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The Journal of Immunology, 2008, 180, 7565 -7573
Copyright © 2008 by The American Association of Immunologists, Inc.
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Mast Cell Cathelicidin Antimicrobial Peptide Prevents Invasive Group A Streptococcus Infection of the Skin1

Anna Di Nardo, Kenshi Yamasaki, Robert A. Dorschner, Yuping Lai and Richard L. Gallo2

Department of Medicine, Division of Dermatology, University of California, San Diego and Veteran's Affairs Medical Center San Diego, CA 92161


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells (MC) express cathelicidin antimicrobial peptides that act as broad-spectrum antibiotics and influence the immune defense of multiple epithelial surfaces. We hypothesized that MC help protect against skin infection through the expression of cathelicidin. The susceptibility of MC-deficient mice (Kit Wsh–/–) to invasive group A streptococcus (GAS) was compared with control mice. Following s.c. injection of GAS, MC-deficient mice had 30% larger skin lesions, 80% more lesional bacteria, and 30% more spleens positive for bacteria. In contrast to results obtained when GAS was injected into skin, no significant differences were noted between MC-deficient mice and control mice after GAS was applied topically, indicating that MC activity is most important after barrier penetration. To determine whether these differences were due to MC expression of cathelicidin, MC-deficient mice were reconstituted with MC derived from either wild-type or cathelicidin-deficient (Camp–/–) mice and challenged with GAS. Forty-eight hours after bacterial injection, mice that did not receive MC had an average lesion size of 200 mm2, mice reconstituted with wild-type MC showed lesions comparable to control mice (25 mm2), while mice reconstituted with Camp–/– MC showed an average lesion size of 120 mm2. Surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) analysis of cathelicidin peptide purified from mast cells defined this as a unique 28-aa peptide. Combined, these results show that MC confer defense against Gram-positive bacterial infection in the skin, a function mediated in part by the expression of a unique cathelicidin peptide.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Mast cells (MC)3 are best known for their initiation and maintenance of anaphylaxis as well as their role in autoimmune diseases such as rheumatoid arthritis, interstitial cystitis, scleroderma, and Crohn's disease (1, 2). Despite these associations with disease, progress has been limited in understanding how MC function as effector cells in innate immune defense (2). Recent insights into the various functions of MC have revealed that they possess the dual ability to kill microbes and to modify classical adaptive immune responses. Members of the cathelicidin family of antimicrobial peptides have been shown to be expressed in MC of both humans and mice (3) as well as at sites of epithelial injury in several mammalian species (4, 5, 6, 7). The localization of cathelicidin peptides, the trigger for their extracellular release, and their capacity to modify inflammatory responses suggest that cathelicidin plays an important role in the capacity of MC to respond to infection. However, few in vivo studies of MC antimicrobial function are available (8, 9, 10, 11, 12, 13). This study aims to extend our understanding of the role of MC antimicrobial peptides in pathogen surveillance.

Mice deficient in MC demonstrate increased susceptibility to gut infection and sepsis (14). Although it is not clear why this occurs, MC are appropriately positioned for tissue defense along interfaces with the external environment at portals of entry for many infectious agents (15). MC recognize bacteria with the aid of opsonins or through expression of TLRs 2, 4, 6, and 8 and the FimH receptor CD48 (15, 16). Thus, MC expresses the necessary pattern recognition receptors to detect a variety of pathogens. In vitro studies have shown that MC release their mediators upon contact with bacteria and initiate a cascade of events leading to vasodilation and increased capillary permeability (17). MC also phagocytose and kill bacteria, although the precise mechanism of killing bacteria remains unclear (18).

MC are derived from a common progenitor of CD34 bone marrow cells and are released into the circulation while immature. They arrive at the periphery early in their life cycle and position themselves within epithelia in close proximity to blood vessels. This position allows them to come in contact with potential pathogens as soon as they cross the skin barrier. In this setting, MC activation can be elicited by different mechanisms that include signaling through TLRs and receptors for endothelins or endogenous ILs. MC are not only the sole producers of IL-4 in the dermis, but are also strongly activated by it. Recently, it has been shown that the capacity of the gut to eliminate nematodes is mostly mediated by the activation of bone marrow-derived cells, especially MC, through IL-4-mediated T cell activation (19). On the contrary, little is known about the capacity of IL-4 to impact the capacity of MC to kill bacteria. MC also release a variety of chemotactic factors such as TNF-{alpha} and leukotrienes B4 and C4. These factors are thought to play a role in host defense against bacterial infections through the recruitment of neutrophils to sites of infection (17, 20). Although MC biology is partially understood, much additional work is necessary to explain the role of MC in human skin diseases.

