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The human cathelicidin antimicrobial peptide acts as an effector molecule of the innate immune system with direct antimicrobial and immunomodulatory effects. The aim of this study was to test whether the cathelicidin LL-37 modulates the response of neutrophils to microbial stimulation. Human neutrophils were exposed to LPS, Staphylococcus aureus and Pseudomonas aeruginosa subsequent to incubation with LL-37 and cytokine release was measured by ELISA. The incubation with LL-37 significantly decreased the release of proinflammatory cytokines from stimulated human neutrophils. ROS production of neutrophils was determined by a luminometric and a flow cytometry method. The peptide induced the production of ROS and the engulfment of bacteria into neutrophils. Peritoneal mouse neutrophils isolated from CRAMP-deficient and WT animals were treated with LPS and TNF-α in the supernatant was measured by ELISA. Antimicrobial activity of neutrophils was detected by incubating neutrophils isolated from CRAMP-knockout and WT mice with bacteria. Neutrophils from CRAMP-deficient mice released significantly more TNF-α after bacterial stimulation and showed decreased antimicrobial activity as compared to cells from WT animals. In conclusion, LL-37 modulates the response of neutrophils to bacterial activation. Cathelicidin controls the release of inflammatory mediators while increasing antimicrobial activity of neutrophils.
Inflammation is an integral part of the reactions of the innate immune system. The innate immune system is responsible for maintaining a functional and physical barrier against microorganisms. Overactive inflammatory responses often cause harm to the organism. Thus, several layers of regulatory mechanisms modulate the effectors of innate immunity to balance between host defense and inflammation. Neutrophils are one of the most prominent effector cells of the innate immune system . They are recruited to the site of infection and are equipped with various antimicrobial effector mechanisms, whose role in host defense has been demonstrated for many pathogens.
Antimicrobial peptides (AMP) are effector molecules of the innate immune system with various activities . In mammals, peptides of the defensin and the cathelicidin families are found. AMP are produced by epithelial and professional host defense cells such as macrophages or neutrophils. AMP directly inactivate microorganisms and, in addition, have diverse activities on various cell types . LL-37/hCAP-18 is the only cathelicidin present in humans  with LL-37 being the C-terminal cleavage product. The precursor hCAP-18 is found in the specific granules of neutrophils and is expressed in many immune and epithelial cells. Cathelicidin has an established role in host defense [5, 6]. In addition to their direct antimicrobial function, cathelicidin peptides are involved in the modulation of repair and tissue homeostasis: augmentation of angiogenesis by a direct effect on endothelial cells , epithelial wound healing , chemoattraction of immune cells , release of inflammatory mediators from epithelial cells by transactivation of the epidermal growth factor receptor (EGFR) pathway .
A number of reports showed that cathelicidins act on myeloid cells. LL-37 significantly up-regulates the endocytic capacity, modifies expression of phagocytic receptors, and increases secretion of Th1-inducing cytokines of dendritic cells (DC) generated from blood monocytes . LL-37 modulates the response of monocytes and DC to toll like receptor (TLR)–ligands [12–14]. Also effects of cathelicidins on neutrophils have been described. One study showed that LL-37 induces the generation of reactive oxygen species (ROS) from human neutrophils . LL-37 has a complex influence on neutrophil apoptosis. The peptide inhibits apoptosis via interaction with P2X7 and G-protein-coupled receptors  and formyl-peptide receptor like 1 (FPRL1) . In contrast, LL-37 also induces secondary necrosis of neutrophils . Neutrophils express many TLR  and respond to the presence of specific ligands by activation of their host defense pathways. LL-37 appears to modulate the response of neutrophils to LPS .
The aim of the current study was to analyze the effect of cathelicidin on the interaction between neutrophils and microbial patterns. We used stimulation experiments applying various microbial stimuli and characterized the effect of cathelicidin. Also the production of ROS was determined. Further, we determined the role of endogenous cathelicidin by using neutrophils isolated from cathelicidin related antimicrobial peptide (CRAMP)-deficient mice.
