antimicrobial peptides human β-defensin (hbd)-3 and hbd-4 activate mast cells and increase skin...

Antimicrobial peptides human b-defensin (hBD)-3 andhBD-4 activate mast cells and increase skin vascularpermeability

Xuejun Chen1,2,3, Fran�ois Niyonsaba2, Hiroko Ushio2, Mutsuko Hara2, Hidenori Yokoi4,Kenji Matsumoto5, Hirohisa Saito5,6, Isao Nagaoka7, Shigaku Ikeda1, Ko Okumura2,8 andHideoki Ogawa2

1 Department of Dermatology, Juntendo University School of Medicine, Tokyo, Japan2 Department of Atopy (Allergy) Research Center, Juntendo University School of Medicine, Tokyo, Japan3 Department of Dermatology, Sichuan Provincial People's Hospital, Chengdu, China4 Department of Otorhinolaryngology, Juntendo University School of Medicine, Tokyo, Japan5 Department of Allergy and Immunology, National Research Institute for Child Healthy and Development, Tokyo,Japan

6 Laboratory of Allergy Transcriptome, Research Center for Allergy and Immunology, RIKEN Yokohama Institute,Yokohama, Japan

7 Department of Host Defense and Biochemical Research, Juntendo University School of Medicine, Tokyo, Japan8 Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan

Antimicrobial peptides human b-defensins (hBD) are mainly produced by epithelia ofseveral organs including skin, and participate in innate immunity by killing invadingpathogens. Besides their microbicidal activities, hBD activate several inflammatory andimmune cells. Since hBD are generated by tissues where mast cells are present, wehypothesized that these peptides could activate mast cells. In this study, wedemonstrated that both hBD-3 and hBD-4 induced mast cell degranulation,prostaglandin D2 production, intracellular Ca2+ mobilization and chemotaxis.Furthermore, hBD-3- and hBD-4-induced activation of mast cells was suppressed bypertussis toxin and U-73122, inhibitors for G protein and phospholipase C, respectively.We further revealed that hBD-3 and hBD-4 increased vascular permeability in the skin,which was dependent on the presence of mast cells, because hBD-3 and hBD-4 failed toenhance vascular permeability in mast cell-deficientWs/Ws rats. We also demonstratedthat hBD-3 and hBD-4 induced phosphorylation of MAPK p38 and ERK1/2, which werefurther required for hBD-mediated mast cell activation, as evidenced by the inhibitoryeffects of p38 and ERK1/2 inhibitors on mast cell degranulation. Together, thesefindings suggest the key role of hBD in inflammatory responses by recruiting andactivating mast cells, and increasing vascular permeability.

Introduction

Antimicrobial peptides are important components of theinnate host defense system against bacteria, viruses andfungi. In humans, these peptides include a- andb-defensins, and cathelicidins. Human a-defensins arefound in neutrophils and intestinal Paneth cells, whereas

Innate immunity

Correspondence: Fran�ois Niyonsaba, Atopy (Allergy) Re-search Center, Juntendo University School of Medicine, 2-1-1Hongo, Bunkyo-ku, Tokyo 113–8421, JapanFax: +81-3-3813-5512e-mail: [email protected]

Received 14/6/06Revised 9/11/06

Accepted 12/12/06

[DOI 10.1002/eji.200636379]

Key words:Antimicrobial peptide� Degranulation � Mast

cell � Migration� Vascular perme-

ability

Abbreviations: BSSA: balanced salt solution containing BSA �[Ca2+]i: intracellular Ca2+ concentration � hBD: humanb-defensin � PGD2: prostaglandin D2 � PLC: phospholipase C �PTx: pertussis toxin

Xuejun Chen et al. Eur. J. Immunol. 2007. 37: 434–444434

f 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.eji-journal.eu

human b-defensins (hBD) are mainly produced byepithelial cells of several organs [1]. Among four hBDidentified in epithelium so far, hBD-1 is generallyconstitutively produced by various epithelial tissuessuch as urogenital and respiratory tracts, and skin [2],whereas the expressions of hBD-2, hBD-3 and hBD-4 areinducible. hBD-2, originally isolated from psoriatic skin,is mainly present in skin, respiratory as well asgastrointestinal tracts [3], and is induced in inflamedskin lesions upon exposure to bacteria and cytokinessuch as TNF-a and IL-1b [3–5]. hBD-3 was discovered byisolation from psoriatic lesion scales and screeninghuman genomic sequences [6, 7]. This peptide isexpressed in epithelia of the respiratory, gastrointestinaland urogenital tracts [6], and keratinocytes [8, 9].Interestingly, hBD-3 is also found in non-epithelialtissues such as tonsils, placenta, adult heart, skeletalmuscle and fetal thymus [7]. Similar to hBD-2, it hasbeen demonstrated that TNF-a, IL-1b and some bacteriainduce the expression of hBD-3 in keratinocytes andtracheal epithelium [6, 7]. As for hBD-4, it has beendetected in the testis, gastric antrum and in the epitheliaof thyroid gland, lung, uterus and kidney [10].Furthermore, hBD-4 gene expression is induced inhuman respiratory epithelial cells upon stimulation withheat-inactivated P. aeruginosa, Staphylococcus pneumo-niae and PMA [10], and in human primary keratinocytesupon exposure to bacteria and inflammatory cytokinessuch as IL-1b, IFN-c or TNF-a [9].

