effect, suggesting a chymotrypsin-like serine protease was most active (Fig. 2C
).
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Surface enzyme activity was next concentrated by centrifugal dialysis and fractionated by reverse-phase chromatography (Fig. 3
A). Fractions numbered 55 to 59 showed the greatest protease activity against a casein substrate and could cleave LL-37 to several smaller peptides (Fig. 3B
). The products cleaved from LL-37 were evaluated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and their protein sequences were deduced from the molecular weights determined in the analysis (Fig. 3C
). Radial diffusion assay revealed that some of these fragments, such as KS-30, KS-27, KR-20, and LL-23, showed antimicrobial activity against S. aureus mprF, whereas other shorter peptides lost antimicrobial activity (data not shown). The deduced cleavage sites producing the peptide patterns in Fig. 3C
suggested that two different serine proteases were involved: one with trypsin-like substrate specificity that hydrolyze after Arg residues and the other with chymotrypsin-like substrate specificity that cleaves at after Phe residues. Therefore, tryptic and chymotryptic serine proteases were considered candidates for skin surface cathelicidin proteases.
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Members of the kallikrein family of serine proteases have been detected in the stratum corneum. Two proteases well established as human skin are stratum corneum tryptic enzyme (SCTE, kallikrein 5/KLK5, hK5) and stratum corneum chymotryptic protease (SCCE, kallikrein 7/KLK7, hK7) (27
, 28)
. To determine whether these enzymes were present in the skin surface preparations and whether they colocalized with cathelicidin in skin, antiserum to each protease was used. SCTE immunoreactivity coeluted with fractions that showed protease activity (Fig. 3A
). Immunohistochemistry of normal human skin demonstrated that SCTE and cathelicidin are colocalized in the granular to spinous layer, and LL-37 alone is detected at the uppermost layer of stratum corneum (Fig. 4
A).
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Next, to determine what proportion of serine protease activity from keratinocytes could be attributed to kallikrein activity, we examined the influence of siRNA targeting of SCTE and SCCE mRNA on the level of caseinolytic activity secreted from keratinocytes. SCTE and SCCE siRNA suppressed SCTE mRNA expression to 56% of control and SCCE mRNA to 72%, respectively (Fig. 5
A), and both SCTE and SCCE siRNA significantly reduced human keratinocytes protease activity (Fig. 5B
).
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Human cathelicidin is initially synthesized as a precursor protein hCAP18 (26)
. To determine whether SCTE and SCCE could cleave hCAP18, recombinant hCAP18 was incubated with recombinant SCTE or SCCE (25)
, and cleavage was examined by Western blot. Polyclonal antibody (pAb) against the cathelin domain (recognizing the N-terminal cathelin domain, but not detecting cleaved C-terminal peptides, ref. 26
) detected bands of 
12 kDa and 10 kDa after SCTE treatment and a 14 kDa band after SCCE treatment (Fig. 6
A). Ab against LL-37 (recognizing the C-terminal 37 amino acid mature peptide domain) revealed that SCTE generated a small fragment of 6 kDa, and SCCE generated an 8 kDa band. To determine the sequence of these small fragments directly, SCTE-treated hCAP18 peptides were isolated by chromatography and the molecular mass were determined by MALDI-MS. The small peptide cleaved by SCTE had a molecular mass of 4491, identifying it as LL-37 (Fig. 6B
). On the other hand, treatment of hCAP18 with SCCE generated multiple peptides whose abundance was insufficient to identify by this approach (data not shown).
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SCTE and SCCE cleave LL-37 to shorter peptides
Unlike the in vitro conditions described in Fig. 6
, the cleaved cathelin domain is rarely detected in vivo. This protein has activity as a protease inhibitor and antimicrobial (26)
, and structural studies suggest the cathelin domain has a pocket in which the C-terminal LL-37 peptide may bind (29)
. As this may protect the peptide from further proteolysis, we next examined conditions that reflect those observed in skin. LL-37 was incubated with recombinant SCTE and SCEE, and the peptide products were determined. As shown in the time course of Fig. 7
A, the major products of SCTE digestion of LL-37 after 1 h of incubation were KS-29, KS-30 (peak g), and KS-22 (peak f). KS-22 was derived from KS-30 by second cleavage eight residues from the C terminus. These three peptides have antimicrobial activity, indicating that at early times of digestion SCTE will generate biologically functional peptides. However, after 3 h the peaks with antimicrobial activity (peaks f, g) began to decline, indicating further processing to small nonfunctional peptides.
