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Original Studies |
Department of Obstetrics and Gynaecology (H.O.D.C., R.L.J., R.G.L., T.A.D., D.T.B.), University of Edinburgh, Medical Research Council Reproductive Biology Unit (R.W.K.), Centre for Reproductive Biology, Edinburgh EH3 9EW; and Department of Pathology (A.R.W.W.), University of Edinburgh, Edinburgh EH8 9AG, United Kingdom
Address all correspondence and requests for reprints to: H. O. D. Critchley, Department of Obstetrics and Gynaecology, University of Edinburgh, Centre for Reproductive Biology, 37 Chalmers Street, Edinburgh EH3 9EW, United Kingdom.
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Abstract |
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 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Monocyte chemoattractant protein-1 (MCP-1), a 76-amino-acid basic protein, is a specific chemoattractant and activator of monocytes (also T cells, NK cells, eosinophils, and basophils). MCP-1 is secreted by a number of cell types: endothelial cells (11), fibroblasts (12), monocytes (13), and lymphocytes (14). Interleukin 8 (IL-8) recruits and activates neutrophils (15) and T lymphocytes (16). IL-8 is produced by a variety of cell types: monocytes, fibroblasts, epithelial and endothelial cells, lymphocytes, and CD56+ large granular lymphocytes (17, 18). The chemokine, IL-8, has been detected in human endometrium (19, 20). Specifically, IL-8 has been immunolocalized to the perivascular cells of blood vessels in endometrium and early decidua (20, 21). Cyclooxygenase-2 (COX-2), the inducible isoform of the enzyme synthesizing PGs, is a further inflammatory mediator that has been localized to perivascular cells and glandular epithelium in endometrium. A significant increase in COX-2 expression has been reported premenstrually and menstrually (21).
The present study has examined the role of P4 (using a model of P4 withdrawal and maintenance in vivo) in the expression of the inflammatory mediators MCP-1, IL-8, and COX-2, and the recruitment of selected leukocyte subpopulations in endometrium before the onset of menstrual bleeding and in early pregnancy decidua.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Twenty fertile women, using barrier contraception, or subjects who had been sterilized were recruited. All women reported regular menstrual cycles (25–35 days). The timing of the onset of the LH surge was determined by subjects themselves using a commercially available home urine LH kit (Conceive; Quidel, San Diego, CA). Women tested urine samples twice daily (0800 and 1800 h) from cycle day 10 (10 days after the start of menses). Detection of the urine LH surge (taken as the day on which doubling of the basal LH levels occurred) thereafter was confirmed by RIA. The women participated in one of four groups as detailed: 1) an endometrial biopsy (Pipelle, Laboratoire CCD, Paris, France) was collected in (group A) the normal midluteal phase (LH peak + 8 to 10), n = 5; 2) menstrual [to time the onset of simulated menstruation accurately, P4 (200 mg Cyclogest, Hoechst UK Ltd, Hounslow, UK; vaginally twice daily) was administered from day LH peak + 8 in the luteal phase for 4 days. An endometrial biopsy (pipelle suction curette) was collected (group B) 24 h (n = 5) and (group C) 48 h (n = 5) after ceasing P4 administration; (3) extended luteal phase (pseudo pregnancy): (group D) the luteal phase was extended for 7 days by extending the lifespan of the corpus luteum by injections of CG from day LH peak + 8 for 14 days to simulate very early pregnancy (n = 5). Incremental doses of CG, ranging from 125–20,000 IU, were administered (22). In addition, early pregnancy decidua was collected from: (E) women with a gestation ectopic to the uterus (fallopian tube, n = 5). Women with an ectopic gestation who described vaginal bleeding were excluded. Women were diagnosed at an early stage by ultrasound and serum CG concentration. (F) Early pregnancy decidua was collected from women undergoing surgical termination of pregnancy (35–63 days), n = 8. Decidua was collected, away from the implantation area, by careful curettage of the uterine wall before vacuum aspiration of the products of conception with a suction catheter. Decidua parietalis (without trophoblast) was subsequently confirmed by examination of hematoxylin and eosin-stained sections of each biopsy. Tissue sections were also stained with cytokeratin (Dako Corp. Laboratories, High Wycombe, UK; code: M0821) to confirm presence or otherwise of trophoblast cells within the decidual tissue.
