ezproxy.upm.edu.my_2058_S0022175999001520_1-s2.0-S0022175999001520-main

Ž .Journal of Immunological Methods 232 1999 45–54www.elsevier.nlrlocaterjim

Neutrophil antibacterial peptides, multifunctional effectormolecules in the mammalian immune system

Gudmundur H. Gudmundsson a,), Birgitta Agerberth b

a Microbiology and Tumorbiology Center, Doktorsringen 13, Karolinska Institutet, S-171 77 Stockholm, Swedenb Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden

Abstract

The bactericidal machinery of mammalian neutrophils is built up of many components with different chemical properties,involving proteins, peptides and oxygen-dependent radicals. All these components work in synergy, leading to destructionand elimination of ingested microbes. During the eighties, it gradually became clear, that cationic peptides are a part of theoxygen-independent bactericidal effectors in phagocytic cells. In mammals, these antimicrobial peptides are represented bytwo families, the defensins and the cathelicidins. These potent broad spectra peptides are included as immediate effectormolecules in innate immunity. The detailed killing mechanism for these effectors is partly known, but nearly all of themhave membrane affinity, and permeate bacterial membranes, resulting in lysis of the bacteria. This peptide–membraneinteraction includes also eukaryotic membranes, that implicates cytotoxic effects on host cells. Studies in vitro haveestablished that the microenvironment is critical for their activities. In connection to cystic fibrosis, the effects ofmicroenvironment changes are apparent, causing inactivation of peptide defences and leading to repeated serious bacterialinfections. Thus, the importance of the microenvironment is also supported in vivo. Additional functions of these peptidessuch as chemotactic, mitogenic and stimulatory in the wound healing process suggest further important roles for thesepeptides. q 1999 Elsevier Science B.V. All rights reserved.

Keywords: Antibacterial peptides; Innate immunity; Microenvironment; Evolutionary variation; Bactericidal synergy

1. Introduction

The phagosomes of neutrophils contain multipleantibacterial components, working in concert onkilling engulfed microorganisms. Already during thesixties, efforts were made to characterize active bac-

) Corresponding author. Tel.: q46-8-728-6685; fax: q46-8-328878; e-mail: [email protected]

tericidal components. Gradually, part of the activitywas found to be oxygen-independent and connected

Žto cationic proteins Zeya and Spitznagel, 1966;.Odeberg and Olsson, 1975 . Further studies con-

firmed the participation of small proteins or peptidesin this antibacterial, oxygen-independent systemŽ .Lehrer et al., 1993 . The first mammalian anti-bacterial peptides were isolated and characterized

Žfrom rabbit alveolar macrophages Selsted et al.,.1983 . The isolation-methods were then established

and similar peptides were found in neutrophils from

0022-1759r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved.Ž .PII: S0022-1759 99 00152-0

( )G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 45–5446

Žseveral mammalian species, including human Lehrer.et al., 1991a . Thus, a peptide based protection as a

general concept in mammalian neutrophils andmacrophages was established. These first peptideswere designated the defensins and found to have ab-sheet structure stabilized by three intrachain disul-

Ž .phide bridges Ganz et al., 1990 . Further peptidepurification and characterization by cDNA cloninglead to isolation of different type of peptides, whichturned out to have a conserved proregion of cathelintype and consequently they were called cathelicidinsŽ .Zanetti et al., 1995 . The active mature peptides of

Žthis family are a heterogenous group Zanetti et al.,.1995 . Thus, in mammalian neutrophils two main

families of antibacterial peptides are present, thecathelicidins and the defensins.

Gradually, it emerged that these broad spectraantimicrobial peptides were a substantial part ofmammalian immediate defences. This gained furthersupport, when the peptides were also found to be

Žexpressed in epithelial cells at mucosal surfaces Di-.amond et al., 1991; Schonwetter et al., 1995 . This

indicated that the peptides constitute a primary bac-tericidal defence barrier, in addition to serve as asecond wave of antibacterial defence effectors, whenneutrophils are recruited to sites of infection andinflammation. The importance of the peptide de-fences in connection to disease has become clear

Ž .through studies on cystic fibrosis Bals et al., 1999 ,where this defence system is impaired, resulting inrepeated infections. However, it is always importantto consider the synergistic effects between differentpeptides, antibacterial proteins and the reactive oxy-gen products, in addition to the complement system.

