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 References Agerberth, B., Gunne, H., Odeberg, J., Kogner, P., Boman, H.G., Gudmundsson, G.H., 1995. FALL-39, a putative human pep- tide antibiotic, is cysteine-free and expressed in bone marrow and testis. Proc. Natl. Acad. Sci. U.S.A. 92, 195199. Agerberth, B., Grunewald, J., Castanos, V.E., Olsson, B., Jornvall, H., Wigzell, H., Eklund, A., Gudmundsson, G.H., 1999. An- tibacterial components in bronchoalveolar lavage fluid from healthy individuals and sarcoidosis patients. Am. J. Respir. Crit. Care Med. 160, 283290. Andreu, D., Rivas, L., 1998. Animal antimicrobial peptides: an overview. Biopolymers 47, 415433. Bals, R., Wang, X., Zasloff, M., Wilson, J.M., 1998. The peptide antibiotic LL-37rhCAP-18 is expressed in epithelia of the human lung where it has broad antimicrobial activity at the airway surface. Proc. Natl. Acad. Sci. U.S.A. 95, 95419546. Bals, R., Weiner, D.J., Wilson, J.M., 1999. The innate immune system in cystic fibrosis lung disease. J. Clin. Invest. 103, 303307. Befus, A.D., Mowat, C., Gilchrist, M., Hu, J., Solomon, S., Bateman, A., 1999. Neutrophil defensins induce histamine secretion from mast cells: mechanisms of action. J. Immunol. 163, 947953. Boman, H.G., 1995. Peptide antibiotics and their role in innate immunity. Annu. Rev. Immunol. 13, 6192. Boman, H.G., Agerberth, B., Boman, A., 1993. Mechanisms of action on Escherichia coli of cecropin P1 and PR-39, two antibacterial peptides from pig intestine. Infect. Immun. 61, 29782984. Cabiaux, V., Agerberth, B., Johansson, J., Homble, F., Goor- maghtigh, E., Ruysschaert, J.M., 1994. Secondary structure and membrane interaction of PR-39, a ProqArg-rich anti- bacterial peptide. Eur. J. Biochem. 224, 10191027. Chertov, O., Michiel, D.F., Xu, L., Wang, J.M., Tani, K., Mur- phy, W.J., Longo, D.L., Taub, D.D., Oppenheim, J.J., 1996. Identification of defensin-1, defensin-2, and CAP37razuroci- din as T-cell chemoattractant proteins released from inter- leukin-8-stimulated neutrophils. J. Biol. Chem. 271, 2935 2940. Couto, M.A., Harwig, S.S., Cullor, J.S., Hughes, J.P., Lehrer, R.I., 1992. Identification of eNAP-1, an antimicrobial peptide from equine neutrophils. Infect. Immun. 60, 30653071. Cowland, J.B., Johnsen, A.H., Borregaard, N., 1995. hCAP-18, a cathelinrpro-bactenecin-like protein of human neutrophil spe- cific granules. FEBS Lett. 368, 173176. Diamond, G., Zasloff, M., Eck, H., Brasseur, M., Maloy, W.L., Bevins, C.L., 1991. Tracheal antimicrobial peptide, a cysteine-rich peptide from mammalian tracheal mucosa: pep- tide isolation and cloning of a cDNA. Proc. Natl. Acad. Sci. U.S.A. 88, 39523956. Eisenhauer, P.B., Lehrer, R.I., 1992. Mouse neutrophils lack defensins. Infect. Immun. 60, 34463447. Engstrom, Y., Kadalayil, L., Sun, S.C., Samakovlis, C., Hultmark, D., Faye, I., 1993. kappa B-like motifs regulate the induction of immune genes in Drosophila. J. Mol. Biol. 232, 327333. Fearon, D.T., Locksley, R.M., 1996. The instructive role of innate immunity in the acquired immune response. Science 272, 5053. Franken, C., Meijer, C.J., Dijkman, J.H., 1989. Tissue distribution of antileukoprotease and lysozyme in humans. J. Histochem. Cytochem. 37, 493498. Frohm, M., Gunne, H., Bergman, A.C., Agerberth, B., Bergman, T., Boman, A., Liden, S., Jornvall, H., Boman, H.G., 1996. Biochemical and antibacterial analysis of human wound and blister fluid. Eur. J. Biochem. 