Immunohistochemistry
Leukocyte subpopulations. All endometrial biopsies were fixed in 10% neutral buffered formalin at 4 C overnight, rinsed, and stored in 70% ethanol and thereafter routinely wax embedded. Sections (5 µm) were cut for immunolocalization using commercially available monoclonal antibodies: 1) CD56+ lymphocytes (Zymed, San Francisco, CA); 2) neutrophils (neutrophil elastase, Dako Corp. Laboratories, code N752); 3) macrophages (CD68, Dako Corp. Laboratories, code M0876); and 4) hematoxylin and eosin staining (H&E) for routine histology.
Tissue sections were dewaxed and rehydrated in descending grades of alcohol. Nonspecific endogenous peroxidase activity was blocked by treatment with 3% hydrogen peroxide in distilled water for 5 min. No antigen retrieval step was necessary for demonstration of neutrophil elastase. Antigen retrieval was necessary for localization of CD56+ lymphocytes and macrophages (as follows).
CD56+ lymphocyte immunostaining. Tissue sections were microwaved at high power in 0.01 mol/L sodium citrate buffer (pH 6.0) for 20 min and then allowed to stand for a further 20 min. Sections were subsequently washed in buffer before a nonimmune block.
Macrophage immunostaining. Tissue sections were subjected to an enzyme digestion with 0.1% trypsin in 0.1% calcium chloride at pH 7.8. The digestion was conducted at 37 C for 15 min; and subsequently, enzyme activity was destroyed by washing in tap water.
All tissue sections were exposed to a nonimmune block with normal horse serum performed for 20 min at room temperature. Tissue sections were then incubated with the appropriate primary antibody for 60 min at 37 C: 1) CD56 dilution, 1 in 250; 2) neutrophil elastase dilution, 1 in 50; and 3) macrophages (CD68) dilution, 1 in 50.
Negative controls were included by replacing the primary antibody with mouse Ig at the same concentration as the primary antibody. Sections were labeled with an avidin-biotin peroxidase detection system (Vector Stain, Vector Laboratories, Inc., Peterborough, UK). Tissue sections were incubated for 2–10 min with diaminobenzidine (DAB) solution (DAB Kit, Vector Laboratories, Inc.) for color development. Thereafter, sections were counterstained with hematoxylin, dehydrated and cleared in xylene, and mounted in Pertex.
Image analysis
Quantification of leukocyte subpopulation immunoreactivity in endometrial stroma employed computerized image analysis (23). The system used has been previously described in detail (23, 24). To represent the amount of positive immunoreactivity, in terms of number of cells, the average dimensions of each leukocyte and stromal cell were estimated. Ratios were then converted by a conversion factor, such that the data could be expressed as percentage of cells in the stroma.
In vitro studies
Samples of endometrium were transported to the laboratory in
ice-cold RPMI 1640 and were cultured for 24 h in RPMI supplemented
with 10% FCS (Gibco, Paisley, UK) and antibiotics (penicillin,
streptomycin, and gentamycin). After 24 h culture in 5%
CO2 in air, media were removed, divided into aliquots, and
stored at -20 C until assayed. Tissue weight was determined after the
incubation period. IL-8 and MCP-1 were assayed by enzyme-linked
immunosorbent assay (ELISA), following the method of Ida et
al. (25) for IL-8 and Ida et al. (26) for MCP-1.
Antisera and standards were the gift of Toray Industries Inc.
(Tokyo, Japan). PG F2
(PG2) and its
metabolite 13, 14 dihydro-15-keto PGF (PGFM) were assayed by ELISA
using specific antibodies and peroxidase-conjugated PGs as the
label.
All concentrations of cytokines were corrected for endometrial weight.
Semiquantitative RT-PCR
The messenger RNA (mRNA) expression levels of the inflammatory
mediators IL-8, MCP-1, and COX-2 were monitored by RT-PCR. Total RNA
was extracted from endometrium from all study groups (n = 16)
using the Ultraspec RNA Isolation system (Biotecx,
Biogenesis, Poole, UK). To obtain quantitative information
about mediator mRNA levels, RT-PCR was first conducted for the
housekeeping gene glyceraldehyde phosphate dehydrogenase (GAP-DH). RNA
samples were reverse transcribed using Avian Myeloblastosis Virus
Reverse Transcriptase (Promega Corp. Ltd, Southampton, UK)
and an antisense oligonucleotide primer specific for GAP-DH, 5' labeled
with biotin (Oswell, Southampton, UK) (see Table 1
). The gene specific complementary DNA
(cDNA) generated was then subjected to PCR using AmpliTaq DNA
Polymerase (Perkin-Elmer Corp., Beaconsfield, UK) with
sense and the same antisense oligonucleotides (described in Table 1)
for 26 cycles of 94 C for 1 min, 60 C for 1 min, and 72 C for 2 min,
followed by a final extension of 10 min at 72 C. This produced a
1103-bp amplicon (see Fig. 3).