Cathelicidin modulates the response of monocytes/macrophages to endotoxin . To investigate whether this effect of cathelicidin is also effective on neutrophils, we added 100 ng/ml LPS to human neutrophils preincubated for 5 min with the human cathelicidin LL-37 in different concentrations. The release of the proinflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α was significantly decreased in presence of LL-37 as compared to the samples stimulated with LPS in the absence of LL-37 (Fig. 1A–D). This effect of LL-37 was dose dependent. LL-37 alone resulted in an increase of cytokine release (Fig. 1A–D). These data show that LL-37 modulates the response of neutrophils to microbial patterns.
To investigate whether the effect of LL-37 is also effective when whole bacteria are used to stimulate neutrophils, we incubated cells with heat inactivated gram-negative Pseudomonas aeruginosa or gram-positive Staphylococcus aureus. TNF-α in the supernatant was quantified by ELISA. Figure 2 A–B shows that the secretion of this cytokine was significantly decreased in presence of LL-37.
It has been demonstrated that LL-37 triggers ROS production in human neutrophils . We assessed ROS production in response to PMA using luminometry and flow cytometry. LL-37 significantly increased the ROS triggered by PMA production in a dose dependent way as determined by luminol chemiluminescence (Fig. 3A). PMA is a stimulus that needs to pass the plasma membrane in order to activate PKC. Thus LL-37 could potentially induce the uptake of PMA. In order to demonstrate that LL-37 also enhances ROS production induced by other agents, we exposed neutrophils to whole bacteria. LL-37 significantly increased the ROS production after stimulation with S. aureus (Fig. 3B). The presence of serum (fetal bovine serum) partly inhibited the effect of LL-37 (Fig. 3C). The application of LL-37 alone resulted in a small increased of ROS production (Fig. 3B). To reveal the activity of endogenous cathelicidin we isolated peritoneal neutrophils from WT and CRAMP-deficient animals using the thioglycollate method as described in the Method section. We found that after stimulation with S. aureus, CRAMP-deficient animals had significantly lower levels of ROS (Fig. 3D).The amplification of ROS by LL-37 was also demonstrated using flow cytometry measurement of the fluorescence intensity of DCFDA-loaded neutrophils. The levels of ROS were significantly increased in the presence of LL-37 (Fig. 3E). This effect of the peptide was dose-dependent. A scrambled form of LL-37 (sLL-37) was used to exclude a nonspecific effect of LL-37; this had no effect on ROS production (Fig. 3E). Lactate dehydrogenase (LDH) release was determined as marker of cytotoxicity; no increased levels were detected up to 30 μg/ml of LL-37 (data not shown).
We next tested whether LL-37 modulates the rate of phagocytic uptake of bacteria into neutrophils, a critical step in the killing of microorganisms  using phagocytosis assays . The cathelicidin peptide significantly increased the uptake of bacteria into neutrophil cells in a dose-dependent manner (Fig. 3F).
These data showed that exposure to the cathelicidin peptide increases host defense activities such as ROS production and engulfment of bacteria.
CRAMP is the homologous molecule of LL-37 in mice . Based on the peptide’s structure, expression pattern and biological activity, this gene and its encoded product CRAMP serve very similar functions as the human counterpart. To test whether endogenousely expressed cathelicidin has similar effects as exogenousely applied peptide, we isolated peritoneal neutrophils from animals deficient in CRAMP. After stimulation with LPS, neutrophils from CRAMP-deficient animals showed significantly increased release of TNF-α (Fig. 4A). To test whether this suppression of inflammatory activation is associated with a breach in antimicrobial activity, we performed bacterial killing assays applying viable P. aeruginosa to neutrophils from CRAMP-deficient and WT control mice. We found that neutrophils from animals deficient in CRAMP have a significantly decreased antimicrobial activity (Fig. 4B). These data show that endogenous cathelicidin modulates the response of neutrophils in responses of the innate immune system. To determine whether CRAMP is released from neutrophils, we performed Western blotting on cell supernatants and found the precursor and the cleaved peptide in supernatants from LPS-stimulated and nonstimulated cells (Fig. 4C).
