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Published as doi: 10.1096/fj.05-4970fje.
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(The FASEB Journal. 2006;20:1701-1702.)
© 2006 FASEB

The novel ß-defensin DEFB123 prevents lipopolysaccharide-mediated effects in vitro and in vivo

Dirk Motzkus1,2, Sandra Schulz-Maronde2, Aleksandra Heitland, Axel Schulz, Wolf-Georg Forssmann, Martin Jübner and Erik Maronde3

IPF PharmaCeuticals, Hannover, Germany

3Correspondence: Institute for Anatomy III, JW Goethe University, Theodor-Stern-Kai-7, Frankfurt D-60590, Germany. E-mail: e.maronde{at}em.uni-frankfurt.de

ABSTRACT

Defensins are a family of secreted antimicrobial peptides proposed to directly interfere with bacterial membranes. Here we show a functional analysis of the novel ß-defensin DEFB123. A peptide comprising the ß-defensin core region was synthesized and used for our analysis. Like other ß-defensins, DEFB123 exerted antimicrobial activity against a broad spectrum of Gram-positive and Gram-negative bacteria, which was assessed by microbroth dilution assay and radial diffusion zone assay. In addition, the peptide showed lipopolysaccharide (LPS)-binding activity in a Limulus amoebocyte lysate (LAL) assay. Moreover, DEFB123 prevented LPS-induced tumor necrosis factor (TNF)-alpha secretion in a murine monocyte cell line (RAW264.7). Accordingly, DEFB123 abolished LPS-mediated MAPK induction in these cells. Protection against LPS-mediated effects was then investigated in a murine model of acute sepsis. Our experiments show that synthetic ß-defensin DEFB123 prevents LPS-induced mortality in C57BL/6 mice in a therapeutic approach. We propose that the physiological role of ß-defensins may include interference with LPS-action on macrophages, a function formerly thought to be restricted to the family of cathelicidins, a structurally unrelated group of antimicrobial peptides.—Motzkus, D., Schulz-Maronde, S., Heitland, A., Schulz, A., Forssmann, W.-G., Jübner, M., and Maronde, E. The novel ß-defensin DEFB123 prevents lipopolysaccharide-mediated effects in vitro and in vivo.


Key Words: antimicrobial peptide • LAL-assay • RAW264.7 • TNF-alpha • murine sepsis model

LIPOPOLYSACCHARIDE (ENDOTOXIN, LPS) is a major component of the outer surface of Gram-negative bacteria and a potent activator of cells of the immune and inflammatory system (1) . In the course of systemic infections caused by Gram-negative bacteria, aggregates of LPS, as well as intact bacterial cells, are rapidly opsonized by LPS-binding protein (LBP), a serum protein synthesized in the liver (2) . LBP effectively catalyzes the transfer of LPS to membrane-bound and soluble forms of CD14 (mCD14 or sCD14). This complex is recognized by the transmembrane signaling unit of Toll-like receptor 4 (TLR-4) and the accessory protein MD-2, which are mainly expressed on monocytes and macrophages (3) . This key process in the recognition of LPS activates the immune system to confine and defeat the invasive organism before it has become widespread. Up-regulation of proinflammatory cytokines, such as TNF-{alpha} is accompanied by induction of high levels of expression of antimicrobial peptides which play a multifunctional role in the innate immunity of animals (4) . A large subgroup of homologous antimicrobial peptides is termed defensins.

Human ß-defensins are cationic peptides with six conserved cysteine residues, which contribute to host defense against bacterial, fungal, and viral infections (4) . The human ß-defensin 1 (DEFB1) was originally isolated from hemofiltrate (5) and displays antimicrobial activity against Gram-positive and Gram-negative bacteria (6) . The corresponding gene, DEFB1 is constitutively transcribed in human epithelia and secretory glands (7 , 8) . Subsequent studies revealed more homologue genes, which were named DEFB2, DEFB3, and DEFB4. Although DEFB2 is highly expressed in epithelia like skin and lung (9) , DEFB3 expression was found in nonepithelial tissues, such as heart or skeletal muscle (10 , 11) . In contrast to the constitutively expressed DEFB1, DEFB2, DEFB3, and DEFB4 are strongly inducible by proinflammatory cytokines (11 , 12 , 13) and are upregulated on infection.

