.

Journal of Immunological Methods 232 1999 4554

www.elsevier.nlrlocaterjim
Neutrophil antibacterial peptides, multifunctional effector
molecules 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, Sweden
b
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 destruction
and elimination of ingested microbes. During the eighties, it gradually became clear, that cationic peptides are a part of the
oxygen-independent bactericidal effectors in phagocytic cells. In mammals, these antimicrobial peptides are represented by
two families, the defensins and the cathelicidins. These potent broad spectra peptides are included as immediate effector
molecules in innate immunity. The detailed killing mechanism for these effectors is partly known, but nearly all of them
have membrane affinity, and permeate bacterial membranes, resulting in lysis of the bacteria. This peptidemembrane
interaction includes also eukaryotic membranes, that implicates cytotoxic effects on host cells. Studies in vitro have
established that the microenvironment is critical for their activities. In connection to cystic fibrosis, the effects of
microenvironment changes are apparent, causing inactivation of peptide defences and leading to repeated serious bacterial
infections. Thus, the importance of the microenvironment is also supported in vivo. Additional functions of these peptides
such as chemotactic, mitogenic and stimulatory in the wound healing process suggest further important roles for these
peptides. q1999 Elsevier Science B.V. All rights reserved.
Keywords: Antibacterial peptides; Innate immunity; Microenvironment; Evolutionary variation; Bactericidal synergy
1. Introduction
The phagosomes of neutrophils contain multiple
antibacterial components, working in concert on
killing engulfed microorganisms. Already during the
sixties, efforts were made to characterize active bac-
)
Corresponding author. Tel.: q46-8-728-6685; fax: q46-8-
328878; e-mail: gudmundur.gudmundsson@mtc.ki.se
tericidal components. Gradually, part of the activity
was found to be oxygen-independent and connected

to cationic proteins Zeya and Spitznagel, 1966;

.
Odeberg and Olsson, 1975 . Further studies con-
firmed the participation of small proteins or peptides
in this antibacterial, oxygen-independent system
.
Lehrer et al., 1993 . The first mammalian anti-
bacterial peptides were isolated and characterized

from 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.
. PII: S0022- 1759 99 00152- 0
( ) G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 4554 46

several mammalian species, including human Lehrer

.
et al., 1991a . Thus, a peptide based protection as a
general concept in mammalian neutrophils and
macrophages was established. These first peptides
were designated the defensins and found to have a
b-sheet structure stabilized by three intrachain disul-
.
phide bridges Ganz et al., 1990 . Further peptide
purification and characterization by cDNA cloning
lead to isolation of different type of peptides, which
turned out to have a conserved proregion of cathelin
type and consequently they were called cathelicidins
.
Zanetti et al., 1995 . The active mature peptides of

this family are a heterogenous group Zanetti et al.,

.
1995 . Thus, in mammalian neutrophils two main
families of antibacterial peptides are present, the
cathelicidins and the defensins.
Gradually, it emerged that these broad spectra
antimicrobial peptides were a substantial part of
mammalian immediate defences. This gained further
support, when the peptides were also found to be

expressed 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 a
second wave of antibacterial defence effectors, when
neutrophils are recruited to sites of infection and
inflammation. The importance of the peptide de-
fences in connection to disease has become clear
.
through studies on cystic fibrosis Bals et al., 1999 ,
where this defence system is impaired, resulting in
repeated infections. However, it is always important
to consider the synergistic effects between different
peptides, antibacterial proteins and the reactive oxy-
gen products, in addition to the complement system.
The resistance of various bacteria to classical
antibiotics is increasing as a serious problem in
health care, therefore the peptides have gained inter-
est as possible candidate components for therapeutic