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), complementary to the internal cDNA sequence
of the GAP-DH-amplified region diluted to 10 nmol/L in Easy Hyb
hybridization buffer (Boehringer Mannheim). After removal of
nonspecifically bound probe, by repeated washing, the probe-amplicon
complex was detected by addition of a sheep antidigoxigenin antibody
conjugated with peroxidase enzyme (Boehringer Mannheim). Substrate
development produced a color change, and the absorbance was measured at
450 nm by a plate reader. A standard curve was plotted of RNA
standards.
Samples were then subjected to RT-PCR for IL-8, MCP-1, and COX-2, using
optimized programs. Oligonucleotide primers were designed for each
mediator (Oswell) (see Table 1). Gene specific RT of 60 ng of template
RNA was conducted, as for GAP-DH, and the total volumes of products
were amplified using the following PCR programs: 1 min at 94 C, 1 min
at X C, and 1 min at 72 C, for a selected number of cycles (see Table 1), followed by a final extension for 10 min at 72 C; where X is the
specific annealing temperature for each primer pair (63 C for IL-8, 60
C for MCP-1 and COX-2). An ELISA was conducted to quantitate RT-PCR
product yield. Menstrual phase endometrial sample was included as a
quality control (QC), and mediator amplicon concentrations were
expressed as a ratio of sample signal to QC signal. Omission of
template served as a negative control for all RT-PCRs. The
reproducibility of RT and PCR stages was assessed, as well as
determining the intra- and interassay variations of the ELISA. All were
determined to be below 7%, based on the variability between a minimum
of 6 samples.
Serum RIAs for P4 and estradiol (E2) concentrations
A venous blood sample was collected at the time of biopsy. Serum was separated and frozen at -20 C for subsequent RIA of E2 and P4 (27). The interassay coefficients of variation for these assays were 11.0 and 10.0%, respectively: interassay coefficients of variation were 8.0 and 8.0%, respectively.
Bleeding profiles
All subjects were requested to keep a diary of menstrual bleeding experience after the attendance for endometrial biopsy and to continue the bleeding record until the cessation of menstruation.
Immunolocalization of inflammatory mediators (IL-8, MCP-1, and COX-2)
Detailed methodology for immunolocalization of the inflammatory mediators IL-8, MCP-1, and COX-2 has already been reported (20, 21).
Briefly, immunolocalization of the chemokines IL-8 and MCP-1 required frozen sections to be lightly fixed in 10% neutral buffered formalin for 10 min at room temperature. Endogenous peroxidase activity was blocked by immersion of slides in 3% hydrogen peroxide in distilled water for 5 min at room temperature. Dilute nonimmune goat serum (Vectastain Elite PK-6101, Vector Laboratories, Inc.) was applied for 20 min in a humidified chamber. Excess serum was removed and either IL-8 rabbit polyclonal antibody (1:500 dilution) was applied for 60 min at 37 C, or MCP-1 rabbit polyclonal antibody (1:400 dilution) was applied for 17 ± 1 h (overnight) at 4 C (both antibodies raised in-house). Sections were thereafter labeled with an avidin biotin peroxidase detection system (Vectastain, Vector Laboratories, Inc.), as previously described. Positive antibody binding was identified by application of DAB solution (DAB Kit, Vector Laboratories, Inc.). Sections were counterstained with hematoxylin.
Immunolocalization of COX-2 was conducted on paraffin sections. A microwave antigen-retrieval step (10 min, sodium citrate buffer, pH 6.0) was required to expose the epitope. Incubation with primary antibody (PG27, rabbit polyclonal, Oxford Biomedical, Biogenesis) at a dilution of 1 in 250 was conducted for 60 min at 37 C. Positive controls were frozen tonsil tissue sections for IL-8 and MCP-1, and third-trimester fetal membranes for COX-2 immunolocalization. Negative controls were included and were either nonimmune rabbit Ig or primary antibody preabsorbed with appropriate synthetic peptide (IL-8 or MCP-1) at 100 µg/mL, substituting for the primary antibody.
Analysis of data
Evaluation of leukocyte immunostaining, as measured by image analysis, ELISA measurements, RIA of E2 and P4 concentrations, and RT-PCR ELISA, were all subjected to an ANOVA, with a Fisher’s protected least significant difference (PLSD) to assign significance, to evaluate whether significant differences were present.