Recent work has provided new insight into basic events involved in host defense. Cathelicidins comprise one family of antimicrobial peptides (AMPs) that has been identified as a critical component of the innate immune system in epithelial tissues and myeloid cells of humans and other mammals (21, 22, 23). Cathelicidin-deficient mice are more susceptible to bacterial infection of the skin, thereby providing the first proof that AMPs are critical to mammalian immune defense (4). Other mouse models of infection, including gut (24, 25, 26), brain (27), and kidney (28) have supported these initial observations and demonstrated the significance of cathelicidin expression in multiple tissues and cell types. A major mechanism of action for cathelicidins is their capacity to directly kill bacteria (29, 30). However, cathelicidins also have been shown to affect cellular immunity through actions as a chemotactic mediator, secretory stimulus, and protease inhibitor (31, 32, 33).

Cathelicidins are produced as propeptides composed of a highly conserved region called the cathelin domain and a variable, species-specific antimicrobial peptide domain (34). Cathelicidins demonstrate broad-spectrum activity against bacteria, fungi, and viruses, as well as in vitro immunomodulatory activity (31, 35, 36). Human cathelicidin (LL-37) and mouse cathelin-related antimicrobial peptide (CRAMP) represent the products of a single cathelicidin gene in each species. These peptides are cationic and amphipathic, properties that promote interactions with biological membranes that allow them to selectively kill microbes. The mature cathelicidin peptide can also be processed into smaller peptides. In the skin, we have demonstrated that these smaller peptides vary in their antimicrobial capacity and immunomodulatory function (33, 37, 38). Moreover, the generation of new peptides is linked to the enzymatic composition of the milieu in which the antimicrobial peptides are immersed.

Studies have been performed in MC-deficient mice to demonstrate their increased susceptibility to peritonitis and sepsis following cecal ligation (14, 39, 40). However, a skin infection model has not been used to demonstrate whether the presence of MC in the skin is able to prevent local infection and lower sepsis potential. The work presented herein demonstrates that the absence of MC in the skin increases susceptibility to localized skin infection and increases the risk of sepsis. In addition, we demonstrate the in vivo importance of cathelicidin activity in MC. Our previous in vitro work has shown that MC derived from wild-type (WT) mice are able to kill bacteria while MC derived from cathelicidin-deficient mice are incapacitated (3). The present study demonstrates that the presence of MC in the skin is important in controlling infection and that cathelicidin activity is crucial for MC function in vivo.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

MC-deficient (Kit Wsh–/–) mice were a donation from Dr P. Besmer's laboratory (Developmental Biology Program, Memorial-Sloan Kettering Cancer Center at Cornell University, NY). The animals were bred at our facility. The Veteran Affairs and Institutional Animal Care and Use Committee approved all animal experiments. These mice have been extensively studied since they were generated (41). Camp–/– C57BL/6 mice were generated in our laboratory as previously described (4). WT C57BL/6 mice were purchased from The Jackson Laboratory.

Cells

Primary MC were generated by extracting bone marrow cells from the femurs of 5- to 8-wk-old mice and culturing cells in RPMI 1640 (Life Technologies) supplemented with 10% inactivated FCS (Life Technologies), 4 mM glutamine, penicillin/streptomycin, and 50 µg/ml 2-ME. Recombinant murine IL-3 (1 ng/ml) and recombinant murine stem cell factor (20 ng/ml), both shown to support the in vitro growth and differentiation of the MC precursor, were also included. After 4 wk, MC were consistently generated as confirmed by the expression of CD117 and Fc{epsilon}RI and by staining with toluidine blue. MC cultures were derived from WT and Camp–/– C57BL/6 mice.

Murine bacterial challenge

Subcutaneuos injections. Invasiveness of GAS in mouse skin was measured as previously described (4). Procedures were approved by the Veterans Affairs (VA) San Diego Healthcare System subcommittee on animal studies. The backs of sex-matched adult littermates were shaved and hair was removed by chemical depilation (Nair; Church & Dwight), then they were injected s.c. with 50 µl of a mid-logarithmic growth phase (A600 = 0.6, ~5 x 107 CFU) of GAS NZ131 complexed to Cytodex beads (Sigma-Aldrich) as a carrier. Lesion sizes were measured daily.

