Antimicrobial Activity As a Chemokine with Broad-Spectrum CCL28 Has

CCL28 Has Dual Roles in Mucosal Immunity as a Chemokine with Broad-Spectrum Antimicrobial Activity

This information is current as Kunio Hieshima, Haruo Ohtani, Michiko Shibano, Dai of September 26, 2021. Izawa, Takashi Nakayama, Yuri Kawasaki, Fumio Shiba, Mitsuru Shiota, Fuminori Katou, Takuya Saito and Osamu Yoshie J Immunol 2003; 170:1452-1461; ;

doi: 10.4049/jimmunol.170.3.1452 Downloaded from http://www.jimmunol.org/content/170/3/1452

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

CCL28 Has Dual Roles in Mucosal Immunity as a Chemokine with Broad-Spectrum Antimicrobial Activity1

Kunio Hieshima,* Haruo Ohtani,2‡ Michiko Shibano,* Dai Izawa,* Takashi Nakayama,* Yuri Kawasaki,* Fumio Shiba,* Mitsuru Shiota,† Fuminori Katou,§ Takuya Saito,¶ and Osamu Yoshie3*

CCL28 is a CC chemokine signaling via CCR10 and CCR3 that is selectively expressed in certain mucosal tissues such as exocrine glands, trachea, and colon. Notably, these tissues commonly secrete low-salt fluids. RT-PCR analysis demonstrated that salivary glands expressed CCL28 mRNA at the highest levels among various mouse tissues. Single cells prepared from mouse parotid glands indeed contained a major fraction of CD3؊B220low cells that expressed CCR10 at high levels and CCR3 at low levels and responded to CCL28 in chemotaxis assays. Morphologically, these cells are typical plasma cells. By immunohistochemistry, acinar epithelial cells in human and mouse salivary glands were strongly positive for CCL28. Furthermore, human saliva and milk Downloaded from were found to contain CCL28 at high concentrations. Moreover, the C terminus of human CCL28 has a significant sequence similarity to histatin-5, a histidine-rich candidacidal peptide in human saliva. Subsequently, we demonstrated that human and mouse CCL28 had a potent antimicrobial activity against Candida albicans, Gram-negative bacteria, and Gram-positive bacteria. The C-terminal 28-aa peptide of human CCL28 also displayed a selective candidacidal activity. In contrast, CCL27, which is most similar to CCL28 and shares CCR10, showed no such potent antimicrobial activity. Like most other antimicrobial peptides, http://www.jimmunol.org/ CCL28 exerted its antimicrobial activity in low-salt conditions and rapidly induced membrane permeability in target microbes. Collectively, CCL28 may play dual roles in mucosal immunity as a chemoattractant for cells expressing CCR10 and/or CCR3 such as plasma cells and also as a broad-spectrum antimicrobial protein secreted into low-salt body fluids. The Journal of Immunology, 2003, 170: 1452–1461.

hemokines are known to play pivotal roles in innate and CCR10 was originally identified as the receptor for CCL27 (also acquired immunity by regulating migration and activa- called IL-11R ␣-locus chemokine or cutaneous T cell-attracting C tion of leukocytes via a group of seven transmembrane G chemokine) (5, 6), whereas CCR3 is the receptor for eotaxin/ protein-coupled receptors (1). In humans, more than 45 members CCL11 and many other chemokines known to act on eosinophils by guest on September 26, 2021 and 18 functional receptors have been identified (1). According to (1). CCL28 transcripts are detected in a variety of tissues but most the arrangement of the amino-terminal conserved cysteine resi- abundantly in trachea, colon, rectum, and exocrine glands such as dues, the chemokines are classified into four subfamilies: CXC, salivary and mammary glands (3, 4). In the colon, CCL28 is se- CC, C, and CX3C (1). Recently, based on the classification of lectively expressed by the epithelial cells (3, 4). Structurally, these four subfamilies, a systematic nomenclature system of the CCL28 is most similar to CCL27, which is expressed highly se- chemokine ligands has been formulated (2). Except for the two lectively in the skin and has been shown to attract cutaneous lym- transmembrane-type chemokines, CX3CL1 and CXCL16, chemo- phocyte Agϩ memory T cells via CCR10 (5Ð8). Consistently, kines are small (8Ð14 kDa), mostly cationic polypeptides with two CCL28 has also been shown to attract cutaneous lymphocyte Agϩ to three intramolecular disulfide bonds (1). CCL28 (also called memory T cells via CCR10 and eosinophils via CCR3 among hu- mucosae-associated epithelial chemokine) is a recently described man peripheral blood leukocytes (3). CC chemokine signaling via CCR10 as well as CCR3 (3, 4). Antimicrobial peptides, now known by Ͼ500 in number, are the diverse family of small, mostly cationic polypeptides found widely Departments of *Microbiology and †Gynecology and Obstetrics, Kinki University in all forms of multicellular organisms that exert a broad spectrum School of Medicine, Osaka, Japan; ‡Department of Pathology, Tohoku University of cytotoxic activity against bacteria, fungi, parasites, and envel- ¤ Graduate School of Medicine, Miyagi, Japan; Department of Oral and Maxillofacial oped viruses (9, 10). In mammals, such peptides are particularly Surgery 1, Tohoku University School of Dentistry, Miyagi, Japan; and ¶Department of Biotechnological Science, Kinki University, Wakayama, Japan abundant in the storage granules of phagocytic cells and on the Received for publication August 26, 2002. Accepted for publication November surface of mucosal tissues. Accumulating evidence points out that 20, 2002. chemokines and antimicrobial peptides have substantially overlap- The costs of publication of this article were defrayed in part by the payment of page ping functions (11). For example, several members of the mam- charges. This article must therefore be hereby marked advertisement in accordance malian antimicrobial proteins are capable of attracting leukocytes with 18 U.S.C. Section 1734 solely to indicate this fact. via interactions with selected seven-transmembrane G protein-cou- 1 This work was supported by grants-in-aid and a High-Tech Research Center Grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan, and pled receptors (12Ð14). Most notably, Yang et al. (15) have re- by Solution Oriented Research for Science and Technology of the Japan Science and cently demonstrated that human ␤-defensins are potent agonists Technology Corporation. for CCR6, the receptor for the chemokine CCL20 (also called liver 2 Current address: Department of Clinical Laboratory Research, National Mito Hos- and activation-regulated chemokine or macrophage inflammatory pital, 3-2-1 Higashihara, Mito, Ibaragi 310-0035, Japan. protein-3␣), which is produced by mucosal epithelial cells and 3 Address correspondence and reprint requests to Dr. Osamu Yoshie, Department of Microbiology, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka- epidermal keratinocytes upon proinflammatory stimulations that Sayama, Osaka 589-8511, Japan. E-mail address: [email protected] selectively attracts immature dendritic cells, memory T cells, and

