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Infect Immun, July 1998, p. 3255-3263, Vol. 66, No. 7
0019-9567/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Potential Role of Epithelial Cell-Derived Histone
H1 Proteins in Innate Antimicrobial Defense in the Human
Gastrointestinal Tract
F. R. A. J.
Rose,1,2
K.
Bailey,3
J. W.
Keyte,3
W. C.
Chan,4
D.
Greenwood,5 and
Y. R.
Mahida1,2,*
Divisions of
Gastroenterology1 and
Microbiology,5
Departments of
Biochemistry3 and
Pharmaceutical
Sciences,4 and
Institute of
Infections and Immunity,2 University of
Nottingham, Nottingham, United Kingdom
Received 30 January 1998/Returned for modification 26 March
1998/Accepted 15 April 1998
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ABSTRACT |
In the human gastrointestinal tract, microorganisms are present in
large numbers in the colon but are sparse in the proximal small
intestine. In this study, we have shown that acid extracts of fresh
human terminal ileal mucosal samples mediate antimicrobial activity.
Following cation-exchange chromatography, one of the eluted fractions
demonstrated antibacterial activity against bacteria normally resident
in the human colonic lumen. This activity was further fractionated by
reverse-phase high-performance liquid chromatography and identified as
histone H1 and its fragments. We have also shown that in tissue
sections, immunoreactive histone H1 is present in the cytoplasm of
villus epithelial cells. In vitro culturing of detached (from the
basement membrane) villus epithelial cells led to the release of
antimicrobial histone H1 proteins, while the cells demonstrated
ultrastructural features of programmed cell death. Our studies suggest
that cytoplasmic histone H1 may provide protection against penetration
by microorganisms into villus epithelial cells. Moreover, intestinal
epithelial cells released into the lumen may mediate antimicrobial
activity by releasing histone H1 proteins and their fragments.
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INTRODUCTION |
Most of the exposure of the human
body to bacteria occurs in the gastrointestinal tract. In the upper
gut, this exposure occurs in association with ingestion of nutrients,
whereas in the colon, there is a large resident population of
microorganisms (estimated to be 1014
[31]). A variety of host responses are required to
mediate protection against invasion by pathogens and to achieve
relative sterility in the small intestine, the lumen of which is rich
in nutrients. These host responses can be divided into innate and adaptive defense systems (17). The latter entails the
development of specific responses embodied in the gastrointestinal
immune system and mediated by secretory immunoglobulin A (IgA)
antibodies (34). Specific secretory IgA-mediated protection
takes time to develop (8); therefore, a preexisting or
rapidly responsive antimicrobial defense is required. Surface
epithelial cells are of critical importance in mediating innate
protection against microbes in the lumen of the gastrointestinal tract.
Recent studies with animals (3, 18-20, 29) and humans
(13, 24, 25) suggested the existence of a novel form of
preexisting innate host protection which is operative in the mucus
layer and the lumen and which is mediated by peptides with
broad-spectrum antibacterial activities. These peptides, of the
defensin family, are expressed by Paneth cells, which are located at
the base of small intestinal crypts (3, 13, 18-20, 24, 29).
In addition to this preexisting innate protection, intestinal
epithelial cells may be capable of mediating antimicrobial activity
following injury.
In studies to investigate the expression of antimicrobial activities in
extracts of human terminal ileal mucosa, we have isolated and
characterized antimicrobial histone H1 proteins and their fragments. In
immunohistochemical studies on tissue sections, histone H1 was found to
be expressed in the cytoplasm of villus epithelial cells. In addition,
detached ileal epithelial cells were shown to release antimicrobial
histone H1 while undergoing apoptosis (programmed cell death). Our
studies suggest that cytoplasmically expressed histone H1 may provide
protection against penetration by microorganisms into villus epithelial
cells. In addition, intestinal epithelial cells released into the lumen
may be capable of releasing antimicrobial histone H1 protein while
undergoing apoptosis.
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MATERIALS AND METHODS |
Mucosal tissue.
Fresh human terminal ileal mucosal samples
were obtained from operation resection specimens (right
hemicolectomies; samples obtained >5 cm from tumor; 38 patients) and
brain-dead organ donors (approved by the Ethics Committee of Nottingham
University Hospitals; 8 donors).