237, 8692. Frohm, M., Agerberth, B., Ahangari, G., Stahle-Backdahl, M., Liden, S., Wigzell, H., Gudmundsson, G.H., 1997. The ex- pression of the gene coding for the antibacterial peptide LL-37 is induced in human keratinocytes during inflammatory disor- ders. J. Biol. Chem. 272, 1525815263. Galli, S.J., Maurer, M., Lantz, C.S., 1999. Mast cells as sentinels of innate immunity. Curr. Opin. Immunol. 11, 5359. Gallo, R.L., Ono, M., Povsic, T., Page, C., Eriksson, E., Klags- brun, M., Bernfield, M., 1994. Syndecans, cell surface heparan sulfate proteoglycans, are induced by a proline-rich antimicro- bial peptide from wounds. Proc. Natl. Acad. Sci. U.S.A. 91, 1103511039. Gallo, R.L., Kim, K.J., Bernfield, M., Kozak, C.A., Zanetti, M., Merluzzi, L., Gennaro, R., 1997. Identification of CRAMP, a cathelin-related antimicrobial peptide expressed in the embry- onic and adult mouse. J. Biol. Chem. 272, 1308813093. Ganz, T., Weiss, J., 1997. Antimicrobial peptides of phagocytes and epithelia. Semin. Hematol. 34, 343354. Ganz, T., Selsted, M.E., Lehrer, R.I., 1990. Defensins. Eur. J. Haematol. 44, 18. Goldman, M.J., Anderson, G.M., Stolzenberg, E.D., Kari, U.P., Zasloff, M., Wilson, J.M., 1997. Human beta-defensin-1 is a salt-sensitive antibiotic in lung that is inactivated in cystic fibrosis. Cell 88, 553560. Gudmundsson, G.H., Agerberth, B., Odeberg, J., Bergman, T., Olsson, B., Salcedo, R., 1996. The human gene FALL39 and processing of the cathelin precursor to the antibacterial peptide LL-37 in granulocytes. Eur. J. Biochem. 238, 325332. Hampton, M.B., Kettle, A.J., Winterbourn, C.C., 1998. Inside the neutrophil phagosome: oxidants, myeloperoxidase, and bacte- rial killing. Blood 92, 30073017. Hancock, R.E., Lehrer, R., 1998. Cationic peptides: a new source of antibiotics. Trends Biotechnol. 16, 8288. Harwig, S.S., Kokryakov, V.N., Swiderek, K.M., Aleshina, G.M., Zhao, C., Lehrer, R.I., 1995. Prophenin-1, an exceptionally proline-rich antimicrobial peptide from porcine leukocytes. FEBS Lett. 362, 6569. Hoffmann, J.A., Reichhart, J.M., Hetru, C., 1996. Innate immu- nity in higher insects. Curr. Opin. Immunol. 8, 813. Huang, H.J., Ross, C.R., Blecha, F., 1997. Chemoattractant prop- erties of PR-39, a neutrophil antibacterial peptide. J. Leuko- cyte Biol. 61, 624629. Johansson, J., Gudmundsson, G.H., Rottenberg, M.E., Berndt, K.D., Agerberth, B., 1998. Conformation-dependent anti- bacterial activity of the naturally occurring human peptide LL-37. J. Biol. Chem. 273, 37183724. Johnsen, A.H., 1998. Phylogeny of the cholecystokininrgastrin family. Front Neuroendocrinol. 19, 7399. ( ) G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 4554 53 Larrick, J.W., Hirata, M., Balint, R.F., Lee, J., Zhong, J., Wright, S.C., 1995. Human CAP18: a novel antimicrobial lipopoly- saccharide-binding protein. Infect. Immun. 63, 12911297. Lehrer, R.I., Ganz, T., Selsted, M.E., 1991a. Defensins: endoge- nous antibiotic peptides of animal cells. Cell 64, 229230. Lehrer, R.I., Rosenman, M., Harwig, S.S., Jackson, R., Eisen- hauer, P., 1991b. Ultrasensitive assays for endogenous antimi- crobial polypeptides. J. Immunol. Methods 137, 167173. Lehrer, R.I., Lichtenstein, A.K., Ganz, T., 1993. Defensins: an- timicrobial and cytotoxic peptides of mammalian cells. Annu. Rev. Immunol. 11, 105128. Lemaitre, B., Nicolas, E., Michaut, L., Reichhart, J.M., Hoff- mann, J.A., 1996. The dorsoventral regulatory gene cassette spatzlerTollrcactus controls the potent antifungal response in Drosophila adults. Cell 86, 973983. Levy, O., 1996. Antibiotic proteins of polymorphonuclear leuko- cytes. Eur. J. Haematol. 