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Modulation by LL-37 of the Responses of Salivary Glands to Purinergic Agonists

Laboratoires de Biochimie et de Biologie Cellulaire (S.P., S.T., S.Q., M.D.L., J.-P.D.) and Microbiologie, Institute of Pharmacy, Université libre de Bruxelles, Brussels, Belgium (M.T.-H., M.D.); Unité de Recherche sur la Structure et la Fonction des Membranes Biologiques, Faculty of Sciences, Université libre de Bruxelles, Brussels, Belgium (M.V.); and Departamento de Bioquimica y Biologia Molecular, Facultad de Ciencias, Universidad del Pais Vasco, Bilbao, Spain (M.G.-M., A.M.)

Received December 7, 2005; accepted March 1, 2006

	Abstract

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The interaction of mice submandibular gland cells with LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES), a cationic peptide with immunomodulatory properties, was investigated. LL-37 at a concentration that did not affect the integrity of the cells increased the uptake of calcium and activated a calcium-insensitive phospholipase A₂ (PLA₂). The small release of ATP induced by LL-37 could not account for this stimulation because apyrase did not significantly block the response to LL-37. The divalent cation magnesium inhibited the response to LL-37, but this inhibition was probably nonspecific because it also inhibited the in vitro bacteriostatic effect of the peptide. The increase of calcium uptake by LL-37 was not affected by 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62), a rather specific inhibitor of P2X₇ receptors in mice. LL-37 also increased [Ca²⁺]_i in cells from mice invalidated for these receptors. LL-37 had no effect on the response to carbachol. It inhibited the increase of [Ca²⁺]_i and the activation of phospholipase D by ATP. It potentiated the activation of the PLA₂ by the nucleotide. Finally, LL-37 increased the fluidity of the plasma membrane of submandibular gland cells. In conclusion, our results suggest that LL-37 is an autocrine regulator of submandibular gland cells. It does not stimulate mouse P2X₇ receptors but modulates their responses.

LL-37 not only kills bacteria but also binds to endotoxin (Bartlett et al., 2004), is chemotactic for human peripheral blood neutrophils, monocytes, and T lymphocytes through formyl peptide receptor-like 1 receptor (Yang et al., 2000) and for mast cells through two other unknown receptors (Niyonsaba et al., 2002). In mast cells, it also promotes the release of histamine (Niyonsaba et al., 2001). It increases the release of chemokines from epithelial cells (Tjabringa et al., 2003) and promotes angiogenesis (Koczulla et al., 2003). It induces keratinocyte migration after epidermal growth factor receptor transactivation (Tokumaru et al., 2005). Binding sites for LL-37 have also been recently described in lung epithelial cells (Lau et al., 2005). It has been reported that LL-37 activates the purinergic P2X₇ receptor expressed by monocytes, leading to the release of interleukine-1 (IL-1) by these cells (Elssner et al., 2004). These conclusions were mostly based on the use of antagonists of the P2X₇ receptors. These observations were at variance with the conclusions of Perregaux et al. (2002). These authors also reported that several antibacterial peptides promoted the release of IL-1 from macrophage, but, according to them, this was best explained by the decrease in potassium content of the macrophages secondary to the increase in permeability of their plasma membrane.

The purpose of our work was to reconsider the possible interaction between LL-37 and P2X₇ receptors. These receptors are present throughout the body and especially in exocrine glands (North, 2002). Considering that the salivary glands express not only P2X₇ receptors but also cathelicidin and that the C-terminal peptide of cathelicidin is present in saliva (Van Nieuw Amerongen et al., 2004), this peptide might have an autocrine effect on salivary glands and regulate the activity of salivary P2X₇ receptors. LL-37, the human peptide, is related (approximately 65%) to the peptide from mouse origin (Pestonjamasp et al., 2001) and recent studies have demonstrated the efficacy of LL-37 in murine cell models (Kurosaka et al., 2005; Zughaier et al., 2005). For those reasons, we decided to use a murine model that gave us the opportunity to compare the response of control mice and of mice with disrupted P2X₇ gene (P2X₇R^-/- mice; Solle et al., 2001). Our results show that LL-37 could mimic some of the responses to P2X₇ agonists [increase of the uptake of extracellular calcium and activation of phospholipase A₂ (PLA₂) activity]. LL-37 also modulated the responses to extracellular ATP: it inhibited the increase of [Ca²⁺]_i and the activation of phospholipase D (PLD), but it potentiated the activation of PLA₂ in response to ATP. LL-37 also increased [Ca²⁺]_i in salivary glands from P2X₇ R^-/- mice. LL-37 could thus elicit some cellular responses independently of P2X₇ receptors but also modulated the responses coupled to these receptors.

	Materials and Methods

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Drugs and Animals. The experiments were carried out on male C57BL/6J control mice and on male P2X₇R^-/- mice kindly supplied by Pfizer, Inc. (Groton, CT) and obtained by homologous recombination (Solle et al., 2001). Breeding P2X₇R^-/- mice males with females was used to maintain the colony of receptor-deficient animals. Mice used in the experiments were between 10 and 16 weeks of age. The animals were fed ad libitum and had free access to water. The care and use of the animals used in this study were approved by the Belgian Ministry of Agriculture in agreement with European regulations.

LL-37 (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was custom synthesized with 99% purity by Genemed Synthesis, Inc. (South San Francisco, CA). Two batches of LL-37 were used for these experiments. Fura-2/AM and 1,6-diphenyl-1,3,5-hexatriene (DPH) were from Invitrogen (Paisley, UK). Collagenase P and bovine serum albumin (BSA) fraction V were from Roche Diagnostics (Mannheim, Germany). The glutamine-free amino acids mixture was from Invitrogen. ATP, HEPES, leupeptin, pepstatin A, aprotinin, phenylmethylsulfonyl fluoride, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and the ATP assay mix were obtained from Sigma-Aldrich (St. Louis, MO). Isooctane was from Fluka (Buchs, Switzerland), 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine (POPC) was from Avanti Polar Lipids (Alabaster, AL), and 9,10-[³H]oleic acid was from American Radiolabeled Chemicals (St. Louis, MO). Silicagel-coated thin layer chromatography (TLC) plates were from Merck (Darmstadt, Germany). Methanol, ethyl acetate, and chloroform were from Labscan Ltd. (Dublin, Ireland). The scintillation solution Ecoscint A was from National Diagnostics (Atlanta, GA).