In addition to their microbicidal abilities, antimicro-bial peptides are multifunctional as mediators ofinflammation with the effects on epithelial andinflammatory cells, influencing cell proliferation,wound healing, cytokine/chemokine production andchemotaxis (reviewed in [11]). For instance, humana-defensins activate monocytes and T cells, and inducemast cell degranulation [12, 13]. Moreover, hBD-2 andhuman cathelicidin LL-37, another antimicrobial peptidegenerated by epithelial cells and neutrophils, have beenreported to participate in innate immunity by activatingand recruiting innate and inflammatory cells such asneutrophils, monocytes, T cells, keratinocytes and mastcells [14–18].

Mast cells are also important effector cells of theinnate immunity. They are particularly abundant inareas of close proximity to epithelial surfaces where theyplay a crucial role in allergy, inflammation and immunesurveillance mechanisms, and their products are pivotalin inflammatory reactions [19]. Upon activation, mastcells release inflammatory mediators including hista-mine, prostaglandin D2 (PGD2) and leukotrienes, whichare known to increase vascular permeability [20].Althoughmast cells are normally distributed throughoutconnective tissues under relatively constant numbers,their numbers increase in local tissues under various

conditions such as in wound healing and in inflamma-tory and allergic diseases [21–23]. The stimulation andrecruitment of mast cells has been attributed to a rangeof stimuli including antimicrobial peptides hBD-2 andLL-37 [17, 18, 24]. However, there is no information onthe effects of hBD-3 and hBD-4 on mast cell activation,and their role in skin inflammatory responses.

Given that hBD are highly expressed in keratinocytesin wounds [8], and in respiratory epithelia [10], wheremast cells have been found to play important roles, wepostulated that these peptides would activate mast cells.Supporting our hypothesis, the current study providedthe evidence that both hBD-3 and hBD-4 induced mastcell chemotaxis, degranulation and PGD2 production,and these activities were via G protein-phospholipase(PL) C-coupled signaling pathways. Moreover, wedemonstrated that hBD-3 and hBD-4 induced thephosphorylation of MAPK p38 and ERK1/2, which werefurther required for mast cell degranulation. Finally, weshowed that the increase of vascular permeability in theskin caused by both hBD-3 and hBD-4 was completelydependent on the presence of mast cells, because thesepeptides failed to enhance vascular permeability in mastcell-deficient rats. Thus, besides their microbicidalproperties, hBD may participate in inflammatoryresponses through recruiting and activating mast cells,and increasing vascular permeability in the skin.

Results

Evaluation of the effects of hBD-3 and hBD-4 onmast cell degranulation

To determine whether hBD-3 and hBD-4 could stimulatemast cell degranulation, we first evaluated the abilitiesof these peptides to induce b-hexosaminidase release,because this enzyme is released in parallel withhistamine, and therefore is a marker of mast celldegranulation [25]. As shown on Fig. 1A and B, bothhBD-3 and hBD-4 induced b-hexosaminidase releasefrom rat peritoneal mast cells in a dose-dependentfashion, with hBD-3 being more potent than hBD-4.Mast cell degranulating abilities of hBD were alsoobserved in a human mast cell line LAD2, which isdescribe positive for histamine, tryptase and chymase,and has functional FceRI and FccRI receptors [26]. Wefound that both hBD-3 and hBD-4 displayed dose-dependent degranulating activities for LAD2 cells. Atconcentrations of 5 lg/mL, hBD-3 almost caused thecomplete degranulation of human mast cells (Fig. 1C),whereas concentrations as high as 20 lg/mL wererequired for hBD-4 to induce approximately 30% ofmastcell degranulation (Fig. 1D). Unpublished observationsshowed that hBD-2, which has been previously demon-

Eur. J. Immunol. 2007. 37: 434–444 Innate immunity 435


strated to induce the histamine release from mast cells[17] was more potent than both hBD-3 and hBD-4.

hBD-3 and hBD-4 induce PGD2 production

Upon stimulation, mast cells release histamine and PGD2

[20]. Since both hBD-3 and hBD-4 induced b-hexos-aminidase release, we hypothesized that these peptidescould also stimulate mast cells to generate PGD2. Werevealed that hBD-3- and hBD-4-stimulated mast cellssignificantly produced PGD2. Fig. 2A shows that PGD2

production was dependent on the increasing doses ofhBD-3 (5–40 lg/mL), whereas the production reached amaximum at 20 lg/mL hBD-4 before decreasing(Fig. 2B). We verified that longer incubation (3–12 h)of mast cells with hBD-3 or hBD-4 did not furtherincrease the amounts of PGD2 production. We furtherconfirmed that these peptides also induced PGD2

generation from LAD2 cells (data not shown).