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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All biopsies conducted in the normal luteal phase (LH + 8 to 10) were consistent with the secretory phase histological features appropriate with LH dating. Endometrium, examined 24 h after P4 withdrawal (simulated menstruation), displayed overall secretory appearances and evidence of some LGL infiltration. PMN leukocytes were not evident. There was spiral artery development in three of the five women biopsied.
Endometrial biopsies, collected from women 48 h after P4 withdrawal, showed spiral vessel development in the majority of subjects and, in two of these women, an infiltrate of LGLs. No PMN leukocyte infiltration was noted with routine histology. Features of tissue breakdown were, however, evident at 48 h after P4 withdrawal, in contrast with the histological appearances of tissue collected in the secretory phase or in early-pregnancy decidua.
Histological appearances of endometrium, where the gestation was ectopic and where endometrium was maintained with CG, showed very similar histological features. Biopsies were described as either late secretory or hypersecretory in appearance. All biopsies showed spiral vessel development and infiltration with LGLs. Stromal decidualization was observed particularly in those biopsies where the gestation was ectopic.
Bleeding profiles
All women biopsied during the normal luteal phase at LH+ 8–10 commenced bleeding attributable to natural luteolysis, within 1–7 days after biopsy. In this context, the cycle length for the five subjects varied between 23 and 30 days. Subjects biopsied after withdrawal of P4 (simulated menstruation) commenced bleeding 24 h or longer after biopsy when the endometrium was sampled 24 h after cessation of P4. Women biopsied 48 h after cessation of P4 all bled (4–6 days) from the day on which the biopsy was conducted. The group of women in whom the luteal phase was maintained for 7 days with exogenous CG all commenced a withdrawal bleed either 24 h (one subject) or 48 h (four subjects) after cessation of CG. The withdrawal bleeding lasted between 2 and 11 days.
Leukocyte subpopulations
Figure 1 displays the distribution
of leukocyte subpopulations (macrophages, CD68; CD56+ lymphocytes;
neutrophils) in endometrium: between days LH + 8 to 10 of the normal
cycle, n = 5 (A); 24 h after withdrawal of P4,
n = 5 (B); 48 h after withdrawal of P4, n =
5 (C); in endometrium maintained with CG for 14 days, n = 5 (D);
in decidua from women with an ectopic pregnancy, n = 5 (E); and in
normal decidua, n = 8 (F). CD56+ cells were the major leukocyte
subtype in the luteal phase (LH + 8 to 10) and remain the major
leukocyte subtype both 24 and 48 h after P4 withdrawal
and in decidua (either exogenously supported by CG, ectopic or normal).
No significant changes in CD56+ large granular lymphocytes (LGLs) were
detected in the model situation in this study. In endometrium collected
from patients where the gestation was ectopic to the uterus, an
increase (P < 0.001) in macrophages was observed,
compared with normal decidua (intrauterine pregnancy). Higher numbers
(P < 0.05) of macrophages (CD68+) were present in
endometrium 48 h after P4 withdrawal (group C) and in
pseudopregnant endometrium (group D), compared with normal decidua.
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A band of 1103 bp, corresponding to the region of the GAP-DH cDNA
amplified, was detected for all samples and standards. The signal
intensity for the standards increased with an increasing amount of
template, producing a linear standard curve of RNA loading (x) plotted
against absorbance of products (y); y = 0.006x + 0.138 (Fig. 2). Equal amounts of RNA (60 ng) were
subsequently amplified for IL-8, MCP-1, and COX-2. The QC (menstrual
endometrium) exhibited a strong signal of the correct molecular weight
for IL-8 (298 bp), MCP-1 (210 bp), and COX-2 (350 bp). Distinct
variations in sample signal intensity were observed (Fig. 3). A significant elevation in IL-8 mRNA
(P < 0.01) was detected 48 h after P4
withdrawal, above control (LH + 8 to 10), and pharmacologically induced
pseudopregnancy (Fig. 4a). There was also
a significant increase (P < 0.05) in COX-2 mRNA
expression at 48 h after P4 withdrawal (Fig. 4c) above
control endometrium (LH + 8–10). A similar trend in MCP-1 mRNA
expression was observed after P4 withdrawal, but
significance was not reached (Fig. 4b). P4 maintenance
(groups D and E) resulted in generally reduced expression of IL-8 and
COX-2 (slightly higher levels of IL-8 mRNA were detected in decidua
collected from ectopic pregnancy). In contrast, MCP-1 mRNA transcript
numbers remained high in both pregnant groups.