Preparation of a Crude Cellular Suspension from Mouse Submandibular Glands. The mice were anesthetized and killed with ether. The submandibular glands were immediately dissected and finely minced. The minced tissue was digested in the presence of 0.4 to 0.5 U/ml collagenase P for 20 min at 37°C under constant shaking in 10 ml of HEPES-buffered saline (HBS) medium containing 24.5 mM HEPES, pH 7.4, 96 mM NaCl, 6 mM KCl, 1 mM MgCl₂, 2.5 mM NaH₂PO₄, 11.5 mM glucose, 5 mM sodium pyruvate, 5 mM sodium glutamate, 5 mM sodium fumarate, 1% (v/v) glutamine-free amino acids mixture, and 0.125% (w/v) BSA. Ten minutes after the beginning of the digestion, the cells were aspirated five times with 10-, 5-, and 2-ml glass pipettes. At the end of the digestion, the crude suspension was mechanically dispersed by gentle pipetting, filtered and washed in an isotonic NaCl solution. The last pellet was resuspended in HBS medium and kept at 4°C until use.

Membrane Preparation. The cellular suspension from three mice was resuspended in 1 ml of ice-cold TEEI buffer (20 mM Tris-HCl, pH 8, 1 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, 2.5 µg/ml pepstatin A, and 2.5 µg/ml leupeptin) and passed through a 27-gauge needle. The suspension was centrifuged at 1000g for 10 min at 4°C. The pellet was extracted with 1 ml of ice-cold TEEI buffer and centrifuged. This extraction was repeated three times. The four supernatants were pooled and centrifuged at 100,000g for 30 min at 4°C.

LDH Assay. LDH activity was measured in a spectrophotometer (Scandinavian Society for Clinical Chemistry and Clinical Physiology, 1974). Two milliliters of Tris-EGTA-NADH (56 mM Tris, 5.6 mM EGTA, and 170 µM NADH) was added to the cuvette of a spectrophotometer. Fifty microliters of the enzyme solution (cellular supernatant or whole-cell extract) was then added. Two minutes later, 200 µl of 14 mM sodium pyruvate was added. The disappearance of the NADH was monitored at 340 nm for 5 min at 37°C.

ATP Assay. The ATP present in the medium was estimated with a bioluminescent assay (Neufeld et al., 1975). Fifty microliters of the cellular supernatant was added to a test tube, which was transferred to the chamber of a Lumat LB9507 luminometer (Berthold Technologies, Wildbad, Germany). Known standards of ATP were prepared in the medium used for the incubations of the cells, and 50-µl aliquots of these standards were assayed. The ATP assay mix was reconstituted according to the recommendations of the manufacturer, and 20 µl of this mix was added to the samples. The emitted light was measured for 15 s.

Measurement of [Ca²⁺]_i. After its isolation, the crude pellet was resuspended in 6 ml of fresh HBS medium in the presence of magnesium chloride, amino acids, and 0.1% albumin. The suspension was kept at 4°C until use. One milliliter of this suspension was incubated in 2 ml of HBS medium in the presence of amino acids, 1 mM MgCl₂, 0.25 mM CaCl₂, and 0.5% (w/v) BSA. The suspension was incubated for 45 min at 25°C in the presence of 2 µM fura-2/AM. At the end of the incubation, 10 ml of isotonic NaCl was added to the tube, which was centrifuged for 1 min at 500g. The supernatant was discarded, and the pellet was resuspended in 4 ml of fresh HBS medium, in the absence of amino acids, magnesium chloride, or albumin but in the presence of 1 mM calcium chloride. Two milliliters of this suspension was transferred in the cuvette of a spectrofluorimeter and constantly agitated. The assay was performed at 25°C. The excitation wavelength was switched every second from 340 to 380 nm (slitwidth 8). The light emitted at 510 nm (slitwidth 16) was recorded. At the end of the assay, the traces were calibrated with the successive addition of 0.1 mM digitonin and 40 mM EGTA, pH 8.5 with Tris. The autofluorescence measured after quenching the fluorescence of the fura-2 by the addition of 100 mM MnCl₂ was subtracted from all the data before calculation of the ratios. The calcium concentration was estimated by the ratio method as described by Grynkiewicz et al. (1985).

Measurement of the Activity of PLA₂. Cells from three mice were resuspended in 1 ml of HBS medium containing the amino acids mixture, 1 mM CaCl₂, and 1 mM MgCl₂, without albumin. The cells were incubated for 2 h at 37°C in the presence of 5 µCi of [³H]oleic acid. At the end of this incubation, the cells were washed twice with 10 ml of isotonic NaCl. They were resuspended in 2 ml of HBS medium containing the amino acids mixture, 0.5 mM calcium chloride, 1 mM magnesium chloride, and 0.5% (w/v) BSA. They were incubated for 1 h at 37°C. At the end of this incubation, the cells were washed twice with 10 ml of isotonic NaCl. The cells were then resuspended in 9 ml of HBS containing the amino acids mixture, 0.5 mM calcium chloride, and 0.1% BSA, but no magnesium chloride. Aliquots of 0.5 ml were incubated for 10 min at 37°C either in control or in the tested condition. Each condition was tested in triplicate. At the end of the incubation, the cells were centrifuged, and 0.4 ml of the supernatant was transferred to scintillation vials. To estimate the amount of [³H]oleic acid present in the medium at the beginning of the incubation (blank values), a similar procedure was applied to nonincubated aliquots of cells. Aliquots of cells were also directly transferred to the scintillation vials to estimate the incorporation of the fatty acid in the cells (total counts). Three milliliters of Ecoscint A was added to each vial, and the radioactivity was measured in a beta-scintillation counter. The blank value was subtracted from the results obtained with the incubated cells, and the results are expressed as percentage of the radioactivity incorporated in the cells.

Measurement of the Activity of PLD. The activity of PLD was assayed by measuring the formation of [³H]phosphatidyl ethanol, which is the product of its specific transphosphatidylation reaction in the presence of ethanol (Pochet et al., 2003). Cells from three mice were resuspended in 1 ml of HBS medium in the presence of 0.5 mM CaCl₂. They were incubated for 90 min at room temperature in the presence of 5 µCi/ml [³H]oleic acid. At the end of the labeling period, the cells were washed three times with isotonic NaCl. The final pellet was resuspended in 1 ml of fresh HBS medium without the tracer and incubated for 30 min at room temperature. The cells were washed and resuspended in 6.5 ml of HBS medium containing 1 mM CaCl₂ but no magnesium. The assays were performed at 37°C under constant shaking. One-milliliter aliquots of the cellular suspension were preincubated for 5 min with 1.5% (v/v) ethanol before being incubated with the tested agent for the indicated period. The reaction was stopped by the addition of 3 ml of a mixture of chloroform/methanol (1:2). Phospholipids were extracted with 1 ml of chloroform and 1 ml of 2.4 N HCl. After 1 h at 4°C, the two phases were separated by centrifugation at 1000g for 10 min. The upper phase was re-extracted with 1 ml of chloroform, and the two lower phases were pooled and washed with 2 ml of a mixture of methanol/1 N HCl (1:1). After centrifugation, the upper phase was discarded and the lower phase was evaporated to dryness. The lipid extract was dissolved in 50 µl of chloroform, and 20 µl was spotted onto a TLC plate. The plate was developed in the upper phase of a mixture of water/ethyl acetate/acetic acid/isooctane (10:13:3:2). The lipids were visualized by exposure to iodine vapor, and phosphatidyl ethanol was identified by comparison with an authentic standard. Phospholipids spots were scraped into scintillation vials containing 1 ml of methanol. Ten milliliters of Ecoscint A was added to each vial, and their radioactivity was determined by beta liquid scintillation. The sum of the radioactivity in these bands was a measure of the incorporation of [³H]oleic acid in total phospholipids. The PLD activity was expressed as the percentage of the radioactivity present in the phosphatidyl ethanol spot compared with the radioactivity in the total phospholipids.