Induction of intracellular Ca2+ mobilization byhBD-3 and hBD-4

It has been reported that Ca2+ is a common keyregulatory agent in the process of histamine release andPG biosynthesis [27, 28]. Therefore, because hBD-3 andhBD-4 induced mast cell b-hexosaminidase release andPGD2 generation, we next examined their ability to

mobilize intracellular Ca2+. After challenging mast cellswith indicated concentrations of hBD-3 and hBD-4,intracellular Ca2+ concentration ([Ca2+]i) rapidlyincreased and reached the maximum within 10–15 safter stimulation, and this effect was dose dependent(Fig. 3A, B). We observed that the increases in [Ca2+]ievoked by hBD-3 and hBD-4 were not statisticallydifferent. Thus, the amplitude of the [Ca2+]i riseselicited by hBD-3 and hBD-4 was not parallel to theirabilities to induce b-hexosaminidase release and PGD2

production, where hBD-3 was more potent than hBD-4.

hBD-3 and hBD-4 induce mast cell chemotaxis

The activities of hBD-3 and hBD-4 on mast cells werefurther investigated by analyzing their ability to inducemast cell migration. As shown in Fig. 4A and B, differentconcentrations of hBD-3 and hBD-4 applied into thelower compartments of chemotaxis chamber resulted inmast cell migration. We observed that the dosedependence of mast cell migration towards hBD-3exhibited a bell-shaped curve (Fig. 4A), while hBD-4-stimulated mast cells showed more increased migrationat higher doses of hBD-4 (Fig. 4B). Both hBD-3 andhBD-4 also exhibited migratory activities on humanmast cell LAD2. The highest migratory response of LAD2cells was observed at the optimal concentrations as lowas 2.5 lg/mL for hBD-3 (Fig. 4A), and 5 lg/mL forhBD-4 (Fig. 4B).

We further conducted a checkerboard analysis toaddress whether the hBD-3- and hBD-4-mediated mast

Figure 1. Mast cell degranulation in response to hBD-3 andhBD-4. Rat mast cells were incubated with indicated concen-trations (1–20 lg/mL) of hBD-3 (A) or hBD-4 (B), whereas humanmast cell line LAD2was stimulatedwith 1–5 lg/mL of hBD-3 (C)or 2.5–20 lg/mL of hBD-4 (D). After 40-min incubation at 37�C,b-hexosaminidase (b-hexo) in the supernatant was measuredas described in theMaterials andmethods. Values are themean�SD of four separate experiments, and are corrected forspontaneous release (3.47 � 1.8% for A and B, and5.15 � 2.1% for C and D).

Figure 2. hBD-3 and hBD-4 induce PGD2 production from mastcells. Cells were stimulated for 1 h at 37�C with hBD-3 (A) andhBD-4 (B) at indicated concentrations. PGD2 released in thesupernatants was quantitated by enzyme immunoassay andcorrected for spontaneous production, which was 185.4 � 0.8pg/mL. Values are mean � SD of four to five separateexperiments.



cell migration was due to chemotaxis or chemokinesis.As found in Table 1, the presence of peptides in only theupper compartments of chemotaxis chamber did notresult in any substantial increase of cell migration,suggesting that the effect was not chemokinetic.However, since a slight dose-dependent migration wasobserved when the identical concentrations of hBD-3 orhBD-4 were present in both upper and lower chambers,we cannot exclude the possibility of minor chemokineticeffect. Nevertheless, the overall results suggest that themigratory effect of hBD-3 and hBD-4 on mast cells waspredominantly chemotaxis rather than chemokinesis.

Effects of pertussis toxin and U-73122 on hBD-3-and hBD-4-induced mast cell activation

Antimicrobial peptides such as hBD-2 and LL-37 havebeen shown to activatemast cells through G protein- andPLC-sensitive pathways [17]. We therefore examinedthe involvement of G protein and PLC in hBD-3- andhBD-4-mediated mast cell activation by evaluating theeffects of pertussis toxin (PTx; inhibitor for G protein)and U-73122 (PLC inhibitor) onmast cell degranulation,PGD2 production, intracellular Ca2+ mobilization andchemotaxis. We observed that 200 ng/mL PTx and

Figure 3. Intracellular Ca2+mobilization induced by hBD-3 and hBD-4. Fura-2-AM-loadedmast cellswere stimulatedwith 1–20 lg/mL hBD-3 (A) or hBD-4 (B) at 37�C as described in theMaterials and methods. Data are representative of four separate experiments.Arrows indicate the addition of stimulant.

Table 1. Checkerboard analysis of hBD-3- and hBD-4-mediated mast cell migrationa)

In lowerchamber(lg/mL)

hBD-3/hBD-4 in upper chamber (lg/mL)

0 5 10 20 40

hBD-3

05102040

31 � 11152 � 38482 � 29996 � 43512 � 42

25 � 958 � 12114 � 19228 � 33102 � 39

42 � 743 � 997 � 22163 � 388 � 27

66 � 1338 � 1147 � 21147 � 1147 � 14

41 � 853 � 737 � 1548 � 333 � 8

hBD-4

05102040

36 � 762 � 16183 � 11381 � 21927 � 18

27 � 449 � 1465 � 899 �15273 � 15

38 � 262 � 1172 � 781 �12125 � 22

19 � 642 � 861 � 1389 � 8112 � 5

38 � 1144 � 953 � 1448 � 3103 � 16

a) hBD-3 or hBD-4 at indicated concentrations was added to the lower chamber and/or upper chamber containing mast cells(5 � 104 cells/500 lL). The migratory response was measured after a 3 h incubation. Each value represents the mean � SD ofthree to four separate experiments.