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A significant increase in IL-8 protein, as measured by
ELISA, was detectable in cultured tissue at 48 h
(P < 0.01) after in vivo P4
withdrawal (group C), when compared with control (LH + 8 to 10)
endometrium (group A), exogenously maintained endometrium (group D),
and normal decidua (group F; Fig. 5). The
increased MCP-1 levels in culture medium 48 h after P4
withdrawal (group C) were elevated but not significantly
different from control (group A; see Fig. 5).
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release from cultured endometrium rose
significantly from 46 ± 12 pg/mL to 170 ± 52 pg/mL
(P < 0.05) and 451 ± 35 pg/mL (P
< 0.001) after withdrawal of P4 for 24 (group B) and
48 h (group C), respectively (Fig. 6). The changes in PGFM parallel the
changes in PGF2 but were comparatively small and were
not able to account for the changes seen in PGF2.
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Inflammatory mediator protein immunohistochemistry
Positive immunostaining for IL-8 protein was evident in a
perivascular location in endometrial biopsies from control endometrium
(LH+8–10) and decidua. Intense immunoreactivity was detected in the
perivascular area, both 24 h (Fig. 7a) and 48 h post P4
withdrawal. Immunostaining was additionally present in glandular
epithelium and stroma 48 h after P4 withdrawal,
corresponding to tissue breakdown. Strong immunoreactivity was also
evident in decidua where the gestation was ectopic or endometrium
maintained with exogenous CG (Fig. 7b). Negative control displayed
absent immunoreactivity (Fig. 7d)
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). COX-2
immunoreactivity was lowest in normal decidua and endometrium
exogenously maintained with CG. No immunoreactivity was observed in
negative control (Fig. 7e). Serum P4 and E2 concentrations
Table 2 represents the serum
P4 (nmol/L) and E2 (pmol/L) concentrations at
the time of endometrial biopsy. Significantly lower serum
P4 concentrations were present, both 24 h
(P = 0.0056) and 48 h (P = 0.0012)
after P4 withdrawal, compared with the luteal-phase control
group. Serum E2 concentrations were significantly higher in
subjects with an ectopic pregnancy (P < 0.01, Group E)
and CG-maintained endometrium (P < 0.02, Group D),
compared with women in the control group (group A) and 24 h (group
C, P < 0.01) and 48 h (group D, P
< 0.01) post P4 withdrawal.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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The major leukocyte subpopulations detected were the CD56+ large granular lymphocytes (LGLs) and macrophages (2). The observations are consistent with reports of leukocyte subpopulations in the literature (4, 33, 34). In this study, the withdrawal of P4 in vivo, in a regimen designed to mimic the physiological fall in P4 levels, was not associated with any detectable changes in CD56+ LGL subpopulations. There were, however, higher numbers of macrophages in ectopic decidua, pseudopregnant endometrium, and endometrium 48 h post P4 withdrawal, when compared with normal decidua. Uterine macrophages regulate, in part, local tissue effects of E2 and P4. Changes in macrophage number in the murine uterus correlate with changes in macrophage colony-stimulating factor 1 (CSF-1) levels and a surge of E2. In the mouse, E2 and P4 have additive effects on macrophage recruitment and activation. Factors other than CSF-1 are involved, and MCP-1 is expressed at higher levels than CSF-1 during early murine pregnancy and is, hence, implicated in macrophage recruitment (35).
In our in vivo model, serum P4 concentrations were significantly lowered at 24 h (mean P4 concentration, 11.3 nmol/L) and at 48 h (mean P4, 3.5 nmol/L) after withdrawal of P4. This observation concerning leukocyte populations is consistent with our earlier study that reported a significant increase in macrophage numbers at 12 h post antigestogen (mifepristone) administration (23). Attention is drawn, however, to a potentially different mechanism, i.e. the physiological withdrawal of P4 that occurs before the onset of menstruation and pharmacological blockade of the P4 receptor with the antagonist mifepristone. Blockage of the receptor is probably a more rapid event than the physiological withdrawal of P4.
