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PAEDIATRIC RESPIRATORY REVIEWS (2001) 2, 306–310
doi:10.1053/prrv.2001.0165, available online at http://www.idealibrary.com on
SERIES: NEW BIOLOGY OF THE AIRWAYS
Epithelial antimicrobial peptides and proteins:
their role in host defence and inflammation
P. S. Hiemstra
Department of Pulmonology, Leiden University Medical Centre, Leiden, The Netherlands
KEYWORDS
epithelium,
antimicrobial peptides,
antimicrobial proteins,
defensins, infection,
inflammation
Summary Various antimicrobial mechanisms act in concert to protect the lung from
infection by forming an efficient host defence system. Most microbial challenges are
counteracted by elements of the innate immune system and antimicrobial peptides and
proteins have been identified as key components of innate immunity. Although phagocytes are an important cellular source of these so-called endogenous antibiotics, it is now
recognized that the airway epithelium is also a major site of synthesis. Antimicrobial peptides and proteins kill a wide variety of micro-organisms. Their importance is illustrated
by the observation that in cystic fibrosis changes in the airway surface fluid may result
in a dysfunction of these components. Recent studies have revealed other functions of
these molecules showing they may link innate and adaptive immunity and appear to be
involved in the regulation of inflammation and tissue repair.
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C 2001 Harcourt Publishers Ltd
INTRODUCTION
An efficient host defence system is required to counteract
the continuous microbial challenge to which the lung is
exposed. The epithelium lining the airways and alveoli is an
essential element in this defence. Epithelial cells provide a
passive barrier, contribute to mucociliary clearance, produce inflammatory mediators which recruit cells to the
site of infection and are a main site of synthesis of antimicrobial substances referred to as antimicrobial peptides
and proteins (or collectively as antimicrobial polypeptides). Therefore the epithelium plays a central role in
innate immunity, a system that is rapidly activated after infection, providing efficient control of the invading
pathogen. Whereas lysozyme and lactoferrin have been
known for some time as key antimicrobial components
of this innate immunity, in the last decade a number of
new epithelial antimicrobial polypeptides have been identified. Together these endogenous antibiotics form an
Correspondence to: P. S. Hiemstra, PhD, Dept. of Pulmonology,
Building 1 C3-P, Leiden University Medical Centre, Albinusdreef
2, P.O. Box 9600, 2300 RC Leiden, The Netherlands. Tel: +31 71
5263848, Fax: +31 71 5266927. e-mail: [email protected]
1526–0542/01/040306 + 05 $35.00/0
antimicrobial ‘soup’, the constituents of which act in concert to kill invading micro-organisms. Research in this area
has intensified after the observation that the increased
susceptibility of patients with cystic fibrosis (CF) to infection may be explained by a loss of the endogenous
epithelial antimicrobial activity against Pseudomonas aeruginosa, as demonstrated in cultured epithelial cells from
CF patients.1 This loss of activity was explained by an increased NaCl concentration in airway surface fluid (ASF)
secreted by these cells which decreased the antimicrobial
activity of epithelial antimicrobial polypeptides. Because
of the difficulty in collecting ASF from patients and assessing its ionic composition in situ, this explanation for bacterial colonisation of the airways in CF is still a controversial
one.2 Recent studies have provided evidence that these
antimicrobial polypeptides may also be involved in inflammation, the generation of adaptive immune responses and
wound repair processes.
ANTIMICROBIAL PEPTIDES
AND PROTEINS
Airway surface fluid contains a variety of antimicrobial factors mainly produced by submucosal glands and surface
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C 2001 Harcourt Publishers Ltd
PEPTIDES AND PROTEINS
Figure 1 Antimicrobial polypeptides produced by the surface
epithelium and submucosal glands. The airway epithelium
provides protection against invading micro-organisms through its
barrier function, mucociliary clearance, regulation of inflammation
and immune responses and production of antimicrobial substances including polypeptides. Cells from the surface epithelium
and the submucosal glands contribute to this production.