10 lM U-73122 significantly suppressed b-hexosamini-dase release induced by hBD-3 (Fig. 5Aa) and hBD-4(Fig. 5Ab). Moreover, PGD2 generation (Fig. 5B),intracellular Ca2+ mobilization (Fig. 5C), and chemo-taxis (Fig. 5D) induced by hBD-3 (a) or hBD-4 (b) weresimilarly inhibited by both PTx and U-73122. Theinactive analogue of U-73122, U-73343 did not affect thehBD-3- and hBD-4-mediated mast cell activation,demonstrating that the effects of U-73122 were specific.

Increase of vascular permeability in the skinby hBD-3 and hBD-4

Because both hBD-3 and hBD-4 induced mast celldegranulation and PGD2 generation, which increasevascular permeability [20], we examined the roles ofhBD-3 and hBD-4 in the acute cutaneous response byevaluating their ability to increase vascular permeabil-ity. The intradermal injection of 5–20 lg/mL(150–600 ng) of hBD-3 (Fig. 6A) and hBD-4 (Fig. 6B)dose dependently increased the skin vascular perme-ability, as evaluated by extravasation of Evans blue dyeat 30 min post injection. The skin reaction was evenobserved within 5 min of hBD injection, and gradually

increased (data not shown). To confirm whether mastcells were required for this acute skin reaction, weadministrated 5 lg/mL (150 ng) of hBD-3 or hBD-4 inmast cell-deficient Ws/Ws rats. Fig. 6C shows that mastcell-deficient Ws/Ws rats exhibited a markedly de-creased Evans blue extravasation, compared to theirwild-type littermates. There was no significant differ-ence between hBD- and normal saline-induced skinreactions in Ws/Ws rats. We confirmed that hBD-2 hadalso similar properties of increasing skin vascularpermeability. This implies that hBD induce vascularpermeability through the activation of skin mast cells.

hBD-3 and hBD-4 induce the phosphorylationof p38 and ERK

Although hBD have been shown to induce phosphoryla-tion of MAPK in keratinocytes [16], it is not clearwhether these peptides also activate MAPK in mast cells.Therefore, cells were stimulated with hBD-3 and hBD-4for 5–30 min, and both the phosphorylated and unpho-sphorylated p38 and ERK1/2 levels were determinedusing Western blot analysis. Incubation of mast cellswith hBD-3 (Fig. 7A) and hBD-4 (Fig. 7B) induced arapid phosphorylation of p38 at 5 min, and phosphor-ylation persisted at 15 min in hBD-3-stimulated cells.The phosphorylation of ERK1/2 was observed at 15 and30 min in both hBD-3- and hBD-4-stimulated mast cells.To determine whether activation of p38 and ERK1/2were required for hBD-3- and hBD-4-induced mast celldegranulation, mast cells were incubated with specificinhibitors for p38 or ERK1/2 for 1 h before stimulationwith hBD-3 and hBD-4. The presence of 20 lMSB203580 (p38 inhibitor) and 20 lM PD98059(ERK1/2 inhibitor), significantly reduced mast celldegranulation induced by hBD-3 (Fig. 8A) and hBD-4(Fig. 8B) (p<0.05, as compared with the absence ofinhibitors). The combination of both SB203580 andPD98059 further significantly decreased the mast celldegranulation by hBD-3 and hBD-4 (p<0.01). Weconfirmed that the treatment of mast cells with MAPKinhibitors did not affect cell viability, as assessed bytrypan blue dye exclusion.

Discussion

The current study has clearly demonstrated the abilitiesof hBD-3 and hBD-4 to induce mast cell degranulation,PGD2 generation and chemotaxis through G protein andPLC pathways. Both hBD-3 and hBD-4 activated MAPKp38 and ERK1/2, which were necessary for mast celldegranulation. Furthermore, we demonstrated thathBD-3 and hBD-4 increased the skin vascular perme-ability that was almost completely dependent on the

Figure 4. hBD-3 and hBD-4 induce mast cell migration. Mastcells (5 � 104 cells/500 lL) in the upper chamber of tissueculture insert were allowed to migrate towards the indicatedconcentrations of hBD-3 (A) or hBD-4 (B) in each well of 24-wellculture plates (lower chamber) for 3 h at 37�C. LAD2 cells (LAD2,diagonal stripes) were stimulated with 2.5 lg/mL hBD-3 (A) or5 lg/mL hBD-4 (B). Values are compared with medium alonewithout peptide (Med). Each bar represents the mean � SD offour separate experiments. * p<0.05.



presence of mast cells. Thus, antimicrobial peptides mayparticipate in inflammatory reactions through theiractivation of mast cells and increase of vascularpermeability in the skin.