56, 263277. Matsui, H., Grubb, B.R., Tarran, R., Randell, S.H., Gatzy, J.T., Davis, C.W., Boucher, R.C., 1998. Evidence for periciliary liquid layer depletion, not abnormal ion composition, in the pathogenesis of cystic fibrosis airways disease. Cell 95, 1005 1015. Medzhitov, R., Janeway, C.A. Jr., 1998. An ancient system of host defense. Curr. Opin. Immunol. 10, 1215. Mirgorodskaya, O.A., Shevchenko, A.A., Abdalla, K.O., Chernu- shevich, I.V., Egorov, T.A., Musoliamov, A.X., Kokryakov, V.N., Shamova, O.V., 1993. Primary structure of three cationic peptides from porcine neutrophils. Sequence determination by the combined usage of electrospray ionization mass spectrom- etry and Edman degradation. FEBS Lett. 330, 339342. Murphy, C.J., Foster, B.A., Mannis, M.J., Selsted, M.E., Reid, T.W., 1993. Defensins are mitogenic for epithelial cells and fibroblasts. J. Cell. Physiol. 155, 408413. Nygaard, S.D., Ganz, T., Peterson, M.W., 1993. Defensins reduce the barrier integrity of a cultured epithelial monolayer without cytotoxicity. Am. J. Respir. Cell Mol. Biol. 8, 193200. Odeberg, H., Olsson, I., 1975. Antibacterial activity of cationic proteins from human granulocytes. J. Clin. Invest. 56, 1118 1124. Oren, Z., Shai, Y., 1998. Mode of action of linear amphipathic alpha-helical antimicrobial peptides. Biopolymers 47, 451 463. Oren, Z., Lerman, J.C., Gudmundsson, G.H., Agerberth, B., Shai, Y., 1999. Structure and organization of the human antimicro- bial peptide LL-37 in phospholipid membranes: relevance to the molecular basis for its non-cell-selective activity. Biochem. J. 341, 501513. Panyutich, A., Ganz, T., 1991. Activated alpha 2-macroglobulin is a principal defensin-binding protein. Am. J. Respir. Cell Mol. Biol. 5, 101106. Panyutich, A.V., Szold, O., Poon, P.H., Tseng, Y., Ganz, T., 1994. Identification of defensin binding to C1 complement. FEBS Lett. 356, 169173. Petersen, U.M., Kadalayil, L., Rehorn, K.P., Hoshizaki, D.K., Reuter, R., Engstrom, Y., 1999. Serpent regulates Drosophila immunity genes in the larval fat body through an essential GATA motif. EMBO J. 18, 40134022. Putsep, K., Branden, C.I., Boman, H.G., Normark, S., 1999. Antibacterial peptide from H. pylori. Nature 398, 671672. Risso, A., Zanetti, M., Gennaro, R., 1998. Cytotoxicity and apoptosis mediated by two peptides of innate immunity. Cell. Immunol. 189, 107115. Robinson, W.E. Jr., McDougall, B., Tran, D., Selsted, M.E., 1998. Anti-HIV-1 activity of indolicidin, an antimicrobial peptide from neutrophils. J. Leukocyte Biol. 63, 94100. Sansonetti, P.J., Arondel, J., Huerre, M., Harada, A., Matsushima, K., 1999. Interleukin-8 controls bacterial transepithelial translocation at the cost of epithelial destruction in experimen- tal shigellosis. Infect. Immun. 67, 14711480. Schonwetter, B.S., Stolzenberg, E.D., Zasloff, M.A., 1995. Ep- ithelial antibiotics induced at sites of inflammation. Science 267, 16451648. Schroder, J.M., Harder, J., 1999. Human beta-defensin-2. Int. J. Biochem. Cell Biol. 31, 645651. Scocchi, M., Skerlavaj, B., Romeo, D., Gennaro, R., 1992. Prote- olytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins. Eur. J. Biochem. 