The present study clearly supports a role for P4 in the modulation of key inflammatory mediators, these being IL-8, MCP-1, and COX-2. This is confirmatory evidence for the sex steroid regulation of these mediators in the human endometrium reported by Jones et al. (21). This is also in agreement with previous reports in the literature. Down-regulation of IL-8 by P4 has been demonstrated in vitro in endometrial, choriodecidual, and cervical cells (30, 36). Similar data have been obtained concerning MCP-1 expression, with a negative regulation by P4 in choriodecidual and breast cancer cells (37), reinforcing previously demonstrated inhibition by glucocorticoids (38). Before the discovery of the COX-2 isoform, studies of uterine PG production revealed that release of P4 premenstrually allowed the full potential of the secretory endometrium to synthesize PGs to be realized (39). Further, when P4 levels are maximal, during pregnancy, basal PG production is reduced (40). This can be mimicked in vitro, with decreased PG release from endometrium in the presence of P4 (41, 42). A possible mechanism was reported by Ishihara et al. (1995) (43) who reported a reduction in COX-2 expression in response to P4.
This study has, for the first time, described a significant elevation
in IL-8 and COX-2 mRNA expression levels in an in vivo
situation designed to mimic the period immediately before onset of
menstruation. Specifically, an elevation in IL-8 and COX-2 mRNA was
observed 48 h post P4 withdrawal. Furthermore, low
numbers of IL-8 and COX-2 mRNA transcripts were observed in
endometrium, either with a trophoblast ectopic to the uterus or where
the corpus luteum had been maintained with exogenous CG. In this
particular context, IL-8 mRNA was marginally higher in endometrium
where the pregnancy was ectopic to the uterus and in endometrium
exposed to exogenous CG administration. This observation coincides with
the slight increase in neutrophil numbers observed in endometrium where
the pregnancy was ectopic to the uterus. These data support the
down-regulation of IL-8 and COX-2 expression by P4. Failure
to detect a significant rise in MCP-1 mRNA may reflect inherent
differences in the role for P4 in the regulation of this
chemokine. It is noted that MCP-1 mRNA transcripts were expressed at a
greater level in endometrium exposed to exogenous or endogenous CG.
P4 might thus have a less inhibitory action on MCP-1
expression, when compared with expression of IL-8. Interestingly, and
consistent with this statement, decidua (although with a trophoblast
ectopic to the uterus) exhibited relatively high levels of MCP-1
immunoreactivity. Differences in the regulation of chemokines IL-8 and
MCP-1 have already been reported, including stimulation of MCP-1 by
platelet-derived growth factor, interferon 
, PRL, and LIF (14, 44, 45, 46). Furthermore, in this context, the high level of MCP-1
expression in the early pregnant uterus correlates with the continued
increase in numbers of LGLs and macrophages recruited into decidual
stroma (33).
The up-regulation of COX-2 mRNA expression occurs at a time point similar to that of increased expression of IL-8. These two local mediators, i.e. IL-8 and PGE2 (product of COX-2), have been reported previously (32, 47, 48) to act in synergy to facilitate leukocyte recruitment.
Immunolocalization of IL-8 protein was evident in a perivascular location in endometrial tissue, both before and after withdrawal of P4 and in early pregnancy decidua. Measurements of IL-8 protein from the supernatant of cultured biopsies (by ELISA) in the timed endometrial biopsies were consistent in also demonstrating a significant increase in IL-8 protein 48 h post P4 withdrawal. The increase in IL-8 protein production coincided with a significant fall in circulating P4 concentrations. There was a nonsignificant increase in MCP-1 protein (as measured by ELISA in culture supernatants) also at 48 h after withdrawal of P4 administration. This observation was consistent with the trend for increased MCP-1 mRNA expression.
The release of PGE and PGF2 by endometrium plays a
critical role in the mechanisms regulating menstruation (28). Secretory
endometrium has a greater capacity for PG synthesis (40, 49) and for PG
metabolism (50). Both of these features are probably dependent on
P4, because persistent proliferative endometrium has the
ability to synthesize PGs but needs arachidonic acid substrate (51).
The effects of P4 withdrawal are not known. This study
shows that P4 withdrawal leads directly or indirectly to an
increase in COX-2 message and PGF2 synthesis. Under the
in vitro conditions used here, there was no clear effect on
metabolism of the PGs. Moreover, P4 stimulates PGDH, the
enzyme responsible for metabolizing PGF2 and PGE to
their inactive 15-keto metabolites. P4 withdrawal reduces
PGDH production perivascularly, hence increasing local concentrations
of PGE (31). This mechanism may be permissive for leukocyte entry into
tissues. Thus, P4 withdrawal may provide an indirect signal
necessary for a leukocyte influx.
