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* Will Rogers Pulmonary Research Laboratory, Departments of Medicine and Pathology, and
Department of Pathology, Geffen School of Medicine, University of California, Los Angeles, CA 90095
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-defensin (HBD)-2 mRNA and peptide, but the induction was
greatly amplified when monocyte-derived cells (MoDeC) acted as
intermediaries between LPS and the epidermis. IL-1R antagonist largely
reversed the effect of MoDeC on epidermal HBD-2, indicating that, from
among the many products of MoDeC, IL-1 was the dominant inducer of
HBD-2 synthesis. In normal fresh human skin, which contains Langerhans
cells and other myeloid cell types, in addition to keratinocytes, LPS
also induced HBD-2 in an IL-1-dependent manner. In DNA microarray
expression studies, HBD-2 was one of the most abundant mRNAs induced in
epidermis by LPS-treated MoDeC, and its induction was reversed by
IL-1Ra. Thus, epidermal response to LPS is potently amplified by MoDeC
through IL-1-mediated signaling, leading to a selective increase in the
synthesis of the antimicrobial peptide HBD-2. This pattern of responses
establishes a key role for both IL-1 and HBD-2 in the host defense
reaction of the epidermis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-defensin
(HBD)3-2 was first
isolated from psoriatic scales (2) and was later shown to
be expressed in other epithelia as well (3). Our
studies have shown that cell differentiation as well as a bacterial or
cytokine signal is required for the induction of HBD-2 expression in
keratinocytes (4, 5). LPS (or endotoxin), an abundant glycolipid of the outer membrane of Gram-negative bacteria, is a well-studied and potent activator of inflammatory and host defense responses (6, 7). In early inflammatory response to LPS, macrophages are the major cellular targets for LPS action. Experiments with mice that lack individual Toll-like receptors (TLR) provide strong evidence that LPS-induced signal transmission depends on TLR4 (8). In a complex with LPS-binding protein and CD-14, LPS binds to TLR4 on macrophages, inducing the production of cytokines and other inflammatory mediators that have widespread effects on the host. Functional TLRs have been detected not only in macrophages but also in epithelia (9, 10, 11, 12, 13, 14, 15). Thus, two kinds of responses can be envisioned. The direct response depends on the detection of pathogen-associated molecules by epithelial TLRs or other receptor proteins and intracellular epithelial signaling. The indirect epithelial response is mediated by macrophages or other myeloid cells that detect pathogen-associated molecules and then signal to the epithelium. The relative roles of direct and indirect epithelial responses to pathogen-associated molecules have not been explored.
IL-1, present in two main agonist forms IL-1
and IL-1, is a
multifunctional cytokine produced by activated macrophages and other
cell types, including keratinocytes. IL-1 mediates a wide spectrum of
inflammatory, metabolic, physiologic, and immunological reactions, both
locally and systemically (16, 17). IL-1R type I, the
principal signaling receptor for IL-1 and IL-1, also belongs to
the Toll-like family of mammalian receptors. In the skin, and
especially in the epidermis, IL-1 induces the transcription of genes
involved in skin inflammation via activation of NF-
B and
mitogen-activated protein kinase pathways (18).
There are several physiological IL-1 inhibitors, among which IL-1R
antagonist (IL-1Ra) is a central regulatory molecule for IL-1 activity
(19, 20). It competes with IL-1 for receptor binding, but
does not activate the receptor, and has no other molecular target as
determined by epistatic genetic analysis (21). IL-1Ra has
been shown to block pathological inflammatory responses induced by IL-1
(22).
We show here that the ordinarily weak effect of LPS on the epidermis is greatly enhanced by MoDeC. The resulting massive and selective induction of antimicrobial peptide HBD-2 is almost entirely mediated by IL-1.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Primary epidermal cultures were obtained from MatTek
(EPI-200-3S; Ashland, MA). The cultures, grown from normal human
epidermal keratinocytes on collagen-coated Millicell CM membranes
(0.45-µm pores, 24-well size; Millipore, Bedford, MA), were
placed in 12-well plates with the medium supplied by the manufacturer
and incubated at 37°C in a 5% CO2-humidified
air atmosphere. On day 4, the epidermal cultures were lifted to the
air-liquid interface and then cultured in air-liquid interface for
another 4 days according to the supplier’s instruction. IL-1 (50
ng/ml; R&D Systems, Minneapolis, MN), Escherichia coli LPS
(100 ng/ml; E. coli 055:B5), or diluted conditioned medium
(CM; produced by incubating monocytes with LPS as detailed below) were
used to stimulate the cultured epidermis with or without IL-1Ra (200
ng/ml; R&D Systems) for 2–48 h.
