Interleukin-8-Derived Peptide Has Antibacterial Activity

Chemokines are inflammatory mediators with effects on diverseprocesses associated with the immune response. Some of the proteinsbelonging to the CXC chemokine subfamily, one of four groupsin the family, possess inherent antibacterial activity againsta wide range of bacteria. The CXC chemokine interleukin-8 (IL-8)has not been ascribed any direct antibacterial activity, butthe fact that several of the amino acids in the carboxy-terminalpart of the protein are identical or similar to those in a bactericidalcecropin-like peptide [Hp(2-20)] from Helicobacter pylori suggeststhat processing of the cytokine might generate peptide fragmentswith antibacterial properties. Synthetic peptides representingthe carboxy-terminal part of IL-8 were investigated for antibacterialactivities. These fragments possessed an antibacterial activityabsent in the full-length IL-8. The antibacterial effects werereduced at increasing salt concentrations whereas the activitywas increased when the pH was lowered. The IL-8-derived peptideshared structural similarity with and was also functionallyadditive to the Hp(2-20) peptide. The IL-8-derived peptide lackedthe proinflammatory effects of the full-length protein. We alsoshowed that acid hydrolysis of IL-8 generated a major peptidefragment corresponding to the antibacterial carboxyl terminusof the protein. The results presented are of special interestwhen put in the context of the suggested importance of antimicrobialpeptides for microbial colonization of the gastric mucosa.

INTRODUCTION

Top

Introduction

Chemokines are inflammatory regulators with effects on a numberof different processes of prime importance for our innate aswell as acquired immune responses, affecting stem-cell survival,development, and homeostasis, as well as chemotaxis and angiogenesis(30). The cytokines are divided into four subfamilies, and basedon their structure and primary amino acid sequence, they belongto the CXC, CC, C, or CX3C group. The CXC subfamily of chemokinesconsists of a number of different monocyte and granulocyte-activatingproteins, including CXCL8, also termed interleukin-8 (IL-8)(30). This interleukin is generated and secreted by severaldifferent types of inflammatory cells as well as by epithelialcells. IL-8 is not only a very potent endogenous monocyte/neutrophilchemoattractant but also a potent secretagogue and activatorof the phagocyte NADPH-oxidase, inducing the release of a largenumber of proteases and reactive oxygen metabolites (2, 23).

Antibacterial peptides are important effector molecules in thedefense system against microorganisms from evolutionarily oldand primitive organisms to highly evolved organisms such asmammals. Accordingly, many chemokines possess, in addition totheir regulatory activities, direct antibacterial properties(11, 35). The formation of a large positively charged area onthe surface of the molecule is a common feature of the antimicrobialchemokines and a characteristic possibly explaining the bacterialinhibitory activities (35).

The CXC chemokine IL-8 is a homologous dimeric molecule, eachmonomer consisting of three antiparallel ß-strandsconnected with loops and one ${alpha}$ -helix made up of the 19 carboxy-terminalresidues (3). The protein has not been linked to any antibacterialactivities, but the fact that the carboxy terminus of IL-8 containsan ${alpha}$ -helical structure suggests that this part of the moleculemight have antibacterial properties even though the full-lengthprotein is devoid of such an activity. It should be noted thatit is a common feature for antibacterial peptides to be activatedupon proteolytic cleavage from larger precursor proteins.

The cecropin-like peptide Hp(2-20), as well as LL-37, whichbelongs to the cathelicidin family of antibacterial peptides,are prominent examples of bactericidal peptides derived throughsuch a cleavage mechanism (15, 22, 34). In addition, analysisof the amino acid sequence of IL-8 revealed similarities withHp(2-20) derived from the gastric pathogen Helicobacter pylori,the causative agent of gastritis and peptic ulcers. This groupof cecropin-like antibacterial peptides, first identified asthe principal component of the humoral immune system of insectsand later found in a variety of organisms (5), have in commonan amphipathic ${alpha}$ -helix structure which is an important structuralfeature. This is illustrated by the fact that amino acid replacementsthat break or stabilize the helical structure will reduce orabolish or increase the antibacterial activity, respectively(5, 14, 29). The cecropins as well as cecropin-like peptidesexercise their antibacterial effect through the formation ofpore structures in phospholipid bilayers, leading to depolarizationof the membrane of the target microbe (13), an activity forwhich the ${alpha}$ -helical structure is decisive (8).

