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*
Laboratory of Mucosal Immunology, Department of Medicine, University of California at San Diego, La Jolla, CA 92093;
Will Rogers Institute Pulmonary Research Laboratory, Department of Medicine, University of California, Los Angeles, CA 90095; and
Magainin Pharmaceuticals Inc., Plymouth Meeting, PA 19462
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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stimulation or infection of those cells with
enteroinvasive bacteria. Moreover, hBD-2 functions as a NF-
B target
gene in the intestinal epithelium as blocking NF-B activation
inhibits the up-regulated expression of hBD-2 in response to IL-1
stimulation or bacterial infection. Caco-2 cells produce two hBD-1
isoforms and a hBD-2 peptide larger in size than previously described
hBD-2 isoforms. Paralleling the in vitro findings, human
fetal intestinal xenografts constitutively express hBD-1, but not
hBD-2, and hBD-2 expression, but not hBD-1, is up-regulated in
xenografts infected intraluminally with Salmonella.
hBD-1 is expressed by the epithelium of normal human colon and small
intestine, with a similar pattern of expression in inflamed colon. In
contrast, there is little hBD-2 expression by the epithelium of normal
colon, but abundant hBD-2 expression by the epithelium of inflamed
colon. hBD-1 and hBD-2 may be integral components of epithelial innate
immunity in the intestine, with each occupying a distinct functional
niche in intestinal mucosal defense. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-defensins,
namely human neutrophil defensins 1–4 (1, 2) as well as
human defensin (HD)4-5
and HD-6 in small-intestinal Paneth cells and the female genital tract,
are known mediators of antimicrobial defense (1, 3, 4, 5, 6).
Less well defined, however, is the distribution and function of two
more recently described members of the defensin family, the human
ß-defensins hBD-1 and hBD-2 (7, 8), that are
predominately expressed at epithelial sites. hBD-1 is constitutively
expressed in respiratory tract, kidney, and urogenital epithelium
(9, 10, 11, 12, 13) and by epithelia in the oral cavity (14, 15), whereas hBD-2 has been identified in the skin (8, 16, 17), respiratory (12, 18, 19), and gingival
epithelium (15). RNA dot-blot screening of a human tissue
panel has also provided evidence for gastrointestinal expression of
hBD-2 mRNA in stomach, small intestine, and colon (18). At
least six hBD-1 isoforms that range in length from 36 to 47 amino
acids, and differ in their microbicidal activity, have been identified
in urine (13). A single isoform of hBD-2, 41 aa in length,
has been isolated from respiratory epithelial secretions and saliva
(12, 15, 18). Purified and recombinant forms of the
ß-defensins in urine and lung epithelial secretions exhibit
antimicrobial activity in vitro against several respiratory,
genitourinary tract, and enteric bacteria, including
Pseudomonas, Escherichia coli, and
Salmonella (14, 18). Unlike hBD-1, which is produced constitutively, hBD-2 in respiratory tract epithelial cells and epidermal and gingival keratinocytes is expressed in response to bacterial infection or proinflammatory agonists, suggesting a role for this ß-defensin in epithelial host defense under those conditions (8, 12, 16, 17, 18, 19). The regulated expression of hBD-2 is consistent with that described for other mammalian ß-defensins. For example, in bovine and murine models, the expression of a number of epithelial ß-defensins, namely bovine enteric ß-defensin in colon crypt epithelium, tracheal antimicrobial peptide and lingual antimicrobial peptide in bovine tracheal and lingual epithelium, and murine ß-defensin (mBD) 3 and Defb2 in respiratory epithelium, are induced or up-regulated as part of the inflammatory response (20, 21, 22, 23, 24, 25).
Despite recent information regarding the expression of ß-defensins at other mucosal sites, little is known about the constitutive or regulated expression of these peptides in the human intestinal tract. The single-cell layer of epithelium that separates intestinal luminal contents from the internal milieu of the host is important for innate host defense by maintaining mucosal barrier function and in the production of an array of proinflammatory immune mediators. These include cytokines and chemokines, NO, and PGs that, together, have the potential to rapidly signal and regulate a coordinated mucosal inflammatory response to microbial infection (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36). Peptide antibiotics, such as the ß-defensins, also may contribute to host defense in the microbe-rich environment of the intestinal tract. As a first step in testing this hypothesis, we applied in vitro and in vivo model systems to determine the expression, regulation, and production of hBD-1 and hBD-2 by human intestinal epithelium.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The human colon epithelial cell lines Caco-2 and HT-29 (26) were cultured in DMEM, supplemented with 10% FBS and 2 mM L-glutamine, and then maintained at 37°C in 5% CO2 and 95% air.
Cytokines, bacteria, and other reagents
Recombinant human (rh) IL-1, rhIFN-
, and rhTNF- were
obtained from Peprotech (Rocky Hill, NJ). Bacterial LPS (E.
coli O111:B4) and the proteasome inhibitor MG-132 were obtained
from Sigma (St. Louis, MO).