Isolation and purification of histone H1.
Terminal ileal
mucosa was dissected from submucosa and extracted with 10% (vol/vol)
acetic acid. The mucosal samples were homogenized, sonicated, and
stirred overnight. After centrifugation (at 67,000 × g
for 60 min at 4°C), the pellets were reextracted with 10% acetic
acid. The supernatants from both extraction procedures were pooled and
applied to a cation-exchange column (SP Sepharose Fast Flow; Pharmacia
Biotech, Uppsala, Sweden), and positively charged molecules were eluted
with a 0 to 1 M NaCl gradient. The eluted fractions were dialyzed with
a 1-kDa-cutoff dialysis membrane (Sectra Por membrane; Pierce & Warriner, Chester, United Kingdom), lyophilized, and tested for
antimicrobial activity. Fractions expressing antimicrobial activity
were purified further by reverse-phase high-performance liquid
chromatography (RP-HPLC) on an Aquapore C4 column (Brownlee
column; Applied Biosystems Ltd., Foster City, Calif.) with a linear
water-acetonitrile gradient that contained 0.1% trifluoroacetic acid.
The purity of the polypeptides was assessed by RP-HPLC (C18
column) and acid urea-polyacrylamide gel electrophoresis (AU-PAGE).
AU-PAGE.
Extracted mucosal samples were analyzed by AU-PAGE
as previously described (15, 21). In brief, acid urea (6.25 M)-15% polyacrylamide minigels were prepared and prerun with 5%
acetic acid for 45 to 60 min at 150 V. Samples (in 3 M urea with 5%
acetic acid) were electrophoresed with 5% acetic acid at 150 V until the methyl green dye front had migrated near the end of the gel. The
gels were subsequently stained with Coomassie blue for 15 min.
For protein sequence analysis, the proteins and peptides were
electrophoretically transferred to polyvinylidene difluoride (PVDF)
membranes (Applied Biosystems Ltd.) (33). In brief, samples were electrophoresed as described above. With a Trans Blot cell (Bio-Rad Laboratories, Hemel Hempstead, United Kingdom), the gel and
the PVDF membrane were sandwiched between filter papers in the transfer
cassette. This was inserted into a tank containing 1% (vol/vol) acetic
acid, with the PVDF membrane facing the cathode. A constant current of
70 mA was applied for 24 h.
Amino acid analysis.
The amino acid compositions of purified
proteins and peptides were determined by use of an Applied Biosystems
420H automated amino acid analyzer. This method was also used to
quantify the amount of histone in a given sample. A known volume was
applied to the analyzer, and the height of the RP-HPLC peak for each
amino acid was recorded. These values were compared to those of
standards; from this comparison, the quantity of each amino acid in the
unknown sample was determined. The total protein in the known volume
was calculated from the amino acid data and thus revealed the
concentration of the sample. Proteins were also quantified by the
Bradford assay (1).
Protein sequencing.
All protein sequencing was carried out
by use of an Applied Biosystems 473A automated sequencer with protocols
and chemicals supplied by the manufacturer and based on Edman
degradation chemistry (2). Large protein fragments were
sequenced directly following their separation by AU-PAGE and subsequent
transfer to PVDF membranes (see above). Visualization of transferred
fragment bands was achieved by staining the membranes with amido black
and destaining with 50% methanol in water.
RP-HPLC-purified proteins, which were assumed to be modified at the N
terminus, were digested with endoprotease Lys-C (Promega
UK Ltd.,
Southampton, United Kingdom). Proteins were digested
overnight at
37°C in 200 mM ammonium bicarbonate. The generated
peptide fragments
were separated by RP-HPLC on an Aquapore C
4 column (Applied
Biosystems Ltd.) with an acetonitrile gradient
of 3 to 40% over 35 min. Selected peptide peaks were collected
and applied to
Briobrene-coated glass fiber discs for subsequent
automated sequencing.
Generated sequence data were analyzed with
Applied Biosystems 610A data
analysis software, and protein sequences
were identified from the
SwissProt database by use of the FASTA
algorithm (
23).
Antimicrobial assays.
