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Bacterially-Induced Preterm Labor and Regulation of Prostaglandin

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Bacterially-Induced Preterm Labor and Regulation of Prostaglandin Powered By Docstoc
					BIOLOGY OF REPRODUCTION 69, 1957–1963 (2003)
Published online before print 6 August 2003.
DOI 10.1095/biolreprod.103.019620


Bacterially-Induced Preterm Labor and Regulation of Prostaglandin-Metabolizing
Enzyme Expression in Mice: The Role of Toll-Like Receptor 41

Hao Wang and Emmet Hirsch2
Department of Obstetrics and Gynecology, Evanston Northwestern Healthcare, Feinberg School of Medicine,
Northwestern University, Evanston, Illinois 60201

ABSTRACT                                                              flammatory stimulus to labor after bacterial exposure re-
                                                                      main incompletely defined.
   Toll-like receptor 4 (TLR-4) is a critical mediator of the cel-       Prostaglandins stimulate uterine contractions and cervi-
lular response to lipopolysaccharide. Our purpose was to ex-          cal ripening during labor. Both prostaglandin E 2 (PGE 2)
amine the role of TLR-4 in parturition and in the regulation of       and prostaglandin F2 (PGF2 ) are produced by maternal
expression of prostaglandin synthase (cyclooxygenase [COX]-1
and COX-2) and 15-hydroxyprostaglandin dehydrogenase
                                                                      and fetal tissues during parturition, and the concentration
(PGDH) following exposure to heat-killed Escherichia coli (HKE)
                                                                      of both increases in the amniotic fluid during labor [9].
in pregnant mice. Inbred TLR-4-mutant C3H/HeJ mice and in-            Administration of prostaglandin synthase inhibitors sup-
bred normal C3HeB/FeJ mice on Day 14.5 of a 19- to 20-day             presses uterine activity and prolongs the length of pregnan-
gestation received intrauterine injection of either HKE or sterile    cy [10]. Primary prostaglandins are formed from arachi-
vehicle (PBS). Preterm or term delivery was recorded for these        donic acid through activity of the cyclooxygenase (COX)
animals. Tissues (myometrium, decidual caps, placentas, fetal         enzyme complex. The COX catalyzes the first committed
membranes, and fetuses) were collected after injection of sterile     step of prostaglandin synthesis, the initial conversion of
vehicle or 5     109 HKE bacteria (n       5 mice per strain per      arachidonic acid to PGH2. Two isoforms of COX have been
treatment per time point). The COX-1, COX-2, and PGDH gene            identified, COX-1 and COX-2, which are also known as
expression was determined by semiquantitative reverse tran-           prostaglandin endoperoxide H synthase (PGHS)-1 and
scription-polymerase chain reaction. We found that 5 109 HKE          PGHS-2, respectively. The COX-1 is constitutively ex-
induced preterm delivery in 100% of TLR-4-normal mice but in          pressed in many tissues with little regulation in synthesis,
0% of TLR-4-mutant mice. The HKE exposure up-regulated ex-            whereas COX-2 is inducible in response to a variety of
pression of COX-2, but not of COX-1, in maternal tissues in both      growth factors and inflammatory stimuli. Mice lacking the
mouse strains. The prostaglandin-catabolizing enzyme PGDH             gene for PGF2 receptor [11] or COX-1 [12] have delayed
was down-regulated in myometrium, fetal membranes, and fe-            onset of labor because of the roles of these factors in reg-
tuses in control mice, but no change was observed in TLR-4-           ulating luteolysis and myometrial expression of oxytocin
mutant mice after HKE treatment. These results demonstrate that       receptors. Recent studies demonstrate a dramatic increase
a functional TLR-4 is essential for HKE-induced preterm labor
and PGDH down-regulation but is not essential for HKE-induced
