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Departments of
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Microbiology and
Gynecology and Obstetrics, Kinki University School of Medicine, Osaka, Japan;
Department of Pathology, Tohoku University Graduate School of Medicine, Miyagi, Japan;
Department of Oral and Maxillofacial Surgery 1, Tohoku University School of Dentistry, Miyagi, Japan; and
¶ Department of Biotechnological Science, Kinki University, Wakayama, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-locus chemokine or cutaneous T cell-attracting chemokine) (5, 6), whereas CCR3 is the receptor for eotaxin/CCL11 and many other chemokines known to act on eosinophils (1). CCL28 transcripts are detected in a variety of tissues but most abundantly in trachea, colon, rectum, and exocrine glands such as salivary and mammary glands (3, 4). In the colon, CCL28 is selectively expressed by the epithelial cells (3, 4). Structurally, CCL28 is most similar to CCL27, which is expressed highly selectively in the skin and has been shown to attract cutaneous lymphocyte Ag+ memory T cells via CCR10 (5, 6, 7, 8). Consistently, CCL28 has also been shown to attract cutaneous lymphocyte Ag+ memory T cells via CCR10 and eosinophils via CCR3 among human peripheral blood leukocytes (3).
Antimicrobial peptides, now known by >500 in number, are the diverse family of small, mostly cationic polypeptides found widely in all forms of multicellular organisms that exert a broad spectrum of cytotoxic activity against bacteria, fungi, parasites, and enveloped viruses (9, 10). In mammals, such peptides are particularly abundant in the storage granules of phagocytic cells and on the surface of mucosal tissues. Accumulating evidence points out that chemokines and antimicrobial peptides have substantially overlapping functions (11). For example, several members of the mammalian antimicrobial proteins are capable of attracting leukocytes via interactions with selected seven-transmembrane G protein-coupled receptors (12, 13, 14). Most notably, Yang et al. (15) have recently demonstrated that human 
-defensins are potent agonists for CCR6, the receptor for the chemokine CCL20 (also called liver and activation-regulated chemokine or macrophage inflammatory protein-3), which is produced by mucosal epithelial cells and epidermal keratinocytes upon proinflammatory stimulations that selectively attracts immature dendritic cells, memory T cells, and B cells (1, 16). In contrast, some chemokines have been shown to have antimicrobial activity. Krijgsved et al. (17) purified antibacterial proteins stored in the -granules of human platelets and determined their amino acid sequences. Surprisingly, these proteins turned out to be CXCL7 (59–126) and CXCL7 (44–126), two related chemokine variants processed from a common precursor platelet basic protein and truncated by 2 aa in the C terminus (17). Furthermore, Cole et al. (18), by examining a panel of 11 chemokines representing all four chemokine subfamilies, demonstrated that the three IFN-inducible non-ELR (Glu-Leu-Arg)-motif CXC chemokines, monokine induced by IFN-
/CXCL9, IFN-inducible protein-10/CXCL10, and IFN-inducible T-cell chemoattractant/CXCL11, had a significant antimicrobial activity against Escherichia coli and Listeria monocytogenes. However, the physiological relevance of the antimicrobial activity of CXCL7 variants present in the platelets or the three non-ELR CXC chemokines commonly inducible by IFN- is still unclear because their antibacterial activity was seen only at high concentrations and also, as with most other antimicrobial peptides, in low salt conditions, which are only possible in certain body surface secretions, but not within the circulation or tissues.
Here we demonstrate that 1) CCL28 is expressed at high levels in mouse salivary glands, 2) the major CD3-B220low plasma cell fraction in mouse salivary glands expresses its receptor CCR10 at high levels and CCR3 at low levels, 3) CCL28 is constitutively produced by the epithelial cells of human and mouse salivary glands, 4) CCL28 is secreted in human saliva and milk at high concentrations, 5) the expended C-terminal region of human CCL28 has a sequence similarity with histatin-5, a histidine-rich candidacidal peptide secreted into human saliva (19), and 6) CCL28 exerts a potent and salt-sensitive antimicrobial activity against a broad spectrum of microbes including Candida albicans, Gram-negative bacteria, and Gram-positive bacteria. Importantly, the tissues that express CCL28 at high levels such as salivary glands, mammary glands, respiratory tract, and distal colon commonly secrete low-salt fluids because of selective reabsorption of sodium ions by the epithelial systems (20). Thus, CCL28 may represent the first chemokine that not only attracts cells expressing its receptors CCR10 and CCR3 into certain mucosal tissues but also is constitutively secreted by epithelial cells in such tissues and functions as a potent antimicrobial factor in physiological settings. CCL28 thus adds further evidence for the close functional and evolutionary relationships between chemokines and antimicrobial peptides (11).