In order to determine the antibacterial potential of IL-8, peptidescorresponding to the carboxy-terminal part of the protein weresynthesized and tested for antibacterial activity. These ${alpha}$ -helicalpeptides possessed antibacterial properties, and acid hydrolysisof the intact IL-8 protein generated a major peptide identicalto the antibacterial fragment.

MATERIALS AND METHODS

Top

Materials and Methods

Reagents. Recombinant human IL-8 was purchased from R&D Systems EuropeLtd. (United Kingdom) in a solution stabilized with bovine serumalbumin (0.5%). The peptide KENWVQRVVEKFLKRAENS (IL-8_81-99)and the peptide corresponding to the acid hydrolysis-generatedfragment, PKENWVQRVVEKFLKRAENS (IL-8_80-99), were synthesizedand high-pressure liquid chromatography purified by KJ ROSS-PETERSENAS (Horsholm, Denmark). No functional difference between thetwo peptides was found, and both are referred to as the IL-8peptide. The cecropin-like peptide Hp(2-20), with a sequencecorresponding to the amino-terminal part of ribosomal proteinL1 in H. pylori (AKKVFKRLEKLFSKIQNDK), was synthesized and high-pressureliquid chromatography purified by Innovagen (Lund, Sweden).The peptides were dissolved in water and stored at –70°Cuntil used. Horseradish peroxidase was from Roche Applied Science(Bromma, Sweden). A-6013 agarose used for casting all inhibitionzone plates was from Sigma Chemical Co. (St. Louis, Mo.) aswere isoluminol, luminol, superoxide dismutase, and the chemotacticpeptide formyl-Met-Leu-Phe (fMLF). Dextran and Ficoll-paquewere purchased from Amersham Biosciences (Uppsala, Sweden).

Inhibition zone assay for determination of antimicrobial activity. Escherichia coli strain MG1655 was grown overnight in LuriaBertani (LB) broth (1) at 37°C on a rotary shaker. A modifiedinhibition zone assay was used for detection of antibacterialactivity (17, 19, 20). In short, standard LB agar (broth supplementedwith 1% [wt/vol] agarose), except when noted, containing bacteria(approximately 5 x 10⁵ CFU in logarithmic growth phase per milliliteragar) was poured into petri dishes, diameter 92 mm. Wells, diameter3 mm and depth 1 mm, were punched in the agar and peptide preparations(3 µl), diluted in distilled water unless otherwise stated,were added to the wells, whereupon the plates were incubatedat ambient temperature for 45 min and then at 37°C overnight.

Peptide concentrations ranging from 50 µM to 2.5 mM wereused in the assays. The inhibition zone was detected as thediameter of the clear zone surrounding the well free of visiblebacteria. The antibacterial effect on fungus and other bacteria(basic characteristics of strains used are given at the www.ccug.gu.sesite), Staphylococcus aureus (CCUG 49245), Candida albicans(CCUG 49242), Proteus mirabilis (CCUG 49244), Salmonella entericaserovar Typhimurium MS 395 and MR10 (a kind gift from Olle Stendahl,University of Linköping, Linköping, Sweden), Klebsiellapneumoniae (CCUG 49243), Bacillus subtilis (strain ATCC 6051,from the Culture Collection, University of Göteborg, Göteborg,Sweden) and Staphylococcus epidermidis (strain ATCC 14990, theCulture Collection, University of Göteborg), were testedin the inhibition zone assay as described above. Streptococcuspyogenes (CCUG 49246) was grown anaerobically (in an anaerobicjar with 25 ml Anaerocult C from Merck [Darmstadt, Germany])in serum broth (3.7% brain heart infusion [Difco 0037] in distilledwater), and for the inhibition zone assays, on agar plates madefrom serum broth. Helicobacter pylori (strain SS1, a kind giftfrom Ingrid Bölin, University of Göteborg, Göteborg,Sweden) was grown microaerophilically (in a jar filled withmicroaerophilic gas) in Brucella broth (base purchased fromSigma Chemical Co., St. Louis, Mo.) and spread onto agar platesmade from Brucella broth in the inhibition zone assay. The assaywas performed with 4 days of incubation in order to get sufficientH. pylori growth.

