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Originally published online as doi:10.1189/jlb.0103024 on June 16, 2003

Published online before print June 16, 2003
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(Journal of Leukocyte Biology. 2003;74:448-455.)
© 2003 by Society for Leukocyte Biology

Many chemokines including CCL20/MIP-3{alpha} display antimicrobial activity

De Yang*, Qian Chen{dagger}, David M. Hoover{ddagger}, Patricia Staley§, Kenneth D. Tucker§, Jacek Lubkowski{ddagger} and Joost J. Oppenheim{dagger},1

* Basic Research Program and
{ddagger} Macromolecular Crystallography and
§ Opportunistic Infection Laboratories, Division of Cancer Treatment and Diagnosis/Developmental Therapeutics Program, Science Applications International Corp., Inc.-Frederick, and
{dagger} Laboratory of Molecular Immunoregulation, Center for Cancer Research, National Cancer Institute at Frederick, Maryland

1Correspondence: LMI, CCR, NCI-at Frederick, Building 560, Room 21-89, Frederick, MD 21702-1201. E-mail: oppenhei{at}mail.ncifcrf.gov


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have demonstrated that ß-defensins exhibit chemotactic activity by sharing the chemokine receptor CCR6 with the CC chemokine ligand CCL20/macrophage-inflammatory protein-3{alpha} (MIP-3{alpha}). Structural analysis of CCL20/MIP-3{alpha} revealed that most of the positively charged residues are concentrated at one area of its topological surface, a characteristic considered to be important for the antimicrobial activity of defensins. Here, we report that similar to defensins, CCL20/MIP-3{alpha} has antimicrobial effects on Escherichia coli, Pseudomonas aeruginosa, Moraxella catarrhalis, Streptococcus pyogenes, Enterococcus faecium, Staphylococcus aureus, and Candida albicans. Additionally, by screening a total of 30 human chemokines, we have identified an additional 17 human chemokines, which exhibit antimicrobial activity in vitro. Collectively, about two-thirds of the chemokines investigated so far has the capacity to kill microorganisms in vitro, suggesting that antimicrobial activity may be another host-defense function for certain chemokines. Comparison of the structural characteristics between antimicrobial and nonantimicrobial chemokines suggests that topological formation of a large, positively charged electrostatic patch on the surface of the molecule is likely to be a common structural feature of antimicrobial chemokines.

Key Words: defensin • macrophage-inflammatory protein • colony-forming assay


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Chemokines are a group of small (8–14 kDa), structurally related proteins, which are important participants in and regulators of diverse physiological and pathological processes, such as development, organogenesis, angiogenesis, inflammation, and immunity, predominantly through regulating cell trafficking [1 , 2 ]. More than 40 chemokines have been identified in humans. Based on the arrangement of the conserved N-terminal cysteine motifs, chemokines have been classified into four subfamilies: CXC, CC, CX3C, and C (or CXCL, CCL, CX3CL, and XCL, respectively, according to the recently proposed nomenclature [3 ]), where "X" represents an amino acid residue other than cysteine. The effects of chemokines on host cells are mediated by their corresponding chemokine receptors, which are also grouped into four groups: CXCR, CCR, CX3CR, and XCR [1 2 3 4 ]. Most, but not all, chemokine receptors can interact with more than one chemokine; CCR6 has only been shown to interact with a single chemokine CCL20/macrophage-inflammatory protein-3{alpha} (MIP-3{alpha}) [5 6 7 ], but it also interacts with several ß-defensins [8 , 9 ].

Defensins are 2–6 kDa cationic, antimicrobial peptides containing three pairs of intramolecular disulfide bonds [10 11 12 ]. Based on the pattern of their disulfide bonding, mammalian defensins are classified into {alpha}, ß, and {theta} categories [10 , 11 ]. Many defensins have broad-spectrum antimicrobial activity against bacteria, fungi, and some enveloped viruses and are thus considered to participate in the first line of innate defense against microbial challenge [10 11 12 ].