Note: the schematic drawing of the submucosal gland is an
oversimplification.
epithelial cells and by macrophages and neutrophils (Fig.
1). Defensins are small, cationic antimicrobial peptides
with a molecular mass of 3.5–4.5 kDa. Based on their
structure (most notably the pairing of disulphide bridges)
human defensins can be divided into the subfamilies of αand β-defensins3 whereas in rhesus monkeys a third class
of defensin has been described. These ϕ-defensins derived their name from their circular molecular structure.4
Human neutrophils store large amounts of four different
α-defensins (Humane Neutrophil Peptide [HNP]-1, -2,
-3 and -4) in their azurophilic granules. These peptides
are released into the phagolysosome where they can kill
ingested micro-organisms or they are secreted into the
extracellular environment. Bronchial and nasal epithelial
cells express one member of the α-defensin subfamily
(human α-defensin 5, HD5;).5 In contrast, all members of
the human β-defensin subfamily identified to date have
been found to be expressed in epithelial cells: hBD-1, -2,
-3, -4, and a splice variant of the human epididymus secretory protein HE2 that encodes a β-defensin consensus
cysteine motif.2−4, 6−8
Cathelicidins are a family of antimicrobial peptides
characterised by a conserved common ‘cathelin-like’ proregion and a C-terminal antimicrobial domain that is
named cathelicidin.2, 4, 6 Whereas various cathelicidins are
present in most mammals, only one human cathelicidin
has been identified. This antimicrobial peptide derives
its name LL-37 from the fact that the two N-terminal
amino acids are leucines and it is composed of 37 amino
acids. The precursor of LL-37, hCAP-18, is produced by
neutrophils, monocytes, NK cells, γδ T cells and B cells,
and by epithelial cells, including those of the bronchial
mucosa.2, 4 Whereas defensins and LL-37 are cationic pep-
307
tides, the airway and alveolar epithelium also produces
anionic antimicrobial peptides.2 Finally, selected members
of the histatin and cystatin polypeptide families display antimicrobial activity but their expression in the respiratory
tract has not yet been intensively studied. Larger polypeptides secreted by airway epithelial cells include lactoferrin
and lysozyme, peptides long recognised as constituents of
various secretions.2, 6
Flemming described the bacteriolytic activity of
lysozyme in nasal secretions 80 years ago. Both lysozyme
and lactoferrin are present in concentrations that are
markedly higher than those observed for the antimicrobial peptides. Another abundant polypeptide present in
airway secretions is secretory leukocyte proteinase inhibitor (SLPI), a protein originally identified as a potent
inhibitor of elastase. Later SLPI was shown also to display
broad-spectrum antimicrobial activity.2, 6 It shares these
properties with elafin, which is present in lower concentrations in airway secretions. Group II phospholipase
A2 is an abundant antimicrobial component in tear fluid
but is also present in airway secretions where it might
contribute to overall antimicrobial activity by degrading
bacterial membranes.6
EXPRESSION
Antimicrobial polypeptides are produced both by surface
epithelial cells and by cells of the submucosal glands. The
relative contribution of these sources and the contribution of the various cell types of the surface epithelium
to polypeptide synthesis are not known. In vitro epithelial
cell culture studies and results from bronchial xenografts
generated from surface epithelial cells, show that the surface epithelium itself may play a major role in the overall synthesis. The expression of antimicrobial polypeptides may either be constitutive, induced or decreased
by a variety of stimuli. Pro-inflammatory cytokines and
bacteria may increase β-defensin expression. From studies in drosophilae, it is likely that pattern-recognition
receptors which recognise pathogen associated molecular patterns (PAMPs) are involved in the stimulatory
effect of micro-organisms on epithelial expression of antimicrobial peptides.9 These receptors, including Toll-like
receptors (TLR), recognise microbial products such as
lipopolysaccharide. Other factors involved in the regulation of β-defensin expression in epithelial cells include
L-isoleucine and its analogues, as demonstrated in bovine
cells.10 It was suggested that isoleucine might serve as a
microbial marker for the presence of bacteria, allowing
the host to mount an adequate response.