12-O-Tetradecanoylphorbol-13-acetate (TPA) was obtained from
Calbiochem (La Jolla, CA). The following bacteria were used:
Salmonella dublin lane (27), an attenuated
aroA aroC strain of Salmonella typhi
(28), and enteroinvasive E. coli (serotype
O29:NM; no. 43892, American Type Culture Collection, Manassas, VA)
(27).
Cytokine stimulation and infection protocols
Caco-2 and HT-29 cells in 6- or 12-well tissue culture plates
(Corning-Costar, Cambridge, MA) were stimulated for 1 h with
rhIL-1 (20 ng/ml), rhTNF- (20 ng/ml), rhIFN- (40 ng/ml), or
LPS (10 µg/ml), after which culture medium was removed and replaced
with fresh medium. For bacterial infection, cells were cultured for
1 h with 1 x 108 bacteria/well, after
which bacteria were removed by washing, and 50 µg/ml of gentamicin
was added to kill any remaining extracellular bacteria, as described
before (27). Supernatants and cells from
agonist-stimulated and bacterially infected cultures were harvested
following an additional 5–6 h incubation. Cells were used for RNA
extraction, and supernatants were stored at –80°C until
use.
Human fetal intestinal xenografts
The human intestinal xenograft model used herein has been
described in detail before (28, 29, 31, 32, 37, 38).
Briefly, human fetal intestine (gestation age, 10–14 wk) was
transplanted s.c. into CB-17 SCID mice, and xenografts were allowed to
develop for 10 wk before use. Xenografts were infected with 
5
x 107 of attenuated aroA
aroC S. typhi in DMEM/F12 medium in a 100-µl
volume injected intraluminally by s.c. injection. Xenograft tissue was
removed 6 h after infection, and mucosal scrapings were prepared
and immediately frozen in liquid nitrogen.
Adenovirus Constructs and Adenovirus Infection
Recombinant adenovirus containing an IB-AA superrepressor
(Ad5IB-A32/36) or the E. coli ß-galactosidase gene
(Ad5LacZ) was constructed as described before (36, 39).
Ad5IB-A32/36 expresses a hemagglutinin epitope-tagged mutant form of
IB in which serine residues 32 and 36 are replaced by alanine
residues (36, 39). The mutant IB cannot be
phosphorylated at positions 32 and 36 and acts as a superrepressor of
NF-B activation (36, 39).
Caco-2 and HT-29 epithelial cells grown to confluence in six-well
tissue culture plates were infected with Ad5IB-A32/36 or Ad5LacZ in
serum-free medium (Opti-MEM; Life Technologies, Grand Island, NY) at a
multiplicity of infection (MOI) of 75 for 16 h. At this MOI,
Ad5IB-A32/36 or Ad5LacZ infected >80% of the cells. Infected cells
expressed IB-A32/36 and ß-galactosidase at high levels as
assessed by staining for ß-galactosidase and immunostaining for
hemagglutinin-tagged IB-A32/36. After infection, adenovirus was
removed by washing, fresh medium containing serum was added, and cells
were incubated for an additional 12 h before bacterial infection
or IL-1 stimulation. The IB-AA superrepressor inhibited
NF-B activation in HT-29 and Caco-2 cells, as assessed by EMSA, and
inhibited TNF--induced up-regulation of IL-8 and ICAM-1 expression
in HT-29 cells but did not alter ß-actin mRNA levels in the same
cells (Ref. 36 and data not shown).
RNA extraction and RT-PCR analysis
Total cellular RNA was extracted from cell lines and xenograft tissue using TRIzol reagent (Life Technologies). RNA (1 µg) was reverse transcribed at 37°C (Life Technologies Superscript kit) with 1 mM each of dATP, dTTP, dCTP, and dGTP and 5 µg/ml of oligo-dT primer in a 20-µl volume as described before (30). Two microliters of each cDNA sample from the reverse transcription reactions was amplified as described before (30), with 4.0 U Taq polymerase (Life Technologies) in a 50-µl volume containing 25 pmol each of the following primers: 5'-CTC TGT CAG CTC AGC CTC-3' (sense) and 5'-CTT GCA GCA CTT GGC CTT CCC-3' (antisense) for hBD-1, 5'-CCA GCC ATC AGC CAT GAG GGT-3' (sense) and 5'-GGA GCC CTT TCT GAA TCC GCA-3' (antisense) for hBD-2, 5'-CCC AGC CAT GAG GAC CAT CG-3' (sense) and 5'-TCT ATC TAG GAA GCT CAG CG-3' (antisense) for HD-5, and 5'-CCA CTC AAG CTG AGG ATG ATC-3' (sense) and 5'-TGA TGG CAA TGT ATG GGA CAC ACA C-3' (antisense) for HD-6 (5). ß-Actin primers were as described before (30). After a hot start, the amplification profile was 35 cycles of 1-min denaturation at 94°C, 1-min annealing at 66°C, and a 1.5-min extension at 72°C. The same amplification profile was used for ß-actin. PCR products were resolved on 1.5% agarose gels (Life Technologies). RT-PCR amplification of hBD-1 and hBD-2 generated products of 272 and 254 bp, respectively.