Mast cells are critical effector cells in the develop-ment of inflammatory reactions, which are mediated byhistamine, eicosanoids, and proinflammatory cyto-kines/chemokines secreted upon activation of mastcells [29, 30]. We found that both hBD-3 and hBD-4induced mast cell b-hexosaminidase release and PGD2

generation; however, these peptides failed to induce theproduction of cytokines such as TNF-a, IL-4, IL-6 orchemokines MCP-1 and RANTES (data not shown).Reportedly, Ca2+ plays a key role in the regulation andrelease of inflammatory mediators such as histamineand PGD2 [27, 28]. Indeed, we have previously showedthat the strength of hBD-2 compared with LL-37 ininducing mast cell degranulation and PGD2 productionwas probably due to the potency of hBD-2 to evokerelatively higher levels of [Ca2+]i [17]. In the present

report, although both hBD-3- and hBD-4-induced risesof [Ca2+]i were almost similar, hBD-3 was more potentthan hBD-4 in inducing mast cell degranulation andPGD2 generation. This suggests that, although intracel-lular Ca2+ is important in the release of inflammatorymediators from mast cells, the amplitude of the [Ca2+]irise is not a determining element for the stimulatorypreferential effect to release more histamine or PGD2.This hypothesis is consistent with a previous report thatin mast cells, compound 48/80 induced intracellularCa2+ mobilization, high secretion of histamine and lowrelease of PGD2, whereas ionomycin evoked high rises in[Ca2+]i, low histamine release and high PGD2 produc-tion [27]. Furthermore, although intracellular Ca2+

mobilization has been found to be important in cellmigration, a recent study has reported that hBD-3- andhBD-4-induced chemotaxis of monocytes but notneutrophils or eosinophils, was not accompanied byintracellular Ca2+ mobilization [8]. Contrasting withthis, the present study revealed that both hBD-3 and

Figure 5. Effects of PTx and U-73122 on hBD-3 andhBD-4-induced mast cell activation. Mast cellswere pretreated with 200 ng/mL PTx for 2 h and10 lM U-73122 or 10 lM U-73343 (U-alg, U-73122analogue) for 1 h. Cells were washed in appro-priate buffers, then challenged with 20 lg/mLhBD-3 (a) or hBD-4 (b) for 40 min for b-hexosami-nidase (b-Hexo) release assay (A); 40 lg/mL hBD-3(a) or 20 lg/mL hBD-4 (b) for 1 h for PGD2

production assay (B); 20 lg/mL hBD-3 (a) or hBD-4(b) for intracellular Ca2+ measurement (C); or with20 lg/mLhBD-3 (a) or 40 lg/mLhBD-4 (b) for 3 h forchemotaxis assay (D). Values are themean � SD offour to five separate experiments. * p<0.05 ascompared between the presence and absence ofinhibitors.



hBD-4 stimulated intracellular Ca2+ mobilization andchemotaxis of mast cells. Therefore, hBD may differen-tially activate their target cells.

In the skin, mast cells are found within the dermis,where they selectively accumulate and become activatedduring inflammation. Besides their participation inallergic and inflammatory reactions, mast cells are alsoindispensable in defense against bacterial infections

[31–33]. These cells are involved in the pathogenesis ofseveral skin disorders, including wounding and lichenplanus [34], where the expression of antimicrobialpeptides such as hBD-3 has been found to be increased[8, 35]. In these two cases, the basal membrane betweendermis and epidermis is impaired, and this may lead tothe direct contact between mast cells and antimicrobialpeptides. Therefore, these peptides may activate mastcells to migrate and release inflammatory mediators.The chemoattractants of mast cells have been detectedduring inflammation in most organs including skinwhere mast cells are present [36], and antimicrobialpeptides have been found at high concentrations inthose tissues. In the skin, the concentrations of hBD-2have been estimated to 3.5–16 lM (15–70 lg/mL) inIL-1a-stimulated epidermal cultures [37] and *20 lM(87 lg/mL, median) in psoriatic skin [38], while LL-37concentrations are 1 lM (4.5 lg/mL) in normal skin[39] and *304 lM (1.38 mg/mL, median) in psoriaticskin lesions [38]. In our study, hBD-3 and hBD-4significantly elicited mast cell chemotaxis at theconcentrations in the range 1–40 lg/mL, equivalentto 0.2–9.2 lM. Since the expression of both hBD-3 andhBD-4 could be increased during inflammation, weassume that these peptides may potentially reach theiroptimal chemotactic concentrations at local inflamma-tory sites. Whereas the high doses of hBD-3 have beendetected in wound healing [8] and psoriatic skin [40,41], current techniques have failed to detect hBD-4peptide in psoriatic or normal skin [41], although itsmRNA expression has been found to increase in humanprimary keratinocytes [9].