Bacterial enumeration from skin. Bacteria were enumerated from the skin as previously described (4) Briefly, the wound site was excised, weighed, and homogenized in distilled water. Ten microliters of the homogenate was serially diluted and plated for enumeration of CFU.

Bacterial enumeration from spleen. On the third day of the experiment, the animals were sacrificed and the spleen was excised, weighed, and homogenized in distilled water. Ten microliters of the homogenate was serially diluted and plated for enumeration of CFU.

Histology sections

On the third day of the experiment when the animals were sacrificed, a skin sample of tissue adjacent to the infection wound was collected, fixed with buffered formalin, and embedded in paraffin for H&E staining. Sections were read at high magnification power with an optical microscope. Neutrophil count was performed on three different fields in an area close to the wound in the upper dermis. Sections were also stained with anti-mouse CD117 (Abcam) developed with peroxidase to stain CD-positive cells and counterstained with hematoxylin Q stain.

Impetigo model

A bacteria invasion assay was performed with group A streptococcus (NZ131). Bacteria were grown to early exponential phase in Todd-Hewitt broth (THB) and adjusted to 1 x 106 CFU bacteria/ml in THB. Ten microliters of bacteria was spotted on Luria-Bertani (LB) agar plates (1% LB broth, 1% agarose, 10 mM phosphate buffer, pH 7.2). After drying up, bacteria with LB agar were cut out with a 3 (or 8)-mm dermal biopsy punch (Miltex) and were taped on the shaved mice backs. Bacteria with gel ({cong}1000 CFU) were kept on mice backs for 3 h and were removed. Skin after bacterial application was excised by a 3 (or 6)-mm punch at a designated time and were thoroughly washed twice with sterile PBS to remove noninvasive bacteria. Bacteria in the skin were recovered in 200 µl of sterile PBS by homogenizing with a MiniBeadBeater (Biospec Products). Bacteria were quantified by plating on THB agar plates and 24 h of incubation at 37°C and represent as CFU per lesion.

Antimicrobial assays

For screening of antimicrobial activity, a liquid assay was used as described previously (42). Lyophilized MC fractions were dissolved in 10 µl of MOPS buffer (pH 7.0) and tested against Staphylococcus aureus. To evaluate the antimicrobial activity of CRAMP and IGE24, CFU assays were performed as described with S. aureus and GAS (NZ131), both isolated from clinical samples (43). Bacteria were washed twice with 20 mM sodium phosphate buffer (20 mM NaH2PO4·H2O, 20 mM Na2HPO4·7H2O) and diluted to a concentration of 2 x 10–6 CFU/ml in RPMI 1640. S. aureus and GAS were incubated for 24 h at 37°C with various concentrations of peptides in a 50-µl total volume in a 96-well round-bottom tissue culture plate (Costar 3799; Corning). After incubation, the cells were diluted and 20 µl of each was plated in triplicate on tryptic soy agar (for S. aureus) or Todd-Hewitt agar (for GAS). The mean CFU/ml was calculated as a measure of the bactericidal activities of the tested reagents.

SELDI-TOF-MS

To quantify the cathelicidin peptides in small samples of normal human skin, we used SELDI-TOF-MS. MC were grown as previously described. Ten million MC were harvested with 1 M acetic acid, lyophilized, and frozen for further analysis.

Protein chips (RS100 protein chip array; Ciphergen Biosystems) were coated with 4 µl of rabbit anti-mCRAMP IgG (0.73 mg/ml) for 2 h at room temperature, followed by blocking with 0.5 M ethanolamine in PBS (pH 8.0). After washing three times with PBS/0.5% Triton X-100, protein chips were assembled in the Bioprocessor reservoir and samples (50 µl dissolved in radioimmunoprecipitation assay buffer) were applied and incubated for 2 h at room temperature. Protein chips were washed three times with 1x radioimmunoprecipitation assay buffer, twice with PBS/0.5% Triton X-100, and three times with PBS, followed by soaking in 10 mM HEPES buffer. After air drying, 0.5 µl of energy absorbance molecule (50% saturated {alpha}-cyano-4-hydroxycinnamic acid in 50% acetonitrile and 0.5% trifluoric acid) was applied twice and all spots were completely dried. Samples were analyzed on a SELDI mass analyzer PBS II with a linear TOF mass spectrometer (Ciphergen Biosystems) using time lag focusing. Specificity and accuracy were confirmed using several synthetic cathelicidin peptides as standards.

