| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Department of Obstetrics and Gynecology II, University of Freiburg, Germany D-79106
Address all correspondence and requests for reprints to: Dr. W. R. Schäfer, Department of Obstetrics and Gynecology, University of Freiburg, Hugstetterstr. 55, 79106 Freiburg i. Br., Germany. E-mail: wschaef{at}frk.ukl.uni-freiburg.de
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Explants from term myometrium were challenged with CRH or LPS (1
µg/mL each) in short-term tissue culture. Interleukin (IL)-1ß,
IL-6, IL-8, and tumor necrosis factor (TNF)
concentrations in the
medium were quantified by enzyme immunoassay. The major cytokines
released after 24 h were IL-6 and IL-8. All cytokines investigated
were stimulated significantly by LPS (P < 0.05)
but not by CRH. Messenger RNA levels of these cytokines were
investigated by RT-PCR. IL-1ß and IL-6 messenger RNA were present in
preterm and term myometrium before and during labor, whereas IL-8 and
TNF were expressed only by myometrium in active labor. Furthermore,
myometrial CRH receptors and macrophages were characterized
immunohistochemically.
We conclude that human term myometrium is a site of production of proinflammatory cytokines and is involved in the inflammation-like reactions mediating the birth process. Cytokine release in term myometrium seems not to be under control of CRH.
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Recently, placental CRH has been suggested to play a key role in the timing of birth in humans (for review, see 4, 5). This hypothesis is based on data demonstrating a steep rise in bioavailable CRH in maternal serum before labor induction (6, 7), a phenomenon that is unique to humans and few anthropoid primates (8). Therefore, no convenient animal model exists to elucidate the biological function of CRH for the timing of birth, underlining the importance of in vitro experiments on human tissue.
To date, two labor-promoting CRH effects are known. First, CRH has been
recognized as a potent vasodilator in the fetoplacental circulation
(9). Second, it has been demonstrated that CRH stimulates
dehydroepiandrosterone sulfate production in human fetal adrenal cells
(10). However, the major portion of placental CRH is
secreted into the maternal circulation (11), and the
expression of both CRH and its receptors has been demonstrated in
myometrium (12, 13, 14). It has been hypothesized that CRH or
the related peptide urocortin (15) may also exert
paracrine or autocrine actions in uterine tissues (4, 5).
Such proinflammatory local effects of CRH have been demonstrated in an
experimental model of chemically induced aseptic inflammation in rats
(16). Although, in recent studies, no direct effect of CRH
on myometrial contractility has been found, CRH has been reported to
enhance the contractile effects of oxytocin and
PGF2 in a synergistic manner (17, 18). A variety of different CRH receptor subtypes that are
expressed differentially during pregnancy has been identified
previously by RT-PCR and immunohistochemistry in human myometrium
(14, 19, 20), pointing to an important role for CRH on
myometrial function.
It is widely accepted that proinflammatory cytokines and chemokines
[e.g. tumor necrosis factor (TNF), interleukin
(IL)-1ß, IL-6, and IL-8] are involved in cervical ripening
(3) and preterm labor (21). Recently, it has
been proposed that cytokines also play a fundamental role during the
physiological birth process representing an inflammation-like response
(2). However, no data exist about interactions of CRH and
the cytokine network in human term myometrium; whereas in mononuclear
cells, a stimulation of IL-6 production by CRH has been demonstrated
(22).
In this study, we investigated possible effects of CRH and lipopolysaccharide (LPS, a well known cytokine stimulant) on the release of proinflammatory cytokines in human term myometrium in vitro. Further, we studied changes in the cytokine expression pattern in preterm and term myometrium before and after the onset of labor.
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
For the investigation of cytokine protein levels in tissue culture experiments, term myometrial biopsies (1.5–2.0 g, 37–40 weeks of gestation) from the upper margin of the lower uterine segment incision were obtained, before the onset of labor, from patients undergoing elective cesarean section because of breech presentation or cephalopelvic disproportion after uncomplicated pregnancies. Additionally, a single biopsy from the myometrial fundus (35 weeks of gestation) was obtained after a hysterectomy for cervical cancer after elective cesarean section.
For the investigation of messenger RNA (mRNA) levels, additional myometrial biopsies from the upper margin of the lower uterine segment incision were used. The tissue samples, obtained before the onset of labor, were divided in preterm (26–36 weeks of gestation; n = 4) and term (38–40 weeks of gestation; n = 5) specimens. Preterm cesarean sections were undertaken for maternal indications excluding preeclampsia. Further, myometrial tissue was collected from secondary cesarean sections after the onset of labor at term (37–40 weeks of gestation; n = 5), because of failure of the normal progress of delivery.
