Download Toll-like receptor 4 and human defensin 5 in normal

Role of Toll-like receptor 4 and human defensin 5 in primary
endocervical epithelial cells
Ma Jing-mei, Yang Hui-xia
Department of OBGYN, Peking University 1st Hospital, Beijing 100034, China
Correspondence to: Dr. Yang Hui-xia, Department of OBGYN, Peking University 1st
Hospital, Beijing 100034, China
(Email:[email protected])
BACKGROUND: Endocervical epithelial cells play early roles in the defense of upper female
genital tract to pathogens. Toll like receptors (TLRs) and human defensins (HD) have recently
been identified as fundamental components of the innate immune responses to bacterial pathogens.
We used in vitro model of human primary endocervical epithelial cells (HPEC) to investigate their
roles in innate immune response of the endocervix.
METHODS: TLR4 expression and distribution in HPEC and endocervix were investigated by
Immunofluorescence (IF). Cultured HPEC were treated by lipopolysaccharide (LPS) for 0, 24 and
48 hours. At each time point, the levels of HD5, IL-6 and TNF-αin supernants were determined by
ELISA and the cell TLR4 and HD5 expression were detected by western blot and/or IF
simultaneously. We also compared HD5 expression pattern between the HeLa cell line and HPEC.
RESULTS: TLR4 were expressed on the surface of endocervix tissue and HPEC. After
incubation with LPS, HPEC expressed higher levels of TLR4 than the untreated group: especially
after 24hours. Then TLR4 expression decreased after 48 hours (P<0.01). LPS could upregulate the
secretion of HD5, IL-6 and TNF- α in a time dependent manner (24hours:P<0.05;
48hours:P<0.01, compared to unstimulated group). Intracellular HD5 decreased over time. HD5
expression patterns in HPEC are different from HeLa.
CONCLUSION: HPEC expressed TLR4. HPEC take part in the mucosal immune response to
LPS by TLR4 functional expression. With LPS stimulation, HPEC can secret HD5. HPEC may be
a better model for immunological study.
Key words:
human primary endocervical epithelial cells; toll-like receptor4; human
defensin 5
Introduction
Sexually transmitted diseases(STDs), for example, Chlamydia trachomatis (CT) and Neisseria
gonorrhoeae, had high prevalence worldwide including China [1]. Although STDs are curable, CT
is one of the major causes behind female pelvic inflammatory disease and female fertility related
disorders as infertility. Single-cell columnar layer of the epithelium in the endocervix are the
targets of these pathogens [2]. At the same time, epithelial cells of the female genital tract are the
first line of defense against the pathogen.
Nearly 20% women infected CT from genital tract is recovered spontaneously without any
serious sequelae, the others may have persistent infections leading to structural damage
[3].
In
higher life forms, survival is dependent on the competence of both innate and adaptive immune
protection. CT, as other pathogen, had evolved immune–evasion mechanisms
[4]
to encounter
host’s immune response, many of which are to reduce host adaptive immune responses. Besides
essential barrier function, epithelia have been found to involve innate immune antimicrobial
functions as well as the ability to modulate the recruitment and activity of immune cells of both
the innate and adaptive immune systems [5-10].
The innate immune system can protect the host at the onset of the infectious microbial
challenge, which relies on germ-line-encoded pattern recognition receptors that recognize
conserved pathogen-associated molecular patterns(PAMP) synthesized only by micro-organisms.
Toll like receptors (TLRs) are one group of receptors that recognize PAMP, which are expressed
on effecter cells including epithelial cells and believed to undergo activation nuclear factor
(NF)-κB and the resultant transcription of immune response genes, including proinflammatory
cytokines and chemokines [11].