FACS analysis

MC were processed for staining with FITC-labeled rat anti-mouse CD 117 (BD Pharmingen) and rat anti-mouse IgE-R (BD Pharmingen). For evaluating CRAMP expression, cells were permeabilized with 0.02% saponin (Sigma-Aldrich) and then incubated with affinity-purified rabbit anti-CRAMP IgG (or rabbit IgG control) at 0.8 µg/ml (17). The secondary Ab was goat anti-rabbit FITC (Cappel Research Products) at 80 µg/ml. For flow cytometry, a FACScan (BD Biosciences), equipped with CellQuest software, was used.

For the CRAMP expression increase experiments, cells were treated with 1 µg/ml LTA, 0.1 µg/ml LPS (Sigma-Aldrich) and 10 ng/ml mouse IL-4 (R&D Systems) for 24 h.

Mast cell degranulation assay

Cells were incubated overnight with anti-DNP IgE (Sigma-Aldrich), washed with medium, and then stimulated with DNP-BSA (Sigma-Aldrich) for 15 min in thyrode buffer. Cell degranulation was monitored by FACS before and after DNP-BSA incubation and with quantification of the D-β-glucosaminidase level in the supernatant with the aim of a colorimetric assay. To identify CRAMP peptide, cells were stained with affinity-purified rabbit anti-CRAMP IgG as previously described.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
MC-deficient mice are more susceptible to GAS skin infection and develop bacteremia

To evaluate the contribution of MC to defense against skin infection, we directly injected WT and MC-deficient mice with GAS and compared the severity of infection. MC-deficient mice and WT littermates were injected with GAS on day 0 and developed a wound at the site of injection after 48 h. The wound area was monitored over time (Fig. 1a). Mice were sacrificed after 4 days and the wound site was excised and cultured for enumeration of bacterial CFU (Fig. 1b). MC-deficient mice developed larger wounds in comparison to their WT littermates and demonstrated a delay in recovery from infection. Significantly higher numbers of bacteria were recovered from the lesions of MC-deficient mice. Furthermore, after the mice were sacrificed, the spleens were recovered, homogenized, diluted, and plated on blood agar. A higher number of bacteria was recovered from the spleens of MC-deficient mice; 66% of the MC-deficient mice had bacteria in the spleen while only 21% of the WT mice had positive bacterial counts in the spleen (Fig. 1c). These data suggest that the presence of MC in the skin provides an important defense against bacteremia.


Figure 1
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  		FIGURE 1. MC-deficient mice are more susceptible to skin infection and develop bacteremia. a, Wound area (mm2) over time in WT and MC-deficient mice infected with GAS.*, p < 0.05 and **, p < 0.01 b, Enumeration of GAS recovered from excised lesions of mice sacrificed on day 4 following s.c. infection, p < 0.001. c, Percentage of mice containing bacteria in the spleen, p < 0.05. d, Enumeration of GAS recovered from the superficial layer of the epidermis following topical infection.

 
To better define the role of MC in controlling bacterial entry into the skin, we designed a model mimicking human impetigo in which bacterial entry occurs from the epidermal surface. A defined number of GAS were grown in a gel and applied to the mouse skin surface for 18 h. The gel was removed and the skin was washed and sampled to enumerate the bacteria that had penetrated below the skin surface. In contrast to the data obtained when the GAS were injected s.c., no significant differences were noticed between groups following topical application (Fig. 1d). These data provide evidence that the absence of MC decreases the skin's capacity to fight bacteria, particularly when the first line of defense, the epidermis, has been breached by potential pathogens.

MC-deficient mice reconstituted with WT and Camp–/– MC in the skin

To investigate the importance of cathelicidin AMP activity in the MC during skin infection, we used an adoptive transfer mouse model. MC-deficient mice were used as a recipient for WT and Camp–/– MC. MC isolated from the bone marrow of WT and Camp–/– mice were differentiated in culture and injected intradermally into the mice upon maturation. Skin biopsy was performed to demonstrate that MC are resident in the skin for at least 3–4 wk after transfer and that reconstitution was similar when mice were injected with WT or Camp–/– MC (data not shown).

