First Line of Defense in Early Human Life
Hiroyuki Yoshio,* Hugo Lagercrantz,† Gudmundur H. Gudmundsson,‡
and Birgitta Agerberth,§
Innate antimicrobial peptides are considered to play an important role in host defense against
microbial invasion. They are expressed in a wide variety of organisms. In the case of human beings,
defensins and the cathelicidin LL-37 appear to be the major microbicidal peptides. With respect to
human neonates, only few investigations have been performed in this context, revealing the presence
of ␣-defensins and LL-37 in neutrophils and vernix caseosa. In addition, ␤-defensins are present in
tracheal aspirates and breast milk, whereas LL-37 has been detected in the skin of the newborn baby.
During recent years, immunomodulatory activities such as chemotaxis have emerged as important
functions of antimicrobial peptides. Thus, these innate effectors may work synergistically to provide
a first line of defense against infection, as well as to promote interactions between the innate and
adaptive immunity in newborn infants.
© 2004 Elsevier Inc. All rights reserved.
he transition of the fetus from the womb
into the open air is not only one of the
most stressful events during life,1 but is also usually associated with the first challenge of microbial invasion. Antimicrobial peptides are of ancient evolutionary origin and exert key
influences on host defense.2 These peptides
have been isolated from a variety of human tissues, including neonatal tissues probably supporting newborn infants to thrive in the extrauterine world.3 Recent experimental evidence
concerning the significance of antimicrobial
peptides associated with primary host defense in
vivo is convincing. Of the antimicrobial peptides
identified to date, the defensins are the most
abundant in humans.4
The newborn infant requires efficient host
defense against infection as it emerges from the
sterile environment in the womb into a world
full of potential hostile pathogens. There has
been limited knowledge on the primary immune
and antiinfective systems in fetus and newborn,
T
From the *National Hospital Organization Okayama Medical Center, Okayama, Japan; †Astrid Lindgren Children’s Hospital, Stockholm, Sweden; ‡Biology Institute, University of Iceland, Reykjavik,
Iceland; and §Department of Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Address reprint requests to Hugo Lagercrantz, Division of Neonatology, Astrid Lindgrens Children Hospital, Karolinska Institute,
Stockholm S-171 76, Sweden; e-mail: hugo.lagercrantz@ks.se
© 2004 Elsevier Inc. All rights reserved.
0146-0005/04/2804-0000$30.00/0
doi:10.1053/j.semperi.2004.08.008
304
but the adaptive immune system, relying on B
and T lymphocytes, is known to be immature
due to shortage of antigen exposure. However,
passive transport of immune components, including immunoglobulins, from the mother is
well documented.
Recent studies confirm the presence of innate
defense effectors in the fetus and the newborn
infant, and these innate components may represent the main defense capacity of neonates.5,6
The present review focuses primarily on the antimicrobial peptides defensins and LL-37 as effectors of innate immunity in human neonates.