Bacteria may also decrease expression of antimicrobial
peptides, as shown by the observation that Shigella spp.
may decrease expression of LL-37 and hBD-1 in intestinal epithelial cells.11 Since, in addition to antimicrobial
peptides and the complement system, phagocytes also
constitute the effector mechanisms of the innate immune
308
system, the observation that neutrophil products such
as neutrophil elastase and defensins modulate SLPI and
elafin production in bronchial epithelial cells suggests another mechanism for fine-tuning the composition of the
antimicrobial screen.12 Epithelial repair may be associated
with increased expression of SLPI in the skin13 and hBD2 in the cornea.14 Thus when the epithelial barrier function is breached, increased expression of antimicrobial
peptides in repairing epithelium may protect the underlying tissue until the protective epithelial barrier is restored. Finally, the observation that glucocorticoids decrease β-defensin expression in bronchial epithelial cells
indicates that anti-inflammatory drugs may alter host defence against infection.15
Whereas transcriptional regulation may be an important mechanism to alter the composition of the antimicrobial soup, post-transcriptional mechanisms may also
be involved. Various antimicrobial polypeptides are produced as proproteins, with a pro-region that blocks the
activity of the mature antimicrobial peptide. Processing
of the pro-protein by proteolytic cleavage is required to
release the active peptide. The importance of this mechanism was illustrated by the observation that mice deficient
in the matrix metalloproteinase matrilysin are not able to
process intestinal prodefensins into the active peptides
and display increased sensitivity to intestinal infections.16
Similar mechanisms may also operate in the lung.
P. S. HIEMSTRA
countermeasures rendering them resistant to the activity
of antimicrobial peptides.19 Therefore, the presence of an
array of polypeptides may be required to prevent colonisation of the respiratory tract by micro-organisms, including those which have evolved resistance mechanisms.
Bacteria do not readily develop resistance against antimicrobial polypeptides in vitro contrary to the development
of resistance against conventional antibiotics. This observation, together with the fact that peptides kill microorganisms which are resistant to conventional antibiotics
has provided a rationale for the development of peptidebased antimicrobial drugs to treat infections.20
The antibacterial activity of antimicrobial polypeptides is the result of an interaction of polypeptides with
bacterial wall components. A major component of the
outer wall of Gram-negative bacteria is lipopolysaccharide (LPS). It is well recognised that LPS displays potent proinflammatory activity by direct interaction with
host cells. Whereas in most cases the outcome of this
interaction of LPS with host cells will be beneficial to
the host, LPS may also cause unwanted inflammatory reactions detrimental to the host. Therefore the capacity of various antimicrobial peptides to inhibit this proinflammatory activity of bacterial components as LPS may
help to shut off the inflammatory reaction initiated by the
infectious process. This property of antimicrobial peptides also makes them attractive for the development of
novel anti-inflammatory treatments.
ANTIMICROBIAL ACTIVITIES
Bacteria, fungi and enveloped viruses are susceptible to
the antimicrobial activity of antimicrobial polypeptides
in vitro. There is also evidence for the in vivo role of antimicrobial peptides as shown in animal models employing over-expression4 or knock-out models, as shown for
matrilysin.16 These peptides kill micro-organisms by disturbing the integrity of microbial membranes by mechanisms such as pore formation, by iron chelation or by
enzymatic disruption of essential microbial target structures. Little is known about the relative contribution
of the various polypeptides to the overall antimicrobial
activity of airway secretions. There are marked differences in the concentration of these components in the
fluid. SLPI may also be a major component in ASF.17
Marked synergistic interactions in the antimicrobial activities of lysozyme, lactoferrin and SLPI have been described,
whereas the activities of hBD-1, hBD-2 and LL-37 were
additive.18 Synergy has also been noted between hBD-4
and lysozyme.7 Antimicrobial polypeptides also display
differences in their activity against various target microorganisms, furthermore, the conditions (e.g. salt concentration and pH) required for optimal activity may differ.