Using bioinformatical approaches more than 30 members of the ß-defensin family have been discovered (14) . Unlike the earlier members located on human chromosome 8, chromosome 20 derived ß-defensins DEFB25 to DEFB29 were almost exclusively expressed in the male genital tract (15) . Accordingly, ß-defensins were also identified by systematic screening for epididymis-specific transcripts (16) . Functional analysis showed that the epididymal peptide DEFB118 (formerly ESC42) is active against bacteria, androgen-regulated, secreted by the epididymal lumen, and induces spermatozoa movement by chemotaxis (17) . Also related to androgenic function, a ß-defensin from rat, Bin1b, can induce progressive sperm motility in immotile immature sperm (18) . No further function was proposed to those novel members of the ß-defensin family. In comparison, most other antimicrobial peptides, and commonly selective bactericides or fungicides, also show direct interactions with innate immune cells. The hCAP-18-derived active peptide, named LL-37, is an antimicrobial agent, but also shows chemotactic activity toward neutrophils, monocytes, T-cells, and mast cells (19 20 21) . To date, in humans only one member of the cathelicidin family was identified, coded by the hCAP-18 gene. Evidence for receptor-mediated functions was also found for the ß-defensin DEFB2, which directly activates the chemokine receptor CCR6 (19) and acts as specific chemoattractant for tumor necrosis factor (TNF)-{alpha}-treated human neutrophils (22) .

MATERIALS AND METHODS

Chemical synthesis of DEFB123 and LL-37 (=CAP18104–140)
DEFB123 (37 amino acids, GTQRCWNLYGKCRYRCSKKERVYVYCINNKMCCVKPK), based on the sequence deduced from the cDNA (NM_153324), and LL-37 (=CAP18104–140) (LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) were assembled using automated Fmoc [N-(9-fluorenyl)methoxycarbonyl] solid-phase synthesis on a peptide synthesizer (model 433A, Applied Biosystems). After TFA cleavage and deprotection from the resin, the linear peptides were purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The synthetic peptides showed single peaks by capillary zone electrophoresis (CZE), and the correct molecular weights (DEFB123: Mr 4523.4 dalton (Da), LL-37: Mr 4502.3 Da) were confirmed on a mass spectrometer (model API100, Applied Biosystems, Foster City, CA, USA).

The annotation "DEFB23" is synonymous to "DEFB123" (this manuscript), as they refer to the identical open reading frame on human chromosome 20q11.1 (see also: http://www.nbn.ac.za/genecards/cgi-bin/carddisp?DEFB123). The alignment by Schutte et al. (14) is based on the deduced amino acid sequences of ß-defensin genes, which have been identified by a bioinformatic approach. The consensus sequence shown by Schutte et al. suggests that two amino acids N-terminally from the first cysteine are conserved (marked as ++), whereas C-terminally from the sixth cysteine no amino acid is conserved (no +). Obviously, this fact is due to a mathematical algorithm of a bioinformatic program and not due to a biologically based prediction (14) . To date, the corresponding native peptide coded by the human DEFB123 gene was not detected with specific antibodies, and the putative biologically active form has not been isolated yet.

According to signalP-server (http://www.cbs.dtu.dk/services/SignalP), the signal peptide of full-length DEFB123 is most likely cleaved between pos. 20 and 21: (-TPG-GT-), which suggests that mature DEFB123 most likely includes the N-terminal dipeptide GT-.

The C-terminal sequence -KPKYQ + 8 shown in the alignment of Schutte et al. (14) implies no prediction of the biological active C-terminal end of DEFB123. We have chosen VKPK as terminal amino acids, as the net charge is comparable to that of the terminal amino acids "KK" of DEFB3, which we used in our experiments (see Fig. 3 ).


Figure 1
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  		Figure 1. Synthetic DEFB123 binds to LPS in a Limulus amoebocyte lysate assay. Binding of synthetic peptides MBI-28, DEFB123, and DEFB3 to lipopolysaccharide (LPS; E. coli O111:B4) in the noted concentrations were determined by chromogenic LAL assay. Data are derived from n = 2 experiments ± average. Note that DEFB3 did not show any effect.


Figure 2
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  		Figure 2. Prevention of LPS-mediated tumor necrosis factor (TNF)-{alpha} release from RAW264.7 cells. Murine macrophage cell line RAW264.7 was preincubated with increasing doses of synthetic DEFB123 for 30 min. Cells were stimulated with LPS or with medium (control) for 5 h. The resulting TNF-{alpha} release was measured with a commercial ELISA. Shown are the inhibitory effects of DEFB123 as calculated from the means of 2 independent experiments done in triplicate ± SD Regression analysis and IC50 determination were calculated using GraphPad Prism 3.1.