We and others [8 , 9 , 13 14 15 ] have previously documented that {alpha}- and ß-defensins are chemotactic for various types of leukocytes. CCR6 is the receptor that mediates chemotactic responses of immature dendritic cells to CCL20/MIP-3{alpha}- and ß-defensins [4 5 6 7 8 9 ]. Recently, the X-ray crystallography and nuclear magnetic resonance structures of human ß-defensin-2 (HBD2) as well as the nuclear magnetic resonance structure of mouse and human CCL20/MIP-3{alpha} have been solved [16 17 18 19 ]. Comparison of their solution structures revealed that albeit ß-defensin-2 and CCL20/MIP-3{alpha} have no apparent similarity at the primary structural level, both have similar topological motifs. An Asp4-Leu9 motif in HBD2, which resembles the Asp5-Leu8 motif of CCL20/MIP-3{alpha}, is considered to be responsible for specific interaction with CCR6, providing a structural basis for the capacity of ß-defensins and CCL20/MIP-3{alpha} to interact with the same receptor [18 ]. Based on the structural similarity and functional overlap between defensins and chemokines, they may also share antimicrobial functions [20 ].

Careful examination of the solution structures of ß-defensins and CCL20/MIP-3{alpha} [17 , 18 ] revealed that they share another common structural characteristic: Their positively charged residues tend to accumulate in one area of their surface, a feature considered to be important for the antimicrobial activity of defensins [10 11 12 ]. The possibility that CCL20/MIP-3{alpha} might also have defensin-like antimicrobial activity was encouraged by the report that interferon-{gamma} (IFN-{gamma})-inducible chemokines, including CXCL9/monokine induced by IFN-{gamma} (MIG), CXCL10/IFN-inducible protein 10 (IP-10), and CXCL11/IFN-inducible T cell-{alpha} chemoattractant (I-TAC), demonstrated antibacterial activity [21 ]. Therefore, we investigated whether CCL20/MIP-3{alpha} also has defensin-like antimicrobial activity. Further evaluation of 30 chemokines belonging to CXC, CC, CX3C, and C subfamilies for their antimicrobial activities provided us with an opportunity to determine the structural characteristics enabling a given chemokine to be antimicrobial.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
All human chemokines used in this study were recombinant proteins purchased from PeproTech (Rocky Hill, NJ). The new and one of the most often-used, traditional nomenclature [3 ] for various chemokines are given. Synthetic HBD2 was purchased from Peptide Institute (Osaka, Japan). Synthetic HBD3 was a generous gift from Drs. Jürgen Harder and Jens M. Schröder (Department of Dermatology, Christian Albrechts University of Kiel, Germany). Recombinant HBD3 (rHBD3) was purchased from PeproTech. Trypticase soy broth (TSB) was purchased from Becton Dickinson Microbiology Systems (Cockeysville, MD). Luria-Bertani (LB) plates were purchased from K-D Medical (Columbia, MD).

Microorganisms and culture conditions
Gram- bacteria used were Escherichia coli (ATCC25922), Pseudomonas aeruginosa (PA01, a conventional laboratory strain), and Moraxella catarrhalis (ATCC43627). For Gram+ bacteria, the following strains were used: Staphylococcus aureus (ATCC29213), Streptococcus pyogenes (ATCC14289), and Enterococcus faecium (1438, a clinical isolate). A single colony of bacteria was inoculated into 50 ml TSB and incubated overnight at 37°C in a shaking incubator (250 rpm). Subsequently, the bacterial culture was diluted with TSB, prewarmed to 37°C at 1/100, and subcultured under the same conditions for another 2–3 h to obtain bacteria in mid-logarithmic growth phase (ABS620<=1.0). The bacteria were centrifuged at 1500 g for 5 min, washed, resuspended, and diluted with 10 mM potassium phosphate buffer (pH 7.4) containing 1% TSB to 107 colony-forming units (CFU)/ml. For each bacterial strain, an ABS620 = 0.2 was equivalent to 5 x 107 CFU/ml.