The antimicrobial activities of
mucosal extracts and their purified fractions were studied by use of
radial diffusion (15) and 96-well microtiter plate
(established in our laboratory) antimicrobial assays. These assays were
performed with Salmonella typhimurium CS015, a
phoP mutant of the wild-type strain (5) (generous gift from S. Miller, Massachusetts General Hospital, Boston); Salmonella typhimurium ATCC 14028 (wild-type strain),
Streptococcus faecium ATCC 19434, and Streptococcus
faecalis ATCC 19433 (from the National Collection of Type
Cultures); Escherichia coli ATCC 25922 (from the National
Collection of Industrial and Marine Bacteria); Escherichia
coli ATCC 29648 (from the American Type Culture Collection); and
Staphylococcus aureus ATCC 9144 and a beta-hemolytic
Streptococcus group G clinical isolate (both generous gifts
from R. Edwards, Queens Medical Centre, Nottingham, United Kingdom).
Radial diffusion assays were performed as previously described
(
14). In brief, bacteria (in the mid-logarithmic phase) were
added to warm (approximately 37°C) 1% (wt/vol)
low-electroendosmosis-type
agarose (Sigma)-0.02% (wt/vol) Tween 20 (Sigma)-0.03% (wt/vol)
Trypticase soy broth (TSB; Becton Dickinson)
in 10 mM sodium phosphate
buffer (pH 7.4). This agarose mixture was
poured onto petri dishes
and allowed to set for 30 to 60 min at 4°C.
Wells (4-mm diameter)
were made in this underlay, and samples (5 to 20 µl in 0.1% [vol/vol]
acetic acid [16 mM]) were applied to each
well. Acetic acid (0.1%)
alone was used as a control. After incubation
for 2 h at 37°C,
the lower layer of agarose was covered with a
nutrient-rich top
layer (1% [wt/vol] agarose-6% [wt/vol] TSB in
distilled water).
Bacteria in the underlay were allowed to grow by
incubation for
18 h at 37°C. Antimicrobial activity in samples
was demonstrated
by the presence of a bacterium-free zone around the
wells. Antimicrobial
activity was expressed in square millimeters and
was calculated
with the formula [(
y 
x)/2]
2, where
y is the diameter of
the zone of bacterial clearance (millimeters)
and
x is the
diameter of the well (4 mm). For example, a clear
zone whose total
diameter was 10 mm (including the sample well)
would represent 9 mm
2 of activity, whereas one whose total diameter was 5 mm
(including
the sample well) would represent 0.25 mm
2 of
activity.
We also used a microtiter plate antimicrobial assay to allow bacterial
growth and its inhibition to be assessed over 5 h.
Bacteria were
grown to the mid-logarithmic phase and diluted 1:2
with fresh broth,
and 10 µl was applied to wells of a 96-well
microtiter plate
(polystyrene, flat bottom, sterile; Costar Corporation,
Cambridge,
Mass.) containing 50 to 80 µl of 10 mM
piperazine-
N,
N'-bis(2-ethanesulfonic
acid)
(PIPES) buffer (pH 7.4) and 10 to 40 µl of sample (in 0.1%
acetic
acid) to achieve a final volume in the well of 100 µl.
Based on the
fact that an optical density at 620 nm (OD
620) of
0.2 was
equal to 5 × 10
7 CFU/ml (
15), a range of
6.25 × 10
7 to 8.75 × 10
7 CFU/ml was
added to each assay well. After incubation of the
plate at 37°C for
1 h, 100 µl of prewarmed (to 37°C) double-strength
(6%
[wt/vol]) TSB was applied to each well (final pH range, 6.8
to 6.9).
The OD
620 of the wells was determined soon after the
application of TSB with a Labsystems iEMS Reader MF. The plate
was
subsequently incubated at 37°C for 5 h, and optical density
readings were taken every hour. The same volume of 0.1% acetic
acid as
in the test sample was used as a control. The antimicrobial
activity of
test samples was expressed as the percent reduction
in bacterial growth
(OD
620) after 5 h of incubation at 37°C compared
to
the bacterial growth (OD
620) of the 0.1% acetic acid
control
with the following equation: antimicrobial activity = [(OD
620 of control
OD
620 of
sample)/OD
620 of control] × 100. For example,
the
antimicrobial activity for a sample with an OD
620 of 0.1 and
a control OD
620 of 0.6 (both after 5 h of
incubation at 37°C)
would be 83.3%.
