Reactive oxygen species mediate leukocyte-endothelium
interactions in prostaglandin F2
-induced luteolysis in
rats
Kazuhiro
Minegishi,
Mamoru
Tanaka,
Osamu
Nishimura,
Shinji
Tanigaki,
Kei
Miyakoshi,
Hitoshi
Ishimoto, and
Yasunori
Yoshimura
Department of Obstetrics and Gynecology, Keio University School
of Medicine, 160-8582 Tokyo, Japan
 |
ABSTRACT |
We
investigated the contribution of neutrophils to prostaglandin
(PG)F2
-induced luteolysis and the role of reactive
oxygen species (ROS) as potential mediators of neutrophil accumulation in regressing corpora lutea of superovulated rats. On day 8 of pseudopregnancy, subcutaneous injection of PGF2
(500 µg) significantly increased rhodamine 6G-labeled leukocyte adhesion in luteal venules, as observed by intravital microscopy. Neutrophil accumulation was confirmed by significantly increased myeloperoxidase (MPO) activity. Pretreatment with anti-leukocyte antibody
(CD18-directed monoclonal antibody, WT-3) significantly inhibited the
PGF2
-induced increases in adherent leukocytes and MPO
activity. Anti-leukocyte antibody also maintained serum progesterone
concentrations. Pretreatment with oxygen free radical scavengers,
superoxide dismutase (50,000 U/kg) and catalase (90,000 U/kg), also
attenuated these PGF2
-induced alterations. Corpora lutea
preloaded with dichlorodihydrofluorescein diacetate succinimidyl ester,
a fluorescent indicator for determining intracellular ROS generation,
exhibited an increase in fluorescence after PGF2
treatment. These findings suggest that leukocyte-endothelium interactions mediated by ROS generation are important in
PGF2
-induced luteolysis in rats.
corpus luteum; neutrophil; CD18 integrin; myeloperoxidase activity; intravital microscopy
 |
INTRODUCTION |
IN MANY SPECIES,
luteolysis is characterized by two closely related events
(21). Loss of the capacity to synthesize and secrete
progesterone (functional luteolysis) is followed by loss of luteal
cells (structural luteolysis). Reactive oxygen species (ROS), including
superoxide anion and hydrogen peroxide, have been implicated in the
luteolytic process. Prostaglandin (PG)F2
induces
functional luteolysis in association with generation of superoxide
anion and hydrogen peroxide in the rat corpus luteum as serum
progesterone concentrations decrease (26, 29, 30). Exogenous hydrogen peroxide has been shown to inhibit progesterone synthesis in human and rat luteal cells (2, 37). We have previously shown in rats that luteal cells might be the source of the
ROS generated in response to PGF2
(36).
Leukocytes, thought to be important in the process of luteal
regression, also are an important source of ROS (3).
Neutrophils, macrophages, and lymphocytes are well known to produce
toxic bursts of ROS, as well as cytokines including interleukins
(IL), interferons (INF), and tumor necrosis factor (TNF). Activated
neutrophils have been shown to inhibit gonadotropin action and
progesterone synthesis by producing ROS, affecting rat luteal cells
(24). These cytokines may be involved in luteolysis, as
expression of mRNAs for IL-1
, INF-
, and TNF-
has been
described in bovine corpora lutea after PGF2
treatment
(25), and a luteolytic effect of the cytokines has been
implicated in suppression of progesterone secretion in bovine or
porcine luteal cells (6, 15, 23). Accordingly, leukocytes
infiltrating the corpus luteum are presumed to be involved in luteal
regression. Influx of neutrophils can contribute to
PGF2
-induced luteolytic events, and ROS generated within
the corpus luteum can act as potential mediators of neutrophil accumulation.
Leukocyte-endothelium interactions are important in the acute
inflammatory response during various pathological processes (9). Circulating leukocytes migrate from the blood vessel
to areas of inflammation by multistep mechanisms involving a process of
rolling, adhesion, and migration. Intravital microscopy permits precise
determination of the time course of these events as well as ongoing
discrimination of the various components of this process. We therefore
used intravital microscopic observation to evaluate PGF2
-induced leukocyte adhesion to venules of the corpus luteum. The present study was designed to assess the contribution of
neutrophils to luteal regression elicited by PGF2
and to
evaluate the role of ROS as possible mediators of neutrophil accumulation via changes in the microcirculation of the regressing rat
corpus luteum.
 |
MATERIALS AND METHODS |
Animal preparation.
