89

Antimicrobial peptides and proteins in the innate defense of the

airway surface
Sue M Travis, Pradeep K Singh and Michael J Welsh
Recent studies have advanced our understanding of innate airways are usually kept sterile. This protection results from
immune mechanisms that protect the airways and maintain a an elaborate and redundant defense network that includes
sterile lung. Multiple antimicrobial peptides and proteins have components of both the innate and acquired immune sys-
been identified in airway secretions and their roles are tems.
beginning to be established in animal models. Moreover,
evidence for coupling between the innate and adaptive When bacteria enter the airway, they encounter the innate
immune systems is beginning to emerge. The understanding of immune system (Figure 1). Mucus acts as a physical barri-
the innate airway defense system offers the opportunity for the er and binds organisms for subsequent removal via
development of novel therapeutic approaches. mucociliary clearance and cough [1]. Below the mucus lies
a thin layer of airway surface liquid (ASL) containing
Addresses antimicrobial factors that kill bacteria. In addition, roaming
Howard Hughes Medical Institute, Departments of Internal Medicine macrophages engulf and destroy bacteria. In so doing, they
and Physiology, and Biophysics, University of Iowa College of and the airway epithelia release cytokines, recruiting neu-
Medicine, 500 Eckstein Medical Research Building, Iowa City,
IA 52242, USA
trophils and other circulating cells [2]. Surfactant protein A
Correspondence: Michael J Welsh; (SP-A) and SP-D, which arise from alveoli, bind to bacte-
e-mail: mjwelsh@blue.weeg.uiowa.edu ria and fungi, further enhancing clearance by neutrophils
and macrophages [3]. When organisms are not rapidly dis-
Current Opinion in Immunology 2001, 13:89–95
posed of, they may activate receptors such as the Toll
0952-7915/01/$ — see front matter receptor on macrophages, dendritic cells and airway
© 2001 Elsevier Science Ltd. All rights reserved. epithelia, thus priming the acquired immune system [4,5].
Abbreviations
ASL airway surface liquid Table 1 contrasts the innate immune system with the more
BAL bronchoalveolar lavage widely appreciated acquired immune system. The evolu-
CF cystic fibrosis
CFTR CF transmembrane conductance regulator
tionarily ancient innate immune system does not require
HBD-1 human β-defensin 1 previous exposure to pathogens or immunologic memory
LPS lipopolysaccharide although expression of some components increases during
SLPI secretory leukoproteinase inhibitor bacterial challenge. The antibacterial peptides and proteins
SP-A surfactant protein A
of the innate immune system are relatively nonspecific and
rapidly kill invading microbes. In contrast, B and T cells
Introduction proliferate and fine-tune the specificity of their response
Throughout the day and night, our airways are exposed to after pathogen exposure; thus, unlike innate responses,
bacteria by inhalation and aspiration. Yet, despite this, the adaptive responses are improved upon re-exposure. The

Figure 1

Mechanisms of innate defense of the airways.

Airway Mucociliary clearance
Bacteria that enter the airway are cleared by a
and cough
combination of defense mechanisms: mucus
acts as a physical barrier and binds organisms Macrophage
for removal by mucociliary clearance and Mucus
cough; below mucus is ASL that contains
antimicrobial factors (from submucosal gland Antimicrobial factors
cells, epithelial cells, neutrophils and other
cells); roaming macrophages can engulf and
Epithelium
destroy bacteria; and macrophages and
epithelia can release cytokines that attract
neutrophils and other cells. If invading
organisms are not rapidly removed, they may
Adaptive
prime the adaptive immune system.
immune
system
Neutrophil
Submucosal
gland
Current Opinion in Immunology
90 Innate immunity

Table 1 a first line of defense against constant exposure to low

levels of bacteria.
Comparison of innate and acquired immunity..

Property Innate immunity Adaptive immunity Many antimicrobial factors have been identified in airway
secretions but the concentrations of factors differ substan-
Agents Peptides and proteins Antibodies and T cells tially (Table 2). Published concentrations should be
Expression Constitutive and inducible Inducible considered rough estimates because of difficulties in col-
Onset of effect Within minutes Within days lecting the minute amounts of ASL, uncertainties in
determining dilution factors, and alterations induced by the
Most effective Small numbers of bacteria Large numbers of
target numbers bacteria
collection procedure itself. In addition, liquid collected from
different parts of the airway could show different concentra-
Targets Broad-spectrum Specific tions of a factor. Nevertheless, the proteins, lysozyme,
Spectrum Fixed Variable lactoferrin and secretory leukoproteinase inhibitor (SLPI)
are by far the most abundant factors. Thus, we speculate
that these may be the most critical to lung defense.
antimicrobial factors in ASL are designed to control small
numbers of bacteria whereas the adaptive immune system is Antimicrobial spectrum
tailored to handle large numbers of bacteria and established The contribution of an individual factor depends in part on
infections. However, the adaptive immune response can its ability to kill the specific bacteria it encounters. For
take days and often produces inflammatory side-effects that example, LL-37 is more potent than HNP1 against a vari-
may damage the airways. Maintenance of healthy airways ety of bacteria including Pseudomonas aeruginosa and
requires both systems. Here, we briefly review recent work Stenotrophomonas maltophilia [6] and HBD-2 is more potent
on a key component of the innate defense system, airway than HBD-1 against P. aeruginosa and Escherichia coli [7]. In
antimicrobial peptides and proteins. addition, killing may depend on bacterial growth phase.