B cells (1, 16). In contrast, some chemokines have been shown to binding to Fc receptors (22). Human embryonic kidney 293T cells were have antimicrobial activity. Krijgsved et al. (17) purified antibac- transiently transfected with the CCL27-Fc and control Fc constructs, and terial proteins stored in the ␣-granules of human platelets and de- CCL27-Fc and control Fc proteins were purified from the culture super- natants by using Protein A-agarose (21). Purified CCL27-Fc and control Fc termined their amino acid sequences. Surprisingly, these proteins were 76 kDa and 55 kDa, respectively, which was in agreement with the turned out to be CXCL7 (59Ð126) and CXCL7 (44Ð126), two predicted molecular mass. By using a panel of L1.2 cell lines covering all related chemokine variants processed from a common precursor human and murine CC chemokine receptors (CCR1Ð10), we confirmed that platelet basic protein and truncated by 2 aa in the C terminus (17). CCL27-Fc specifically bound to human and murine CCR10 (data not shown). Furthermore, Cole et al. (18), by examining a panel of 11 chemokines representing all four chemokine subfamilies, demonstrated Preparation of single cells from mouse parotid glands that the three IFN-inducible non-ELR (Glu-Leu-Arg)-motif CXC Female BALB/c mice were purchased from CLEA Japan (Osaka, Japan). chemokines, monokine induced by IFN-␥/CXCL9, IFN-inducible After cervical dislocation, parotid glands were excised from 12- to 16-wk- protein-10/CXCL10, and IFN-inducible T-cell ␣ chemoattractant/ old mice, carefully avoiding adjacent cervical lymph nodes. Excised pa- CXCL11, had a significant antimicrobial activity against Esche- rotid glands were gently disrupted by a pair of glass slides, and cell sus- richia coli and Listeria monocytogenes. However, the physiolog- pensions were passed through nylon mesh to remove aggregates and cell debris. After washing, single cells were suspended in a medium appropriate ical relevance of the antimicrobial activity of CXCL7 variants for subsequent experiments. present in the platelets or the three non-ELR CXC chemokines commonly inducible by IFN-␥ is still unclear because their anti- RT-PCR analysis bacterial activity was seen only at high concentrations and also, as This was conducted essentially as described previously (16). In brief, var-

with most other antimicrobial peptides, in low salt conditions, ious tissues were carefully excised from 12- to 16-wk-old female BALB/c Downloaded from which are only possible in certain body surface secretions, but not mice. Total RNA was prepared from freshly isolated tissues by using Trizol reagent (Life Technologies, Gaithersburg, MD) and RNeasy (Qiagen, within the circulation or tissues. Hilden, Germany). Reverse transcription of total RNA (1 ␮g) was con-

Here we demonstrate that 1) CCL28 is expressed at high levels ducted using oligo(dT)18 primer and SuperScript II reverse transcriptase in mouse salivary glands, 2) the major CD3ϪB220low plasma cell (Life Technologies). First-strand DNA (20 ng total RNA equivalent) and fraction in mouse salivary glands expresses its receptor CCR10 at original total RNA (20 ng) were amplified in a final volume of 20 ␮l containing 10 pmol of each primer and1UofEx-Taq polymerase (Takara, high levels and CCR3 at low levels, 3) CCL28 is constitutively Kyoto, Japan). The primers used were as follows: ϩ5Ј-CATACTTC http://www.jimmunol.org/ produced by the epithelial cells of human and mouse salivary CCATGGCCTCC-3Ј and Ϫ5Ј-GAGAGGCTTCGTGCCTGTG-3Ј for mouse glands, 4) CCL28 is secreted in human saliva and milk at high CCL28 (mCCL28); ϩ5Ј-AGAGCTCTGTTACAAGGCTGATGTC-3Ј and concentrations, 5) the expended C-terminal region of human Ϫ5Ј-CAGGTGGTACTTCCTAGATTCCAGC-3Ј for mCCR10; ϩ5Ј-TTG Ј Ϫ Ј CCL28 has a sequence similarity with histatin-5, a histidine-rich CAGGACTGGCAGCATT-3 and 5 -CCATAACGAGGAGAGGAAGA GCTA-3Ј for mCCR3; ϩ5Ј-GCCAAGGTCATCCATGACAACTTTGG-3Ј candidacidal peptide secreted into human saliva (19), and 6) and Ϫ5Ј-GCCTGCTTCACCACCTTCTTGATGTC-3Ј for G3PDH. The am- CCL28 exerts a potent and salt-sensitive antimicrobial activity plification conditions, which were carefully chosen to obtain signals in a linear against a broad spectrum of microbes including Candida albicans, amplification range, were denaturation at 94¡C for 30 s (5 min at the first Gram-negative bacteria, and Gram-positive bacteria. Importantly, cycle), annealing at 60¡C for 30 s, and extension at 72¡C for 30 s (5 min at the