Similar to hBD-2 and LL-37, antimicrobial peptideshBD-3 and hBD-4 activate mast cells via at least Gprotein-coupled receptors and PLC-dependent pathway,as demonstrated by the inhibitory effects of PTx andU-73122 against hBD-3- and hBD-4-induced mast celldegranulation, PGD2 generation, intracellular Ca2+

mobilization and chemotaxis. Although CCR6 has beendemonstrated as a common receptor for hBD-1 and hBD-2 in dendritic and T cells [15], and for hBD-3 inmonocytes [42], the receptors for hBD in mast cells havenot yet been identified. However, it seems that hBD donot utilize CCR6 as a functional receptor on mast cellmembrane, because it has been demonstrated that thesole ligand for CCR6, MIP-3a, neither activates mastcells nor competes for hBD-2 high-affinity binding siteson mast cells [18]. This implies the possibility that mastcells do not express CCR6 as a functional receptor, andthat hBD act through additional receptor other thanCCR6. To prove possible usage of receptor(s) by hBD-3and hBD-4 on rat mast cells, it would be helpful tostimulate these cells with rat orthologs of hBD-3 andhBD-4, and compare their potency and efficacy.However, only two rat b-defensins rBD-1 and rBD-2,

Figure 6. hBD-3 and hBD-4 increase the skin vascularpermeability, which is dependent on the presence of mastcells. Wild-type rats were intravenously injected with 1 mL0.5% Evans blue before intradermal injection of 5–20 lg/mL(150–600 ng) of hBD-3 (A) or hBD-4 (B) into one side of the ear,and vehicle saline (Saline) into the other ear. The rats werekilled 30 min after hBD-3, hBD-4 or saline injection, and Evansblue dye contents in the ear tissuesweremeasured. The resultsare expressed as Evans blue ng/mg tissue. Data shown aremean� SD of four wild-type rats, and compared between hBD-3 or hBD-4 administration and normal saline administration(Saline). * p<0.05. (C)Wild-type (dots) ormast cell-deficientWs/Ws (open bars) rats were intradermally injected with 30 lLhBD-3 and hBD-4 (each 5 lg/mL, equivalent to 150 ng) ornormal saline (Saline) for 30 min as described above. Values arecompared between four wild-type rats and four mast cell-deficient Ws/Ws rats injected with hBD-3 or hBD-4. * p<0.05.



which are homologous to hBD-1 and hBD-2, respec-tively, have been isolated so far [43]. Alternatively, theobservation that hBD-3 and hBD-4 induced human mastcell LAD2 migration and degranulation (for hBD-3) atconcentrations much lower than those required for ratmast cells suggests that these peptides may activatehuman mast cells via selective receptor(s). However,since hBD are cationic peptides, they might act either atsome non-selective membrane receptors or mightdirectly bind to and activate G proteins sensitive toPTx and coupled to PLC, as has been shown for most ofbasic secretagogues of rat peritoneal mast cells [44].

To further understand the downstream eventsinvolved in hBD activation pathway, we evaluated theeffects of hBD on MAPK activation. We demonstratedthat hBD-3 and hBD-4 induced the phosphorylation ofMAPK p38 and ERK1/2, and that the inhibitors for p38and ERK significantly decreased hBD-3- and hBD-4-mediated mast cell degranulation, suggesting theinvolvement of MAPK in mast cell activation uponhBD stimulation. In contrast, JNK was not activated byhBD-3 or hBD-4, and its specific inhibitor (SP600125)did not affect mast cell degranulation (data not shown).The link between G protein and MAPK in antimicrobialpeptide-mediated cell activation has not been consis-tently demonstrated. For instance, it has been reportedthat LL-37 phosphorylated p38 and ERK in humanmonocytes and bronchial epithelial cells [45]. However,this phosphorylation was independent of the G protein-coupled receptors. Thus, antimicrobial peptides likelyact on target cells through different signaling pathways.

Mast cells play an important role in physiologicalchanges during allergic and anaphylactic responses[46]. We found that hBD-3 and hBD-4 caused the in vitrorelease of pro-inflammatory mediators known toprovoke vasodilatation, and revealed that these peptidesrapidly induced the increase of vascular permeability inthe skin. To date, only mast cell classical stimuli such asIgE, compound 48/80 and substance P were reported toinduce vascular permeability [47–49]. To our know-ledge, this is the first study to demonstrate that epithelialcell-derived antimicrobial peptides hBD increase theskin vascular permeability. The hBD-induced vascularpermeability was almost totally dependent on thepresence of mast cells as evidenced by the markedlyreduced levels of vascular permeability in mast cell-deficient Ws/Ws rats. Thus, antimicrobial peptides mayparticipate in acute cutaneous inflammation throughactivation of mast cells. However, in contrast to hBD-3,hBD-4 peptide has not been found in skin tissues [41].Therefore, the in vivo relevance of hBD-4 as a mast cellstimulant requires further investigation.

In conclusion, although hBD are believed to beinvolved in innate host defense by killing invadedpathogenic microorganisms, the present study clearly

Figure 7. hBD-3 and hBD-4 induce the activation of MAPK p38and ERK. Mast cells (2 � 106 cells/mL) were stimulated with20 lg/mL of hBD-3 (A) and hBD-4 (B) for the indicated timeperiods, and phosphorylated p38 (p-p38) or ERK1/2 (p-ERK1/2)and unphosphorylated p38 (p38) or ERK1/2 (ERK1/2) levels incellular lysates were determined by Western blot analysis.Shown is one representative of five independent experimentswith similar results.