applications and drug development Hancock and

.
Lehrer, 1998 . In this context, additional functions
for the peptides are of importance. These functions
include antifungal, antiviral, chemotactic, mitogenic
and stimulatory activities as have been described for
the a-defensins, which are the best studied mam-
.
malian peptides Lehrer et al., 1993 . Some of these
activities have also been noted for the cathelicidin
peptides. 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 mammalian
species. The a-defensins HNP 13 seem to be the
major bactericidal peptides identified in human neu-
trophils, with an estimation of 30%50% of total
.
protein in azurophil granules Ganz et al., 1990 . In
addition, a-defensin HNP-4 with a different anti-
bacterial spectrum compared to HNP 13 is present
.
but at lower concentrations Wilde et al., 1989 . In
contrast to human, neutrophils from horse and mouse
do not contain the a-defensins as major antibacterial

peptides 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 of
a-defensins genes are expressed, but with different
tissue distribution, sofar mainly located in the gastro-
.
intestinal tract Selsted et al., 1992 . The mouse

a-defensins have been designated cryptdins Selsted

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

cidins Mirgorodskaya et al., 1993; Harwig et al.,

.
1995 . The cathelicidins constitute a family of anti-
bacterial pro-peptides with a conserved cathelin
proregion, containing a variant C-terminal anti-
.
bacterial domain Zanetti et al., 1995 . Hence, the
cathelin proregion seems to serve as a carrier for
several different antibacterial peptides. In mouse and
human neutrophils, the cathelicidins are only repre-

sented by one member, called CRAMP Gallo et al.,

.
1997 and hCAP18rLL-37 Cowland et al., 1995;
.
Larrick et al., 1995; Gudmundsson et al., 1996 ,
respectively. It appears that the variation is not only
due to divergence in amino acid sequences, but also
applies to the number and abundance of expressed
gene products of the two main families coding for
antibacterial peptides in neutrophils, i.e., the de-
fensins and the cathelicidins. In the light of this
variation, it is clear that these defence effectors are
fast evolving entities as compared with for example
peptide hormones, that represent another group of
bioactive peptides, that have been conserved through
.
evolution Johnsen, 1998 . Most likely the variation
of antibacterial peptides reflects the character of their
( ) G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 4554 47
targets, i.e., the microbes that are fast evolving, and
exhibit tremendous variation. In addition, there seems
to be extensive flexibility for the peptide microbe
interactions, since substitutions of charge equivalent
amino acids in the peptides do not alter their affinity
to microbe membranes or their lethal effects. Thus,
the peptides probably reflect rapid adaptive evolu-
tionary changes, regarding hostmicrobe interplay,
and represent different evolutionary history with re-
spect to interaction with variable targets of the natu-
ral flora and evolutionary bottlenecks caused by
severe pathogens.
3. Ancient defence system
Antibacterial peptides are widespread in nature as
defence effectors, and have been found in plants,

invertebrates, vertebrates and also in bacteria Bo-

.
man, 1995; Putsep et al., 1999 . In addition, to
represent an ancient defence system that probably
was well adapted already among early eukaryotic
cells in the form of defence effectors, these peptides
probably also participated in degrading microorgan-
isms as a source for nutrition. Most likely the pep-
tides were early a part of the effector armament in
phagocytosis and have remained as such through
evolution. In human, they are important effector
molecules of oxygen-independent defences in the
main phagocytic cells, the neutrophils and the
macrophages.
The first antibacterial peptides were characterized

in insects during the early eighties Steiner et al.,

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

peptides of diverse insect species Andreu and Rivas,

.
1998 . In addition, their selective inducibility and
regulatory pathways have been partially resolved

Engstrom et al., 1993; Lemaitre et al., 1996; Pe-

.
tersen et al., 1999 . The base for unravelling the
regulatory pathways has been the well-defined
model system, combining genetic and biochemical
approaches, available in the fruitfly, Drosophila
.
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 pathways
are not at all defined. Interestingly, the proteins in
the regulatory pathways identified in insects have

counterparts 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. Resolving
the signal pathways for the expression of anti-
bacterial peptides in mammals will certainly increase
the insight into regulation of innate immunity and
may open up therapeutic alternatives for combating
infectious diseases.
4. Multifunctional activities
During evolution, antibacterial peptides have
adopted additional functions apart from the broad
spectra bactericidal activities. Lethal activities against
other microbes mainly fungi and viruses have been
reported for several of the mammalian neutrophil