After 2 wk, the reconstituted mice and two control groups, including MC-deficient mice and WT C57BL/6 littermates, were injected with GAS. Mice developed edema at the site of injection after 24 h (Fig. 2, a and b). The MC-deficient mice demonstrated the largest area of edema while mice reconstituted with WT MC showed the smallest. The group reconstituted with Camp–/– MC demonstrated intermediate edema in comparison to the two nonreconstituted groups (Fig. 2b). These data suggest the presence of MC in the skin limits the initial damage provoked by the presence of pathogens and that reconstitution with MC containing cathelicidin limits this even further. Edema at 24 h developed into erosion formation by 48 h (Fig. 2c). Although differences were still evident between MC-deficient mice and WT littermates, MC-deficient mice reconstituted with WT MC (containing cathelicidin) presented a wound lesion compatible with the WT controls (Fig. 2c, column 1 vs column 4). Finally, we analyzed spleens from the same mice and found that MC-reconstituted mice had fewer bacteria in the spleen (Fig. 2d). All reconstituted groups were significantly different from WT that did not show bacteria in the spleen. Only 50% of reconstituted mice had bacteria detected in their spleen while 100% of mice not reconstituted were positive.


Figure 2
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  		FIGURE 2. MC-deficient mice (Kit Wsh–/–) reconstituted with WT and Camp–/– MC in the skin. a, Representative lesions from MC-deficient mice (Kit Wsh–/–), MC-deficient mice (Kit Wsh–/–) reconstituted with Camp–/– (Kit Wsh–/– plus Camp–/– MC) MC, MC-deficient mice (Kit Wsh–/–) reconstituted with WT MC (Kit Wsh–/– plus WT MC), and WT mice at 24 h following GAS infection. Arrows indicate the margins of edema visible on the skin surface. b, Measurement of visible edema on skin (mm2) 24 h after GAS infection in WT, MC-deficient mice (Kit Wsh–/–), and MC-deficient mice receiving adoptive transfer of WT MC (Kit Wsh–/– +WT MC) or MC derived from cathelicidin-deficient mice (+Camp–/– MC). c, Measurement of GAS-erosive lesion size (mm2) 48 h after bacterial injection in WT mice, MC-deficient mice (Kit Wsh–/–), and MC-deficient mice receiving adoptive transfer of WT MC (Kit Wsh–/– plus WT MC) or MC derived from cathelicidin-deficient mice (+Camp–/– MC). d, Number of bacteria in spleens containing bacteria at 72 h postinfection in WT mice and MC-deficient mice (Kit Wsh–/–) receiving adoptive transfer of WT MC (+WT), no transplant (none) or MC derived from cathelicidin-deficient mice (+Camp–/–), *, p < 0.05.

 
MC modify the recruitment of neutrophils to the site of infection

To better understand the relationship between the MC and the inflammatory response, we sectioned and stained the skin area close to the wound (Fig. 3, a and b). Neutrophils were present in all mice but the distribution inside the dermis, especially in the area close to the wound, was very different between WT mice and the MC-deficient mice (Kit Wsh–/–). Fig. 3c shows the results of quantification of the upper dermal neutrophilic infiltrate and demonstrates a decrease in the inflammatory cell infiltrate in MC-deficient mice (Kit Wsh–/–). This infiltrate was restored in MC-reconstituted mice when compared with nonreconstituted mice and a small, but statistically significant decrease in the infiltrate observed in Kit Wsh–/– mice reconstituted with Camp–/– MC compared with WT. Because the number of MC increases after infection (44, 45, 46), we also counted the number of CD117-positive cells at the site of inflammation. The results are shown in Fig. 3, d and e. The number of CD117 cells parallels the increase of neutrophils at the same site in the different type of mice, but in the reconstituted they do not reach the same concentration that they have in the WT. The number of MC in the WT-reconstituted mice is not different from the number of the MC in the Camp–/– reconstituted mice. Therefore, the difference in MC activity between the two reconstituted mice is related to the presence of cathelicidin and at its direct influence on inflammation.


Figure 3
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  		FIGURE 3. The presence of MC during infection modifies neutrophil recruitment in the upper dermis. a, H&E staining of skin in an area adjacent to the infected lesion in a WT mouse. b, H&E staining of skin in an area adjacent to the infected lesion in MC-deficient mice (Kit Wsh–/–). c, Measurement of the neutrophil infiltrate in the upper dermis at 72 h. Cell infiltrate was manually counted in three adjacent sections next to the site of infection. Data shown are cell number observed in WT mice, MC-deficient mice (Kit Wsh–/–) without transplant (none) and MC-deficient mice receiving adoptive transfer of WT MC (+WT) or MC derived from cathelicidin-deficient mice (+Camp–/–), *, p < 0.05; **, p < 0.01; and ***, p < 0.001. d, CD117 immunoperoxidase staining of skin in an area adjacent to the infected lesion in MC-deficient mice (Kit Wsh–/–) reconstituted with MC derived from cathelicidin-deficient mice. The arrows indicate positive cells. e, Measurement of the CD117-positive infiltrate in the dermis at 72 h. Cell infiltrate was manually counted in three adjacent sections next to the site of infection. Data shown are cell number observed in WT mice, MC-deficient mice (Kit Wsh–/–) without transplant, and MC-deficient mice receiving adoptive transfer of WT MC (+WT) or MC derived from cathelicidin-deficient mice (+Camp–/–).