Therefore, synergistic actions, different target cell selectivity and requirements for optimal activity may explain
the need for a wide variety of polypeptides in the ASF.
Another explanation may be that bacteria have developed
ROLE IN INFLAMMATION
AND IMMUNITY
Cell recruitment
Recruitment of leukocytes is an essential element in
the defence against infections. It results in the influx
of phagocytes which ingest and kill the invading microorganisms and includes cells, essential in the initiation
and propagation of a specific immune response. Inflammatory cells may cause tissue injury. Chemoattractants
such as chemokines play a central role in the regulation of cell recruitment. Recent data suggest that not
only chemokines and other chemoattractants but also
α-defensins, β-defensins and LL-37 display chemotactic
activity for various cell types (Fig. 2).21 Interestingly these
peptides were shown to employ host chemoattractant
receptors to mediate this effect. Soon after the characterisation of α-defensins in human neutrophils in the mid1980s chemotactic activity of these peptides for monocytes was described.22 Subsequent studies showed that
neutrophil α-defensins are also chemotactic for immature dendritic cells, for resting CD4 T cells, and for
CD8 T cells.21 The receptor involved in this activity
was not identified but evidence was obtained suggesting it is a chemokine receptor. The direct involvement
of chemokine receptors in signalling by antimicrobial
PEPTIDES AND PROTEINS
309
chemotactic activity, other functions are also likely. Members of the defensin and cathelicidin families and lactoferrin, may have growth-promoting activities which could
play a role in the restoration of epithelial integrity. Epithelial repair is a complex biological process involving
epithelial cell migration, proliferation and differentiation.
Further evidence for a role of antimicrobial polypeptides
in epithelial wound repair comes from a study showing that mice deficient in SLPI have a deficiency in cutaneous wound repair that is associated with increased
inflammation.24 The relative contribution of the antimicrobial, proteinase inhibitory or other activities of SLPI
in wound healing was not studied in detail.
Figure 2 Role of epithelial antimicrobial polypeptides in regulation of the influx of inflammatory and immune cells following microbial challenge. Contact of the epithelium with bacteria
will result in the production of antimicrobial polypeptides and
chemokines causing chemotaxis of antigen-presenting dendritic
cells, monocytes, T cells and neutrophils. Once recruited, neutrophils may cause epithelial injury and further amplify cell recruitment by releasing α-defensins, LL-37 and chemokines. In addition,
peptides such as neutrophil-derived α-defensins will stimulate production of chemokines such as IL-8 resulting in a positive feedback
loop for neutrophil recruitment to the site of infection.
peptides was demonstrated in a study describing that
β-defensins employ the chemokine receptor CCR6 to
attract immature dendritic cells and memory T cells.23
Subsequent studies revealed that selected β-defensins
(hBD-3 and -4) also attract monocytes.7 In contrast to the
defensins, the cathelicidin LL-37 lacks chemotactic activity for dendritic cells but does attract neutrophils, monocytes and T cells. This activity is mediated by another receptor for known chemoattractants, the formyl peptide
receptor-like 1 (FPRL1). Finally, other neutrophil-derived
antimicrobial polypeptides display chemotactic activity for
leukocytes.21 Another mechanism by which antimicrobial
polypeptides may attract leukocytes to the epithelium, is
local induction of chemokine production in the epithelium. Such a mechanism has been demonstrated in vitro
for neutrophil α-defensins. This provides evidence for the
possibility that antimicrobial peptides not only serve to
protect against infection by their antimicrobial activity but
may also regulate the influx of inflammatory cells. These
polypeptides also appear to bridge the innate and adaptive immune systems, by attracting the cellular elements
required for orchestrating an immune response.
