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Published ahead of print on January 24, 2005, doi:10.1165/rcmb.2004-0250OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 32, pp. 443-452, 2005
© 2005 American Thoracic Society
DOI: 10.1165/rcmb.2004-0250OC

The Antimicrobial Antiproteinase Elafin Binds to Lipopolysaccharide and Modulates Macrophage Responses

Jonathan W. McMichael, Ali Roghanian, Lu Jiang, Robert Ramage and Jean-Michel Sallenave

Rayne Laboratory, MRC Centre for Inflammation Research, Edinburgh University Medical School; and Albachem Ltd, Chemistry Department, University of Edinburgh, Edinburgh, Scotland, United Kingdom

Correspondence and requests for reprints should be addressed to Jean-Michel Sallenave, Rayne Laboratory, MRC Centre for Inflammation Research, Edinburgh University Medical School, Teviot Place, Edinburgh, Scotland EH8 9AG, UK. E-mail: J.Sallenave{at}ed.ac.uk


   Abstract
Top
 Abstract
Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
Lipopolysaccharides (LPS) of the outer membrane of Gram-negative bacteria represent a primary target for innate immune responses. We demonstrate here that the antimicrobial/anti–neutrophil elastase full-length elafin (FL-EL) is able to bind both smooth and rough forms of LPS. The N-terminus was shown to bind both forms of LPS more avidly. We demonstrate that the lipid A core-binding proteins polymyxin B (PB) and LPS-binding protein (LBP) compete with elafin for binding, and that LBP is able to displace prebound elafin from LPS. When PB, FL-EL, N-EL, and C-EL were pre-incubated with LPS before addition to immobilized LBP, PB was the most potent inhibitor of LPS transfer to LBP. These data prompted us to examine the biological consequences of elafin binding to LPS, using tumor necrosis factor (TNF)-{alpha} release by murine macrophages. In serum-containing conditions, N-EL had no effect, whereas both C-EL and FL-EL inhibited TNF-{alpha} production. In serum-free conditions, however, all moieties had a stimulatory activity on TNF-{alpha} release, with C-EL being the most potent at the highest concentration. The differential biological activity of elafin in different conditions suggests a role for this molecule in either LPS detoxification or activation of innate immune responses, depending on the external cellular environment.

Key Words: elafin • lipopolysaccharide • LPS-binding protein • macrophages • innate immunity


   Introduction
Top
 Abstract
 Introduction
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The immune system detects and eliminates invading pathogenic microorganisms by discriminating between self and infectious nonself. Adaptive immunity relies on gene rearrangement and clonal expansion upon detection of specific antigens of the invading pathogens, whereas innate immune responses are not pathogen-specific, but rather rely on recognition of conserved molecular patterns exhibited by the pathogens. Of particular importance in this regard is lipopolysaccharide (LPS) or endotoxin, a ubiquitous component of the outer membrane of Gram-negative bacteria. LPS is a potent agonist of the innate immune system and mediates its effects via the CD14/TLR4 receptor complex (1). The rapid response against LPS can be of benefit to the host in local sites of infection and in moderate levels by promoting inflammation and priming the immune system to eradicate the invading pathogens; however, an excessive or systemic response to LPS (for example, when LPS enters the bloodstream) can lead to endotoxemia and a potential systemic inflammatory condition characterized by multiple organ failure, shock, and potentially death (2). The discovery of novel strategies aimed at modulating the inflammatory response to LPS is therefore of great interest. One potential approach to addressing therapeutically the pathophysiologic sequelae induced by LPS is to target the molecule directly and sequester its agonistic activities. In this regard, cationic antimicrobial peptides of the innate immune system are particularly attractive (3–4). Among these, our group is chiefly interested in the four-disulphide core proteins secretory leukocyte proteinase inhibitor (SLPI) and elafin (5–6). The latter is produced in a variety of epithelial and mucosal sites throughout the body, including the lung (7–10), and elafin has been found to constitute ~ 20% of the total antielastase activity retrieved from bronchoalveolar lavage fluid in normal subjects (11). Similar to SLPI, early inflammatory "alarm signals" such as LPS, tumor necrosis factor (TNF), interleukin (IL)-1, and human neutrophil elastase, can switch on the production of elafin (for reviews see Refs. 5 and 6), suggesting a role in the initial stages of the innate immune response to infection. Previous studies from us and others have shown that elafin and its two domains (N-terminus and C-terminus) have antibacterial activities in vitro (12–13) as well as in vivo (14). In addition to their antibacterial activity, some of these antimicrobial molecules have also been shown to bind LPS and downregulate its proinflammatory activity (15–17). This was shown recently to be the case for SLPI (18), which we have also used in the present investigation. Using a variety of biochemical and immunologic methods, this manuscript describes for the first time the interaction of elafin (and its domains) with LPS and the biological consequences of this interaction on the proinflammatory activities of LPS on murine RAW 264.7 macrophages.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Synthetic Human Elafin Peptides and SLPI
Human elafin peptides were produced synthetically and have previously been described by us (12). Three peptides were used for these studies, namely full-length elafin, henceforth referred to as FL-EL (H2N-1AVT.....95Q-COOH), the NH2-terminal domain N-EL (H2N-1AVT.....50K-COOH), and the COOH-terminal domain, C-EL (H2N-51PGS.....95Q-COOH). The molecular weights of elafin moieties, determined by mass spectrometry, were 9,925 D for FL-EL, 5,172 D for N-EL, and 4,776 D for C-EL.

