Chemokines in
the Female
Reproductive
Tract
Laboratory Report
Carolann Waddell
Word count: 1993
Abstract
Introduction: Inflammatory responses are associated with reproductive
processes. Chemokines help to regulate these inflammatory pathways by
mediating leukocyte recruitment into reproductive tissues. The purpose of this
study was to examine the expression of the chemokines CXCL1, 2, 3 and 5 in
the vagina and uterine horn of CXCR2-/- and CXCR2 +/+ mice and also to
determine CCL27 protein expression in the ovary of C57/BL6 mice.
Methods: C57BL/6, BALB/C and CXCR2 -/- (BALB/C background) mice were
used in experiments. mRNA expression of CXCL1, 2, 3 and 5 was determined
using qRT-PCR and CCL27 protein expression was determined by ELISA.
Results: There were no statistically significant differences between
expression of the CXC chemokines in the vagina or uterine horn of CXCR2-/compared with CXCR2+/+ mice. CCL27 protein was highly expressed in the
C57BL/6 mouse ovary, and was approximately 10-fold higher than CCL27
protein concentration in the skin, uterine horn and vagina, p<0.05.
Conclusions: CXC chemokine expression in the murine vagina and uterine
horn suggests a role in the recruitment of neutrophils to these sites. CCL27,
which is involved in memory T cell recruitment, may function to modulate T
cell trafficking to the ovary. The presence and trafficking of neutrophils and T
cells to the vagina and ovary respectively, warrants further investigation.
Carolann Waddell
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Introduction
Almost all female reproductive processes including ovulation, menstruation,
implantation, placentation, labour and post partum remodelling are associated
with inflammatory responses. [1, 2] Depending on the tissue and process
involved, a unique repertoire of immune cells, pathways and inflammatory
mediators are expressed. They function to initiate and execute reproductive
processes as well as to repair and remodel reproductive tissues to preserve
future reproductive function. This occurs in a cyclical fashion throughout a
female’s reproductive life cycle under the influence of the ovarian steroid
hormones progesterone and oestrogen. Locally, inflammatory responses are
controlled and regulated by a host of inflammatory mediators such as
prostaglandins, growth factors, cytokines and chemokines. Importantly,
aberrant inflammatory pathways are thought to contribute to a variety of
pathologies of female reproductive function such as polycystic ovarian
syndrome (PCOS), ovarian cancer, premature ovarian failure, unexplained
infertility, recurrent miscarriage, endometriosis, pre-eclampsia and preterm
labour. [1]
Chemokines are small polypeptides, typically 8-14kDa in length. They are
secreted by a variety of cells and are primarily involved in recruitment of
leukocytes into tissues. Approximately 50 human chemokines have been
identified. They bind to G-coupled transmembrane receptors, of which around
19 are known. [3] Generally one chemokine will bind many receptors and one
receptor will bind many chemokines, making it difficult to define their specific
roles. To complicate matters further it is likely that the sequential or
combinatorial action of multiple chemokines is necessary for the recruitment
of specific populations of immune cells. Moreover it is now recognised that
chemokines have non-immune functions such as promoting angiogenesis,
differentiation and proliferation in tissues. [3] A number of studies have
identified important physiological roles for chemokines in the reproductive
tract. [2]
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28 November 2012
Recent work in our laboratory (unpublished) found that the CXC chemokines
CXCL1, 2, 5 and 7 were highly expressed in the cervix and vagina of C57BL/6
mice compared to other solid tissues. This also corresponded with high levels
of Neutrophil Granule Protein (NGP) in these tissues, a known neutrophil
marker. Neutrophils are the predominant CXCR2+ leukocytes in peripheral
blood and CXCL1, 2, 5 and 7 mediate neutrophil recruitment through
activation of this receptor. In addition, we also found that mRNA levels of the
chemokine, CCL27 were high in the ovary. This was a notable finding
because other chemokines examined had low levels of expression in this
tissue type.