For example, bacteria are more susceptible to secretory
Antimicrobial factors in airway surface liquid phospholipase A2 when they are actively dividing [8•].
Concentration and location These differences in specificity underscore the need for
ASL contains a rich diversity of antimicrobial proteins multiple antimicrobial factors in ASL.
and peptides; Table 2 shows several. Submucosal gland
serous cells produce the bulk of these factors, with airway The environment of airway surface liquid
epithelia and neutrophils contributing to the mixture. The environment of ASL probably exerts a major influ-
The antibacterial factors present in the airway include ence on the activity of antimicrobial factors. For example,
proteins, such as lysozyme and lactoferrin, and peptides, the activity of many ASL antimicrobial factors is dimin-
such as human β-defensin 1 (HBD-1), HBD-2, LL-37 (a ished in high salt concentrations [9–11,12••]. Such
cathelicidin), HNPs (human neutrophil peptides) and concentrations also disrupt the synergistic activity of
acidic peptides as well as other small molecules. lysozyme, lactoferrin and SLPI [12••]. In addition, some
Antimicrobial factors are not unique to ASL; they defend divalent cations strongly inhibit antibacterial factors [6,10];
the body wherever there is contact with the external killing activity is often most effective near neutral pH [13].
environment, including the gastrointestinal tract, the uro- Unfortunately, at present, absolute ion concentrations and
genital tract, eyes, mouth and skin. These factors provide pH in proximal and distal airways remain uncertain (see

Table 2

Antimicrobial factors in ASL.

Factor Sources Estimated concentration in human ASL

Lysozyme Submucosal glands, neutrophils, macrophages 250–500 µg/ml in nasal secretions [14••], 7 µg/ml in BAL fluid [50]
Lactoferrin Submucosal glands, neutrophils 80–200 µg/ml in nasal secretions [14••], 12 µg/ml in BAL fluid [50]
SLPI Submucosal glands, epithelia, neutrophils 10–80 µg/ml in nasal secretions [14••], 0.1 µg/ml in BAL fluid [51,52]
Secretory Neutrophils, epithelia 0.6 µg/ml in nasal secretions [53]
phospholipase A2
HBD-1 Neutrophils, epithelia ≤2 ng/ml in BAL fluid [7]
HBD-2 Neutrophils, epithelia 0.3–4.0 µg/ml in nasal secretions [14••], >0.1 ng/ml in BAL fluid [7]
Anionic peptides Epithelia 1.1 µg/ml in BAL fluid [54]
Cathelicidin LL-37 Neutrophils, epithelia Unknown
 Antimicrobials in innate defense of the airways Travis, Singh and Welsh
below). Two other ASL components, mucus and elastase,
do not inhibit activity of the abundant antimicrobial factors
[10]. However ASL is a complex mixture of proteins, lipids
and carbohydrates, and we have much to learn about how
its components might influence airway defense.
Antimicrobial factors in airway surface liquid
are part of an integrated defense system
Although most studies of ASL antimicrobial factors have
focused on their individual action, it is apparent that ASL
factors function together and with other components of the
innate and adaptive immune system. These integrated
actions are likely to increase antimicrobial potency and
provide mechanisms to recruit additional components of
the immune system if initial innate defenses are inade-
quate. Empirical support for the importance of multiple
antimicrobial factors comes from studies of nasal secretions
[14••]; antimicrobial activity in human nasal secretions was
reduced by boiling (among other effects, this removes
lysozyme and lactoferrin) and it could not be entirely
reconstituted by adding back the most abundant antibac-
terial proteins, lysozyme and lactoferrin. This result
suggests that the antimicrobial potency of nasal secretions
may depend upon multiple factors and their interactions.
The combined action of antimicrobial factors
Antimicrobial factors can work additively, antagonistically
(i.e. their combined activity is less than the sum of their
individual activities) or synergistically (i.e. combined activ-
ity is greater than the sum of individual activities). The
time-kill method evaluates combined antimicrobial action
by measuring the effect of a subinhibitory concentration
(i.e. less than that able to inhibit/kill bacteria) of one agent
on the killing ability of another over time (Figure 2). Using
this test, it was shown that three factors, lactoferrin, SLPI
and LL-37, had synergistic activity with lysozyme [12••].