Immunohistochemical analysis.
Cryostat sections (6 µm
thick) of human small intestine and cytospin preparations of
EDTA-isolated terminal ileal epithelial cells (see below) were air
dried, fixed in acetone for 10 min, and labelled with anti-human
histone H1 monoclonal antibodies (Cambio, Cambridge, United Kingdom).
Bound antibodies were detected with biotinylated anti-mouse
immunoglobulin G followed by an avidin-biotinylated horseradish
peroxidase complex (Vectastain Elite ABC kit; Vecta Laboratories,
Burlingame, Calif.). Peroxidase activity was developed with
3'-diaminobenzidine tetrahydrochloride, but nuclear counterstaining was
not performed. Control sections and cytospin preparations were
incubated with phosphate-buffered saline (PBS) (pH 7.4) instead of the
primary anti-human histone antibodies. All other steps were identical.
Dot blot analysis.
Samples (10 µl) of acetic acid extracts
of supernatants of detached and cultured terminal ileal epithelial
cells (see below) were applied to PVDF membranes by use of a
microfiltration apparatus attached to a water-vac system. The PVDF
membranes were subsequently incubated with anti-human histone H1
antibodies or control buffer (PBS [pH 7.4]). Bound antibodies were
detected with the Vectastain Elite ABC kit as described above.
Isolation of epithelial cells.
Villus epithelial cells of
fresh human terminal ileal mucosal samples were detached with EDTA as
described previously (16). In brief, strips (approximately 5 by 2 mm) of mucosa were incubated with 1 mM EDTA in a shaking water
bath at 37°C for 30 min. The detached epithelial cells were washed
three times with PBS (pH 7) by centrifugation at 400 × g for 10 min each time. After the third wash, the cells were
resuspended in PBS (pH 7) and incubated at 37°C in 5%
CO2 for 3 or 18 h.
Cell culture supernatants were obtained by centrifugation (at
13,400 ×
g for 30 min at 4°C). These were
subsequently extracted
with 10% (vol/vol) acetic acid by overnight
stirring, followed
by centrifugation (at 13,400 ×
g
for 30 min at 4°C). The extracts
were lyophilized, resuspended in
0.1% acetic acid, and subsequently
used in antimicrobial assays,
AU-PAGE, and immunoblot studies
(see above). Cultured epithelial cells
were studied by transmission
electron microscopy (TEM) (see below).
Cytospin preparations of
detached epithelial cells were also made
(approximately 50,000
cells per slide), fixed in acetone, and stored at
20°C before
immunohistochemical studies (see above).
Electron microscopy.
Cultured epithelial cells (see above)
were obtained for TEM after each incubation period. These cells were
fixed in 2.5% glutaraldehyde (in 0.1 M cacodylate buffer [pH 7.4])
for 24 h at 4°C and processed according to standard procedures
(28). Sections (80 nm) were mounted on copper grids and
stained with uranyl acetate and lead citrate prior to examination on a
JEOL 1010 transmission electron microscope.
Immunoelectron microscopy.
Sections (15 µm) of normal
human small intestine were incubated with anti-human histone H1
monoclonal antibodies. Labelled antibodies were detected with the
Vectastain Elite ABC kit as described above. After development of
peroxidase activity, sections were fixed in 2.5% glutaraldehyde (in
0.1 M cacodylate buffer [pH 7.4]), postfixed, dehydrated in ascending
grades of alcohol, and infiltrated with Taab resin (28). A
resin-filled capsule was inverted over the section at the region of
interest and allowed to polymerize at 60°C for 18 h. After
removal of the capsules, 0.5-µm sections were cut and stained with
1% toluene blue. Areas containing peroxidase-positive epithelial cells
were selected, and ultrathin sections (which were not counterstained
with toluidine blue or hematoxylin) were examined by TEM.
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RESULTS |
Unfractionated terminal ileal mucosal extracts.