Isolation of monocytes and cell culture
Human monocytes were isolated from three healthy volunteer donors under a protocol approved by the University of California, Los Angeles, Institutional Review Board. Freshly obtained heparinized whole blood was diluted with 0.2 volume of 1x PBS and centrifuged through Ficoll-Paque Plus (Amersham Pharmacia Biotech, Piscataway, NJ). The buffy coat cells were collected and washed with 1x PBS to remove platelets. The monocytes were enriched by centrifugation on a step 46% iso-osmotic Percoll (Sigma-Aldrich, St. Louis, MO) gradient overlaid with RPMI 1640 with 10% FCS, recovered from the interface between the Percoll and the medium, washed with 1x PBS, and counted and cultured at a concentration of 1.2 x 106/ml in RPMI 1640 with 20% autologous serum. The monocytes were 80–85% pure as judged by blood stain morphology. To prepare CM, LPS (100 ng/ml; E. coli 055:B5) was added into the culture medium and incubated with monocytes for 4 days. The cell-free supernatant is referred to as CM.
Normal human skin culture and stimulation
The use of human skin samples in this study was approved by UCLA
Institutional Review Board. Normal human skin specimens (from reduction
mammoplasties) were obtained immediately after surgery. After removing
the fat tissues and part of the dermis, the skin was then cut into
small strips 
1 mm wide and 5 mm long. The samples were incubated in
keratinocyte basal medium (Invitrogen, San Diego, CA) containing
2.5% human serum, and stimulated with LPS (100 ng/ml) with or without
IL-1Ra (200 ng/ml) for 24 h.
Northern blot analysis
Total RNA was isolated by using TRIzol (Invitrogen). The RNA was
further purified by using RNeasy minipreps (Qiagen, Valencia, CA),
according to the manufacturer’s instructions. Ten micrograms of the
purified total RNA from each sample was loaded onto 1% agarose
formaldehyde denaturing gel, and electrophoresis was performed at 5
V/cm for 23 h. After rinsing the gel in distilled water and 10x
SSC, the gel was transferred to GeneScreen Plus (NEN, Boston, MA)
overnight. The resulting blots were rinsed with 2x SSC, and then
stained with 0.04% methylene blue in 0.3 M sodium acetate for 1 min
and destained with distilled water for 2 min. After photography, the
blots were baked for 2 h at 80°C before hybridization. The
full-length HBD2 cDNA was labeled and hybridized to the blots at 42°C
in a solution containing 50% formamide, 5x SSC, 5x Denhardt
solution, 1% SDS, and 100 µg/ml denatured salmon sperm DNA.
Membranes were washed twice with 0.2x SSC/0.1% SDS at 4255°C and
exposed at -80°C.
Affymetrix GeneChip arrays and analysis
The Affymetrix GeneChip HuGene U133 set (Affymetrix, Santa
Clara, CA) was used to define gene expression profiles of the epidermis
stimulated by IL-1, LPS, or CM from LPS-exposed monocytes, with or
without IL-1Ra. Total RNA was prepared as for Northern blots, from
triplicate or quadruplicate epidermal samples to average differences
between individual wells. RNA labeling and microarray hybridization was
performed by University of California, Los Angeles, Microarray Core
Facility. Briefly, 15 µg of total RNA was used to synthesize
biotinylated cRNAs. The GeneChip sets were hybridized with 15 µg of
fragmented cRNA probes and then washed. The staining was performed with
streptavidin-PE. Images were scanned at 3-µm resolution by using a
GeneArray Scanner made for Affymetrix by Hewlett-Packard (Palo Alto,
CA). Analysis was performed using GeneChip Analysis Suite 5.0 software
(Affymetrix). To highlight potential changes in global gene expression,
no scaling or normalization of chip signals was performed.