 
MC use cathelicidin to kill GAS intracellularly

To investigate the mechanism used by MC to kill bacteria, we inhibited phagocytosis of MC with cytochalasin. Cytochalasin substantially decreased MC capacity to kill GAS (Fig. 4a). Furthermore, to determine whether cathelicidin inside granules contributed to MC antimicrobial activity, MC were incubated with DNP-anti-DNP IgE to stimulate degranulation. The degranulated cells lost antimicrobial activity (Fig. 4b). Cathelicidin expression was monitored by FACS analysis, which confirmed that cathelicidin inside the granules was released during IgE-mediated degranulation (Fig. 5). This demonstrated the importance of cathelicidins within MC granules for antimicrobial activity and further supports earlier observations of a significant difference in the killing capacity of MC derived from WT and Camp–/– mice (3).


Figure 4
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  		FIGURE 4. MC use cathelicidin to kill GAS intracellularly. a, Enumeration of GAS following incubation with MC treated with or without cytochalasin for 3 h. *, p < 0.05 and **, p < 0.01. b, Enumeration of GAS incubated with degranulated MC, *, p < 0.05.

 

Figure 5
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  		FIGURE 5. MC degranulation assay and cathelicidin release. MC degranulation was confirmed by two different systems. FACS analysis of side scatter show cells granularity before (left a) and after degranulation (left b). Before degranulation, cells are bigger and present higher side scatter level. Cathelicidin content was measured simultaneously in the same cell populations with FITC anti-Cramp (right a and b). c, After degranulation, medium was analyzed to confirm degranulation by D-β-glucosaminidase assay; results were read with a spectrophotometer and plotted as {Delta}OD.

 
Stimulated MC up-regulate cathelicidin expression and process cathelicidin to smaller peptides. We have previously shown that LPS-mediated stimulation of MC induces cathelicidin mRNA expression (3). In the present studies, we stimulated MC with various activators including LTA, LPS, and IL-4 and measured cathelicidin protein expression by FACS analysis. All three stimuli induced cathelicidin expression in MC, but IL-4 was by far the most potent (Fig. 6a), and the addition of LTA or LPS to IL-4 was not able to further increase cathelicidin expression (data not shown).


Figure 6
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  		FIGURE 6. Stimulated MC increase cathelicidin expression and process cathelicidin to smaller peptides. a, FACS analysis of cathelicidin expression in MC stimulated with IL-4 (10 ng/ml). The dotted line represents IL-4-treated cells, the gray line the untreated, and the black the IgG-negative control. b, Identification of a novel processed form of MC cathelicidin, IGE24, by SELDI-TOF-MS. MC were stimulated with IL-4, harvested with acetic acid, lyophilized, resuspended in MOPS buffer, and analyzed by SELDI-TOF using a polyclonal cathelicidin Ab. Mass of peptide indicated by arrow is 2805 kDa, corresponding to sequence IGEKLKKIGQKI KNFFQKLVPQ PEQ. In the control lane of the SELDI-TOF, peaks are visible corresponding to the CRAMP peptide and to the full-length peptide mCAP18. c, Minimal inhibitory concentration of synthetic IGE24 peptide against GAS following 5-h incubation in RPMI 1640/10% FCS. d, Minimal inhibitory concentration of synthetic IGE24 peptide against S. aureus following 5-h incubation in RPMI 1640/10% FCS.