Thus, the aim of this study was to investigate the role of the CXC chemokines
in the female reproductive tract, by comparing the expression of CXCL1, 2, 5
and 7 in CXCR2 +/+ and CXCR2 -/- mice using qRT-PCR. We also aimed to
confirm the presence of CCL27 protein in the ovary, and quantify this
expression by ELISA, using lysates of cervix, ovary, skin, uterine horn and
vagina collected from C57/BL6 mice.
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Methods
Animals
Animal work was carried out under UK Home Office licenses. C57BL/6 mice,
BALB/C mice and CXCR2 -/- (BALB/C background) mice were maintained in
specific pathogen-free conditions at the University of Glasgow’s Central
Research Facility. Stage of estrus cycle was determined by observation of
vaginal appearance and by microscopic examination of cell types within the
vaginal washout.
Number at each stage of estrus cycle in each strain of mouse used
Proestrus
Estrus
Metestrus
Diestrus
BALB/C CXCR2 +/+ (n=5)
/
4
1
/
BALB/C CXCR2 -/- (n=7, 1 N/A)
4
2
/
/
C57BL/6 (n=12)
2
2
4
4
Table 1. Numbers of mice from each strain at each stage of the estrus cycle when tissues
were collected.
RNA extraction
Cervix, ovary and skin were removed from BALB/C and CXCR2-/- (BALB/C
background) mice, snap frozen in liquid nitrogen and stored at -80oC until
required for RNA extraction. Mouse tissue was homogenised in 1ml of Trizol
(Invitrogen) using an Omni µH homogenizer and total RNA was isolated
according to the manufacturer’s instructions. Quantity of RNA (ng/ul) and
260/280 absorbance of each RNA preparation were measured using
Nanodrop nd-100 spectrophotometer (Nanodrop Technologies Inc.).
Preparation of cDNA
5µg of RNA was DNAase treated using the DNA-freeTM kit (Ambion, Austin,
TX, USA) according to manufacturer’s instructions. cDNA was synthesised
from DNAase treated RNA using the High-Capacity cDNA Reverse
Transcription Kit (Applied Biosystems, Warrington, UK) according to
manufacturer’s instructions. cDNA was stored at -20oC until required.
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Quantitative Real-Time PCR (qRT-PCR)
qRT-PCR was performed using the ABI Prism 7900HT system (Applied
Biosystems). Each reaction contained 12.5ul of 2x Taqman mastermix
(Applied Biosystems), 10.25ul of DEPC-treated water, 1.25ul of 20x target
assay or endogenous control assay (Applied Biosystems) and 1ul of sample
cDNA. One microliter of 1:10 dilution of cDNA was added to each well that
included the endogenous control gene, GAPDH. Probes and control assays
used are shown in Table 2. The thermal cycler conditions were 50oC for 2
minutes, 95oC for 10 minutes then 40x 95oC for 15 seconds and 60oC for 1
minute. Data was analysed using sequence detection software 2.3, which
calculated the threshold cycle (Ct) values. Target gene expression was
normalised by subtracting the Ct value of GAPDH, the endogenous control,
from the Ct value of the target assay. Fold increase relative to the control was
obtained using the formula 2-ΔCt and expressed as a percentage.
Applied Biosystems
NCBI reference
assay ID
sequence
Cxcr2
Mm99999117_s1
NM_009909.3
Mouse CXCL1
Cxcl1
Mm00433858_g1
NM_008176.3
Mouse CXCL2
Cxcl2
Mm00436450_m1
NM_009140.2
Mouse CXCL5
Cxcl5
Mm00436451_g1
NM_009141.2
Mouse CXCL7
Ppbp
Mm01347901_g1
NM_023785.2
Mouse GAPDH
Gapdh
Mm99999915_g1
NM_008084.2
Gene
Gene symbol
Mouse CXCR2
Table 2. Target assay mixes and endogenous control probes used in qRT-PCR
Collection of protein lysates
Cervix, ovary, skin, uterine horn and vagina were removed from C57BL/6
mice, snap frozen in liquid nitrogen and stored at -80oC until required for
protein lysate preparation. Tissues were weighed and 400µl of Cell Lytic MT
solution (Sigma) containing protease inhibitors (Roche) added to each tissue
sample. Tissues were homogenised and lysates cleared of debris by
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centrifugation at 13000rpm for 10 minutes at 4 oC. Supernatant containing
protein was collected and stored at -20oC until required.