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Materials and Methods |
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Recombinant human and mouse chemokines were all purchased from R&D Systems (Minneapolis, MN). Mouse monoclonal anti-human CCL28 (clone no. 62705; IgG1), goat polyclonal anti-human CCL28, biotin-labeled goat polyclonal anti-human CCL28, and goat polyclonal anti-mouse CCL28 were also purchased from R&D Systems. Isotype-matched IgG controls were purchased from DAKO Japan (Kyoto, Japan). The C-terminal 28 aa of CCL28 (CCL28-C; HRKKHHGKRNSNRAHQGKHETYGH KTPY)4 was chemically synthesized and purified by reverse-phase HPLC to >98% purity by Sawady Technology (Tokyo, Japan).
Generation of CCL27-Fc chimera protein
To detect surface expression of CCR10, we used the technique of a chemokine-Fc chimera protein as described previously (21). In brief, the coding sequence of human CCL27 was amplified from pDREF-ILC-Flag by using PCR (8) and was subcloned into a pcDNA3.1-Fc cassette, which encodes the human IgG1 Fc domain with a mutation that abolishes its binding to Fc receptors (22). Human embryonic kidney 293T cells were transiently transfected with the CCL27-Fc and control Fc constructs, and CCL27-Fc and control Fc proteins were purified from the culture supernatants by using Protein A-agarose (21). Purified CCL27-Fc and control Fc were 76 kDa and 55 kDa, respectively, which was in agreement with the predicted molecular mass. By using a panel of L1.2 cell lines covering all human and murine CC chemokine receptors (CCR1–10), we confirmed that CCL27-Fc specifically bound to human and murine CCR10 (data not shown).
Preparation of single cells from mouse parotid glands
Female BALB/c mice were purchased from CLEA Japan (Osaka, Japan). After cervical dislocation, parotid glands were excised from 12- to 16-wk-old mice, carefully avoiding adjacent cervical lymph nodes. Excised parotid glands were gently disrupted by a pair of glass slides, and cell suspensions were passed through nylon mesh to remove aggregates and cell debris. After washing, single cells were suspended in a medium appropriate for subsequent experiments.
RT-PCR analysis
This was conducted essentially as described previously (16). In brief, various tissues were carefully excised from 12- to 16-wk-old female BALB/c mice. Total RNA was prepared from freshly isolated tissues by using Trizol reagent (Life Technologies, Gaithersburg, MD) and RNeasy (Qiagen, Hilden, Germany). Reverse transcription of total RNA (1 µg) was conducted using oligo(dT)18 primer and SuperScript II reverse transcriptase (Life Technologies). First-strand DNA (20 ng total RNA equivalent) and original total RNA (20 ng) were amplified in a final volume of 20 µl containing 10 pmol of each primer and 1 U of Ex-Taq polymerase (Takara, Kyoto, Japan). The primers used were as follows: +5'-CATACTTCCCATGGCCTCC-3' and -5'-GAGAGGCTTCGTGCCTGTG-3' for mouse CCL28 (mCCL28); +5'-AGAGCTCTGTTACAAGGCTGATGTC-3' and -5'-CAGGTGGTACTTCCTAGATTCCAGC-3' for mCCR10; +5'-TTGCAGGACTGGCAGCATT-3' and -5'-CCATAACGAGGAGAGGAAGAGCTA-3' for mCCR3; +5'-GCCAAGGTCATCCATGACAACTTTGG-3' and -5'-GCCTGCTTCACCACCTTCTTGATGTC-3' for G3PDH. The amplification conditions, which were carefully chosen to obtain signals in a linear amplification range, were denaturation at 94°C for 30 s (5 min at the first 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, and 27 cycles for G3PDH. Amplification products (10 µl each) were loaded onto 2% agarose, run by electrophoresis, and visualized by staining with ethidium bromide.
Flow cytometric analysis
Single cells prepared from mouse parotid glands were suspended in ice-cold PBS containing 3% FBS and 0.1% sodium azide (staining medium). All the following steps were conducted on ice. For detection of surface CCR10, cells were incubated with 1 µg/ml of CCL27-Fc or control Fc for 30 min. After washing, cells were incubated with biotin-labeled goat anti-human Ig (Vector Laboratories, Burlingame, CA) for 30 min. After washing, 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 Jose, CA) for 30 min. After washing, cells were analyzed on a FACSCalibur (BD Biosciences, Mountain View, CA). For detection of surface CCR3, cells were incubated with rabbit polyclonal anti-mCCR3 (BD PharMingen) or control rabbit IgG for 30 min. After washing, cells were stained with a cocktail of FITC-labeled anti-rabbit IgG, CyChrome-labeled anti-B220, and PE-labeled anti-CD3 for 30 min. After washing, cells were analyzed on a FACSCalibur.