                                                                      of COX-1, but not of COX-2, transcripts and activity in the
COX-2 gene up-regulation. The TLR-4 may mediate bacterially           uterus [13] and fetal membranes [14] in mice during late
induced preterm labor via regulation of prostaglandin degra-          gestation. In one report, both COX-1 and COX-2 expres-
dation rather than prostaglandin synthesis.                           sion within the uterus were significantly altered within 2 h
                                                                      of lipopolysaccharide (LPS) administration, with COX-2
gene regulation, parturition, pregnancy uterus                        increasing and COX-1 decreasing [15].
                                                                         The NAD -dependent 15-hydroxyprostaglandin dehy-
INTRODUCTION                                                          drogenase (PGDH) is responsible for the initial inactivation
                                                                      of prostaglandins, catalyzing the conversion of primary
    Preterm birth occurs in 5–10% of all pregnancies and              prostaglandins to their biologically inactive 15-keto deriv-
accounts for 70–75% of early neonatal morbidity and mor-              atives. Expression and activity of PGDH have been dem-
tality [1]. Approximately 30–40% of preterm births are as-            onstrated in fetomaternal tissues of different species. Re-
sociated with an underlying infectious process. Systemic              duced PGDH expression and activity in myometrium and
and local intrauterine infections have been implicated in the         chorion have been suggested in association with term and
pathogenesis of preterm labor and delivery [2, 3]. Indeed,            preterm birth in humans [9]. However, in the mouse, PGDH
either local or systemic exposure to microbial products               mRNA increased in placentas and fetal membranes [16]
leads to preterm birth in several animal models [4–8]. How-           and decreased in uterus [13] during late gestation. These
ever, the signaling pathways leading from the inciting in-            studies suggest that COX and PGDH in the fetomaternal
                                                                      environment may play important roles in the initiation of
1
 Supported by a grant from March of Dimes (6-FY99-908) and NIH        labor, but the relative contributions of the maternal and the
(1RO1HD41689).                                                        fetal tissues to COX and PGDH activity during infection
2
 Correspondence: Emmet Hirsch, Department of Obstetrics and Gyne-     are still incompletely defined.
cology, Evanston Northwestern Healthcare, 2650 Ridge Avenue, Evans-
ton, IL 60201. FAX: 847 733 5083; e-mail: e-hirsch@northwestern.edu
                                                                         We have reported a model of infection-induced preterm
                                                                      birth in mice following intrauterine inoculation with live or
                                                                      heat-killed Escherichia coli (HKE). Intrauterine inoculation
Received: 28 May 2003.
First decision: 14 June 2003.
                                                                      of pregnant CD-1 mice with HKE on Day 14.5 of a 19- to
Accepted: 6 August 2003.                                              20-day gestation leads to dose-dependent preterm delivery.
   2003 by the Society for the Study of Reproduction, Inc.            This process mimics human infection-associated preterm
ISSN: 0006-3363. http://www.biolreprod.org                            labor in many important ways, such as the expression of
                                                                  1957
1958                                                             WANG AND HIRSCH