For deciding how basic experimental conditions, such as variedsalt concentrations and pH, might influence the antibacterialactivity of the peptides, different concentrations of ionicsalts (15 to 350 mM; NaCl, Na₂SO₄, MgCl₂, MgSO₄, CaCl₂ and CaSO₄)were added to LB agar without NaCl before the inhibition zoneplates were cast. The pH was set to 5.0, 7.0, 7.4, or 9.0 (measuredwith an Orion pH meter) by adding NaOH or HCl to the LB agar,and the inhibition zone assays were performed as described above.

Isolation of human neutrophils. Human neutrophils were isolated by a standard technique (6,7) from buffy coats obtained from healthy blood donors at theSahlgrenska Hospital, Göteborg, Sweden. In short, dextransedimentation was performed at 1 x g, where after hypotoniclysis of the remaining erythrocytes, and centrifugation in aFicoll-Paque gradient, the neutrophils were washed twice andresuspended (10⁷/ml) in Krebs-Ringer phosphate buffer containingglucose (10 mM), Ca²⁺ (1 mM), and Mg²⁺ (1.5 mM) (KRG, pH 7.3).The cells were kept on melting ice until use.

Neutrophil chemotaxis assay. Neutrophil chemotaxis was determined using ChemoTx multiwellchambers (Neuroprobe Inc. Gaithersburg, MD) according to instructionsprovided by the manufacturer. In short, neutrophil samples (30µl of 10⁶ cells/ml) suspended in KRG supplemented withbovine serum albumin (0.3% [wt/vol]) were placed on top of 3-µmpolycarbonate filters and allowed to migrate through the filters.Various concentrations of IL-8, fMLF (positive control for chemotaxis),and IL-8 peptide were applied to the lower compartments. Theaccumulation of cells in the lower compartments was determinedafter incubation at 37°C for 90 min by quantifying the contentof myeloperoxidase by adding a peroxidase substrate (o-phenylenediamine;Dako A/S, Glostrup, Denmark) after cell lysis.

Neutrophil NADPH-oxidase activity. The production and release of superoxide anions in neutrophilswere measured in a luminol- or isoluminol-enhanced chemiluminescenceassay described in detail elsewhere (12). In short, the chemiluminescenceactivity was measured in a six-channel Biolumat LB 9505 apparatus(Berthold Co., Wildbad, Germany), using 4-ml polypropylene tubeswith a 400-µl reaction mixture containing 4 x 10⁵ neutrophils,horseradish peroxidase (a cell impermeable peroxidase; 4 U),and isoluminol (a cell impermeable CL substrate; 2 x 10^–5M) for extracellular measurements. Tubes used for measurementof intracellular generation of reactive oxygen species contained4 x 10⁵ neutrophils, superoxide dismutase (a cell impermeablescavenger for O₂^–; 50 U), catalase (a cell impermeablescavenger for H₂O₂; 2,000 U), and luminol (a cell permeableCL substrate; 2 x 10^–5 M). The tubes were equilibratedin the Biolumat at 37°C, after which the stimulus (40 µl)was added and the light emission was recorded continuously.