Human SLPI (11.7 kD) was obtained from R&D Systems (Abingdon, UK).

LPS and Reagents Related to LPS-Binding Studies
Escherichia coli O55:B5 LPS (smooth-form serotype) and polymyxin B sulfate (PB) were from Sigma (Poole, UK). Biotinylated E. coli O55:B5 LPS, recombinant human LPS-binding protein (LBP), and anti-LBP monoclonal antibody (mAb) HM2 were kind gifts from Dr. Anita Vreugdenhil (University of Maastricht, The Netherlands). E. coli K12, R2, and R3 LPS (representative of three of the five classes of rough-form serotypes) were kind gifts from Dr. Richard Gibbs (Department of Medical Microbiology, University of Edinburgh, UK). Human LBP peptide (1.8 kD), natural human LBP, and monoclonal murine antibody to PB were obtained from HyCult Biotechnology (Uden, The Netherlands).

Polyacrylamide Gel Electrophoresis for the Investigation of LPS Electrophoretic Migration (pH 8.8)
The interaction between peptides (FL-EL and PB) and LPS was investigated using polyacrylamide gel electrophoresis (PAGE) analysis (pH 8.8), as described before for SLPI (18). Sample buffer and gels were SDS-free and non-denaturing, whereas SDS was included in the electrode buffer. Peptides were incubated alone or with E. coli O55:B5 LPS for 30 min at 37°C in dH2O, and mixed with sample buffer to a total volume of 25 µl before electrophoresis. Electrophoresis was performed on 15% gels at 70–110 V for 2–3 h in a vertical electrophoresis tank Mini Protean II system (Bio-Rad, Hemel Hempstead, UK). Protein bands were detected either by silver stain (Bio-Rad) or by immunoblotting. In the latter case, proteins were transferred onto Hybond ECL nitrocellulose membrane (Amersham Pharmacia, Cambridge, UK) at 110 V for 1 h. Nonspecific protein binding sites were blocked overnight at 4°C with phosphate-buffered saline (PBS)/0.1% Tween 20 containing 5% skim milk. Elafin was then detected with rabbit anti-elafin IgG, followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin, as described before (19). PB was detected with the murine anti-PB monoclonal antibody (see above), followed by goat anti-mouse immunoglobulin (Dako, Glostrup, Denmark). Membranes were developed using the ECL reagent (Western Lightning Chemiluminescence Reagent Plus; Amersham Pharmacia).

PAGE for the Investigation of SLPI, FL-EL, N-EL, and C-EL Electrophoretic Migration (pH 4.5)
Peptides and LPS were diluted to the appropriate concentration in dH2O, and incubated together for 30 min at 37°C. Where one peptide was incubated with LPS before the addition and incubation of a second peptide with the reaction mixture, each incubation was performed for 30 min at 37°C. In all cases, the final sample volume was 24 µl. Six microliters of sample buffer was added to each sample before electrophoresis. Electrophoresis was performed on 15% potassium hydroxide-acetic acid gels at 80 V for 2–3 h in a vertical electrophoresis tank Mini Protean II system, using a procedure modified from Reisfeld and coworkers (20). Protein bands were detected by Coomassie staining.

Enzyme-Linked Immunosorbent Assay for the Investigation of the Inhibition of the LPS–LBP Interaction
The enzyme-linked immunosorbent assay (ELISA) technique used here measures the binding of biotinylated LPS to immobilized LBP. This technique was performed according to the method of Scott and colleagues (21). Briefly, the anti-LBP mAb HM21 was diluted to 10 µg/ml in PBS and adsorbed onto a 96-well assay plate (Corning Costar, High Wycombe, UK) overnight at 4°C. The plate was then blocked with PBS containing 1% bovine serum albumin (BSA; Sigma) for 1 h at room temperature and washed with 0.1% Tween 20 in dH2O. Recombinant LBP (25 ng/ml) diluted in PBS/0.1% BSA was added to the plate for 1.5 h at room temperature. After washing, biotinylated E. coli O55:B5 LPS was added in the presence or absence of peptides (30 min incubation at 37°C in PBS). After 1 h incubation at room temperature and washing, binding of biotinylated LPS to immobilized LBP was detected using HRP-conjugated streptavidin (Dako) diluted 1:2,000 in PBS/0.1% BSA. The plate was incubated for 1 h at room temperature, washed, and 3,3',5'5-tetramethylbenzidine (TMB; Boehringer Mannheim UK, Lewes, UK) added 1:50 in H2O2/sodium acetate citrate (pH 4.9) for 15 min. The reaction was stopped by addition of 1 M H2SO4 (BDH, Poole, UK) and the optical density read at 490 nm (MRX II microplate reader; Dynex Technologies, Billinghurst, UK).