proinflammatory cytokines [17–19]. We have demonstrated                         or HKE suspended in PBS were injected into the midsection of the right
significant increases in interleukin (IL)-1, IL-6, and tumor                    uterine horn at a site between two adjacent fetuses. The abdominal incision
                                                                               was then closed in two layers using interrupted 5-0 coated vicryl sutures
necrosis factor (TNF) within uteri and fetal membranes fol-                    through the peritoneum and staples at the skin.
lowing HKE treatment in this mouse model [19], as has                              To establish the dose-response relationship between bacterial exposure
been demonstrated in humans. However, several groups                           and preterm delivery, a group of 40 pregnant C3HeB/FeJ (TLR-4-normal)
have shown that blockade of IL-1 and/or TNF by using IL-                       mice and 20 pregnant C3H/HeJ (TLR-4-mutant) mice were monitored af-
1-receptor antagonist, soluble TNF-receptor Fc fusion pro-                     ter inoculation of variable amounts of HKE. These animals underwent
tein, or IL-1 and IL-1-receptor knockout mice does not pre-                    twice-daily observations in which health status was recorded. Mice that
                                                                               delivered prematurely (defined as the finding of at least one pup in the
vent preterm delivery in mice after administration of bac-                     cage or the lower vagina within 48 h of surgery) or delivered at term
teria or LPS [20–22]. Thus, it remains uncertain whether                       underwent autopsy when fetuses were found in the cage. Some mice not
inflammatory cytokines are critical mediators of the signals                    delivered prematurely underwent autopsy 72 h after surgery to determine
by which bacterial exposure causes labor.                                      fetal status.
   A gram-negative bacterial cell wall component, LPS can                          For measurement of COX and PGDH transcripts within fetomaternal
induce expression of inflammatory cytokines and prosta-                         tissues, a second group of pregnant C3HeB/FeJ and C3H/HeJ mice were
                                                                               inoculated with either pyrogen-free PBS or HKE sufficient to cause pre-
glandins and leads to preterm birth in pregnant mice [6, 8]                    term delivery in 100% of C3HeB/FeJ mice (5 109 organisms). Animals
and rats [23] following intraperitoneal injection or intra-                    were killed with carbon dioxide gas, and tissues were collected at 0, 1, 2,
uterine infusion. The current consensus is that LPS signal-                    and 4 h after surgery for C3HeB/FeJ mice or at 4 h after surgery for C3H/
ing occurs through a heterotrimeric receptor complex, of                       HeJ mice (5 animals per group per time point). Harvest times early during
which the toll-like receptor 4 (TLR-4) protein is a critical                   the course of infection were selected, because previous data showed that
component. A point mutation in the intracytoplasmic region                     levels of cytokines, enzymes, and transcription factors change in myo-
                                                                               metrium in CD-1 mice within 4 h of inoculation with high-dose HKE
of TLR-4 is responsible for the 20- to 40-fold LPS hypo-                       [18]. The abdomen was opened, and the injected uterine horn was cut
responsiveness of C3H/HeJ mice [24]. The defective Lps                         away from its mesometrium and then incised longitudinally along the an-
allele affects the functions of several cell types, including                  timesenteric border. The gestational sacs and placentas were shelled out,
the macrophage, which on activation normally secretes a                        and the right uterine horns were then washed in ice-cold PBS. The decid-
large array of proinflammatory factors and is critical for the                  ual cap at each implantation site was removed by sharp dissection, leaving
innate immune response to bacterial pathogens.                                 behind the myometrium. The individual sacs surrounding each fetus were
                                                                               cut away, and fetus, fetal membranes, and placentas were washed in cold
   In the present study, we used inbred pregnant C3HeB/                        PBS. These specimens were then minced and immediately frozen in liquid
FeJ mice (TLR-4-normal) and C3H/HeJ (TLR-4-mutant)                             nitrogen. Decidual caps, fetal membranes, placentas, and fetuses were
mice to test the hypothesis that E. coli induces preterm                       pooled by tissue for each pregnancy.
labor via TLR-4 and to characterize the role of TLR-4 in
the fetal and maternal expression of prostaglandin meta-                       RNA extraction
bolic enzymes during the early phase of bacterially induced
preterm labor.                                                                     Total RNA was extracted from tissue specimens after homogenization
                                                                               in TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufac-