Human defensins (HD) have been identified as another fundamental component of the innate
immune responses to bacterial pathogens. Studies
[12]
have found that HD expression appeared to
be correlated with local TLRs stimulation. HD5, one of the human defensin, was discovered to be
secreted by specialized Paneth cells resident in the crypts of the small intestines
[13]
and in other
epithelial tissues. One report [14] observed HD5 peptides at various sites in human female tracts
and found high levels of HD5 in vaginal wash materials.
Previous studies have shown that TLR4 may be expressed by endocervical epitheium. It is
expected that endocervical epithelial cells play a role in innate immunity as well as constituting a
physical barrier. In this study, we used human primary endocervical epithelial cells (HPEC) to
confirm TLR4 expression and test function of TLR4 and HD5 in the endocervix after LPS
stimulation.
METHOD
Isolation and primary cell culture of HPEC
HPEC were obtained from normal endocervix (n=10). Written informed consent was obtained
prior to the collection of samples, and the study was approved by the local institutional Research
Ethics Committee. All subjects were of premenopausal age (41-47, median 45 years old) and
underwent hysterectomy for benign gynaecological conditions. None of them were under
hormonal therapy or had recent history of infection, including HIV, HCV, HBsAg, HPV and other
genital tract infections. The Pap smear was normal. All specimens were uterus benign conditions
and free of intraepithelial neoplasia, according to histopathology evaluation. Fresh human
endocervix tissues were selected from areas away from the squamocolumnar junction, and
separated as previously described [15]. The tissue was cut into 0.5×0.5 cm pieces and placed onto
25 cm2 culture plates. 3 ml of keratinocyte serum-free medium (ker-sfm; GIBICO/Life
Technologies, USA) was added to each plate. Cultures were re-fed with 3-5 ml media every 3 days
for 2-3 weeks. When these HPEC reached 90% confluence, they were trypsinized, counted and
passaged. The light microscopic and transmission electron microscopic examination of the cells
revealed keratinocyte-like cells (Fig 1). Since the cytokeratin and vimentin is the marker of
epithelial cells and fibroblast respectively, immunostaining was used to assess purity of cell
preparations and had typically demonstrated greater than 98% purity. The cells of passages 3-5
were used for study.
Fig1a. Light microscopic(original magnification × 200)
Fig1b. Transmission electron microscopic examination of the cells revealed keratinocyte-like cells.
HeLa cell lines were cultured in 1640 medium with 10% FBS.
LPS (from Salmonella enterica serotype minnesota; catalog number L4641, Sigma, USA)
(100μg/ml) was added to HPEC medium. At 0, 24 and 48h time points, the supernatants were
collected and stored in -80℃. The cell protein was collected simultaneously.
Immunofluorescence (IF)
TLR4 was detected on frozen cryostat sections of normal endocervix biopsy specimens by
epifluorescent microscopy. Briefly, cryostat sections (4 m) were fixed with 4% PFA for 30 min.
Sections were blocked for 90 min with 1% BSA before incubation with primary mAb against
TLR4 (sc-13593, Santa Cruz, USA, 1:200), or the corresponding isotype control Ab for 60 min,
37℃. The sections were then incubated with secondary FITC anti-mouse IgG for 60 min at
37℃.After using Hoechst/PI to dye nuclear for 2 min, the slides were mounted in crystal
mounting medium. The cryosections were prepared and stored at -80℃ until used. Positive
controls used PBMC.
Localization of TLR4 on HPEC cell was used by Laser scanning method (confocal microscopy).
The subconfluent monolayer of cells were rinsed and fixed with prewarmed 2%PFA for 15min.
Fixed cells were then incubated for 30min with 1% BSA. Anti-TLR4 mouse monoclonal antibody
was diluted in 1:200. FITC-conjugated secondary antibody was incubated with the cells over night
at 4℃.After using Hoechst/PI to dye nuclear for 2 min, the slides were mounted in DABCO. The
images were acquired by ZEISS, CSM510 META.
Expression of TLR4 or HD5 was examined for at least three independent endocervix specimens
and each experiment was repeated twice.