Synergistic combinations were not only more potent but
also increased the rate of bacterial killing. This may lessen
the time available for bacteria to multiply and develop
more resistant phenotypes, such as those associated with
biofilm formation. The mechanisms responsible for syner-
gism are not known; however, factors with complementary
mechanisms may be more likely to act synergistically. For
example, lysozyme hydrolyzes the peptidoglycan layer
whereas lactoferrin deprives bacteria of iron and causes
lipopolysaccharide (LPS) release from the outer mem-
brane [15]. Synergy may result from lactoferrin damaging
LPS in the bacterial outer membrane, making the pepti-
doglycan layer more accessible to lysozyme. In contrast,
the activities of HBD-1 and LL-37 are simply additive
[12••]. Although their exact mechanisms are not entirely
clear, both peptides disrupt the bacterial membrane and
two factors that attack the same bacterial target may not
have more activity in combination.
The fact that ASL factors have multiple antimicrobial mech-
anisms may contribute significantly to the enhanced activity
of some of these factors in combination. The multiplicity of
91
Figure 2
100 Lactoferrin
Bacterial survival
(% control)
L.U. % Control
50 Lysozyme
Lysozyme plus
lactoferrin
0
0 2 4
Time (hours)
Antimicrobial action of lysozyme and lactoferrin measured by the time-
kill method. The graph shows the effect of combining lysozyme (at a
concentration that kills approximately 50% of the bacteria after 4 hours
when used alone) with a subinhibitory concentration of lactoferrin.
Open triangles show antimicrobial activity of lysozyme alone; open
squares show lactoferrin alone; filled circles show the combined effect
of lysozyme and lactoferrin. Reproduced, with permission, from [12••].
mechanisms may also increase the spectrum of organisms
susceptible to these factors and may prevent the emergence
of strains resistant to a given antimicrobial target. In vivo, the
presence of multiple factors may increase potency further.
For example, secreted phospholipase A2 killing activity
appears to be due to phospholipid degradation [8•]. It will be
interesting to learn if this activity is synergistic with other air-
way antimicrobial factors.
Constitutive and inducible production of antimicrobial
factors
Some ASL antimicrobial factors are constitutively
expressed, providing an ever-present defense, whereas oth-
ers show little constitutive expression but then increase
rapidly with infectious stimuli [16]. The β-defensins pro-
vide an example of both constitutive and inducible
expression [17]: in airway epithelia, HBD-1 is constitutive-
ly produced and inflammatory stimuli have little apparent
effect on its production [7]; in contrast, HBD-2 expression
is dramatically increased by the proinflammatory cytokine
IL-1β and, in some systems by TNF-α, mucoid P. aerugi-
nosa and bacterial LPS [7,18•,19•]. SLPI expression is also
regulated by inflammatory stimuli and SLPI secretion is
increased by neutrophil defensins [20•]. Antibacterial activ-
ity is also increased in bronchoalveolar lavage (BAL) fluid
from individuals with pulmonary inflammatory diseases
such as sarcoidosis [21•]. The increase in antibacterial activ-
ity may explain why these individuals rarely develop severe
airway infections despite abnormal pulmonary function.
A role for macrophages in regulating defensin production was
suggested in a study that examined production of HBD-2
[22]. Production of this factor by differentiated human airway
epithelia increased 10-fold with cytokine stimulation but
heat-killed bacteria and LPS had no effect in this system.
However, when LPS was added to the surface of the epithe-
lia in the presence of human pulmonary macrophages,
92 Innate immunity
HBD-2 mRNA increased 150-fold. These data suggest that,
like other components of the airway immune response, pro-
duction of epithelium-derived defense peptides depends in
part on interactions with immune cells.
It is interesting to speculate about the role inducible fac-
tors may play in lung defense. They would presumably not
be present to kill bacteria when an organism first enters the
airway. Instead, they may be needed to help quell an
emerging infection, acting in concert with phagocytes and
the adaptive immune system.
Defensins link innate to adaptive immunity
The signaling capabilities of the antimicrobials HBD-1
and HBD-2 were recently demonstrated in a study that
linked the innate and adaptive immune responses. At sub-
micromolar concentrations, HBD-1 and HBD-2 were
chemotactic for immature dendritic cells and memory
T cells [23••]. It appears that the defensins stimulated
these cells via the CCR6 chemokine receptor. This
intriguing finding suggests a mechanism by which the
defensins could promote the development of adaptive
immunity at the site of infection. This work also provides
an additional function for the defensins, which may be pre-
sent at very low concentrations that are not bactericidal on
their own. However, it seems surprising that the constitu-
tively expressed HBD-1 and the inducible HBD-2 were
both chemoattractants with relatively similar potencies.
One might have predicted that the constitutively produced
HBD-1 would not activate chemotaxis whereas HBD-2
would be the better signaling molecule for this response.
Perhaps this puzzle could be explained if HBD-1 is secret-
ed exclusively onto the apical surface — then only with
injury and disruption of epithelial integrity would it reach
submucosal dendritic and T cells. Further work in this
interesting area is clearly warranted.