For immunohistochemical characterizations, term myometrial biopsies (37–40 weeks of gestation; n = 6) were used.
Ethical approval was obtained from the ethical committee of the University Hospital of Freiburg, and patients gave their informed written consent to the study.
Myometrial short-term tissue culture
All myometrial samples were placed into HBSS (Biochrom, Berlin, Germany) immediately after delivery and then washed several times in Dulbecco’s PBS (Life Technologies, Inc., Karlsruhe, Germany) to remove excessive blood.
For tissue culture experiments, the myometrium was cut into small pieces (10–30 mg wet weight). Three myometrial explants were combined for 1 incubation and placed into 1 well of a 24-well plate containing 1 mL culture medium (Iscove’s modified Dulbecco’s medium; PAA Laboratories, Cölbe, Germany) complemented with 1% antibiotic and antimycotic solution containing 100 U/mL penicillin, 100 µg/mL streptomycin, and 250 ng/mL amphotericin (Sigma-Aldrich Corp., Deisenhofen, Germany). This basal medium was checked for endotoxins using a pyrocheck kit (DPC Biermann, Bad Nauheim, Germany) and found to be endotoxin-negative. After preincubation at 37 C for 30–60 min in a humidified atmosphere of 95% O2-5% CO2, the explants were transferred to a new well containing fresh culture medium and incubated for 24 h under the same conditions. Tissue samples were incubated with CRH (Bachem Biochemica GmbH, Heidelberg, Germany) or with LPS (Escherichia coli 026; B6, Sigma-Aldrich Corp.), respectively (1 µg/mL each). Control experiments were run without addition of CRH or LPS. Each set of experiments was carried out in triplicate. After incubation, the supernatants were collected and stored at -20 C. For time course studies, 18 myometrial explants were used for 1 incubation and placed in 1 well of a 6-well plate containing 6.3 mL of the same culture medium as described above. Aliquots were taken after 0, 1, 4, 6, 8, 21, and 23 h. Tissue viability was checked at the end of the incubation period by the lactate dehydrogenase assay (23).
Cytokine quantification by enzyme immunoassays (EIAs)
Cytokine concentrations in culture medium were measured using an
automated chemiluminescent EIA (Immulite, DPC Biermann). Protein levels
of IL-1ß and TNF were determined in undiluted medium; whereas for
IL-6 or IL-8 quantification, samples were diluted 1:200 in diluents
provided by the manufacturer (DPC Biermann).
The coefficients of intraassay variability were 2.9% for IL-8
(n = 8), 2.3% for IL-6 (n = 7), 3.1% for IL-1ß (n =
7), and 8.3% for TNF (n = 8); and the coefficients of
interassay variability were 4.7% for IL-8 (n = 18), 8.3% IL-6
(n = 15), 2.6% for IL-1ß (n = 8), and 13.7% for TNF
(n = 9).
RNA isolation and RT-PCR
For RT-PCR experiments investigating basal cytokine expression, myometrial tissue was snap-frozen in liquid nitrogen immediately after washing in Dulbecco’s PBS and was stored at -80 C. Additionally, myometrial explants from tissue cultures were snap-frozen in liquid nitrogen at the end of incubation, immediately after determination of wet weight, and stored at -80 C.
Total RNA was prepared, from frozen tissue specimens, by the guanidinium-isothiocyanate/phenol/chloroform method (24). Complementary DNA (cDNA) synthesis from total RNA (1–2 µg) was carried out in a reaction vol of 30 µL, including 50 mmol/L Tris-HCl (pH 8.3), 75 mmol/L KCl, 3 mmol/L MgCl2, 10 mmol/L dithiothreitol, 25 ng/µL oligo(dT) primer, 2.7 mmol/L deoxynucleoside triphosphate, and 10 U/µL Superscript II reverse transcriptase (all reagents from Life Technologies, Inc.). Initially, the RNA was denatured at 85 C for 5 min, then the reaction mixture was added, and the RT was performed at 42 C for 90 min. The reaction was stopped by denaturing the enzyme at 85 C for 15 min. The cDNA was diluted, in ribonuclease-free water, to an end vol of 90 µL. For PCR 1 µL of the cDNA solution was used. If RT was not followed immediately by PCR, cDNA was stored at -20 C.