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Cleavage of LL-37 by SCCE was less efficient than by SCTE, as the LL-37 peak disappeared much more slowly in the incubation containing SCCE than in that containing SCTE. Consistent with its predicted specificity, SCCE cleaved LL-37 after Phe residues (Fig. 7D
). Although SCCE generated peptides RK-31 and KR-20 (peaks o and n in Fig. 7C
, respectively), which had antimicrobial activity and were seen on the surface of skin, the existence of these peptides appeared to be short-lived. Their quantities showed a detectable change with time, and were no longer present after 24 h of incubation. Thus, their production appears highly transient, with digestion to smaller fragment proceeding faster than their generation.
Serine protease activity controls antimicrobial function in skin
We next tested cathelicidin proteolysis in an in vivo model. Lympho-epithelial Kazal-type related inhibitor (LEKTI) is a serine protease inhibitor encoded by SPINK5 (serine protease inhibitor Kazal-type 5) gene (30)
. LEKTI is expressed in the epidermis, and mutations of SPINK5 gene are found in the human condition, Netherton syndrome, leading to a disruption in skin barrier function (31)
. The activities of SCTE and SCCE are increased in SPINK5-deficient mice, and these mice mimic the epidermal dysfunctions in Netherton syndrome (32)
. We extracted skin of neonatal SPINK5-deficient mice and of WT littermates and confirmed that protease activity in skin extracts from SPINK5-deficient mice was higher than WT littermates (Fig. 8
A). Direct analysis of the effect of recombinant SCTE and SCCE on bacterial growth showed that these enzymes are not inherently antimicrobial. Furthermore, the abundance of total immunoreactive cathelicidin expressed in SPINK5-deficient mice was not significantly different from WT littermates (Fig. 8B
). Despite the similarity in the expression of cathelicidin, skin extracts from SPINK5-deficient mice inhibited growth of S. aureus mprF whereas skin extracts from WT littermates did not (Fig. 8C
). Other Gram-positive and negative bacterial species were similarly inhibited by SPINK5-deficient, but not WT mice including WT S. aureus, methycillin-resistant S. aureus, group A Streptococcus, and Pseudomonas aeruginosa (data not shown). Immunoabsorption of skin extracts with anti-CRAMP Ab effectively removed mCRAMP and precursor protein based on HPLC and SELDI-TOF analysis (data not shown) and eliminated antimicrobial activity detected in the in SPINK5-deficient mice (Fig. 8A
). SELDI-TOF-MS analysis confirmed that mCRAMP in SPINK5-deficient mouse skin was processed to shorter peptides (data not shown). The mass of the major peak in SPINK5-deficient mouse was 3878 Da and matched with the mCRAMP1 (GLL-34) peptide (33)
. In WT littermates, major peaks of 17 and 20 kDa matched the size of mCRAMP proprotein without and with signal peptide, respectively. These data suggested that processing of cathelicidin in SPINK5-deficient mice skin augmented antimicrobial activity.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. In the present study we show that cathelicidin, in either the proform hCAP18 or the peptide form LL-37 released upon neutrophil recruitment, is susceptible to proteolytic processing by serine proteases belonging to the tissue kallikrein family. This processing generates novel peptides with antimicrobial activity and suggests that cathelicidins in skin are more diverse than previously reported. Their function ultimately depends on the activity of proteases at the epithelial surface. Inhibitors of serine proteases such as LEKTI (25)
and anti-leukoprotease/SLPI (34)
regulate this process (Fig. 9
). Although not directly evaluated here, these findings also suggest that other serine protease inhibitors such as SKALP/elafin, a potent proteinase 3 inhibitor expressed in human epidermis (35)
, may affect hCAP18 proteolysis through the regulation of proteinase 3 activity.