The resistance of various bacteria to classicalantibiotics is increasing as a serious problem inhealth care, therefore the peptides have gained inter-est as possible candidate components for therapeutic

Žapplications and drug development Hancock and.Lehrer, 1998 . In this context, additional functions

for the peptides are of importance. These functionsinclude antifungal, antiviral, chemotactic, mitogenicand stimulatory activities as have been described forthe a-defensins, which are the best studied mam-

Ž .malian peptides Lehrer et al., 1993 . Some of theseactivities have also been noted for the cathelicidinpeptides. Thus, it is justified to refer to these pep-tides as multifunctional effector molecules.

2. Variation among mammalian neutrophil bacte-ricidal peptides

Evolutionary, it is of interest to note the pro-nounced variation between bactericidal peptides pre-sent in the neutrophils of different mammalianspecies. The a-defensins HNP 1–3 seem to be themajor bactericidal peptides identified in human neu-trophils, with an estimation of 30%–50% of total

Ž .protein in azurophil granules Ganz et al., 1990 . Inaddition, a-defensin HNP-4 with a different anti-bacterial spectrum compared to HNP 1–3 is present

Ž .but at lower concentrations Wilde et al., 1989 . Incontrast to human, neutrophils from horse and mousedo not contain the a-defensins as major antibacterial

Žpeptides Couto et al., 1992; Eisenhauer and Lehrer,.1992 . However, this fact does not indicate the ab-

sence of genes encoding these potent effector com-ponents in these species. In the mouse, a number ofa-defensins genes are expressed, but with differenttissue distribution, sofar mainly located in the gastro-

Ž .intestinal tract Selsted et al., 1992 . The mouseŽa-defensins have been designated cryptdins Selsted

.et al., 1992 . Porcine neutrophils also appear to bedeficient of a-defensins but are abundant in catheli-

Žcidins Mirgorodskaya et al., 1993; Harwig et al.,.1995 . The cathelicidins constitute a family of anti-

bacterial pro-peptides with a conserved cathelinproregion, containing a variant C-terminal anti-

Ž .bacterial domain Zanetti et al., 1995 . Hence, thecathelin proregion seems to serve as a carrier forseveral different antibacterial peptides. In mouse andhuman neutrophils, the cathelicidins are only repre-

Žsented by one member, called CRAMP Gallo et al.,. Ž1997 and hCAP18rLL-37 Cowland et al., 1995;

.Larrick et al., 1995; Gudmundsson et al., 1996 ,respectively. It appears that the variation is not onlydue to divergence in amino acid sequences, but alsoapplies to the number and abundance of expressedgene products of the two main families coding forantibacterial peptides in neutrophils, i.e., the de-fensins and the cathelicidins. In the light of thisvariation, it is clear that these defence effectors arefast evolving entities as compared with for examplepeptide hormones, that represent another group ofbioactive peptides, that have been conserved through

Ž .evolution Johnsen, 1998 . Most likely the variationof antibacterial peptides reflects the character of their

( )G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 45–54 47

targets, i.e., the microbes that are fast evolving, andexhibit tremendous variation. In addition, there seemsto be extensive flexibility for the peptide microbeinteractions, since substitutions of charge equivalentamino acids in the peptides do not alter their affinityto microbe membranes or their lethal effects. Thus,the peptides probably reflect rapid adaptive evolu-tionary changes, regarding host–microbe interplay,and represent different evolutionary history with re-spect to interaction with variable targets of the natu-ral flora and evolutionary bottlenecks caused bysevere pathogens.