Figure 8. Involvement of MAPK p38 and ERK1/2 in hBD-3- andhBD-4-mediated mast cell degranulation. Mast cells were pre-incubated with 20 lM p38-specific inhibitor SB203580 (SB),20 lM ERK1/2-specific inhibitor PD98059 (PD), 20 lM SB203580and PD98059 (SB + PD), or medium alone (Ctrl, control) for 1 h,after which the cellswere stimulated for 40 minwith 20 lg/mLof hBD-3 (A) and hBD-4 (B). b-Hexosaminidase in the super-natants was measured as described in the Materials andmethods. Values are the mean � SD of four separate experi-ments. * p<0.05, ** p<0.01 as compared between the presenceand absence of each MAPK inhibitor.



demonstrated that these antimicrobial peptidesmay alsomodulate the inflammatory reactions by recruiting andactivating mast cells, and increasing the vascularpermeability in the skin.

Materials and methods

Reagents

Synthetic hBD-2, hBD-3 and hBD-4 were purchased fromPeptide Institute (Osaka, Japan), and their connectivities ofcysteine bridges are Cys8-Cys37, Cys15-Cys30, and Cys20-Cys38

for hBD-2; Cys11-Cys40, Cys18-Cys33, and Cys23-Cys41 for hBD-3; and Cys6-Cys33, Cys13-Cys27, and Cys17-Cys34 for hBD-4. U-73122 {1-[6-([(17b)-3-methoxyestra-1, 3, 5 (10)-trien-17-yl]amino) hexyl]-1H-pyrrole-2.5-dione}, U-73343 {1-[6-[(17b)-3-methoxyestra-1, 3, 5 (10)-trien-17-yl] amino] hexyl)-2,5-pyrrolidinedione} and PTx were from Sigma (St Louis, MO).Fura-2 acetoxy-methylester (fura-2-AM) was obtained fromDojindo Laboratories (Kumamoto, Japan). Enzyme immu-noassay kit for PGD2 was from Cayman Chemical Company(Ann Arbor, MI). Rabbit polyclonal anti-phosphorylated p38and ERK1/2 Ab, and p38 and ERK1/2 Ab were purchased fromCell Signaling Technology (Beverly, MA). The inhibitorsSB203580 (Sigma-Aldrich) and PD98059 (Cell SignalingTechnology) were used to study the MAPK pathway involvedin the activation of mast cells.

Mast cell preparation and culture

Rat peritoneal mast cells, representative of connective tissuemast cells mainly found in skin and peritoneal cavity [50],were obtained from male Sprague-Dawley rats by lavage ofperitoneal cavity with PBS containing 0.1% BSA. Followingcentrifugation of recovered lavage fluids, collected cells weresuspended in MEM with 10% FCS, layered on 75% Percollsolution, and further centrifuged at 600 � g for 25 min atroom temperature. The purity of mast cells was �95% and theviability 99%. LAD2 cells were a kind gift from Dr Dean D.Metcalfe (NIH, Bethesda, MD). They were cultured in serum-free media (StemPro-34, Life Technologies, Rockville, MD)supplemented with 2 mM L-glutamine, 50 IU/mL penicillin,50 lg/mL streptomycin, and 100 ng/mL recombinant humanstem cell factor (Peprotech, Rocky Hill, NJ). The LAD2 celldensity was maintained at 5 � 105 cells/mL, and the cellculture was diluted every 2 weeks. LAD2 cell experimentswere performed at the Research Center for Allergy andImmunology, RIKEN Yokohama Institute. All experiments wereperformed according to the approved manual of JuntendoUniversity.

b-Hexosaminidase release assay

Mast cells (1 � 105 cells/well) were suspended in Tyrode'sbuffer as reported before [19], and then stimulated withvarious concentrations of hBD-3 and hBD-4 for 40 min at 37�C.The b-hexosaminidase in the supernatants and total cell lysatessolubilized with Triton X-100 was quantified by hydrolysis of p-

nitrophenyl-N-acetyl-b-D-glucopyranoside in 0.1 M sodiumcitrate buffer for 90 min at 37�C. The percentage ofb-hexosaminidase release was calculated using the formula:% release = (ODstimulated supernatant – ODunstimulated super-

natant) � 100 / (ODtotal cell lysate – ODunstimulated supernatant).In some experiments, mast cells were pre-treated with

20 lMp38 inhibitor, SB203580 or ERK1/2 inhibitor, PD98059for 1 h, washed, and then stimulated with hBD-3 or hBD-4.b-Hexosaminidase in the supernatant was measured asdescribed above.

Measurement of PGD2 production

Aliquots of 150 lL mast cells (1 � 106 cells/mL) suspended inRPMI 1640 without phenol red were incubated with hBD-3 orhBD-4 at the indicated concentrations for 1 h at 37�C. Afterstimulation, cells were centrifuged at 220 � g for 10 min at4�C, and the supernatants were used for PGD2 quantificationby enzyme immunoassay kit according to the manufacturer'sinstructions.