In vivo studies of antimicrobial factors
Nearly all studies of ASL antimicrobial peptides have been
done using in vitro assays. This, plus the plethora of factors
in ASL, raises the question of which factors are important to
airway defense in vivo. Presumably all are important but evi-
dence to support this assumption is minimal. One way to
study the importance of individual factors would be to gen-
erate mice with disruptions in the genes encoding various
airway antimicrobial factors. We expect that reports of such
experiments will soon emerge. However, there may be sig-
nificant obstacles inherent in this approach. Because there
are so many factors, it would not be surprising if deleting a
single factor had little discernable consequence. It may be
necessary to disrupt multiple genes in a single animal to see
an effect. In addition, the applicability of the results to
humans may not be straightforward, given the variation in
ASL factors between species and differences in the
pathogens that infect various species. As an example of the
potential difficulties, mice do not make neutrophil defensins
and therefore would not provide a good model for some
human antimicrobial factors.
Although knockout animals deficient in specific airway
antimicrobial factors have not been reported, there have
been complementary approaches. For example, lysozyme
was overexpressed in mice under control of a lung-specific
promoter [24•]. When bacteria were administered intratra-
cheally, the rate of clearance of bacteria was increased,
there was less systemic spread of bacteria and survival of
mice improved in the transgenics, relative to control mice.
Another study examined the role of overexpressed LL-37.
Overexpression of LL-37 in mouse airway reduced the
TNF-α response to bacterial infection (which may reduce
harmful effects of inflammation); systemic expression of
LL-37 increased mouse survival after exposure to bacteria
or LPS [25•]. A role for SP-A was shown in studies of SP-A
knockout mice [26]. These mice showed defective uptake
of bacteria by macrophages and showed decreased bacteri-
al killing. A recent study of sheep airway defense
demonstrated a role for lactoperoxidase, an enzyme that
generates the biocide, hypothiocyanite [27•]. In vivo inhibi-
tion of lactoperoxidase slowed the normally rapid
pulmonary clearance of aerosolized Pasteurella haemolytica, a
sheep pathogen. Moreover, the clearance rate was normal-
ized when exogenous lactoperoxidase was administered
prior to bacterial challenge.
Recent studies of intestinal innate defense demonstrate
regulation of defensin production in vivo. In the intestine,
Paneth cells secrete both an α-defensin (cryptidin) precur-
sor and matrilysin, the protease that cleaves the precursor
to generate activate cryptidin peptide (Figure 3).
Matrilysin deficient mice failed to generate mature cryp-
tidin [28••]. As a result, orally administered bacteria
survived in greater numbers and mouse survival fell.
Interestingly, matrilysin and cryptidin production were
regulated in Paneth cells; addition of E. coli or bacterial
factors stimulated matrilysin and cryptidin production
[29••,30•]. It seems likely that this theme of multiple reg-
ulatory steps will be repeated at a number of sites where
defensin function is important.
Abnormalities of antimicrobial killing in disease
A genetic disruption of the antimicrobial defense system
has been proposed to account for chronic airway infections
in cystic fibrosis (CF) [31,32]. Airway infections begin early
in the course of disease with many different organisms, and
then with time P. aeruginosa and Staphylococcus aureus pre-
dominate. In CF, the loss of the CFTR (CF transmembrane
conductance regulator) Cl– channel may cause a higher
ASL salt concentration that reduces antimicrobial potency,
thereby impairing the innate immune system and predis-
posing airways to infection [31,32]. Using models of the
airway, several studies have shown that steady-state NaCl
concentrations in ASL are lower than those in serum, and
ASL NaCl concentrations are abnormally elevated in CF
[32,33]. However, uncertainty persists as to whether this is
the case in vivo in humans [34,35] because it is difficult to
collect and analyze the small amount of ASL without intro-
ducing a number of artifacts [36].

Figure 3
Cryptidin activation by matrilysin in Paneth
cells. (a) The cryptidin precursor is activated
when cleaved by the protease, matrilysin;
activated cryptidin has antimicrobial activity.
Mice deficient in matrilysin do not produce
mature cryptidin peptide and they are more
susceptible to bacterial infections [28••].
Expression of both (b) matrilysin and
(c) cryptidin precursor is regulated by (+)
exposure to bacteria [29••,30•]. Cryptidin
precursor
Matrilysin
(+)
Unfortunately, this issue has also been difficult to evaluate
in mice; animals with a disrupted CFTR gene do not
develop CF-like lung disease, and the ion transport prop-
erties and ASL salt concentrations differ from those in
humans. Moreover the loss of CFTR Cl– channels in the
intestinal epithelium and the resulting intestinal disease
and malnutrition may affect the airways; other mouse mod-
els have shown that both malnutrition and intestinal
inflammation compromise lung defenses [37•].
Might loss of ASL antimicrobial factors disrupt airway
defense? Interestingly, a study of nasal secretions found
that antimicrobial activity varies among individuals [14••].
Some individuals were naturally colonized with S. aureus
and their nasal secretions showed a reduction in the abili-
ty to kill S. aureus in vitro. We speculate that genetic
variability in the production of ASL antimicrobial factors
may predispose some individuals to airway infections.