3. Ancient defence system

Antibacterial peptides are widespread in nature asdefence effectors, and have been found in plants,

Žinvertebrates, vertebrates and also in bacteria Bo-.man, 1995; Putsep et al., 1999 . In addition, to

represent an ancient defence system that probablywas well adapted already among early eukaryoticcells in the form of defence effectors, these peptidesprobably also participated in degrading microorgan-isms as a source for nutrition. Most likely the pep-tides were early a part of the effector armament inphagocytosis and have remained as such throughevolution. In human, they are important effectormolecules of oxygen-independent defences in themain phagocytic cells, the neutrophils and themacrophages.

The first antibacterial peptides were characterizedŽin insects during the early eighties Steiner et al.,

.1981 . Continued intensive research on insect immu-nity has lead to characterization of many different

Žpeptides of diverse insect species Andreu and Rivas,.1998 . In addition, their selective inducibility and

regulatory pathways have been partially resolvedŽEngstrom et al., 1993; Lemaitre et al., 1996; Pe-

.tersen et al., 1999 . The base for unravelling theregulatory pathways has been the well-definedmodel system, combining genetic and biochemicalapproaches, available in the fruitfly, Drosophila

Ž .melanogaster Hoffmann et al., 1996 .In mammals, the expression of antibacterial genes

takes place in differentiating neutrophils, macro-phages and epithelial cells, but the control pathwaysare not at all defined. Interestingly, the proteins inthe regulatory pathways identified in insects have

Žcounterparts in mammals Medzhitov and Janeway,.1998 . However, it remains to find out if these

regulatory proteins control the same or similar effec-tor molecules in mammals as in insects. Resolvingthe signal pathways for the expression of anti-bacterial peptides in mammals will certainly increasethe insight into regulation of innate immunity andmay open up therapeutic alternatives for combatinginfectious diseases.

4. Multifunctional activities

During evolution, antibacterial peptides haveadopted additional functions apart from the broadspectra bactericidal activities. Lethal activities againstother microbes mainly fungi and viruses have beenreported for several of the mammalian neutrophil

Žantibacterial peptides Lehrer et al., 1993; Robinson.et al., 1998 . Other functions reported for the pep-

tides that are of relevance in immunity are, chemo-taxis, histamine releasing effect on mast cells, stimu-

Žlation of repair in wounds and apoptosis Andreu and.Rivas, 1998 . Chemotactic activity for T cells has

been demonstrated for human a-defensins HNP 1–2Ž .Chertov et al., 1996 , porcine PR-39 for neutrophilsŽ .Huang et al., 1997 and human LL-37 for both T

Žcells and neutrophils Agerberth et al., manuscript in.preparation . An interesting interplay seems to occur

Ž .between the chemokine interleukin-8 IL-8 and a-defensins, leading to an increase of the recruitmentresponse and thus, enhancing the potency for mi-crobe elimination. IL-8 is released by epithelial cells

Žsometimes as a response to pathogens Sansonetti et.al., 1999 , thereby attracting neutrophils and stimu-

Ž .lates secretion of a-defensins Chertov et al., 1996 .The a-defensins would then attack microbes that arepresent but in addition alarm T-cells and induce the

Ž .synthesis of IL-8 Van Wetering et al., 1997 . Thisinterplay could exemplify a positive signal loop, thatcertainly involves additional control components. Itis apparent that close links exist between the innateand the adaptive immune systems. Innate immunityeffectors like cytokines have an instructive role forthe highly specific lymphocytes of the adaptive im-

Ž .mune system Fearon and Locksley, 1996 . How-ever, lymphocytes alarming and lethal activities are


Ž .combined in the a-defensin molecule HNP 1–2 ,hence a role for the peptides in activating adaptiveimmunity is possible, leading to increased clearancecapacity against invading microbes.

Growth promoting activities have been demon-strated for neutrophilic antibacterial peptides, thatmay be involved in wound healing. For the a-de-fensins, a mitogenic effect for fibroblast and epithe-

Žlial cells has been demonstrated in vitro Murphy et.al., 1993 . More detailed studies have been per-

formed on the porcine cathelicidin PR-39 that hasbeen found to induce the heparansulfate extracellular

Žmatrix proteins, syndecan 1 and 4 Gallo et al.,.1994 . PR-39 mediated induction of syndecans mim-

ics their expression in the wound healing process,indicating a double role for this peptide in keepingthe wound sterile and promoting the healing. Neu-trophilic peptides are known to be present in human

Ž .wound fluid Frohm et al., 1996 , but the humancounterpart of PR-39 has not yet been identified.