The main finding of the current study is that the cathelicidin peptide LL-37 modulates the activation of neutrophils stimulated by bacterial patterns. Exogenousely applied peptide as well as endogenousely, physiologically expressed peptide have this effect on neutrophils. Ex vivo analysis of neutrophils isolated from CRAMP-deficient animals were used as genetic model. These data support the role of cathelicidin peptide in the modulation of innate immune responses.
Cathelicidin peptides are known to impact on the inflammatory mechanisms relevant in infection and sepsis [23–25]. Peptide application and overexpression of the peptides’ genes have been used in various sepsis models and demonstrated that cathelicidins inhibit overreactive responses to microbial patterns [13, 23, 25, 26]. The underlying mechanisms likely involve the modulation of the responses of the host defense cells to microbial patterns. While it has been known that cathelicidin modulates the response of macrophages [13, 27] and DC [12, 14] to TLR-ligands, the effect on neutrophil activation has been less clear. Our results show that cathelicidin modulates the response of neutrophils to endotoxin and to whole bacteria. The release of a variety of proinflammatory mediators (IL-1β, IL-6, IL-8, and TNF-α) was significantly decreased suggesting that the peptide protects from the development of a cytokine storm. This effect was also detectable when whole Gram-positive and–negative bacteria where applied, indicating that the cathelicidin’s effect is also effective in the interaction of neutrophils with biologically relevant microorganisms. Interestingly, endogenous cathelicidin also had this modulatory effect on cytokine release as shown in the experiments using neutrophils from CRAMP-deficient mice. The study of Zheng et al.  found increased IL-8 release at high concentration of LL-37 (20 and 40 μg/ml). This study did not investigate the combination of LPS and LL-37. In the current study, we did not find an increased release of cytokines at concentrations of 5–20 μg/ml or evidence of cell lysis as measured by LDH release. The application of LL-37 alone resulted in the release of cytokines from neutrophils, possibly related to binding of the peptide to GAPDH .
In addition to the release of inflammatory mediators, cathelicidin modulates other cellular processes including cell death pathways and the production of ROS. Cathelicidin has a complex effect on neutrophil apoptosis. Cathelicidin modulates apoptosis via the receptors FPRL1  and P2X7 , while different methodology revealed induction of secondary apoptosis . LL-37 appears to also functionally bind to cxcr2 . Cathelicidin is known to interfere with the pathways that regulate the production of ROS in myeloid cells including macrophages  and neutrophils . We confirmed the observations that application of LL-37 results in amplification of PMA-triggered ROS production. ROS are considered important effector molecules in the direct or indirect bactericidal activities of neutrophils . The significantly decreased ROS production in CRAMP-deficient animals underscores the role of the endogenous peptide in this process. We therefore investigated whether the presence of cathelicidin is necessary for the antimicrobial activity of neutrophils and found that CRAMP-deficient neutrophils showed significantly less antibacterial activity. While these data do not discriminate between different antimicrobial pathways of neutrophils, they show that cathelicidin increases antimicrobial activities of neutrophils while decreasing the release of proinflammatory mediators. Phagocytosis assay also revealed increased uptake of bacteria into neutrophils. ROS production and antimicrobial activity were dependent on the endogenous peptide as demonstrated in the experiments with murine cells. The fact that LL-37 increases phagocytosis of bacteria could help to explain why peptide-treated neutrophils produce higher levels of ROS. Since the precursor and active form are released by neutrophils it is difficult to speculate about the site of activity. In the living organisms, cathelicidin peptide from different sources could contribute to the described effects on neutrophils. Several cell types produce the peptide, including epithelial  and immune cells . Also, peptide synthesized in and secreted from neutrophils likely contributes. The peptide’s effect on ROS production is partly inhibited by serum components. This could indicate that the described mechanisms is not active in the blood; however it is relevant for neutrophils that have immigrated into tissue and body surfaces.
The mechanism by which cathelicidin modulates the function of myeloid cell types is largely unclear. The peptide is able to bind to LPS and decreases its detection by the receptor complexes [34, 35]. Additionally, the peptide, likely interacts with cellular receptors, including FPRL1 , P2X7 , or epidermal growth factor receptor (EGFR) , with possible downstream effects on inflammatory pathways . The effectiveness of an all-D-form of the peptide, together with observations of altered cell membrane function and structure implies a more complex mechanism including interaction with biomembranes and modulation of the function of membrane associated proteins .