last cycle) for 33 cycles for mCCL28, 38 cycles for mCCR10 and mCCR3, by guest on September 26, 2021 the tissues that express CCL28 at high levels such as salivary and 27 cycles for G3PDH. Amplification products (10 ␮l each) were loaded glands, mammary glands, respiratory tract, and distal colon com- onto 2% agarose, run by electrophoresis, and visualized by staining with monly secrete low-salt fluids because of selective reabsorption of ethidium bromide. sodium ions by the epithelial systems (20). Thus, CCL28 may Flow cytometric analysis represent the first chemokine that not only attracts cells expressing its receptors CCR10 and CCR3 into certain mucosal tissues but Single cells prepared from mouse parotid glands were suspended in ice- also is constitutively secreted by epithelial cells in such tissues and cold PBS containing 3% FBS and 0.1% sodium azide (staining medium). All the following steps were conducted on ice. For detection of surface functions as a potent antimicrobial factor in physiological settings. CCR10, cells were incubated with 1 ␮g/ml of CCL27-Fc or control Fc for CCL28 thus adds further evidence for the close functional and 30 min. After washing, cells were incubated with biotin-labeled goat anti- evolutionary relationships between chemokines and antimicrobial human Ig (Vector Laboratories, Burlingame, CA) for 30 min. After wash- peptides (11). ing, cells were incubated with a cocktail of APC-labeled streptavidin (BD PharMingen, San Diego, CA), CyChrome-labeled anti-B220 (RA3-6B2; BD PharMingen), and PE-labeled anti-CD3 (KT3; Beckman Coulter, San Materials and Methods Jose, CA) for 30 min. After washing, cells were analyzed on a FACSCali- Reagents bur (BD Biosciences, Mountain View, CA). For detection of surface CCR3, cells were incubated with rabbit polyclonal anti-mCCR3 (BD Recombinant human and mouse chemokines were all purchased from R&D PharMingen) or control rabbit IgG for 30 min. After washing, cells were Systems (Minneapolis, MN). Mouse monoclonal anti-human CCL28 stained with a cocktail of FITC-labeled anti-rabbit IgG, CyChrome-labeled (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-la- anti-B220, and PE-labeled anti-CD3 for 30 min. After washing, cells were beled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse analyzed on a FACSCalibur. CCL28 were also purchased from R&D Systems. Isotype-matched IgG controls were purchased from DAKO Japan (Kyoto, Japan). The C-termi- May-Gru¨nwald-Giemsa staining of CCR10-expressing cells nal 28 aa of CCL28 (CCL28-C; HRKKHHGKRNSNRAHQGKHETYGH KTPY)4 was chemically synthesized and purified by reverse-phase HPLC Single cells prepared from mouse parotid glands were incubated with 1 ␮ to Ͼ98% purity by Sawady Technology (Tokyo, Japan). g/ml of CCL27-Fc or control Fc on ice for 30 min. After washing, cells were stained with FITC-labeled goat anti-human IgG (Beckman Coulter) Generation of CCL27-Fc chimera protein for 30 min. After washing, cells were placed on glass slides, air dried, and fixed with methanol. Cells were further stained with May-Gru¬nwald- To detect surface expression of CCR10, we used the technique of a che- Giemsa. Finally, cells in the same fields were examined on a fluorescent mokine-Fc chimera protein as described previously (21). In brief, the cod- microscope under UV and visible lights. ing sequence of human CCL27 was amplified from pDREF-ILC-Flag by using PCR (8) and was subcloned into a pcDNA3.1-Fc cassette, which Chemotaxis assay encodes the human IgG1 Fc domain with a mutation that abolishes its This was conducted using Transwell plates with 5- or 8-␮m pore inserts (Corning Costar, Cambridge, MA). In brief, single cells prepared from 6 4 Abbreviations used in this paper: CCL28-C, C-terminal 28 aa of CCL28; m, mouse; mouse parotid glands were suspended at 2 ϫ 10 /ml in RPMI 1640 con- PPB, potassium phosphate buffer; PI, propidium iodide. taining 1 mg/ml BSA (Sigma-Aldrich, St. Louis, MO) and 20 mM HEPES 1454 ANTIMICROBIAL ACTIVITY OF CCL28

(pH 7.4) and applied to upper wells (100 ␮l/well). The same medium We also analyzed candidacidal activity of CCL28 by flow cytometry. without or with chemokine was applied to lower wells (600 ␮l/well). After Candia cells in mid-logarithmic growth phase were suspended in 1 mM 4hat37¡C, inserts were removed and a known number of counting beads PPB (pH 7.2) and treated without or with various concentrations of CCL28 (BD PharMingen) were added to each well. The content of each well was at 37¡C for indicated lengths of time. After washing, cells were incubated transferred to a polypropylene pointed-bottom tube. The beads and cells with FITC-labeled annexin V (Wako, Osaka, Japan) for 10 min. After were pelleted by centrifugation at 200 ϫ g for 5 min, resuspended in the washing, cells were resuspended in buffer containing 2 ␮g/ml propidium staining medium (see above), and stained with CyChrome-labeled anti- iodide (PI) and were immediately analyzed on a FACSCalibur. B220 and PE-labeled anti-CD3 as described above. After washing, cells were analyzed on a FACSCalibur. All assays were done in duplicate. Scanning electron microscopy Immunohistochemistry Microbes suspended in 1 mM PPB (pH 7.2) were mock treated or treated with 10 ␮M CCL28 at room temperature. Microbes were immobilized to Tissues of human submandibular gland with no pathologic changes were plastic cover slips coated with poly(L-lysine) by centrifugation at 3000 rpm obtained from two patients with advanced oral cancer during surgical re- for 7 min. After fixing with 2% glutaraldehyde for1hatroom temperature, section. Written informed consents were obtained from both patients. Im- samples were rinsed twice in distilled water and dehydrated in a graded munohistochemistry was conducted as described previously (23). In brief, series of ethanol. After treatment with isoamyl acetate, samples were dried periodate-lysine-4% paraformaldehyde-prefixed frozen sections were re- in a critical-point drying apparatus (HCP-1; Hitachi Koki, Tokyo, Japan) acted with mouse monoclonal anti-CCL28 (clone no. 62705; IgG1). As with liquid carbon dioxide. Dried samples were coated with 3-nm-thick negative controls, isotype-matched mouse IgG1 was used (DAKO Japan). platinum-paradium by evaporation in a magnetron sputter coater (E-1030; After that, sections were treated with a goat anti-mouse Envisionϩ/HRP kit Hitachi, Ibaraki, Japan). Specimens were observed on an S-900 ultra-high (DAKO Japan). In the case of staining mouse salivary glands, periodate- resolution scanning electron microscope (Hitachi) with accelerating volt- lysine-4% paraformaldehyde-prefixed frozen sections were first treated age of 10 kV. ϩ with anti-CD16/32 FcR block (Immunotech, Marseille, France) 10% Downloaded from normal rabbit serum and were further treated with the Biotin Blocking Results System (DAKO Japan). Then, sections were successively reacted with goat ϩ anti-mouse CCL28 or normal goat IgG, biotinylated rabbit anti-goat IgG Strong expression of CCL28 and presence of CCR10 cells in (Vector Laboratories), and Vectastain ABC/HRP kit (Vector Laboratories). mouse salivary glands Immunoelectron microscopy CCL28 has been shown to be expressed in a variety of human tissues but most abundantly in colon, trachea, and exocrine glands

This was conducted by adopting the pre-embedding immunoperoxidase such as salivary and mammary glands (3, 4). Therefore, we con- http://www.jimmunol.org/ method as described previously (23). In brief, sections adjacent to those used for immunohistochemistry were processed the same way, except that ducted semiquantitative RT-PCR analysis for expression of