Because of the redundancy in this system, the propensity
may be subtle, only observed with some organisms or only
manifest in the presence of another injury to the airways.
Opportunities for new therapeutic approaches
in the airways
The rapid emergence of bacterial strains resistant to conven-
tional antibiotics has hastened the search for new
antimicrobial agents that could be used in the airways and
elsewhere. Antimicrobial peptides are promising candidates:
they kill a broad spectrum of microbes, they kill rapidly, they
are relatively nonimmunogenic and resistant bacteria are
uncommon [38]. Potential therapeutic peptides could be
based on the defensins, cathelicidin-derived peptides or other
known antimicrobial peptides from a variety of organisms
[38–41,42•,43•]. Importantly, some naturally occurring pep-
tides are salt-insensitive; an example is porcine protegrins
[44]. Such peptides make particularly attractive candidates for
pharmaceutical development. In addition, understanding the
mechanisms by which peptides kill bacteria and the rules that
Antimicrobials in innate defense of the airways Travis, Singh and Welsh 93
Intestinal
Paneth cells
(c)
Cryptidin Mature
precursor cryptidin
Bacterium
(a)
Matrilysin
(b)
Current Opinion in Immunology
govern potency may provide guidance in drug design. A
recent study of linear antimicrobial peptides suggested that a
hydrophobicity gradient in the amphipathic α helix was asso-
ciated with increased potency [45]. However, this hypothesis
needs to be tested directly with appropriately designed pep-
tides. These and other observations are an important starting
point for future drug development.
The salt-sensitivity of most ASL antimicrobials suggests
another strategy to enhance airway defenses. It may be
possible to increase activity of endogenous antimicrobial
factors by lowering the ASL salt concentration with a non-
ionic osmolyte such as xylitol. This osmolyte has a low
transepithelial permeability, is poorly metabolized by sev-
eral pathogenic bacteria and, when applied to the apical
surface, lowers the ASL salt concentration in vitro [46•].
When sprayed onto the nasal mucosa, it also reduced the
number of nasal coagulase-negative Staphylococci. Although
an osmolyte such as xylitol might be of value in preventing
airway infections, it is not likely to relieve established
infections because endogenous antimicrobial factors are
more effective against small numbers of bacteria. Potential
applications of this strategy may include prevention of air-
way infections in patients whose airway defenses are
compromised by mechanical ventilation, by other injury or
by CF. In regard to CF, aerosolized hypertonic saline and
mannitol have been used to stimulate cough and clearance
of mucus [47,48]; perhaps hypertonic xylitol would serve a
similar purpose and also enhance bacterial killing.
Conclusions and future prospects
It has been almost 80 years since Alexander Fleming
described the bactericidal activity of lysozyme in airway
secretions [49]. The renewed interest in this area now
poses several questions and presents several opportunities.
We do not know all the components of ASL and how they
work in concert. ASL is a complex soup; we need to know
what the ‘meat and potatoes’ are and how the various
94 Innate immunity
‘spices’ interact and enhance the mix. We must also under-
stand how ASL components interact with epithelial,
subepithelial and inflammatory cells.
We wonder if ASL defenses can be enhanced to prevent and
treat pulmonary infections. A better understanding of ASL
antimicrobial factors and their mechanisms of action should
help in the development of new therapeutics. When the
innate defenses are breached by a genetic defect, by a tran-
sient, acquired defect or by an exceptionally large challenge,
the ensuing infection and inflammation can lead to chronic
lung disease — CF is a good example. However, infection may
be involved to a greater extent than is currently appreciated in
the pathogenesis of many other chronic lung diseases marked
by inflammation. A better understanding of the relationship
between the innate immune system and the acquired immune
system and inflammation may yield insights that will provide
new ways to treat or prevent lung disease.
References and recommended reading
Papers of particular interest, published within the annual period of review,
have been highlighted as:
• of special interest
•• of outstanding interest
1. Widdicombe J: Relationships among the composition of mucus,
epithelial lining liquid, and adhesion of microorganisms. Am J
Respir Crit Care Med 1995, 151:2088-2093.
2. Zhang P, Summer WR, Bagby GJ, Nelson S: Innate immunity and
pulmonary host defense. Immunol Rev 2000, 173:39-51.
3. Crouch E, Hartshorn K, Ofek I: Collectins and pulmonary innate
immunity. Immunol Rev 2000, 173:52-65.
4. Imler J-L, Hoffmann JA: Signaling mechanisms in the antimicrobial
host defense of Drosophila. Curr Opin Microbiol 2000, 3:16-22.
5. Medzhitov R, Janeway C Jr: Innate immune recognition:
mechanisms and pathways. Immunol Rev 2000, 173:89-97.
6. Turner J, Cho Y, Dinh NN, Waring AJ, Lehrer RI: Activities of LL-37, a
cathelin-associated antimicrobial peptide of human neutrophils.
Antimicrob Agents Chemother 1998, 42:2206-2214.