Preparation of cell protein extracts
106 cells were suspended in 1ml ice-cold PBS and pelleted by centrifugation at 1,000 rpm for 5
min. Supernatants were removed and the pellet was resuspended in 150 l lysis buffer (50mm pH
7.0 Tris-HCl, 2% SDS, 5 g/ml aprotinin, chymostatin, leupeptin, pristane, and trypsin inhibitor)
for 20min, then the protein extracts were separated by centrifugation at 14,000 rpm, 20 min. All
prepare procedures were carried out at 4℃. Protein concentration were determined by Bradford
method and stored at -20℃ until used.
Western Blot
Extracts (30μg of proteins) were separated by electrophoresis on 12.5% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose membrane. The
membrane was blocked with 5% non-fat milk and PBS containing 0.05% tween-20 and then
incubated mouse monoclonal antibody overnight at 4℃ (MAB1478, R&D, USA, 1:300). HD5
protein was detected using rabbit anti-HD5 (HDEFA1-A, ADL, USA, 1 g/ml), followed by
incubation with horseradish peroxidase conjugated anti-mouse/rabbit IgG for 1 hour at room
temperature. Chemiluminescent LumiGlo reagent A and peroxide reagent B according to the
manufacturer’s instructions. The density ratio value represented the level of each subunit protein.
Positive controls used human peripheral blood mononuclear cells. (PBMC)
ELISA
HD5, IL-6 and TNF-α quantification were performed using a commercial sandwich
enzyme-linked immunosorbent assay (ELISA) (ADL, Sun Biomedical Technology, Beijing Co.
Ltd) according to the manufacturer’s instructions. The assay sensitivity was less than 0.01 g/ml
(HD5) or 10 pg/ml (others). Samples were taken in duplicate. Variability between separate
samples run in parallel was minimal as shown in the error bars in Figs 3. The data were obtained
by measuring A450/492 nm using the plate reader.
Statistical analysis
The data is presented as the mean±standard error of the mean. Dunnett’s post test was performed
with one-way analysis of variance .We used SPSS 10.0 to analysis the data. P≤0.05 was
considered statistically significant.
Result
1. TLR4 expression in endocervix and HPEC
With frozen cryostat sections of normal endocervix biopsy specimens, intense staining by
anti-TLR4 antibody was seen in the epithelium and cells lining the endocervical glands (Fig 1a).
The membrane and cytosol of HPEC both stained positive for TLR4 (Fig 1b).
2a
2b
Fig2 expression of TLR4 (a) in the normal endocervix, (b) in HPEC
Green: FITC-conjugated anti-TLR4 antibody; blue: Hoechst - nuclear; red: PI- nuclear
1(a)At ×200 magnification 1 ; 1(b)At ×400 magnification
After LPS stimulation, TLR4 expression in HPEC changed:
Compared to the control group, the co-cultivation of HPEC and LPS induced the significant
changes of TLR4 expression in HPEC confirmed by IF and Western blot. At different time points
during persistent LPS stimulation, TLR4 expression was upregulated significantly (P<0.01), after
48 hour, TLR4 expression decreased significantly (P<0.01.)
The TLR4 antibody in western blot was different from the TLR4 antibody used in IF method. We
observed the same pattern of expression as with our microscopy studies (Fig3).
89kDa TLR4
43kDa actin
+
0h
24h
48h
Relative protein level of TLR4
TLR4 changes as LPS stimulating HPEC
Fig 3. Western blot show that after LPS stimulation, TLR4 expression in HPEC increased at 24h; in contrast, after
48h, TLR4 expression decreased
The results provided that the inducenment of LPS to HPEC was related to time. When it lasted too
long, the function became self-limiting.
2. Detection of IL-6, TNF-α and HD-5
140
TNF-α
（pg/ml）
control
TNF-α
（pg/ml）
LPS
IL-6
（pg/ml
control
IL-6
（pg/ml
LPS
120
*
100
pg/ml
80
60
40
20
0
0h
24h
48h
time(hour)
**
0.