Three laboratory strains of fungi including Candida albicans (99-788, azole-resistant), C. albicans (90028, azole-sensitive), and Cryptococcus neoformans (96-1043) were used to test the antifungal activity of certain chemokines. To obtain yeast cells in mid-logarithmic growth phase, C. albicans and C. neoformans were cultured for 22 h at 37°C with shaking (100 rpm) in a 125-ml flask containing 20 ml yeast-malt (YM) broth. The yeast cells were diluted with 10 mM potassium phosphate buffer (pH 7.4) containing 1% TSB-106 CFU/ml. For these strains of fungi, an ABS600 = 0.3 was equivalent to 1.5 x 107 CFU/ml.

Antimicrobial assay
The standard colony-forming assay (CFA) was used with slight modification [22 ]. Briefly, test microorganisms (105 CFU) were mixed with chemokines or HBD3 to reach the final concentration, as specified in 0.1 ml 10 mM potassium phosphate buffer (pH 7.4) containing 1% TSB. For the antibacterial assay, the mixture was incubated for 3 h at 37°C in an environmental shaking incubator (250 rpm). In some experiments, NaCl at various concentrations was supplemented to test the effect of salt on the antimicrobial activiy of CCL20/MIP-3{alpha}. After serial dilution with 10 mM potassium phosphate buffer (pH 7.4) containing 1% TSB, the diluted mixture was plated in triplicate on LB plates. The plates were incubated overnight (18–20 h) at 37°C, and the colonies formed were counted. For antifungal assay, the mixture was incubated for 3 h at 37°C without shaking. At the end of the 3-h incubation, the mixture was serially diluted with YM broth and plated on YM agar plates, which were then incubated for 22–28 h at 37°C before counting the colonies. The results were documented as CFU/ml.

Structure analysis
The isoelectric points (pI) of selected chemokines were calculated by the use of DNASIS software (Hitachi Software Engineering America, South San Francisco, CA), based on the amino acid sequences of mature proteins. The charged residue profile and hydropathicity index plot for a given chemokine were generated by the use of ProtScale software, available online on the ExPASy homepage (http://ca.expasy.org/cgi-bin/protscale.pl). For the calculation of hydrophathicity indices, the Kyte and Doolittle [23 ] scaling model was used. To visualize the three-dimensional structure of chemokine, the GRASP program [24 ] was used. The backbone of the monomeric form of chemokine was depicted as "tube worm" (in gray), and the electrostatic surfaces were contoured at +4 kt (in blue) and -4 kt (in red). The structural data for chemokines were retrieved from the Protein Data Bank with the following codes: 1IL8 [CXCL8/interleukin-8 (IL-8)], 1F2L (the chemokine domain of CX3CL1/fractalkine), 1DOL [CCL2/monocyte chemoattractant protein-1 (MCP-1)], 1B53 (CCL3/MIP-1{alpha}), 1ESR (CCL8/MCP-2), 1LV9 (CXCL10/IP-10), 1A15 [CXCL12/stromal cell-derived factor-1{alpha} (SDF-1{alpha})], 1J9O (XCL1/lymphotactin), 1EL0 (CCL1/I-309), and 1M8A (CCL20/MIP-3{alpha}).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
CCL20/MIP-3{alpha} is antimicrobial with a particular spectrum for target microorganisms
CCL20/MIP-3{alpha} was initially tested for antimicrobial activity against Gram- E. coli using the standard CFA after coincubation of 105 CFU bacteria in 0.1 ml 10 mM potassium phosphate buffer (pH 7.4) with various concentrations of CCL20/MIP-3{alpha}. Human and mouse (data not shown) CCL20/MIP-3{alpha} killed E. coli in a dose-dependent manner with a lethal dose (LD)50 (the dose that achieves 50% reduction of CFU) of 0.4 µg/ml (ranging from 0.1 to 0.4 µg/ml in three independent experiments) for human CCL20/MIP-3{alpha} (Fig. 1A ). In parallel experiments, HBD2 and HBD3 showed a LD50 ranging from 0.4 to 1 µg/ml (data not shown), similar to previous reports [22 , 25 ]. Thus CCL20/MIP-3{alpha} is more potent than (or at least equally potent as) ß-defensins in terms of killing E. coli.