7. Singh PK, Jia HP, Wiles K, Hesselberth J, Liu L, Conway BD,
Greenberg EP, Valore EV, Welsh MJ, Ganz T et al.: Production of
β-defensins by human airway epithelia. Proc Natl Acad Sci USA
1998, 95:14961-14966.
8. Foreman-Wykert AK, Weinrauch Y, Elsbach P, Weiss J: Cell-wall
• determinants of the bactericidal action of group IIA
phospholipase A2 against Gram-positive bacteria. J Clin Invest
1999, 103:715-721.
This study describes the effects of bacterial growth stage, antibiotics and
autolysins on the activity of phospholipase A2. The results suggest that
areas of the bacterial envelope that are actively growing are the target for
phospholipase A2.
9. Bals R, Wang X, Wu Z, Freeman T, Bafna V, Zasloff M, Wilson JM:
Human β-defensin 2 is a salt-sensitive peptide antibiotic
expressed in human lung. J Clin Invest 1998, 102:874-880.
10. Travis SM, Conway BD, Zabner J, Smith JJ, Anderson NN, Singh PK,
Greenberg EP, Welsh MJ: Activity of abundant antimicrobials of the
human airway. Am J Respir Cell Mol Biol 1999, 20:872-879.
11. Kalfa VC, Brogden KA: Anionic antimicrobial peptide-lysozyme
interactions in innate pulmonary immunity. Int J Antimicrob Agents
1999, 13:47-51.
12. 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 2000, 279:L799-L805.
This comprehensive report shows that lysozyme is synergistic in combination
with lactoferrin or secretory leukoproteinase inhibitor (SLPI), and the triple
combination is even more synergistic. This synergy was lost in the presence
of high salt concentrations.
13. Lehrer RI, Selsted ME, Szklarek D, Fleischmann J: Antibacterial
activity of microbicidal cationic proteins 1 and 2, natural peptide
antibiotics of rabbit lung macrophages. Infect Immun 1983,
42:10-14.
14. Cole AM, Dewan P, Ganz T: Innate antimicrobial activity of nasal
•• secretions. Infect Immun 1999, 67:3267-3275.
This careful study shows that antimicrobial activity of nasal secretions varies
among donors, and some donor secretions are unable to kill
Staphylococcus aureus. This is one of few studies that quantitates the lev-
els of antimicrobial factors in airway secretions.
15. Ellison RT III, Giehl TJ, LaForce FM: Damage of the outer membrane
of enteric Gram-negative bacteria by lactoferrin and transferrin.
Infect Immun 1988, 56:2774-2781.
16. Diamond G, Legarda D, Ryan LK: The innate immune response of
the respiratory epithelium. Immunol Rev 2000, 173:27-38.
17. Diamond G, Bevins CL: ß-defensins: endogenous antibiotics of the
innate host defense response. Clin Immunol Immunopath 1998,
88:221-225.
18. Harder J, Meyer-Hoffert U, Teran LM, Schwichtenberg L, Bartels J,
• Maune S, Schröder JM: Mucoid Pseudomonas aeruginosa, TNF-α α,
and IL-1ββ, but not IL-6, induce human β-defensin-2 in respiratory
epithelia. Am J Respir Cell Mol Biol 2000, 22:714-721.
In this thorough study of HBD-2 expression, the authors purified HBD-2
from airway epithelial cells, and showed that HBD-2 expression can be
increased by bacteria or cytokines.
19. Becker MN, Diamond G, Verghese MW, Randell SH: CD14
• dependent LPS-induced β-defensin-2 expression in human
tracheobronchial epithelium. J Biol Chem 2000, 275:29731-29736.
The authors show that lipopolysaccharide (LPS) increases HBD-2 expres-
sion in tracheobronchial epithelial cells. LPS induction of HBD-2 requires
the LPS co-receptor CD14. This report provides new information about the
pathways of LPS recognition.
20. van Wetering S, van der Linden AC, van Sterkenburg MAJA,
• de Boer WI, Kuijpers ALA, Schalkwijk J, Hiemstra PS: Regulation of
SLPI and elafin release from bronchial epithelial cells by
neutrophil defensins. Am J Physiol Lung Cell Mol Physiol 2000,
278:L51-L58.
The authors show that neutrophil defensins stimulate secretory leukopro-
teinase inhibitor (SLPI) protein release from airway cells, without affecting
SLPI mRNA levels. This suggests a new role for defensins in the airway.
21. Agerberth B, Grunewald J, Castanos-Velez E, Olsson B, Jörnvall H,
• Wigzell H, Eklund A, Gudmundsson GH: Antibacterial components
in bronchoalveolar lavage fluid from healthy individuals and
sarcoidosis patients. Am J Respir Crit Care Med 1999,
160:283-290.
This report shows that BAL fluid from people with sarcoidosis has higher
antimicrobial activity than BAL fluid from healthy individuals. This might
explain the relatively low frequency of bacterial infections in this disease.