May-Grünwald-Giemsa staining of CCR10-expressing cells
Single cells prepared from mouse parotid glands were incubated with 1 µg/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) for 30 min. After washing, cells were placed on glass slides, air dried, and fixed with methanol. Cells were further stained with May-Grünwald-Giemsa. Finally, cells in the same fields were examined on a fluorescent microscope under UV and visible lights.
Chemotaxis assay
This was conducted using Transwell plates with 5- or 8-µm pore inserts (Corning Costar, Cambridge, MA). In brief, single cells prepared from mouse parotid glands were suspended at 2 x 106/ml in RPMI 1640 containing 1 mg/ml BSA (Sigma-Aldrich, St. Louis, MO) and 20 mM HEPES (pH 7.4) and applied to upper wells (100 µl/well). The same medium without or with chemokine was applied to lower wells (600 µl/well). After 4 h at 37°C, inserts were removed and a known number of counting beads (BD PharMingen) were added to each well. The content of each well was transferred to a polypropylene pointed-bottom tube. The beads and cells were pelleted by centrifugation at 200 x g for 5 min, resuspended in the staining medium (see above), and stained with CyChrome-labeled anti-B220 and PE-labeled anti-CD3 as described above. After washing, cells were analyzed on a FACSCalibur. All assays were done in duplicate.
Immunohistochemistry
Tissues of human submandibular gland with no pathologic changes were obtained from two patients with advanced oral cancer during surgical resection. Written informed consents were obtained from both patients. Immunohistochemistry was conducted as described previously (23). In brief, periodate-lysine-4% paraformaldehyde-prefixed frozen sections were reacted with mouse monoclonal anti-CCL28 (clone no. 62705; IgG1). As negative controls, isotype-matched mouse IgG1 was used (DAKO Japan). After that, sections were treated with a goat anti-mouse Envision+/HRP kit (DAKO Japan). In the case of staining mouse salivary glands, periodate-lysine-4% paraformaldehyde-prefixed frozen sections were first treated with anti-CD16/32 FcR block (Immunotech, Marseille, France) + 10% normal rabbit serum and were further treated with the Biotin Blocking System (DAKO Japan). Then, sections were successively reacted with goat anti-mouse CCL28 or normal goat IgG, biotinylated rabbit anti-goat IgG (Vector Laboratories), and Vectastain ABC/HRP kit (Vector Laboratories).
Immunoelectron microscopy
This was conducted by adopting the pre-embedding immunoperoxidase method as described previously (23). In brief, sections adjacent to those used for immunohistochemistry were processed the same way, except that 0.3% H2O2/0.1% NaN3 in PBS was used after reaction with primary Ab. Sections were then fixed in 0.2% glutaraldehyde for 30 s before the enzymatic reaction. After incubation with diaminobenzidine solution (Dojin, Kumamoto, Japan), sections were embedded in Epon.
ELISA
Whole and parotid saliva were collected from healthy subjects. Whole saliva was collected under resting conditions. Parotid saliva was collected upon gustatory stimulation with 1 mg of vitamin C by using a Teflon catheter. Samples were cleared by centrifugation and stored at -20°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 polyvinylidene difluoride membrane (Millipore, Bedford, MA). Filters were successively treated with biotinylated polyclonal goat anti-human CCL28 (0.5 µg/ml in TBS-0.05% Tween 20), streptavidin-HRP (5 µg/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). In brief, microbial cells in mid-logarithmic growth phase were resuspended at 
105 cells/ml in 1 mM potassium phosphate buffer (PPB) (pH 7.2), 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 plates at room temperature for 2 h with brief mixing every 15 min. After appropriate dilutions with 1 mM PPB, cells were plated on agar plates and grown overnight at 37°C. Colonies were counted and percentage viability was obtained. All assays were done at least in triplicate.
We also analyzed candidacidal activity of CCL28 by flow cytometry. Candia cells in mid-logarithmic growth phase were suspended in 1 mM PPB (pH 7.2) and treated without or with various concentrations of CCL28 at 37°C for indicated lengths of time. After washing, cells were incubated with FITC-labeled annexin V (Wako, Osaka, Japan) for 10 min. After washing, cells were resuspended in buffer containing 2 µg/ml propidium iodide (PI) and were immediately analyzed on a FACSCalibur.