ELISA
CCL27 concentration of each sample was measured using a DuoSet ELISA
kit (R&D systems) according to the manufacturer’s instructions. Optical
density of each sample was measured at 450nm (with correction) and
analysed on a microplate-reader.
CCL27 concentration is presented as
amount of chemokine (pg) per mg of tissue.
Statistical analysis
Data was analysed using GraphPad Prism version 5.04 software. MannWhitney U-test or Kruskal-Wallis test (with Dunn’s multiple comparison test)
was performed as appropriate, p < 0.05 was accepted as significant.
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Results
Expression of CXC chemokines in female reproductive tissues
Despite previous studies in C57BL/6 mice demonstrating that the CXC
chemokines were highly expressed in both the cervix and vagina, only uterine
horn and vaginal tissues from CXCR2-/- and CXCR2+/+ were available for
analysis. Cervical tissue from these mice is currently being collected. There
were no statistically significant differences between expression of the CXC
chemokines in the vagina or uterine horn of CXCR2-/- compared with
CXCR2+/+ mice (Fig 1).
1.0
0.5
0.0
10
5
0
6
4
2
0
1000
500
0
KO
60
40
20
0
KO
1500
W
T
Relative Expression (%) +/- SE
0
2000
KO
20
W
T
80
W
T
40
KO
0
Relative Expression (%) +/- SE
2
60
CXCL7 VAGINA
CXCL5 VAGINA
2500
KO
Relative Expression (%) +/- SE
4
KO
W
T
KO
W
T
W
T
CXCL2 VAGINA
80
W
T
Relative Expression (%) +/- SE
KO
W
T
0
1.5
CXCL1 VAGINA
6
5
2.0
0.0
CXCR2 VAGINA
10
Relative Expression (%) +/- SE
0.5
KO
0.0
8
15
W
T
0.2
Relative Expression (%) +/- SE
0.4
1.0
KO
Relative Expression (%) +/- SE
0.6
W
T
Relative Expression (%) +/- SE
0.8
CXCL7 UHORN
CXCL5 UHORN
2.5
15
Relative Expression (%) +/- SE
CXCL2 UHORN
CXCL1 UHORN
1.5
Relative Expression (%) +/- SE
CXCR2 UHORN
1.0
Figure 1. qRT-PCR analysis of CXC chemokine (CXCL1, 2, 5 and 7) expression in vagina/
uterine horn in CXCR2-/- (n=7) compared with CXCR2+/+ (n=5) mice. Expression was
normalised to GAPDH. Data is presented as mean +/- SEM.
No statistically significant
differences in chemokine expression were found between CXCR2-/- and CXCR2 +/+ mice, as
determined by Mann-Whitney U-test.
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Quantification of CCL27 protein in female reproductive tissues
CCL27 protein was highly expressed in the mouse ovary, and was
approximately 10-fold higher than CCL27 protein concentration in the skin,
uterine horn and vagina, p<0.05 (Fig 2).
CCL27
*
*
*
150
pg/mg of tissue
*
100
50
A
IN
G
R
O
H
U
VA
N
Y
O
VA
R
C
ER
VI
X
SK
IN
0
Figure 2. CCL27 protein in lysates of cervix, ovary, skin, uterine horn and vagina from
C57/BL6 mice (n=12), measured by ELISA. Data is presented as mean +/- SEM. *p<0.05,
Kruskal-Wallis test with Dunn’s Multiple Comparison Test.