ELISA for the Investigation of Elafin/LBP Competition for LPS Binding
An ELISA technique was devised to measure binding of elafin to immobilized LPS. LPS alone binds poorly to assay plates, therefore LPS was prebound to PB to form complexes that are stable when coated onto microplates; this method was adapted from Scott and Barclay (22). Briefly, equal volumes of 36 µg/ml E. coli O55:B5 LPS and 20 µM PB (both diluted in dH2O) were mixed with continuous shaking for 30 min at room temperature. The mixture was then dialyzed overnight against dH2O using a 3.5-kD pore size, to remove excess unbound PB. Fifty microliters per well of this mixture was coated onto a 96-well assay plate overnight at 4°C. The plate was then blocked with PBS containing 1% BSA for 1 h at room temperature and washed with 0.1% Tween 20 in dH2O. FL-EL was added for 1.5 h at room temperature (final concentration, 156 nM). Alternatively, 25 ng/ml of recombinant LBP was added to wells simultaneously (final concentration, 0.42 nM) with elafin, or 45 min after addition of FL-EL, and then incubated for a further 45 min. After washing, rabbit anti-elafin IgG diluted 1:1,000 in PBS/0.1% BSA was added and incubated for 1 h at room temperature. The plate was washed, then incubated for 1 h at room temperature with HRP-conjugated goat anti-rabbit immunoglobulin diluted 1:2,000 in PBS/0.1% BSA. After washing, the plate was developed and read as described previously.

Investigation of the Effects of Elafin Peptides on RAW 264.7 Macrophage TNF-{alpha} Production after Stimulation with LPS
RAW 264.7 murine macrophages (obtained from the ATCC collection; ATCC, Manassas, VA) were seeded at 5 x 105 cells/well in 48-well plates (Corning Costar) and incubated overnight at 37°C. Cells were washed twice with PBS, and 500 µl of either serum-free Dulbecco's modified Eagle's medium (DMEM) or DMEM containing 0.2% fetal calf serum (Labtech, Ringmer, UK) was added. A quantity of 50 ng/ml of E. coli O55:B5 LPS was incubated with cells for 4 h at 37°C either alone or in the presence of elafin peptides. In the latter case, 2.5-µl samples of LPS were incubated with 5-µl samples of elafin peptides for 30 min at 37°C before addition to cells. Total sample volume was 7.5 µl, and both LPS and elafin were diluted to test concentrations in dH2O. In all cases, cell supernatants were analyzed for TNF-{alpha} prodution using a commercial ELISA kit (Duoset; R&D Systems) in accordance with the manufacturer's instructions. The standard curve measured between 15.6 pg/ml and 1,000 pg/ml protein, and test samples were diluted accordingly in PBS containing 1% BSA.

Statistical Analysis
Results are reported as pooled data from a series of n separate experiments, each performed in duplicate or triplicate, and presented as mean ± SE. Statistical significance was analyzed by one-way ANOVA with comparisons between groups made using the Bonferroni Multiple Comparison Test. Statistical significance was assigned to data returning a P value < 0.05.


   RESULTS
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
PAGE Analysis
Inhibition of SLPI and elafin electrophoretic migration after LPS incubation. To assert that elafin and its constituent moieties interact directly with smooth-form LPS, a native acidic (pH 4.5) PAGE system was used, which allowed us to visualize the cationic migration of SLPI and elafin. SLPI and elafin peptides were incubated with LPS and were applied to this gel system as shown in Figure 1. FL-EL and SLPI were shown to bind LPS as evidenced by the gradual disappearance of the characteristic protein bands in a dose-responsive manner with increasing LPS concentrations (lanes 1–7 and corresponding densitometry). Both N-EL and C-EL bound to LPS, although the affinity of the N-EL/LPS interaction was greater, demonstrating specificity in our system. It must be noted that the affinity of LPS with FL-EL versus that of its constituent domains cannot be compared directly in Figure 1, because of the different amounts of peptides required for detection by silver staining (2.5 µg for FL-EL and SLPI versus 5.0 µg for N-EL and C-EL). In addition, we show (Figure 2) that N-EL was able to displace smooth-form LPS from C-EL (and hence compete for LPS binding), when added after C-EL/LPS incubation (lanes 4 and 5), or when all reagents were added simultaneously (lane 6), as demonstrated in both cases by the "recovery" of the C-EL band (compared with lane 3; see densitometry). We then tested the binding of peptides to rough-form E. coli LPS serotypes E. coli K12, R2, and R3, which consist solely of the lipid A-core moiety. For illustrative purposes, SDS was added in the PAGE (pH 8.8) electrode buffer (Figure 3, upper left) to demonstrate the distinction between smooth and rough LPS; otherwise, other gels in Figure 3 (native PAGE, pH 4.5) are strictly SDS-free. FL-EL was found to bind strongly to K12 (Figure 3, upper right, lanes 2 and 3). Binding to R2 and R3 was also demonstrated by a dose-responsive weakening and upwards shift of FL-EL (perhaps caused by a decrease in the overall net positive charge after interaction with LPS), but did not appear to be as strong as the interaction with K12 (lanes 4–7). SLPI appeared to also have a very high affinity for rough-form LPS serotypes (Figure 3, lower left). When studying the interaction of N-EL and C-EL with K12, R2, and R3, qualitatively similar results were obtained, compared with FL-EL: K12 was bound efficiently by N-EL and C-EL, in comparison with R2 and R3 (Figure 3, lower right). Because the molecular weights of these rough-form LPS serotypes are similar (23–24), and considering that even less K12 was required to inhibit peptide migration, it is evident that, on a molar ratio basis, elafin peptides bind K12 more avidly than R2 or R3.