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Evidence to support the role of serine proteases to modify the proinflammatory activity of cathelicidin comes from observations of their distribution in the epidermis and their biochemical activity. SCTE and cathelicidin are colocalized in the granular layer, but cathelicidin is dominant in the cornified layer of normal human skin. Since cathelicidin and kallikreins are both stored in lamellar granules and secreted from keratinocytes (36
, 37)
, their localization in skin suggests that after secretion, hCAP18 can be efficiently cleaved by kallikreins and subsequently released at the surface to form an antimicrobial barrier. If processed to forms that are not inherently chemotactic or capable of stimulating host cells to release chemotactic factors such as IL-8, this mechanism explains why the constitutive presence of cathelicidins on the skin is not inflammatory under normal conditions or in transgenic models (38)
despite the potent capacity of LL-37 or mCRAMP to induce cytokine release. Kallikrein in the normal human epidermis has the potential to both activate and degrade cathelicidin to inactive peptides.
Analysis of the enzymatic processing of cathelicidin suggests that two steps are required: activation from the precursor hCAP18 and subsequent processing to modify activity. We found that two cleavage patterns were generated in the skin from cathelicidin precursor proteins: cleavage C-terminal to arginine (R), and cleavage C-terminal to phenylalanine (F). SCTE was effective in generating LL-37 from the precursor hCAP18 and can subsequently produce KS-30, KS-22, and LL-29 from LL-37. RK-31 and KR-20 peptides are cleaved by SCCE from LL-37. The ability to generate these antimicrobial peptides quickly and in high amounts relative to other peptides suggests that the cleavage sites producing these peptides are preferred by SCTE. SCTE has trypsin-like activity, and the preference of SCTE for arginine over lysine has been noted in model substrates (39)
. Thus, SCTE prefers to cleave C-terminal to arginine residues in LL-37, although LL-37 has several arginine and lysine residues in its sequence and may best be considered a generator of antimicrobial activity rather than a degrader of LL-37. Conversely, data obtained with SCCE suggested this enzyme might be considered a degrader of LL-37 rather than a generator of antimicrobial peptides. SCCE has been shown to prefer tyrosine over phenylalanine and to be less able to cleave C-terminal to phenylalanine in model substrates (34)
. Therefore, SCCE is less effective than SCTE in cleaving LL-37 to generate smaller peptides, and smaller peptides may be easier to be accessed and degraded by SCCE. Thus, SCCE may serve as an inactivator, an important role when the chemotactic activity of LL-37 is no longer beneficial to the defense process.
It is debatable whether only SCTE and SCCE digest hCAP18 and generate cathelicidin antimicrobial peptides at the complex human skin surface. Human skin epidermis and appendages express several kallikrein proteases, and these proteases may act together (40)
. Treatment of human keratinocytes with siRNA for SCTE and SCCE showed a decrease of serine protease activity, suggesting that SCTE and SCCE are major serine proteases in culture. Purification of crude skin extracts was detected SCTE in fractions that cleaved LL-37. The chymotryptic serine protease SCCE is also a candidate because protease activity in the skin surface was suppressed the most by the chymotrypsin-specific inhibitor chymostatin, and both chymotrypsin-like cleavage patterns and trypsin-like cleavage patterns were observed with LL-37 as substrate. However, unlike SCTE, we did not detect SCCE in protease-active fractions with commercially available anti-SCCE antibodies (data not shown). This may reflect a relative lack in the sensitivity of this Ab, as our other data based on RNAi and uses of recombinant enzymes suggest that SCTE and SCCE can both cleave cathelicidin. Alternatively, assays of total proteolytic activity from the skin surface based on casein substrate may not detect either SCCE or SCTE, and may actually detect another chymotrypsin. Furthermore, in the current study SELDI-TOF-MS revealed that diverse cathelicidin peptides exist in skin, but these were not identical to the peptides isolated earlier by standard HPLC approaches from human sweat (24)
. Because the current samples were extracted from whole skin, these may have been generated by proteases from various cells in skin such as keratinocytes, endothelial cells, fibroblast, dendritic cells, and some inflammatory cells, and not only surface proteases such as SCTE and SCCE. Alternatively, the purification methods may have influenced these results. However, taken together with results derived from tissues and recombinant enzymes, the current observations strongly support a major role for both SCTE and SCCE in processing cathelicidin in skin.