                                                                               turer’s instructions. The quantity and quality of the RNA was verified by
MATERIALS AND METHODS                                                          spectrophotometry and formaldehyde gel electrophoresis, respectively.
Animals
                                                                               Reverse Transcription-Polymerase Chain Reaction
    All animals were treated in accordance with the Guide for Care and
Use of Laboratory Animals and with the approval of the Animal Care and             Five to eight micrograms of total RNA were used as a template for
Use Committee of Evanston Northwestern Healthcare and Northwestern             cDNA synthesis. The cDNA was prepared using random primers and the
University. Inbred C3HeB/FeJ mice and C3H/HeJ mice (Jackson Labo-              Moloney murine leukemia virus reverse transcriptase system (Invitrogen).
ratory, Bar Harbor, ME) were housed at an ambient temperature of 72 F          Polymerase chain reaction (PCR) primers were designed and synthesized
and a 12L:12D photoperiod. Animals had free access to food and water.          on the basis of reported mouse cDNA sequences for COX-1, COX-2,
Females (age, 8–14 wk) were mated with fertile males. Mating was veri-         PGDH, and -actin. Sequences of the primers and amplicon lengths were
fied by the presence of a vaginal plug. On Day 14.5 after plugging ( 75%        as follows: for COX-1, 5 -gcatgtggctgtggatgtca-3 (forward) and 5 -ggt
of the typical 19- to 20-day gestation), surgery was performed (see below).    cttggtgttgaggcaga-3 (reverse), with an amplicon of 388 base pairs (bp)
                                                                               corresponding to nucleotides 1395–1782 in the mouse COX-1 cDNA
Preparation of HKE                                                             (GenBank accession no. BC005573.1); for COX-2, 5 -acactctatcactggca
                                                                               ccc-3 (forward) and 5 -gaagggacaccccttcacat-3 (reverse), with an ampli-
    Escherichia coli bacteria (American Type Culture Collection no.            con of 585 bp corresponding to nucleotides 1229–1813 in the mouse
12014) were grown to log phase at 37 C in Luria-Bertani broth (Invitro-        COX-2 cDNA (GenBank accession no. NM011198.1); for PGDH, 5 -
gen, Carlsbad, CA) and concentrated by centrifugation. They were then          atttcggaagattggatattttggtc-3 (forward) and 5 -ttcaatgagatctattaatccattgg-3
washed three times with phosphate-buffered saline (PBS) and suspended          (reverse), with an amplicon of 461 bp corresponding to nucleotides 269–
in PBS. Serial dilutions of the E. coli suspension were plated in triplicate   729 in the mouse PGDH cDNA (GenBank accession no. NM008278.1);
to determine the concentration of bacteria by overnight culture. Immedi-       and for -actin, 5 -attgtgatggactccggtgacgg-3 (forward) and 5 -atcttgatctt-
ately after plating these dilutions, the E. coli within the PBS suspension     catggtgctagg-3 (reverse), with an amplicon of 536 bp corresponding to
were killed by boiling in water for 5 min, and the suspension was then         nucleotides 373–908 in the mouse -actin cDNA (GenBank accession no.
frozen at 20 C. Bacterial killing was verified by lack of growth overnight      M12481.1). Each PCR reaction was performed in a 30- l mixture con-
in broth and solid media. After the concentration of the bacterial suspen-     taining 1.0 l of cDNA, 10 pmol of each primer, 0.25 mM dNTP, and
sion was determined, the HKE stock was thawed and diluted to a concen-         1.25 U of Taq DNA polymerase (Roche Applied Science, Indianapolis,
tration of 1     1010 organisms/ml. This latter suspension was vortexed,       IN). Cycling conditions were as follows: denaturation for 40 sec at 94 C,
aliquoted, and frozen at 80 C. Before each experiment, one of the frozen       annealing for 30 sec at 62 C, and extension for 60 sec at 72 C. After
killed bacterial aliquots was thawed, vortexed, and diluted as necessary to    sequencing of COX-1, COX-2, PGDH, and -actin PCR products to con-
the desired concentration.                                                     firm the gene specificity of these fragments, the optimal number of am-
                                                                               plification cycles was determined in pilot reactions and were as follows:
Inoculation procedure and specimens                                            22 cycles for -actin, 28 cycles for COX-1, 30 cycles for COX-2, and 38
                                                                               cycles for PGDH.
    Pregnant mice were anesthetized with 16–18 g/g body weight of a                After amplification, the PCR products were resolved by 1.2% agarose
mixture of 2.5% (w/v) tribromoethyl alcohol and 2.5% (v/v) tert-amyl           gel electrophoresis and visualized by staining with SYBR green-1 (Mo-
alcohol (Aldrich Chemical, Milwaukee, WI) in PBS. A 1.5-cm midline             lecular Probes, Eugene, OR). Cycle numbers were determined empirically
incision was made in the lower abdomen, and 100 L of pyrogen-free PBS          to yield amplicon bands of moderate intensity that represented a linear
                                     TLR-4, COX, AND PGDH IN BACTERIALLY-INDUCED LABOR                                                          1959