The strategy for quantitative RT-PCR analysis using internal standard
RNAs and the construct for the quantitation of human ß-actin mRNA
were described before (28). For the quantitation of hBD-1
and hBD-2, a plasmid construct was developed incorporating 5' and 3'
primer sites for hBD-1 and hBD-2 separated by an intervening insert of
the mouse Ig constant region (28). Briefly, a 260-bp
fragment of a mouse CH cDNA clone was
amplified with the primers 5'-CCA GCC ATC AGC CAT GAG GGT GAA CAG
TGG CGC ATC ATT CAA GTC-3' (sense) and 5'-CTT GCA GCA CTT GGC CTT
CCC GCA ACA CGC TTG TCA CCA GGT AGG-3' (antisense). Regions
corresponding to mouse CH are underlined. The
resulting product was further PCR amplified using the primers 5'-CGA
AGC AAG CTT CTC GTC AGC TCA GCC CCC AGC CAT CAG CCA TGA GGG
T-3' (sense) and 5'-CGA AGC TCT AGA GGA GCC CTT TCT GAA TCC
GCA CTT GCA GCA CTT GGC CTT CCC-3' (antisense). HindIII
(sense primer) and XbaI (antisense primer) restriction sites
are underlined. The resulting PCR product was ligated into the
XbaI and HindIII sites of plasmid pBATNOT, which
contains a promoter for T7 RNA polymerase upstream and a
d(pA)16 sequence and a NotI
restriction site downstream of the cloned fragment. Plasmid linearized
using NotI was in vitro transcribed using T7 RNA polymerase
(T7 polymerase kit, Life Technologies) to yield a 430-nt standard RNA.
Numbers of standard RNA transcripts were calculated on the basis of the
RNA concentration and the size of the standard RNA as described before
(28). For quantitation, known amounts of standard RNA
transcripts were added to constant amounts of cellular RNA as described
before (30). cDNA from the standard RNA transcripts
included in quantitative RT-PCR yielded fragments of 368 bp for hBD-1
and 371 bp for hBD-2.
hBD-1 or hBD-2 mRNAs were regarded as not expressed when PCR
amplification yielded no product or <103
transcripts/µg cellular RNA, because the latter are not likely to be
paralleled by biologically meaningful protein production (i.e., because
105 cells yield 1 µg cellular RNA,
103 transcripts/µg cellular RNA is equivalent
on average to 1 transcript per 100 cells).
Detection of hBD-1 and hBD-2 by immunoblot
Supernatants were harvested from unstimulated or
IL-1-stimulated HT-29 cells after 6 h incubation. Cationic
peptides from cell-culture supernatants were extracted using the weak
cation exchange matrix MacroPrep CM (carboxymethyl) support (Bio-Rad
Laboratories, Hercules, CA). Briefly, matrix MacroPrep CM was added to
the culture supernatant at a v/v ratio of 1:75, incubated overnight at
4°C with stirring, sedimented at 1000 x g for 5 min,
and washed three times for 5 min with 100 volumes of 25 mM ammonium
acetate (pH 6.8). Peptides bound to the matrix were eluted with four
matrix volumes of 10% acetic acid for 30 min, followed by subsequent
elutions with 5% acetic acid. Acetic acid eluates were pooled, and
aliquots were lyophilized before electrophoresis on acetic acid urea
gels and immunoblot analysis, as described before (13).
The transfer time was 20 min with fixation in a formalin vapor chamber
for 20 min. Blots were developed using a 1:1000 dilution of polyclonal
rabbit anti-hBD-1 or hBD-2 (12, 13, 15) as primary Ab,
alkaline phosphatase-conjugated goat anti-rabbit IgG as secondary
Ab, and bromochloroindolyl phosphate/nitroblue tetrazolium as substrate
as described before (13). rhBD-1 and rhBD-2 peptides were
used as standards (12, 13, 15).