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  		Figure 1. Anti-E. coli activity of CCL20/MIP-3{alpha}. (A) Dose-response of CCL20/MIP-3{alpha} by standard CFA with a LD50 0.4 µg/ml (ranging from 0.1 to 0.4 µg/ml in three independent experiments). (B) Salt sensitivity of the anti-E. coli activity of CCL20/MIP-3{alpha} (10 µg/ml) was examined in the absence (•) or presence ({blacktriangleup}) of increasing concentration of NaCl. The 50% and 90% inhibition doses for NaCl were 90 mM and 130 mM, respectively, when the CFU of E. coli treated in the absence of CCL20/MIP-3{alpha} and NaCl ({blacksquare}) was considered 100%. Shown was the mean ± SD (error bars) of triplicated plates (error bars<=size of the symbol if not evident).

 
The antimicrobial activity of defensins can be inhibited by the presence of 100 mM NaCl [10 11 12 , 25 ]. We explored whether the anti-E. coli activity of CCL20/MIP-3{alpha} was also sensitive to the presence of salt by incubating E. coli and CCL20/MIP-3{alpha} at various concentrations of NaCl. At 10 µg/ml, CCL20/MIP-3{alpha} killed more than 99.9% of E. coli; however, NaCl blunted its bactericidal activity in a dose-dependent manner (Fig. 1B) . The 50% and 90% inhibitory concentrations of NaCl were 90 and 130 mM, respectively. Thus, like most defensins, the antimicrobial activity of CCL20/MIP-3{alpha} is attenuated by a high concentration of salt.

The capacity of CCL20/MIP-3{alpha} to kill S. aureus and C. albicans was also examined using HBD3 as a positive control. CCL20/MIP-3{alpha} demonstrated anti-S. aureus activity with a LD50 of 10 µg/ml, thus appears to be more potent than HBD2 but less than HBD3 (Fig. 2A and refs. [22 , 25 ]). CCL20/MIP-3{alpha} also displayed some anti-C. albicans (strain 99-788) activity at higher concentrations with a LD50 of ~35 µg/ml (Fig. 2B) . These results suggested that CCL20/MIP-3{alpha} might be a defensin-like antimicrobial factor with a selective target spectrum.



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  		Figure 2. Antimicrobial activity of CCL20/MIP-3{alpha} against S. aureus (A) and C. albicans (strain 99-788; B), as investigated by standard CFA. Synthetic and rHBD3 (50 µg/ml) were used as positive controls for anti-S. aureus (A) and anti-C. albicans (B) assays, respectively. Shown was the mean ± SD (error bars) of triplicated (A) or quadruplicated (B) plates (error bars<=size of the symbol if not evident).

 
To further investigate the target spectrum of CCL20/MIP-3{alpha}, we examined the antimicrobial activity of CCL20/MIP-3{alpha} against six additional strains of bacteria and fungi (Table 1) . Among the bacteria examined, Gram+ S. pyogenes (LD50=0.2 µg/ml) was very sensitive to the antimicrobial activity of CCL20/MIP-3{alpha}, followed by two Gram- bacteria, M. catarrhalis (LD50=0.9 µg/ml) and P. aeruginosa (LD50=1.1 µg/ml). The Gram+ E. faecium was less sensitive to CCL20/MIP-3{alpha} with a LD50 of 4.5 µg/ml (Table 1) . In agreement with Figure 2B , much higher concentrations of CCL20/MIP-3{alpha} were required to kill C. albicans (90028) and C. neoformans (the LD50 was 25 and 80 µg/ml, respectively). Thus, CCL20/MIP-3{alpha}, a defensin-like antimicrobial chemokine, is more effective against bacteria than fungi.