Analysis of the acid hydrolysis products from IL-8. Acid hydrolysis was performed as described by Lin et al. (25).In short, recombinant human IL-8 (12 µM) was diluted (1/20)in trifluoroacetic acid (0.6% [vol/vol], pH 1), and the mixturewas incubated for 16 h at 65°C. The sample was purifiedon a ZipTip-C₁₈ (Millipore) according to standard proceduresavailable at www.millipore.com. The sample was analyzed usinga MALDI-LR (Micromass, Manchester, United Kingdom) matrix-assistedlaser desorption ionization-time of flight (MALDI-TOF) in reflectronmode; 0.5 µl of the sample was mixed with 0.5 µlmatrix solution (12 mg/ml ${alpha}$ -cyano-4-OH-cinnamic acid in acetonitrile/water1:1, 0.1% trifluoroacetic acid) directly on the MALDI probeand allowed to dry at ambient conditions. ACTH (18-39) MH⁺ 2465.199was used as the external and tryptic autodigest and MH⁺ 2211.105as the internal lock mass. The sample was eluted in 3.5 µlof actonitrile/water (1:1) containing 0.1% formic acid, andthe peak of interest identified in MALDI-TOF was fully sequencedby electrospray ionization-dual mass spectrometry (ESI-MS/MS),on a Q-TOF Ultima API (Micromass, Manchester, United Kingdom).Fragment ion data were acquired by nanoflow electrospray usingargon collision gas. The analyses were performed by the SweGeneProteomics Centre at the University of Göteborg.

Statistical analysis. A two-sided Student's t test was utilized to determine statisticalsignificance. P < 0.05 was considered significant.

RESULTS

Top

Results

Physicochemical properties of the IL-8 peptide and sequencealignment with the cecropin-like peptide Hp(2-20). The sequenceof IL-8 was compared to that of the antibacterial cecropin-likepeptide Hp(2-20), derived from Helicobacter pylori. The carboxylterminus of IL-8 showed a high degree of similarity with Hp(2-20),manifested both as identity at the amino acid level and similarityin physicochemical properties and exchangeability of the constitutingamino acids (Fig. 1A). The sequence alignments were performedusing the Clustal W (1.81) software provided by the open accessWeb-site http://bio.lundberg.gu.se. The computer-based estimatedthree-dimensional structure of the carboxyl terminus of IL-8revealed an ${alpha}$ -helical structure, and the truncated molecule couldbe plotted as an almost perfect amphipathic wheel (the calculationwas performed at the http://bioinf.man.ac.uk/ $~$ gibson/HelixDraw/helixdraw.htmlsite with the HelixDraw v.1.00 software), suggesting structuralsimilarities with antibacterial peptides such as the cecropins(Fig. 1B). In order to determine if the structural similaritybetween the carboxyl end of IL-8 and Hp(2-20) was accompaniedby any functional similarities, we synthesized peptides correspondingto the carboxy-terminal ${alpha}$ -helical part of the IL-8 molecule.

View larger version (32K):
[in this window]
[in a new window]
FIG. 1. Structure and sequence similarities between the cecropin-like Hp(2-20) and the 20-amino-acid-long carboxyl terminus of IL-8. (A) The IL-8 peptide had amino acid sequence similarities to Hp(2-20). The stars (*) denote total amino acid identity, the two dots (:) represent amino acids with high similarity regarding physicochemical properties where exchangeability with preservation of structure and function is likely, and one dot (.) denotes a low degree of similarity. Space means no significant similarity. (B) The ${alpha}$ -helical wheel constituting the IL-8 peptide was amphipathic with hydrophobic amino acids concentrated to one side of the ${alpha}$ -helix (pink residues) and polar or charged amino acids to the other side (yellow residues).

Antimicrobial activity of synthetic IL-8-derived peptides against E. coli. An inhibition zone assay was employed to determine the antibacterialactivity of the synthetic peptides. The synthetic IL-8 peptideshad dose-dependent antibacterial properties similar to thoseof Hp(2-20) (Fig. 2a), but no such activity was evident whenthe full-length IL-8 molecule was applied at similar concentrations(Fig. 2b). The medium control (water) did not give rise to anyinhibition zone at all (data not shown).