Based on the current study and former data it is still difficult to shortly summarize the role of cathelicidin in host defense and immunity. In vitro studies revealed a direct antimicrobial activity [32, 37, 38] that is supported by the results of infection models in CRAMP-deficient animals [5, 6]. Cathelicidin interacts with a number of cell types such as endothelial cells , epithelial cells , mast cells , macrophages , and DC [11, 12]. As shown in the current study, cathelicidin modulates neutrophil functions by suppressing the release of proinflammatory mediators and increasing antimicrobial activity. Animal studies showed that cathelicidin protects from exaggerated inflammatory reactions , however cathelicidin also has proinflammatory roles in skin . Based on the current data, cathelicidin is an effector molecule of innate immunity with diverse functions, including antimicrobial activity and modulation of wound repair and inflammation.
CRAMP-deficient mice in a 129/SVJ background  and their WT controls were used for neutrophil isolation. The animals were kept under specific pathogen free conditions at the animal center of the University of Marburg. The animal experiments were approved by the responsible authorities (Regierungspräsidium Giessen). 1 ml of sterile 4% thioglycollate broth (BD Difco, Heidelberg, Germany) was intraperitoneally injected, the animals were euthanized after 4 h and the peritoneal cavity was lavaged twice with 10 ml of PBS. The peritoneal lavage was centrifuged and red blood cells were lyzed. After washes in PBS, the cells were resuspended in RPMI 1640 medium (Gibco, Grand Island, NY). The purity of neutrophils was determined using cytospin preparations, the viability of the cells was tested using trypan blue staining. For measurement of the neutrophils’ antimicrobial activity, opsonized bacteria and neutrophils were incubated together at a ratio of 1:1 (shaking at 200 rpm, 37°C). 20 μl aliquots were removed after 0, 30, 60, and 90 min and neutrophils were lyzed by adding Triton 0.1 %. Dilutions in PBS were plated onto agar plates and bacterial colonies were counted after 24 hrs. Western blots using an polyclonal antiserum against CRAP was applied to supernatants as described earlier .
Buffy coats made from 500 ml blood of healthy volunteers donor were obtained from the blood bank of the University hospital Marburg and diluted 1:1 with PBS containing 2 mM of EDTA. 9 ml of this suspension was layered onto 15 ml of FicollSeparating Solution 1.077 (Biochrom AG, Berlin, Germany), and centrifugated at 1500 rpm for 30 min. The cell pellet, containing granulocytes and red blood cells was resuspended in 25 ml dextran 4% and incubated at room temperature for 40 min. The neutrophils in the upper phase were collected after lysis of red blood cells and resuspended in RPMI medium containing 0.5 % serum albumin. The neutrophils were counted, the purity of the cell preparation was determined by Wright–Giemsa staining, and the viability was tested using Trypan blue staining. The purity of isolated neutrophils with this method was more than 95%.
Staphylococcus aureus 113 wt (Dr. A. Peschel, University of Tubingen, Germany). Pseudomonas aeruginosa NH57388A and NH57388C (Dr. Niels Hoiby, University of Copenhagen, Denmark) were handled as described earlier . The number of bacteria in suspension was adjusted based on measurements of the OD 600 nm and using a reference dilution. The medium was replaced with PBS. Complement-inactivated human serum was used to opsonize the bacteria.
The LL-37 peptide (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLV-PRTES) and its scrambled form sLL-37 (RSLEGTDRFPFVRLKN-SRKLEFKDIKGIKREQFVKIL), were chemically synthesized (Charité, Humboldt-Universität, Berlin, Germany). 1×104 CFU/ ml bacteria were heat-inactivated (95°C for 1 h), opsonized with human serum, and incubated with human neutrophils (1×106 cell/ml, overnight). The supernatants were collected after centrifugation at 2000 rpm for 10 min. The samples were pre-incubated with different concentrations of LL-37 (5, 10, 15, and 20 μg/ml) for 30 min. Then, LPS (100 ng/ml, 12 h) was used to stimulate neutrophils. All experiments were performed in the absence of serum except where indicated.