Immunohistochemistry
Human colon and small-intestinal mucosa from individuals undergoing partial colectomy, small bowel resection, or colon or small bowel endoscopic biopsy was embedded in OCT compound and snap-frozen in isopentane/dry ice. Cryostat sections (5 µm) were air dried, fixed with 4% paraformaldehyde, and then blocked with 0.1 M glycine. Sections were overlaid for 1 h with a 1:1000 dilution of rabbit antisera to hBD-1 or hBD-2 (13, 15) or an identical concentration of control preimmune sera from the same rabbits, after which endogenous biotin was blocked (Avidin/Biotin Blocking Kit; Zymed, South San Francisco, CA). Specific binding of rabbit IgG was detected using the LSAB 2 kit against primary rabbit IgG (Dako, Carpinteria, CA) according to the manufacturer’s instructions and streptavidin-Cy3 to visualize specific binding.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether human colon epithelial cell lines
constitutively express ß-defensins, RNA from unstimulated Caco-2 and
HT-29 cells was analyzed by RT-PCR using hBD-1- and hBD-2-specific
primers. As shown in Fig. 1
, Caco-2 and
HT-29 cells constitutively expressed mRNA for hBD-1, whereas there was
little if any constitutive expression of hBD-2 mRNA. hBD-1 transcript
levels in the cell lines ranged from 105 to
106 transcripts/µg cellular RNA in three
repeated experiments as assessed by quantitative RT-PCR.
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stimulation or infection
with enteroinvasive bacteria, whereas hBD-1 mRNA levels are not
affected
hBD-1 mRNA expression was not up-regulated by stimulation of
Caco-2 or HT-29 cells with IL-1, TNF-, IFN-, LPS, or TPA, or
by infection of those cells with the enteroinvasive bacteria, S.
dublin or enteroinvasive E. coli, as shown in Fig. 1
and confirmed by quantitative RT-PCR. In contrast, hBD-2 mRNA
expression was induced by stimulation of those cells with IL-1 or by
infection with S. dublin or enteroinvasive E.
coli. Maximal expression of hBD-2 mRNA occurred by 4–6 h after
IL-1 stimulation or bacterial infection (peak expression ranged from
105 to 106 transcripts/µg
cellular RNA in three repeated experiments), with transcript levels
returning toward baseline by 8 h. TNF- as well as IFN-, LPS,
and TPA had little to no effect on hBD-2 expression (Fig. 1). Neither
cell line expressed mRNA for either human Paneth cell -defensin,
HD-5 or HD-6 (data not shown).
Production of hBD-1 and hBD-2 peptides by Caco-2 cells
To determine whether expression of hBD-1 and hBD-2 mRNA was
paralleled by the production of those proteins, hBD-1 and hBD-2 were
assayed in supernatants of unstimulated and IL-1-stimulated Caco-2
cells by immunoblot analysis. Consistent with its constitutive mRNA
expression, hBD-1 was present in culture supernatants from unstimulated
Caco-2 cells (Fig. 2). The hBD-1 isoforms
produced by Caco-2 cells appeared to be 47 and 44 aa in length, as
assessed by comparison with hBD-1 peptide standards resolved in
parallel. In some, but not all, experiments, hBD-1 release into culture
supernatants increased after IL-1 stimulation, as depicted in Fig. 2. After 6 h, approximate hBD-1 concentrations in supernatants of
unstimulated cells ranged from 10 to 15 ng/ml as determined by
comparison with known amounts of hBD-1 standards run in parallel. We
note this may underestimate the actual hBD-1 concentration in culture
supernatants because of possible losses of protein in the isolation and
preparative steps.
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-stimulated cells. A 41-aa isoform of hBD-2 has been
identified in saliva, airway surface fluid, and bronchoalveolar lavage
fluid (12, 15, 18). However, the hBD-2 isoform present in
Caco-2 culture supernatants migrated somewhat more slowly than a known
hBD-2 peptide standard of this size, suggesting a peptide larger than
41 aa (Fig. 2). After 6 h, the approximate hBD-2 concentration in
culture supernatants was 5 ng/ml.
hBD-2 is a NF-B target gene
NF-B has a key role in regulating the transcription of several
members of a proinflammatory gene program in intestinal epithelial
cells that is induced in response to inflammation or infection with
enteroinvasive bacteria (e.g., IL-8, ICAM-1, inducible NO synthase,
COX-2, and growth-related oncogene ) (26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36). The
identification of a NF-B consensus sequence in the proximal promoter
of the hBD-2 gene (15, 16) suggested that hBD-2 might be a
component of this epithelial NF-B target gene program. Because IL-1,
S. dublin, and enteroinvasive E. coli are known
to activate NF-B in intestinal epithelial cells (36, 39, 40), we assessed whether the IL-1 and bacterially induced
expression of hBD-2 was mediated by an NF-B-dependent mechanism.
As shown in Fig. 3A,
inhibiting NF-B activation in HT-29 and Caco-2 cells with a
proteasome inhibitor, MG-132, or by infection with a recombinant
adenovirus expressing an IB superrepressor (Ad5IB-A32/36),
completely blocked the IL-1-induced expression of hBD-2 mRNA.