View larger version (37K):
[in this window]
[in a new window]
FIG. 2. Antibacterial activity of the IL-8 peptide. (A) Inhibition zone diameters (diameter of well subtracted from the results) for the IL-8 peptides and Hp(2-20) are shown in one representative experiment. Results from several different experiments have been summarized and presented as mean ± standard error of the mean of the lethal concentration, as defined by Hultmark (D. Hultmark, A. Engstrom, K. Andersson, H. Steiner, H. Bennich, and H. G. Boman, EMBO J. 2:571-576, 1983); n = 4 (inset). (B) The IL-8 peptide at 2.5 mM inhibited the surrounding E. coli as visualized by a zone free of bacterial growth around the well (right well). No such zone was obtained for 2.5 mM of the IL-8 protein (left well). (C) Hp(2-20) (2.0 mM) was combined with various concentrations of IL-8 peptide (0 to 1,000 µM). The inhibition zone diameters were related to the inhibition zone diameter obtained by adding only Hp(2-20) and the relation on the y axis was calculated according to the equation [inhibition zone diameter of 2.0 mM Hp(2-20) + x µM IL-8 peptide]/[inhibition zone diameter of 2.0 mM Hp(2-20)], where x ranged from 0 to 1,000 µM, n = 4, and bars show mean + standard error of the mean.

The inhibitory activity of antibacterial peptides is often influencedby the basic experimental conditions. This was the case forthe IL-8 peptide, as illustrated by the fact that the antibacterialactivity increased when the sodium chloride concentration wasreduced, while the activity decreased when the concentrationwas raised. At around 350 mM NaCl the antibacterial activitywas lost. In contrast, the H. pylori peptide Hp(2-20) was equallypotent when the growth medium was supplemented with sodium chlorideup to concentrations of 100 mM and the activity was still pronouncedat 350 mM (data not shown). The differences between the IL-8peptide and Hp(2-20) were quantified and the data are shownin Table 1.

View this table:
[in this window]
[in a new window]
TABLE 1. Antibacterial activities of both the IL-8 peptide and Hp(2-20) are negatively influenced by raised NaCl concentrations while decreased pH enhances the antibacterial effect of the IL-8 peptide^a

The same pattern of sensitivity, including the 350 mM cutoff,was observed when increasing concentrations of Na₂SO₄ were addedto the growth medium, whereas no difference was obtained betweenthe IL-8 peptide and Hp(2-20) with respect to the sensitivityto increasing concentrations of the divalent cations Mg²⁺ andCa²⁺. Concentrations of Mg²⁺ or Ca²⁺ exceeding 15 mM totallyabolished the antibacterial activity of the peptides (data notshown).

In addition, the pH was of importance for the antibacterialactivity of the IL-8 peptide. When the pH was lowered, a significantenhancement of antibacterial activity was obtained, whereasno such effect was observed for Hp(2-20) (Table 1). We couldnot use pH levels below 5.0 owing to the fact that the solidificationof the agarose was lost at these levels. An increment of pHto 9.0 reduced the antibacterial activity of both Hp(2-20) andthe IL-8 peptide (data not shown).

Combined antibacterial effects of the IL-8 peptide and Hp(2-20). Despite the fact that the inhibition zone assay does not allowfor a direct addition of one zone diameter to another when theconcentration of a drug is increased or when two antimicrobialagents are combined, the technique can be used to determineadditivity/synergy. Combined action of the IL-8 peptide andHp(2-20) increased the antimicrobial activity, and we concludedthat the effects of the two peptides were additive. This conclusionwas based on the fact that the zone-diameter obtained by Hp(2-20)could be increased through an addition the IL-8 peptide, evenwhen the latter was present in concentrations that were subinhibitorywhen acting alone (Fig. 2C).