antibacterial 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-

lation of repair in wounds and apoptosis Andreu and

.
Rivas, 1998 . Chemotactic activity for T cells has
been demonstrated for human a-defensins HNP 12
.
Chertov et al., 1996 , porcine PR-39 for neutrophils
.
Huang et al., 1997 and human LL-37 for both T

cells and neutrophils Agerberth et al., manuscript in

.
preparation . An interesting interplay seems to occur
.
between the chemokine interleukin-8 IL-8 and a-
defensins, leading to an increase of the recruitment
response and thus, enhancing the potency for mi-
crobe elimination. IL-8 is released by epithelial cells

sometimes as a response to pathogens Sansonetti et

.
al., 1999 , thereby attracting neutrophils and stimu-
.
lates secretion of a-defensins Chertov et al., 1996 .
The a-defensins would then attack microbes that are
present but in addition alarm T-cells and induce the
.
synthesis of IL-8 Van Wetering et al., 1997 . This
interplay could exemplify a positive signal loop, that
certainly involves additional control components. It
is apparent that close links exist between the innate
and the adaptive immune systems. Innate immunity
effectors like cytokines have an instructive role for
the highly specific lymphocytes of the adaptive im-
.
mune system Fearon and Locksley, 1996 . How-
ever, lymphocytes alarming and lethal activities are
( ) G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 4554 48
.
combined in the a-defensin molecule HNP 12 ,
hence a role for the peptides in activating adaptive
immunity is possible, leading to increased clearance
capacity against invading microbes.
Growth promoting activities have been demon-
strated for neutrophilic antibacterial peptides, that
may be involved in wound healing. For the a-de-
fensins, a mitogenic effect for fibroblast and epithe-

lial cells has been demonstrated in vitro Murphy et

.
al., 1993 . More detailed studies have been per-
formed on the porcine cathelicidin PR-39 that has
been found to induce the heparansulfate extracellular

matrix 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 keeping
the wound sterile and promoting the healing. Neu-
trophilic peptides are known to be present in human
.
wound fluid Frohm et al., 1996 , but the human
counterpart of PR-39 has not yet been identified.
Further examples of intercellular signaling for
antibacterial peptides include defensin stimulation of
.
histamine release from mast cells Befus et al., 1999 .
The enigmatic mast cell has been suggested to be a
regulator of innate response, through the production
.
of cytokines Galli et al., 1999 . The effect of
defensins on mast cells might be of coordinating
character for innate immunity and further increases
the role of defensins in immunity. Finally, some
antibacterial neutrophilic peptides, i.e., the bovine
BMAP-27 and BMAP-28 have been proposed to
induce apoptosis at similar concentration as for the
.
antibacterial effect Risso et al., 1998 . Whether this
applies to other defence peptides is not known, but
these observations could indicate an active role for
the peptides in elimination of tumor cells, virus
infected cells and cells infected with intracellular
bacteria.
5. Mechanism of action and cytotoxicity
Most of the antibacterial peptides interact with the
bacterial membrane by disrupting the order of the
phospholipid bilayer, causing loss of membrane in-
tegrity. Destruction of the energy gradient across the
membrane occurs and increased membrane damage
leads to lysis of the bacteria. Two main mechanisms
have been suggested for peptide permeation of the
.
bacterial membrane: i the barrel-stave mechanism,
where bundles of peptides form transmembrane pores
through the bacterial membrane, as is proposed for
. .
the a-defensins White et al., 1995 and ii the
carpet-like mechanism, where membrane destruc-
tionrsolubilization occurs via parallel binding of the
peptides to the bacterial membrane, covering the
.
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 been
published on these mechanisms in connection to
.
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 and
an antiparallel b-sheet structure, LL-37 forms a lin-
ear amphipathic a-helix and the protegrins fold into
a loop structure with one disulphide bridge. Despite
these structural variations the bactericidal peptides
have certain common denominators; in being of ba-
sic character and in the folded form the charged
amino acids are gathered at one side of the molecule
and the neutral residues on the other side, thereby
creating a strong dipole moment of amphiphilic char-
acter. This character is probably the basis for the
affinity 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 the
target cells. PR-39 can adopt a lefthanded polypro-
.
line helix Cabiaux et al., 1994 and kills bacteria by
inhibiting DNA synthesis and protein translation by
.
an unknown mechanism Boman et al., 1993 . In
some cases, the peptidemicrobe interaction might
shift the equilibrium of the peptide folding from an