The PCR was performed in a 50-µL reaction vol containing 20 µmol/L Tris-HCl (pH 8.4), 50 mmol/L KCl, 1.5 mmol/L MgCl2, 800 µmol/L deoxynucleoside triphosphate, 0.025 U/µL Taq polymerase (all from Life Technologies, Inc.), and 4 µmol/L of each specific primer (synthesized by TIB MOLBIOL, Berlin, Germany). The reaction started with an initial denaturation step at 94 C for 5 min, followed by 35 cycles at 94 C for 30 sec, 60 C for 40 sec, and 72 C for 90 sec, and a final elongation step at 72 C for 5 min.
For detection of cytokine mRNA, the primers described by Bouaboula
et al. (25) were used, resulting in a 427-bp
fragment for TNF, a 263-bp fragment for IL-1ß, a 260-bp fragment
for IL-6, and a 247-bp fragment for IL-8. The efficiency of the whole
procedure was controlled by amplification of the mRNA of the
housekeeping gene human cyclophilin A (GenBank accession number Y00052)
using the same conditions as for amplification of the cytokine
mRNAs. A sense primer (5'-AGGGTGGTGACTTCACACGCCAT-3') and an antisense
primer (5'-GTCTTGCCATTCCTGGACCCAAA-3') were used and resulted in a
267-bp fragment after amplification. All primer pairs are situated on
different exons to distinguish between amplification of cDNA and
genomic DNA.
The resulting PCR fragments were resolved on 2% agarose gels and stained with ethidium bromide. Bands were visualized under ultraviolet light, and photographs were taken by an electronic camera (Raytest, Straubenhardt, Germany). The DNA size marker used was the 1Kb Plus ladder (Life Technologies, Inc.).
The identity of the PCR products was confirmed by restriction enzyme analysis and controlled by the Big Dye Terminator cycle sequencing on an ABI Prism 377 sequencer (PE Biosystems, Weiterstadt, Germany). The sequencing was performed by the Core Facility of the Department of Internal Medicine I at the University of Freiburg. Sequence data were analyzed using the program Basic Blast 2.0 from the National Center for Biotechnology Information (Washington DC).
Immunohistochemistry
Paraffin sections (5-µm) were used to investigate the distribution of tissue macrophages and CRH receptors. Antigens were exposed by treatment with pronase (0.1%; LINARIS, Wertheim-Bettingen, Germany) for 15 min. Serial sections were then incubated with either anti-CD68 (PG-M1; 1:50; DAKO Corp., Hamburg, Germany) or anti-CRH-R (1:75; Santa Cruz Biotechnology, Inc., Heidelberg, Germany) overnight at 4 C. The bound antibody was detected with the biotin-streptavidin-peroxidase system (Vectastain-ABC-kit; Serva, Heidelberg, Germany) using diaminobenzidine (DAB; Sigma-Aldrich Corp.) as chromogen. After counterstaining with hemalum, the slides were mounted with Entellan (Merck Eurolab, Darmstadt, Germany). The sections were evaluated by conventional light microscopy. Negative controls were performed by omitting the primary antibodies.
Statistical analysis
IL-1ß, IL-6, IL-8, and TNF concentrations in the
supernatants, after myometrial tissue culture, are expressed as the
mean ± SD. To determine the differences among all
experimental groups (control, CRH, LPS), a Kruskal-Wallis one-way ANOVA
on ranks was performed, followed by Dunnett’s post hoc test
to isolate the group(s) that differed from the control group.
Significance for stimulating effects of CRH or LPS, compared with
controls, was assessed at the P < 0.05 value.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The major cytokines released from term myometrium in the absence
of CRH or LPS, after 24 h of culture, were IL-6 and IL-8, whereas
only low levels of IL-1ß and TNF were found (Table 1
). No significant effect of CRH on the
release of either cytokine was observed, whereas incubation experiments
with LPS showed significant stimulation of IL-8 (P <
0.01), IL-6 (P < 0.01), IL-1ß (P <
0.05), and TNF protein release (P < 0.05) for all
tissues investigated (Fig. 1). In a
single myometrial sample from the fundus, the release of all cytokines
investigated was strongly stimulated by LPS but not by CRH (Table 1).
|
|
). No CRH effect
on IL-8 release was observed at all times, whereas LPS showed the same
stimulating effect on production of IL-8 as described above (Fig. 2).