Effect of the IL-8 peptide and Hp(2-20) on different bacteria. The antibacterial potential and inhibition profile differedbetween the IL-8 peptide and Hp(2-20) (Table 2). The two peptideswere equally potent in inhibiting the test strain of E. coli,but this was not the case when E. coli was replaced by othergram-negative bacteria (Table 2). There was also a differencebetween the peptides with respect to the capacity to inhibitH. pylori; while the bacteria were resistant to Hp(2-20), alow degree of antibacterial activity was obtained with the IL-8peptide. The two peptides efficiently inhibited streptococci(Streptococcus pyogenes) whereas other gram-positive bacteriatested were largely unaffected by either peptide, as was thefungus tested (Table 2).

View this table:
[in this window]
[in a new window]
TABLE 2. The IL-8 peptide and Hp(2-20) differ in antibacterial properties against different bacteria

Proinflammatory activities of the IL-8 peptide. Binding of full-length IL-8 to its neutrophil cell surface receptor(CXCR) gives rise to a proinflammatory signaling cascade thatresults in activation of the phagocyte NADPH-oxidase (21). Thisactivation in turn results in production and release of reactiveoxygen species (Fig. 3). When the IL-8 peptide replaced thefull-length protein, no production of reactive oxygen speciescould be detected (Fig. 3). Higher concentrations of the IL-8peptide did not result in any activity either (data not shown).

View larger version (20K):
[in this window]
[in a new window]
FIG. 3. Lack of proinflammatory activity of the IL-8 peptide. Activation of the neutrophil NADPH-oxidase leading to an extracellular release of superoxide anions was determined following addition of IL-8 or the IL-8 peptide. The figure shows one representative experiment and the time point for addition of stimuli is indicated by the arrow. Abscissa, time of study; ordinate, superoxide release (MCPM = 10⁶ cpm). The inset shows chemotaxis relative to fMLF; fMLF (10 nM), IL-8, and IL-8 peptide (1.2 nM).

The full-length IL-8 molecule (1.2 x 10^–9-1.2 x 10^–10M) was also a potent neutrophil chemoattractant, but no suchactivity was induced when the IL-8 peptide (1.2 x 10^–8-1.2x 10^–10 M) was introduced in the chemotaxis assay system(Fig. 3, inset). Accordingly, the IL-8 peptide was (in contrastto full-length IL-8) unable to activate neutrophils in termsof oxidase activity and induction of chemotaxis and had no pro-inflammatoryactivities similar to those earlier described for Hp(2-20) (7).

Identification of one major cleavage fragment generated after acid hydrolysis of IL-8. A major cleavage peptide with a molecular weight of 2,459 Dawas identified and this fragment was subsequently found to beidentical to the carboxy-terminal part of the full-length IL-8molecule, i.e., a 20-amino-acid-long peptide of amino acids80 to 99 with the sequence PKENWVQRVVEKFLKRAENS (Fig. 4). Infact, the fragment generated through acid hydrolysis containedthe complete ${alpha}$ -helix (ranging from amino acid 81 through 97).

View larger version (19K):
[in this window]
[in a new window]
FIG. 4. MALDI-TOF and electrospray ionization-dual mass spectrometry spectra of acid-hydrolyzed IL-8. The major 2,459-Da fragment was identified in MALDI-TOF. This fragment was further identified as consisting of the 20 amino acids of the carboxyl terminus in full-length IL-8 with the sequence PKENWVQRVVEKFLKRAENS in electrospray ionization-dual mass spectrometry.

DISCUSSION

Top

Discussion

We show here that the ${alpha}$ -helical carboxy-terminal end of the cytokineIL-8 has antibacterial capacity that is absent in the full-lengthIL-8 molecule. The antibacterial potency was affected by ioncomposition and pH, and the antibacterial effect against E.coli was additive to that of a cecropin-like peptide from H.pylori, Hp(2-20). The peptide derived from IL-8 was unable toinduce the proinflammatory activities ascribed to Hp(2-20) (9)or full-length IL-8.