Western-blot analysis of HBD-2
Cultured human epidermis was harvested, homogenized, and
extracted with 30% acetic acid overnight. The acid extractions were
lyophilized and resuspended in 1 M HCl and 1% trifluoroacetic acid.
After removing insoluble debris, supernatants were transferred to new
tubes and aliquots of the solution were lyophilized completely. The
resulting protein pellets were dissolved in SDS sample buffer overnight
at 4°C. Samples and HBD-2 standard were boiled for 5 min and then
loaded onto a 16.5% SDS-tricine polyacrylamide gel (23).
Transfer to Immobilon-PSQ membranes (Millipore) was performed for
1 h at 0.18 A in 0.05 M sodium borate (pH 9.0) with 20%
methanol and 0.05% SDS. Blots were fixed for 30 min with 0.5%
glutaradehyde in TBS (500 mM NaCl and 20 mM Tris; pH 7.5), blocked for
30 min in 0.75% Blotto (nonfat powdered milk) in Blotto PBS (0.9%
NaCl and 10 mM sodium phosphate buffer; pH 7.4), then incubated for
18 h in 1:1000 rabbit anti-HBD-2 serum (5)
diluted in Ab dilution buffer (0.25% Blotto in PBS containing 0.01%
thimerosal as a preservative). The blots were washed in 0.1% BSA in
Blotto TBS (0.9% NaCl and 20 mM Tris-HCl; pH 4.55.0) three times
for 10 min each. The membranes were incubated in a 1/2000 dilution of
alkaline phosphatase (AP)-conjugated goat anti-rabbit IgG (Pierce,
Rockford, IL) in Ab dilution buffer for 1 h, then washed three
times as before and developed in AP development solution
(bromochloroindolyl phosphate/nitroblue tetrazolium).
Immunocytochemistry
Deparaffinized human epidermal sections were rehydrated and subsequently washed with TBS (500 mM NaCl and 20 mM Tris; pH 7.5). The slides were incubated with a 1/1000 dilution of rabbit polyclonal serum in 1% gelatin TBS, 0.05% Tween 20 (Sigma-Aldrich), and 0.01% thimerosal for 18 h at room temperature. After three 20-min washes in TBS with 0.05% Tween 20, the slides were incubated with AP-conjugated goat anti-rabbit IgG (Pierce) diluted 1/2000 in the same buffer as the first Ab and incubated for overnight followed three 20-min washes. Color development was conducted with Fast Red chromogen (Sigma-Aldrich) in Tris buffer for 3 min and then counterstained with Harris hematoxylin.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human epidermal cultures (Epiderm; MatTek) formed all the
morphological layers of human epidermis: a basal layer, a spinous
layer, a granular layer, and a cornified layer (Fig. 1
and H&E stain, not shown). After
exposure of the epidermis to medium conditioned by LPS-treated MoDeC
(CM), HBD-2 was detected by immunostaining (4) in spinous
and granular layers of epidermal cultures, in a pattern similar to that
seen with IL-1-stimulated epidermal cultures (Fig. 1). Western blots of
epidermal extracts (Fig. 2) confirmed the
appropriate immunoreactive peptide band in CM-treated epidermis but not
in unstimulated epidermis. HBD-2 peptide was detectable 12 h after
stimulation with CM, and continued to accumulate thereafter. By
comparison to standards, the amount of HBD-2 of stimulated epidermal
culture after 48 h induction was estimated at 68 ng, i.e.,
10 µg/g of tissue, a concentration previously shown sufficient for
antimicrobial activity (4). After stimulation with CM,
there was a large increase in HBD-2 mRNA concentration, with a strong
hybridization signal present already at 2 h (Fig. 3), peaking at 24 h and decreasing
somewhat at 48 h. IL-1 was present in CM at active concentrations:
IL-1, 380 ± 44 pg/ml, and IL-1, 4649 ± 662 pg/ml
(mean ± SD, n = 3 donors).
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Substantial enhancement of HBD-2 mRNA induction (over the weak
induction observed with LPS alone) was seen even at 1/32 dilution of CM
in 1 ml of keratinocyte medium (Fig. 4),
corresponding to the output of 4.8 x 104
monocytes/cm2 of epidermis. The addition of
IL-1Ra at a concentration of 200 ng/ml completely or nearly completely
blocked HBD-2 mRNA induction (Fig. 4) indicating that IL-1 is the main
inducer of HBD-2 expression in CM.