Human Antimicrobial Peptides
Mammalian antimicrobial peptides are classified
into two large families: the defensins and the
cathelicidins.4,7 The defensins, which can further be divided into two main subgroups, ␣- and
␤-defensins, are a family of 3- to 4-kDa cationic
peptides, forming a triple-stranded ␤-sheet structure with three intrachain disulfide bridges,
where ␣- and ␤-defensins differ in the bridging
of their conserved cysteines.8
Six human ␣-defensins have been characterized, including human neutrophil peptide 1 to 4
(HNP1-4) mainly found in neutrophils9,10 and
human defensins 5 to 6 (HD5-6) expressed by
Paneth cells in the small intestine.11,12 The four
␤-defensins (HBD1-4) so far characterized are
expressed primarily by epithelial cells.13-16
Seminars in Perinatology, Vol 28, No 4 (August), 2004: pp 304-311
First Line of Defense in Early Life
Genomic analysis has recently revealed 28 putative genes for human ␤-defensins, although it is
not yet known whether all of them are functional.17,18
The cathelicidins have been thought to be
expressed only in mammals.7 However, recently,
a cathelicidin was identified in hagfish.19 This
family of highly variant antimicrobial peptides
contains a conserved proregion designated
cathelin with a variable antimicrobial C-terminal
domain, which is cleaved off by processing enzyme(s), liberating the active antimicrobial peptide.7 LL-37 is a 37-residue peptide with an amphipathic ␣-helical conformation, which is
critical for its broad antimicrobial spectra and is
the only member of the cathelicidins present in
humans.20,21
Mechanism of Action
The cationic properties of antimicrobial peptides are essential for their affinity to the negatively charged microbial membranes. Studies in
vitro have shown that the peptides are able to kill
Gram-positive and Gram-negative bacteria,
fungi, parasites such as trypanosomes and plasmodia, certain enveloped viruses, and even cancer cells.22 The interaction of antimicrobial peptides with models of bacterial membranes has
been extensively studied. Most of these peptides
have affinity to the bacterial outer membrane
with negatively charged phospholipid headgroups, and it has been suggested that their
305
hydrophobic properties can integrate into the
bacterial membranes, resulting in destabilization of the membrane with subsequent lysis of
the bacteria.23 In contrast, the outer layer of the
membrane of the host cells is composed primarily of lipids with no net charges, leading to
weaker affinity between these membranes and
antimicrobial peptides.2
Expression and Function of Antimicrobial
Peptides in Neonates
Multiple antimicrobial peptides are expressed by
many organs in the human body (Table 1).
␣-Defensins are present primarily in neutrophils, but also in Paneth cells of the small intestine. The ␤-defensins are expressed primarily in
epithelial tissues such as the skin, the respiratory
and gastrointestinal tracts, the urogenital system, but also in the kidney, the pancreas, and
the placenta.13,24,25 The ␣-defensins and ␤-defensin-1 (HBD1) are constitutively expressed,
whereas the three ␤-defensins, HBD2 to 4, are
inducible.4 LL-37 is encoded by the CAMP
(cathelicidin antimicrobial peptide) gene on
chromosome 3p21.3,26 and is widely distributed
throughout the epithelial linings.27 In addition,
LL-37 is stored in both neutrophils and specific
mononuclear cells,28 and works synergistically
with lactoferrin and lysozyme.29,30 Although the
distribution of these microbicidal peptides in
infants may be similar to that in adults, relatively
Table 1. Human Antimicrobial Peptides
Peptides
␣-Defensins
HNP1-3
HNP4
HD5,6
␤-defensins
HBD1
HBD2
HBD3
HBD4
Cathelicidin
LL-37
Others
Histatin
Hepcidin
Distribution
granulocytes, lymphocytes, spleen, cornea, thymus, vernix, amniotic fluid
granulocytes
paneth cells of the intestine
kidney, pancreas, salivary gland, lung, skin, placenta, thymus, gut, testis, small intestine,
mammary gland, breast milk
skin, lung, kidney, small intestine, colon, stomach, pancreas, thymus, uterus, testis, liver
skin, tonsil, lung, thymus, uterus, kidney
testis, gastric antrum
granulocytes, lymphocytes, lung, skin, colon, saliva, vernix, amniotic fluid
saliva
liver
306
Yoshio et al
few investigations have been performed in human neonates.
Neutrophils
Neutrophils are the first cells recruited to sites of
infection and inflammation, where they play a
major role in host defense, utilizing a variety of
first-line antimicrobial action. Of the different
peptides stored in the granules of human neutrophils, ␣-defensins (HNP1-3) appear to be the
major bactericidal components, accounting for
5-7% of the total cellular proteins in these
cells.31 Lactoferrin, secretory phospholipase A2,
lysozyme, and LL-37 are examples of other bactericidal protein/peptides constituents of neutrophils.9 The fourth ␣-defensin (HNP-4) is also
found in azurophilic granules of neutrophils but
at lower concentrations than HNP1 to 3.10 In
response to stimulation of neutrophils, these effectors are activated within vacuoles of the cell
or released into the bloodstream and cooperate
in killing microbes.