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  		Table 1. The Antimicrobial Activity of CCL20/MIP-3{alpha} against Various Strains of Bacteria and Fungi

 
Many chemokines have antimicrobial activity
The antimicrobial activity of CCL20/MIP-3{alpha} together with the fact that IFN-{gamma}-inducible chemokines are also antimicrobials prompted us to screen whether other chemokines also have microbicidal activity. A total of 30 chemokines were tested at a single dose (10 µg/ml) against E. coli, S. aureus, and C. albicans, using CCL20/MIP-3{alpha} and the three IFN-{gamma}-inducible chemokines (CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC) as positive controls. Among the 30 chemokines screened, only several chemokines, including CXCl2/growth-related ß (Groß), CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1{alpha}, CCL11/eotaxin, and CCL13/MCP-4, demonstrated low anti-C. albicans activity at 10 µg/ml. In addition to CCL20/MIP-3{alpha}, CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC, another 17 chemokines demonstrated antibacterial activities with varying potency (Table 2) . These include six CXC subfamily members (CXCL1/Gro{alpha}, CXCL2/Groß, CXCL3/Gro{gamma}, CXCL12/SDF-1, CXCL13/BCA-1, and CXCL14/BRAK), 10 CC subfamily members (CCL1/I-309, CCL8/MCP-2, CCL11/eotaxin, CCL13/MCP-4, CCL17/TARC, CCL18/PARC, CCL19/MIP-3ß, CCL21/SLC, CCL22/MDC, and CCL25/TECK), and XCL1/lymphotactin, the only identified member of the C subfamily. Several points are worth mentioning: Most bactericidal chemokines, in particular CXCL1, CXCL2, CXCL3, CXCL12/SDF-1, CXCL13/BCA-1, CCL1/I-309, CCL13/MCP-4, CCL19/MIP-3ß, CCL20/MIP-3{alpha}, and XCL1/lymphotactin, were more potent against Gram- E. coli than against Gram+ S. aureus. The striking difference was observed between the antibacterial activity of CCL19/MIP-3ß and CCL21/SLC, although both are similar in size and charge and use the same chemokine receptor, CCR7 [1 2 3 4 ]. CCL19/MIP-3ß was active against E. coli with no detectable anti-S. aureus activity under the conditions tested; however, CCL21/SLC, albeit less potent against E. coli than CCL19/MIP-3ß, demonstrated potent anti-S. aureus activity. In contrast to the report that XCL1/lymphotactin has no antimicrobial activity [21 ], we consistently detected potent antibacterial activity for this chemokine. The reason for this discrepancy is not completely clear but is most likely a result of the use of different antimicrobial assays (radial diffusion assay vs. CFA) or different strains of bacteria, as the XCL1/lymphotactin samples in that study and ours were from the same source (PeproTech).


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  		Table 2. The Antimicrobial Activities of Selected Chemokines

 
What makes a chemokine antimicrobial?
Most chemokines, such as defensins, are positively charged at neutral pH, exhibiting a pI higher than 8.0. All of the antimicrobial chemokines tend to have a higher pI, and no chemokine with a pI lower than 9.0 demonstrated anti-E. coli activity (Table 2) , indicating that cationicity is an important feature for antimicrobial chemokines. However, cationicity is not likely to be the sufficient feature to distinguish antimicrobial from nonantimicrobial chemokines. For example, CCL1/I-309 and CCL7/MCP-3 have a pI of ~10.0, yet only the former is antimicrobial (Table 2) . Conversely, CCL22/MDC, albeit with lower pI (~9.0), has potent antimicrobial activity (Table 2) . Furthermore, the potency of antimicrobial chemokines does not directly correlate with their cationicity (Table 2) . Therefore, in addition to cationicity, other structural characteristic(s) may endow a given chemokine with antimicrobial activity.