Figure 3
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  		Figure 3. DEFB123 inhibits LPS-mediated MAPK phosphorylation. Macrophages (RAW264.7) were incubated with combinations of LPS and synthetic DEFB123, DEFB3, MBI-28, or left untreated, cellular proteins were extracted, separated by PAGE, and blotted onto PVDF membrane. LPS signaling via MAP kinase p42/44 (A) and MAP kinase p38 (B) was detected using phospho-specific antibodies. Figure shows the phospho-specific bands (top) of the respective kinase in comparison to total MAP kinase (middle) and ß-actin as controls (bottom). Note that DEFB3 did not show an inhibitory effect.

Antimicrobial activity assays
Conventional inhibition zone assay on agar dishes and microbroth dilution assays were prepared as described previously (23 ,24) . The following bacterial strains were purchased from the Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ; Braunschweig, Germany): Staphylococcus aureus ATCC25923, Streptococcus pneumoniae DSM11865, Escherichia coli DSM 96440, Escherichia coli DSM 1103, Klebsiella pneumoniae DSM681 and Pseudomonas aeruginosa DSM 1128. Staphylococcus carnosus TM300 was provided by F. Goetz, University of Munich. In brief, all bacterial strains were grown to midlogarithmic phase in 10 mM potassium phosphate/1% tryptic soy broth (pH 7.2). For the determination of the MIC, two-fold serial dilutions of peptides starting with 200 or 300 µg/ml were prepared in 1x and 1/4x Mueller–Hinton broth medium (MHB; Difco, Heidelberg, Germany) on 96-well cell culture plates (Costar, Corning, Corning, NY, USA). Inocula of 1–5 x 105 colony forming units (CFU) of bacteria were added to each well. The number of the CFUs used to determine the MIC was verified by spreading 2 x 100 µl of diluted bacteria onto agar plates and incubating for 18 to 24 h at 37°C. Grown colonies were counted and the number of CFUs used for the experiment was calculated. According to the NCCLS guidelines (25) , only experiments within a range of 2–5 x 105 CFUs per well were analyzed. After incubation for 18 ± 2 h at 37°C, the bacterial growth was determined by measuring the optical density (OD) at OD570 with a Microplate reader (Dynatech Laboratories, Sullyfield, VA, USA). The MIC was defined as the lowest concentration, which led to no increase in OD570 after 18 ± 2 h.

Radial diffusion assay
The radial diffusion assay method of Lehrer and coworkers (26) was used with slight modifications. In brief, bacterial strains were grown in 5 ml of growth medium at 37°C for 18 h, until they reached a OD600= 0.8. Subsequently, 500 µl of the bacteria were diluted in 100 ml agar [30 mg Tryptic-Soy-Broth (Sigma T-8261), 800 mg NuSieve GTG Agarose (Biozym), 200 µl Tween-20 (10%), 10 mM sodium phosphate buffer, pH 7.2]. Sterile petri dishes were filled with 25 ml agar and a series of 3-mm diameter cavities were punched after agar solidification. The peptides were applied in these cavities in different concentrations (2 µg, 10 µg, 20 µg). As a positive control, 10 µg synthetic MBI-28 were applied in cavities on the same plates. Antibacterial activity was identified as a clear zone around the cavity, the absence of microbial growth, after 18 h incubation at 37°C. The diameter of the inhibition zone around the wells was measured using the metric scale (0.1 mm increments) and net clearing was expressed in units (0.1 mm diameter inhibition=1 U) minus the well diameter.