Repair
The expression of epithelial antimicrobial polypeptides
may be increased at sites of epithelial injury and repair.
Whereas this may help in the protection of the exposed
tissue against infection by direct antimicrobial activity and
Miscellaneous functions
Antimicrobial polypeptides may display activities distinct
from those described above. α-defensins also regulate
processes such as complement activation, fibrinolysis,
mast cell degranulation and regulation of endogenous cortisol production.3 Neutrophil α-defensins stimulate the
binding of selected bacteria to epithelial cells.25 This may
facilitate bacterial killing as it exposes these bacteria to
high concentrations of epithelial antimicrobial polypeptides or it may allow bacteria to colonise the respiratory
tract. The outcome may depend on local conditions such
as the salt content of the ASF. It is likely that further studies will reveal other functions for these peptides as well.
A recent example of this is the observation that hBD-2
and LL-37 stimulate mast cells to release histamine and
produce prostaglandin D2 .26
CONCLUSIONS
During the last decade, antimicrobial peptides have been
closely studied leading to the discovery of several such
molecules and to the reappraisal of the function of others such as SLPI. Several new functions have now been
ascribed to these components which has led to exciting
new views on the link between innate and adaptive immunity. How can we use this information to understand the
pathogenesis of lung disease and to optimise treatment of
patients? Whereas relatively little is known about the role
of these peptides in disease, it is known from a limited
number of studies that their expression may be increased
in inflammatory lung disease and their activity may be decreased in CF. Modulation of peptide expression in the
lung may offer new treatment strategies. Understanding
the role of Toll-like receptors in the regulation of innate
immunity may be one of the keys towards the development of such innovative strategies. In addition, the peptides themselves or peptides based on the structure of
endogenous antibiotics are being developed and evaluated in clinical trials to treat infections. Aerosol therapy
using antimicrobial polypeptides designed on the basis of
310
structure-function studies of endogenous antibiotics may
become a novel form of topical treatment of respiratory
infections. As new functions of antimicrobial polypeptides
are discovered, we may begin to consider other therapeutic applications for these peptides such as enhancement
of wound repair and modulation of immune responses.
RESEARCH DIRECTIONS
• Evaluation of the expression and activity of
antimicrobial polypeptides in health and disease.
• Studies directed to clarify the primary function
of each molecule in vivo.
• Modulate the expression and activity of
endogenous antimicrobial polypeptides in
patients.
• Evaluation of the clinical efficacy of peptide
antibiotics based on the structure of
endogenous antimicrobial polypeptides.
REFERENCES
1. Smith JJ, Travis SM, Greenberg EP, Welsh MJ. Cystic fibrosis airway
epithelia fail to kill bacteria because of abnormal airway surface fluid.
Cell 1996; 85: 229–236.
2. Travis SM, Singh PK, Welsh MJ. Antimicrobial peptides and proteins
in the innate defense of the airway surface. Curr Opin Immunol 2001;
13: 89–95.
3. van Wetering S, Sterk PJ, Rabe KF, Hiemstra PS. Defensins: key
players or bystanders in infection, injury, and repair in the lung?
J Allergy Clin Immunol 1999; 104: 1131–1138.
4. Bals R. Epithelial antimicrobial peptides in host defense against infection. Resp Res 2000; 1: 141–150.
5. Frye M, Bargon J, Dauletbaev N, Weber A, Wagner TO, Gropp R.
Expression of human alpha-defensin 5 (HD5) mRNA in nasal and
bronchial epithelial cells. J Clin Pathol 2000; 53: 770–773.
6. Ganz T. Antimicrobial proteins and peptides in host defense. Semin
Respir Infect 2001; 16: 4–10.
7. Conejo Garcia JR, Krause A, Schulz S, Rodriguez-Jimenez F-J,
Klüver E, Adermann K, et al. Human β-defensin 4: a novel inducible
peptide with a specific salt-sensitive spectrum of antimicrobial activity. FASEB J 2001;15: 1819–1821.