Recruitment of neutrophils usually occurs at
the time of birth. Thus, the neutrophil function
is very important for newborn infants at birth.
Although there are many reports on neonatal
neutrophilic activity, including immature adherence, chemotaxis, and phagocytosis,32,33 little is
presently known about the activity of bactericidal peptides of these cells in neonates. However, a 55-kDa protein, the cationic bactericidal/
permeability-increasing protein (BPI), exhibits
remarkably specific cytotoxic activity toward
Gram-negative bacteria, reflecting its high affinity for bacterial lipopolysaccharides.34,35 The average BPI level in neutrophils derived from cord
blood of full-term newborns has been demonstrated to be much lower than in adult blood,
although with some individual variations.36 In
another study, the plasma levels of BPI in newborns with clinical sepsis were found to be
higher than those of both healthy term infants
and adults, whereas preterm infants had lower
capacity to release BPI.37 The binding capacity
of neutrophils, originating from neonatal cord
blood, to lipopolysaccharide (LPS) was also
weaker than that of neutrophils derived from
adults.38 These findings suggest that some newborns, especially preterm infants, are at high risk
to develop serious sepsis or meningitis, when
exposed to Gram-negative bacteria. However,
further investigations, including prospective
clinical studies, are required to establish the contribution of BPI in the protection against Gramnegative bacterial infection at birth. Recently,
administration of recombinant BPI to cord
blood proved to exert a beneficial effect on newborns against Gram-negative bacterial infections.39
LL-37 is expressed not only by human neutrophils, but also by B cells, NK cells, ␥␦T cells,
and monocytes.28 LL-37 has been detected in
neutrophils that have migrated into the skin of
the newborn, indicating an active role of this
peptide in primary host defense for newborn
infants.40
Skin
Several antimicrobial peptides are expressed in
adult human skin, where LL-37 and ␤-defensins
appear to be important effectors for cutaneous
defense against microbial invasion.41 HBD1 is
produced constitutively, while HBD2, HBD3,
and LL-37 are induced in inflamed skin, where
LL-37 is upregulated in keratinocytes of the epidermis in certain inflammatory skin disorders.42
Both HBD1 and HBD2 exhibit bactericidal activity predominantly against Gram-negative bacteria such as E. coli and P. aeruginosa,43 whereas
HBD3 is highly effective against the Gram-positive bacteria S. aureus.15
Skin, respiratory, and gastrointestinal tracts
are sites for microbial invasion also in newborns.
Interestingly, the expression of both LL-37 and
HBD2 in the newborn foreskin is strong when
compared with adult skin.44 These two peptides
exhibit bactericidal activity against group B
streptococcus (GBS), a frequent neonatal pathogen.44
Erythema Toxicum Neonatorum (ETN),
which is a well known benign, inflammatory skin
disorder of neonates at early days after birth,
contains several immune cells such as dendritic
cells and eosinophils, expressing LL-37, while
there is low expression in the normal skin of the
newborns.40 At present, the etiology and physiological significance of ETN is unknown. However, these data suggest that, even though it
might be in localized skin area, these cells and
First Line of Defense in Early Life
LL-37 may serve as active skin protectors of newborn infants after birth.
Vernix caseosa (vernix) is a lipid-rich white substance that covers the skin of the fetus and the
newborn. The integral composition and the
physiologic role of vernix is gradually becoming
evident. The characteristic composition of vernix suggests limited interactions with hydrophilic liquids which suggest that in uterus vernix
acts as a hydrophobic barrier, waterproofing the
fetal skin against amniotic fluid.45 Recently, we
discovered that vernix contains several antimicrobial peptides such as HNP1 to 3, lysozyme,
LL-37, and psoriasin.40,46 Notably, the mucous
plug that is formed at the cervix also contains
some of the same antimicrobial peptides.47 Furthermore, in amniotic fluid, similar microbicidal
peptides are present, ie, HNP1 to 3, lysozyme,
and LL-37, that partly could be derived from the
fetal skin detachments and respiratory elements.46 These findings suggest that vernix
works as a natural biofilm for obstruction of
microbial passage, interacting with amniotic
fluid, and hence contributes to skin defense of
the fetus and the newborn against microbial
invasion at birth.