0.3% H2O2/0.1% NaN3 in PBS was used after reaction with primary Ab. CCL28 and its receptors CCR10 and CCR3 in various mouse tis- Sections were then fixed in 0.2% glutaraldehyde for 30 s before the enzy- sues. As shown in Fig. 1, all three salivary glands expressed matic reaction. After incubation with diaminobenzidine solution (Dojin, CCL28 at levels much higher than those in other mucosal tissues. Kumamoto, Japan), sections were embedded in Epon. Notably, however, CCR10 signals were not proportionally ele- ELISA vated in the salivary glands compared with other mucosal tissues. Furthermore, CCR3 signals were lower in the salivary glands than Whole and parotid saliva were collected from healthy subjects. Whole in other mucosal tissues. saliva was collected under resting conditions. Parotid saliva was collected by guest on September 26, 2021 upon gustatory stimulation with 1 mg of vitamin C by using a Teflon catheter. Samples were cleared by centrifugation and stored at Ϫ20¡C until use. Samples of human milk during midlactation were also obtained. To measure CCL28, we developed a sandwich-type ELISA essentially as described previously (16). We used mouse anti-human CCL28 mAb (clone no. 62705) as capturing Ab, biotinylated polyclonal goat anti-human CCL28 (R&D Systems) as detecting Ab, and streptavidin-HRP conjugate (Vector Laboratories) to detect biotinylated second Abs. The detection range of the present ELISA was typically between 1 and 20 ng/ml. Immunoblot Saliva and milk samples were mixed with SDS sample buffer containing 2-ME, heated at 95¡C for 5 min, and run on a 15% polyacrylamide gel. Size-fractionated proteins were electrophoretically transferred to a polyvi- nylidene difluoride membrane (Millipore, Bedford, MA). Filters were successively treated with biotinylated polyclonal goat anti-human CCL28 (0.5 ␮g/ml in TBS-0.05% Tween 20), streptavidin-HRP (5 ␮g/ml in TBS-0.05% Tween 20), and ECL kit (Amersham Pharmacia Biotech, Little Chalfont, U.K.). Antimicrobial assay C. albicans from a clinical isolate was cultured on Sabouraud dextrose agar. Klebsiella pneumoniae NCTC 9632, Pseudomonas aeruginosa ATCC 10145 and Staphylococcus aureus ATCC 6538P were cultured on soybean-casein digest agar plates. Streptococcus mutans ATCC 25175 and Streptococcus pyogenes Cook strain were cultured on trypticase soy agar containing 5% defibrinated sheep blood. Antimicrobial assays were conducted in low ionic conditions essentially as described previously (24, 25). FIGURE 1. Semiquantitative RT-PCR analysis for expression of In brief, microbial cells in mid-logarithmic growth phase were resuspended CCL28, CCR10, and CCR3 in various mouse tissues. Total RNA samples ϳ 5 at 10 cells/ml in 1 mM potassium phosphate buffer (PPB) (pH 7.2), were prepared from salivary glands, colon, appendix, small intestine with- mixed with an equal volume of 1 mM PPB without or with test samples in twofold serial dilutions, and incubated in a U-bottom 96-well microtest out Payer’s patches, and Payer’s patches obtained from 12- to 16-wk-old plates at room temperature for 2 h with brief mixing every 15 min. After BALB/c mice. RT-PCR was conducted as described in Materials and appropriate dilutions with 1 mM PPB, cells were plated on agar plates and Methods. Representative results from three separate experiments are grown overnight at 37¡C. Colonies were counted and percentage viability shown. Relative signal intensities obtained by normalization with G3PDH was obtained. All assays were done at least in triplicate. are shown in the lower panels as mean Ϯ SD. The Journal of Immunology 1455

To demonstrate the role of CCL28 in the salivary glands as a 2A (left), single cells from parotid glands were divided into two chemoattractant for cells expressing CCR10 and/or CCR3, we ex- major fractions: CD3ϪB220low and CD3ϪB220Ϫ. We called the amined single cells prepared from mouse parotid glands for sur- former B220low in comparison with B220high cells observed in face expression of CCR10 and CCR3. For this purpose, we first splenocytes (data not shown). Only a small number of cells were established a binding assay using human CCL27-Fc chimera pro- CD3ϩ. As shown in Fig. 2A (right), the CD3ϪB220low fraction tein. Chemokines fused to the Fc domain of IgG are often used to specifically bound CCL27-Fc at high levels. In contrast, the measure surface expression of chemokine receptors (21). We CD3ϪB220Ϫ fraction hardly bound CCL27-Fc. We were unable to chose CCL27 because it is a monospecific ligand for CCR10 (5, 6). analyze CD3ϩ cells because of their very low numbers. In the case Using a panel of mouse L1.2 stable transfectants expressing human of CCR3, cells from the CD3ϪB220low fraction, but not those from and mouse CCRs, we confirmed that CCL27-Fc specifically bound the CD3ϪB220Ϫ fraction, were also weakly reactive with anti- to human and mouse CCR10 (data not shown). Therefore, we mCCR3. Consistently, even though requiring relatively high con- stained cells from mouse parotid glands for CD3, B220, and centrations, cells from the CD3ϪB220low fraction vigorously mi- CCR10 and analyzed them by flow cytometry. As shown in Fig. grated to mCCL28 and mCCL27 (Fig. 2B, filled bars). Notably, Downloaded from http://www.jimmunol.org/ by guest on September 26, 2021

