Epithelial cell-derived human ß-defensin-2 acts as a chemotaxin for mast cells through a pertussis toxin-sensitive and phospholipase C-dependent pathway
François Niyonsaba1,
Kazuhisa Iwabuchi1,
Hiroshi Matsuda3,
Hideoki Ogawa2 and
Isao Nagaoka1
Departments of 1 Biochemistry and 2 Dermatology, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan 3 Department of Veterinary Clinic, Faculty of Agriculture, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
Correspondence to: I. Nagaoka; E-mail: nagaokai{at}med.juntendo.ac.jp
Transmitting editor: T. Taniguchi
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Abstract
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Mast cells are known to accumulate at the sites of inflammation in response to chemoattractants generated in the local milieu. Since human ß-defensin-2 (hBD-2) is generated in several epithelial tissues where mast cells are present and because we have recently reported that this human antibacterial peptide induces mast cell degranulation, we thus hypothesized that hBD-2 could be a mast cell chemotaxin. Here we report that hBD-2 directly and specifically induces mast cell migration with an optimal concentration of 3 µg/ml. Checkerboard analysis showed that the migration was more chemotactic rather than chemokinetic. Moreover, Scatchard analysis using 125I-labeled hBD-2 revealed that mast cells have at least two classes of receptors, high- and low-affinity receptors, for this peptide. Moreover, the competitive binding assay suggested that hBD-2 is unlikely to utilize CCR6, a functional receptor for hBD-2-mediated dendritic and T cell migration, on mast cells. In addition, treatment of mast cells with G protein inhibitor, pertussis toxin, and phospholipase C inhibitor, U-73122, abolished the cell chemotaxis in response to hBD-2, indicating that the G proteinphospholipase C signaling pathway is involved in hBD-2-induced mast cell activation. Thus, we suggest that hBD-2, which was originally believed to be involved in innate host defense, may participate in the recruitment of mast cells to inflammation foci.
Keywords: antibacterial peptide, defensin, chemotaxis, G protein-coupled receptor
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Introduction
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Antimicrobial peptides are found in insects, plants and mammals where they exhibit antibacterial activities against both Gram-positive and Gram-negative bacteria, fungi, and viruses (13). In humans, over a dozen of these peptides have been identified, including salivary histatins, granulysin, lactoferricin,
- and ß-defensins, and human cathelicidin hCAP18 (human cationic antibacterial protein of 18 kDa)-derived LL-37 (1). The major family of human antibacterial peptides that has been well characterized is defensins. The human defensins include
- and ß-defensins that differ with respect to the placement and connectivity of six conserved cysteine residues, the structures of their precursors, and their patterns of expression (1,3). In contrast to
-defensins that are produced by neutrophils and intestinal Paneth cells, ß-defensins are mainly expressed in epithelial tissues (4,5). Members of human ß-defensins (hBDs) include hBD-1, hBD-2 and hBD-3. First, hBD-1 was purified as a trace peptide from human hemofiltrate obtained from patients with end-stage renal diseases, and is constitutively expressed in the skin, airway epithelium and genitourinary tracts (68). hBD-2, as well as hBD-3, was isolated from extracts of lesional scales from patients with psoriasis. These two basic polypeptides are inducibly expressed in inflamed skin and lung epithelium upon treatment with bacterial lipopolysaccharide, and proinflammatory cytokines such as tumor necrosis factor (TNF)-
and IL-1ß (912).
Mast cells are normally distributed throughout connective tissues and their numbers are relatively constant. However, mast cell numbers increase at local tissues under various conditions such as wound healing, tumors, and inflammatory and allergic diseases including asthma, allergic rhinitis and rheumatoid arthritis (1316). Although the specific homing mechanisms that lead to mast cell recruitment are poorly understood, directed migration of mast cells within tissues may be an important mechanism for rapidly increasing local mast cell numbers. This hypothesis has been supported by the finding that nerve growth factor, stem cell factor, transforming growth factor-ß family, and the anaphylatoxins C3a and C5a, which are generated in the local milieu, are acting as mast cell chemoattractants (1720).
Recent investigations have revealed that besides their antibacterial activities by killing invading microorganisms, hBDs possess the ability to promote both innate and adaptive immune responses by inducing the chemotaxis of memory T cells and immature dendritic cells (21). However, there is no evidence of these peptides on mast cell migration.