Airway
The first mammalian epithelial antimicrobial
peptide was isolated from bovine tracheal mucosa, and subsequently characterized as a ␤-defensin with inducible properties.48 Several investigations have focused on antimicrobial peptides
in the respiratory tract. HBD1 to 3 and LL-37 are
found in the epithelial lining of the airway and
play a pivotal role in intrinsic mucosal immune
defense.13,15,24,29,49 HBD4 transcripts have also
been detected in human lung, however with a
low expression.16
Pulmonary infection is common in newborns
and often takes a serious clinical course with
ventilator care. In general, the more premature
the newborn is, the stronger severity of disease.
The direct activity of antimicrobial peptides to
microbes as the first line of defense in neonatal
respiratory system is thought to be essential, but
little is known about its intrinsic role in the
newborn infant. There is limited knowledge
about the developmental regulation of antimicrobial peptides in the human fetus. Although
307
HBD1 is developmentally regulated postnatally
in lung parenchyma, the expression of HBD1
could not be detected by the sensitive RNaseprotection assay in samples from fetal tissue at
15 and 22 weeks of gestation.50 In another study,
it was demonstrated that HBD1 to 2 and LL-37
were detected by an antigen capture assay in
neonatal tracheal aspirates during ventilator
support of term and preterm infants. The concentrations of the peptides were similar in all
infants studied, and the levels of the peptides
were enhanced on infection and correlated with
inflammatory parameters such as IL-8 and TNF␣.51 Recently, we have detected bactericidal activity by an inhibition zone assay in bronchoalveolar lavage of infected newborns after birth,
and this activity was shown to correlate positively
with systemic inflammation.52 These data suggest that rapid response or production of antimicrobial peptides seems to occur after microbial challenge after birth.
Gastrointestinal Tract
Human enteric ␣-defensins (HD5 and HD6) are
expressed in Paneth cells.11,12 Both HD5 and
HD6 seem to be developmentally regulated,
since their transcripts are detected by PCR at the
gestation of 13.5 weeks in the small intestine and
for HD5 mRNA also in the colon. At 17 weeks
gestation, the expression of HD5 is only located
in the small intestine.53 By Northern blot analysis and immunohistochemistry, the expression
of both these enteric peptides was detectable at
24 weeks gestation, confirming the presence of
HD5 to 6 in the Paneth cells of the fetus. At 24
weeks gestation, both the number of Paneth
cells and the level of mRNA are significantly
lower than those in adult, which may predispose
preterm newborns to serious enteric infection.
Necrotizing enterocolitis (NEC) is a critical
cause of morbidity and mortality among preterm
infants. The etiology is still unclear and enteric
infections are generally involved and associated
with a premature local innate defense.54 The
increase in both enteric defensin mRNA expression and Paneth cell number was demonstrated
in subjects with NEC compared with a control
group.55 The upregulation of the expression of
enteric defensins may be a consequence of the
pathologic process of NEC. In colonic biopsies
308
Yoshio et al
Antimicrobial action
Recruitment of inflammatory
cells
Promote microbe ingestion
Neutrophils adherence,
Non-opsonic phagocytosis
Neutrophils, T cells
Antimicrobial peptides
Wound repair
Fibroblast growth and adherence,
Angiogenesis
Stimulate release of
pro- and anti-inflammatory factors
Histamine release out of mast cells,
Cytokine release
derived both from adult and children with Shigella infection, epithelial downregulation of both
LL-37 and HBD1 was demonstrated.56 Interestingly, LL-37 is upregulated in colon epithelial
cells by short chain fatty acids, including butyrate,57 which may have potential in therapeutic
use for this disorder. Butyrate in the colon originates from fermentation of fibers and hence the
normal flora is responsible for its production.
These results suggest that antimicrobial peptides
might be key effectors in a complicated situation
in the human enteric tract in which pathogens
and commensal bacteria interact with each
other.