The actual identity (size and sequence) of cathelicidin peptides at various locations has only recently come into question. Subsequent to the initial cloning of human cathelicidin from a human bone marrow library, a 39 aa peptide designated FALL-39 was predicted (41)
. Subsequent purification and direct sequencing from human neutrophils identified the actual peptide as a 37aa peptide, LL-37, and proteinase 3, an elastase-like serine protease stored in neutrophil granules, was suggested to be the enzyme responsible for the processing of precursor protein hCAP18 to generate LL-37 (21)
. More recently, urogenital epithelium was found to process hCAP18 to ALL-38 by the prostate-derived protease gastricsin (22)
. In contrast to these longer peptides, several alternate cathelicidin antimicrobial peptides were detected at the skin surface (23)
, an interface that has been shown to rely on the expression of cathelicidin for defense against microbial invasion (4)
. These observations suggested that previous conclusions claiming LL-37 was the sole human cathelicidin were an oversimplification.
The significance of variable processing of cathelicidin to peptides other than LL-37 lies in the observations that LL-37 induces chemokine release and can function in host cell stimulation through direct receptor-ligand interactions such as the stimulation of formyl peptide receptor-like 1 (42)
or through transactivation of the epidermal growth factor (EGF) receptor (43)
. A comparison of the ability of LL-37 and shorter peptides to induce CXCL8 secretion has shown that shorter peptides are less able to induce inflammatory reactions but are more potent antimicrobials (19)
. LL-37 acts as a chemoattractant of neutrophils, monocytes, T cells, and mast cells (42
, 44
, 45)
. Human cathelicidin peptide LL-37 and mouse cathelicidin CRAMP also show angiogenic properties (46)
. However, smaller cathelicidin peptides such as KR-20, RK-31, and KS-30 have greater antimicrobial activity and are less able to induce CXCL8 (19
, 23)
. Additional cathelicidin peptides were found at the skin surface in the present work (LL-23, LL-29, and KS-27), confirming the antimicrobial activity of such shorter cathelicidins.
Based on these observations, it is reasonable to hypothesize that after infection or wounding, cathelicidin expression in skin will shift from the short form cathelicidins generated and inactivated by the kallikreins toward a predominance of the LL-37 form released from neutrophils. This would act to amplify the recruitment of neutrophils through its action as a chemoattractant and host cell stimulant. Upon resolution of the microbial challenge or injury, epidermal protease activity will process LL-37 to forms lacking proinflammatory activity and thus re-establish an effective microbial shield without further increasing tissue damage as a result of inflammation.
The importance of maintaining normal activity of serine proteases for control of antimicrobial activity was shown directly in the LEKTI-deficient mice. A lack of this serine protease inhibitor increased the balance of serine protease activity, increased antimicrobial activity, and increased the proportion of mature cathelicidin in the skin. The increase of cathelicidin was not due to an increase in leukocyte recruitment or keratinocyte synthesis since these newborn mice were euthanized within 8 h of birth, and there was neither infection nor inflammatory cell infiltration at that stage. However, the increase in the antimicrobial properties of the skin of SPINK5-deficient mice is not sufficient to compensate for the increased susceptibility to infection due to the severe disruption in barrier properties of the epidermis. Similarly, patients with Netherton syndrome develop frequent and sometimes severe skin infections (47)
.
Several antimicobial molecules in addition to cathelicidin are produced in skin and may be subject to similar enzymatic regulation. These include defensins and SKALP/elafin (48)
. Human ß-defensin 2 (HBD-2) and SKALP/elafin have been reported to be strongly expressed in the epidermis of Netherton syndrome (49)
. Like cathelicidins, HBD-2 and SKALP/elafin are inducible by bacterial infection and inflammatory reactions, but low in normal skin (48
, 50)
. Reports of high HBD-2 and SKALP/elafin in Netherton syndrome may reflect the consequence of repeated skin infection and inflammation due to barrier disruption (51)
. In the current mouse model, the use of neonatal mice minimized this effect and permitted evaluation of antimicrobial function as regulated by enzymatic processing distinct from the quantity of the gene expressed.
Taken together, these data show that the balance of serine protease activity in the skin leads directly to the control of innate antimicrobial activity. All components of this system, their triggers, and individual modifying agents remain to be elucidated. Our findings show that kallikreins are at least one important part of this system, and demonstrate that several cathelicidin antimicrobial peptides are generated at the barrier between the host and the external environment. The balance of proteolytic activity will modulate cathelicidin function to direct diverse antimicrobial and stimulatory activities; this is a previously unrecognized mechanism for regulation of immune defense. Furthermore, these findings show that control of microbial growth and an inflammatory response may rest in the balance of epithelial proteolytic activity.
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ACKNOWLEDGMENTS |
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Received for publication March 7, 2006. Accepted for publication May 15, 2006.
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