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Discussion
Previous studies, which utilised C57BL/6 mice, found the expression of
CXCL1, 2, 5 and 7 to be high in the vagina and cervix. In addition, expression
of these chemokines was influenced by estrus cycle stage with all
chemokines showing highest levels of expression during the metestrus and
diestrus stages. The CXCR2-deficient mice available for use were on a
different mouse background (BALB/c) to those used in previous studies.
Different strains of mice could potentially express different repertoires of
chemokines, so studies should be repeated in both strains. The primary aim
of this study presented, was to determine the effect that a deficiency in
expression of CXCR2, the receptor for CXCL 1, 2, 5 and 7, has on the
chemokine profile within the female reproductive tissues.
We found no
statistical differences in expression of CXCL1, 2, 5, and 7 in the vagina or
uterine horn of CXCR2-/- and CXCR2+/+ mice.
Although stage of estrus cycle in all mice was determined, we did not take
account of this in our analysis because numbers in each group would be too
small to yield reliable results. This could explain the large spread of data in
each group and why we were unable to detect significantly statistical
differences between the CXCR2-/- and CXCR2+/+ mice. We aim to include
stage of estrus cycle in our analysis once sufficient numbers of mice have
been obtained. This will allow us to understand the impact that the estrus
cycle has on the expression profile of these chemokines, and may give some
insight into their functions in the vagina and uterine horn.
Future studies should confirm protein expression of CXCL 1, 2, 5 and 7 in the
reproductive tissues because mRNA and protein expression do not always
correlate. Chemokine protein expression could also be localised using
immunohistochemistry in order to characterise the distribution of these
chemokines in these tissues. CXCL 1, 2, 5 and 7 are known to mediate
neutrophil recruitment via CXCR2. Using immunohistochemistry, we plan to
localise and quantify neutrophils in reproductive tissues and compare
Carolann Waddell
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differences in number and distribution between CXCR2-/- and CXCR+/+ mice.
This will provide insight regarding the role of CXCR2 in recruitment of
neutrophils to reproductive tissues, the vagina in particular. The vagina is a
mucosal tissue and is therefore exposed to the external environment.
Neutrophil recruitment to this site is essential in order to provide
immunological protection against potential pathogens.
In addition to studying the CXC chemokines, we wished to further study the
CC chemokine, CCL27. We found CCL27 protein to be highly expressed in
the mouse ovary compared to other tissues examined, consistent with our
previous findings in which high levels of CCL27 mRNA were expressed in this
tissue. Future studies will examine the spatial distribution of this protein in the
murine ovary using immunohistochemistry. Estrus cycle stage was not
accounted for in these studies as CCL27mRNA did not exhibit differential
expression, however, as previously mentioned stage of estrus cycle can
influence chemokine abundance and distribution. This will also be a focus of
future experiments.
The function of CCL27 in the ovary is unknown; however the high abundance
we observed in our study suggests an important role in ovarian physiology.
During skin inflammation CCL27 mediates memory T-cell recruitment via
CCR10. [4] With this in mind CCL27 could be involved in T-cell trafficking to
the ovary. Memory T cells have been found to be significantly reduced in the
ovaries of women with PCOS compared to non-PCOS ovaries, implicating
memory T cells in the pathogenesis of the disease. [5] Thus, understanding
the role of CCL27 in T-cell trafficking to the ovary could also have important
implications for understanding ovarian pathology in humans.
There is increasing evidence implicating chemokines in reproductive
processes. They are also thought to be involved in the pathogenesis of many
diseases of the reproductive system, making them candidates for therapy.
However, the repertoire of chemokines involved, and the function they
perform has not been fully characterised. We have shown mRNA expression
of CXCL 1, 2, 5 and 7 in the murine vagina and uterine horn as well CCL27
Carolann Waddell
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mRNA and protein expression in the murine ovary. Further work is required to
depict the role of these chemokines in these tissues and their contributions to
reproductive processes.
However, when interpreting these results it is
important to note that this is a snapshot of a complex dynamic biological
system. It is likely that the sequential or combinatorial action of multiple
chemokines is necessary for the recruitment of specific populations of
immune cells and to drive specific inflammatory pathways.
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References
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