Some of the CXC cytokine family members, for example GRO- ${alpha}$ /CXCL-1,are antibacterial, whereas others are devoid of such activities(11, 35). Cationicity seems to be an important feature sharedby the antibacterial chemokines, as well as by many other antibacterialpeptides/proteins. The cationic criterion is fulfilled by thefull-length IL-8 molecule, and this feature is also conferredon the IL-8 peptide possessing antibacterial activities. Thesemolecules have isoelectric points of 8.9 and 9.7, respectively,and a pI value above 8.0 is a prerequisite, but not sufficient,for antibacterial activity (35). The topological formation ofa large positively charged area on the protein surface appearsto be an important feature in antibacterial chemokines (35),and this property is also shared by the IL-8 molecule. Manyphysicochemical characteristics thus suggest that the full-lengthIL-8 should also possess antibacterial activities. A possibleexplanation to why the full-length IL-8 cytokine lacks antibacterialeffect might be that the amphipathic parts at the carboxy-terminalend are hidden or disturbed by other parts of the molecule.

As illustrated by the fact that all members of the cathelicidinfamily of antimicrobial peptides are derived from the commoncathelin precursor, many antimicrobial peptides are cleavedoff from larger precursor proteins (15). Matrix metalloproteinasessuch as gelatinase B and neutrophil collagenase have been shownto cleave members of the CXC chemokine family (33), suggestingthat processing of the carboxy terminus constitutes an importantregulatory mechanisms. Proteinases with specificity for theamino-terminal side of aspartic/cysteic acid residues or thecarboxy side of proline residues are abundant in human cells.During interaction with IL-8, these types of proteinases should,according to the amino acid sequence, generate fragments suchas IL-8_79-99 and IL-8_81-99 that contain the ${alpha}$ -helical part ofthe molecule, suggesting that antibacterial peptides can begenerated from IL-8 by several different mechanisms. In lightof the results that also acid hydrolysis of IL-8 generates anantibacterial peptide; studies of the peptide in relation toantibacterial/inflammatory activities in the acidic environmentof the intestine would be of interest. This is further emphasizedwhen considering the similarity and cooperative mode of actionfor the H. pylori derived peptide Hp(2-20) and the IL-8 peptide.

In addition to IL-8, there may be other CXC chemokines withantibacterial activity residing from the carboxy-terminal partof the protein. For example GRO- ${alpha}$ , -ß, and - ${gamma}$ all have ${alpha}$ -helical parts similar to the one found in IL-8, suggestingthat the antibacterial property of these three proteins mightbe derived from this part of the molecule. This remains to beshown, however.

It is well known that increased ionic strength results in reductionof the activity exerted by many antibacterial peptides. Theantibacterial peptide LL-37, for example, loses activity asthe sodium chloride concentration is raised (4). The IL-8 peptidewas more sensitive to increased sodium chloride concentrationsthan Hp(2-20). A positive correlation between peptide foldingand antibacterial activity has been observed, and importantdeterminants for proper folding of certain antimicrobial peptidesseem to be anions such as SO₄^2–, HCO₃^– and CFCOO^–(16). However, in this study the effect of anions did not seemto be crucial for the antibacterial properties of the IL-8 peptide.Very small effects on the antibacterial activity were obtainedthrough varying the concentration of NaCl or Na₂SO₄ whereasincreasing concentrations of MgCl₂, MgSO₄, CaCl₂ or CaSO₄ totallyabolished the antibacterial effects of the IL-8 peptide, suggestingthat divalent cations are of great importance. This phenomenonhas been described earlier (24).

The negative effect on the IL-8 peptide activity upon additionof divalent cations might emanate from unfavorable conformationchanges in the peptide or from the effect on cellular factorsin the bacteria that makes it more resistant to the inhibitoryaction of the peptide. Yet another plausible explanation thatpreviously has been proposed is the effect exerted by divalentcations on lipopolysaccharide molecules in gram-negative bacteria(24, 32). Cationic antibacterial peptides cross the outer membraneof gram-negative by the self-promoted pathway and the initialstep in this process is assumed to be a high affinity bindingof the peptide to lipopolysaccharides in the bacteria. Thiscauses a displacement of divalent cations that stabilize adjacentlipopolysaccharide molecules. The displacement is hypothesizedto destabilize the outer membrane, resulting in uptake of thepeptide across the outer membrane followed by channel formationin the cytoplasmic membrane, the primary target in both gram-positiveand gram-negative bacteria, and bacterial death. The actionsof cationic antibacterial peptides are inhibited at this initialstep by high concentration of divalent cations (24, 32).