All acetic
acid extracts (some of which were pooled) of terminal ileal mucosal
samples obtained from a total of 38 resected intestines demonstrated
potent activity against S. typhimurium CS015 (mean ± standard deviation [SD] reduction in bacterial OD620 at
5 h, 90.7% ± 6.0% [n = 29]; Fig.
1) and against two strains of E. coli, ATCC 25922 (reduction in bacterial OD620, 89.3% ± 6.1%) and ATCC 29648 (reduction in bacterial OD620,
66.1% ± 3.5%). Bacterial growth was always observed in the control
sample (containing the same volume of 0.1% acetic acid), as shown in
the representative experiment in Fig. 1.
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FIG. 1.
Activity against S. typhimurium CS015 by acid
extracts of human terminal ileal mucosa. The mucosal extract (in 0.1%
acetic acid) ( ) and the control (0.1% acetic acid only) ( ) were
applied, together with bacteria, to 96-well microtiter plates, and
bacterial growth was assessed hourly. The data represent one of 29 experiments performed.
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Activity against
S. typhimurium CS015 was also observed in
the radial diffusion assay (17.0-mm
2 zone of bacterial
clearance).
Fractionation of mucosal extracts by cation-exchange
chromatography.
Aqueous acetic acid extracts of terminal ileal
mucosal samples were applied to a cation-exchange column incorporated
into a fast protein liquid chromatography apparatus. Bound, positively charged proteins were eluted with a 0 to 1 M NaCl gradient. After extensive dialysis, four eluted fractions demonstrated activity against
S. typhimurium CS015. One of the active fractions
(designated fraction 15) was used for further studies as it
demonstrated impressive antimicrobial activity and distinct bands on
Coomassie blue-stained AU-PAGE. This fraction also demonstrated
activity against E. coli ATCC 25922, E. coli ATCC
29648, and S. faecium ATCC 19434 (each tested once) and
against S. faecalis ATCC 19433 and S. aureus ATCC
9144 (each tested twice) (Table 1).
Although the fraction demonstrated activity against the phoP
mutant of S. typhimurium (CS015), no activity was present
against wild-type S. typhimurium ATCC 14028 or against a
beta-hemolytic Streptococcus group G clinical isolate (each
tested twice) (Table 1).
This active fraction (fraction 15) was applied to an Aquapore
C
4 RP-HPLC column, and of the fractions eluted, three
demonstrated
activity against
S. typhimurium CS015 (Fig.
2a). Analysis of one
of these (fraction
H) on an Aquapore C
18 RP-HPLC column and by
AU-PAGE
demonstrated the presence of a single entity (Fig.
2b).
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FIG. 2.
(a) RP-HPLC fractionation of semipurified ileal mucosal
extract. Antimicrobial fraction 15 eluted from the cation-exchange
column (1 M NaCl) was applied to an Aquapore C4 column, and
of the eluted fractions, F, G, and H demonstrated antimicrobial
activity. (b) AU-PAGE of fraction 15 (lane 1), fraction H (lane 3), and
synthetic magainin (lane 2). Of the many positively charged peptides
and proteins present in fraction 15, one was purified by RP-HPLC
(fraction H in panel a).
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Amino acid analysis of the purified protein showed that it was rich in
lysine and alanine (Table
2). N-terminal
sequencing
failed to provide data, suggesting that it was N terminally
blocked.
The purified protein was therefore digested with Lys-C, and
three
peptide fragments were subsequently purified by RP-HPLC. Amino
acid sequence analysis of these fragments showed them to be 100%
homologous to human histones H1b, H1c, and H1d (Fig.
3).
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TABLE 2.
Amino acid composition of an antimicrobial protein
purified from acid extract of human terminal
ileal mucosaa
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FIG. 3.
C4 RP-HPLC separation of fraction H (Fig. 2)
following digestion with Lys-C. Eluted peaks (1, 2, and 3) were
collected and sequenced. Peak 1 (retention time, 9.45 min) gave a
single unequivocal sequence. Peak 2 (retention time, 11.86 min) gave
two signals for some cycles of sequence analysis, indicating a mixture
of two peptides. However, these two signals were of sufficiently
different intensities to allow the two sequences to be assigned (2a and
2b). Peak 3 gave no sequence, suggesting that it contained the
undigested N terminally blocked protein. The amino acid sequence (amino
acids 31 to 80) of human histone H1d is shown. Amino acid sequences of
peptides 1, 2a, and 2b showed 100% homology to histone H1d. In
addition, these peptides also showed 100% sequence homology to
histones H1b and H1c.