Further examples of intercellular signaling forantibacterial peptides include defensin stimulation of

Ž .histamine release from mast cells Befus et al., 1999 .The enigmatic mast cell has been suggested to be aregulator of innate response, through the production

Ž .of cytokines Galli et al., 1999 . The effect ofdefensins on mast cells might be of coordinatingcharacter for innate immunity and further increasesthe role of defensins in immunity. Finally, someantibacterial neutrophilic peptides, i.e., the bovineBMAP-27 and BMAP-28 have been proposed toinduce apoptosis at similar concentration as for the

Ž .antibacterial effect Risso et al., 1998 . Whether thisapplies to other defence peptides is not known, butthese observations could indicate an active role forthe peptides in elimination of tumor cells, virusinfected cells and cells infected with intracellularbacteria.

5. Mechanism of action and cytotoxicity

Most of the antibacterial peptides interact with thebacterial membrane by disrupting the order of thephospholipid bilayer, causing loss of membrane in-tegrity. Destruction of the energy gradient across themembrane occurs and increased membrane damageleads to lysis of the bacteria. Two main mechanisms

have been suggested for peptide permeation of theŽ .bacterial membrane: i the barrel-stave mechanism,

where bundles of peptides form transmembrane poresthrough the bacterial membrane, as is proposed for

Ž . Ž .the a-defensins White et al., 1995 and ii thecarpet-like mechanism, where membrane destruc-tionrsolubilization occurs via parallel binding of thepeptides to the bacterial membrane, covering the

Ž .membrane in a carpet like manner Shai, 1995 .Biophysical analyses of several a-helical anti-bacterial peptides support their carpet-like mecha-nism of action. A thorough review has recently beenpublished on these mechanisms in connection to

Ž .a-helical peptides Oren and Shai, 1998 .Primary and secondary structures of the peptides

exhibit pronounced variation, for example the de-fensins have three intrachain disulphide bridges andan antiparallel b-sheet structure, LL-37 forms a lin-ear amphipathic a-helix and the protegrins fold intoa loop structure with one disulphide bridge. Despitethese structural variations the bactericidal peptideshave certain common denominators; in being of ba-sic character and in the folded form the chargedamino acids are gathered at one side of the moleculeand the neutral residues on the other side, therebycreating a strong dipole moment of amphiphilic char-acter. This character is probably the basis for theaffinity of the peptides to negatively charged bacte-rial membranes. The best studied exception is PR-39,that is not membrane active and does not lyse thetarget cells. PR-39 can adopt a lefthanded polypro-

Ž .line helix Cabiaux et al., 1994 and kills bacteria byinhibiting DNA synthesis and protein translation by

Ž .an unknown mechanism Boman et al., 1993 . Insome cases, the peptide–microbe interaction mightshift the equilibrium of the peptide folding from an

Žunordered random coil to the folded stage Steiner et.al., 1988 . For LL-37, we have shown that the

microenvironment is a determinant for folding with acorrelation of the a-helix formation to the anti-bacterial activity. These observations indicate thatthe membrane driven folding is of minor importance

Ž .for LL-37 Johansson et al., 1998 . The initial attrac-tion between the positively charged peptides and thenegatively charged bacterial surfaces seems to bebased on electrostatic forces. In bacteria, the outerleaflet of the cell membrane is more negativelycharged than in eukaryotic cells. Most likely this fact


partially explains the different effects of the peptideson prokaryotic cells compared to eukaryotic cells,whose membranes contain a mixture of negativelycharged and zwitterionic phospholipids. The differ-ence in membrane character with respect to phospho-lipid composition and charge could be the basis formembrane selectivity and avoidance of self destruc-