Scanning electron microscopy
Microbes suspended in 1 mM PPB (pH 7.2) were mock treated or treated with 10 µM CCL28 at room temperature. Microbes were immobilized to plastic cover slips coated with poly(L-lysine) by centrifugation at 3000 rpm for 7 min. After fixing with 2% glutaraldehyde for 1 h at room temperature, samples were rinsed twice in distilled water and dehydrated in a graded series of ethanol. After treatment with isoamyl acetate, samples were dried in a critical-point drying apparatus (HCP-1; Hitachi Koki, Tokyo, Japan) with liquid carbon dioxide. Dried samples were coated with 3-nm-thick platinum-paradium by evaporation in a magnetron sputter coater (E-1030; Hitachi, Ibaraki, Japan). Specimens were observed on an S-900 ultra-high resolution scanning electron microscope (Hitachi) with accelerating voltage of 10 kV.
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Results |
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CCL28 has been shown to be expressed in a variety of human tissues but most abundantly in colon, trachea, and exocrine glands such as salivary and mammary glands (3, 4). Therefore, we conducted semiquantitative RT-PCR analysis for expression of CCL28 and its receptors CCR10 and CCR3 in various mouse tissues. As shown in Fig. 1, all three salivary glands expressed CCL28 at levels much higher than those in other mucosal tissues. Notably, however, CCR10 signals were not proportionally elevated in the salivary glands compared with other mucosal tissues. Furthermore, CCR3 signals were lower in the salivary glands than in other mucosal tissues.
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Collectively, the mouse parotid gland contained a large number of CD3-B220low 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 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 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 CCL28 might also have a role different from cell recruitment. To explore this possibility, we first conducted immunohistochemical staining of CCL28 in human and mouse salivary glands to identify cells producing CCL28 (Fig. 3). In human submandibular glands (n = 2), serous acinar epithelial cells were strongly positive for CCL28, whereas duct epithelial cells were usually negative (Fig. 3A). Occasionally, secreted materials within the ducts were 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, D–F). The epithelial cells in the parotid gland, which is mostly serous, showed a strong and almost uniform CCL28 immunoreactivity (Fig. 3D). The epithelial cells in the submandibular gland, which is the mixed type, showed a rather heterogeneous pattern of CCL28 immunoreactivity (Fig. 3E), which was quite similar to that of the human submandibular gland (Fig. 3A). The epithelial cells in the sublingual gland, which is mostly mucinus, were apparently filled with mucus granules, and CCL28 immunoreactivity was confined to the basolateral sides (Fig. 3F). Again, no such immunoreactivity was observed with control Ab (Fig. 3, G–I). We further examined the subcellular localization of CCL28 in the human submandibular gland by immunoelectron microscopy. As shown in Fig. 3J, CCL28 immunoreactivity was localized to the secretory granules within the epithelial cells. Again, no such immunoreactivity was observed with control Ab (Fig. 3K). Collectively, these results strongly suggest that CCL28 is constitutively produced and probably secreted by the acinar epithelial cells of the salivary glands.
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To examine secretion of CCL28 into saliva, we next measured CCL28 in whole saliva and vitamin C-stimulated parotid secretions collected from healthy adult subjects by using a sandwich-type ELISA. As shown in Fig. 4A, we indeed detected CCL28 at high concentrations: 30–63 nM in whole saliva (n = 16) and 65–232 nM in parotid secretions (n = 5). In the same donors, parotid secretions consistently contained much higher concentrations of CCL28 than did whole saliva. We further conducted immunoblot analysis of CCL28 in parotid secretions. As shown in Fig. 4B, immunoblot analysis also confirmed the presence of CCL28 in parotid secretions at 50–200 nM. In nonreducing conditions, CCL28 was mostly dimer (data not shown). We also detected CCL28 in human milk samples (Fig. 4B). CCL28 concentrations in milk samples obtained during midlactation (mature milk) were 13–34 nM (n = 8). Collectively, CCL28 is indeed secreted in the salivary and lacteal secretions at high concentrations.