Measurement of the Membrane Fluidity. The fluidity of the membranes was estimated by fluorescence anisotropy (Van Laethem et al., 2003). The membrane pellet was resuspended in HBS (10 mM HEPES, pH 7.4, and 150 mM NaCl) and sonicated for 5 s at 15-µm amplitude. The membranes were incubated in the absence or in the presence of 10 µM LL-37. Aliquots of these two membrane suspensions were labeled with the fluorescent probe DPH by adding the probe in tetrahydrofuran at a 1 µg/ml final concentration and incubating at 37°C for approximately 15 min. The measurements were performed in a SLM 8000C spectrofluorimeter with Glan-Thompson polarizers placed in T-geometry. Excitation was performed at 360 nm, and emission was recorded at 430 nm. For each experiment, 2 ml of HS with the labeled vesicles was transferred in a 10 x 10 x 45-mm acrylic cuvette (Kartell, Noviglio, Italy) placed in a thermostatic chamber. Polarization measurement was made by simultaneously measuring the vertical and horizontal components of the polarized emission. Correction for the background polarization due to sample turbidity was made using unlabeled samples. The ratio of the intensities in the vertically and horizontally polarized detectors was measured with vertically and horizontally polarized excitation, giving, respectively, the R_vert and R_horiz ratios. Polarization (P) was calculated as P = (R_corr - 1)/(R_corr + 1), where R = R_vert/R_horiz. Anisotropy was derived from P using R = 2P/(3 - P). Similar measurements were performed on POPC and DPPC multilamellar liposomes prepared as described by Bangham et al. (1965).

Hemolysis of Murine Red Blood Cells. Mice were anesthetized with ether. Blood was collected by venipuncture of the inferior vena cave just above the junction of the renal veins, and 2 mg/ml EDTA was added. Red blood cells were collected by centrifugation and washed three times with phosphate-buffered saline. The final pellet was resuspended with PBS at a 10% hematocrit. LL-37 (final concentration 10 µM) or 2 µl of Triton X-100 was added to 98 µl of the cellular suspension, and the cells were incubated for 30 min at 37°C. At the end of the incubation, the tubes were centrifuged, and the hemoglobin present in the supernatant was estimated by measuring the absorbance at 540 nm.

Bactericidal and Bacteriostatic Effect of LL-37. The assay was performed in a 96-well microplate. The bacteria (Escherichia coli strain ATCC 10536) were grown to the mid-logarithmic phase. Approximately 10,000 colony-forming units were incubated in the presence of 100 µl of Mueller-Hinton medium and of serial dilutions of LL-37 (concentration range 30 nM-20 µM). The plate was incubated for 16 h at 37°C. The plate was then scanned with the GS-690 imaging densitometer from Bio-Rad (Hercules, CA). To evaluate the bactericidal property of the peptide, aliquots were removed from these wells and transferred to a Petri dish with the Mueller-Hinton medium. The presence of colonies was examined after an overnight incubation at 37°C.

Statistical Analysis. Results are expressed as means ± S.E.M. of the number of experiments indicated. Statistical significance between various conditions was assessed with Student's t test.

	Results

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LL-37 is generated by digestion of human cathelicidin by elastase or protease-3. The peptide has 37 amino acids with two leucines at its N-terminal side. With its five acidic and 11 basic residues, it is positively charged at physiological pH (pI = 10.6). It forms a perfect amphipathic -helix between residues 11 and 31 (Agerberth et al., 1995). This peptide is highly toxic for bacteria but has also some deleterious effects on the plasma membrane of eukaryotic cells (Oren et al., 1999). In preliminary experiments, the release of LDH from a cellular suspension of submandibular glands exposed to LL-37 was examined (Fig. 1). After a 15-min incubation at 37°C, LL-37 did not promote the efflux of LDH when tested at concentrations up to 10 µM (from 1.8 ± 0.4% in the absence to 2.9 ± 0.5% in the presence of 10 µM LL-37; n = 3; P > 0.05). A higher concentration of LL-37 (20 µM) significantly increased the release of LDH after 15 min (to 11.5 ± 1.9%; P < 0.05; n = 3). After a 60-min incubation LL-37 significantly increased the release of LDH at 10 µM (to 7.9 ± 1.0%; P < 0.05) and at 20 µM (23.9 ± 3.1%; P < 0.01). The peptide was then tested at a 10 µM concentration on the hemolysis of murine red blood cells. After a 30-min incubation at 37°C, the hemolysis averaged only 2% of the maximal hemolysis measured with Triton X-100. It was thus decided to test the peptide at concentrations not exceeding 10 µM.

View larger version (12K): [in this window] [in a new window]   Fig. 1. Effect of LL-37 on the release of LDH from submandibular gland cells. A crude cellular suspension was prepared from mice submandibular glands and incubated for 15 or 60 min in the presence of various concentrations of LL-37. At the end of the incubation, the cells were centrifuged, and the LDH activity of the supernatant was estimated. An aliquot of the cellular suspension was homogenized and its LDH activity was assayed (total activity of the cell extract). The results are expressed as percentage of the total LDH activity released in the medium during the experiment. They are the means ± S.E.M. of three experiments. *, P < 0.01; **, P < 0.005.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effect of carbachol, ATP, and LL-37 on [Ca2+]i in cells from mice submandibular glands. A crude cellular suspension was prepared from submandibular glands of P2X7R+/+ (top) or P2X7R-/- (bottom) mice. The cells were loaded with fura-2/AM, and after washing they were resuspended in HBS medium, in the absence of amino acids, MgCl2, and BSA but in the presence of 1 mM CaCl2. They were transferred in the cuvette of a spectrofluorimeter and incubated at 25°C under constant agitation. Two minutes after the start of the incubation, the cells were exposed to either 100 µM carbachol, 1 mM ATP, or 10 µM LL-37. At the end of the incubation, the traces were calibrated as described under Materials and Methods. Results are the means ± S.E.M. of three to eight experiments.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Effect of various concentrations of LL-37 on [Ca2+]i. Cells from submandibular glands loaded with fura-2 were exposed to 0, 1, 3, or 10 µM LL-37. Results are the variation of [Ca2+]i 1 min before and 1 min after the addition of DMSO or LL-37 to the medium. Results are the means ± S.E.M. of four to 11 experiments. *, P < 0.05; **, P < 0.01.