Ž .tion Oren and Shai, 1998; Oren et al., 1999 . How-ever, many of the antibacterial peptides are cyto-toxic, but at elevated concentrations compared to theminimal concentrations needed for killing bacteria.This has clearly been shown for a-defensins,

ŽBMAP-27, BMAP-28 and LL-37 Lehrer et al., 1993;.Skerlavaj et al., 1996; Johansson et al., 1998 . Inter-

estingly, a structural connection to cytotoxcity hasbeen demonstrated for BMAP-27 and BMAP-28,where the C-terminal hydrophobic tail is essential forthe cytotoxic, but not for the antibacterial effectŽ .Skerlavaj et al., 1996 . We have shown that thecytotoxic concentration for LL-37 is only three tofive times higher than the minimal concentration

Ž .needed for killing bacteria Johansson et al., 1998 ,hence one can envision that the concentration ofpeptides must be under tight control. Indeed, thepeptides are all synthesized as inactive pro-proteins.For cathelicidins, in neutrophils the correspondinggenes are transcribed during differentiation of pre-cursor cells and stored as pro-proteins in neutrophilicgranules, ready for fast activation by an enzymatic

Žcleavage Scocchi et al., 1992; Verbanac et al.,.1993 . The precursor for the human peptide LL-37

Ž .hCAP18rLL-37 has even been detected at highconcentrations in plasma, showing that the proform

Ž .can also be secreted Sorensen et al., 1997b . Tofurther diminish the potential harmful effect of cyto-toxicity, scavenger-proteins for the peptides are pre-sent in the circulation. The effect of a-defensins isneutralized by a2-macroglobulin and activated C1

Žcomplement Panyutich and Ganz, 1991; Panyutich.et al., 1994 and apolipoprotein A-1 binds and in-

hibits the antibacterial and cytotoxic activity of LL-37Ž .Wang et al., 1998; Sorensen et al., 1999 . PR-39 isnot lytic and most likely not cytotoxic, therefore noprotection is needed, and accordingly no inhibitory

Žeffect of porcine plasma was detected Johansson et.al., 1998 . Upon neutrophil recruitment, that occurs

early in infection or during inflammation, the totalconcentration of these peptides might locally reach

high levels. Furthermore, upregulation of certainantibacterial peptides takes place, as has been shownfor LL-37 in keratinocytes during inflammatory skin

Ž .disorders Frohm et al., 1997 and HBD-2 in lungŽ .infection Singh et al., 1998 supporting that local

high concentrations can be reached. The suggestionthat these peptides are involved in immunopatho-

Žgenic tissue damages is therefore plausible Nygaard.et al., 1993 . However, nothing is reported on the

turn over of the active peptides, but one role for themany proteases that are activated during inflamma-tion and infections could be to titrate the concentra-tions of these lethal peptides.

6. Antimicrobial assays

The main assays used for screening antibacterialŽactivity are the inhibition zone assay or the radial

.diffusion assay and modifications thereof, like theŽ .ultrasensitive diffusion assay Lehrer et al., 1991b .

Ž .The minimal inhibitory concentration MIC valueestimated in solution is a more accurate method toevaluate the activity of pure components. The disad-vantage of the MIC value method is the requirementof large quantities of active material and therefore itis not suitable as a screening method.

For screening chromatographic fractions, in orderto detect antibacterial activity we have used a modi-fied inhibition zone assay in thin agarose platesŽ .Gudmundsson et al., 1996 . The plates are poured in

Ž .standard Luria Bertani LB medium seeded withapproximately 6=104 colony forming units permilliliter in logarithmic growth phase. Depending onthe starting material, the assay medium is with orwithout medium E, which is a physiological saltmedium originally worked out for culturing Es-

Ž .cherichia coli Vogel and Bonner, 1956 . This assayis rapid and does not consume large amounts ofmaterial. The test bacteria used in our assays areeither the Gram negative bacterium E. coli, strainD21 or the Gram positive bacterium Bacillus mega-terium, strain Bm11. These two laboratory strains aresensitive and suitable in screenings to detect anti-bacterial activities. In general, we start by estimatingthe total activity in a peptiderprotein concentrate ofthe starting material during different assay conditionsand then select the strain and conditions for furtherscreening during the purification procedure.