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Antimicrobial activity of purified histone H1 protein.
The
purified histone H1 protein demonstrated potent activity against
S. typhimurium CS015 (mean ± SD reduction in bacterial OD620 at 5 h, 93.7% ± 11.9%; tested 10 times; MIC,
3.47 to 6.95 µg/ml) but not against wild-type S. typhimurium ATCC 14028 (mean ± SD reduction in bacterial
OD620 at 5 h, 10.4% ± 6.3%; tested 3 times).
Histone H1 fragments mediate antimicrobial activity.
Antimicrobial activity from another human terminal ileal mucosal
extract was determined by cation-exchange chromatography and RP-HPLC as
described above. One fraction demonstrated potent activity against
S. typhimurium CS015 (mean ± SD reduction in bacterial
OD620 at 5 h, 90.3% ± 2.0%; tested three times).
AU-PAGE showed that this fraction contained four distinct protein
entities. Following transfer to a PVDF membrane, amino acid sequence
analysis of these four proteins showed them to be 100% homologous to
histone H1 proteins (Fig. 4).
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FIG. 4.
AU-PAGE of an antimicrobial fraction of human ileal
mucosal extract. This fraction was obtained by cation-exchange
chromatography, followed by RP-HPLC. AU-PAGE analysis demonstrated the
presence of four distinct protein bands (stained by Coomassie blue).
Following transfer to a PVDF membrane, amino acid sequence analyses of
these four proteins revealed 100% homology to histone H1 proteins.
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Expression of histone H1 proteins in human terminal ileal
mucosa.
In order to investigate the expression of histone H1
proteins in human terminal ileal mucosa in vivo, immunohistochemical analysis was performed on tissue sections with a monoclonal antibody to
histone H1. As expected, these studies (in which no nuclear counterstaining was performed) showed the presence of immunoreactive histone H1 in nuclei of epithelial cells and cells in the lamina propria. In addition, immunoreactivity was also seen in the cytoplasm of some villus epithelial cells. These cells were present at the villus
tip and on the lateral aspect of some villi (Fig.
5). Studies by electron microscopy showed
that immunoreactive histone H1 was present in the cytoplasm of the
epithelial cells (Fig. 6).
Immunohistochemical studies on cytospin preparations of detached villus
epithelial cells also demonstrated the presence of immunoreactive
histone H1 in the cytoplasm of some cells (Fig.
7).
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FIG. 5.
Expression of histone H1 in human terminal ileal mucosa.
Immunohistochemical analysis was performed with an anti-histone H1
monoclonal antibody, but a nuclear stain was not used. (a) Histone H1
immunoreactivity was seen in the cytoplasm of epithelial cells, at the
tip and on the lateral aspect of villi (arrows), as well as in nuclei.
(b) Villus (from the left margin of panel a) at a high magnification.
Arrows indicate immunoreactivity. Approximate magnifications: a, ×224;
b, ×389.
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FIG. 6.
Immunoelectron microscopy of a section of normal human
small intestinal mucosa labelled with an anti-human histone H1
monoclonal antibody by the peroxidase technique. Two epithelial cells
near the villus tip are shown. (a) In one, immunoreactive histone H1
(arrows) was present in the cytoplasm below the microvilli. (b) The
cytoplasm of an adjacent epithelial cell did not contain immunoreactive
histone H1.
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FIG. 7.
Expression of immunoreactive histone H1 in detached
villus epithelial cells of human terminal ileal mucosa. Cytospin
preparations of epithelial cells were made following detachment with
EDTA (and subsequent washing with PBS). Photomicrographs of the same
cytospin preparation were taken. Strong histone H1 immunoreactivity was
seen in nuclei. Strong histone H1 immunoreactivity was seen in the
cytoplasm (arrows) of some epithelial cells (b). Magnification,
×590.
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Extracts of detached villus epithelial cells express antimicrobial
activity.