Since hBDs are produced in the epithelial tissues where mast cells are present and because we have recently reported that hBD-2 can stimulate mast cell degranulation (22), we thus hypothesized that this peptide may also act as a mast cell chemotaxin. Supporting this hypothesis, here we provide the evidence that hBD-2 induces mast cell chemotaxis through specific receptor(s) coupled to G proteinphospholipase C (PLC) signaling.
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Methods
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Reagents
hBD-1, hBD-2 and hBD-2 antiserum were purchased from Peptide Institute (Osaka, Japan). U-73122 (1-[6-([(17ß)-3-methoxyestra-1,3,5(10)-trien-17-yl] amino)hexyl]-1H-pyrrole-2.5-dione) was obtained from Sigma (St Louis, MO). Pertussis toxin (PTx) was from List Biological (Campbell, CA) and Na125I from ICN Biomedicals (Irvine, CA). Rat recombinant macrophage inflammatory protein (MIP)-3
and mouse recombinant TNF-
that is active in both mouse and rat systems were obtained from Genzyme/Techne (Cambridge, MA).
Preparation of rat mast cells and neutrophils
Mast cells were obtained from male Sprague-Dawley rats weighing 300400 g by lavage of the peritoneal cavity as described by Sawada et al. (17). Briefly, rats were injected i.p. with PBS supplemented with 0.1% BSA and the peritoneal lavage fluids were recovered after abdominal massage for 3 min. After centrifugation, cells were suspended in 10 ml of modified Eagles medium (MEM) with 10% FCS, layered on 15 ml 75% Percoll solution (Pharmacia Biotech, Uppsala, Sweden) and further centrifuged at 600 g for 25 min at room temperature. Purified mast cells (>95% purity and 99% viability) were washed twice and resuspended at a concentration of 1 x 105 cells/ml in MEM containing 1% BSA, 50 IU/ml penicillin and 50 µg/ml streptomycin (assay medium). Neutrophils were isolated from rat peritoneal cavities 1315 h after i.p. injection of 0.17% glycogen in saline as described previously (23). The purity of isolated neutrophils was >90%.
Chemotaxis assay
Various concentrations of hBD-2 (500 µl) or the assay medium alone were applied into each well of 24-well culture plates. Then, a 10-mm tissue culture insert with an 8-µm pore size polycarbonate membrane (Nalge Nunc, Roskilde, Denmark) was placed into each well and 5 x 104 cells (500 µl) were added into the insert. Cells were incubated for 3 h at 37°C in 5% CO2 atmosphere. Following aspiration of non-adherent cells from the insert, cells adherent to the upper surface of the membrane were removed by scraping with a cotton bud. Migrated cells on the lower surface of the membrane were fixed with methanol for 5 min and stained with 0.1% toluidine blue. The membranes were mounted on glass slides by routine histological methods. The total number of mast cells that migrated across the membrane was counted under a light microscope. In some experiments hBD-2 antiserum was added to the chemoattractant in the lower compartment and incubated for 30 min before addition of cells into the insert. For the chemotaxis of neutrophils towards MIP-3
, cells at the concentration of 5 x 106 cells/ml were cultured for 6 h in RPMI 1640 supplemented with 10% FCS in the absence or presence of 2.5 ng/ml TNF-
(26). After incubation, cells were washed 3 times and then added to the upper wells of a 48-well microchemotaxis chamber (Neuroprobe, Cabin John, MD). The upper wells of the chamber and lower wells containing chemoattractants were separated by a polycarbonate membrane with 3-µm diameter pores. The number of migrated cells during a 60-min incubation was counted under a light microscope.
Treatment with PTx and U-73122
The effects of PTx and U-73122 were investigated by pretreating mast cells (2 x 105 cells/ml) with various concentrations of PTx (25200 ng/ml) in assay medium for 2 h or U-73122 (0.011 µM) for 1 h at 37°C. Cells were then washed twice and resuspended at 1 x 105 cells/ml in the same medium before chemotaxis assay.
Iodination of hBD-2 and MIP-3
hBD-2 and MIP-3
were iodinated with 1 and 0.5 mCi Na125I (100 mCi/ml) respectively using Iodo-Beads (Pierce, Rockford, IL) at room temperature for 15 min. The 125I-labeled hBD-2 or 125I-labeled MIP-3
was separated from free Na125I by gel filtration through a Sephadex G-10 column (Amersham Pharmacia) equilibrated with 0.01% acetic acid. The sp. act. of the labeled material was 80 Ci/mmol for hBD-2 and 1075 Ci/mmol for MIP-3
. In preliminary experiments, we confirmed that the migration of mast cells to labeled hBD-2 or neutrophils to MIP-3
was the same as that to unlabeled peptide (data not shown), indicating these chemoattractants were not degraded during the labeling procedure.