Animals were housed and cared for in the fully accredited facilities of
the Division of Animal Care of Keio University. All procedures were
conducted in accordance with the US National Institutes of Health (NIH)
Guidelines for the Care and Use of Laboratory Animals and
were approved by the Animal Care Committee of Keio University. Immature
female Sprague-Dawley rats (26-28 days old; Sankyo Laboratory
Service, Tokyo, Japan) were injected with 50 IU of pregnant mare serum
gonadotropin (Teikokuzouki Pharmaceuticals, Tokyo, Japan),
followed 56 h later with 50 IU of human chorionic gonadotropin
(hCG; Mochida Pharmaceuticals, Tokyo, Japan) to induce ovulation and
corpus luteum formation. Rats were studied for 8 days after hCG
injection, encompassing the luteal phase of the ovarian cycle. To
initiate luteal regression, 500 µg of PGF2
(Dinoprost;
a generous gift from Ono Pharmaceutical, Tokyo, Japan) were injected on
day 8 of pseudopregnancy (26, 30).
Intravital observation of microcirculation in the corpus luteum.
Animals were initially anesthetized with ether. Then, the left femoral
artery was cannulated under general halothane anesthesia (3% for
induction, followed by 1.0-1.5% for maintenance). Systemic arterial pressure was continuously monitored with a pressure transducer connected to a femoral artery cannula, as described previously (20). The left femoral vein was cannulated for drug
administration. Rats were placed in a left supine position on a
microscope stage, where their backs were opened via a small incision.
The ovary was carefully exposed by incision of the ovarian bursa and
then mounted on a plastic support for intravital microscopy. The
preparation was kept at 37°C, and the ovarian microcirculation was
observed under superfusion with Krebs-Henseleit bicarbonate-buffered
solution (pH 7.4) saturated with a 95% N2-5%
CO2 gas mixture.
The microcirculation of the corpus luteum was visualized through an
intravital microscope (×20 water immersion objective lens; Nikon,
Tokyo, Japan) via a digital color charge-coupled device camera (C5810;
Hamamatsu Photonics, Shizuoka, Japan). A video camera mounted on the
microscope projected the image onto a TV monitor (PVM-1444Q; Sony,
Tokyo, Japan), and the images were recorded for playback analysis using
a videocassette recorder (SVO-260, Sony). A video time generator
(VTG-55; For-A, Tokyo, Japan) projected the time and stopwatch
functions onto the monitor.
For visualization of leukocytes, the in vivo fluorescent marker
rhodamine 6G (0.3 mg/kg; Sigma Chemical, St. Louis, MO) was injected
intravenously 5 min before experiments and 60 min thereafter (12). To visualize blood flow, erythrocytes from separate
donor rats were stained with fluorescein isothiocyanate (FITC; Isomer I; Sigma) in vitro according to a modification of the method of Zimmerhackl et al. (38). FITC-labeled erythrocytes were
injected intravenously (0.2 ml/body) 10 min before experiments. For
epi-illumination, a 100-W mercury lamp was attached to a Nikon
illuminator with a G-2A filter block (excitation, 510-560 nm;
emission,
590 nm; Nikon) and a B-2A filter block (excitation,
450-490 nm; emission,
520 nm; Nikon). Rhodamine 6G-stained
leukocytes were identified using the G-2A filter block, whereas
FITC-labeled erythrocytes were identified using the B-2A filter block.
Thus the system allowed separate visualization of erythrocytes and leukocytes.
Single unbranched venules with diameters ranging between 35 and 55 µm
and lengths >150 µm were selected for this study. Vessel diameter
was measured directly off the videotape on playback by use of
previously videotaped calibrations (28). The number of adherent leukocytes was also determined off-line during playback of
taped images. A leukocyte was considered to be adherent to the venular
endothelium if it remained stationary for 10 s or longer. Adherent
cells were expressed as the number per 100-µm length of venule. Each
of these variables was recorded in three to five unbranched venules and
presented as the mean value for each experiment. In some experiments,
erythrocyte velocity was calculated from the time required for a
labeled red cell to traverse a measured length of vessel during
videotape playback. Mean erythrocyte velocity was obtained by averaging
the velocities of each red cell over a selected venule length at the
midpoint of the flow observation period (28).
Myeloperoxidase activity in ovarian tissue.
Tissue-associated myeloperoxidase (MPO) was used as a marker for the
evaluation of ovarian neutrophil infiltration. Tissue-associated MPO
activity was determined by a modification of the method of Grisham et
al. (10). Briefly, both ovaries were dissected and then
stored at
80°C until analysis. The tissue was then weighed and
homogenized in 10 volumes of 0.02 M potassium phosphate buffer (pH 7.4)
and centrifuged at 16,000 g for 20 min at 4°C. The
supernatant was discarded, and the pellet was sonicated for 10 s
in a further homogenization step with an equivalent volume of 0.05 M
potassium phosphate buffer (pH 6.0) containing 0.5% (wt/vol)
hexadecyltrimethylammonium bromide (Sigma) and then recentrifuged. MPO
activity was assessed by measuring hydrogen peroxide-dependent
oxidation of 3,3',5,5'-tetramethylbenzidine. One unit of enzyme
activity was defined as the amount of MPO that caused a change in
absorbance of 1.0/min at 655 nm and 37°C. Enzyme activity was
expressed as units per gram of tissue wet weight.