Villus epithelial cells were detached by treatment with
EDTA and extracted with acetic acid. These extracts demonstrated
activity against S. typhimurium CS105 in the radial
diffusion assay (5.9 ± 0.6 mm2).
Detached villus epithelial cells release antimicrobial histone H1
while undergoing apoptosis.
Following treatment with EDTA,
detached villus epithelial cells were cultured at 37°C in 5%
CO2 for 3 or 18 h. Acetic acid extracts of the
supernatants of the cultured epithelial cells demonstrated activity
against S. typhimurium CS015 (mean ± SD reduction in
bacterial OD620 at 5 h, 98.3% ± 2.4% and 97% ± 4% for 3-h and 18-h supernatants, respectively) (Fig.
8). Immunoblotting studies demonstrated
the presence of histone H1 in the supernatants (Fig.
9).
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FIG. 8.
Acid extracts (in 0.1% acetic acid) of supernatants of
detached and cultured (for 3 h [] or for 18 h [ ])
villus epithelial cells mediate antimicrobial activity. Microtiter
plate antimicrobial assays were performed with S. typhimurium CS015, and 0.1% acetic acid only () was used as a
control. The data represent one of three experiments performed.
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FIG. 9.
Dot blot analysis demonstrating the presence of
immunoreactive histone H1 in supernatants of detached and cultured (for
18 h) terminal ileal villus epithelial cells. Acid extracts were
applied to PVDF membranes, which were subsequently incubated with an
anti-human histone H1 monoclonal antibody (b) or control buffer (a).
Strong histone H1 immunoreactivity was seen with the monoclonal
antibody.
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In TEM studies, the detached and cultured villus epithelial cells
demonstrated ultrastructural features of cells undergoing
apoptosis
(Fig.
10).
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FIG. 10.
Transmission electron micrograph of detached villus
epithelial cells cultured for 3 h. Apoptotic bodies (small arrows)
and an apoptotic epithelial cell (large arrow) are shown.
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DISCUSSION |
There is a very large and complex resident population of bacteria
(approximately 1012 per g) in the human colonic lumen
(31). In contrast, the lumen of the small intestine is
relatively sterile. A marked change in the enteric flora occurs across
the ileocecal valve, with up to a 106-fold decrease in the
number of microorganisms per g (32). In the lumen of the
more proximal small intestine (jejunum), microorganisms are either
absent or present in very small numbers (32).
A number of factors are likely to be responsible for the maintenance of
this state of relative sterility of the small intestinal lumen. The
roles of gastric acid and peristaltic activity have long been
recognized, but recent studies have begun to investigate protective
defense mechanisms which are operative at the mucosal level and which
can be divided into innate immunity and adaptive immunity
(17). Innate protection is broad spectrum and provides either preexisting or rapidly responsive antimicrobial defense. Recent
studies with animals have suggested that Paneth cells, which are
normally restricted to the small intestine, are important in mediating
innate antimicrobial defense. In addition to lysozyme (4),
these cells have been shown to express antimicrobial phospholipase A2
(PLA2s) (10) and antimicrobial peptides of the defensin
family (3, 13, 18-20, 24, 25, 29). Other components of
innate antimicrobial defense likely are operative at the mucosal level.
In order to isolate and characterize antimicrobial peptides and
proteins expressed in the human terminal ileal mucosa, we have
investigated extracts of fresh mucosal samples. Acetic acid extracts of
terminal ileal mucosa demonstrated potent antimicrobial activity, which
appeared to be mediated by a number of antimicrobial peptides and
proteins. We have isolated and purified one of these entities and
identified it as histone H1. In addition, fragments of histone H1 were
also shown to express antimicrobial activity. The activities were
demonstrated against S. typhimurium CS015 and bacteria
normally resident in the human colonic lumen. Wild-type S. typhimurium appears to be resistant to histone H1 proteins and
their fragments. The relative resistance of wild-type S. typhimurium (compared to the phoP mutant strain) has
also been demonstrated against murine PLA2s (10). Thus,
wild-type S. typhimurium may be capable of invading the host
by its ability to overcome different innate host defense mechanisms. We
have also recently purified human PLA2s from our terminal ileal mucosal
extracts (unpublished observations).