unordered 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 a
correlation of the a-helix formation to the anti-
bacterial activity. These observations indicate that
the membrane driven folding is of minor importance
.
for LL-37 Johansson et al., 1998 . The initial attrac-
tion between the positively charged peptides and the
negatively charged bacterial surfaces seems to be
based on electrostatic forces. In bacteria, the outer
leaflet of the cell membrane is more negatively
charged than in eukaryotic cells. Most likely this fact
( ) G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 4554 49
partially explains the different effects of the peptides
on prokaryotic cells compared to eukaryotic cells,
whose membranes contain a mixture of negatively
charged and zwitterionic phospholipids. The differ-
ence in membrane character with respect to phospho-
lipid composition and charge could be the basis for
membrane selectivity and avoidance of self destruc-
.
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 the
minimal concentrations needed for killing bacteria.
This has clearly been shown for a-defensins,

BMAP-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 has
been demonstrated for BMAP-27 and BMAP-28,
where the C-terminal hydrophobic tail is essential for
the cytotoxic, but not for the antibacterial effect
.
Skerlavaj et al., 1996 . We have shown that the
cytotoxic concentration for LL-37 is only three to
five times higher than the minimal concentration
.
needed for killing bacteria Johansson et al., 1998 ,
hence one can envision that the concentration of
peptides must be under tight control. Indeed, the
peptides are all synthesized as inactive pro-proteins.
For cathelicidins, in neutrophils the corresponding
genes are transcribed during differentiation of pre-
cursor cells and stored as pro-proteins in neutrophilic
granules, ready for fast activation by an enzymatic

cleavage Scocchi et al., 1992; Verbanac et al.,

.
1993 . The precursor for the human peptide LL-37
.
hCAP18rLL-37 has even been detected at high
concentrations in plasma, showing that the proform
.
can also be secreted Sorensen et al., 1997b . To
further diminish the potential harmful effect of cyto-
toxicity, scavenger-proteins for the peptides are pre-
sent in the circulation. The effect of a-defensins is
neutralized by a2-macroglobulin and activated C1

complement Panyutich and Ganz, 1991; Panyutich

.
et al., 1994 and apolipoprotein A-1 binds and in-
hibits the antibacterial and cytotoxic activity of LL-37
.
Wang et al., 1998; Sorensen et al., 1999 . PR-39 is
not lytic and most likely not cytotoxic, therefore no
protection is needed, and accordingly no inhibitory

effect of porcine plasma was detected Johansson et

.
al., 1998 . Upon neutrophil recruitment, that occurs
early in infection or during inflammation, the total
concentration of these peptides might locally reach
high levels. Furthermore, upregulation of certain
antibacterial peptides takes place, as has been shown
for LL-37 in keratinocytes during inflammatory skin
.
disorders Frohm et al., 1997 and HBD-2 in lung
.
infection Singh et al., 1998 supporting that local
high concentrations can be reached. The suggestion
that these peptides are involved in immunopatho-

genic 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 the
many 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 antibacterial