Neither MG-132 nor the IB superrepressor affected hBD-1 or
ß-actin mRNA levels in those cells. To assess whether blocking
NF-B activation also abrogated the expression of hBD-2 in response
to S. dublin or enteroinvasive E. coli infection,
Caco-2 cells that were not infected with adenovirus and Caco-2 cells
infected either with adenovirus expressing the IB superrepressor
(Ad5IB-A32/36) or ß-galactosidase (Ad5LacZ) were infected with
S. dublin or enteroinvasive E. coli O29:NM. As
shown in Fig. 3B, the S. dublin- and
enteroinvasive E. coli-induced expression of hBD-2 mRNA was
decreased in cells expressing the IB superrepressor, but not in
Ad5LacZ-infected cells, whereas hBD-1 and ß-actin mRNA levels
remained unchanged. Parallel results were obtained also using HT-29
cells (data not shown).
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The mucosa of human intestinal xenografts contains surface and
crypt epithelium that is strictly of human origin (28, 29, 31, 32, 37, 38). To confirm the in vitro data generated in colon
epithelial cell lines in an in vivo infection model, human intestinal
xenografts implanted s.c. on the backs of SCID mice were infected
intraluminally with an attenuated aroA aroC
strain of S. typhi for 6 h or were left uninfected as
controls, after which xenografts were harvested and mRNA for hBD-1 and
hBD-2 from mucosal scrapings of the xenografts was amplified by RT-PCR.
As shown in Fig. 4, control xenografts
and S. typhi-infected xenografts expressed mRNA for hBD-1,
whereas the S. typhi-infected xenografts, but not the
controls, also expressed hBD-2 mRNA.
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-defensins HD-5 and HD-6 (1, 3, 4, 5). To
assess whether Paneth cells in the xenografts also express HD-5 and
HD-6 mRNA and whether expression of those defensins is up-regulated in
response to bacterial infection, HD-5 and HD-6 mRNA from control and
Salmonella-infected xenografts was amplified by RT-PCR. As
shown in Fig. 4, xenograft mucosa constitutively expressed mRNA for
HD-5 and HD-6, and expression of these -defensins was similar after
Salmonella infection. hBD-1 and hBD-2 expression by human intestinal epithelial cells in vivo
To determine whether the expression of hBD-1 and hBD-2 by human
colon epithelial cell lines and fetal intestinal xenografts is
representative of adult human intestine, frozen sections of human
intestine were immunostained with polyclonal rabbit antisera to hBD-1
and hBD-2, using preimmunization sera from the same rabbits as a
control. As shown in Fig. 5, hBD-1 was
equally expressed by the epithelium of uninflamed healthy colon (Fig. 5A) and inflamed colon obtained at surgery from a patient
with ulcerative colitis (Fig. 5C). hBD-1 was also equally
expressed by the epithelium of normal (Fig. 5E) and inflamed
duodenal mucosa (Fig. 5G) obtained at the time of endoscopy
from a patient with acid-peptic disease. hBD-1 immunostaining was
present in both surface and crypt epithelial cells of the colon and in
villous and crypt cells of the small intestine. As shown in Fig. 6, there was little to no expression of
hBD-2 by the epithelium of uninflamed colon (Fig. 6A), but
hBD-2 was expressed by surface and crypt cells in the epithelium of
inflamed colon (Fig. 6, C and E).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-defensins, of which the
expression in the human intestine is restricted to Paneth cells in the
crypts of the small bowel (1, 3, 4, 5). Consistent with
ß-defensin expression in respiratory and gingival epithelia and in
the skin, the expression of intestinal epithelial hBD-1 and hBD-2 is
differentially regulated. Our findings suggest that enteric
ß-defensins are integral components of constitutive and regulated
innate host defense.
hBD-2 was expressed by intestinal epithelial cells in response to
IL-1 stimulation or bacterial infection and by the epithelium of
inflamed colonic mucosa in vivo. The up-regulated expression of hBD-2
in intestinal epithelium in response to mucosal inflammation is similar
to that noted in several other tissues. For example, hBD-2 peptide was
not detected in bronchoalveolar lavage fluid from normal individuals
but was present in the fluid from patients with pneumonia, cystic
fibrosis, or inflammatory lung disease (12, 18, 19). In
epidermal tissue, hBD-2 was only expressed in sites surrounding areas
of inflammation, whereas adjacent, noninflamed regions did not express
hBD-2 protein (17). Similarly, hBD-2 mRNA was only
significantly expressed in inflamed gingival epithelial tissue
(15). Our data identify IL-1 as a major agonist for
hBD-2 induction in intestinal epithelial cell lines. Consistent with
this finding, expression of hBD-2 mRNA and protein in cultured tracheal
epithelial cells (12), and hBD-2 mRNA expression in
gingival keratinocytes was induced following IL-1 stimulation
(15).