In the absence of extracellular calcium, carbachol increased [Ca²⁺]_i from 27 ± 5 to 62 ± 8 nM (Fig. 4, inset; n = 3; P < 0.05), confirming that the muscarinic agonist could mobilize intracellular pools of calcium. ATP had no effect on [Ca²⁺]_i, confirming that the nucleotide did not stimulate a metabotropic receptor coupled to the hydrolysis of polyphosphoinositides. LL-37 significantly decreased [Ca²⁺]_i from 57 ± 2 to 44 ± 3 nM after 90 s (n = 3; P < 0.05). After a preincubation for 2 min in control conditions and in the absence of calcium, the addition to the medium of 1 mM calcium significantly increased [Ca²⁺]_i from 49 ± 2 to 113 ± 9 nM (n = 6; P < 0.05) within 10 s (Fig. 4). When the cells had been exposed for 2 min to ATP, the addition of calcium to the medium increased [Ca²⁺]_i to 165 ± 9 nM after 5 s and to 321 ± 30 nM (n = 4; P < 0.001) after 3 min. LL-37 (10 µM) also increased the uptake of extracellular calcium: [Ca²⁺]_i averaged 206 ± 26 nM after 5 s and remained stable (205 ± 30 nM after 3 min; n = 3; P < 0.01). From these results, it could be concluded that ATP and LL-37 did not mobilize intracellular pools of calcium but rather increased the uptake of extracellular calcium.

View larger version (23K): [in this window] [in a new window]   Fig. 4. Effect of ATP and LL-37 on the mobilization of intracellular pools of calcium and on the uptake of extracellular calcium. Cells from submandibular glands loaded with fura-2 were resuspended in a calcium-free medium. Thirty seconds after the beginning of the measurement, 100 µM EGTA was added to the medium (A), and 30 s later the cells were exposed to either DMSO or to 1 mM ATP or 10 µM LL-37 (B). Two minutes later, 1 mM CaCl2 was added to the medium (C). Inset, cells were incubated in the same conditions but were exposed to 100 µM carbachol at 1 min. Results are the means ± S.E.M. of three experiments.

Interaction between LL-37 and Muscarinic or Purinergic Agonists on [Ca²⁺]_i. The interaction between LL-37 and carbachol or ATP was next explored. The cells were exposed for 5 min to 10 µM LL-37 before stimulation with either 100 µM carbachol or 1 mM ATP. The preincubation with LL-37 had no effect on the response to carbachol (Fig. 5, top). The response to 1 mM ATP was inhibited by LL-37 (Fig. 5, bottom). In the absence of LL-37, the variation of [Ca²⁺]_i after 2-min exposure to the nucleotide averaged +323 ± 38 nM (n = 6) in cells preincubated with DMSO and +198 ± 16 nM (n = 4) in cells preincubated with 10 µM LL-37 (P < 0.05).

View larger version (20K): [in this window] [in a new window]   Fig. 5. Effect of LL-37 on the variation of [Ca2+]i in response to carbachol and ATP. Cells from submandibular glands loaded with fura-2 were resuspended in a medium containing 1 mM CaCl2. Two minutes after the beginning of the measurement, the cells were exposed to either 0.5% DMSO (open symbols) or to 10 µM LL-37 (closed symbols). Five minutes later, 100 µM carbachol (Cb, top) or 1 mM ATP (bottom) was added to the medium. The results are expressed as the variation of [Ca2+]i compared with [Ca2+]i at 6 min. They are the means ± S.E.M. of three to six experiments.

Contribution of ATP and Purinergic Receptors in the Response to LL-37. The time course of the response to LL-37 and its dependence toward extracellular calcium suggested that an ionotropic purinergic receptor might be involved in the response to the peptide. Considering that cationic peptides can permeabilize the plasma membrane of eukaryotic cells (Oren et al., 1999), we considered the possibility that the response to LL-37 was merely the consequence of the release of ATP induced by the peptide. The cells were incubated with 10 µM LL-37. After centrifugation, the ATP content of the medium was estimated with a luciferin-luciferase mixture (Neufeld et al., 1975). Cells were incubated in a test tube in the chamber of the luminometer and preincubated with the ATP assay mix before the addition of LL-37 (Fig. 6). In these conditions, a significant increase (P = 0.015; n = 6) of the light emitted was noted. This increase was transient and significantly lower 15 s after the addition of the peptide (P = 0.029; n = 6). The contribution of this small release of ATP in the response to a lower (3 µM) concentration of LL-37 was examined in the presence of apyrase or magnesium ion. Apyrase is an ATP diphosphohydrolase that catalyzes the degradation of ATP; magnesium ion forms a complex with ATP and decreases the concentration of ATP^4-, the true agonist of P2X receptors. Apyrase had no effect on the increase of [Ca²⁺]_i in response to carbachol (data not shown). The enzyme did not significantly affect the acute response to 300 µM ATP (Fig. 7, top) but fully suppressed its late effect [from +118 ± 9 nM (n = 4) in the absence of apyrase to +34 ± 4 nM (n = 5) in the presence of the enzyme, 4 min after the addition of ATP; P < 0.0001, nonpaired t test]. The response to 3 µM LL-37 was not inhibited by the enzyme [from +89 ± 19 nM (n = 5) in the absence to +56 ± 15 nM (n = 4) in the presence of 5 U/ml apyrase, after a 4-min exposure to LL-37; P = 0.2574] (Fig. 7, bottom). As shown in Fig. 8, the addition of 5 mM magnesium to the extracellular medium inhibited by more than 80% the response to 1 mM ATP (from +148 ± 17, n = 8 to +27 ± 4 nM, n = 7; P < 0.0001) and by 50% the response to LL-37 [from +81 ± 7 nM (n = 3) to +37 ± 14 nM (n = 3); P = 0.048]. Because the results obtained with apyrase and magnesium were not consistent, we decided to test the effect of magnesium on the "bactericidal" activity of LL-37. Five millimolar magnesium blocked the bacteriostatic and bactericidal effects of LL-37 (data not shown), confirming that magnesium inhibited the peptide itself. From these results and considering that apyrase did not inhibit the increase of [Ca²⁺]_i in response to LL-37, we concluded that released ATP did not mediate the effect of LL-37 on calcium.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Effect of LL-37 on the release of ATP. Cells from submandibular glands were resuspended in a medium without calcium. Aliquots (190 µl) were transferred to a test tube containing 10 µl of the ATP assay mix. The test tube was placed in the chamber of the luminometer, and the light emitted was integrated for 15 s (-15 0). Ten microliters of DMSO or LL-37 (final concentration 10 µM) was added, and the light was measured for the next 30 s (0 +15 and +15 +30). Results are the means ± S.E.M. of six experiments. *, P < 0.05.