It is clear that the microenvironment, such asionic strength, ionic composition, pH and agarosequality have variant influence on the activity ofdifferent peptides. As an example, medium E addedto LB medium has pronounced positive influence for

Ž .the activity of LL-37 Agerberth et al., 1995 ,whereas defensins exhibit better activity in low ionic

Ž .strength medium Lehrer et al., 1993 . Another prob-lem frequently encountered during chromatographicseparations is the loss of synergistic effect betweendifferent components and hence reduced activity inisolated fractions.

7. Synergism between defence effector molecules

Despite the ancient origin and the potent broadspectrum activity, antibacterial peptides are no soloplayers in the immune system, but rather intertwinedin the complex network of effectors in innate andadaptive immunity. Even as mediators of the lethalhit to bacteria the peptides certainly act in synergywith various proteins, like lactoferrin, lysozyme, bac-

Ž .tericidalrpermeability-increasing protein BPI ,Žphospholipase A2 and antileukoproteas ALP, also

called secretory leukocyte proteinase inhibitorŽ ..SLPI in neutrophils and the complement cascade

Žin the circulation Levy, 1996; Ganz and Weiss,.1997 . In the oxygen depending pathways, superox-

ide is produced through the action of NADPHoxidase and several reactive radicals are made

Ž .by myeloperoxidase MPO for attacking bacteriaŽ .Hampton et al., 1998 . For the same purpose, nitricoxide and peroxynitrite are produced through the

Žaction of nitric oxide synthase Hampton et al.,.1998 . After neutrophil attraction, all these bacterici-

dal effectors can be collected to fight invading bacte-ria. Thus, the bacterial intruders are bombarded in adefensive attack. How can then pathogens or oppor-tunistic intruders survive at all? Indeed, their visit isoften transient and they are eliminated in most cases.Defining the weak spots in the system utilized bypathogens as immune escape strategies or impairedspots, resulting in persistent infections will certainlybe one of the future challenges in this research field.

Synergistic action of different components poses apractical problem during isolation procedures of ac-tive peptides. The synergy is broken up during sepa-

ration, leading to loss of activity and results indifficulties to detect and isolate active components.Synergism has been demonstrated between LL-37

Ž .and lactoferrin in vitro Bals et al., 1998 . The fact,that LL-37 and lactoferrin are colocalized in the

Žsecretory granules of neutrophils Sorensen et al.,.1997a and in the serous cells of submucosal glands

Ž .of the lung Bals et al., 1998 , is an indication thatthe synergistic effects also takes place in vivo. Inaddition, one can assume that lysozyme play a piv-otal role in synergism with other components, be-cause of its unique action of breaking up bacterialcell walls. Thus, lysozyme paves the way for otherfactors, giving access to the membrane of the mi-crobe in question. The expression and localization oflysozyme in many body fluids and tissues is also astrong indication for its key role in host defenceŽ .Franken et al., 1989 .

8. Microenvironment

It is evident that the microenvironment affects theactivity of antibacterial peptides. The activity ofa-defensins is dependent on ionic strength, resultingin pronounced reduction of the activity at high salt

Ž .concentration Lehrer et al., 1993 . Similar effectshave been noted for the epithelial b-defensin, HBD-1Ž .Goldman et al., 1997; Singh et al., 1998 . We haveobserved that the total antibacterial activity in BALFŽ .broncoalveolar lavage fluid is reduced as the NaCl

Ž .concentration increases Agerberth et al., 1999 . In-terestingly, the microenvironment in connection toantibacterial activity has recently been highlighted inrelation to the immunocompromised lung in cysticfibrosis. The original finding showed inactivation ofantibacterial activity, presumably dependent on apeptide-like component, in secreted material derivedfrom cultures of lung epithelial cells from cystic