                                                                                                       FIG. 1. Expression of COX-1 and COX-2
                                                                                                       transcripts in maternal tissues of C3HeB/
                                                                                                       FeJ (TLR-4-normal) mice receiving PBS
                                                                                                       (Control) or HKE. The COX-1, COX-2, and
                                                                                                         -actin transcripts were detected by RT-
                                                                                                       PCR. Typical gels are shown (A). Ratios of
                                                                                                       the relative signal intensities of COX-1/ -
                                                                                                       actin (B) and COX-2/ -actin (C) in myo-
                                                                                                       metrium and decidual caps (n        5 in each
                                                                                                       group) are depicted. *P      0.05 compared
                                                                                                       with the value of vehicle-treated controls.




relationship between the number of cycles and the logarithm of the number     RESULTS
of target molecules. The density of each DNA band was evaluated with a
STORM-860 PhosphorImager and analyzed using the ImageQuantTM soft-            Dose-Response Relationship between HKE and Incidence
ware package (both from Molecular Dynamics, Sunnyvale, CA) as re-             of Preterm Delivery in TLR-4-Normal (C3HeB/FeJ) Mice
ported previously [25]. The ratios of the signals for COX-1, COX-2, and
PGDH to that of -actin were used as to determine the relative level of           Six of six LPS-sensitive C3HeB/FeJ mice treated with
transcription expression. We have shown that -actin levels remain rela-       vehicle delivered normal litters at term. Intrauterine injec-
tively stable within gestational tissues following HKE exposure in our        tion of HKE demonstrated a dose-response relationship be-
mouse model over at least 4 h (unpublished data).
                                                                              tween inoculum size and incidence of preterm delivery (Ta-
                                                                              ble 1). Administration of the higher inocula (5     109 or 1
                                                                                 10 10 bacteria) resulted in 100% preterm delivery within
Statistical Analysis
                                                                              an average of 17.5 h after surgery. All offspring delivered
    All values in the figures and text are expressed as the mean      SEM.     preterm were dead. Among animals treated with the lower
Data sets were examined by one-way analysis of variance, and individual
group means were compared with the Student unpaired t-test. For preterm
                                                                              HKE inocula, intrauterine demise was observed in 20% of
delivery or fetal death, chi-square analyses was used with the Fisher exact   fetuses exposed to 1 107 bacteria (48 live fetuses and 12
correction when necessary. Differences between groups were considered         dead fetuses in a total of six mothers killed on Day 3 after
to be significant when P       0.05.                                           surgery) and in 55% exposed to 1       108 bacteria (17 live