View larger version (21K): [in this window] [in a new window]   Fig. 7. Effect of apyrase on the variation of [Ca2+]i in response to ATP or LL-37. Cells from submandibular glands loaded with fura-2 were resuspended in the presence of 1 mM CaCl2 in the absence (open symbols) or in the presence (closed symbols) of 5 U/ml apyrase. One minute after the start of the measurement, the cells were exposed to 300 µM ATP (top) or 3 µM LL-37 (bottom). Results are expressed as the variation of [Ca2+]i compared with the initial [Ca2+]i. They are the means ± S.E.M. of four or five experiments.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Effect of magnesium on the variation of [Ca2+]i in response to ATP or LL-37. Cells from submandibular glands loaded with fura-2 were resuspended in the presence of 1 mM CaCl2 in the absence (open symbols) or in the presence (closed symbols) of 5 mM MgCl2. One minute after the start of the measurement, the cells were exposed to 1 mM ATP (top) or 3 µM LL-37 (bottom). Results are expressed as the variation of [Ca2+]i compared with initial [Ca2+]i. They are the means ± S.E.M. of three to eight experiments.

It has been recently reported that LL-37 could activate by itself human P2X₇ receptors (Elssner et al., 2004). We thus tested the effect of KN-62, a calmidazolium derivative that is a strong inhibitor of some P2X₇ receptors (Virginio et al., 1997). Fig. 9, top, shows that 10 µM KN-62 inhibited the response to 1 mM ATP by 50% (from +130 ± 23 nM 2 min after ATP in control cells to +65 ± 7 nM in cells treated with KN-62; n = 4; P = 0.038). KN-62 had no significant effect on the response to LL-37 (Fig. 9). These results suggested that the P2X₇ receptors were probably not involved in the increase of [Ca²⁺]_i in response to LL-37. This was confirmed using cells isolated from salivary glands of P2X₇R^-/- mice (Solle et al., 2001). Fig. 2, bottom, shows that these cells responded normally to carbachol. Their response to extracellular ATP was deeply blunted and had a totally different time course compared with cells from wild-type animals. ATP only transiently increased [Ca²⁺]_i from 99 ± 6 to 125 ± 8 nM (n = 3). The variation of [Ca²⁺]_i in response to LL-37 was not affected by the absence of P2X₇ receptors (from 92 ± 2 to 233 ± 14 nM; n = 7).

View larger version (23K): [in this window] [in a new window]   Fig. 9. Effect of KN-62 on the variation of [Ca2+]i in response to ATP or LL-37. Cells from submandibular glands loaded with fura-2 were resuspended in the presence of 1 mM CaCl2 and preincubated for 10 min in the absence (open symbols) or in the presence (closed symbols) of 10 µM KN-62. One minute after the start of the measurement, the cells were exposed to 1 mM ATP (top) or 3 µM LL-37 (bottom). Results are expressed as the variation of [Ca2+]i compared with initial [Ca2+]i. They are the means ± S.E.M. of four experiments.

Effect of LL-37 on the Activity of Phospholipases and on the Fluidity of the Plasma Membrane. The P2X₇ receptor regulates the activity of various phospholipases in salivary glands (Alzola et al., 1998; Pochet et al., 2003). The activity of PLD was estimated by measuring the production of Peth in cells labeled with [³H]oleic acid and incubated in the presence of ethanol. Fig. 10, top, shows that the purinergic agonist and carbachol increased the production of Peth 3.5- and 2-fold, respectively. By itself, 10 µM LL-37 had no effect on the concentration of this phospholipid (90 ± 5%; n = 5 compared with control). LL-37 inhibited the activation of PLD by ATP from 348 ± 16% (n = 4) in the absence to 196 ± 16% (n = 4) in the presence of LL-37. The peptide had no effect on the stimulation of PLD by carbachol (Fig. 10, top).

View larger version (19K): [in this window] [in a new window]   Fig. 10. Effect of ATP, carbachol, and LL-37 on the PLD and PLA2 activities of submandibular cells. Cells from mice submandibular glands were labeled with 5 µCi/ml [3H]oleic acid for 90 min and allowed to equilibrate for 30 min. Top (PLD), cells were resuspended in HBS medium with 1 mM CaCl2 but without magnesium. One-milliliter aliquots were preincubated with 1.5% ethanol for 5 min. Cells were then incubated for 30 min at 37°C in the presence of 100 µM carbachol or 1 mM ATP, in the absence or in the presence of 10 µM LL-37. At the end of the incubation, the lipids were extracted, separated by TLC, and [3H]phosphatidyl ethanol (Peth) formation was measured. Bottom (PLA2), cells were resuspended in HBS medium without magnesium but with 1 mM CaCl2 or without CaCl2 but with 100 µM EGTA. The cells were incubated at 37°C for 10 min in the presence of 0.5% DMSO or 1 mM ATP or 100 µM carbachol or 10 µM LL-37. The radioactivity present in the medium at the beginning of the experiment was estimated in nonincubated samples and subtracted from the incubated samples. An aliquot of cells was counted to estimate the total radioactivity of cells. Results are expressed as percentage of control (PLD) or as the total radioactivity in the cells (PLA2) and are the means ± S.E.M. of three to five experiments (top) or of three experiments (bottom). *, P < 0.05 compared with the control condition (absence of ATP); **, P < 0.01 compared with the control condition (absence of LL-37).

To measure the activity of PLA₂, the cells were labeled with [³H]oleic acid and after a washout period, the release of the fatty acid in the medium was estimated. Fig. 10, bottom, shows that carbachol had no significant effect on this activity. Extracellular ATP increased the release of oleic acid 2.5-fold. Removal of the extracellular calcium had no effect on the basal release of oleic acid, but it inhibited by 60% (from +170 to +70%) the stimulation by ATP. LL-37 also promoted the release of oleic acid. In the presence of extracellular calcium the response to 10 µM LL-37 was very similar to the response to ATP. Removal of calcium had no effect on the response to LL-37 [from 7.1 ± 0.8% (n = 5) in the presence of calcium to 7.4 ± 0.5% (n = 4) in the absence of the ion]. The combination of the two stimuli further increased the release of oleic acid. The synergism was better observed in the absence of calcium: more than 15% of the total oleic acid incorporated in the lipids was released when ATP and LL-37 were added together. Activation of the muscarinic receptors did not affect the release of oleic acid either in basal conditions or in the presence of LL-37. The effect of LL-37 on the fluidity of the plasma membrane was next tested. Membranes were isolated and incubated with a fluorescent probe (DPH). The movement of the probe was estimated by measuring the anisotropy at various temperatures in control membranes or in membranes preincubated with 10 µM LL-37. The anisotropy of micelles DPPC at 20°C (0.315 ± 0.008; n = 3) was much higher than the anisotropy of micelles of POPC (0.117 ± 0.009; n = 3). The anisotropy decreased at higher temperatures in both micelles (data not shown). This result is consistent with the fact that the presence of an unsaturated fatty acid or a high temperature increases the fluidity of the micelles. By comparison, the anisotropy of control membranes was intermediate (0.183 ± 0.006 at 20°C; n = 6). The pretreatment with LL-37 significantly decreased the anisotropy (0.163 ± 0.004 at 20°C; n = 6, P < 0.05 compared with control membranes). This small effect was consistent in the six experiments and significant at every temperature tested.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
Our results show that in submandibular glands from control mice, LL-37 increased [Ca²⁺]_i and stimulated a calcium-insensitive PLA₂. These responses were observed at concentrations of LL-37 that had no effect on the response to carbachol. This last result confirmed that LL-37 did not affect the integrity of the muscarinic receptors, their coupling to a PPI-PLC, the filling state of intracellular pools of calcium, and their mobilization by inositol phosphates. LL-37 did not activate a receptor coupled to the activation of a PPI-PLC because LL-37 did not mobilize intracellular pools of calcium but only increased the uptake of extracellular calcium.