In preliminary experiments, the CRH-related peptide urocortin did not
display any stimulating effects on either IL-6 or IL-8 production in
myometrial tissue cultures (n = 3; data not shown).
|
Tissue samples directly snap-frozen after primary cesarean
sections were investigated for their basal cytokine mRNA expression. In
term myometrium obtained before the onset of labor, only IL-1ß and
IL-6 mRNA were found (Fig. 3). In term
myometrium obtained after the onset of labor, additionally IL-8 mRNA
was found in four, and TNF mRNA in two of five, specimens (Table 2). In tissues from preterm cesarean
sections, IL-1ß mRNA was detected in all four samples, whereas IL-6
mRNA was present only in the three samples obtained after 33–36 weeks
of gestation but not in a sample collected after 26 weeks of gestation
(Table 2). All tissues investigated showed a prominent band for the
housekeeping gene cyclophilin that served as a positive control (Fig. 3). The identity of all bands was controlled by restriction enzyme
analysis and confirmed by sequencing.
|
|
mRNA) was
found (n = 9; Fig. 3
). No further change of the cytokine
expression pattern could be observed by this qualitative method in
myometrial explants incubated with either CRH or LPS (results not
shown). Immunohistochemical characterization of macrophages and CRH receptors in term myometrium
A widespread distribution of CRH receptors, predominantly on
leiomyocytes and endothelial cells, in term myometrium was demonstrated
by immunohistochemistry (Fig. 4A). The
antibodies used did not differentiate between CRH receptor subtypes.
Resident macrophages in term myometrium were characterized by a CD68
antibody. Positive staining was predominantly restricted to connective
tissue areas (Fig. 4B). Negative controls, excluding primary
antibodies, did not show any positive cells (Fig. 4C).
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
after 24 h of tissue culture. Time course
experiments revealed a continuously rising release of IL-8 protein
during 23 h of incubation. The synthesis of myometrial IL-6, IL-8,
IL-1ß, and TNF was stimulated significantly by LPS, but not by
CRH, under these conditions. Regarding the stimulation of
proinflammatory cytokines by LPS, we conclude that the cytokine network
in human term myometrium is challenged by bacterial endotoxins and thus
may also be involved in the pathomechanisms contributing to preterm
deliveries. No stimulation of cytokine release has been observed in the presence of CRH in our tissue culture experiments, which contradicts the hypothesis that CRH might exert autocrine or paracrine effects on the cytokine network in term myometrium. This assumption was based on findings that CRH exerts local proinflammatory effects in an animal model (16) and that a variety of CRH receptors are expressed on myometrial smooth muscle cells, endothelial cells, and fibroblasts (14, 19, 20), as well as on bone marrow-derived cells possibly invading the myometrium (26). We conclude that myometrial cytokines are not directly involved in positive feedback loops triggered by CRH at the time of birth.
The cellular source of myometrial cytokines remains to be identified. Resident macrophages may significantly contribute to myometrial cytokine formation, because they are known to produce cytokines in other tissues (e.g. decidua; 27). Recently, the infiltration of the myometrium by leukocytes during human parturition has been reported (28). The presence of CD68-positive macrophages in term myometrium has also been demonstrated in this study. Further, fibroblasts have been recognized as a site of cytokine formation in the lower segment of the human uterus (29). Additionally, cytokines might also be released by myometrial leiomyocytes, because smooth muscle cells from other organs (e.g. airway smooth muscle cells) have been shown to express and secrete cytokines during inflammatory processes (30).
Several reasons led us to the decision to use a tissue culture rather than a cell culture design for the investigation of effects on the myometrial cytokine network. First, the possible contribution of different cell types to cytokine production is better represented by a tissue model than by isolated cells, where cell function and composition of cell populations may be altered during the digestion and isolation procedure. Second, in tissue culture (but not in cell culture) experiments, indirect effects by paracrine cell-to-cell signaling can be recognized. Third, Lonsdale et al. observed a strong activation of the cytokine production by tissue digestion. Therefore, they recommended use of tissue culture instead of cell culture in investigations of the role of the cytokine network at the fetomaternal interface (31).
It cannot be completely ruled out that CRH is inactivated in our experimental setting, e.g. by proteases, before it has penetrated to its target cells. To minimize this problem, we have investigated very small biopsies, incubated with an excessive amount of CRH, and washed out proteases by medium exchange after a preincubation period before adding CRH.
Additional data about the differential expression of the cytokine
network in human myometrium, from the third trimester before and after
the onset of labor, were obtained by RT-PCR using biopsies immediately
snap-frozen after cesarean section. In these experiments, we have
demonstrated that mRNA for IL-1ß and predominantly also for IL-6 are
present throughout the third trimester of pregnancy. Both IL-8 and
TNF mRNA have been found exclusively in biopsies obtained from
patients that had undergone labor. In myometrial cell cultures, both
IL-1ß and TNF have been shown to induce cyclooxygenase-2
(32), thus stimulating the formation of the uterotonic
agents PGE2 and PGF2, as
well as matrix metalloproteinases (33), which are involved
in cervical ripening, implicating the importance of both cytokines for
the onset of labor and delivery. Furthermore, using an in
vitro model, myometrial IL-8, IL-1 and TGFß have been shown to
interact in a complex manner and have been proposed to be integrated
into a system of autocrine signals during parturition
(34).