TABLE 1. Relationship between inoculum size of HKE and incidence of preterm delivery in C3HeB/FeJ (normal) and C3H/HeJ (TLR-4-mutant) mice
treated on Day 14.5 of 20-day gestation.a

HKE in 100 l of PBS                                  0            1    107        1    108       1    109          5    109          1     1010
C3HeB/FeJ mice (TLR-4 normal)                        6                 6               8              7               7                    6
Preterm delivery                                    0%                0%              25%            71%            100%                 100%
C3H/HeJ mice (TLR-4 mutant)                          5                                                                8                    7
Preterm delivery                                    0%                                                                0%                  28%
a   Preterm delivery was defined as expulsion of one or more pups within 48 h of surgery.
1960                                                           WANG AND HIRSCH

FIG. 2. Expression of COX-1 and COX-2
transcripts in fetal tissues of C3HeB/FeJ
(TLR-4-normal) mice receiving PBS (Con-
trol) or HKE. The COX-1, COX-2, and -
actin transcripts were detected by reverse
transcription-PCR. Typical gels are shown
(A). Ratios of the relative signal intensities
of COX-1/ -actin (B) and COX-2/ -actin
(C) in placentas, fetal membranes, and fe-
tuses (n    5 in each group) are depicted.
*P     0.05 compared with the value of ve-
hicle-treated controls.




fetuses and 21 dead fetuses in a total of four mothers killed                was observed in 7% of fetuses exposed to 5 109 bacteria
on Day 3 after surgery). In general, animals that were ad-                   (26 live fetuses and two dead fetuses in a total of five moth-
ministered a delivery inoculum at or below 5 109 organ-                      ers killed on Day 3 after surgery) and in 34% exposed to
isms appeared to be healthy or mildly ill (exhibiting mild                   1 1010 bacteria (23 live fetuses and 12 dead fetuses in a
piloerection, decreased mobility, and anorexia). Recovery                    total of five mothers killed on Day 3 after surgery). Ma-
after delivery appeared to be complete in most cases. The                    ternal illness was not seen with 5     109 bacteria and was
HKE inoculation at a dose of 1       1010 organisms caused                   only mild with 1      1010 bacteria.
maternal death or severe illness requiring five of six mice
to be killed.                                                                Effects of HKE on COX-1, COX-2, and PGDH Transcripts
                                                                             in Maternal and Fetal Tissues
Effect of TLR-4 on Bacterially Induced Preterm Delivery
                                                                                To determine whether transcription of the rate-limiting
   To test the hypothesis that HKE induces preterm deliv-                    enzymes of prostaglandin synthesis (COX-1, COX-2, and
ery via TLR-4, C3H/HeJ (TLR-4-mutant) mice were in-                          PGDH) is regulated via TLR-4 during the early phase of
oculated with quantities of HKE sufficient to cause preterm                   bacterially induced preterm labor, semiquantitative reverse
delivery in 100% of control C3HeB/FeJ mice. As shown                         transcription-PCR was performed in pregnancy tissues ob-
in Table 1, preterm delivery occurred in none of eight TLR-                  tained within 4 h after administration of either PBS or 5
4-mutant mice after treatment with 5       109 HKE and in                    109 HKE. In both mouse strains, bacterial exposure caused
two of seven after treatment with 1      1010 HKE. Among                     significant increases in COX-2 mRNA in myometrium and
TLR-4-mutant mice treated with HKE, intrauterine demise




FIG. 3. Comparison of COX-2 transcripts in fetomaternal tissues of nor-      FIG. 4. Comparison of COX-1 transcripts in fetomaternal tissues of nor-
mal (C3HeB/FeJ) and TLR-4-mutant (C3H/HeJ) mice 4 h after HKE injec-         mal (C3HeB/FeJ) and TLR-4-mutant (C3H/HeJ) mice 4 h after HKE injec-
tion. For purposes of standardization, all ratios are expressed as fold-     tion. For purposes of standardization, all ratios are expressed as fold-
change from the baseline measurement in control fetuses. *P 0.05 com-        change from the baseline measurement in control fetuses. No significant
pared with the value of vehicle-treated controls for the same tissue type.   differences are present between the two treatment groups.
                              TLR-4, COX, AND PGDH IN BACTERIALLY-INDUCED LABOR                                               1961