These responses to LL-37 were not secondary to the release of ATP. Indeed, the measurement of the ATP content in the incubation medium revealed that LL-37 evoked a very small and transient release of ATP. It must be stressed, however, that these measurements probably underestimate the concentration of ATP at the vicinity of the purinergic receptors. The contribution of ATP in the response to LL-37 was evaluated by the addition of apyrase in the incubation medium. This enzyme, which degrades ATP to AMP and pyrophosphate, strongly inhibited the response to exogenous ATP. It did not inhibit the response to LL-37. Similar results were obtained with cells incubated in the presence of glucose and hexokinase (data not shown). These results are not consistent with those of Elssner et al. (2004) who also reported that LL-37 could indeed provoke an efflux of ATP from lipopolysaccharide-primed monocytes but that apyrase inhibited the release of IL-1 by these cells in response to LL-37.

Because apyrase (and hexokinase) did not inhibit the response to LL-37 and because Elssner et al. (2004) reported that the peptide could stimulate P2X₇ receptors, the effect of KN-62, a rather specific antagonist of these receptors was tested on the response to LL-37. This calmidazolium derivative inhibited the response to ATP without affecting the response to LL-37. This is at variance with the results of Elssner et al. (2004) who reported that KN-62 could block the activation of macrophages by LL-37 and suggests that, in mouse salivary glands, the response to LL-37 was not mediated by P2X₇ receptors. Our results were confirmed in mice lacking the P2X₇ receptor. ATP only slightly increased [Ca²⁺]_i in cells from the salivary glands of these mice, and this small response was strictly dependent on the presence of calcium in the medium, suggesting that it involved another P2X receptor (S. Pochet, personal communication). Cells from P2X₇R^-/- mice that were thus devoid of P2X₇ receptors normally responded to LL-37 with respect to [Ca²⁺]_i. This result confirmed that the mouse P2X₇ receptors were not required to observe an increase of [Ca²⁺]_i in response to LL-37.

It has been reported that LL-37 could increase [Ca²⁺]_i in other cells such as mast cells (Niyonsaba et al., 2001) or neutrophils (Yang et al., 2000). In these systems, LL-37 activated a PPI-PLC and mobilized intracellular pools of calcium after activating a plasma membrane receptor coupled to a GTP-binding protein such as formyl peptide receptor-like 1 (Yang et al., 2000) or another receptor not yet defined (Niyonsaba et al., 2002). This mechanism is very unlikely in our model: LL-37 did not mobilize intracellular pools of calcium because it did not increase [Ca²⁺]_i in a calcium-free medium. More recently, Tjabringa et al. (2003) reported that LL-37 could stimulate epidermal growth factor receptor in epithelial cells. This stimulation was blocked by G6001, an inhibitor of metalloproteases. They concluded that LL-37 might activate a metalloprotease and release an endogenous activator of EGFR. The response to LL-37 was not affected by either an activator or an inhibitor of the protease-activated receptor type 2, the protease-activated receptor expressed by the salivary glands (J. P. Dehaye, unpublished results), suggesting that this receptor was not involved in the response to LL-37. From all these negative results, it can be concluded that the uptake of calcium induced by LL-37 in mouse submandibular gland was not secondary to the activation of a murine purinergic- or protease-activated receptor or a receptor coupled to a G protein activating a PPI-PLC.

Salivary glands express various phospholipases A₂ (Alzola et al., 1998) and D (Pochet et al., 2003) among which some are activated by an increase of [Ca²⁺]_i. Both ATP and LL-37 increased PLA₂ activity. The activation of PLA₂ by ATP is absent in cells from P2X₇R^-/- mice, confirming that P2X₇ receptors are involved in this response (E. Kabré, personal communication). The response to LL-37 was not mediated by these receptors because it was present in cells from P2X₇R^-/- mice (E. Kabré, personal communication). The activation by LL-37 was not secondary to the increase of [Ca²⁺]_i because it could still be observed in the absence of extracellular calcium. In these ionic conditions, the combination of ATP and LL-37 led to a much larger increase of the PLA₂ activity. The effect of LL-37 on the activity of PLA₂ has never been measured directly but it has been previously reported that LL-37, at the opposite of human defensin, did not promote the release of prostaglandins from mast cells (Niyonsaba et al., 2001). Yet the modulation of the PLA₂ activity by amphipathic peptides is a well known phenomenon: mellitin, a strong activator of PLA₂, shares with LL-37 a high content of basic residues and the formation of an amphipathic -helix (Bucki et al., 2004). More recently it has been reported that antibacterial peptides with a high affinity for phosphatidyl cholines can activate a secretory PLA₂ (Zhao and Kinnunen, 2003). LL-37 had no effect on the activity of the phospholipase D. This result was unexpected considering that other cationic antimicrobial peptides like mellitin or dermaseptin are activators of PLD, respectively, in human monocytic leukemia cells (Saini et al., 1999) and in polymorphonuclear leukocytes (Ammar et al., 1998). LL-37 specifically interfered with the purinergic regulation of PLD: it inhibited by nearly 50% the response to ATP without affecting the response to carbachol.