The proximal promoter of hBD-2 has consensus NF-B binding sites, and
activation of NF-B appears to be necessary for optimal hBD-2 gene
expression in human colon epithelial cell lines. Consistent with a role
of NF-B, blocking NF-B activation with a proteasome inhibitor,
MG-132, or a mutant IB that acts as a superrepressor of NF-B
activation, abrogated IL-1 and bacterially induced expression of
hBD-2. Nonetheless, activation of NF-B does not appear to be
sufficient for high level hBD-2 expression, as TNF- was a weak
inducer of hBD-2, although it maximally activated NF-B and the
expression of other NF-B target genes (e.g., IL-8 and ICAM-1) in the
same cells (data not shown) in which there was little, if any,
induction of hBD-2. This finding indicates that transcriptional
regulation of hBD-2 differs from that of several other intestinal
epithelial cell NF-B target genes.
The six isoforms of hBD-1 identified in urine differ by truncations at
the amino terminus and range in length from 36 to 47 aa
(13). These hBD-1 isoforms vary in microbicidal activity,
and there are significant interindividual variations in the relative
proportions of the isoforms present in urine (13).
Nonetheless, two hBD-1 isoforms of 44 and 40 aa predominate. Immunoblot
analysis revealed that two isoforms of hBD-1, 47 and 44 aa in
length, were produced by Caco-2 cells. However, it is possible that
additional forms of enteric hBD-1 may be generated intraluminally by
N-terminal processing in vivo. hBD-1 was constitutively expressed by
colon epithelial cell lines, intestinal xenograft tissue, and adult
human colon tissue. Nonetheless, increased release of hBD-1 peptide was
observed in some experiments following IL-1 stimulation of Caco-2
cells. This indicates that IL-1 can increase translation and/or
release of hBD-1 peptide into the extracellular environment, but
whether parallel events occur in vivo is not known.
Bronchoalveolar lavage fluid and airway surface fluid from cultured
airway epithelia contains a 41-aa isoform of hBD-2 (12, 18). The same hBD-2 isoform was identified in saliva
(15). In contrast, the hBD-2 peptide produced by
IL-1-stimulated Caco-2 cells migrated slower than a 41-aa hBD-2
peptide standard, suggesting a larger isoform. This peptide may be a
precursor of the 41-aa variant that, under physiological conditions,
may be subjected to N-terminal processing to yield a shorter
peptide(s), just as the 47-aa hBD-1 is cleaved to yield a range of
shorter isoforms of hBD-1 (13). As processing conditions
at each mucosal site could be distinct in vivo, there may be
differences in cleavage of epithelial cell-derived peptides,
particularly between luminal environments as different as the lung and
intestine. Further, it is possible that N-terminal variations may
result in differences in peptide stability and microbicidal activity
within different microenvironments.
LPS did not induce the expression of hBD-2 in colon epithelial cell
lines. However, LPS has been shown to induce hBD-2 in other mucosal
tissues, namely gingival and epidermal keratinocytes (8, 15), suggesting that there are differences in the regulation of
ß-defensin expression among mucosal sites. In bovines, both LPS and
TNF- up-regulated tracheal antimicrobial peptide and lingual
antimicrobial peptide mRNA expression by cultured tracheal epithelial
cells (20, 22, 23). DefB1 or mBD-1 and Defb2/mBD-3 are
murine homologues of hBD-1 and hBD-2, respectively (24, 25, 41, 42). In respiratory epithelia in vivo, Defb1/mBD-1 expression is
constitutive (41, 42), whereas expression of Defb2 and
mBD-3 mRNA is induced following infection with Pseudomonas
aeruginosa or following LPS stimulation (24, 25).
Differences in ß-defensin regulation between different sites and
species may reflect the adaptation of epithelial cells to the
microenvironment in which the ß-defensins function as components of
host defense. Thus, the failure of LPS to induce hBD-2 expression in
intestinal epithelial cell lines may reflect an adaptation of the
intestinal epithelial cells to the high levels of bacterial LPS that
are normally present in the human colon compared with the lower levels
in the oral mucosa and airway epithelium. Alternatively, there may be
differences in the LPS responsiveness of the cell lines compared with
intestinal epithelium in vivo.
The genes encoding hBD-1 and hBD-2 have 39% identity and appear to
have evolved by duplication of a single ancestral gene (16, 17). Our data suggest that the products of these genes occupy
distinct functional niches with the NF-B consensus sequence
distinguishing an inducible (hBD-2) from a constitutive (hBD-1) form of
intestinal epithelial peptide antibiotic. Constitutively expressed
hBD-1 could mediate epithelial interactions with the commensal flora,
whereas hBD-2 may participate in the host defense response to enteric
microbes that can breach the epithelial barrier. The discovery of
structurally and functionally homologous forms of constitutive and
inducible epithelial ß-defensins in the mouse (24, 25, 41, 42) may allow these important issues to be tested in the context
of an animal model.
hBD-1 and hBD-2 have bactericidal activity against a spectrum of
Gram-positive and Gram-negative enteric, urinary tract, and respiratory
bacteria in vitro. However, we note that bactericidal activity of the
ß-defensins, like that of the -defensins and cathelicidins in
humans, is salt sensitive (1, 4, 11, 13, 18, 43, 44), and
exact salt concentrations at the putative site of action of these
peptides in vivo are not known. To the extent that these peptides act
in microenvironments at the interface between the intestinal epithelial
membrane, which has ion pumps and channels, and the intestinal lumen,
regional variations in salt concentrations may be present and
ultimately determine the in vivo microbicidal activity of the
intestinal epithelial cell ß-defensins.