                                                                                         FIG. 5. Comparison of PGDH transcripts
                                                                                         in fetomaternal tissues of normal (C3HeB/
                                                                                         FeJ) and TLR-4-mutant (C3H/HeJ) mice 4 h
                                                                                         after HKE injection. The PGDH and -ac-
                                                                                         tin transcripts were detected by reverse
                                                                                         transcription-PCR. Typical gels are shown
                                                                                         (upper panels) as well as quantitative anal-
                                                                                         ysis of relative signal intensities of PGDH/
                                                                                           -actin ((lower panels); n     5 in each
                                                                                         group). *P     0.05 compared with the val-
                                                                                         ue of vehicle-treated controls.




decidual caps but not in placentas and fetuses (Figs. 1–3).     sheep, increased COX-2 expression in endometrium, pla-
After HKE treatment, COX-2 mRNA becomes higher in               centa, and myometrium was tightly associated with the on-
maternal tissues (myometrium and decidual caps) than in         set of betamethasone-induced premature labor as well as
fetal tissues in C3HeB/FeJ (normal) mice but not in C3H/        spontaneous term labor [30]. In the baboon, COX-2 ex-
HeJ (LPS-resistant) mice (Fig. 3). Basal levels of COX-1        pression was increased in the lower uterine segment, cervix,
mRNA in maternal tissues are higher than in fetal tissues       and decidua but not in the uterine fundus, chorion, and
in C3H/HeJ mice but not in C3HeB/FeJ mice. Levels of            placenta during late pregnancy and labor [31]. In humans,
COX-1 remained unchanged in all tested maternal and fetal       a large increase in COX-2 mRNA was found throughout
tissues in both strains (Figs. 1, 2, and 4).                    late gestation in fetal tissues [32]. Both COX-1 and COX-
    In normal (C3HeB/FeJ) mice, basal levels of PGDH            2 have been reported to decrease or remain unchanged in
were detectable but were lower in maternal tissues (myo-        the myometrium at the onset of labor [33] and to increase
metrium and decidual caps) than in fetal tissues (Fig. 5).      in the amnion during term labor [34].
Levels of PGDH decreased significantly after HKE treat-             The present results show that the levels of COX-2 tran-
ment in fetuses, fetal membranes, and myometrium, re-           scripts in myometrium and decidua increase sharply in re-
spectively, compared to those after control injections. In      sponse to HKE treatment in both C3HeB/FeJ and C3H/HeJ
decidual caps, however, PGDH mRNA increased over its            mice, whereas COX-1 mRNA levels remain unchanged in
low basal level. In TLR-4-mutant (C3H/HeJ) mice, basal          the tissues tested for both mouse strains. These data suggest
levels of PGDH were detectable in fetal tissues but not in      that TLR-4 signaling may not be essential for bacterially
maternal tissues (Fig. 5). Levels of PGDH remained un-          induced COX-2 gene expression, and they support the no-
changed in fetal tissues after HKE treatment.                   tion that COX-2, not COX-1, is the enzyme primarily re-
                                                                sponsible for increased prostaglandin biosynthesis during
DISCUSSION                                                      bacterially induced preterm labor. Several studies have
   The present study demonstrates that TLR-4 is a critical      shown that COX-2 antagonism may inhibit preterm labor
mediator of labor signals in the murine bacterial infection     in different species, such as human [35], sheep [36], rat
model. Although HKE induces premature delivery in nor-          [37], and mice [15, 38]. This conclusion is also supported
mal mice in a dose-dependent manner, this phenomenon            by other data, such as a report that pretreatment of pregnant
does not occur, or is greatly obtunded, in TLR-4-mutant         mice with COX-2 inhibitor, but not COX-1 inhibitor, pre-
mice. The small effect observed with very high numbers          vented LPS-induced preterm labor [15].
of HKE may be caused either by residual signaling via the          To our knowledge, this is the first study to report that
mutant TLR-4 or other receptors or by bacterial factors oth-    the basal levels of PGDH mRNA in mid- to late-pregnancy
er than LPS. Parallel observations were also made in rela-      mice are higher in fetal tissues (fetus, fetal membranes, and
tion to maternal illness and fetal demise resulting from bac-   placenta) than in maternal uterine tissues in both C3HeB/
terial exposure. We have previously demonstrated that fetal     FeJ and C3H/HeJ mice. This tissue distribution of PGDH
death per se in the absence of a bacterial stimulus does not    may allow for finely controlled regulation of prostaglandin
result in labor in the mouse [19].                              activity in individual tissues during pregnancy. After HKE
   What is the mechanism by which bacteria cause labor?         treatment, PGDH mRNA decreases significantly in the fe-
Previous studies have suggested a central role for prosta-      tus, fetal membranes, and myometrium in TLR-4-normal
glandins and their synthetic and catabolic enzymes in this      mice but not in TLR-4 mutant mice. These data suggest
process. Variable reports have appeared concerning changes      that TLR-4 signaling may be involved in HKE-induced
in COX expression at parturition in different species and in    PGDH down-regulation. The role of prostaglandins gener-
different circumstances. In the mouse, increased COX-1,         ated in the fetus itself during gestation is still not clear.
but not COX-2, mRNA was reported during late gestation          When the fetus is infected, fetal cortisol, cytokine, and
in the uterus [13] and fetal membranes [14], with increased     prostaglandin production are increased [39, 40]. The cur-
COX-2 expression during term labor as well as ethanol-          rently accepted hypothesis is that PGDH represents a met-
and LPS-induced preterm labor [26, 27]. In the rat, one         abolic barrier in fetal tissues, either to prevent the passage
study found that both COX-1 and COX-2 increased in myo-         of prostaglandins generated in these tissues to the uterus or
metrium with the onset of labor [28], but a different study     to prevent prostaglandins generated in other tissues from
reported increased expression of COX-2 only [29]. In            damaging the fetus. During HKE-induced preterm labor,
1962                                                             WANG AND HIRSCH