The previous results emphasized the fact that the interactions between LL-37 and purinergic responses were complex. By itself, LL-37 did not reproduce all the responses to ATP (it did not activate a PLD). When combined with ATP, LL-37 inhibited some responses (increase of [Ca²⁺]_i and activation of PLD) but potentiated others (activation of PLA₂). These contradictory effects of LL-37 on cellular responses induced by P2X₇ receptors exclude a direct activation of the receptor by LL-37. The responses to LL-37 might be explained by the formation of a pore in the plasma membrane, and the subsequent influx of calcium and efflux of potassium responsible for the activation of a PLA₂ (Andrei et al., 2004). The interaction of the peptide with the lipid phase of the plasma membrane might also account for its effects on the regulation by ATP of phospholipase A₂ (activation) and PLD (inhibition). Indeed, according to Rao (1992), membrane-active peptides activate PLA₂ and inhibit PLD activity when tested on unilamellar vesicles and these responses are best explained by a modification of the packaging of the lipid. It should be noted that LL-37 slightly increased the fluidity of the plasma membrane. LL-37 might thus regulate the cellular function by modifying the physicochemical state of the membrane rather than by interacting with a plasma membrane receptor. Zughaier et al. (2005) have proposed such a model. According to these authors, the synergy between endotoxin and LL-37 on the release of reactive oxygen species is mediated by a better interaction of the peptide with the plasma membrane and not by a direct interaction with TLR4, the receptor for lipopolysaccharides. This model is in agreement with the general model proposed by Perregaux et al. (2002) who proposed that the cationic peptides activate macrophages by perturbing their plasma membrane. It is also consistent with the recent results of Lau et al. (2006). These authors reported that concentrations of LL-37 similar to the concentrations used in our study could induce apoptotic lesions in thymocytes. We observed that LL-37 provoked a small release of ATP and a slight efflux of calcium when the cells were incubated in a calcium-free medium. A significant release of LDH was observed at high concentrations of LL-37. From these results, we cannot exclude that the responses of salivary glands to LL-37 were secondary to proapoptotic lesions provoked by the peptide.

In conclusion, our results show that LL-37 regulates the activity of submandibular gland cells. This result implies that salivary glands contribute to the host defense mechanisms not only by secreting LL-37 (Murakami et al., 2002; Woo et al., 2003) but also by the cellular responses like the activation of PLA₂ locally triggered by the peptide. The response of submandibular glands to LL-37 does not require the expression of P2X₇ receptors, but the peptide can nevertheless modulate the responses to this receptor probably by its effect on the properties of the plasma membrane.

	Acknowledgements

 
We thank Dr. C. Gabel (Pfizer, Inc.) for the generous gift of P2X₇R^-/- mice. The scintillation counter was a gift from Sopar Pharma (Brussels, Belgium).

	Footnotes

 
This work was supported by grant 3.4506.00 from the Fonds de la Recherche Scientifique Médicale of Belgium and a grant from the Emile Defay Fund (to J.-P.D.) and by "Fundación benefico-docente Jesus Gangoiti-Barrera" and grants 9/UPV00042.310-15941/2004 from the University of the Basque (to A.M.) S.P. is a postdoctoral researcher of the Fonds National de la Recherche Scientifique of Belgium.

Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

doi:10.1124/mol.105.021444.

ABBREVIATIONS: IL, interleukin; PLD, phospholipase D; PLA₂, phospholipase A₂; AM, acetoxymethyl ester; DPH, 1,6-diphenyl-1,3,5-hexatriene; BSA, bovine serum albumin; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; POPC, 1-palmitoyl, 2-oleyl-sn-glycero-3-phosphocholine; TLC, thin layer chromatography; HBS, HEPES-buffered saline; LDH, lactate dehydrogenase; PPI-PLC, polyphosphoinositide-specific phospholipase C; DMSO, dimethyl sulfoxide.

Address correspondence to: Dr. J. P. Dehaye, Laboratoire de Biochimie et de Biologie Cellulaire, Université libre de Bruxelles-Institut de Pharmacie C.P. 205/3, Boulevard du Triomphe, B1050 Bruxelles, Belgium. E-mail: jdehaye{at}ulb.ac.be

	References

Top Abstract Materials and Methods Results Discussion References

 
Agerberth B, Gunne H, Odeberg J, Kogner P, Boman HG, and Gudmundsson GH (1995) FALL-39, a putative human peptide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc Natl Acad Sci USA 92: 195-199.[Abstract/Free Full Text]

Alzola E, Pérez-Etxebarria A, Kabré E, Fogarty DJ, Métioui M, Chaïb N, Macarulla JM, Matute C, Dehaye JP, and Marino A (1998) Activation by P2X₇ agonists of two phospholipases A₂ (PLA₂) in ductal cells of rat submandibular gland. Coupling of the calcium-independent PLA₂ with kallikrein secretion. J Biol Chem 273: 30208-30217.[Abstract/Free Full Text]

Ammar B, Perianin A, Mor A, Sarfati G, Tissot M, Nicolas P, Giroud JP, and Roch-Arveiller M (1998) Dermaseptin, a peptide antibiotic, stimulates microbicidal activities of polymorphonuclear leukocytes. Biochem Biophys Res Commun 247: 870-875.[CrossRef][Medline]

Andrei C, Margiocco P, Poggi A, Lotti LV, Torrisi MR, and Rubartelli A (2004) Phospholipases C and A₂ control lysosome-mediated IL-1 secretion: implications for inflammatory processes. Proc Natl Acad Sci USA 101: 9745-9750.[Abstract/Free Full Text]

Bals R and Wilson JM (2003) Cathelicidins - a family of multifunctional antimicrobial peptides. Cell Mol Life Sci 60: 711-720.[CrossRef][Medline]

Bangham AD, Standish MM, and Miller N (1965) Cation permeability of phospholipids model membranes: effect of narcotics. Nature (Lond) 208: 1295-1297.[CrossRef][Medline]

Bartlett KH, McCray PB Jr, and Thorne PS (2004) Reduction in the bactericidal activity of selected cathelicidin peptides by bovine calf serum or exogenous endotoxin. Int J Antimicrob Agents 23: 606-612.[CrossRef][Medline]

Bowdish DM, Davidson DJ, Scott MG, and Hancock RE (2005) Immunomodulatory activities of small host defense peptides. Antimicrob Agents Chemother 49: 1727-1732.[Abstract/Free Full Text]

Bucki R, Pastore JJ, Randhawa P, Vegners R, Weiner DJ, and Janmey PA (2004) Antibacterial activities of rhodamine B-conjugated gelsolin-derived peptides compared to those of the antimicrobial peptides cathelicidin LL37, magainin II and melittin. Antimicrob Agents Chemother 48: 1526-1533.[Abstract/Free Full Text]

Elssner A, Duncan M, Gavrilin M, and Wewers MD (2004) A novel P2X₇ receptor activator, the human cathelicidin-derived peptide LL37, induces IL-1 processing and release. J Immunol 172: 4987-4994.[Abstract/Free Full Text]

Grynkiewicz G, Poenie M, and Tsien RY (1985) A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440-3450.[Abstract/Free Full Text]

Koczulla R, von Degenfeld G, Kupatt C, Krötz F, Zahler S, Gloe T, Issbrücker K, Unterberger P, Zaiou M, Lebherz C, et al. (2003) An angiogenic role for the human peptide antibiotic LL-37/hCAP-18. J Clin Investig 111: 65-1672.