22. Singh PK, Kline JN, Jia HP, Ganz T, Welsh MJ, Tack BF, McCray PB Jr:
Macrophages mediate HBD-2 production by airway epithelia
[abstract]. Am J Respir Crit Care Med 1999, 159:A18.
23. Yang D, Chertov O, Bykovskaia SN, Chen Q, Buffo MJ, Shogan J,
•• Anderson M, Schröder JM, Wang JM, Howard OMZ, Oppenheim JJ:
β-defensins: linking innate and adaptive immunity through
dendritic and T cell CCR6. Science 1999, 286:525-528.
This reports the novel finding that human β-defensins (HBDs) are chemotac-
tic for dendritic cells and memory T cells. HBDs act through the chemokine
receptor CCR6 and may function to recruit cells to sites of infection.
24. Akinbi HT, Epaud R, Bhatt H, Weaver TE: Bacterial killing is
• enhanced by overexpression of lysozyme in the lungs of
transgenic mice. J Immunol 2000, 165:5760-5765.
This study demonstrates that increased lysozyme expression in mouse air-
ways can improve bacterial clearance in vivo. This is the first demonstration
of a role for lysozyme in vivo.
25. Bals R, Weiner DJ, Moscioni AD, Meegalla RL, Wilson JM:
• Augmentation of innate host defense by expression of a
cathelicidin antimicrobial peptide. Infect Immun 1999,
67:6084-6089.
This interesting paper reports that overexpression of LL-37 in airway protects
mice from bacterial infection. In addition, systemic expression protected the
mice from sepsis.
26. LeVine AM, Whitsett JA, Gwozdz JA, Richardson TR, Fisher JH,
Burhans MS, Korfhagen TR: Distinct effects of surfactant protein A
or D deficiency during bacterial infection on the lung. J Immunol
2000, 165:3934-3940.
 Antimicrobials in innate defense of the airways Travis, Singh and Welsh
27. Gerson C, Sabater J, Scuri M, Torbati A, Coffey R, Abraham JW,
• Lauredo I, Forteza R, Wanner A, Salathe M et al.: The
lactoperoxidase system functions in bacterial clearance of
airways. Am J Respir Cell Mol Biol 2000, 22:665-671.
This paper shows that lactoperoxidase is an important contributor to innate
defense in sheep airways. In vivo inhibition of airway lactoperoxidase
impaired clearance of Pasteurella haemolytica.
28. Wilson CL, Ouellette AJ, Satchell DP, Ayabe T, López-Boado YS,
•• Stratman JL, Hultgren SJ, Matrisian LM, Parks WC: Regulation of
intestinal α-defensin activation by the metalloproteinase
matrilysin in innate host defense. Science 1999, 286:113-117.
This important study demonstrates the role of defensins in the mouse intes-
tine in vivo. The authors generated a mouse strain that was deficient in
matrilysin, the protease that activates intestinal defensins. These mice lack
mature defensin peptides, and are less able to clear bacteria and survive
intestinal infection.
29. López-Boado YS, Wilson CL, Hooper LV, Gordon JI, Hultgren SJ,
•• Parks WC: Bacterial exposure induces and activates matrilysin in
mucosal epithelial cells. J Cell Biol 2000, 148:1305-1315.
This important paper reports that bacteria stimulate matrilysin production by
epithelial cells, including airway epithelial cells. Bacterial stimulation also
increased the bactericidal activity released from epithelial cells.
30. Ayabe T, Satchell DP, Wilson CL, Parks WC, Selsted ME,
• Ouellette AJ: Secretion of microbicidal α-defensins by intestinal
Paneth cells in response to bacteria. Nat Immunol 2000, 1:113-118.
This interesting study shows that bacteria, and bacterial products, stimulate
secretion of defensins from intestinal crypts. Significantly, the authors estimated
that the defensins in the crypt lumen are present at bactericidal concentrations.
31. 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.
32. Goldman MJ, Anderson GM, Stolzenberg ED, Kari UP, Zasloff M,
Wilson JM: Human ß-defensin-1 is a salt-sensitive antibiotic in
lung that is inactivated in cystic fibrosis. Cell 1997, 88:553-560.
33. Zabner J, Smith JJ, Karp PH, Widdicombe JH, Welsh MJ: Loss of
CFTR chloride channels alters salt absorption by cystic fibrosis
airway epithelia in vitro. Mol Cell 1998, 2:397-403.
34. Joris L, Dab I, Quinton PM: Elemental composition of human
airway surface fluid in healthy and diseased airways. Am Rev
Respir Dis 1993, 148:1633-1637.
35. Knowles MR, Robinson JM, Wood RE, Pue CA, Mentz WM, Wager GC,
Gatzy JT, Boucher RC: Ion composition of airway surface liquid of
patients with cystic fibrosis as compared with normal and disease-
control subjects. J Clin Invest 1997, 100:2588-2595.
36. Erjefält I, Persson CGA: On the use of absorbing discs to sample
mucosal surface liquids. Clin Exp Allergy 1990, 20:193-197.