FIGURE 2. Surface expression of CCR10 and CCR3 and migration to CCL28 of CD3ϪB220low cells isolated from mouse parotid glands. A, Flow cytometric analysis. Single cells were prepared from parotid glands excised from 12- to 16-wk-old BALB/c mice. For detection of cells expressing CCR10, cells were first incubated with CCL27-Fc or control Fc. After washing, cells were incubated with biotin-labeled goat anti- human IgG. After washing, cells were stained with a cocktail of APC-labeled streptavidin, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. For detection of cells expressing CCR3, cells first were incubated with rabbit polyclonal anti-mCCR3 or control rabbit IgG. After washing, cells were incubated with a cocktail of FITC-labeled anti-rabbit IgG, CyChrome-labeled anti-B220, and PE-labeled anti-CD3. Finally, cells were analyzed on FACSCalibur. Representative results from six independent experiments are shown. B, Chemotaxis assay. Single cells prepared from parotid glands were added to the inserts of Transwell plates, with lower wells containing medium without or with indicated concentrations of mCCL2, mCCL28, or mCCL27. After4hat37¡C, cells migrated into lower wells were harvested. Original cells and cells migrated into lower wells were stained with PE-labeled anti-CD3 and CyChrome-labeled anti-B220 and were analyzed by flow cytometry in the presence of a known number of counting beads. Filled bars, CD3ϪB220low cells; open bars, CD3ϪB220Ϫ cells. The data are mean Ϯ SD from three separate experiments. C, Plasma cell morphology of CCR10-expressing cells. Single cells prepared from mouse parotid glands were incubated with CCL27-Fc or control Fc. After washing, cells were stained with FITC-labeled goat anti-human IgG. Cells were then placed on glass slides, fixed with methanol, and further stained with May-Gru¬nwald-Giemsa. Cells in the same fields were observed on a fluorescent microscope under UV (Ca) and visible (Cb) lights. No FITC-staining cells were seen by control Fc (data not shown). Magnification, ϫ1000. 1456 ANTIMICROBIAL ACTIVITY OF CCL28 mCCL28, the ligand of both mCCR10 and mCCR3 (3, 4), tended to induce migration of CD3ϪB220low cells much more efficiently than mCCL27, the ligand of mCCR10 alone (5, 6). In contrast, cells from the CD3ϪB220Ϫ fraction significantly migrated to JE/ mCCL2 but not to mCCL28 or mCCL27 (Fig. 2B, open bars). To gain an insight into CD3ϪB220low cells expressing CCR10 and CCR3, we stained single cells from mouse salivary glands for CCR10 by using CCL27-Fc and FITC-labeled anti-human IgG. The cells were then fixed on glass slides and stained with May- Gru¬nwald-Giemsa. As shown in Fig. 2C, FITC-labeled cells (Fig. 2Ca) consistently displayed the typical morphology of plasma cells such as an eccentric nucleus, low nuclear-to-cytoplasmic ra- tio, a relatively dark basophilic cytoplasm, and a pale perinuclear compartment (Fig. 2Cb). Collectively, the mouse parotid gland contained a large number of CD3ϪB220low cells that expressed CCR10 at high levels and CCR3 at low levels and were capable of responding to CCL28 in chemotaxis assays. Furthermore, parotid cells expressing CCR10 were morphologically plasma cells. These results suggest that Downloaded from CCL28 is involved in the recruitment of plasma cells into the salivary glands. Constitutive expression of CCL28 by epithelial cells of salivary glands

From the above results, CCL28 is likely to play a major role in the http://www.jimmunol.org/ recruitment of plasma cells expressing CCR10 and CCR3 into the salivary glands. Nevertheless, we felt that the levels of expression of CCL28 in the salivary glands were out of proportion to the levels of recruitment of cells expressing CCR10 and/or CCR3 in comparison with other mucosal tissues (Fig. 1). This suggests that FIGURE 3. Immunohistochemistry and immunoelectron microscopy of CCL28 might also have a role different from cell recruitment. To CCL28 in human and mouse salivary glands. Periodate-lysine-4% parafor- explore this possibility, we first conducted immunohistochemical maldehyde-prefixed frozen sections of human submandibular gland (AÐC, staining of CCL28 in human and mouse salivary glands to identify J, and K), mouse parotid gland (D and G), mouse submandibular gland (E cells producing CCL28 (Fig. 3). In human submandibular glands and F), and mouse sublingual gland (F and I) were stained with goat anti- by guest on September 26, 2021 human CCL28 (A, B, and J), goat anti-mouse CCL28 (DÐF), or normal (n ϭ 2), serous acinar epithelial cells were strongly positive for goat IgG (C, GÐI, and K). Immunohistochemistry (AÐI): scale bar, 100 ␮m acinus; ૾, duct. Immunoelectron microscopy (J ,ء ;(CCL28, whereas duct epithelial cells were usually negative (Fig. (AÐC) and 20 ␮m(DÐI 3A). Occasionally, secreted materials within the ducts were and K): scale bar, 1 ␮m; arrow, immunoreactive granule; Lu, lumen. strongly positive for CCL28 (Fig. 3B). No such immunoreactivity was seen with the control Ab (Fig. 3C). The mouse salivary glands were also strongly positive for CCL28 (Fig. 3, DÐF). The epithelial 232 nM in parotid secretions (n ϭ 5). In the same donors, parotid cells in the parotid gland, which is mostly serous, showed a strong secretions consistently contained much higher concentrations of and almost uniform CCL28 immunoreactivity (Fig. 3D). The ep- CCL28 than did whole saliva. We further conducted immunoblot ithelial cells in the submandibular gland, which is the mixed type, analysis of CCL28 in parotid secretions. As shown in Fig. 4B, showed a rather heterogeneous pattern of CCL28 immunoreactiv- immunoblot analysis also confirmed the presence of CCL28 in ity (Fig. 3E), which was quite similar to that of the human sub- parotid secretions at 50Ð200 nM. In nonreducing conditions, mandibular gland (Fig. 3A). The epithelial cells in the sublingual CCL28 was mostly dimer (data not shown). We also detected gland, which is mostly mucinus, were apparently filled with mucus CCL28 in human milk samples (Fig. 4B). CCL28 concentrations in granules, and CCL28 immunoreactivity was confined to the baso- milk samples obtained during midlactation (mature milk) were lateral sides (Fig. 3F). Again, no such immunoreactivity was ob- 13Ð34 nM (n ϭ 8). Collectively, CCL28 is indeed secreted in the served with control Ab (Fig. 3, GÐI). We further examined the salivary and lacteal secretions at high concentrations. subcellular localization of CCL28 in the human submandibular gland by immunoelectron microscopy. As shown in Fig. 3J, Similarity between the C terminus of CCL28 and histatin-5 CCL28 immunoreactivity was localized to the secretory granules Constitutive secretion of a large amount of CCL28 in saliva and within the epithelial cells. Again, no such immunoreactivity was milk suggested a role of CCL28 different from cell recruitment. observed with control Ab (Fig. 3K). Collectively, these results The mature protein of CCL28, which constitutes 105 aa with six strongly suggest that CCL28 is constitutively produced and prob- cysteine residues instead of the standard four, has an extended C ably secreted by the acinar epithelial cells of the salivary glands. terminus (3, 4). We noticed that the 28-aa C-terminal segment after the conserved 4th cysteine residue contains as many as eight his- Secretion of CCL28 in saliva and milk tidine residues. This is quite striking because other chemokines To examine secretion of CCL28 into saliva, we next measured have only a small number of histidine resides after the 4th cysteine CCL28 in whole saliva and vitamin C-stimulated parotid secre- residue; the second highest histidine content is three in CCL27 and tions collected from healthy adult subjects by using a sandwich- CCL25 in human chemokines. Such a high histidine content of the type ELISA. As shown in Fig. 4A, we indeed detected CCL28 at C-terminal segment of CCL28 reminded us of its potential simi- high concentrations: 30Ð63 nM in whole saliva (n ϭ 16) and 65Ð larity to human histatins, which are a family of at least 12 small, The Journal of Immunology 1457 Downloaded from