The presence of IL-1ß and IL-6 mRNA in preterm and term myometrium
before the onset of labor suggests that these cytokines are involved in
the preparation of the myometrium for labor. In contrast, IL-8 and
TNF, whose mRNA expression is restricted to myometrial biopsies that
were obtained after the onset of labor, seem to play a role in later
stages of the birth process or to be a consequence of it. Our results
support recent findings that cytokines may contribute to
inflammation-like processes of normal term labor. In this context, the
release of proinflammatory cytokines has also been reported in the
cervix, amnion, chorion, decidua, and placenta obtained at term after
uncomplicated pregnancies (29, 35, 36, 37).
We have further demonstrated that after short-term tissue culture of myometrial explants obtained before the onset of labor, the mRNA levels of IL-1ß, IL-6, and IL-8 are elevated, compared with the basal expression in uncultured biopsies. Therefore, all cultured tissue samples from nonlaboring women show a cytokine expression pattern similar to uncultured biopsies from patients that had undergone labor. This marked expression of cytokines, after culture in a low endotoxin medium, shows that the myometrial cytokine network is very susceptible to exogenous irritations. Thus, we cannot exclude that a possible CRH effect, significantly smaller than the stimulating effect of LPS, has been superimposed by the culture conditions in our short-term tissue culture experiments. However, in preliminary incubation experiments, run in a balanced salt solution (HBSS), where the basal IL-8 release was much lower than in a parallel incubation using a complete culture medium (Iscove’s modified Dulbecco’s medium), IL-8 production was also not affected by CRH but highly stimulated by LPS (unpublished data).
One should keep in mind that our data were obtained from human myometrium of the lower uterine segment and that these findings might not be representative for the whole uterus, because of topographical differences in myometrial tissue constitution. It has been reported previously that receptors for CRH (14), PGs (38), and oxytocin (39), as well as leukocytes (28), are distributed inhomogeneously in the myometrium. Functionally, the lower segment, which is more susceptible to ascending infections, is thought to relax under birth, to allow the passage of the fetus (14), whereas the fundus is the origin of the contracting forces for the expulsion of the baby. However, the release of proinflammatory cytokines in a single fundal specimen included in our study was not stimulated by CRH either.
In summary, we have demonstrated, on the mRNA and the protein level, that human myometrium from the lower uterine segment, obtained after uncomplicated pregnancies, expresses proinflammatory cytokines at term. Cytokine release is stimulated by LPS but not by CRH. mRNA expression of the cytokine network is up-regulated after the onset of labor.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received June 5, 2000.
Revised August 2, 2000.
Accepted August 18, 2000.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
activity
on human myometrium in vitro. Am J Obstet Gynecol. 171:126–131.[Medline]
This article has been cited by other articles:
 |
|
 |
|
  A. Dubicke, A. Akerud, M. Sennstrom, R. Rafik Hamad, B. Bystrom, A. Malmstrom, and G. Ekman-Ordeberg Different secretion patterns of matrix metalloproteinases and IL-8 and effect of corticotropin-releasing hormone in preterm and term cervical fibroblasts Mol. Hum. Reprod., November 1, 2008; 14(11): 641 - 647. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M Lindstrom and P. R Bennett The role of nuclear factor kappa B in human labour Reproduction, November 1, 2005; 130(5): 569 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Oger, C. Mehats, M. S. Barnette, F. Ferre, D. Cabrol, and M.-J. Leroy Anti-Inflammatory and Utero-Relaxant Effects in Human Myometrium of New Generation Phosphodiesterase 4 Inhibitors Biol Reprod, February 1, 2004; 70(2): 458 - 464. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Young, A. J. Thomson, M. Ledingham, F. Jordan, I. A. Greer, and J. E. Norman Immunolocalization of Proinflammatory Cytokines in Myometrium, Cervix, and Fetal Membranes During Human Parturition at Term Biol Reprod, February 1, 2002; 66(2): 445 - 449. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Helmer, U. Tretzmuller, M. Brunbauer, A. Kaider, P. Husslein, and M. Knofler Production of Oxytocin Receptor and Cytokines in Primary Uterine Smooth Muscle Cells Cultivated Under Inflammatory Conditions Reproductive Sciences, January 1, 2002; 9(1): 15 - 21. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