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  		Figure 1. Inhibition of elafin and SLPI electrophoretic migration after incubation with smooth-form LPS (native PAGE, pH 4.5). Peptides (SLPI, FL-EL, N-EL, C-EL) were incubated alone or with smooth-form E. coli O55:B5 LPS serotypes for 30 min at 37°C in 24 µl total sample volume; 6 µl of sample buffer (SDS-free) was mixed with samples before loading. Electrophoresis was performed in 15% gels at 80 V for 2–3 h, and peptides were detected by Coomassie staining. Densitometry was performed to assess the intensity of peptide bands (arbitrary units). Each gel is representative of three independent experiments.

 


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  		Figure 2. N-EL competes favorably with C-EL for binding of LPS (native PAGE, pH 4.5). Peptides were incubated alone (lanes 1–2, 7) or with smooth-form O55:B5 LPS (lanes 3–6). Alternatively, C-EL was successively incubated with LPS (30 min at 37°C) and with varying amounts of N-EL (lanes 4–5), or was incubated with LPS and N-EL simultaneously (lane 6). In all cases, electrophoresis was performed in 15% gels at 80 V for 2–3 h, and peptides were detected by Coomassie staining. Densitometry was performed to assess the intensity of peptide bands (arbitrary units). Each gel is representative of three independent experiments.

 


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  		Figure 3. Inhibition of elafin and SLPI electrophoretic migration after incubation with rough-form LPS (native PAGE, pH 4.5). Peptides (SLPI, FL-EL, N-EL, C-EL) were incubated alone or with rough-form E. coli LPS serotypes K12, R2, or R3 for 30 min at 37°C in 24 µl total sample volume; 6 µl of sample buffer (SDS-free) was mixed with samples before loading. Electrophoresis was performed in 15% gels at 80 V for 2–3 h, and peptides were detected by Coomassie staining. To illustrate the difference in banding between smooth- and rough-form LPS, SDS was included in the electrode buffer in pH 8.8 PAGE analysis (upper left): O55: smooth-form LPS ; K12, R2, R3: rough-form LPS. The other gels are strictly SDS-free. Each gel is representative of three independent experiments.

 
Interaction of elafin with LPS lipid A-core portion. Inhibition of elafin and SLPI migration after incubation with rough-form LPS (Figure 3) was strongly suggestive of binding of these peptides to LPS via the lipid A-core portion. We strove to demonstrate this by incubating LPS and FL-EL and analyzing the complex by using a PAGE analysis (pH 8.8) compatible with LPS migration (see Figure 4). Because elafin did not bind R2 as well as K12, we elected to further test the binding of elafin to the former serotype in a different system, making use of the increased sensitivity of the detection analysis (antibodies against elafin with PB [a cationic cyclic polypeptide antibiotic isolated from Bacillus polymyxa, already known to bind to lipid A with high affinity and to neutralize many of the biological effects of LPS (25)] as a control). Gels stained with silver stain show the electrophoretic pattern of LPS serotypes incubated with either PB (Figure 4A), or with FL-EL (Figure 4C). Western blot analysis allowed us to show that FL-EL bound to the lipid A core portion of both smooth and rough forms of LPS (Figure 4D, lanes 3 and 5, respectively), as did PB (Figure 4B, lanes 3 and 5, respectively). Noticeably, when FL-EL alone was applied, a small proportion (as evidenced by the presence of a weak band) was still able to migrate toward the anode in this system (Figures 4C and 4D, lane 1), presumably because of the presence of SDS in the electrode buffer, which could still be able to charge negatively a proportion of FL-EL. In addition, we performed competition assays and show in Figure 5 that PB incubation with LPS before incubation with elafin peptides (FL-EL, N-EL, and C-EL) reduced the interaction of the peptides with LPS (left panels; compare lanes 3 and 2). Similarly, we demonstrated that addition of increasing amounts of a 1.8-kD peptide from LBP (another well-characterized lipid A–binding protein involved in transferring LPS to CD14/TLR4 receptors and initiating the inflammatory responses [1]) to LPS inhibited subsequent binding of FL-EL and N-EL. Again, C-EL binding to LPS was weaker, demonstrating further the specificity of the interaction (Figure 5, right panels; compare lanes 3–5 with lane 2). These experiments, performed with the following molar ratios: LBP:FL-EL = 5:1; 10:1; 15:1 (lanes 3–5); LBP: N-EL /C-EL = 1:1; 3:1; 5:1 (lanes 3–5), suggested a stronger interaction between LBP and LPS than between elafin and LPS. We therefore designed two ELISA assays to study further the dynamics of the LPS/LBP/elafin interaction.