Kurosaka K, Chen Q, Yarovinsky F, Oppenheim JJ, and Yang D (2005) Mouse cathelin-related antimicrobial peptide chemoattracts leukocytes using formyl peptide receptor-like 1/mouse formyl peptide receptor-like 2 as the receptor and acts as an immune adjuvant. J Immunol 174: 6257-6265.[Abstract/Free Full Text]

Lau YE, Bowdish DM, Cosseau CC, Hancock RE, and Davidson DJ (2006) Apoptosis of airway epithelial cells: human serum sensitive induction by the cathelicidin LL-37. Am J Respir Cell Mol Biol 34: 399-409.[Abstract/Free Full Text]

Lau YE, Rozek A, Scott MG, Goosney DL, Davidson DJ, and Hancock RE (2005) Interaction and cellular localization of the human host defense peptide LL-37 with lung epithelial cells. Infect Immun 73: 583-591.[Abstract/Free Full Text]

Murakami M, Ohtake T, Dorschner RA, and Gallo RL (2002) Cathelicidin antimicrobial peptides are expressed in salivary glands and saliva. J Dent Res 81: 845-850.[Abstract/Free Full Text]

Neufeld HA, Towner RD, and Pace J (1975) A rapid method for determining ATP by the firefly luciferin-luciferase system. Experientia 31: 391-392.[CrossRef][Medline]

Niyonsaba F, Iwabuchi K, Someya A, Hirata M, Matsuda H, Ogawa H, and Nagaoka I (2002) A cathelicidin family of human antibacterial peptide LL-37 induces mast cell chemotaxis. Immunology 106: 20-26.[CrossRef][Medline]

Niyonsaba F, Someya A, Hirata M, Ogawa H, and Nagaoka I (2001) Evaluation of the effects of peptide antibiotics human -defensins-1/-2 and LL-37 on histamine release and prostaglandin D₂ production from mast cells. Eur J Immunol 31: 1066-1075.[CrossRef][Medline]

North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82: 1013-1067.[Abstract/Free Full Text]

Oren Z, Lerman JC, Gudmundsson GH, Agerberth B, and Shai Y (1999) Structure and organization of the human antimicrobial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem J 341: 501-513.[CrossRef][Medline]

Panyutich A, Shi J, Boutz PL, Zhao C, and Ganz T (1997) Porcine polymorphonuclear leukocytes generate extracellular microbicidal activity by elastase-mediated activation of secreted proprotegrins. Infect Immun 65: 978-985.[Abstract]

Pestonjamasp VK, Huttner KH, and Gallo RL (2001) Processing site and gene structure for the murine antimicrobial peptide CRAMP. Peptides 22: 1643-1650.[CrossRef][Medline]

Perregaux DG, Bhavsar K, Contillo L, Shi J, and Gabel CA (2002) Antimicrobial peptides initiate IL-1 posttranslational processing: a novel role beyond innate immunity. J Immunol 168: 3024-3032.[Abstract/Free Full Text]

Pochet S, Métioui M, Grosfils K, Gomez-Munoz A, Marino A, and Dehaye JP (2003) Regulation of phospholipase D by muscarinic receptors in rat submandibular ductal cells. Cell Signal 15: 103-113.[CrossRef][Medline]

Rao NM (1992) Differential susceptibility of phosphatidylcholine small unilamellar vesicles to phospholipases A₂, C and D in the presence of membrane active peptides. Biochem Biophys Res Commun 182: 682-688.[CrossRef][Medline]

Ritonja A, Kopitar M, Jerala RA, and Turk V (1989) Primary structure of a new cysteine proteinase inhibitor from pig leucocytes. FEBS Lett 255: 211-214.[CrossRef][Medline]

Saini SS, Chopra AK, and Peterson JW (1999) Melittin activates endogenous phospholipase D during cytolysis of human monocytic leukemia cells. Toxicon 37: 1605-1619.[Medline]

Scandinavian Society for Clinical Chemistry and Clinical Physiology (1974) Recommended methods for the determination of four enzymes in blood. Scand J Clin Lab Investig 33: 291-306.[Medline]

Solle M, Labasi J, Perregaux DG, Stam E, Petrushova N, Koller BH, Griffiths RJ, and Gabel CA (2001) Altered cytokine production in mice lacking P2X₇ receptors. J Biol Chem 276: 125-132.[Abstract/Free Full Text]

Sorensen OE, Follin P, Johnsen AH, Calafat J, Tjabringa GS, Hiemstra PS, and Borregaard N (2001) Human cathelicidin hCAP-18 is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3. Blood 97: 3951-3959.[Abstract/Free Full Text]

Tjabringa GS, Aarbiou J, Ninaber DK, Drijfhout JW, Sorensen OE, Borregaard N, Rabe KF, and Hiemstra PS (2003) The antimicrobial peptide LL-37 activates innate immunity at the airway epithelial surface by transactivation of the epidermal growth factor receptor. J Immunol 171: 6690-6696.[Abstract/Free Full Text]

Tokumaru S, Sayama K, Shirakata Y, Komatsuzawa H, Ouhara K, Hanakawa Y, Yahata Y, Dai X, Tohyama M, Nagai H, et al. (2005) Induction of keratinocyte migration via transactivation of the epidermal growth factor receptor by the antimicrobial peptide LL-37. J Immunol 175: 4662-4668.[Abstract/Free Full Text]

Van Laethem F, Liang X, Andris F, Urbain J, Vandenbranden M, Ruysschaert JM, Resh MD, Stulnig TM, and Leo O (2003) Glucocorticoids alter the lipid and protein composition of membrane rafts of a murine T cell hybridoma. J Immunol 170: 2932-2939.[Abstract/Free Full Text]

Van Nieuw Amerongen A, Bolscher JG, and Veerman EC (2004) Salivary proteins: protective and diagnostic value in cariology? Caries Res 38: 247-253.[CrossRef][Medline]

Virginio C, Church D, North RA, and Surprenant A (1997) Effects of divalent cations, protons and calmidazolium at the rat P2X₇ receptor. Neuropharmacology 36: 1285-1294.[CrossRef][Medline]

Woo J-S, Jeong JY, Hwang YJ, Chae SW, Hwang SJ, and Lee HM (2003) Expression of cathelicidin in human salivary glands. Arch Otolaryngol Head Neck Surg 129: 211-214.[Abstract/Free Full Text]

Yang D, Chen Q, Schmidt AP, Anderson GM, Wang JM, Wooters J, Oppenheim JJ, and Chertov O (2000) LL-37, the neutrophil granule- and epithelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) as a receptor to chemoattract human peripheral blood neutrophils, monocytes and T cells. J Exp Med 192: 1069-1074.[Abstract/Free Full Text]

Yeaman MR and Yount NY (2003) Mechanisms of antimicrobial peptide action and resistance. Pharmacol Rev 55: 27-55.[Abstract/Free Full Text]

Zhao H and Kinnunen PKJ (2003) Modulation of the activity of secretory phospholipase A₂ by antimicrobial peptides. Antimicrob Agents Chemother 47: 965-971.[Abstract/Free Full Text]

Zughaier SM, Shafer WM, and Stephens DS (2005) Antimicrobial peptides and endotoxin inhibit cytokine and nitric oxide release but amplify respiratory burst response in human and murine macrophages. Cell Microbiol 7: 1251-1262.[CrossRef][Medline]

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