activity are the inhibition zone assay or the radial

.
diffusion assay and modifications thereof, like the
.
ultrasensitive diffusion assay Lehrer et al., 1991b .
.
The minimal inhibitory concentration MIC value
estimated in solution is a more accurate method to
evaluate the activity of pure components. The disad-
vantage of the MIC value method is the requirement
of large quantities of active material and therefore it
is not suitable as a screening method.
For screening chromatographic fractions, in order
to detect antibacterial activity we have used a modi-
fied inhibition zone assay in thin agarose plates
.
Gudmundsson et al., 1996 . The plates are poured in
.
standard Luria Bertani LB medium seeded with
approximately 6=10
4
colony forming units per
milliliter in logarithmic growth phase. Depending on
the starting material, the assay medium is with or
without medium E, which is a physiological salt
medium originally worked out for culturing Es-
.
cherichia coli Vogel and Bonner, 1956 . This assay
is rapid and does not consume large amounts of
material. The test bacteria used in our assays are
either the Gram negative bacterium E. coli, strain
D21 or the Gram positive bacterium Bacillus mega-
terium, strain Bm11. These two laboratory strains are
sensitive and suitable in screenings to detect anti-
bacterial activities. In general, we start by estimating
the total activity in a peptiderprotein concentrate of
the starting material during different assay conditions
and then select the strain and conditions for further
screening during the purification procedure.
( ) G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 4554 50
It is clear that the microenvironment, such as
ionic strength, ionic composition, pH and agarose
quality have variant influence on the activity of
different peptides. As an example, medium E added
to LB medium has pronounced positive influence for
.
the activity of LL-37 Agerberth et al., 1995 ,
whereas defensins exhibit better activity in low ionic
.
strength medium Lehrer et al., 1993 . Another prob-
lem frequently encountered during chromatographic
separations is the loss of synergistic effect between
different components and hence reduced activity in
isolated fractions.
7. Synergism between defence effector molecules
Despite the ancient origin and the potent broad
spectrum activity, antibacterial peptides are no solo
players in the immune system, but rather intertwined
in the complex network of effectors in innate and
adaptive immunity. Even as mediators of the lethal
hit to bacteria the peptides certainly act in synergy
with various proteins, like lactoferrin, lysozyme, bac-
.
tericidalrpermeability-increasing protein BPI ,

phospholipase A2 and antileukoproteas ALP, also

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

in the circulation Levy, 1996; Ganz and Weiss,

.
1997 . In the oxygen depending pathways, superox-
ide is produced through the action of NADPH
oxidase and several reactive radicals are made
.
by myeloperoxidase MPO for attacking bacteria
.
Hampton et al., 1998 . For the same purpose, nitric
oxide and peroxynitrite are produced through the

action 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 a
defensive attack. How can then pathogens or oppor-
tunistic intruders survive at all? Indeed, their visit is
often transient and they are eliminated in most cases.
Defining the weak spots in the system utilized by
pathogens as immune escape strategies or impaired
spots, resulting in persistent infections will certainly
be one of the future challenges in this research field.
Synergistic action of different components poses a
practical problem during isolation procedures of ac-
tive peptides. The synergy is broken up during sepa-
ration, leading to loss of activity and results in
difficulties to detect and isolate active components.
Synergism has been demonstrated between LL-37
.
and lactoferrin in vitro Bals et al., 1998 . The fact,
that LL-37 and lactoferrin are colocalized in the

secretory granules of neutrophils Sorensen et al.,

.
1997a and in the serous cells of submucosal glands
.
of the lung Bals et al., 1998 , is an indication that
the synergistic effects also takes place in vivo. In
addition, one can assume that lysozyme play a piv-
otal role in synergism with other components, be-
cause of its unique action of breaking up bacterial
cell walls. Thus, lysozyme paves the way for other
factors, giving access to the membrane of the mi-
crobe in question. The expression and localization of
lysozyme in many body fluids and tissues is also a
strong indication for its key role in host defence
.
Franken et al., 1989 .
8. Microenvironment
It is evident that the microenvironment affects the
activity of antibacterial peptides. The activity of
a-defensins is dependent on ionic strength, resulting
in pronounced reduction of the activity at high salt
.
concentration Lehrer et al., 1993 . Similar effects
have been noted for the epithelial b-defensin, HBD-1
.
Goldman et al., 1997; Singh et al., 1998 . We have
observed that the total antibacterial activity in BALF
.
broncoalveolar lavage fluid is reduced as the NaCl
.
concentration increases Agerberth et al., 1999 . In-
terestingly, the microenvironment in connection to
antibacterial activity has recently been highlighted in
relation to the immunocompromised lung in cystic
fibrosis. The original finding showed inactivation of
antibacterial activity, presumably dependent on a
peptide-like component, in secreted material derived
from cultures of lung epithelial cells from cystic
.
fibrosis patients Smith et al., 1996 . Further studies
claimed the importance of high salt concentration in
cystic fibrosis lung and more specific an inactivation
of the salt-sensitive antibacterial peptide HBD-1
.
Goldman et al., 1997 . It is now clear that the
immediate surface defences in the lung are depen-
dent on several components, including the peptides