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  		Figure 4. FL-EL and PB bind to the lipid A core portion of LPS. FL-EL (C and D) or PB (A and B) were incubated alone or with 4 µg of smooth-form O55:B5 LPS or 2 µg of rough-form R2 LPS (incubation of 30 min at 37°C in 20 µl total sample volume). These mixtures were subjected to PAGE analysis (pH 8.8) using SDS in the electrode buffer only. Electrophoresis was performed in 15% gels at 70–110 V for 2–3 h and was followed by silver staining (A and C) or Western blotting with either anti-PB monoclonal antibody (B) or anti-elafin polyclonal antibody (D). The double-headed horizontal arrow shows the position of the lipid A core. Each gel is representative of three independent experiments.

 


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  		Figure 5. Inhibition of elafin-LPS binding by preincubation of LPS with LBP peptide or PB (native PAGE, pH 4.5). A fixed amount of elafin peptides (FL-EL, N-EL, C-EL) was incubated alone (lanes 1) or with 50 or 100 µg of smooth-form E. coli O55:B5 LPS for 30 min at 37°C (lanes 2) in 24 µl total sample volume; alternatively, increasing amounts of LBP peptide (right panels, lanes 3–5) or a fixed amount of polymyxin B (left panels, lane 3) were incubated with LPS (as above) for 30 min at 37°C before addition of elafin peptides, as illustrated by: (+FL-EL), (+N-EL), (+C-EL). These mixtures were then further incubated for 30 min at 37°C. Six microliters of sample buffer (SDS-free) was mixed with samples before loading. Electrophoresis was performed in 15% gels at 80 V for 2–3 h, and peptides were detected by Coomassie staining. Densitometry was performed to assess the intensity of peptide bands. Each gel is representative of three independent experiments.

 
Measurement of Elafin/SLPI Inhibition of LPS–LBP interaction by ELISA Techniques
LPS/polymyxin B–coated ELISA plates. LPS was pre-complexed with PB before coating onto microtiter plates, to enhance adherence of LPS to the wells. Although it was thought theoretically possible that PB would occupy binding sites on LPS required for elafin binding (Figure 4), significant binding of elafin to LPS could still be detected under the employed conditions, suggesting that the concentration of PB was probably sufficient to stabilize LPS molecules but insufficient to occupy the entire binding domain. No nonspecific effects such as binding of elafin to LBP, PB, blocking agent, or binding of secondary antibody to the wells were detected (not shown). When FL-EL and LBP were added in succession, the latter was able to displace 45% of FL-EL bound to LPS (Figure 6A, middle bar). Furthermore, simultaneous addition of FL-EL and LBP induced a 90% inhibition of FL-EL binding to LPS (right bar), as compared with binding in the absence of LBP (left bar). These findings confirm those observed during PAGE studies (Figure 5), and demonstrate that LBP indeed binds LPS more avidly than does FL-EL.



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  		Figure 6. ELISA-based assays for binding studies. (A) LBP outcompetes elafin binding to LPS. FL-EL (final concentration, 156 nM) was added to 96-well plates containing 0.9 µg of E. coli O55:B5 LPS immobilized using PB. Alternatively, 25 ng/ml LBP was added to wells containing FL-EL preincubated with LPS, or FL-EL and LBP were added to wells simultaneously. Binding of FL-EL to LPS was detected using anti-elafin polyclonal antibody followed by an HRP-conjugated secondary antibody; absorbance was read at 490 nm. Major ELISA components are depicted diagrammatically beneath the graph. Values represent mean ± SE of n = 4 experiments, each performed in duplicate (*significant difference, P < 0.001, compared with "FL-EL alone"; **significant difference, P < 0.001, compared with "FL-EL then LBP"). (B) Elafin peptides inhibit the binding of LPS to LBP. Biotinylated E. coli O55:B5 LPS or mixtures of biotinylated LPS preincubated (30 min at 37°C) with increasing concentrations of peptides were added to wells containing 25 ng/ml recombinant LBP, immobilized using anti-LBP monoclonal antibody. Binding of biotinylated LPS was detected using HRP-conjugated streptavidin, and absorbance was read at 490 nm. Major ELISA components are depicted diagrammatically beside the graph. Values represent mean ± SE of n = 3 experiments, each performed in duplicate.