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Acknowledgments |
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Footnotes |
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2 Current address: La Jolla Institute for Allergy and Immunology, San Diego, CA 92121.
3 Address correspondence and reprint requests to Dr. Martin F. Kagnoff, University of California, San Diego, Department of Medicine (0623D), 9500 Gilman Drive, La Jolla, CA 92093-0623. E-mail address:
4 Abbreviations used in this paper: HD, human defensin; rh, recombinant human; TPA, 12-O-tetradecanoylphorbol-13-acetate; MOI, multiplicity of infection; mBD, murine ß-defensin.
Received for publication August 13, 1999. Accepted for publication October 6, 1999.
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K. Hase, L. Eckmann, J. D. Leopard, N. Varki, and M. F. Kagnoff Cell Differentiation Is a Key Determinant of Cathelicidin LL-37/Human Cationic Antimicrobial Protein 18 Expression by Human Colon Epithelium Infect. Immun., February 1, 2002; 70(2): 953 - 063. [Abstract] [Full Text] [PDF]
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T. G. A. M. Wolfs, W. A. Buurman, A. van Schadewijk, B. de Vries, M. A. R. C. Daemen, P. S. Hiemstra, and C. van 't Veer In Vivo Expression of Toll-Like Receptor 2 and 4 by Renal Epithelial Cells: IFN-{gamma} and TNF-{alpha} Mediated Up-Regulation During Inflammation J. Immunol., February 1, 2002; 168(3): 1286 - 1293. [Abstract] [Full Text] [PDF]
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Y. Tsutsumi-Ishii and I. Nagaoka NF-{kappa}B-mediated transcriptional regulation of human {beta}-defensin-2 gene following lipopolysaccharide stimulation J. Leukoc. Biol., January 1, 2002; 71(1): 154 - 162. [Abstract] [Full Text] [PDF]
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S. Krisanaprakornkit, J. R. Kimball, and B. A. Dale Regulation of Human {beta}-Defensin-2 in Gingival Epithelial Cells: The Involvement of Mitogen-Activated Protein Kinase Pathways, But Not the NF-{kappa}B Transcription Factor Family J. Immunol., January 1, 2002; 168(1): 316 - 324. [Abstract] [Full Text] [PDF]
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N. S. Goncalves, M. Ghaem-Maghami, G. Monteleone, G. Frankel, G. Dougan, D. J. M. Lewis, C. P. Simmons, and T. T. MacDonald Critical Role for Tumor Necrosis Factor Alpha in Controlling the Number of Lumenal Pathogenic Bacteria and Immunopathology in Infectious Colitis Infect. Immun., November 1, 2001; 69(11): 6651 - 6659. [Abstract] [Full Text] [PDF]
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D. Yang, O. Chertov, and J. J. Oppenheim Participation of mammalian defensins and cathelicidins in anti-microbial immunity: receptors and activities of human defensins and cathelicidin (LL-37) J. Leukoc. Biol., May 1, 2001; 69(5): 691 - 697. [Abstract] [Full Text]
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C. Zhao, T. Nguyen, L. Liu, R. E. Sacco, K. A. Brogden, and R. I. Lehrer Gallinacin-3, an Inducible Epithelial {beta}-Defensin in the Chicken Infect. Immun., April 1, 2001; 69(4): 2684 - 2691. [Abstract] [Full Text] [PDF]
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M. Cecilia Berin, L. Eckmann, D. H. Broide, and M. F. Kagnoff Regulated Production of the T Helper 2-Type T-Cell Chemoattractant TARC by Human Bronchial Epithelial Cells In Vitro and in Human Lung Xenografts Am. J. Respir. Cell Mol. Biol., April 1, 2001; 24(4): 382 - 389. [Abstract] [Full Text]
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 A. Izadpanah, M. B. Dwinell, L. Eckmann, N. M. Varki, and M. F. Kagnoff Regulated MIP-3{alpha}/CCL20 production by human intestinal epithelium: mechanism for modulating mucosal immunity Am J Physiol Gastrointest Liver Physiol, April 1, 2001; 280(4): G710 - G719. [Abstract] [Full Text] [PDF]
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 C. M. Steppan, E. J. Brown, C. M. Wright, S. Bhat, R. R. Banerjee, C. Y. Dai, G. H. Enders, D. G. Silberg, X. Wen, G. D. Wu, et al. A family of tissue-specific resistin-like molecules PNAS, January 16, 2001; 98(2): 502 - 506. [Abstract] [Full Text] [PDF]