A comparison of the secondary structure of IFN-{gamma}-inducible antimicrobial chemokines (CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC) with some nonantimicrobial CXC chemokines (CXCL5/epithelial-neutrophil activating peptide-78 and CXCL8/IL-8) revealed that IFN-{gamma}-inducible chemokines contain a C-terminal segment uniquely rich in positively charged amino acids [21 ]. As many antimicrobial peptides such as defensins interact with the anionic moieties on the surface of bacteria before disrupting their membrane [10 11 12 ], it was proposed that the heavily cationic tail of IFN-{gamma}-inducible CXC chemokines is responsible for their antimicrobial activity [21 ]. To determine if this is the common structural feature for all antimicrobial chemokines, we analyzed the charge distribution and hydropathicity of all chemokines investigated in this study (Fig. 3 ). Among the chemokines that demonstrated marked anti-E. coli activity (>=++), CXCL9/MIG and CCL21/SLC have the most cationic tails, followed by CXCL11/I-TAC, CXCL10/IP-10, CCL1/I-309, CCL19/MIP-3ß, CCL20/MIP-3{alpha}, CXCL14/BRAK, CCL13/MCP-4, CCL17/TARC, CCL11/eotaxin, and CXCL2/Groß (Fig. 3A) . However, CXCL1/Gro{alpha}, CXCL13/BCA-1, CXCL12/SDF-1{alpha}, XCL1/lymphotactin, CCL18/PARC, CCL22/MDC, and CXCL3/Gro{gamma}, albeit demonstrated marked anti-E. coli activity, do not have significant cationic tails (Fig. 3A) . Furthermore, the cationicity of the tails of chemokines does not seem to correlate with their anti-E. coli potency (Fig. 3A) . With respect to the hydropathicity, there is no apparent pattern in terms of hydropathicity profiling (Fig. 3B) that can distinguish nonantimicrobial from antimicrobial chemokines.



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  		Figure 3. The profiles of charged residues (A) and hydropathicity indices plots (B) of chemokines using ProtScale of the ExPASy program. (A and B) The sequences of mature proteins were used for the analysis. The plots of chemokines were arranged in a descending manner according to their anti-E. coli activity. For CX3CL1/fractalkine (Fract.), only the chemokine domain was analyzed. lymphot., Lymphotactin.

 
The fundamental, structural principal common to all or many antimicrobial peptides including defensins appears to be the topological (rather than linear) amphipathic design, where clusters of hydrophobic and cationic amino acids are organized in discrete surface areas [12 ]. Therefore, we compared the three-dimensional structures of the 10 selected chemokines with a particular focus on the topological distribution of the electrostatic charges on the surfaces of the molecules (Fig. 4 ). Common to the five chemokines with marked anti-E. coli activity, most of the cationic residues appear to be accumulated in a discrete surface area, allowing the formation of a large, positively charged electrostatic patch on their surface (Fig. 4 , lower panel). The rest of the molecular surface is mostly hydrophobic with spotted negative electrostatic charges. Therefore, the three-dimensional structures of the five antimicrobial chemokines can be approximated as consisting globally of a "cationic domain" and a "hydrophobic domain." In clear contrast, in chemokines that demonstrated no or very low (e.g., CCL8/MCP-2) antimicrobial activity, no such cationic domain is present (Fig. 4 , upper panel). CCL3/MIP-1{alpha} does not have many positive charges on its surface. In CXCL8/IL-8, CX3CL1/fractalkine, CCL2/MCP-1, and CCL8/MCP-2, the formation of a large, positive electrostatic patch seems to be interrupted by anionic residues. CCL8/MCP-2 has a calculated pI higher than that of CCL1/I-309 or CCL20/MIP-3{alpha} (Table 2) , yet it did not demonstrate marked antimicrobial activity, perhaps as a result of the failure to form a large, positively charged electrostatic patch on its surface (Fig. 4) . Thus, topological formation of a large, positively charged electrostatic patch on the surface is likely to be an additional structural characteristic for antimicrobial chemokines.