 
Anti-LBP antibody-LBP–coated ELISA plates. LPS and either SLPI or elafin peptides (concentration ranging from 9.75–156 nM) were incubated before administration to anti-LBP mAb–LBP complexes (Figure 6B). At these concentrations, all SLPI and elafin peptides had a similar inhibitory activity on LPS–LBP interaction across the concentration range; maximal inhibition was obtained at 156 nM and at this concentration all three elafin peptides were equally efficient (~ 45% inhibition). The Kds calculated for the peptides' interaction with LPS (using the GraphPad Prism program) were very similar (between 2.2 and 7.9 nM). Of note, PB was the most powerful inhibitor overall (inhibition of LPS–LBP binding of 62% at 156 nM), although the Kd was of the same order of magnitude (7.1 nM). These values are within the same range as those published for the LPS/LBP, LPS/PB, and LPS/MD2 interactions (26–27).

Effects of Elafin Peptides on the TNF-{alpha} Response of RAW 264.7 Murine Macrophages to LPS
To translate in a biological system the biophysical/biochemical findings regarding LPS/elafin interaction (see above), we studied the properties of elafin peptides on the TNF-{alpha} responses of RAW 264.7 macrophages upon stimulation with LPS. These studies were performed either in serum-free medium, to mimic the situation in the lung alveolar compartment where serum components are scarce, or in serum-containing medium, to mimic events in the circulating blood compartment. In the latter system, we titered down the concentration of serum and found that 0.2% of fetal calf serum had the same stimulating effect as purified 25 ng/ml LBP, when LPS was added to RAW cell cultures (not shown). Elafin peptides did not elicit a TNF-{alpha} response when added on their own in serum-free or serum-containing conditions (not shown), nor did they cause any discernible damage to the cell layer, as assessed by light microscopy and trypan blue exclusion (not shown). As expected, because serum is well known to potentiate LPS responses through binding of LPS to the LBP protein and transfer to CD14 (1), TNF output was much lower in serum-free conditions, as compared with serum-containing conditions (4,500 and 25,000 pg/ml, respectively). Interestingly, in serum-containing conditions, while N-EL had no effect, incubation of LPS with FL-EL and C-EL before addition to cells significantly reduced (at 10 and 100 nM) the TNF-{alpha} response (Figure 7A). Of note, the inhibitory effects of both FL-EL and C-EL disappeared when LPS and the peptides were not incubated together but were added simultaneously to the macrophage cell culture (not shown), indicating again that the relative lower avidity of elafin peptides for LPS, compared with LBP, translates similarly in this biological system. However, the effect of incubating elafin peptides with LPS in serum-free conditions was in stark contrast to that observed in serum-containing conditions: pre-incubating FL-EL (10 and 100 nM), N-EL, and C-EL (100 nM) with LPS significantly enhanced (and not decreased) the proinflammatory activity of LPS (Figure 7B). C-EL (100 nM) exerted a greater activity than N-EL and FL-EL, suggesting that elafin terminal domains do not function synergistically and that the observed pro-inflammatory activity of the FL-EL molecule may be regulated by an interaction between its constituent domains.



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  		Figure 7. Effect of elafin on LPS stimulation of RAW 264.7 macrophages, in the presence or absence of serum. RAW 264.7 murine macrophages were seeded at 5 x 105 cells/well of a 48-well plate overnight, washed twice with PBS and 500 µl of serum-containing (A) or serum-free DMEM (B) added. A quantity of 50 ng/ml of E. coli O55:B5 LPS was incubated with or without elafin peptides (30 min at 37°C). These mixtures were then added to RAW cells and media were analyzed 4 h later for mTNF-{alpha} content by ELISA. Values represent mean ± SE of n = 6 experiments, each performed in triplicate (*significant difference, P < 0.05, compared with LPS alone; **significant difference, P < 0.01, compared with LPS alone; ***significant difference, P < 0.001, compared with LPS alone).

 

   DISCUSSION
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
Antimicrobial peptides are evolutionarily ancient elements of the host defense system against infections, and can be found in animals, plants, and bacteria (28). They are small (< 120 amino acids), often cationic, and structurally diverse. In excess of 700 of these molecules have been isolated (http://www.bbcm.univ.trieste.it/~tossi/pag1.htm). Most molecules display broad-spectrum antimicrobial activity against a range of bacteria, fungi, and viruses, and they show selective toxic activity toward microorganisms, probably because of fundamental differences in the composition of the microbial and host cell membrane (29). In addition to restricting microbial growth, other activities have been identified, indicating that these peptides may act as multifunctional mediators of immunity, inflammation, and wound repair. In particular, many studies have suggested roles for cationic antimicrobial peptides in dampening macrophage responses to LPS, such as those describing activity of peptides derived from silk moth cecropin and bee melittin (30–31); Limulus anti-LPS factor (LALF) and bactericidal/permeability increasing protein (BPI [32]), PB (33), cathelicidins CAP18 and CAP11 (34), peptides derived from human lactoferrin (35), and the human defensins HNP-1 and HBD-2 (21).