Human milk
It is well known that breast milk contains several
antiinfective components, such as lactoferrin,
the bifidus factor, lysozyme, lactoperoxidase,
and oligosaccharides, which are suggested to
protect newborns against a variety of infection.58
Lactoferrin, an iron-binding protein abundant
in human milk, absorbs enteric iron in the presence of IgA and bicarbonate, thus preventing
microbes from obtaining the iron needed for
survival. Recently, the expression of HBD1 was
found in human breast milk and mammary
gland epithelia,59,60 which could provide mucosal defenses in both mother and newborn. The
HBD1 expression is usually documented to be
constitutive. However, higher HBD1 immunoreactivity was demonstrated in breast tissue during
lactation compared with that during nonlacta-
Apoptosis
Infected host cells
Figure 1. Suggested functions of antimicrobial peptides.
tion.59 Furthermore, enhanced expression was
detected in the urinary tracts of pregnant women.25 These data suggest that the expression of
HBD1 may be upregulated by hormones during
pregnancy. Although HBD1 is highly effective
against Gram-negative bacteria, it may work synergistically with other antibiotic peptides against
a broad range of microbes such as Staphylococcus
aureus, which is a main cause of lactational mastitis. Further studies remain to be conducted to
clarify the regulation of HBD1 expression in
breast milk, and the influence of breastfed-mediated antimicrobial peptides in newborn during or after lactation.
Multifunction and Link to Disease
Additional functions for antimicrobial peptides
other than direct microbicidal activity have been
established in recent years, such as chemotaxis,
histamine release from mast cells, wound repairing, and apoptosis61,62 (Fig 1). Chemotactic activity for T cells has been reported for HNP1 to
2, with induction of IL-8 synthesis.63,64 The
HBD2 expression is induced by IL-1␣, IL-1␤, and
TFN␣ stimulation, with activation through
NF-␬B (nuclear factor ␬B), and HBD1 to 2 attracts CD4 T cells and dendritic cells through
interactions with the chemokine receptor
CCR6.65,66 LL-37 is able to attract CD4 T cell,
monocytes, and neutrophils via the formyl peptide receptor-like 1 (FPRL-1).28,67 Thus, antimicrobial peptides are multifunctional with activi-
First Line of Defense in Early Life
ties that enhance our defense barrier with
reference to both adaptive and innate defenses.
It is clinically important to investigate the alteration of antimicrobial peptides expression associated with disease.3 The knock-out mouse for the
CRAMP gene, the homologue to the human
CAMP gene encoding LL-37 is very sensitive to skin
infections by Streptococcus group A.68 In human, the
Kostmann syndrome, which is a severe congenital
neutropenia from the first day of life and is usually
fatal due to serious bacterial infections, can be
treated with infusions of granulocyte-colony stimulating factor (G-CSF).69,70 A recent study revealed
that LL-37 was missing both in granulocytes and
saliva of Kostmann patients.71 One patient, who
after a bone-marrow transplantation restored LL37, did not exhibit severe periodontal disease, a
common feature in Kostmann patients.71 LL-37
might be one relevant component to the defects in
Kostmann syndrome and can be considered for
therapeutical use. Atopic dermatitis is a common
allergic skin disorder, often manifested with cutaneous infections by Staphylococcus aureus. Decreased expressions of both HBD2 and LL-37 in
the skin lesions of atopic dermatitis were demonstrated compared with the inflamed lesions of psoriasis.72 This finding may explain the high susceptibility of S. aureus in the skin of atopic dermatitis.
It is likely that antimicrobial peptides are intrinsic components of barrier defenses in early
human life like they are in adults. Future research must consider defects in expression and
activity of innate antimicrobial peptides as one
possible cause for immunological disorders that
affect the fetus or the newborn infant. There is
limited knowledge on variations in the levels of
antimicrobial peptides related to innate defenses. The peptides represent first line defenses, and low expression levels might be linked
to susceptibility to infections that are often fatal
for newborns. Furthermore, the immune modulatory activities of the peptides might be connected to adjuvant activity, where the peptides
might be initiators of important immune functions to train the system for later challenges.
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