FIGURE 4. Exocrine secretion of CCL28. A, Measurement of CCL28 in whole and parotid saliva by ELISA. Whole saliva and parotid secretions were obtained from healthy adult donors. All assays were done in triplicate and mean values were calculated. For details, see Materials and Methods. Whole and parotid saliva samples obtained from the same donors were connected by lines. B, Immunoblot analysis for CCL28. Recombinant http://www.jimmunol.org/ CCL28, parotid saliva samples (10 ␮l), and mature milk samples (20 ␮l) were loaded as indicated. For details, see Materials and Methods. Repre- sentative results from two separate experiments are shown.

neutral to basic, histidine-rich peptides secreted into saliva that have a potent antimicrobial activity against C. albicans (20, 26, 27). In Fig. 5A, the amino acid sequences of CCL28-C and hista- by guest on September 26, 2021 tin-5 are compared. The similarity calculated by the BestFit pro- gram from the GCG package is 53%. An important feature of histatin-5 is the presence of a zinc-binding motif HExxH at residues 15Ð19 (Fig. 5A). It has been reported that histatin-5 is capable of fusing negatively charged vesicles only in the presence of zinc FIGURE 5. Analysis of the amino acid sequence of human CCL28. A, (28). CCL28-C also has a similar HExxxH motif at residues 19Ð24 Amino acid comparison of histatin-5 and CCL28-C. The zinc-binding mo- (Fig. 5A), which can also be a zinc-binding site (29). Fig. 5B shows tifs HExxH and HExxxH are underlined. B, Hydrophobicity plot of hista- the hydrophobicity plots generated by the Kyte & Doolittle’s tin-5 and CCL28-C. C, A phylogenic tree of chemokines and antimicrobial method. Histatin-5 and CCL28-C have a similar overall molecular peptides. MEC/CCL28 and CCL28-C are boxed. arrangement of hydrophobic and polarized regions. In a phylogenic tree consisting of chemokines and antimicrobial peptides, histatin-5 and CCL28-C align side by side (Fig. 5C). Most antimicrobial peptides are known to be ineffective in high salt concentrations (10). Therefore, we examined the effect of salt Antimicrobial activity of CCL28 concentrations on the antimicrobial activity of CCL28. As shown The similarity of the C terminus of CCL28 with histatin-5 in Fig. 7, the killing activity of CCL28 against C. albicans and P. prompted us to test antimicrobial activity of CCL28 and its C- aeruginosa was indeed seen only in low salt concentrations. terminal peptide. As shown in Fig. 6A, human CCL28 effectively killed C. albicans, P. aeruginosa and Streptococcus mutans. Rapid induction of microbial membrane permeability by CCL28 Mouse CCL28 also effectively killed C. albicans and Streptococ- We next examined the mechanism of CCL28 antimicrobial activity cus mutans but was less effective against P. aeruginosa. Human against C. albicans by using flow cytometry. We monitored mem- and mouse CCL28 also killed K. pneumoniae, Streptococcus pyo- brane permeability by uptake of PI and alteration in the membrane genes, and Staphylococcus aureus (Table I and data not shown). In phospholipid asymmetry by annexin V. As shown in Fig. 8A, upon contrast, human CCL27 tested in parallel showed no significant treatment with CCL28 at 10 ␮M, 40% of fungal cells were already bactericidal activity and killed C. albicans only at high concentra- PI-positive by 5 min, and Ͼ90% of fungal cells were PI-positive tions. We also compared CCL28-C and histatin-5 for antimicrobial by 15 min. Cells stained with annexin V appeared less rapidly than activity. As shown in Fig. 6B, CCL28-C showed a candidacidal did PI-positive cells. Fig. 8B shows the dose-dependent effects of activity, which was even more potent than that of histatin-5, es- CCL28 determined by the flow cytometric analysis, which were pecially at lower concentrations. CCL28-C and histatin-5, how- highly consistent with those obtained by the colony-forming assay ever, were mostly ineffective in killing bacteria. All these results (Fig. 6). Thus, CCL28 rapidly induced membrane permeability in are summarized in Table I. C. albicans. 1458 ANTIMICROBIAL ACTIVITY OF CCL28 Downloaded from http://www.jimmunol.org/

FIGURE 6. Antimicrobial assay. CCL28, mCCL28, CCL27, CCL28-C, and histatin-5 were examined for antimicrobial activity against C. albicans, P. aeruginosa, and Streptococcus mutans by using the CFU assay. For details, see Materials and Methods. All assays were done in triplicate. Vertical bars indicate SD. Representative results from three separate experiments are shown.

Mode of action of CCL28 analyzed by scanning electron Discussion by guest on September 26, 2021 microscopy Antimicrobial peptides are the evolutionary ancient weapons of mul- To directly visualize the effects of CCL28 on microbial cells, mi- ticellular organisms against a wide range of microbes, including bac- crobial cells mock treated or treated with CCL28 at 10 ␮M were teria, fungi, protozoa, and enveloped viruses (9, 10). These molecules observed by scanning electron microscopy (Fig. 9). Whereas are expressed in a cocktail in each species and are considered to play mock-treated C. albicans had a smooth surface (Fig. 9, A and D), important roles in the innate immunity against microbes. Structurally, those treated with CCL28 for 30 min clearly showed numerous antimicrobial peptides are grouped roughly into four classes, which surface blebs with frequent projections of cellular debris arising comprise anti-parallel ␤-sheet peptides stabilized by two to three di- from the cytoplasm (Fig. 9, B and E). In 60 min, most cells were sulﬁde bridges, amphipathic ␣-helical peptides, proline-rich coiled burst (Fig. 9, C and F). We observed similar morphological peptides, and looped or cyclic peptides (10). The fundamental struc- changes in P. aeruginosa and Streptococcus mutans treated with tural principal underlying all classes of antimicrobial peptides seems CCL28 (Fig. 9, H and J, respectively) compared with mock-treated to be the amphipathic design, in which clusters of hydrophobic and cells (Fig. 9, G and I, respectively). These observations are con- cationic amino acids are organized in discrete sectors (10). The basis sistent with the notion that CCL28 directly attacks the plasma of the selectivity of these peptides against microbes is considered to membrane of microbial cells and generates pores, leading to leaks be due to the relative abundance of negative charges in the microbial of cellular contents and eventual burst of target cells. cell membranes compared with those of the host’s cells (10). Thus,