Binding of [125I]hBD-2 to mast cells
Aliquots of 50 µl of mast cell suspensions (2 x 106 cells/ml) were incubated with 2 µl of labeled hBD-2 in 100 µl MEM containing 1% heat-inactivated BSA for 30 min at 25°C. The final concentration of [125I]hBD-2 was 1.10 x 1010 to 5.56 x 108 M. After incubation, the mixture was layered over 500 µl 10% sucrose/PBS in a 1.5 ml polypropylene tube and then centrifuged at 400 g for 5 min at 4°C. After aspirating the supernatant, the tube was cut 23 mm above the cell pellet and the cell-associated [125I]hBD-2 was counted using a
-counter (Pharmacia; model 1270 Rack Gamma II). Non-specific binding was determined in parallel experiments in the presence of a 100-fold excess of unlabeled peptide. Specific binding was calculated by subtracting the non-specific binding from total counts. The data were curve fitted with the computer program LIGAND to determine the affinity and number of binding sites as has been described previously (24). For the binding of [125I]MIP-3
to neutrophils, 50 µl of TNF-
(2.5 ng/ml, 6 h)-pretreated cell suspensions (2 x 106 cells/ml) was incubated with [125I]MIP-3
(6 x 1010 M) in 100 µl RPMI 1640 containing 1% heat-inactivated BSA, for 30 min at 25°C, and the assay was performed as described above for hBD-2. In some experiments, mast cells (1 x 106 cells/ml) were incubated in 100 µl MEM containing 1% heat-inactivated BSA with 6 x 109 M labeled hBD-2 for 30 min at 25°C in the presence of various amounts (from 2.4 to 620 nM) of unlabeled hBD-1, hBD-2 or MIP-3
to evaluate the competition of binding with [125I]hBD-2. The number of bound ligands was determined as described above.
Statistical analysis
Statistical analysis was performed using ANOVA and P < 0.05 was considered to be significant. The results are shown as mean ± SD.
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Results
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hBD-2-induced mast cell migration
We first evaluated the abilities of hBD-2 to induce the migration of mast cells. As shown in Fig. 1, when various concentrations of hBD-2 were applied into the lower compartment, mast cells migrated towards this antibacterial peptide. The dose-dependence of mast cell migration to hBD-2 exhibited a bell-shaped curve with higher concentrations resulting in a loss of migration. The optimal concentration of hBD-2 for the maximal migration of mast cells was 3 µg/ml. The medium alone without stimuli had no effect on migration of mast cells. In contrast to hBD-2, hBD-1 that did not induce histamine release and prostaglandin D2 production from mast cells (22), was unable to chemoattract mast cells at various concentrations tested (120 µg/ml).

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Fig. 1. Mast cell migration in response to hBD-2. Mast cells (5 x 104 cells/500 µl) placed in the culture inserts (upper compartment) were allowed to migrate towards 120 µg/ml hBD-1 (circles) or 110 µg/ml hBD-2 (diamonds) in each well of 24-well culture plates (lower compartment) for 3 h at 37°C. Mast cell migration was assessed by counting the number of cells through the polycarbonate membrane. The spontaneous cell migration (medium alone without peptide) was 20.2 ± 6.9 cells. Each bar represents the mean ± SD of four separate experiments.
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Checkerboard analysis of hBD-2-induced mast cell migration
We conducted experiments to determine whether the mast cell migratory response induced by hBD-2 is due to directional (chemotaxis) or random (chemokinesis) activation. As shown in Table 1, checkerboard analysis with various concentrations of hBD-2 in the lower compartments demonstrated the gradient-dependent migration of mast cells. The presence of peptide in only the upper compartment did not induce any substantial increase of cell migration. However, a slight dose-dependent migration with a bell-shaped curve was observed when the same concentrations of hBD-2 were present in both the upper and lower chambers. Thus, we concluded that hBD-2 induced predominant chemotaxis and slight chemokinesis of mast cells.
Specificity of hBD-2-induced chemotaxis
To determine the specific effect of hBD-2 on mast cell chemotaxis, hBD-2 antiserum was added to the assay medium containing the optimal dose of hBD-2 (3 µg/ml) in the lower compartment (Fig. 2). The addition of 100-fold diluted hBD-2 antiserum abolished the hBD-2-induced chemotaxis, whereas rabbit control serum had no effect on it.