Cytokine levels in culture supernatants were determined using commercially available DuoSet ELISA Development kits for IL-6, IL-8, TNF-α and IL-1β according to the manufacturer’s instructions (R&D Systems Minneapolis, USA).
To test whether LL-37 or sLL-37 (0.5–50 μg/ml) have cytotoxic effects on neutrophils, the release of LDH into the medium was determined by colorimetric quantification (Cytotoxicity Detection Kit, Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions.
The luminol chemiluminescence assay was used to determine the production of reactive oxygen species (ROS). Krebs-ringer buffer with serum albumin (0.5 g/100 ml) was used to resuspend the isolated neutrophils. Luminol was dissolved in 10 ml DMSO and added to 1 l of 0.1 M NaOH to 50 mM. Each 1 ml of reaction mixture was prepared containing luminol (0.5 mM), SOD (5000 U/ml), catalase (200 000 U/ml), and neutrophil cells (1×106 cells). The samples were equilibrated to 37°C and different concentrations of LL-37 (5, 10, 20, and 30 μg/ml) were added. The emission of luminescent light was recorded using a luminometer (Magellan; Chantilly, VA, USA) after triggering ROS production with adding PMA (100 nM) (Sigma-Aldrich, Taufkirchen, Germany) or 104 CFU/ml Staphylococcus aureus opsonized with human serum and washing for 3 min. The emission of light was recorded for 1 hour (one measurement each 3 minutes) using a luminometer under shaking and 37C°.
Isolated neutrophils were labeled using FITC-labeled mouse anti-human CD66 and PerCP-labeled mouse anti-human CD45 (Becton-Dickinson, Heidelberg, Germany). 1 ml of cells suspension with 1×106 cells/ml was preincubated for 15 min at 37°C with 100 μM 2′,7′ dichlorofluorescin diacetate (DCFDA; Fluka, Steinheim, Germany) and then various concentrations of LL-37 or sLL-37 were applied for 30 min followed by stimulation with PMA. After washes (3 min), the cells were analyzed by flow cytometry (Becton-Dickinson, Heidelberg, Germany). A 488 nm argon laser beam was used for excitation. The mean fluorescence intensity of at least 105 neutrophil cells was calculated by the CELLQUEST software (Becton-Dickinson, Heidelberg, Germany). For each experiment, unstained cells served as controls.
106/ml neutrophils were resuspended in Krebs-Ringer phosphate buffer (KRG, pH 7.3) containing glucose (10 mM), Ca2+ (1 mM), and Mg2+(1.5 mM), and 0.3% BSA and preincubated at 37°C for 10 min in the presence of different concentrations of LL-37 (1, 5, 10 and 20 μg/ml). Staphylococcus aureus was added at a ratio of 1: 25 = neutrophils : bacteria. After incubation at 37°C for 1 h, the cells were subjected to repeated washes in PBS and resuspended in 0.5 ml of RPMI 1640 medium supplemented with 10% heat-inactivated FBS. Samples were taken for cytospin and the uptake of bacteria was determined microscopically by a blinded investigator. Cytopreparations were air dried, fixed in methanol, Giemsa stained, and mounted. Phagocytosis is expressed as mean number of bacteria (bound or ingested)/cell. Positive and negative controls were done (negative control = neutrophils were fixed with 3.7% formaldehyde before adding bacteria; positive = active neutrophils were incubated with bacteria without preincubation with LL-37).
For all experiments, at least triplicate determinations were made for each experimental condition. In indicated cases, the results of representative experiments were shown. All data are expressed as mean and standard deviation (SD). Comparisons between two experimental groups were performed using Student’s t test, for multiple group comparisons we used ANOVA with Tukey’s post test. Results were considered statistically significant for P values less than 0.05.
This work was supported by grants of the Deutsche Forschungsgemeinschaft to RB (Ba 1641/8, SFB/TR 22). SA is a fellow of the Syrian Arab Republic. We thank Thomas Damm and Annette Püchner for excellent technical support. We thank Dr. Melanie L. Conrad and Hani Harb for support.
Conflict of interest: The authors declare no financial or commercial conflict of interest.