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  		Figure 4. The three-dimensional structure of selected nonantimicrobial and antimicrobial chemokines. The structures of selected chemokines were generated using the program GRASP, and the backbone in tube worm form (in gray) and the electrostatic surfaces were contoured at +4 kt (in blue) and -4 kt (in red). The structure graphs were intentionally arranged to place the relatively more cationic part upward (the first column). The second, third, and fourth columns were obtained by rotating the first column of graphs 90, 180, and 270 degrees, respectively, along the putative vertical axis. The structural data for chemokines were retrieved from the protein database (details in Materials and Methods). For CX3CL1/fractalkine, only the chemokine domain was analyzed. For XCL1/lymphotactin, the C-terminal uncharged tail (from residues 77 to 93) was omitted to generate structural graphs of similar size.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Chemokines have been studied predominantly as mediators and regulators of the trafficking of various types of cells. Recently, CXCL7 (connective tissue activation peptide-3 and neutrophil activating peptide-2, products of the same SCYB7 gene) and the three IFN-{gamma}-inducible chemokines (CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC) have been shown to have in vitro antimicrobial activity [21 , 26 ]. In the present study, we tested 30 human chemokines and identified 18 additional antimicrobial chemokines including CCL20/MIP-3{alpha} (Table 2) . Thus, of the 33 chemokines studied to date, 22 demonstrate in vitro antimicrobial activity, suggesting that antimicrobial activity may be another property for many chemokines.

A recent publication reported that CCL5/RANTES derived from platelets demonstrated antimicrobial activity against E. coli, S. aureus, C. albicans, and C. neoformans [27 ]. In contrast, we could not detect any antimimicrobial activity against E. coli, S. aureus (Table 2) , or fungi (C. albicans and C. neoformans; data not shown) for recombinant CCL5/RANTES, in complete agreement with the report by Cole et al. [21 ]. The discrepancy is most likely a result of the differences in assay conditions. In our study, CCL5/RANTES was tested at 10 µg/ml in potassium phosphate buffer at pH 7.4. The antimicrobial activity of CCL5/RANTES became evident only when tested at a pH below 6.5 and at concentrations equal to or higher than 50 nmol/ml (equivalent to ~390 µg/ml) [27 ]. Such a high concentration of CCL5/RANTES and low pH are unlikely to occur in vivo.

Analysis of the structures of chemokines has revealed that the formation of a large patch of positive electrostatic charges on the surface appears to be a characteristic that distinguishes antimicrobial from nonantimicrobial chemokines. This conclusion is based on the analysis of antimicrobial chemokines whose tertiary structures have been reported. A comprehensive analysis would have to wait until the structures of other antichemokines (e.g., CCL18/PARC, CCL22/MDC, CCL17/TARC, CCL19/MIP-3ß, CCL21/SLC, CCL25/TECK, CXCL9/MIG, CXCL11/I-TAC, etc.) are solved. However, the size of the positively charged electrostatic surface area does not seem to correlate with the anti-E. coli potency of antimicrobial chemokines. Notably, CCL1/I-309, albeit possessing the largest cationic electrostatic patch (Fig. 4) , is not the most potent anti-E. coli chemokine (Table 2) . Therefore, the antimicrobial potency of a given antimicrobial chemokine may be affected by factors other than the size of the cationic electrostatic patch, such as the species and strain of microorganisms. For example, CCL20/MIP-3{alpha} demonstrates difference in terms of potency against various strains of bacteria and fungi (Table 1) and so do INF-{gamma}-inducible chemokines [21 ].