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Constitutive secretion of a large amount of CCL28 in saliva and milk suggested a role of CCL28 different from cell recruitment. The mature protein of CCL28, which constitutes 105 aa with six cysteine residues instead of the standard four, has an extended C terminus (3, 4). We noticed that the 28-aa C-terminal segment after the conserved 4th cysteine residue contains as many as eight histidine residues. This is quite striking because other chemokines have only a small number of histidine resides after the 4th cysteine residue; the second highest histidine content is three in CCL27 and CCL25 in human chemokines. Such a high histidine content of the C-terminal segment of CCL28 reminded us of its potential similarity to human histatins, which are a family of at least 12 small, 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 histatin-5 are compared. The similarity calculated by the BestFit program from the GCG package is 53%. An important feature of histatin-5 is the presence of a zinc-binding motif HExxH at residues 15–19 (Fig. 5A). It has been reported that histatin-5 is capable of fusing negatively charged vesicles only in the presence of zinc (28). CCL28-C also has a similar HExxxH motif at residues 19–24 (Fig. 5A), which can also be a zinc-binding site (29). Fig. 5B shows the hydrophobicity plots generated by the Kyte & Doolittle’s method. Histatin-5 and CCL28-C have a similar overall molecular 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).
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The similarity of the C terminus of CCL28 with histatin-5 prompted us to test antimicrobial activity of CCL28 and its C-terminal peptide. As shown in Fig. 6A, human CCL28 effectively killed C. albicans, P. aeruginosa and Streptococcus mutans. Mouse CCL28 also effectively killed C. albicans and Streptococcus mutans but was less effective against P. aeruginosa. Human and mouse CCL28 also killed K. pneumoniae, Streptococcus pyogenes, and Staphylococcus aureus (Table I and data not shown). In contrast, human CCL27 tested in parallel showed no significant bactericidal activity and killed C. albicans only at high concentrations. We also compared CCL28-C and histatin-5 for antimicrobial activity. As shown in Fig. 6B, CCL28-C showed a candidacidal activity, which was even more potent than that of histatin-5, especially at lower concentrations. CCL28-C and histatin-5, however, were mostly ineffective in killing bacteria. All these results are summarized in Table I.
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We next examined the mechanism of CCL28 antimicrobial activity against C. albicans by using flow cytometry. We monitored membrane permeability by uptake of PI and alteration in the membrane phospholipid asymmetry by annexin V. As shown in Fig. 8A, upon treatment with CCL28 at 10 µM, 40% of fungal cells were already PI-positive by 5 min, and >90% of fungal cells were PI-positive by 15 min. Cells stained with annexin V appeared less rapidly than did PI-positive cells. Fig. 8B shows the dose-dependent effects of CCL28 determined by the flow cytometric analysis, which were highly consistent with those obtained by the colony-forming assay (Fig. 6). Thus, CCL28 rapidly induced membrane permeability in C. albicans.
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To directly visualize the effects of CCL28 on microbial cells, microbial cells mock treated or treated with CCL28 at 10 µM were observed by scanning electron microscopy (Fig. 9). Whereas mock-treated C. albicans had a smooth surface (Fig. 9, A and D), those treated with CCL28 for 30 min clearly showed numerous surface blebs with frequent projections of cellular debris arising from the cytoplasm (Fig. 9, B and E). In 60 min, most cells were burst (Fig. 9, C and F). We observed similar morphological changes in P. aeruginosa and Streptococcus mutans treated with CCL28 (Fig. 9, H and J, respectively) compared with mock-treated cells (Fig. 9, G and I, respectively). These observations are consistent with the notion that CCL28 directly attacks the plasma membrane of microbial cells and generates pores, leading to leaks of cellular contents and eventual burst of target cells.