Table I. Summary of antimicrobial activitya

␮ IC50 ( M)

Microbe CCL28 mCCL28 CCL27 CCL28-C Histatin-5

P. aeruginosa 0.4 Ϯ 0.1 1.7 Ϯ 0.1 Ͼ10 Ͼ10 Ͼ10 K. pneumoniae 0.3 Ϯ 0.1 1.6 Ϯ 0.1 Ͼ10 Ͼ10 3.0 Ϯ 0.7 S. mutans 1.7 Ϯ 0.4 1.5 Ϯ 0.3 Ͼ10 Ͼ10 Ͼ10 S. pyogenes 3.0 Ϯ 0.2 4.5 Ϯ 0.4 Ͼ10 Ͼ10 Ͼ10 S. aureus 0.9 Ϯ 0.1 0.9 Ϯ 0.1 Ͼ10 7.0 Ϯ 1.2 Ͼ10 C. albicans 0.7 Ϯ 0.2 1.3 Ϯ 1.0 5.0 Ϯ 1.9 1.6 Ϯ 0.4 3.5 Ϯ 1.6

a Colony-forming assay was carried out as described in Materials and Methods, and IC50 was obtained. Data represent the mean Ϯ SD from at least three independent experiments. The Journal of Immunology 1459 Downloaded from

FIGURE 7. Salt-sensitive antimicrobial activity of CCL28. The effects http://www.jimmunol.org/ FIGURE 9. Antimicrobial activity of CCL28 analyzed by scanning of NaCl concentrations on the antimicrobial activity of CCL28 against C. electron microscopy. C. albicans (AÐF) was mock treated (A and D)or albicans and P. aeruginosa were examined by using CFU assay. For de- treated with 10 ␮M CCL28 for 30 min (B and E)or60min(C and F). P. tails, see Materials and Methods. All assays were done in triplicate. Ver- aeruginosa (G and H) and Streptococcus mutans (I and J) were mock tical bars indicate SD. Representative results from two separate experi- treated (G and I) or treated with 10 ␮M CCL28 for2h(H and J). Microbes ments are shown. were immobilized, dried, coated with 3-nm thick platinum-paradium, and observed by a scanning electron microscope. Scale bars: A,2␮m; D, 200 nm; and G, 300 nm. these peptides are selectively attracted to the microbial plasma membrane by the mechanism of electrostatic interactions, which explains by guest on September 26, 2021 why most antimicrobial peptides are ineffective at high salt concen- an important component of innate host defense against skin infec- trations. Subsequently, most peptides are considered to be spontane- tion by Group A streptococcus in mice. ously inserted into the membrane and to generate physical holes that In the present study, we have demonstrated that mouse salivary cause cellular contents to leak out (10). For a long time, despite glands express CCL28 mRNA at extremely high levels (Fig. 1). ample demonstrations of the presence of various antimicrobial pep- We have further shown that mouse parotid glands contain a large Ϫ low tides in a wide variety of species, the definitive proof of their in fraction of CD3 B220 cells that express CCR10 at high levels vivo roles in host defense against microbial invasions has been dif- and CCR3 at low levels and vigorously respond to CCL28 in che- ficult to obtain. Recently, however, Nizet et al. (30) have generated motaxis assays (Fig. 2). Morphologically, these cells are mostly mice with targeted disruption of Cnlp encoding cathelicidin antimi- plasma cells (Fig. 2). This is the first time that plasma cells have crobial peptides and have demonstrated that cathelicidins are indeed been shown to express CCR10 and CCR3 (31). Thus, the salivary glands attract plasma cells obviously by producing CCL28. How- ever, the very high levels of expression of CCL28 in salivary glands may suggest that CCL28 also has a role in salivary glands that is different from cell recruitment. In this context, we have demonstrated that 1) CCL28 is produced by acinar epithelial cells of human and mouse salivary glands (Fig. 3), 2) CCL28 is secreted into human saliva and milk at high concentrations (Fig. 4), 3) the extended C terminus of CCL28 has a sequence similarity to candidacidal peptide histatin-5 (Fig. 5), and 4) human and mouse CCL28 have a potent antimicrobial activity against a broad spectrum of microbes including C. albicans, Gram-positive bacteria, and Gram-negative bacteria (Fig. 6; Table I). Given the particularly strong and selective expression of CCL28 mRNA in certain mucosal tissues such as salivary glands, mammary glands, trachea, and large intestine (3, 4), which commonly secrete low-salt fluids because of reabsorption of sodium ions by the epithelial cells ex- FIGURE 8. Flow cytometric analysis on CCL28 antimicrobial activity. C. albicans were treated with CCL28 as indicated. After washing, cells pressing the epithelial sodium channel (19), one important function were stained with FITC-labeled annexin V for 10 min and were washed of CCL28 in these tissues may be that it is apically secreted as an again. After addition of PI at 2 ␮g/ml, cells were immediately analyzed on antimicrobial protein and protects the mucosal surfaces against FACSCalibur. The representative results of three independent experiments colonizing microbes. Chemokines are well known to bind to hepa- are shown. ran sulfate (32), an abundant component of epithelial cell surface 1460 ANTIMICROBIAL ACTIVITY OF CCL28 and extracellular matrix. This ability is considered to keep chemo- peptides, especially the family of defensins, have diverged from a kines locally concentrated in the vicinity of producing cells and to common primordial molecule. Thus, some old-type chemokines form a gradient within the tissue (1). The same ability is also likely such as CCL28 still retain antimicrobial activities and function as to help secreted CCL28 to be highly concentrated on the mucosal such. However, it is rather striking that CCL27, the chemokine surfaces to form a barrier shield against colonizing microbes. most similar to CCL28, hardly displays a significant anti-microbial Thus, even though the concentrations of CCL28 found free in sa- activity (Table I). Thus, another possibility is that the antimicrobial liva and milk are relatively low compared with its IC50 for various activity of CCL28, as well as its histatin-like C terminus, has been microbes, its high surface concentrations on the mucosal surfaces fortuitously generated through a convergent evolution of a chemo- together with synergistic effects with other antimicrobial factors kine (CCL28) to be an antimicrobial protein as well. In either case, rich in mucosal secretions (33) may provide a highly effective the functional and evolutionary relationships between chemokines barrier protection against a wide spectrum of microbes. Notably, and antimicrobial peptides in innate and adaptive immunity will be autologous skin flaps transplanted into the oral cavity to recon- an interesting subject in future studies (11). struct large tissue defects after radical resection of oral cancer are In conclusion, CCL28 has dual functions in mucosal immunity known to be frequently infected with C. albicans (23). Skin sur- as a chemokine attracting cells expressing CCR10 and/or CCR3 faces, which may be less effectively covered by CCL28 than nor- and also as an apically secreted molecule with a potent antimicro- mal oral mucosa, may be easily colonized by C. albicans even in bial activity against a broad spectrum of microbes. Future studies the presence of high concentrations of histatins and other antimi- using CCL28-knockout mice or neutralization of CCL28 activity crobial agents in the saliva. in vivo will further define its roles in innate and adaptive immunity