Limulus amoebocyte lysate assay
The Limulus Amoebocyte Lysate (LAL) assay was performed with a quantitative chromogenic LAL kit (No. 50–648U, BioWhittaker, Europe), according to the supplier’s instructions. Incubations were performed in flat-bottom, nonpyrogenic 96-well tissue culture plates (Costar, catalog no. 3596). In brief, a solution containing LPS from E. coli O111:B4, including 1 endotoxin units (EU)/ml, was vortexed for 120 s before experimentation and used to perform dilution series from 0.1 to 1.0 EU/ml. Stock solutions of synthetic peptides DEFB3, MBI-28, and DEFB123 were prepared in endotoxin-free acidified water (0.01% acetic acid) and serially diluted. Samples of each dilution (1 to 50 µg/ml) were transferred to the 96-well plates and lyophilized. Fifty microliters of an endotoxin dilution (0.75 EU/ml) were added to each peptide-containing well. The plate was incubated for 10 min at 37°C to permit binding of the peptide to LPS. After addition of 50 µl LAL reagent and 10 min further incubation, two volumes of chromogenic substrate (acetyl–Ile–Glu–Ala–Arg–p-nitroanilide) were added, and the plate was incubated for another 6 min at 37°C. The reaction was stopped by addition of 50 µl 25% acetic acid. Liberation of p-nitroaniline was monitored at 405 nM with a SpectraMax 250 Kinetic Microplate Spectrophotometer (Molecular Devices). LPS binding was calculated by linear regression using the GraphPad PRISM V3.02 program.

Cells and conditions
The murine macrophage cell line RAW 264.7 was obtained from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium (Invitrogen, Carlsbad, CA, USA) supplemented with 100 U/ml penicillin and 100 mg/ml streptomycin (Invitrogen), 2 mM L-glutamine (Invitrogen), and 10% fetal calf serum (Biochrom, Berlin, Germany). Cells were grown at 37°C and 5% CO2 in humidified air and subcultured 3 times a week. Cells were seeded in 96-well plates at 4 x 105 cells/ml or 6-well plates at 4 x 106 cells/ml. RAW 264.7 cells were preincubated with or without synthetic ß-defensin in cell culture medium for 30 min before the addition of LPS (E. coli, O111:B4, Sigma-Aldrich, Germany) and further incubation for 5 h. For MAPK determination experiments, cells were stimulated for 15 min. TNF-{alpha} levels per 50 µl cell culture supernatant was determined using a mouse cytokine ELISA kit (Pierce, Rockford, IL, USA) according to the manufacturers instructions. The corresponding dose-response curve was normalized according to the total amount of TNF-{alpha} release in relation to medium alone (=100%).

Immunoblot analysis
After stimulation of RAW264.7, the cells were lysed, electrophoresed, and blotted as described (27 ,54) . Membranes were incubated with antisera against pMAPK42/44 or p38 (1:2000, New England BioLabs, Beverly, MA, USA) overnight at 4°C. Signals were detected as described (28) .

Murine model
All experiments have complied with all relevant federal guidelines and institutional policies. 7- to 8-wk-old male C57BL/6 mice with a body wt ranging from 20–23 g were purchased from Charles River Laboratories (Sulzfeld, Germany). The animals were housed for at least 3 days in single cages before experimentation. D-Galactosamine (D-GalN) and LPS derived from E. coli, strain O111:B4, were obtained from Sigma-Aldrich. All dilutions were done in 0.9% NaCl (w/v) in endotoxin-free water (Sigma-Aldrich). Before experimentation, LPS was diluted in saline and vortexed for 120 s before injection. C57BL/6 mice were intraperitoneally (i.p.) injected with D-GalN (40 mg/0.2 ml saline), LPS (100 ng/0.2 ml saline) and either DEFB123 (dilution in 0.3 ml saline) or saline alone (0.3 ml) within 30 s, always changing the side of the peritoneum starting with D-GalN and LPS before the injection of the synthetic peptide. Deaths due to LPS-induced sepsis were recorded every 24 h until day 7 postinfection. All deaths occurred within 48 h of infection. All mice that did not die within 48 h survived the entire 7 days.

RESULTS

Identification and synthesis of DEFB123
We were analyzing a genomic cluster (Acc. no. AL121751.12) on human chromosome 20q11.1 containing tandemly orientated members of the ß-defensin family (15) . One of the genes, named DEFB123 [NP_697019, synonym: DEFB23, also identified by (14) ]), shows an average sequence homology similar to the known members of the ß-defensin family. After cloning and confirmation of the corresponding cDNA sequence (NM_153324), we generated a synthetic peptide comprising the cysteine core region (GTQRCWNLYGKCRYRCSKKERVYVYCINNKMCCVKPK), which was used for all experiments.