LL-37 and HBD-2 in addition to HBD-1 Agerberth

.
et al., 1999; Schroder and Harder, 1999 . The domi-
( ) G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 4554 51
nance of the salt effect has recently been questioned,
and other factors like mucin or DNA have been
proposed to inhibit the bactericidal activity and could

be 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 different
molecules at the epithelial surface is difficult also in
.
model systems Bals et al., 1999 . However, the
relation between antibacterial peptides and cystic
fibrosis underlines the importance of the microenvi-
ronment, while the molecular details giving the com-
plete mechanisms in this disease are not clear. We
have analysed the effects of the microenvironment
on folding and activity of the antibacterial peptide
.
LL-37 in some details Johansson et al., 1998 . By
.
combining structural CD circular dichroism mea-
surements of folding and antibacterial activity, we
have observed a positive correlation between the
degree of folding and the antibacterial activity. The
main determinants for the folding were certain an-
ions as SO
2y
, HCO
y
, and CFCOO
y
. In contrast, the
4 3
Cl
y
anion and cations in general had much less
effect on the folding. Others have reported that high
NaCl concentrations reduce the antibacterial activity
of LL-37 in a complex media, where for example
3y
.
PO was included Turner et al., 1998 . These
4
results seem contradictory, but most likely the Cl
y
is
competing out a stronger folding promoting anion
such as PO
3y
and thus, reducing the activity of the
4
peptide.
9. Epithelial expression and recruitment
As mentioned above, several of the antibacterial
peptides have adopted additional functions apart from
the microbicidal activity. Antibacterial peptides have
also been found to be expressed in epithelial cells
and secreted on to mucosal surfaces, in the lungs,

gastrointestinal and urogenital tracts Bals et al.,

.
1998; Valore et al., 1998; Agerberth et al., 1999 .
Thus, these peptides are an integral part of the
epithelial defence barrier. Epithelial cells are often
the initial contact with microbes and therefore repre-
sent the outpost of our defences, while the neu-
trophils constitute a second wave of defence as being
the primary recruited cells. There seems to be an-
other set up of defensins at the epithelial surfaces
compared to neutrophils, the a-defensins are substi-
tuted with the b-defensins, whereas LL-37 is unique
in being expressed by both neutrophils and epithelial
cells. In addition, epithelial cells also have alarming

function upon pathogen contact Sansonetti et al.,

.
1999 by synthesizing the chemokine IL-8, that at-
tracts neutrophils and stimulates them to secrete
.
a-defensins Chertov et al., 1996 . Furthermore, the
secreted a-defensins from the recruited neutrophils
attracts more neutrophils together with T-cells. This
interplay might constitute an important link between
the innate and adaptive immunity.
10. Conclusion
The research field concerning antibacterial pep-
tides has expanded during the last years and the
number of characterized peptides is constantly
.
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 activities
other than bacterial killing have been described for
the peptides and probably we only see the tip of
the iceberg in this context. Recent development of
biochemical methods for characterization, where
less material is needed, will certainly promote this
progress. The relevance of the peptides to different
inflammatory disorders and infections must be fur-
ther investigated to evaluate their importance in im-
munity. In mammals, important parts of the peptide
defences are still missing like the regulatory circuits
for the expression of these peptides, their processing,
their potential receptors in signaling and also their
role in regulating the natural flora. Future research in
the field will certainly focus on these aspects.
Acknowledgements
Support is acknowledged from The Swedish Med-
ical Research Council, The Swedish Heart Lung

Foundation; Magnus Bergvalls Foundation; and Ake

Wibergs Foundation.
( ) G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 4554 52
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