0.4
5
*
50.
0.3
4
**
50.
ug/m
3
l 0.2
**
50.
HD-5(ug/ml
contro
)
HD-5(ug/ml
l
)
0.1
2
50.
0.0
1
LPS
5 0
0
h
24
Time (hour)
h
48
h
Fig4(a,b) Comparison of TNF-a, IL-6 and HD-5 from HPEC after LPS (100ng/ml) Treated to Untreated.
* P＜0.05, * * P ＜ 0.01
In order to test TLR4 function, the response to the specific TLR4 ligand, HPEC and LPS was
co-cultrued in the HPEC. Compared with the control group, the concentration of IL-6 and TNF-α
increased significantly (Fig 4a).
Meanwhile, we found that median HD5 can be upregulated with by LPS. HD5 production pattern
seemed to like those of the pro-inflammatory protein (Fig 4b).
HD5 changes in the cell protein extracts with western blot. The results showed that after 24 hours
of LPS stimulation, HD5 in HPEC could not be detected with this method (Fig 5), and suggest
HD5 is primarily secreted into the lumen to join the host immune responses to LPS.
43kD ACTIN
0h
Fig5.
24h
48h
5kD HD5
5
After LPS stimulation (100 μg/ml), effects of LPS on the level of HD-5 proteins in HPEC.
3. Comparison of HPEC and the HeLa cell line
The immunological difference between HPEC and HeLa were evaluated for the expression of
protein for HD5 by western blot. As seen in Fig6, HPEC express protein for HD5, while
expression of HD5 was hardly observed in HeLa cells. IL-6 secretion of HeLa cell lines was
different from NEE cells expression pattern.
43kDa actin
5kDa HD-5
HPEC
HeLa
Fig6. Western blot results show the difference expression of HD-5 from the HPEC and HeLa cell
lines extracts.
Discussion
The female reproductive tract is open to the outside environment and must have sufficient
immunological mechanisms of defence to avoid infection. The lumen of the lower reproductive
tract (vagina and ectocervix) is lined with squamous epithelium, whereas the upper reproductive
tract (endocervix, uterus and Fallopian tubes) is lined with columnar epithelium. Endocervical
epithelial is the target cell layer to encounter microbes ascending to the female reproductive tract.
While both types of cells form the physical barriers to invading microbes, the means by which
they mediate innate immune protection remains unclear. The presence of TLRs in the epithelium
are integral to initiation of, and communication between, the innate and adaptive immune
responses.
We used both tissues and in vitro primary cell models to demonstrate TLR4 expression.
LPS increased the TLR4 expression and the release of HD5, IL-6 and TNF-α. Our data suggested
that TLR4 signaling pathways are involved in the innate response against LPS by HPEC.
TLRs expression is site-specific. TLR4’s ligand
[16]
includes LPS from Gram-negative
bacteria, heat-shock protein 60 from CT. The roles of TLRs in female genital tract have recently
been given growing attention.There are still controversies on TLR4 expressions in primary
endocervical epithelial cells and in cervicovaginal epithelium which is lack of TLR4. Fichorova [17]
et al found that epithelial cells from normal human endocervix failed to express mRNA for TLR4.
While Fazeli [18] et al characterized the TLR in the lower female reproductive tract in humans and
found that TLR4 was only present in the endocervix and absent in the vagina and ectocervix. In
our research, there were TLR4 expressions in the endocervix. We used both tissues and in vitro
primary cell models to demonstrate TLR4 expression, which was similar to Fazeli et al’s study.
We compared the sample election of these three papers. Fichorova et al used the primary
endocervical epithelial cells purchased from BioWhittaker/Clonetics. While Fazeli et al and our
study used tissue from patients undergoing hysterectomy for benign gynaecological conditions.