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and
data not shown). Unlike the induction of HBD-2 by IL-1 or CM, this
effect was not consistent and two of four samples failed to generate
detectable HBD-2 mRNA in response to LPS. In contrast to the CM-treated
samples, this direct effect of LPS, when detectable, was not inhibited
by the addition of IL-1Ra into the culture (data not shown), indicating
that it did not depend on the intermediate generation of IL-1 by
keratinocytes.
To confirm the expected effects of CM, LPS, and IL-1Ra on the levels of
HBD-2 peptide, we performed Western analysis on human epidermal
cultures. As shown in Fig. 5, a specific
HBD-2 band was detectable in epidermal extracts after exposure to CM
but the induction of HBD-2 peptide by CM was suppressed by IL-1Ra.
Although in a parallel experiment we observed significant increase of
HBD-2 mRNA after LPS induction, no HBD-2 peptide was detectable after
48 h of direct LPS stimulation. These data confirm that LPS alone
is a weak stimulus for HBD-2 peptide synthesis in the epidermis.
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or CM
Analysis of gene expression in epidermal cultures collected after
24 h of stimulation with 50 ng/ml of IL-1 showed that HBD-2
became one of the most abundant mRNAs in stimulated epidermis (Fig. 6). In CM-stimulated epidermis, HBD-2 is
also among the most abundant inducible mRNAs (Fig. 6), and the
induction was largely reversed by IL-1Ra. We confirmed the high
abundance of HBD-2 mRNA after induction with CM or IL-1 in an
independent experiment with CM from another donor and another sample of
epidermis (Table I). In that experiment,
global suppression of gene expression was seen after 24 h of CM
and to a lesser extent after IL-1 stimulation (data not shown). The
suppressive effect of CM on the expression of abundant genes was
largely reversed by the addition of IL-1Ra (200 ng/ml) indicating that
IL-1 was principally responsible for this effect as well. The
biological determinants and reasons for the variability of the global
suppressive effect of CM on gene expression remain to be
determined.
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):
psoriasin (also called S100A7, a member of the S100 calcium-binding
protein family) and small proline-rich protein 2B (SPRR-2B) (Table I).
In contrast to IL-1 and CM, LPS was a weak inducer of HBD-2 mRNA
(Fig. 6), an observation consistent with Northern blot analysis
(Fig. 4).
Comparing the expression levels among known human antimicrobial
peptides (defensins and the cathelicidin LL-37), Fig. 7 shows that HBD-2 is the only
antimicrobial peptide that is robustly induced by IL-1 or CM. This
finding strongly supports the hypothesis that, among the various
antimicrobial peptides, HBD-2 plays a key biologic role in skin
defense.
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), and this induction
was abrogated by the simultaneous addition of IL-1Ra (200 ng/ml).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-defensin, tracheal
antimicrobial peptide, and the transcription is accompanied by NF-B
activation and binding to the tracheal antimicrobial peptide promoter
sequence (25). The local induction of defensin mRNA in
response to local injury or infection was also observed in the
keratinocytes that form the epithelium of the bovine tongue
(26). In principle, the epithelial responses could be
directly triggered by epithelial pattern recognition receptors such as
the TLR. In support of this concept, TLRs have been detected on various
epithelia by both functional and histological assays (11, 13, 14, 27). However, we noticed that the direct response to LPS,
demonstrable in studies with epithelial cell lines transfected with
NF-B reporter constructs, was relatively weak compared with the
response of the same cells to cytokines such as IL-1 (28).