Antimicrobial activity of synthetic DEFB123
Defensins have been defined so far as small cationic peptides that are main components of the innate immune system. To examine the antimicrobial properties of DEFB123, we tested the peptide against reference bacteria comprising a set of Gram-positive and Gram-negative bacterial strains (Table 1 ). First, we determined the minimal inhibitory concentration (MIC) of DEFB123 by a serial dilution test. We found that synthetic DEFB123 showed growth inhibitory properties against Klebsiella pneumoniae, Streptococcus pneumoniae, Pseudomonas aeroginosa, and against Escherichia coli and Staphylococcus species (Table 1) . To confirm our data, we tested the antimicrobial activity of DEFB123 against the same panel of bacteria by RDA. Our results show that synthetic DEFB123 inhibits bacterial growth against most of the tested strains in a dose-dependent manner (Table 1) .


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  		Table 1. Antimicrobial activity of synthetic DEFB123

LPS-binding properties of DEFB123
Antimicrobial peptides are known to bind with the negatively charged LPS-leaflet of Gram-negative bacteria. To test whether DEFB123 binds to LPS, a quantitative chromogenic Limulus amoebocyte lysate (LAL) assay was performed. Serial dilutions of synthetic DEFB3 (formerly hBD3) and DEFB123 were tested for their interference with the effects of LPS (E. coli O111:B4) in the LAL assay. To compare our results with other LPS-binding peptides we used synthetic MBI-28 (29) as a reference. Our results show that DEFB123 was able to bind 50% of free LPS (Fig. 1 ). Interestingly, DEFB3 showed no effect in the used assay. The binding capacity was only 1.7-fold lower than that of the synthetic peptide MBI-28. We therefore decided to further investigate this unusual property of DEFB123.

DEFB123 interferes with LPS-mediated effects on RAW264.7 cells
We wondered whether the binding of DEFB123 to LPS could also inhibit endotoxin effects on living cells. Clinical sepsis is currently viewed as an inflammatory dysregulation arising when the host is unable to successfully contain an infection (30) . The pathophysiological mechanisms underlying sepsis is accompanied by a progressive inflammatory response, but the exact mechanism is not completely understood. Studies on animal models have revealed that high amounts of TNF-{alpha} are released in response to LPS, leading to multiple organ failure and death, which, at least, resembles the situation in human sepsis (30) . To test whether DEFB123 can inhibit LPS-induced immune response, we used the murine macrophage cell line RAW 264.7 cells as a model. Cells were incubated with or without micromolar concentrations of synthetic DEFB123, before they were stimulated with LPS. The corresponding TNF-{alpha} release was recorded by ELISA after 5 h of stimulation. Our study shows that DEFB123 inhibits LPS-mediated TNF-{alpha} release in a dose-dependent manner (Fig. 2 ). According to our data, the half inhibitory concentration was calculated to be below 10 µM, which corresponds to 45 µg/ml.

To further validate this effect, we tested whether earlier events in LPS signaling were inhibited by DEFB123 action. Using the same macrophage model, we tested whether endotoxin-induced MAP kinase activation was also inhibited by our synthetic ß-defensin. Using phosphospecific antibodies that detect activated (phosphorylated) kinases, we confirmed that high levels of pMAPK42/44 and p38 were completely abolished by preincubation of DEFB123 with the RAW264.7 cells before LPS stimulation. This effect was seen with DEFB123 but not with ß-defensin DEFB3 (Fig. 3 ).

DEFB123 protects D-GalN-sensitized mice from the lethal toxicity of LPS
Because our data showed evidence that DEFB123 binds to and intervenes with LPS-mediated effects in vitro and in cell culture models, we investigated its interference properties in vivo. Antimicrobial peptides have demonstrated endotoxin-neutralizing activities in the murine sepsis model (31) , but to our knowledge, no member of the ß-defensin family has been tested so far. To assure that DEFB123 itself had no toxic effects in vivo, we injected 1 mg synthetic peptide in the tail vein of three C57BL/6 mice, respectively. The mice did not show signs of intoxication in our recording time of more than 30 days (data not shown).