Both of last two studies emphasized that the tissue were free from infection situations.
In our primary HPEC cell model, after LPS stimulation, TLR4 expression showed a quick
reaction. In the 24h after stimulation, TLR4 showed an increased expression, 48h later, the
expression decreased sharply. This phenomenon showed that as a member of the innate immune
system, TLR4 react to the invading factors quickly as research in other organs[19] and could be
downregulated which may be a benefit for HPEC from cytokine overproduction. This finding also
may give an explanation to the different results of TLR4 expression.
The cell responses induced by TLRs activation may share many features with the IL-1R and
TNFR pathway, including the involvement of NF-κB activation. When NF-κB activation occurs,
pro-inflammatory and immunoregulatory cytokines, chemokines and costimulating molecules,
including IL-6 and TNF-α, which are necessary for further immune responses, are subsequently
produced. . We investigated LPS responsiveness in HPEC using IL-6, TNF-α protein production
as a surrogate of LPS responsiveness. Our studies indicated that stimulation of HPEC with LPS
induced the expression of IL-6 and TNF-α. .
The pro-inflammatory cytokine and human defensin can influence the course of diseases but
their overproduction may cause serious inflammatory reactions and long term complications.
Recent studies have been focused to target the cytokine secretion pathway as treatment. In our
primary HPEC cell model, after LPS stimulation, TLR4 expression changed which may be a
benefit for HPEC from cytokine overproduction.
HD5 is a member of antimicrobial peptides, which are part of the innate local host response
of multicellular organisms. They are kind of microbicidal which are towards Gram-positive and
Gram-negative bacteria, various pathogens including fungi and even some enveloped viruses.
Among human α-defensin family, HD5 has the strongest bactericidal activity. This phenomenon
may be associated to the strongest cationic properties and unique distribution of charged residues
on the molecular surface of HD5
[20].
HD5 is expressed as a propeptide that is proteolytically
processed to yield a variety of mature HD5 isoforms. Quayle et al
[13]
have found that HD-5 were
found in cervicovaginal secretions and definitely localize HD5 to intracellular within the stratified
epithelium with no evidence of secretion. In this circumstance, commensals may be resistant to
concentrations of antimicrobial components normally found in vaginal secretions. So there may be
differences between endocervix and ectocervix in HD5 production. In our study, we testified that
HD5 can be induced by LPS from HPEC.
The therapeutic potential of antimicrobial peptides to prevent or treat infection has been clear
since the discovery of their bactericidal properties
[21].
Besides the most straitforward method of
treatment by direct application to the site of action, an alternative possibility for using
antimicrobial peptides would be induce or augment their natrual production.
In our study, we showed that after LPS stimulation, HD5 in the cell protein extracts went
down. Meanwhile, HD5 in the supernatants increased, which means that HD5 was a quick
response to the pathogen. If pathogens were much more or strong enough to survives, HD5 may
exhaust, and could not be protective any more. Schaefer et al [22] recently demonstrated that uterine
epithelial cells can express TLR3，and TLR3 stimulation induced HBD1 and HBD2 expression.
There are many papers on TLR and human defensin
[12, 23]
. Our study gave a new sight of TLR4
and HD5 expression relation in HPEC.
Finally, we provide some information on the cellular responses with HD5 of HeLa cells, a
cell model which has been widely used in the studies of host defense [24]. There is another model
for cervical epithelial cells immunology which was evaluated by Alison in 1997. The latter model
is transfected by HPV. Our data showed that HeLa cell lines have some difference with primary
endocervical epithelial cells for immune responses study. After too many generations, HeLa cells
may lose some important characteristics of immunology. Also, as human tumour cell line, HeLa
cells might have some special immunology property. While the primary endocervical cells are
easy to be obtained and cultured, keeping its immunological characteristics completely, so that
HPEC maybe better in research on drug and immunological response to the pathogens and this
may affect the prognosis.
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