Ž .fibrosis patients Smith et al., 1996 . Further studiesclaimed the importance of high salt concentration incystic fibrosis lung and more specific an inactivationof the salt-sensitive antibacterial peptide HBD-1Ž .Goldman et al., 1997 . It is now clear that theimmediate surface defences in the lung are depen-dent on several components, including the peptides

ŽLL-37 and HBD-2 in addition to HBD-1 Agerberth.et al., 1999; Schroder and Harder, 1999 . The domi-


nance of the salt effect has recently been questioned,and other factors like mucin or DNA have beenproposed to inhibit the bactericidal activity and could

Žbe equally important in this context Matsui et al.,.1998 . The innate defense of the lung is a complex

system and to measure the concentration of differentmolecules at the epithelial surface is difficult also in

Ž .model systems Bals et al., 1999 . However, therelation between antibacterial peptides and cysticfibrosis underlines the importance of the microenvi-ronment, while the molecular details giving the com-plete mechanisms in this disease are not clear. Wehave analysed the effects of the microenvironmenton folding and activity of the antibacterial peptide

Ž .LL-37 in some details Johansson et al., 1998 . ByŽ .combining structural CD circular dichroism mea-

surements of folding and antibacterial activity, wehave observed a positive correlation between thedegree of folding and the antibacterial activity. Themain determinants for the folding were certain an-ions as SO2y, HCOy, and CFCOOy. In contrast, the4 3

Cly anion and cations in general had much lesseffect on the folding. Others have reported that highNaCl concentrations reduce the antibacterial activityof LL-37 in a complex media, where for example

3y Ž .PO was included Turner et al., 1998 . These4

results seem contradictory, but most likely the Cly iscompeting out a stronger folding promoting anionsuch as PO3y and thus, reducing the activity of the4

peptide.

9. Epithelial expression and recruitment

As mentioned above, several of the antibacterialpeptides have adopted additional functions apart fromthe microbicidal activity. Antibacterial peptides havealso been found to be expressed in epithelial cellsand secreted on to mucosal surfaces, in the lungs,

Žgastrointestinal and urogenital tracts Bals et al.,.1998; Valore et al., 1998; Agerberth et al., 1999 .

Thus, these peptides are an integral part of theepithelial defence barrier. Epithelial cells are oftenthe initial contact with microbes and therefore repre-sent the outpost of our defences, while the neu-trophils constitute a second wave of defence as beingthe primary recruited cells. There seems to be an-

other set up of defensins at the epithelial surfacescompared to neutrophils, the a-defensins are substi-tuted with the b-defensins, whereas LL-37 is uniquein being expressed by both neutrophils and epithelialcells. In addition, epithelial cells also have alarming

Žfunction upon pathogen contact Sansonetti et al.,.1999 by synthesizing the chemokine IL-8, that at-

tracts neutrophils and stimulates them to secreteŽ .a-defensins Chertov et al., 1996 . Furthermore, the

secreted a-defensins from the recruited neutrophilsattracts more neutrophils together with T-cells. Thisinterplay might constitute an important link betweenthe innate and adaptive immunity.

10. Conclusion

The research field concerning antibacterial pep-tides has expanded during the last years and thenumber of characterized peptides is constantly

Ž .increasing Andreu and Rivas, 1998 . Detailed struc-tural analysis combined with studies on the mecha-nism of action are required for developing the pep-tides as drugs for therapeutic use. Potent activitiesother than bacterial killing have been described forthe peptides and probably we only see the tip ofthe iceberg in this context. Recent development ofbiochemical methods for characterization, whereless material is needed, will certainly promote thisprogress. The relevance of the peptides to differentinflammatory disorders and infections must be fur-ther investigated to evaluate their importance in im-munity. In mammals, important parts of the peptidedefences are still missing like the regulatory circuitsfor the expression of these peptides, their processing,their potential receptors in signaling and also theirrole in regulating the natural flora. Future research inthe field will certainly focus on these aspects.

Acknowledgements

Support is acknowledged from The Swedish Med-ical Research Council, The Swedish Heart Lung

˚Foundation; Magnus Bergvall’s Foundation; and AkeWiberg’s Foundation.


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