this functional barrier may break down, perhaps accounting                   18. Muhle RA, Pavlidis P, Grundy WN, Hirsch E. A high-throughput
for the fetal inflammatory response that has been observed                        study of gene expression in preterm labor with a subtractive microar-
                                                                                 ray approach. Am J Obstet Gynecol 2001; 185:716–724.
during infection [40, 41].                                                   19. Mussalli GM, Blanchard R, Brunnert SR, Hirsch E. Inflammatory cy-
   In summary, the present study suggests that TLR-4 sig-                        tokines in a murine model of infection-induced preterm labor: cause
naling is a critical factor in bacterially induced preterm la-                   or effect? J Soc Gynecol Investig 1999; 6:188–195.
bor. During bacterially induced preterm labor, TLR-4 sig-                    20. Reznikov LL, Fantuzzi G, Selzman CH, Shames BD, Barton HA, Bell
naling mediates PGDH gene down-regulation but is not es-                         H, McGregor JA, Dinarello CA. Utilization of endoscopic inoculation
sential for COX-2 gene up-regulation. Bacterially induced                        in a mouse model of intrauterine infection-induced preterm birth: role
                                                                                 of interleukin 1beta. Biol Reprod 1999; 60:1231–1238.
prostaglandin activity may be mediated primarily by in-                      21. Fidel PL Jr, Romero R, Cutright J, Wolf N, Gomez R, Araneda H,
creased synthesis in maternal tissues and decreased degra-                       Ramirez M, Yoon BH. Treatment with the interleukin-I receptor an-
dation in fetal tissues.                                                         tagonist and soluble tumor necrosis factor receptor Fc fusion protein
                                                                                 does not prevent endotoxin-induced preterm parturition in mice. J Soc
ACKNOWLEDGMENT                                                                   Gynecol Investig 1997; 4:22–26.
                                                                             22. Hirsch E, Muhle RA, Mussalli GM, Blanchard R. Bacterially induced
   The authors thank Yana Filipovich for technical assistance.                   preterm labor in the mouse does not require maternal interleukin-1
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