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Discussion |
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-sheet peptides stabilized by two to three disulfide bridges, amphipathic -helical peptides, proline-rich coiled peptides, and looped or cyclic peptides (10). The fundamental structural principal underlying all classes of antimicrobial peptides seems to be the amphipathic design, in which clusters of hydrophobic and cationic amino acids are organized in discrete sectors (10). The basis of the selectivity of these peptides against microbes is considered to be due to the relative abundance of negative charges in the microbial cell membranes compared with those of the host’s cells (10). Thus, these peptides are selectively attracted to the microbial plasma membrane by the mechanism of electrostatic interactions, which explains why most antimicrobial peptides are ineffective at high salt concentrations. Subsequently, most peptides are considered to be spontaneously inserted into the membrane and to generate physical holes that cause cellular contents to leak out (10). For a long time, despite ample demonstrations of the presence of various antimicrobial peptides in a wide variety of species, the definitive proof of their in vivo roles in host defense against microbial invasions has been difficult to obtain. Recently, however, Nizet et al. (30) have generated mice with targeted disruption of Cnlp encoding cathelicidin antimicrobial peptides and have demonstrated that cathelicidins are indeed an important component of innate host defense against skin infection by Group A streptococcus in mice. In the present study, we have demonstrated that mouse salivary glands express CCL28 mRNA at extremely high levels (Fig. 1). We have further shown that mouse parotid glands contain a large fraction of CD3-B220low cells that express CCR10 at high levels and CCR3 at low levels and vigorously respond to CCL28 in chemotaxis assays (Fig. 2). Morphologically, these cells are mostly plasma cells (Fig. 2). This is the first time that plasma cells have been shown to express CCR10 and CCR3 (31). Thus, the salivary glands attract plasma cells obviously by producing CCL28. However, 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 expressing the epithelial sodium channel (19), one important function of CCL28 in these tissues may be that it is apically secreted as an antimicrobial protein and protects the mucosal surfaces against colonizing microbes. Chemokines are well known to bind to heparan sulfate (32), an abundant component of epithelial cell surface and extracellular matrix. This ability is considered to keep chemokines locally concentrated in the vicinity of producing cells and to form a gradient within the tissue (1). The same ability is also likely to help secreted CCL28 to be highly concentrated on the mucosal surfaces to form a barrier shield against colonizing microbes. Thus, even though the concentrations of CCL28 found free in saliva and milk are relatively low compared with its IC50 for various microbes, its high surface concentrations on the mucosal surfaces together with synergistic effects with other antimicrobial factors rich in mucosal secretions (33) may provide a highly effective barrier protection against a wide spectrum of microbes. Notably, autologous skin flaps transplanted into the oral cavity to reconstruct large tissue defects after radical resection of oral cancer are known to be frequently infected with C. albicans (23). Skin surfaces, which may be less effectively covered by CCL28 than normal oral mucosa, may be easily colonized by C. albicans even in the presence of high concentrations of histatins and other antimicrobial agents in the saliva.
Based on the amino acid sequence similarity between the C-terminal region of CCL28 and histatin-5 (Fig. 5), we originally expected that the antimicrobial mechanism of CCL28 was similar to that of histatin-5, which selectively binds to the mitochondrial membrane after internalization into yeast cells (34, 35). However, we now consider this unlikely because of the following observations: 1) CCL28 shows a much broader spectrum of antimicrobial activity than does histatin-5, which is mainly effective against Candida species (26), 2) CCL28 induces a rapid membrane permeability in target microbes, in contrast with the reported relatively slow antimicrobial effect of histatin-5 (34), and 3) FITC-labeled CCL28 mainly stained cell surface of C. albicans (data not shown), in contrast with the reported association of histatin-5 to mitochondria membrane within yeast cells (25). Like most other antimicrobial peptides (10), CCL28 apparently exerts its antimicrobial activity by spontaneous membrane insertion and pore formation in target microbes (Figs. 8 and 9). It remains to be seen whether the mode of action of CCL28-C, the 28-aa C-terminal peptide of CCL28, which has a sequence similarity with histatin-5 and shows a selective killing of C. albicans like histatin-5 (Fig. 6), is similar to that of histatin-5.
Because most antimicrobial peptides are generated from a larger precursor protein through proteolytic processing (10), CCL28 may also function as a precursor of smaller antimicrobial peptides such as CCL28-C. So far, however, we have obtained no evidence supporting proteolytic processing of CCL28. For example, the immunoblot analysis clearly demonstrated that natural CCL28 in parotid and lacteal secretions was identical with recombinant CCL28 in size (Fig. 4). Furthermore, CCL28 showed a much broader spectrum of antimicrobial activity than did CCL28-C (Table I). Thus, CCL28 itself is likely to be a natural antimicrobial protein, and the whole molecule of CCL28 is important for its efficient antimicrobial activity.
What could have been an evolutionary process that has led CCL28 to be both a chemokine and an antimicrobial protein? We generated a phylogenic tree from the amino acid sequences of chemokines and antimicrobial peptides using parallel prrp and phylp programs (36). As shown in Fig. 5C, CCL28 and CCL27 are closely related to the Zebrafish CC chemokine. Thus, these two chemokines may represent the most primordial type CC chemokines in mammals. It should also be noted that the extended C-terminal domain of CCL28 (CCL28-C), which aligns side by side with histatin-5 (Fig. 5C), is not encoded by a separate exon in the CCL28 gene. Thus, CCL28 is not a chimeric protein generated through a new combination of exons during evolution. Therefore, one possibility is that the chemokine family and the antimicrobial peptides, especially the family of defensins, have diverged from a common primordial molecule. Thus, some old-type chemokines such as CCL28 still retain antimicrobial activities and function as such. However, it is rather striking that CCL27, the chemokine most similar to CCL28, hardly displays a significant anti-microbial activity (Table I). Thus, another possibility is that the antimicrobial activity of CCL28, as well as its histatin-like C terminus, has been fortuitously generated through a convergent evolution of a chemokine (CCL28) to be an antimicrobial protein as well. In either case, the functional and evolutionary relationships between chemokines and antimicrobial peptides in innate and adaptive immunity will be an interesting subject in future studies (11).