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Fig. 2. Inhibition of hBD-2-induced mast cell chemotaxis by hBD-2 antiserum. Mast cells were allowed to migrate towards 3 µg/ml hBD-2 after hBD-2 antiserum (1:100 dilution) was added to the chemoattractant in the lower compartment. Supernatants from the mast cell mixtures were obtained after incubation of mast cells with 3 µg/ml hBD-2 for 3 h at 37°C, whereas the supernatants from the lower chamber were obtained by centrifuging the lower chamber contents after a 3-h chemotaxis assay. These supernatants were further applied in the lower chamber to chemoattract mast cells in the absence or presence of specific antiserum. Values are compared between the presence and absence of hBD-2 antiserum. **P < 0.005. Each bar represents the mean ± SD of three separate experiments.
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It is possible that hBD-2 may trigger the production of some chemotactic agents that may induce chemotaxis of mast cells. Thus, we obtained the supernatants from the mast cell mixtures stimulated by hBD-2 for 3 h at 37°C or supernatants from the lower chambers after mast cell chemotaxis to this peptide and tested their chemotactic activities against mast cells in the presence or absence of hBD-2 antiserum. As shown in Fig. 2, the presence of hBD-2 antiserum almost completely abolished mast cell migration towards these hBD-2-containing supernatants, indicating that hBD-2 acts directly and specifically as a mast cell chemotaxin.
Effects of PTx and U-73122 on hBD-2-induced chemotaxis
To speculate the signaling pathway for hBD-2, we examined the effects of PTx, a reagent known to selectively interfere with signals mediated through Gi-type G proteins, on hBD-2-induced mast cell migration. Treatment of mast cells with various concentrations of PTx inhibited hBD-2-induced mast cell migration in a dose-dependent fashion (Fig. 3), suggesting that hBD-2 utilizes Gi protein-coupled receptors to activate mast cell migration.

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Fig. 3. Effects of PTx and U-73122 on hBD-2-induced chemotaxis. Mast cells were pretreated with PTx for 2 h or U-73122 for 1 h at 37°C at the indicated concentrations before performing the chemotaxis assay using 3 µg/ml hBD-2 or medium alone (without peptide). Values are compared between without and with PTx- or U-73122-treatment. **P < 0.005. Values are the mean ± SD of three separate experiments.
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Next, we investigated the possible involvement of PLC in the migration of mast cells to hBD-2. Pretreatment of mast cells with a PLC inhibitor, U-73122, dose-dependently inhibited the subsequent migration of mast cells in response to hBD-2 (Fig. 3). Together these observations suggest the involvement of G proteinPLC pathway in the actions of hBD-2 on mast cell migration.
Scatchard analysis and binding interactions of mast cells to hBD-2
To clarify whether mast cells have specific receptors (binding sites) for hBD-2 and to characterize those receptors, we performed Scatchard analysis. As seen in Fig. 4, the binding of 125I-labeled hBD-2 to mast cells indicates that there are at least two types of receptors on mast cells: high-affinity receptors with a Kd of 0.236 ± 0.001 µM and a density of 1468 ± 11 sites/cell, and low-affinity receptors with a Kd of 32.481 ± 0.207 µM and a density of 15,253 ± 651 sites/cell (mean ± SD of three experiments).

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Fig. 4. Scatchard plot analysis of hBD-2 binding to mast cells. Specific binding of [125I]hBD-2 to mast cells was determined as described in Methods. Data were analyzed in terms of a two-site model using a modified LIGAND program (24). Solid lines were predicted by linear regression of the data, and used for the calculation of Kd values and the numbers of receptors (binding sites), and broken lines represent the computerized curvilinear best fits for a two-site model. Each point is the average of two determinations in a single experiment. Data are representative of three to five separate experiments.
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Competitive binding of hBD-2 to mast cells
Recently, hBD-1 and hBD-2 were reported to utilize CCR6 as a functional receptor on dendritic and T cells (21). To test whether CCR6 is acting as the receptor of hBDs on mast cells, we performed competition binding assays between [125I]hBD-2 and unlabeled MIP-3
(LARC), a sole chemokine ligand for CCR6 (25). As can be seen in Fig. 5, unlabeled hBD-2 could dose-dependently inhibit the binding of [125I]hBD-2 to mast cells, whereas unlabeled MIP-3
could not significantly displace the binding of iodinated hBD-2; a 100-fold excess of unlabeled MIP-3
decreased the binding of [125I]hBD-2 at <5%. Furthermore, we demonstrated that hBD-1, which could not induce mast cell migration (Fig. 1), was unable to compete with [125I]hBD-2. Moreover, we revealed that [125I]MIP-3
could not specifically bind to mast cells (data not shown). In separate experiments, we tested whether MIP-3
was active and thus could bind to CCR6 by using TNF-
-activated rat neutrophils, because TNF-
is reported to increase the expression of CCR6 on neutrophils (26). The results showed that TNF-
-activated but not untreated neutrophils migrated towards MIP-3
and [125I]MIP-3
could specifically bind to these cells (data not shown). In addition, the binding of MIP-3
to neutrophils was competed by the addition of 100-fold excess of hBD-2 (data not shown). These results suggest that MIP-3
used in this study was active and could bind to CCR6 on rat neutrophils, and that hBD-2 utilizes CCR6 as a receptor as shown by D. Yang et al (21).