In our organotypic epidermal cultures, the induction of HBD-2 mRNA in
direct response to LPS was sometimes detectable, but IL-1 consistently
induced much more HBD-2 production (4) than did relatively
large doses of LPS. This raised the possibility that the epidermal
responses to LPS in vivo could, under some circumstances, be amplified
by other cell types that produce IL-1 in response to LPS. Such indirect
mechanism would allow a graded response: colonization of the epidermal
surface or minor disruption of epithelial integrity by pathogens would
elicit low level activation, but a penetrating infection would expose
nonepithelial cell types to the pathogens and would result in full
activation of the epidermis. The molecular basis of the relative
unresponsiveness of the epidermis to LPS was not directly examined in
this study, but we anticipate that, like in recently studied epithelial
cell lines (28), epidermal keratinocytes may express only
low levels of TLRs and accessory molecules required for their
function. In earlier unpublished studies, we explored the effects of direct addition of LPS and monocytes to the apical surface of intact or abraded organotypic epidermal cultures (data not shown). In these experiments, we observed marked potentiation by monocytes of the LPS-mediated HBD-2 induction, but the magnitude of this effect was highly variable, probably due to the variable survival of monocytes under these conditions.
In the current study, we modeled the modulating effects of
nonepithelial cell types on the epidermis by comparing the effects of
LPS alone to those of LPS acting through the intermediate of the
LPS-MoDeC-CM. Compared with the effects of LPS alone, exposure of the
epidermis to CM increased the induction of HBD-2 mRNA as well as of
other transcripts, some of which are also implicated in host defense
responses. The near complete inhibition of the effect of CM by IL-1Ra,
and the presence of active range concentrations of IL-1 and IL-1
in the CM supported our hypothesis that IL-1 is the key intermediate
activator of the epidermal defensive response. Although we did not
examine LPS-induced responses in other epithelia, our findings raise
important questions about the relative magnitude, role, and mechanisms
of direct vs indirect responses in all epithelia.
Like IL-1, CM induced the expression of mRNAs for HBD-2 as well as
psoriasin, a nondefensin protein with a proposed role in host defense
and inflammation (29). The mRNA for SPRR-2B was also
abundant and inducible by both IL-1 and CM (Table I). Both psoriasin
and SPRR-2B are located in a genomic cluster (30) encoding
proteins involved in barrier properties of the skin. In view of the
inducibility of these mRNAs by IL-1, the potential host defense role of
these two proteins deserves further examination.
In the epidermis in vivo, both keratinocytes and Langerhans cells are
capable of synthesizing IL-1 (31), but keratinocytes lack
caspase-1 and thus cannot activate IL-1 (32), while
IL-1 is not readily released from keratinocytes except by mechanical
stress or other injurious stimuli (33). Moreover, the
release of IL-1 during mechanical strain is accompanied by the
release of comparatively large amounts of IL-1Ra (33).
Thus, it appears that Langerhans cells and, during later phases of the
inflammatory response, recruited monocytes and macrophages, constitute
significant sources of active IL-1 in the infected human epidermis. The
density of Langerhans cells in normal human skin is
105/cm2 of epidermis
(34), which is somewhat higher than the density of
monocytes necessary for the potentiation of epidermal LPS response in
our experiments. Even if Langerhans cells are less active producers of
IL-1 than are monocytes (35), their IL-1 production in
response to microbial stimulation could contribute to the activation of
epidermis and the early induction of HBD-2 synthesis in infected
epidermis.
In experiments with fresh whole human skin, we confirmed that normal skin responds to LPS by inducing HBD-2 mRNA and that this response is also dependent on the intermediate generation of IL-1. Because only keratinocytes produce high levels of HBD-2 mRNA, and several other abundant cell populations (e.g., fibroblasts and vascular endothelial cells) are present in the whole skin, we cannot directly compare the intensity of the response of the whole skin to that of the epidermis. The high concentration of HBD-2 in psoriatic skin (2) could be due to the higher IL-1 concentration generated in inflamed skin by myeloid cells (monocytes, macrophages, and neutrophils) recruited into the lesion.
Erdag and Morgan (36) recently reported that IL-1 or
IL-6 stimulation enhanced epidermal antimicrobial properties of
cultured keratinocyte grafts maintained on devitalized dermis. IL-1
was more effective than IL-6 against E. coli and
Pseudomonas aeruginosa but was less effective against
Staphylococcus aureus. RT-PCR assays of antimicrobial
peptide mRNAs showed that IL-1 but not IL-6 treatment induced HBD-2
expression in that system. Because S. aureus is resistant to
HBD-2 (2, 4), this antimicrobial profile would be expected
if HBD-2 peptide contributed appreciably to the antimicrobial
properties of epidermis. Further studies of the mechanisms of
enhancement of antimicrobial properties of grafts by cytokines are
certainly indicated.