Since histone H1 proteins are normally expressed predominantly in
eucaryotic nuclei, we performed further studies to investigate the
likely functional significance of our findings. Studies on tissue
sections by immunohistochemistry and electron microscopy showed that in
addition to being present in nuclei, histone H1 proteins were also
present in the cytoplasm of intestinal villus epithelial cells. These
cells could therefore mediate host protection in vivo following
penetration by microorganisms through the cell membrane. Detached
villus epithelial cells also released immunoreactive histone H1 protein
during culturing while undergoing apoptosis. Moreover, extracts of
supernatants of these cultured epithelial cells demonstrated
antimicrobial activity. Since we had previously demonstrated that
purified histone H1 protein and its fragments express antimicrobial
activity, it is likely that the antimicrobial activity in the
supernatants of the cultured detached villus epithelial cells is
mediated by the released histone H1 protein. It is possible that other
antimicrobial molecules are also present in these supernatants.
In the normal small intestine, there is a rapid turnover of villus
epithelial cells, with estimates of 2 × 1011 and
1.9 × 108 cells per day lost into the lumen in humans
(27) and mice (26), respectively. Although the
mechanism(s) of epithelial cell loss into the lumen is not fully
understood, studies with rodents and humans have suggested an important
role for apoptosis (7, 9, 30). Epithelial cells exfoliated
into the lumen of the human small intestine also demonstrate
characteristic features of cells undergoing apoptosis (30).
From our studies, we postulate that antimicrobial histone H1 proteins
and their fragments are released into the human intestinal lumen in
vivo by exfoliated apoptotic epithelial cells.
We previously showed that following exposure to Clostridium
difficile toxin A, intestinal epithelial cells detach from the basement membrane and undergo apoptosis (16). Such injured
and detached cells are released into the lumen in vivo and may
represent an important form of rapidly responsive innate protection
against resident luminal microorganisms. There has been recent interest in the bactericidal potential of cells undergoing apoptosis in host
defense (14, 36). Our studies suggest a novel mechanism by
which intestinal epithelial cells undergoing apoptosis may mediate
antimicrobial activity. Such activity would most likely be released
into the lumen to provide innate protection against luminal
microorganisms.
A potent antimicrobial peptide with amino acid sequence similarity to
histone H2A was recently isolated from gastric tissue of the Asian toad
Bufo bufo gargarizans (22). Thus, peptide fragments of histone proteins, which may be released by cells undergoing apoptosis and which may also arise following proteolytic digestion in the intestinal lumen, could mediate potent antimicrobial activity. Histone H2B fragments have also been demonstrated in extracts
of human wound fluid (6). Histone and histone-like antimicrobial proteins have been extracted from a murine macrophage cell line and shown to be expressed in the cytoplasm of these cells
(11). In addition, cytoplasmic expression of histone H1 in
mouse liver cells has also been reported (35). Thus,
although the antimicrobial activity of calf thymus histone has been
known for many years (12), our study and other recent
studies suggest potential in vivo sites of activity for this family of
proteins, which were previously thought to be present only in nuclei.
In conclusion, we have isolated from extracts of human terminal ileal
mucosa antimicrobial proteins which have been identified as histone H1
and its fragments. Immunohistochemical studies suggest that in vivo,
histone H1 is expressed in the cytoplasm of villus epithelial cells.
Our studies also suggest that intestinal epithelial cells detached into
the lumen release antimicrobial histone H1 proteins and their fragments
while the cells are undergoing apoptosis. Thus, histone proteins and
their fragments derived from epithelial cells lost into the lumen may
represent an important innate antimicrobial defense against luminal
bacteria in the human intestine.
| |
ACKNOWLEDGMENTS |
This study was supported by grants from the Medical Research
Council and The Wellcome Trust. The electron microscopy studies used
equipment funded by grant 048326 from The Wellcome Trust.
We thank Trevor Gray for assistance in the electron microscopy studies.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Division of
Gastroenterology, University Hospital, Queens Medical Centre,
Nottingham NG7 2UH, United Kingdom. Phone: 115 970 9973. Fax: 115 942 2232. E-mail:
muzyrm{at}mmn1.medical.nottingham.ac.uk.
Editor: P. J. Sansonetti
| |
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Abstract
Introduction