209, 589595. Selsted, M.E., Brown, D.M., DeLange, R.J., Lehrer, R.I., 1983. Primary structures of MCP-1 and MCP-2, natural peptide antibiotics of rabbit lung macrophages. J. Biol. Chem. 258, 1448514489. Selsted, M.E., Miller, S.I., Henschen, A.H., Ouellette, A.J., 1992. Enteric defensins: antibiotic peptide components of intestinal host defense. J. Cell Biol. 118, 929936. Shai, Y., 1995. Molecular recognition between membrane-span- ning polypeptides. Trends Biochem. Sci. 20, 460464. Singh, P.K., Jia, H.P., Wiles, K., Hesselberth, J., Liu, L., Conway, B.A., Greenberg, E.P., Valore, E.V., Welsh, M.J., Ganz, T., Tack, B.F., McCray, P.B. Jr., 1998. Production of beta-de- fensins by human airway epithelia. Proc. Natl. Acad. Sci. U.S.A. 95, 1496114966. Skerlavaj, B., Gennaro, R., Bagella, L., Merluzzi, L., Risso, A., Zanetti, M., 1996. Biological characterization of two novel cathelicidin-derived peptides and identification of structural requirements for their antimicrobial and cell lytic activities. J. Biol. Chem. 271, 2837528381. Smith, J.J., Travis, S.M., Greenberg, E.P., Welsh, M.J., 1996. Cystic fibrosis airway epithelia fail to kill bacteria because of abnormal airway surface fluid. Cell 85, 229236. Sorensen, O., Arnljots, K., Cowland, J.B., Bainton, D.F., Borre- gaard, N., 1997a. The human antibacterial cathelicidin, hCAP- 18, is synthesized in myelocytes and metamyelocytes and localized to specific granules in neutrophils. Blood 90, 2796 2803. Sorensen, O., Cowland, J.B., Askaa, J., Borregaard, N., 1997b. An ELISA for hCAP-18, the cathelicidin present in human neutrophils and plasma. J. Immunol. Methods 206, 5359. Sorensen, O., Bratt, T., Johnsen, A.H., Madsen, M.T., Borregaard, N., 1999. The human antibacterial cathelicidin, hCAP-18, is ( ) G.H. Gudmundsson, B. AgerberthrJournal of Immunological Methods 232 1999 4554 54 bound to lipoproteins in plasma. J. Biol. Chem. 274, 22445 22451. Steiner, H., Hultmark, D., Engstrom, A., Bennich, H., Boman, H.G., 1981. Sequence and specificity of two antibacterial proteins involved in insect immunity. Nature 292, 246248. Steiner, H., Andreu, D., Merrifield, R.B., 1988. Binding and action of cecropin and cecropin analogues: antibacterial pep- tides from insects. Biochim. Biophys. Acta 939, 260266. Turner, J., Cho, Y., Dinh, N.N., Waring, A.J., Lehrer, R.I., 1998. Activities of LL-37, a cathelin-associated antimicrobial pep- tide of human neutrophils. Antimicrob. Agents Chemother. 42, 22062214. Valore, E.V., Park, C.H., Quayle, A.J., Wiles, K.R., McCray, P.B. Jr., Ganz, T., 1998. Human beta-defensin-1: an antimicrobial peptide of urogenital tissues. J. Clin. Invest. 101, 16331642. Van Wetering, S., Mannesse-Lazeroms, S.P., Van Sterkenburg, M.A., Daha, M.R., Dijkman, J.H., Hiemstra, P.S., 1997. Effect of defensins on interleukin-8 synthesis in airway epithelial cells. Am. J. Physiol. 272, L888L896. Verbanac, D., Zanetti, M., Romeo, D., 1993. Chemotactic and protease-inhibiting activities of antibiotic peptide precursors. FEBS Lett. 317, 255258. Vogel, H.J., Bonner, D.M., 1956. Acetylornithinase of Es- cherichia coli: partial purification and some properties. J. Biol. Chem. 218, 97106. Wang, Y., Agerberth, B., Lothgren, A., Almstedt, A., Johansson, J., 1998. Apolipoprotein A-I binds and inhibits the human antibacterialrcytotoxic peptide LL-37. J. Biol. Chem. 273, 3311533118. White, S.H., Wimley, W.C., Selsted, M.E., 1995. Structure, func- tion, and membrane integration of defensins. Curr. Opin. Struct. Biol. 5, 521527. Wilde, C.G., Griffith, J.E., Marra, M.N., Snable, J.L., Scott, R.W., 1989. Purification and characterization of human neu- trophil peptide 4, a novel member of the defensin family. J. Biol. Chem. 264, 1120011203. Zanetti, M., Gennaro, R., Romeo, D., 1995. Cathelicidins: a novel protein family with a common proregion and a variable C- terminal antimicrobial domain. FEBS Lett. 374, 15. Zeya, H.I., Spitznagel, J.K., 1966. Antimicrobial specificity of leukocyte lysosomal cationic proteins. Science 154, 1049 1051.