 
Cathelicidin is processed and activated extracellularly in neutrophils by elastase or proteinase 3 (47). The resulting fragments (CRAMP and LL-37) are able to either activate or inactivate dendritic cells in response to the environment (48, 49). Active cathelicidin can be further processed to shorter peptides with increased antimicrobial activity, as has been demonstrated in the epidermis (33). Enzymes released from MC could potentially process cathelicidin and release additional active molecules. Therefore, SELDI-TOF-MS analysis was used to further characterize MC cathelicidin and discriminate different peptide fragments. We compared the cathelicidin peptide profile of mature MC and MC activated by IL-4 with an expected theoretical profile based on predicted enzymatic digestion and with the known neutrophil cathelicidin. The results of this experiment show that activated MC are able to process cathelicidin into a smaller peptide that is distinct from those found during inflammation in keratinocytes and neutrophils (Fig. 6b). Both IL-4-activated and control MC had the full-length peptide mCramp while the precursor protein from which these peptides are derived was seen best in the unstimulated MC evaluated by SELDI-TOF. We named this peptide IGE24 according to its size and N-terminal sequence. We tested IGE24 against major skin pathogens S. aureus and GAS and found that IGE24 has antibacterial activity with a minimal bactericidal concentration of 8 µM against GAS and 4 µM against S. aureus (Fig. 6, c and d).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
MC are resident in various tissues but are most commonly found at sites that serve as portals of entry for pathogens such as the skin and mucous membranes. MC were initially recognized for their potential in unleashing histamine and provoking urticaria. Recent studies indicate that MC also have the capacity to respond to pathogens. Herein, we provide in vivo evidence that MC serve a role in blocking the progression of Gram-positive sepsis from the skin. In a mouse model of GAS skin infection, MC were found to significantly inhibit bacterial dissemination. Furthermore, by studying the results of adoptive transfer experiments in MC-deficient mice and cathelicidin function in cultured MC, we provide evidence to indicate that AMP synthesis and processing represents one potential mechanism by which MC exert control over bacterial infection.

A novel aspect of the present study was the investigation of the sentinel role of MC against invasive skin infection. This was accomplished by using MC-deficient mice in a well-established experimental model of GAS skin infection. Reconstitution of these mice with cathelicidin AMP-deficient MC provided an ideal model for studying the antimicrobial activity of cathelicidin. We noted large differences in lesion size and bacterial content between WT and MC-deficient mice, a finding consistent with findings with other less common bacterial infections (8). Our findings represent the first description for GAS, a common invasive skin pathogen, and demonstrate the critical role for cathelicidin. One striking observation was the increased percentage of splenic involvement in the absence of MC. Topical application of GAS to MC-deficient mice did not result in increased bacterial penetration of the skin, thereby suggesting that MC act after the skin barrier has been broken. The present study clearly demonstrates that MC represent sentinel cells in the upper dermis, as their presence not only fights infection locally but also prevents bacterial spread into the bloodstream.

Reconstitution of skin with WT MC, but not Camp–/– MC, reduced edema formation and wound extension, suggesting that cathelicidin acts during the initial phase of bacterial attack. MC activity in preventing edema formation has been already described in a contact sensitization model (50). In this study, mice deficient in MC developed more edema than their WT littermates after contact sensitization. They explained this difference through the expression of IL-10 but also indicated that other mediators in MC could have this anti-inflammatory activity. In our model, mice reconstituted with Camp+/+ cells inhibited edema to a higher extent than the mice reconstituted with Camp–/– MC, indicating that cathelicidin might have a modulating effect on the anti-inflammatory cytokine production. Moreover, we were able to confirm previous observations that MC are able to modulate neutrophilic infiltration (8, 11, 51). This effect may be partially modified by the presence of cathelicidin inside MC. This observation is consistent with the observation that at later time points, MC reconstitution protects mice from bacterial dissemination to the bloodstream in a cathelicidin-independent manner.