In epidermis stimulated by either IL-1 or CM, HBD-2 was among the most abundant induced transcripts detected by DNA microarrays and the only antimicrobial peptide that was substantially induced by these stimuli. In general, at least micromolar concentrations of antimicrobial peptides are required for activity against microbes. In neutrophils and Paneth cells, high concentrations of defensins are generated by release of preformed defensins from granules (37). Inside epidermal keratinocytes, HBD-2 is located predominantly in lamellar bodies that transport lipids and other substances to cell membranes and intercellular spaces (38). Compared with the granules of neutrophils and Paneth cells, lamellar bodies represent a very small storage compartment. In view of the limited storage compartment in the epidermis, rapid generation of high concentrations of peptides would require high rates of peptide synthesis and correspondingly high levels of peptide mRNAs. Based on its abundance, HBD-2 is a leading candidate among antimicrobial peptides as an effector of antimicrobial activity in the inflamed epidermis.
There is increasing evidence that antimicrobial peptides contribute significantly to the resistance of the skin to infections. Most recently, mice with ablation of the gene that encodes the murine cathelicidin cathelin-related antimicrobial peptide were found to have decreased resistance to cutaneous infection with group A streptococcus (39). In human dermatoses, the high level of HBD-2 expression in psoriatic lesions may explain the surprising resistance of these lesions to superinfection (40), and conversely, the lack of antimicrobial peptide expression in atopic dermatitis may predispose to infections (41). In the aggregate, the clinical and experimental observations point to the central role of IL-1-induced defensin HBD-2 in the host defense response of the epidermis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Tomas Ganz, CHS 37-055, Department of Medicine, Geffen School of Medicine, University of California, Los Angeles, CA 90095-1690. E-mail address: tganz{at}mednet.ucla.edu
3 Abbreviations used in this paper: HBD, human -defensin; MoDeC, monocyte-derived cell; IL-1Ra, IL-1R antagonist; TLR, Toll-like receptor; CM, conditioned medium; AP, alkaline phosphatase; SPRR-2B, small proline-rich protein 2B.
Received for publication July 3, 2002. Accepted for publication October 25, 2002.
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References |
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-defensins by human airway epithelia. Proc. Natl. Acad. Sci. USA 95:14961.-defensin-2 production in keratinocytes is regulated by interleukin-1, bacteria, and the state of differentiation. J. Invest. Dermatol. 118:275.[Medline]
-defensin HBD-2 gene and its expression at sites of inflammation. Gene 222:237.[Medline]
and TNF- mediated up-regulation during inflammation. J. Immunol. 168:1286.-defensin gene expression in tracheal epithelial cells. Infect. Immun. 68:113.: different strategies of IL-1 production and processing in monocytes and keratinocytes. J. Clin. Invest. 87:1066.
and IL-1 receptor antagonist. J. Immunol. 159:5084.[Abstract]
induces Langerhans cell migration in humans. Br. J. Dermatol. 141:192.[Medline]
and interleukin-6 enhance the antibacterial properties of cultured composite keratinocyte grafts. Ann. Surg. 235:113.[Medline]
-defensin-2 is packagedin lamellar
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O. E. Sorensen, D. R. Thapa, A. Rosenthal, L. Liu, A. A. Roberts, and T. Ganz Differential Regulation of {beta}-Defensin Expression in Human Skin by Microbial Stimuli J. Immunol., April 15, 2005; 174(8): 4870 - 4879. [Abstract] [Full Text] [PDF]
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J. Harder and J.-M. Schroder Psoriatic scales: a promising source for the isolation of human skin-derived antimicrobial proteins J. Leukoc. Biol., April 1, 2005; 77(4): 476 - 486. [Abstract] [Full Text] [PDF]
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T. D. Starner, B. Agerberth, G. H. Gudmundsson, and P. B. McCray Jr. Expression and Activity of {beta}-Defensins and LL-37 in the Developing Human Lung J. Immunol., February 1, 2005; 174(3): 1608 - 1615. [Abstract] [Full Text] [PDF]
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