We then tested the benefit of synthetic DEFB123 in a murine model of acute sepsis according to the procedure Nagaoka and colleagues (32) . Groups of C57BL/6 mice were sensitized with D-Galactosamine (D-GalN), which induces high susceptibility to LPS-induced lethality (33) . Endotoxemia was induced by 100 ng LPS/mouse. In our first approach, we preincubated the synthetic peptide with LPS under permissive binding conditions and subsequently injected the solution in groups of mice (Table 2 ). As a control, the single injection of LPS induced lethality in 5 out of 6 mice within 48 h. Although our mice were age- and sex-matched, one survivor in our control group was seen in two independent experiments. Coincubation of LPS and an amount of 1000 µg of synthetic DEFB123 resulted in the survival of all animals, while the usage of 100 µg synthetic DEFB123 had no effect (Table 2) . We hypothesized that the survival effect could be mainly due to physical binding of DEFB123 to LPS, induced by the preincubation step. We further investigated the protective effect of DEFB123 by a more therapeutic approach. After i.p. administration of a lethal dose of 100 ng LPS, we injected synthetic DEFB123 on the opposite site of the peritoneum without prior preincubation of the agents. Administration of 1000 µg showed survival rates of 75%, which was reproducible in 3 independent experiments with n ≥ 6 animals per group (Table 2) . For comparison of the potency of DEFB123, we used different doses of synthetic LL-37/CAP18104–140 peptide, an optimized cathelicidin-derivative, with a high potential to prevent LPS-mediated lethality in the sepsis model used in these experiments (33) . Synthetic LL-37/CAP18104–140 showed similar protective effects against endotoxemia at approximately 2 orders of magnitude lower amounts of peptide. Mice that survived due to peptide administration were monitored for at least 8 days, showing no obvious signs of septic shock. After 8 days, mice were sacrificed and livers were dissected. No obvious anatomical changes were seen, whereas the mice that died from LPS-treatment showed a broad range of abnormalities in most of the liver lobes (data not shown).


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  		Table 2. Human beta-defensin blocks LPS-induced lethality in D-GalN sensitized C57BL/6 mice

DISCUSSION

Most animal species, from insects to humans, have developed a diverse array of peptide defense systems to counter microbial invasion and infection (34) . Antimicrobial peptides, believed to specifically build a first barrier of the immune system, were summarized according to their bacterial membrane-permeabilizing properties. However, beside their common function in the inhibition of bacterial growth in vitro, all peptides show marked differences in tissue expression, gene regulation, structural properties, and biological activities on the immune system. The members of the ß-defensin family were identified according to a conserved pattern of six cysteine residues. To date, 34 human genes coding for members of the ß-defensin family were assigned (35) . We have analyzed the antimicrobial properties of the epididymally expressed ß-defensin DEFB123, located at the centromere of human chromosome 20. For our investigation, we chemically synthesized the corresponding ß-defensin core region. Like all other members of the ß-defensin family tested so far, DEFB123 revealed a broad range of antimicrobial activity against Gram-negative and Gram-positive bacteria. The derived MIC values resemble those of other ß-defensins. Antimicrobial activity is known to depend on the intrinsic properties of the peptide, including its three-dimensional structure and the positioning of charges and hydrophobic residues along the peptide chain (36) . In addition, the N- and C-termini play a role in bacterial strain inhibition, which was described for DEFB1 (formerly hBD1), with differing antimicrobial activities of peptide fragments ranging from 36 to 47 amino acids (37) .

Different modes of action were also reported from HE2-derived ß-defensins, originally isolated as a epididymis-specific transcript (38) . Consistent with the ability to kill bacteria, full-length HE2 proteins and C-terminal peptides caused rapid dose-dependent permeabilization of outer and cytoplasmic membranes of E. coli (39) . In comparison, a much longer exposure time was required for human ß-defensin-1 (hBD1)-mediated permeabilization of membranes, suggesting a difference in the mode of action of the corresponding peptides (39) .

Many of the host defense peptides that exert antimicrobial activity are also able to bind to and neutralize LPS; however, these two activities do not necessarily correlate (31) . Structurally related peptides can confer different efficiencies in LPS neutralization without alteration of the binding affinity for LPS (40) .

Interestingly, we have found LPS-binding properties of DEFB123 in a LAL-assay, whereas the structurally similar ß-defensin DEFB3 (i.e., hBD3) fails to bind, even in concentrations of up to 50 µM (Fig. 1) . In contrast to DEFB3, the putative ß-sheet peptide, DEFB123, shows comparable activities to that of MBI-28 (formerly, CEMA), a designed {alpha}-helical antimicrobial peptide derived from parts of silk moth cecropin and bee melittin (29) .