Measurement of [Ca2+]i

Mast cells (5 � 105 cells/mL) were suspended in a balancedsalt solution containing 0.05% BSA (BSSA), and the measure-ment of [Ca2+]i was assayed as reported previously [17]. Cellswere loaded with 3 lM fura-2-AM solution for 30 min at 37�C,washed twice with BSSA and then challenged with hBD-3 orhBD-4 at the indicated concentrations. The ratios of maximumandminimum fluorescence were determined by the addition of1% Triton X-100 and 15 mM EGTA. The measurements of[Ca2+]i were performed and taken for 300 s as before [15].

Chemotaxis assay

The chemotaxis assay was performed as described previously[18]. Briefly, different concentrations of hBD-3 or hBD-4diluted in 500 lL MEM containing 1% BSA were applied intoeach well of 24-well culture plates. Then, a 10-mm tissueculture insert with an 8-lm pore-size polycarbonate mem-brane was placed into each well, and aliquots of 500 lL of cells(5 � 104 cells/well) were added into the insert. After a 3 hincubation period at 37�C, migrated cells on the lower surfaceof the membrane were fixed with methanol and stained with0.1% toluidine blue. The membranes were mounted and themigrated cells were counted under a light microscope (in fourhigh power fields, HPF).

Treatment with PTx and U-73122

The inhibitory effects of PTx, U-73122 and U-73343 weredetermined by pre-treating mast cells with 200 ng/mL PTx for2 h, 10 lM U-73122 or 10 lM U-73343 for 1 h at 37�C. Cellswere washed twice with Tyrode's buffer for b-hexosaminidaserelease assay, with RPMI 1640 for PGD2 production assay, withBSSA for measurement of [Ca2+]i or with MEM for migrationassay. Cells were then resuspended in the appropriate bufferfollowed by stimulation with hBD. b-Hexosaminidase release,PGD2 production, measurement of [Ca2+]i and cell migrationassays were performed as described above.



Induction of vascular permeability in the skin

The induction of vascular permeability in the skin by hBD-3and hBD-4 was investigated as described elsewhere [19] withslight modifications, using male mast cell-deficient rats (Ws/Ws) and their mast cell-competent littermates (+/+) obtainedfrom Japan SLC (Shizuoka, Japan). Rats were lightlyanesthetized, and then intravenously injected with 1 mL0.5% Evans blue 5 min before intradermal injection of 30 lL ofvarious concentrations of hBD-3 and hBD-4 into one side of theear, and vehicle 0.9% normal saline into the other ear. After30 min, rats were killed, and the ears were removed, weighed,and then dissolved in 300 lL 1 N KOH overnight at 37�C.Then, 1.8 mL of a mixture of 0.6 N H3PO4 and acetone (5:13)was added. After shaking, the supernatant was collected bycentrifugation at 1920 � g for 10 min and the OD wasmeasured at 620 nm. The amount of dye was calculatedaccording to the standard curve of known concentrations ofEvans blue, and the results were expressed as ng Evans blue/mg tissue.

Western blot analysis

Mast cells (2 � 106 cells/mL) were stimulated with 20 lg/mLof hBD-3 or hBD-4 for the indicated time periods. Afterstimulation, the lysates were obtained by lysing cells in lysisbuffer (50 mM Tris-HCl, pH 8, 150 mM NaCl, 0.02% NaN3,0.1% SDS, 1% NP 40, containing 1 lM PMSF, 10 lg/mLleupeptin, 10 lg/mL pepstatin-A, 50 lg/mL aprotinin and2 mM sodium orthovanadate). The amount of total proteinwas determined by BCA Protein Assay (Pierce Chemical,Rockford, IL), and equal amounts of total protein fromstimulated or non-stimulated cells were analyzed by Westernblot. The cell lysates were separated by SDS-PAGE on 15%polyacrylamide gel onto Immobilon-P membrane (Millipore,Bedford, MA). Nonspecific binding sites were blocked, and theblots were incubated with polyclonal Ab against phosphory-lated p38 or ERK1/2, and unphosphorylated p38 or ERK1/2overnight according to the manufacturer's instructions. Themembrane was developed with an enhanced chemilumines-cence detection kit (Amersham Pharmacia Biotech, Piscat-away, NJ).

Statistical analysis

The statistical analysis was performed using Student's t-test,and p<0.05 was considered to be significant. The results areshown as mean � SD.

Acknowledgements: We would like to thank themembers of Atopy (Allergy) Research Center andDepartment of Immunology of Juntendo UniversitySchool of Medicine for their excellent discussion,comments and encouragement. We thank Dr Yoshimi-chi Okayama of RIKEN Yokohama Institute for helpfulsuggestions and discussion, and Dr. Takahisa Ogasa-wara of Department of Allergy and Immunology,National Research Institute for Child Healthy andDevelopment for skillful technical assistance. We are

also very grateful to Michiyo Matsumoto for secretarialassistance. This work was supported in part by Grant-in-Aid for Scientific Research from the Ministry ofEducation, Culture, Sports, Science and Technology,Japan to F. N. (16790660), and Atopy (Allergy) ResearchCenter, Juntendo University, Tokyo, Japan.

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antimicrobial peptides human β-defensin (hbd)-3 and hbd-4 activate mast cells and increase skin...

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