8. Jia HP, Schutte BC, Schudy A, et al. Discovery of new human betadefensins using a genomics-based approach. Gene 2001; 263: 211–
218.
9. Medzhitov R, Janeway C, Jr. Innate immunity. N Engl J Med 2000;
343: 338–344.
P. S. HIEMSTRA
10. Fehlbaum P, Rao M, Zasloff M, Anderson GM. An essential amino
acid induces epithelial beta-defensin expression. Proc Natl Acad Sci
USA 2000; 97: 12723–12728.
11. Islam D, Bandholtz L, Nilsson J, et al. Downregulation of bactericidal
peptides in enteric infections: a novel immune escape mechanism
with bacterial DNA as a potential regulator. Nat Med 2001; 7: 180–
185.
12. van Wetering S, van der Linden AC, van Sterkenburg MA, Rabe
KF, Schalkwijk J, Hiemstra PS. Regulation of secretory leukocyte
proteinase inhibitor (SLPI) production by human bronchial epithelial
cells: increase of cell-associated SLPI by neutrophil elastase. J Investig
Med 2000; 48: 359–366.
13. Wingens M, van Bergen BH, Hiemstra PS, et al. Induction of SLPI
(ALP/HUSI-I) in epidermal keratinocytes. J Invest Derm 1998; 111:
996–1002.
14. McDermott AM, Redfern RL, Zhang B. Human beta-defensin 2 is
up-regulated during re-epithelialization of the cornea. Curr Eye Res
2001; 22: 64–67.
15. Duits LA, Rademaker M, Ravensbergen B, et al. Inhibition of hBD-3,
but not hBD-1 and hBD-2, mRNA expression by corticosteroids.
Biochem Biophys Res Commun 2001; 280: 522–525.
16. Wilson CL, Ouellette AJ, Satchell DP, et al. Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in
innate host defense. Science 1999; 286: 113–117.
17. Cole AM, Dewan P, Ganz T. Innate antimicrobial activity of nasal
secretions. Infect Immun 1999; 67: 3267–3275.
18. Singh PK, Tack BF, McCray PB, Jr., Welsh MJ. Synergistic and additive
killing by antimicrobial factors found in human airway surface liquid.
Am J Physiol Lung Cell Mol Physiol 2000; 279: L799–L805.
19. Ganz T. Fatal attraction evaded. How pathogenic bacteria resist
cationic polypeptides. JEM 2001; 193: F31–F34.
20. Hancock REW. Peptide antibiotics. Lancet 1997; 349: 418–422.
21. Yang D, Chertov O, Oppenheim JJ. Participation of mammalian defensins and cathelicidins in anti-microbial immunity: receptors and
activities of human defensins and cathelicidin (LL-37). J Leukoc Biol
2001; 69: 691–697.
22. Territo MC, Ganz T, Selsted ME, Lehrer R. Monocyte-chemotactic
activity of defensins from human neutrophils. J Clin Invest 1989; 84:
2017–2020.
23. Yang D, Chertov O, Bykovskaia SN, et al. Beta-defensins: linking
innate and adaptive immunity through dendritic and T cell CCR6.
Science 1999; 286: 525–528.
24. Ashcroft GS, Lei K, Jin W, et al. Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal
wound healing. Nat Med 2000; 6: 1147–1153.
25. Gorter AD, Hiemstra PS, de Bentzmann S, van Wetering S, Dankert
J, van Alphen L. Stimulation of bacterial adherence by neutrophil
defensins varies among bacterial species but not among host cell
types. FEMS Immunol Med Microbiol 2000; 28: 105–111.
26. Niyonsaba F, Someya A, Hirata M, Ogawa H, Nagaoka I. Evaluation
of the effects of peptide antibiotics human beta-defensins- 1/-2 and
LL-37 on histamine release and prostaglandin D(2) production from
mast cells. Eur J Immunol 2001; 31: 1066–1075.