The antibacterial effect of the IL-8 peptide was enhanced bylowering of the pH. This potentiating effect on antibacterialactivity in mildly acidic environments was not applicable toHp(2-20) but has earlier been shown also for the defensin groupof antimicrobial peptides and platelet microbicidal proteins(31, 36). The induction of positive conformational changes and/orinfluence on net charge by protonation of specific amino acidsin the peptide might be possible explanations.

The synergistic action of antibacterial peptides when combinedis a well-known phenomenon in the protection of epithelial surfaces,and it has for example been described for neutrophil defensinsand cathelicidins (27), two representative families of neutrophilantibacterial peptides. The heterogenic group constituting theantibacterial peptides has diverse properties and the exactmechanisms of killing are unknown. One essential, contributingeffect seems to be the microbe cell membrane lysis accomplishedthrough peptide-peptide and peptide-membrane interaction, inwhich the amphipathicity is very important (37). One importantstep in this process is the threshold concentration that hasto be reached before the microbe membrane can be lysed. Twoantibacterial peptides resembling each other should be ableto interact cooperatively with the target membrane. Such aninteraction would add antimicrobial effects of two peptides,as observed for the IL-8 peptide and Hp(2-20). The IL-8 peptideand Hp(2-20) seem to have slightly different mechanisms of actionsince they vary in their antibacterial properties against bacteriaother than E. coli. One could hypothesize that they bind to/interactwith different structures on the bacterial membrane, resultingin various antibacterial capacity depending on the particularbacterial strain investigated.

The IL-8-derived peptides were devoid of the proinflammatoryactivities ascribed to full-length IL-8. This is not so surprisingsince the proinflammatory effects exerted on neutrophils byIL-8 derives from the amino-terminal end of the protein. Forexample, the ELR motif, positions 31 to 33 in unprocessed IL-8(1-99) and positions 4 to 6 in the most common, processed formof IL-8 (28-99), is essential for the chemotactic activity exertedon neutrophils by the protein (10).

It is also worth noticing that H. pylori, the bacterium associatedwith peptic ulcers, is unique in its ability to adapt to long-termsurvival in the acidic environment of the human stomach. Interactionbetween the bacteria and host mucosal cells results in an inductionof transcription and local production of the precursor to theIL-8-derived antimicrobial peptides (18, 26, 28) and the persistenceof the bacteria in the mucosa has been suggested to be facilitatedby antibacterial products giving a competitive advantage overother microorganisms (29). Such antibacterial peptides are producedby the bacteria themselves but locally produced host-derivedantibacterial peptides originating from inflammatory cytokinesmight also contribute to the antibacterial activity. Consideringthe results presented here, induction of IL-8 production andcleavage of the cytokine should aid the innate immune clearanceof invading bacteria, but also the colonization and persistencein the stomach mucosa of pathogenic microbes such as H. pylori,since the antibacterial capacity of the IL-8 peptide is muchmore pronounced against other bacteria compared to against H.pylori.

ACKNOWLEDGMENTS

This work was supported by the Swedish Medical Research Council,the Swedish Society for Medical Research, and the King GustafV 80-year foundation.

FOOTNOTES

* Corresponding author. Mailing address: Department of Rheumatology and Inflammation Research, University of Göteborg, Guldhedsgatan 10, S-413 46 Göteborg, Sweden. Phone: 46 31 342 49 27. Fax: 46 31 82 88 98. E-mail: ase.bjorstad{at}rheuma.gu.se.

REFERENCES

Top