In conclusion, CCL28 has dual functions in mucosal immunity as a chemokine attracting cells expressing CCR10 and/or CCR3 and also as an apically secreted molecule with a potent antimicrobial activity against a broad spectrum of microbes. Future studies using CCL28-knockout mice or neutralization of CCL28 activity in vivo will further define its roles in innate and adaptive immunity as a chemokine and as an antimicrobial protein.
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Acknowledgments |
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Footnotes |
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2 Current address: Department of Clinical Laboratory Research, National Mito Hospital, 3-2-1 Higashihara, Mito, Ibaragi 310-0035, Japan.
3 Address correspondence and reprint requests to Dr. Osamu Yoshie, Department of Microbiology, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan. E-mail address: o.yoshie{at}med.kindai.ac.jp
4 Abbreviations used in this paper: CCL28-C, C-terminal 28 aa of CCL28; m, mouse; PPB, potassium phosphate buffer; PI, propidium iodide.
Received for publication August 26, 2002. Accepted for publication November 20, 2002.
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References |
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-locus chemokine (ILC), which is located on chromosome 9p13 and a potential homologue of a CC chemokine encoded by Molluscum contagiosum virus. FEBS Lett. 460:544.[Medline]
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  M. A. Crawford, Y. Zhu, C. S. Green, M. D. Burdick, P. Sanz, F. Alem, A. D. O'Brien, B. Mehrad, R. M. Strieter, and M. A. Hughes Antimicrobial Effects of Interferon-Inducible CXC Chemokines against Bacillus anthracis Spores and Bacilli Infect. Immun., April 1, 2009; 77(4): 1664 - 1678. [Abstract] [Full Text] [PDF]
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 C. Maerki, S. Meuter, M. Liebi, K. Muhlemann, M. J. Frederick, N. Yawalkar, B. Moser, and M. Wolf Potent and Broad-Spectrum Antimicrobial Activity of CXCL14 Suggests an Immediate Role in Skin Infections J. Immunol., January 1, 2009; 182(1): 507 - 514. [Abstract] [Full Text] [PDF]
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 H. M. Linge, M. Collin, P. Nordenfelt, M. Morgelin, M. Malmsten, and A. Egesten The Human CXC Chemokine Granulocyte Chemotactic Protein 2 (GCP-2)/CXCL6 Possesses Membrane-Disrupting Properties and Is Antibacterial Antimicrob. Agents Chemother., July 1, 2008; 52(7): 2599 - 2607. [Abstract] [Full Text] [PDF]
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A.-K. Shirakawa, D. Nagakubo, K. Hieshima, T. Nakayama, Z. Jin, and O. Yoshie 1,25-Dihydroxyvitamin D3 Induces CCR10 Expression in Terminally Differentiating Human B Cells J. Immunol., March 1, 2008; 180(5): 2786 - 2795. [Abstract] [Full Text] [PDF]
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 J. L. Hermsen, F. E. Gomez, Y. Maeshima, Y. Sano, W. Kang, and K. A. Kudsk Decreased Enteral Stimulation Alters Mucosal Immune Chemokines JPEN J Parenter Enteral Nutr, January 1, 2008; 32(1): 36 - 44. [Abstract] [Full Text] [PDF]
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 M. Tohyama, K. Sayama, H. Komatsuzawa, Y. Hanakawa, Y. Shirakata, X. Dai, L. Yang, S. Tokumaru, H. Nagai, S. Hirakawa, et al. CXCL16 is a novel mediator of the innate immunity of epidermal keratinocytes Int. Immunol., September 1, 2007; 19(9): 1095 - 1102. [Abstract] [Full Text] [PDF]
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J. H. Lee, S. G. Kang, and C. H. Kim FoxP3+ T Cells Undergo Conventional First Switch to Lymphoid Tissue Homing Receptors in Thymus but Accelerated Second Switch to Nonlymphoid Tissue Homing Receptors in Secondary Lymphoid Tissues J. Immunol., January 1, 2007; 178(1): 301 - 311. [Abstract] [Full Text] [PDF]