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Discussion
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Following the initiation of an inflammatory process, mast cell numbers increase at a specific site within hours. It is likely that directed migration of mast cells in tissues is an important mechanism for local mast cell accumulation in inflammation. The present work provides novel evidence that epithelial cell-derived peptide hBD-2 is a direct and specific chemoattractant for mast cells. This epithelial cell-derived antibacterial peptide mainly induced mast cell chemotaxis rather than chemokinesis and this mechanism involves the G proteinPLC pathway. In addition, we show that mast cells express at least high- and low-affinity receptors for hBD-2, which are distinct from CCR6.
Mast cell chemotactic factors have been detected during inflammation in most organs including skin and lungs where mast cells are present (27). In addition, epithelial tissue-derived antibacterial peptides have been found at high concentrations in those tissues. For example, hBD-2 is detected in airway surface liquid at
10 µg/ml in bronchoalveolar lavage liquid (28). Assuming that the expression of hBD-2 could be increased during inflammation, it could be speculated that hBD-2 may potentially reach its optimal chemotactic concentration at local inflammatory sites. Interestingly, in psoriatic skin where the expression of hBD-2 is significantly up-regulated (9), mast cell accumulation is observed and likewise is assumed to be implicated in the pathophysiology of the disease (13).
hBD-1 and hBD-2 have been shown to induce dendritic and T cell migration at similar potency and efficacy, and use CCR6 as a common receptor (21). However, we have recently reported that hBD-2 but not hBD-1 could stimulate mast cells to release histamine, mobilize intracellular Ca2+ or generate prostaglandin D2 (22). Moreover, the present study showed that only hBD-2 could chemoattract mast cells. Thus, we speculate that the receptor for hBD-2 may not be CCR6 on mast cells. This was approved by the finding that neither hBD-1 nor MIP-3
, the sole CCR6 ligand, could not compete the hBD-2 binding to mast cells. In addition, we observed that MIP-3
could neither induce Ca2+ mobilization nor chemotaxis for mast cells at various concentrations tested (0.055 µg/ml) (data not shown). Furthermore, the fact that [125I]MIP-3
was unable to bind to mast cells also supported the possibility that mast cells do not express CCR6 as a functional receptor.
In this study, the binding assay indicated the presence of two classes of mast cell receptors for hBD-2: high- and low-affinity receptors with Kd values of 0.236 ± 0.001 and 32.481 ± 0.207 µM respectively. Furthermore, hBD-2 induced a maximal migration of mast cells at 3 µg/ml (equivalent to 0.7 µM), which was approximate to the Kd value of receptors detected on mast cells. Thus, these data support our hypothesis that mast cells may possess specific receptors for hBD-2. Further investigations will be necessary to identify these specific receptors.
The present study revealed that hBD-2 serves as a potent chemoattractant for mast cells via specific receptors on this cell population, and likely suggests an important link between epithelial cell-derived antibacterial peptides and mast cells. Thus, mast cell migration towards antibacterial peptide hBD-2, which was originally believed to be involved in innate host defense, appears to represent a novel mechanism in the recruitment of mast cells to inflammation foci and may propose a role for human antibacterial peptides in inflammatory response.
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Acknowledgements
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We wish to thank Dr A. Itakura (Tokyo University of Agriculture and Technology) for her kind advice in performing the chemotaxis assay. This work was supported in part by grants from the Atopy (Allergy) Research Center, Juntendo University, Tokyo, Japan, and the Ministry of Education, Culture, Sports, Science and Technology, Tokyo, Japan.
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Abbreviations
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hBDhuman ß-defensin
MEMmodified Eagles medium
MIPmacrophage inflammatory protein
PLCphospholipase C
PTxpertussis toxin
TNFtumor necrosis factor
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