Thus, there are increasing lines of evidence to support the immune endocrine interactions in the regulation of normal endometrial functions. The features of menstruation and early pregnancy share much in common with an inflammatory reaction, specifically, traffic movement of leukocytes and production of local inflammatory mediators. Data provided herein strongly support a role for IL-8, COX-2, and indeed MCP-1, in the modulation of menstruation and remodeling in endometrial tissues.
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Acknowledgments |
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Footnotes |
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Received April 1, 1998.
Revised September 18, 1998.
Accepted September 23, 1998.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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and PGE2 from separated cells of human
endometrium and decidua. Prostaglandins Leukot Essent Fatty Acids. 33:91–96.[CrossRef][Medline]
and E2 in the endometrium
throughout the menstrual cycle and after the administration of
clomiphene or an oestrogen-progestogen pill and in early pregnancy. J
Endocrinol. 77:361–371.. Am J Pathol. 145:913–921.[Abstract]
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 C. J. Lockwood, P. Matta, G. Krikun, L. A. Koopman, R. Masch, P. Toti, F. Arcuri, S.-T. J. Huang, E. F. Funai, and F. Schatz Regulation of Monocyte Chemoattractant Protein-1 Expression by Tumor Necrosis Factor-{alpha} and Interleukin-1{beta} in First Trimester Human Decidual Cells: Implications for Preeclampsia Am. J. Pathol., February 1, 2006; 168(2): 445 - 452. [Abstract] [Full Text] [PDF]
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 F. Modugno, R. B. Ness, C. Chen, and N. S. Weiss Inflammation and Endometrial Cancer: A Hypothesis Cancer Epidemiol. Biomarkers Prev., December 1, 2005; 14(12): 2840 - 2847. [Abstract] [Full Text] [PDF]
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 N. J. Hannan, R. L. Jones, H. O. D. Critchley, G. J. Kovacs, P. A. W. Rogers, B. Affandi, and L. A. Salamonsen Coexpression of Fractalkine and Its Receptor in Normal Human Endometrium and in Endometrium from Users of Progestin-Only Contraception Supports a Role for Fractalkine in Leukocyte Recruitment and Endometrial Remodeling J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6119 - 6129. [Abstract] [Full Text] [PDF]
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S. K. Dey, H. Lim, S. K. Das, J. Reese, B. C. Paria, T. Daikoku, and H. Wang Molecular Cues to Implantation Endocr. Rev., June 1, 2004; 25(3): 341 - 373. [Abstract] [Full Text] [PDF]
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C. J. Lockwood, P. Kumar, G. Krikun, S. Kadner, P. Dubon, H. Critchley, and F. Schatz Effects of Thrombin, Hypoxia, and Steroids on Interleukin-8 Expression in Decidualized Human Endometrial Stromal Cells: Implications for Long-Term Progestin-Only Contraceptive-Induced Bleeding J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1467 - 1475. [Abstract] [Full Text] [PDF]
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 K. Tsuboi, A. Iwane, S. Nakazawa, Y. Sugimoto, and A. Ichikawa Role of Prostaglandin H2 Synthase 2 in Murine Parturition: Study on Ovariectomy-Induced Parturition in Prostaglandin F Receptor-Deficient Mice Biol Reprod, July 1, 2003; 69(1): 195 - 201. [Abstract] [Full Text] [PDF]
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O. Yoshino, Y. Osuga, Y. Hirota, K. Koga, T. Hirata, T. Yano, T. Ayabe, O. Tsutsumi, and Y. Taketani Endometrial Stromal Cells Undergoing Decidualization Down-Regulate Their Properties to Produce Proinflammatory Cytokines in Response to Interleukin-1{beta} via Reduced p38 Mitogen-Activated Protein Kinase Phosphorylation J. Clin. Endocrinol. Metab., May 1, 2003; 88(5): 2236 - 2241. [Abstract] [Full Text] [PDF]
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N. Mulayim, S. F. Palter, U. A. Kayisli, L. Senturk, and A. Arici Chemokine Receptor Expression in Human Endometrium Biol Reprod, May 1, 2003; 68(5): 1491 - 1495. [Abstract] [Full Text] [PDF]
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D. K. Hapangama, H. O. D. Critchley, T. A. Henderson, and D. T. Baird Mifepristone-Induced Vaginal Bleeding Is Associated with Increased Immunostaining for Cyclooxygenase-2 and Decrease in Prostaglandin Dehydrogenase in Luteal Phase Endometrium J. Clin. Endocrinol. Metab., November 1, 2002; 87(11): 5229 - 5234. [Abstract] [Full Text] [PDF]