We have shown that elafin is a molecule which is part of the "anti–neutrophil elastase shield" in the lung, along with {alpha}-1 proteinase inhibitor and SLPI. Whereas {alpha}-1 proteinase inhibitor is synthesized principally in the liver and released into the circulation, SLPI and elafin are produced locally at mucosal sites, constitutively and in response to early "alarm signals" (5). Elafin constitutes ~ 20% of the total antielastase activity retrieved from bronchoalveolar lavage fluid in normal subjects (11). In addition, we have shown that it is an antimicrobial molecule which is able to modulate LPS activities in vitro and in vivo (36–37). We studied here whether elafin could physically bind to LPS and investigated the characteristics and biological consequences of this LPS interaction.

The present studies compared first the activity of elafin with that of SLPI, which has previously been shown to bind LPS (18). Although it is not clear whether FL-EL is more effective than SLPI at binding smooth-form LPS (Figure 1), on investigation of the individual domains of elafin, it was apparent that N-EL was able to bind LPS more strongly than C-EL and that, in accordance, N-EL was able to compete favorably with C-EL for LPS binding (Figure 2). We have shown in another study the related information that it was also N-EL which displayed greater antimicrobial activity against the Gram-negative Pseudomonas aeruginosa (although, interestingly, this was not the case against the Gram-positive Staphylococcus aureus [12]). The site of interaction between SLPI and elafin peptides with LPS was further refined by showing that all peptide species could bind to the rough form of LPS, which lack the O-specific polysaccharide side chain (Figure 3). Specifically, we studied three of the five core oligosaccharide types in E. coli (K12, R2, and R3, the other two being R1 and R4) and showed, interestingly, that elafin peptides bound K12 much more strongly than either R2 or R3. Although the reason for this is not entirely clear, this could be due to the slightly different organization of the outer core in these subtypes: in R2 and R3, one of the outer core sugars is substituted with a positively charged N-acetyl glucosamine residue, while one of the K12 sugars is instead substituted with a neutral heptose residue, the latter potentially also substituted with a negatively charged phosphate group (38). The preferential interaction of elafin peptides with K12 rather than R2 or R3 could therefore be mediated by electrostatic interactions (binding to phosphate groups in K12 and repulsion caused by N-acetyl glucosamine residues in R2 or R3), although a similar interaction with K12 was noted between N-EL (+5 net positive charge) and C-EL (+2 net positive charge). Together, this array of data suggested that binding of SLPI and elafin occurred within the lipid A oligosaccharide core of LPS, and not the O-specific polysaccharide side chain. Well-characterized LPS-binding molecules such as LBP, BPI, and PB are known to bind to the conserved lipid A portion of LPS (23, 25, 39–44). Although the precise binding sites of these molecules within the lipid A structure are unclear, most studies concur that the minimal LPS structure to which LBP, BPI, and PB bind is the acylated glucosamine disaccharide unit of lipid A (23, 40). The binding site for PB also appears to overlap with those of LBP and BPI, because binding of BPI to LPS is inhibited by PB (45). We also showed here that FL-EL can bind directly to the lipid A core (Figure 4). This prompted us to study whether elafin peptides were able to compete with LBP and PB for binding to LPS. Indeed, preincubation of LPS with PB (Figure 5, left panels) or LBP (Figure 5, right panels) or incubation of LPS with simultaneously added LBP and elafin (Figure 6A) greatly inhibited interaction of LPS with elafin peptides, particularly FL-EL and N-EL. Interestingly, although LBP does not require incubation with LPS to prevent elafin binding and outcompetes the latter, elafin peptides require incubation with LPS to partially compete out LBP binding to LPS (Figures 6A and 6B). The suggestion that net charges may have a role in the interactions described above have been put forward in various studies: the cationic N-terminus of LBP (amino acids 1–197) is known to possess the LPS-binding properties of the holo-molecule, whereas the COOH-terminal domain (amino acids 198–456) is important for transfer of LPS to CD14 (46–47). A similar situation exists with BPI (not studied here) in which the cationic N-terminus domain (which share 45% sequence homology at the amino acid level with LBP) was shown to have the LPS antagonistic activity (48–49). However, charge does not entirely account for this phenomenon, because PB, the most potent test peptide, has only a +5 net positive charge, whereas FL-EL, a comparatively less effective peptide (Figure 6), has a net positive charge of +7. It is therefore likely that other molecular determinants, such as hydrophobicity, may play a role in the binding of LPS. At a primary sequence level, although recombinant LBP and BPI have an homology of 45%, there is very little homology between these sequences and that of elafin. Likewise, PB, being a cyclic decapeptide, has very little homology with either LBP, BPI, or elafin.