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B. Eksteen, A. Miles, S. M. Curbishley, C. Tselepis, A. J. Grant, L. S. K. Walker, and D. H. Adams Epithelial Inflammation Is Associated with CCL28 Production and the Recruitment of Regulatory T Cells Expressing CCR10 J. Immunol., July 1, 2006; 177(1): 593 - 603. [Abstract] [Full Text] [PDF]
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 A. Dongari-Bagtzoglou and P.L. Fidel Jr. The Host Cytokine Responses and Protective Immunity in Oropharyngeal Candidiasis Journal of Dental Research, November 1, 2005; 84(11): 966 - 977. [Abstract] [Full Text] [PDF]
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 A. E. John, M. S. Thomas, A. A. Berlin, and N. W. Lukacs Temporal Production of CCL28 Corresponds to Eosinophil Accumulation and Airway Hyperreactivity in Allergic Airway Inflammation Am. J. Pathol., February 1, 2005; 166(2): 345 - 353. [Abstract] [Full Text] [PDF]
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D. Alvarez, G. Harder, R. Fattouh, J. Sun, S. Goncharova, M. R. Stampfli, A. J. Coyle, J. L. Bramson, and M. Jordana Cutaneous Antigen Priming via Gene Gun Leads to Skin-Selective Th2 Immune-Inflammatory Responses J. Immunol., February 1, 2005; 174(3): 1664 - 1674. [Abstract] [Full Text] [PDF]
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 T. Hirata, Y. Furukawa, B.-G. Yang, K. Hieshima, M. Fukuda, R. Kannagi, O. Yoshie, and M. Miyasaka Human P-selectin Glycoprotein Ligand-1 (PSGL-1) Interacts with the Skin-associated Chemokine CCL27 via Sulfated Tyrosines at the PSGL-1 Amino Terminus J. Biol. Chem., December 10, 2004; 279(50): 51775 - 51782. [Abstract] [Full Text] [PDF]
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 H. Ogawa, M. Iimura, L. Eckmann, and M. F. Kagnoff Regulated production of the chemokine CCL28 in human colon epithelium Am J Physiol Gastrointest Liver Physiol, November 1, 2004; 287(5): G1062 - G1069. [Abstract] [Full Text] [PDF]
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 E. Wilson and E. C. Butcher CCL28 Controls Immunoglobulin (Ig)A Plasma Cell Accumulation in the Lactating Mammary Gland and IgA Antibody Transfer to the Neonate J. Exp. Med., September 20, 2004; 200(6): 805 - 809. [Abstract] [Full Text] [PDF]
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K. Hieshima, Y. Kawasaki, H. Hanamoto, T. Nakayama, D. Nagakubo, A. Kanamaru, and O. Yoshie CC Chemokine Ligands 25 and 28 Play Essential Roles in Intestinal Extravasation of IgA Antibody-Secreting Cells J. Immunol., September 15, 2004; 173(6): 3668 - 3675. [Abstract] [Full Text] [PDF]
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T. Shimaoka, T. Nakayama, K. Hieshima, N. Kume, N. Fukumoto, M. Minami, K. Hayashida, T. Kita, O. Yoshie, and S. Yonehara Chemokines Generally Exhibit Scavenger Receptor Activity through Their Receptor-binding Domain J. Biol. Chem., June 25, 2004; 279(26): 26807 - 26810. [Abstract] [Full Text] [PDF]
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P. Schaerli, L. Ebert, K. Willimann, A. Blaser, R. S. Roos, P. Loetscher, and B. Moser A Skin-selective Homing Mechanism for Human Immune Surveillance T Cells J. Exp. Med., May 3, 2004; 199(9): 1265 - 1275. [Abstract] [Full Text] [PDF]
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A. Zaballos, R. Villares, J. P. Albar, C. Martinez-A, and G. Marquez Identification on Mouse Chromosome 8 of New {beta}-Defensin Genes with Regionally Specific Expression in the Male Reproductive Organ J. Biol. Chem., March 26, 2004; 279(13): 12421 - 12426. [Abstract] [Full Text] [PDF]
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H. Hanamoto, T. Nakayama, H. Miyazato, S. Takegawa, K. Hieshima, Y. Tatsumi, A. Kanamaru, and O. Yoshie Expression of CCL28 by Reed-Sternberg Cells Defines a Major Subtype of Classical Hodgkin's Disease with Frequent Infiltration of Eosinophils and/or Plasma Cells Am. J. Pathol., March 1, 2004; 164(3): 997 - 1006. [Abstract] [Full Text] [PDF]
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, duct. Immunoelectron microscopy (J and K): scale bar, 1 µm; arrow, immunoreactive granule; Lu, lumen.