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P. Caballero-Campo, F. Dominguez, J. Coloma, M. Meseguer, J. Remohi, A. Pellicer, and C. Simon Hormonal and embryonic regulation of chemokines IL-8, MCP-1 and RANTES in the human endometrium during the window of implantation Mol. Hum. Reprod., April 1, 2002; 8(4): 375 - 384. [Abstract] [Full Text] [PDF]
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N. Sugino, A. Karube-Harada, S. Kashida, S. Takiguchi, and H. Kato Differential regulation of copper-zinc superoxide dismutase and manganese superoxide dismutase by progesterone withdrawal in human endometrial stromal cells Mol. Hum. Reprod., January 1, 2002; 8(1): 68 - 74. [Abstract] [Full Text] [PDF]
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A. Karube-Harada, N. Sugino, S. Kashida, S. Takiguchi, H. Takayama, Y. Yamagata, Y. Nakamura, and H. Kato Induction of manganese superoxide dismutase by tumour necrosis factor-{alpha} in human endometrial stromal cells Mol. Hum. Reprod., November 1, 2001; 7(11): 1065 - 1072. [Abstract] [Full Text] [PDF]
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 O.D. Slayden, K. Chwalisz, and R.M. Brenner Reversible suppression of menstruation with progesterone antagonists in rhesus macaques Hum. Reprod., August 1, 2001; 16(8): 1562 - 1574. [Abstract] [Full Text] [PDF]
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S.A. Milne, A. Rakhyoot, T.A. Drudy, S. Brechin, S.C. Riley, and H.O.D. Critchley Co-localization of matrix metalloproteinase-1 and mast cell tryptase in the human uterus Mol. Hum. Reprod., June 1, 2001; 7(6): 559 - 565. [Abstract] [Full Text] [PDF]
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C. E. Gargett, F. Lederman, B. Heryanto, L. S. Gambino, and P. A.W. Rogers Focal vascular endothelial growth factor correlates with angiogenesis in human endometrium. Role of intravascular neutrophils Hum. Reprod., June 1, 2001; 16(6): 1065 - 1075. [Abstract] [Full Text] [PDF]
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A. E. King, H. O.D. Critchley, and R. W. Kelly The NF-{{kappa}}B pathway in human endometrium and first trimester decidua Mol. Hum. Reprod., February 1, 2001; 7(2): 175 - 183. [Abstract] [Full Text] [PDF]
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A. E. King, R. W. Kelly, H. O. D. Critchley, A. Malmstrom, M. Sennstrom, and R. P. Phipps CD40 Expression in Uterine Tissues: A Key Regulator of Cytokine Expression by Fibroblasts J. Clin. Endocrinol. Metab., January 1, 2001; 86(1): 405 - 412. [Abstract] [Full Text]
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R.L. Jones, L.A. Salamonsen, H.O.D. Critchley, P.A.W. Rogers, B. Affandi, and J.K. Findlay Inhibin and activin subunits are differentially expressed in endometrial cells and leukocytes during the menstrual cycle, in early pregnancy and in women using progestin-only contraception Mol. Hum. Reprod., December 1, 2000; 6(12): 1107 - 1117. [Abstract] [Full Text] [PDF]
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N. R. Nayak, H. O. D. Critchley, O. D. Slayden, A. Menrad, K. Chwalisz, D. T. Baird, and R. M. Brenner Progesterone Withdrawal Up-Regulates Vascular Endothelial Growth Factor Receptor Type 2 in the Superficial Zone Stroma of the Human and Macaque Endometrium: Potential Relevance to Menstruation J. Clin. Endocrinol. Metab., September 1, 2000; 85(9): 3442 - 3452. [Abstract] [Full Text]
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D. Ghosh, P.G.L. Lalitkumar, V. J. Wong, A.G. Hendrickx, and J. Sengupta Preimplantation embryo morphology following early luteal phase anti-nidatory treatment with mifepristone (RU486) in the rhesus monkey Hum. Reprod., January 1, 2000; 15(1): 180 - 188. [Abstract] [Full Text] [PDF]
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S. A. Milne, H. O. D. Critchley, T. A. Drudy, R. W. Kelly, and D. T. Baird Perivascular Interleukin-8 Messenger Ribonucleic Acid Expression in Human Endometrium Varies across the Menstrual Cycle and in Early Pregnancy Decidua J. Clin. Endocrinol. Metab., July 1, 1999; 84(7): 2563 - 2567. [Abstract] [Full Text]
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