The biological consequences of elafin binding to LPS were then studied in a macrophage culture system. Interestingly, in serum-containing medium, unlike N-EL, both FL-EL and C-EL had a significant inhibitory effect on TNF production when incubated with LPS (Figure 7A). This contrasts with the gels/ELISA data presented above, where N-EL was able to bind to smooth LPS as well (Figure 6B) or better (Figures 1, 2, 3, and 5) than C-EL; this observation implies that the N-EL domain may be important in binding LPS, but does not dictate the ultimate fate of the complex. This is reminiscent of the case of BPI, whose C-domain (which contains, like C-EL, a hydrophobic core region) has been shown to be important for the modulation of host cell functions (10, 12, 40–44). In stark contrast with the serum-containing situation, preincubation of elafin peptides with LPS in the absence of serum led to a greatly enhanced TNF-{alpha} production in macrophages (Figure 7B). Although all three elafin peptides increased the cytokine production in these conditions, C-EL was again the most active at 100 nM. It is therefore tempting to speculate that the elafin molecule may undergo proteolytic cleavage in tissues low in serum (such as the airways) to release a more biologically active (C-EL) domain. Of interest in this regard, elafin has been isolated in a variety of proteolytic fragments (50–53), which may indeed reflect a conversion from an anti-inflammatory molecule in serum to a proinflammatory one in mucosal or epithelial secretions. Irrespective of the higher activity of C-EL, the FL-EL LPS binding activity suggests that in serum (and hence LBP)-deficient conditions (although LBP has also been shown to be produced in vitro in lung epithelial cells [54]), elafin may act to replace LBP as a carrier molecule for LPS to initiate innate immune responses. We have indeed recently shown that adenovirus-mediated overexpression of elafin in murine airways augmented TNF-{alpha} and macrophage inflammatory protein 2 (MIP-2) levels in bronchoalveolar fluid after intratracheal LPS administration, and that the increases were associated with airway neutrophilia (36). Furthermore, in a different system, we showed that after intratracheal LPS instillation, transgenic mice expressing human elafin exhibited higher bronchoalveolar/serum ratios of proinflammatory cytokines (TNF-{alpha}, MIP-2, and monocyte chemotactic protein 1) than wild-type mice, with a concurrent increase in neutrophil and macrophage influx (37). Interestingly, and possibly related to the in vitro results described here, when LPS was given systemically, elafin transgenic mice were hyporesponsive to LPS (37), suggesting again that in serum-rich conditions (compared with relatively low serum concentrations in BALF), elafin has a dampening effect on LPS responses. The reasons for this differential effect of elafin (anti-inflammatory in serum-containing medium and proinflammatory in the absence of serum) are unclear. However, it may be that, in the former case, elafin present in high enough concentrations leads to overriding of LBP and detoxification of LPS (by partially segregating it from LBP and/or transferring it to scavenging lipoproteins [55, 56]). By contrast, in serum-deficient conditions, elafin may be able to shuttle LPS directly onto the cellular macrophage transduction machinery, in the relative absence of "professional" shuttle proteins such as LBP, and induce a beneficial proinflammatory response. Indeed, elafin is upregulated in the lung by proinflammatory signals (11, 19, 57) and an LPS–elafin complex may serve as an amplifier of innate responses in serum-poor alveolar sites.

In conclusion, we show here for the first time that elafin, an important antimicrobial molecule with antielastase activity, is able to interact directly with Gram-negative bacterial LPS. This interaction appears to take place within the conserved lipid A core domain, with direct, albeit contrasting, biological consequences on the initiation of macrophage responses. Current work in our laboratory is aiming at the dissection of the pro- and anti-inflammatory pathways induced by LPS–elafin complexes, under these different conditions.


   Acknowledgments
 
The authors are grateful to Prof. I. Poxton and Dr. R. Gibbs (Department of Medical Microbiology, Edinburgh) for providing LPS samples and for helpful guidance and comments, and to Dr. A. Vreugdenhil (Department of General Surgery, Maastricht University, Maastricht, The Netherlands) for the kind gift of ELISA reagents. They also thank many local colleagues for invaluable advice in preparation of this work, particularly M. Marsden, and Drs. T. I. Brown, P. A. Henriksen, G. McLachlan, and J. King (all from the Rayne Laboratory). They are also indebted to Drs. M. Si-Tahar (Unite de Defense Innee et Inflammation, INSERM E336, France) and C. Erridge (Edinburgh University) for critically appraising the manuscript. This work was supported by the MRC (studentship to J.W.M.) and the Salvesen Emphysema Research Fund.


   Footnotes
 
Conflict of Interest Statement: J.W.M. has no declared conflicts of interest; A.R. has no declared conflicts of interest; L.J. has no declared conflicts of interest; R.R. has no declared conflicts of interest; and J-M.S. has no declared conflicts of interest.

Received in original form August 5, 2004

Received in final form January 17, 2005


   References
Top
 Abstract
 Introduction
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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