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V. Kaiser and G. Diamond Expression of mammalian defensin genes J. Leukoc. Biol., December 1, 2000; 68(6): 779 - 784. [Abstract] [Full Text]
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 R. Krzysiek, E. A. Lefevre, J. Bernard, A. Foussat, P. Galanaud, F. Louache, and Y. Richard Regulation of CCR6 chemokine receptor expression and responsiveness to macrophage inflammatory protein-3alpha /CCL20 in human B cells Blood, October 1, 2000; 96(7): 2338 - 2345. [Abstract] [Full Text] [PDF]
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M. G. Scott, C. M. Rosenberger, M. R. Gold, B. B. Finlay, and R. E. W. Hancock An {alpha}-Helical Cationic Antimicrobial Peptide Selectively Modulates Macrophage Responses to Lipopolysaccharide and Directly Alters Macrophage Gene Expression J. Immunol., September 15, 2000; 165(6): 3358 - 3365. [Abstract] [Full Text] [PDF]
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D. A. O'Neil, S. P. Cole, E. Martin-Porter, M. P. Housley, L. Liu, T. Ganz, and M. F. Kagnoff Regulation of Human beta -Defensins by Gastric Epithelial Cells in Response to Infection with Helicobacter pylori or Stimulation with Interleukin-1 Infect. Immun., September 1, 2000; 68(9): 5412 - 5415. [Abstract] [Full Text] [PDF]
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 K.S. Sahasrabudhe, J.R. Kimball, T.H. Morton, A. Weinberg, and B.A. Dale Expression of the Antimicrobial Peptide, Human {beta}-defensin 1, in Duct Cells of Minor Salivary Glands and Detection in Saliva Journal of Dental Research, September 1, 2000; 79(9): 1669 - 1674. [Abstract] [PDF]
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R N CUNLIFFE and Y R MAHIDA Antimicrobial peptides in innate intestinal host defence Gut, July 1, 2000; 47(1): 16 - 17. [Full Text] [PDF]
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J. Krijgsveld, S. A. J. Zaat, J. Meeldijk, P. A. van Veelen, G. Fang, B. Poolman, E. Brandt, J. E. Ehlert, A. J. Kuijpers, G. H. M. Engbers, et al. Thrombocidins, Microbicidal Proteins from Human Blood Platelets, Are C-terminal Deletion Products of CXC Chemokines J. Biol. Chem., June 30, 2000; 275(27): 20374 - 20381. [Abstract] [Full Text] [PDF]
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M. N. Becker, G. Diamond, M. W. Verghese, and S. H. Randell CD14-dependent Lipopolysaccharide-induced beta -Defensin-2 Expression in Human Tracheobronchial Epithelium J. Biol. Chem., September 15, 2000; 275(38): 29731 - 29736. [Abstract] [Full Text] [PDF]
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J. Harder, J. Bartels, E. Christophers, and J.-M. Schroder Isolation and Characterization of Human beta -Defensin-3, a Novel Human Inducible Peptide Antibiotic J. Biol. Chem., February 16, 2001; 276(8): 5707 - 5713. [Abstract] [Full Text] [PDF]
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K.-i. Ogushi, A. Wada, T. Niidome, N. Mori, K. Oishi, T. Nagatake, A. Takahashi, H. Asakura, S.-i. Makino, H. Hojo, et al. Salmonella enteritidis FliC (Flagella Filament Protein) Induces Human beta -Defensin-2 mRNA Production by Caco-2 Cells J. Biol. Chem., August 3, 2001; 276(32): 30521 - 30526. [Abstract] [Full Text] [PDF]
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Y. Yamaguchi, S. Fukuhara, T. Nagase, T. Tomita, S. Hitomi, S. Kimura, H. Kurihara, and Y. Ouchi A Novel Mouse beta -Defensin, mBD-6, Predominantly Expressed in Skeletal Muscle J. Biol. Chem., August 17, 2001; 276(34): 31510 - 31514. [Abstract] [Full Text] [PDF]
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Y. S. Lopez-Boado, C. L. Wilson, and W. C. Parks Regulation of Matrilysin Expression in Airway Epithelial Cells by Pseudomonas aeruginosa Flagellin J. Biol. Chem., October 26, 2001; 276(44): 41417 - 41423. [Abstract] [Full Text] [PDF]
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P. Fehlbaum, M. Rao, M. Zasloff, and G. M. Anderson An essential amino acid induces epithelial beta -defensin expression PNAS, November 7, 2000; 97(23): 12723 - 12728. [Abstract] [Full Text] [PDF]
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