The concentrations required for CCL20/MIP-3{alpha} to exert significant antibacterial activity were different depending on the species and strains of bacteria, and LD50 ranged from 0.2 to 10 µg/ml (Figs. 1A and 2 and Table 1 ). These concentrations are higher than that for inducing leukocyte migration (optimal dose, {approx}100 ng/ml; data not shown). This is also the case for IFN-{gamma}-inducible CXC chemokines [21 ] and possibly for all antimicrobial chemokines. Can these concentrations of chemokines be achievable in vivo? IFN-{gamma}-inducible chemokines have been shown to be able to reach several hundred ng/ml levels in patients with severe melioidosis [28 ]. As far as CCL20/MIP-3{alpha} is concerned, epithelial cells and keratinocytes can generate 3–5 ng CCL20/MIP-3{alpha} per 1 million cells upon stimulation in vitro by proinflammatory cytokines [29 , 30 ]. The concentration of cells required for the generation of one LD50 of CCL20/MIP-3{alpha} would be ~108 cells/ml, which could be achieved at inflammatory sites in epithelium or skin, given the size of keratinocytes and epithelial cells. A recent study also indicates that within a lung granuloma, CCL22/MDC can reach up to 1700 ng per gram of tissue [31 ]. Furthermore, many antimicrobial chemokines and antimicrobial peptides are produced at sites of microbial entry such as in psoriatic skin lesions [22 , 25 , 32 33 34 ], and there are likely to be additive or synergistic effects between various antimicrobial chemokines or between chemokines and other antimicrobial peptides.

Similar to HBD1 and -2 [25 , 35 36 37 ], the antimicrobial activity of CCL20/MIP-3{alpha} in vitro is inhibited by NaCl at concentrations >=130 mM (Fig. 1B) . Salt sensitivity is also observed for IFN-inducible chemokines. This raises the concern that the antimicrobial activity of chemokines may not be effective in vivo. However, intestinal epithelial cells and skin keratinocytes produce CCL20/MIP-3{alpha}, and its expression can be enhanced by inflammatory stimuli, including proinflammatory cytokines and lipopolysaccharide [29 , 30 , 38 , 39 ]. Skin biopsies from patients with psoriasis, contact dermatitis, and mycosis fungoides show abundant CCL20/MIP-3{alpha} expression [32 , 38 ]. Therefore, CCL20/MIP-3{alpha} may exert antimicrobial activity on the surface of skin and certain epithelia, where a low salt condition may exist [40 ]. Given the fact that human beings already possess scores of antimicrobial peptides, what is the need for chemokines with antimicrobial activity? One possible explanation may be that any given antimicrobial protein (defensins, chemokines, cathelicidins, etc.) may have evolved to be mostly effective against a few potentially harmful microorganisms, although many have overlapping target spectra. For example, CCL20/MIP-3{alpha} demonstrates a different microbicidal spectrum from that of HBD1, -2, or -3 [22 , 25 , 35 , 36 ] and is most potent against S. pyogenes among nine microorganisms examined in the present study (Figs. 1A and 2 and Table 1 ). Another possibility is that the antimicrobial redundancy among chemokines, defensins, and other types of antimicrobial proteins may provide additional means for coping with massive microbial invasion during infection.

In conclusion, many chemokines, including CCL20/MIP-3{alpha}, similar to many antimicrobial peptides such as defensins, have antimicrobial activity at least in vitro under low salt conditions used in our experiments. Characteristics common to all antimicrobial chemokines appear to be high cationicity and topological formation of a large, positive electrostatic patch on the surface of the molecule. Additional studies using a combination of a chemokine-knockout and its corresponding receptor knockout mice or neutralizing antibodies will be required to more clearly define the contribution of chemokines in direct antimicrobial defenses.


   ACKNOWLEDGEMENTS
 
This project has been funded in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. NO1-CO-12400. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. The publisher or recipient acknowledges right of the U.S. Government to retain a nonexclusive, royalty-free license in and to any copyright covering the article. J. L. and J. J. O. are funded in part by CPA grant. We thank Drs. J. Harder and J. M. Schröder (Department of Dermatology, Christian Albrechts University of Kiel, Germany) for providing us with synthetic HBD3. The support of the laboratory manager Mrs. C. Fogle-Lamb and secretarial assistance of Ms. C. Nolan are gratefully appreciated.

Received January 16, 2003; revised March 31, 2003; accepted April 10, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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