37. Yu H, Nasr SZ, Deretic V: Innate lung defenses and compromised
• Pseudomonas aeruginosa clearance in the malnourished mouse
model of respiratory infections in cystic fibrosis. Infect Immun
2000, 68:2142-2147.
This interesting study shows that mice lacking cystic fibrosis transmembrane
conductance regulator (CFTR) are malnourished and that these malnour-
ished mice have compromised innate lung defenses.
38. Hancock REW, Lehrer R: Cationic peptides: a new source of
antibiotics. Trends Biotechnol 1998, 16:82-88.
39. Boman HG: Gene-encoded peptide antibiotics and the concept of
innate immunity: an update review. Scand J Immunol 1998,
48:15-25.
40. Huttner KM, Bevins CL: Antimicrobial peptides as mediators of
epithelial host defense. Ped Res 1999, 45:785-794.
95
41. Lehrer RI, Ganz T: Antimicrobial peptides in mammalian and insect
host defence. Curr Opin Immunol 1999, 11:23-27.
42. Porter EA, Wang X, Lee HS, Weisblum B, Gellman SH: Non
• haemolytic beta-amino-acid oligomers. Nature 2000, 404:565.
The authors designed and synthesized a new peptide using β amino acids.
This peptide is microbicidal and nonhemolytic, and may provide a basis for
new antimicrobial peptides.
43. Yu Q, Lehrer RI, Tam JP: Engineered salt-insensitive alpha
• defensins with end-to-end circularized structures. J Biol Chem
2000, 275:3943-3949.
The authors synthesized several circularized defensin peptides, on the basis
of the sequence of a rabbit defensin. The circularized defensins are bacteri-
cidal and, unlike other defensins, they are active in high salt concentrations.
44. Harwig SSL, Waring A, Yang HJ, Cho Y, Tan L, Lehrer RI:
Intramolecular disulfide bonds enhance the antimicrobial and lytic
activities of protegrins at physiological sodium chloride
concentrations. Eur J Biochem 1996, 240:352-357.
45. Travis SM, Anderson NN, Forsyth WR, Espiritu C, Conway BD,
Greenberg EP, McCray PB Jr, Lehrer RI, Welsh MJ, Tack BF:
Bactericidal activity of mammalian cathelicidin-derived peptides.
Infect Immun 2000, 68:2748-2755.
46. Zabner J, Seiler MP, Launspach JL, Karp PH, Kearney WR, Look DC,
• Smith JJ, Welsh MJ: The osmolyte xylitol reduces the salt
concentration of airway surface liquid and may enhance bacterial
killing. Proc Natl Acad Sci USA 2000, 97:11614-11619.
This paper demonstrates that xylitol can be used to alter the salt concentra-
tion of ASL. In addition, nasal administration of xylitol decreased nasal
Staphylococcus levels. This is the first report of a potential therapy to alter
ASL salt concentrations.
47. Robinson M, Regnis JA, Bailey DL, King M, Bautovich GJ, Bye PT:
Effect of hypertonic saline, amiloride, and cough on mucociliary
clearance in patients with cystic fibrosis. Am J Respir Crit Care
Med 1996, 153:1503-1509.
48. Robinson M, Daviskas E, Eberl S, Baker J, Chan HK, Anderson SD,
Bye PT: The effect of inhaled mannitol on bronchial mucus
clearance in cystic fibrosis patients: a pilot study. Eur Respir J
1999, 14:678-685.
49. Fleming A: On a remarkable bacteriolytic element found in tissues
and secretions. Proc R Soc Lond Biol 1922, 3:306-317.
50. Thompson AB, Bohling T, Payvandi F, Rennard SI: Lower respiratory
tract lactoferrin and lysozyme arise primarily in the airways and
are elevated in association with chronic bronchitis. J Lab Clin Med
1990, 115:148-158.
51. Vogelmeier C, Hubbard RC, Fells GA, Schnebli HP, Thompson RC,
Fritz H, Crystal RG: Anti-neutrophil elastase defense of the normal
human respiratory epithelial surface provided by the secretory
leukoprotease inhibitor. J Clin Invest 1991, 87:482-488.
52. Kouchi I, Yasuoka S, Ueda Y, Ogura T: Analysis of secretory
leukocyte protease inhibitor (SLPI) in bronchial secretions from
patients with hypersecretory respiratory diseases. Tokushima J
Exp Med 1993, 40:95-107.
53. Aho HJ, Grénman R, Sipilä J, Peuravuori H, Hartikainen J,
Nevalainen TJ: Group II phospholipase A2 in nasal fluid, mucosa
and paranasal sinuses. Acta Otolaryngol 1997, 117:860-863.
54. Brogden KA, Ackermann MR, McCray PB Jr, Huttner KM: Differences
in the concentrations of small, anionic, antimicrobial peptides in
bronchoalveolar lavage fluid and in respiratory epithelia of
patients with and without cystic fibrosis. Infect Immun 1999,
67:4256-4259.