Using a macrophage cell line (RAW 264.7), DEFB123 showed dose-dependent abolishment of LPS-mediated induction of p38 and p42/44 MAPK phosphorylation, as well as LPS-induced TNF-{alpha} release. TNF-{alpha} is considered to be a primary mediator of endotoxemia (41) . In comparison, synthetic DEFB3 did not reduce LPS-mediated activation of MAPK, albeit DEFB2 (i.e., hBD2) is known to have a similar effect as DEFB123 (42) . The inhibition of LPS and the blocking of TNF-{alpha} was also reported for the {alpha}-helical peptide MBI-28 (43) , which is in the same MW range. In addition, the structurally unrelated cathelicidin CAP18/LL-37 shows similar behavior (33) .

To complete our study, we have investigated the LPS-inhibitory action of DEFB123 in vivo using a murine sepsis model. DEFB123 efficiently inhibited LPS-mediated lethality. A high lethality-decreasing effect was also seen when LPS and peptide were injected on different sides of the peritoneum. Although used in a comparably high dose, DEFB123 showed no toxic effect on the mice. Thirty days after a 1-mg intravenous (i.v.) injection of synthetic DEFB123, mice were indistiguishable from uninjected mice. In vivo capacity to protect against death in the murine sepsis model was also demonstrated for the cathelicidins (44) . Supported by our data, ß-defensin peptides also show protective action against LPS-mediated effects, although they are structurally unrelated to the cathelecidins. The mechanism of this action remains unclear.

Our data suggest that DEFB123 exerts LPS eradication capacity, which may not solely be explained by LPS-binding properties. Interestingly, LL-37-derived peptides bind to the RAW264.7 membrane surface, inhibit LPS binding protein-mediated transport of LPS to CD14+ cells (45) , and inhibit the LPS-induced suppression of neutrophil apoptosis by blocking the binding of LPS to target cells (22) . Epididymally expressed defensins, like DEFB123, bind to the spermatozoan membrane. One can speculate that binding to target cells functions as a preventive effect, building a passive defense against LPS, which on sepsis, increases too fast for effective innate or adaptive immune response.

A physiological role of the epididymally expressed DEFB123 in the inhibition of septic shock seems unlikely. It is known that LPS toxicity in D-galactosamine-treated mice actually results from severe apoptotic liver injury caused by TNF-{alpha} induced hepatocyte apoptosis (46) . In either case, effective inhibition of LPS-mediated effects in vivo by a ß-defensin hints to a more complex involvement of the peptides in the immune system. To date, millions of peptides have been synthesized and analyzed for the use as antibiotics. Well-documented structure-activity relationship studies for bacterial clearance are lacking, as are systematic studies on variations of biological functions. Thousands of antimicrobial peptides with variable lengths and sequences (http://www.bbcm.univ.trieste.it/~tossi/search.htm) have been described, all of which are active at similar concentrations, suggesting a general mechanism for killing bacteria rather than a specific mechanism that requires preferred active structures (47) . Finally, evaluating individual functions of the ß-defensins could assist in justifying the reported number of defensins discovered in this family, since true redundancy of function is rare in nature.

The determination of whether our findings are reflective of DEFB123 activity in vivo will require isolation and confirmation of the secondary structure of the native peptide. Interestingly, ß-defensins are characterized by an intricate tertiary structure with a core of three antiparallel ß sheet components resembling chemokines (48 , 49) . This may be reflected by the chemotactic recruitment of neutrophils to inflammatory sites by DEFB2 (22) and by modulation of dendritic cells induced by LL-37 (50) .

Our studies suggest, that the group of ß-defensins show immunological properties in the inhibition of LPS-mediated effects in vitro and in vivo. The induction of antimicrobial peptides by LPS also points to an influence of the innate and adaptive immune system (51 , 52 , 53) . Because ß-defensins are known to bind to cells and to the spermatozoan membrane, systemic inhibition of LPS might mirror a more specific effect in the immune system, involving both bacteria and cells of the immune system.

ACKNOWLEDGMENTS

We are very grateful for the excellent technical assistance of Gabriele Walkling and Rainer Schreeb. We greatly appreciate discussions and advice of Dr. Knut Adermann for the selection of the DEFB123 peptide sequence. We would also like to thank the anonymous reviewers who helped tremendously in improving the quality of the manuscript. This work was supported by a grant from the Lower Saxony Ministry of Economics, Technology and Transport.

FOOTNOTES

1 Current address: Deutsches Primatenzentrum GmbH, Kellnerweg 4, Göttingen 37077, Germany.

2 These authors contributed equally to this work.

Received for publication October 6, 2005. Accepted for publication March 31, 2006.

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