Based on the amino acid sequence similarity between the C- as a chemokine and as an antimicrobial protein. Downloaded from terminal region of CCL28 and histatin-5 (Fig. 5), we originally expected that the antimicrobial mechanism of CCL28 was similar Acknowledgment to that of histatin-5, which selectively binds to the mitochondrial We thank Dr. Hiroshi Shiraishi for collecting saliva samples. membrane after internalization into yeast cells (34, 35). However, we now consider this unlikely because of the following observa- References tions: 1) CCL28 shows a much broader spectrum of antimicrobial 1. Yoshie, O., T. Imai, and H. Nomiyama. 2001. Chemokines in immunity. Adv. http://www.jimmunol.org/ activity than does histatin-5, which is mainly effective against Immunol. 78:57. Candida species (26), 2) CCL28 induces a rapid membrane per- 2. Zlotnik, A., and O. Yoshie. 2000. Chemokines: a new classification system and their role in immunity. Immunity 12:121. meability in target microbes, in contrast with the reported rela- 3. Pan, J., E. J. Kunkel, U. Gosslar, N. Lazarus, P. Langdon, K. Broadwell, tively slow antimicrobial effect of histatin-5 (34), and 3) FITC- M.A. Vierra, M. C. Genovese, E. C. Butcher, and D. Soler. 2000. A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal labeled CCL28 mainly stained cell surface of C. albicans (data not tissues. J. Immunol. 165:2943. shown), in contrast with the reported association of histatin-5 to 4. Wang, W., H. Soto, E. R. Oldham, M. E. Buchanan, B. Homey, D. Catron, mitochondria membrane within yeast cells (25). Like most other N. Jenkins, N. G. Copeland, D. J. Gilbert, N. Nguyen, et al. 2000. Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2). J. Biol. Chem. antimicrobial peptides (10), CCL28 apparently exerts its antimi- 275:22313. crobial activity by spontaneous membrane insertion and pore for- 5. Homey, B., W. Wang, H. Soto, M. E. Buchanan, A. Wiesenborn, D. Catron, by guest on September 26, 2021 mation in target microbes (Figs. 8 and 9). It remains to be seen A. Muller, T. K. McClanahan, M. C. Dieu-Nosjean, R. Orozco, et al. 2000. Cutting edge: the orphan chemokine receptor G protein-coupled receptor-2 whether the mode of action of CCL28-C, the 28-aa C-terminal (GPR-2, CCR10) binds the skin-associated chemokine CCL27 (CTACK/ALP/ peptide of CCL28, which has a sequence similarity with histatin-5 ILC). J. Immunol. 164:3465. and shows a selective killing of C. albicans like histatin-5 (Fig. 6), 6. Jarmin, D. I., M. Rits, D. Bota, N. P. Gerard, G. J. Graham, I. Clark-Lewis, and C. Gerard. 2000. Cutting edge: identification of the orphan receptor G-protein- is similar to that of histatin-5. coupled receptor 2 as CCR10, a specific receptor for the chemokine ESkine. Because most antimicrobial peptides are generated from a larger J. Immunol. 164:3460. 7. Morales, J., B. Homey, A. P. Vicari, S. Hudak, E. Oldham, J. Hedrick, R. Orozco, precursor protein through proteolytic processing (10), CCL28 may N. G. Copeland, N. A. Jenkins, L. M. McEvoy, and A. Zlotnik. 1999. CTACK, also function as a precursor of smaller antimicrobial peptides such a skin- associated chemokine that preferentially attracts skin-homing memory T as CCL28-C. So far, however, we have obtained no evidence sup- cells. Proc. Natl. Acad. Sci. USA 96:14470. 8. Ishikawa-Mochizuki, I., M. Kitaura, M. Baba, T. Nakayama, D. Izawa, T. Imai, porting proteolytic processing of CCL28. For example, the immu- H. Yamada, K. Hieshima, R. Suzuki, H. Nomiyama, and O. Yoshie. 1999. Mo- noblot analysis clearly demonstrated that natural CCL28 in parotid lecular cloning of a novel CC chemokine, interleukin-11 receptor ␣-locus che- and lacteal secretions was identical with recombinant CCL28 in mokine (ILC), which is located on chromosome 9p13 and a potential homologue of a CC chemokine encoded by Molluscum contagiosum virus. FEBS Lett. 460: size (Fig. 4). Furthermore, CCL28 showed a much broader spec- 544. trum of antimicrobial activity than did CCL28-C (Table I). Thus, 9. Ganz, T., and R. I. Lehrer. 1994. Defensins. Curr. Opin. Immunol. 6:584. 10. Zasloff, M. 2002. Antimicrobial peptides of multicellular organisms. Nature 415: CCL28 itself is likely to be a natural antimicrobial protein, and the 389. whole molecule of CCL28 is important for its efficient antimicro- 11. Yang, D., A. Biragyn, L. W. Kwak, and J. J. Oppenheim. 2002. Mammalian bial activity. defensins in immunity: more than just microbicidal. Trends Immunol. 23:291. 12. Territo, M. C., T. Ganz, M. E. Selsted, and R. Lehrer. 1989. Monocyte-chemo- What could have been an evolutionary process that has led tactic activity of defensins from human neutrophils. J. Clin. Invest. 84:2017. CCL28 to be both a chemokine and an antimicrobial protein? We 13. Chertov, O., D. F. Michiel, L. Xu, J. M. Wang, K. Tani, W. J. 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