Cathelicidin is produced in high concentrations (10–30 µM) by neutrophils, MC, and epithelial cells during inflammation and bacterial invasion (52). Cathelicidin also possesses chemotactic properties toward neutrophils and macrophages that have been associated with activating formyl peptide receptor-like 1, which belongs to the GI protein-coupled receptor family (53, 54). Therefore, MC cathelicidin activity could be mediated by neutrophil chemotaxis and TNF-{alpha} release. In this study, we found that cathelicidin is able to directly kill bacteria, even when it was not detectable extracellularly under culture conditions. Studies using cell culture models have shown that MC respond to bacteria through pattern recognition receptors and through ingestion of bacteria (55). We demonstrated that treatment with cytochalasin blocks the ability of MC to kill GAS. In addition, we showed that MC subjected to massive degranulation, in which cathelicidin was released, lost the capacity to kill bacteria. These experiments demonstrate that cathelicidin is able to kill GAS inside of MC. This result contrasts with a recent publication that described a phagocytosis-independent pathway for MC by means of extracellular trap formation using cathelicidin and DNA structures to trap bacteria (56). In that article, the authors suggest that in their in vitro system MC kill GAS extracellularly. Our results conflict with this as we show intercellular killing is also important for the MC. These differences between our results can be explained by the different conditions in which the experiments were performed. In the prior report, MC were exposed to a very large number of bacteria, leading to cell death and release not only of their antimicrobial peptides, but also of their DNA. In our system, the number of bacteria used was lower and more compatible with the number of MC. In this case, MC survival was an essential condition for performing the experiment. This condition is consistent with the in vivo situation, since images during tissue infection confirm that MC are vital, and with intact nuclei. Nonetheless, we cannot exclude that cathelicidin released by degranulation may also be responsible for chemoattraction and neutrophil engagement in vivo during severe infection, and that the cathelicidin-DNA trapping of bacteria might be an additional mechanism whereby dying mast cells kill bacteria when intracellular killing is not sufficient.

Cathelicidins are biologically inactive without extensive posttranslational structural modifications. Our recent research has suggested that cell- and environment-specific posttranslational adaptations are essential for cathelicidin antimicrobial potency and spectrum of activity (38). In this study, we demonstrated that MC cathelicidin expression can be increased by stimulation with IL-4 and known activators of TLR2 and TLR4, although the combination of IL-4 and a TLR2/4 activator did not further increase total cathelicidin content. Therefore, IL-4 is a good candidate for being an activator of cathelicidin expression in MC. IL-4 is a well-known activating factor for MC, is required for MC activation during nematode infestation (19, 57), and preincubation with it induces MC MCP-1 and IL-8 production (58). Furthermore, MC activation was shown here to generate a novel cathelicidin peptide that has not been previously identified in other cell types. This peptide demonstrated potent antimicrobial activity against S. aureus and GAS. Further work is required to determine how this novel peptide may influence chemotactic or other actions attributed to the MC in these settings.

The ability of MC to interact with pathogens occurs either through direct activation of TLRs and the FimH receptor CD48 or through indirect activation of Fc receptors, complement activation, and cytokine release. The current data suggest a novel mechanism by which MC can subsequently directly fight bacterial infection or indirectly increase inflammation through the activity of cathelicidin AMP. Our in vitro data make an important connection by demonstrating that a Th2 cytokine can activate MC and modify their activity during infection. The in vivo implications of this finding will be investigated in future studies.

To our knowledge, our data provide the first in vivo evidence that MC cathelicidin modulates tissue responsiveness to bacterial infection and suggests two possible explanations for this observation. First, acting as a natural antibiotic in MC, the presence of cathelicidin may protect the skin from invasive infection by directly killing bacteria. Second, the presence of cathelicidin in MC may act to facilitate recruitment of neutrophils, thus indirectly providing enhanced protection against infection. These findings, combined with previous studies, now firmly establish the MC as crucial for immune defense against skin infection. MC help to prevent the extension of localized Gram-positive infections of the skin and provide a critical barrier to further dissemination of infection and systemic disease. These conclusions lead to new strategies for the treatment of localized and systemic infections by controlling the AMP function of the MC.


   Acknowledgments
 
We thank Dr. Marissa Braff for editing work and Dr. Antonella Vitiello for suggestions during the manuscript preparation.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
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.

1 This work was supported by National Institutes of Health Grants R01AR052728 and R01 AI052453, a Veteran's Affairs Merit Award (to R.L.G.), a Skin Research Grant from the Johnson & Johnson Skin Research Center, and a Career Development Grant from Dermatology Foundation (to A.D.). Back

2 Address correspondence and reprint requests to Dr. Richard L Gallo, Department of Medicine, Division of Dermatology, University of California, 3350 La Jolla Village Drive, San Diego, CA 92161. E-mail address: Rgallo{at}ucsd.edu Back

3 Abbreviations used in this paper: MC, mast cell; AMP, antimicrobial peptide; GAS, group A Streptococcus; THB, Todd-Hewitt; LB, Luria-Bertani; SELDI-TOF-MS, surface-enhanced laser desorption/ionization time-of-flight mass spectrometry; LTA, lipoteichoic acid; SELDI-TOF-MS, surface-enhanced laser desorption/ionization time-of-flight-mass spectrometry; WT, wild type; CRAMP, cathelin-related antimicrobial peptide. Back

Received for publication June 8, 2007. Accepted for publication March 28, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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