Determination of serum progesterone.
Serum progesterone concentrations were measured as an indicator of the
occurrence of luteal regression. Blood samples were collected from the
inferior vena cava of each animal when the ovaries were removed.
Progesterone concentrations were measured using a commercial
radioimmunoassay kit (Diagnostic Products, Los Angeles, CA). All
samples and standards (100 µl) were assayed in duplicate. Inter- and
intra-assay coefficients of variation were 4.8 and 4.5%, respectively.
Effects of PGF2
on leukocyte accumulation in the
rat corpus luteum.
In all experiments, preparations for observation were finished within
15 min to minimize the influence of manipulation; then a 15-min
stabilization period was allowed before recording. Intravital microscopic observation and recording were carried out before and 30, 60, and 120 min after PGF2
administration. Control rats
underwent a sham operation with cannulation. To ensure the stage of
neutrophil infiltration during luteal regression, ovaries were resected
for MPO assay before and after 30, 60, and 120 min of
PGF2
treatment. At the same time points
(n = 6 for each time), blood samples were collected
from the inferior vena cava for determination of serum progesterone.
Because these results indicated that PGF2
induced a
significant increase in neutrophil infiltration and a decrease in serum
progesterone concentration at 120 min, inhibitory experiments
concerning neutrophil infiltration and luteal regression were performed
120 min after PGF2
administration.
Effects of anti-CD18 antibody treatment on PGF2
-induced
leukocyte accumulation and luteolysis.
To determine specific inhibition of leukocyte adhesion in our model, a
mouse monoclonal antibody against the rat CD18 molecule (WT-3, 0.8 mg/kg; Seikagakukogyo, Tokyo, Japan) (20, 35) was administered via the femoral vein 30 min before PGF2
treatment (n = 6). Intravital microscopic observation
then was performed before and 30, 60, and 120 min after
PGF2
administration. Because this monoclonal antibody
represented a complete mouse IgG1 molecule, the same amount of mouse
nonspecific IgG1 (Sigma) was used as a control (n = 6).
In another set of experiments, the ovaries and the blood samples were
taken 120 min after the PGF2
injection. MPO activity in
the ovaries and serum progesterone concentrations were measured, as
described above, to investigate the effects of WT-3 (n = 10) or nonspecific mouse IgG1 (n = 10) pretreatment.
Effects of oxygen free radical scavenger treatment on
PGF2
-induced leukocyte accumulation and luteolysis.
To determine the possible role of ROS in mediating
leukocyte-endothelium response to PGF2
, rats received
subcutaneous injection of superoxide dismutase (SOD, 50,000 U/kg;
Sigma) and catalase (90,000 U/kg; Sigma) 60 min before
PGF2
administration (n = 6). These doses
of SOD and catalase reportedly scavenge ROS produced by
ischemia-reperfusion injury (13). In another set of experiments, SOD and catalase also were administered at the doses
described above 60 min before PGF2
treatment. MPO
activity in the ovaries and serum progesterone concentrations were
evaluated 120 min after PGF2
injection
(n = 6).
Intravital microscopic observation of hydrogen peroxide formation
after PGF2
treatment in the rat corpus luteum.
Time course and spatial distributions of oxidative changes with or
without PGF2
treatment in the corpus luteum were
evaluated by the fluorescence microscopic system (×10 water immersion
objective lens, Nikon). Dichlorodihydrofluorescein (DCFH) diacetate
succinimidyl ester (Molecular Probes, Eugene, OR) was used to detect
hydrogen peroxide generated in an intracellular space (31,
32). DCFH diacetate succinimidyl ester can enter cells, where it
is hydrolyzed by esterase to yield DCFH succinimidyl ester, a
fluorogenic precursor that binds to lysine residues of intracellular
proteins. Intracellular DCFH can be oxidized by inorganic and organic
hydrogen peroxide into dichlorofluorescein (DCF), a fluorescent
product. In these experiments, rats were intravenously injected with 50 µM DCFH diacetate succinimidyl ester via the femoral vein over a
30-min loading period. DCF fluorescence images were monitored with a silicon-intensified target camera (C2400-08; Hamamatsu Photonics) in a superficial region of the corpus luteum with a fixed-gain control
setting. The exposure time was limited to less than 1 s by a
shutter to prevent a photobleaching effect. All fluorescence images
were recorded on videotape. Gray levels (0-256) of
the recorded fluorographs were analyzed as fluorescence intensity by
the image digitizer (NIH Image 1.61 with a Power Macintosh computer) in
the corpus luteum, avoiding large blood vessels, and the mean intensity
was calculated. Data are expressed as percent change from the baseline.
These intensity changes were compared between
PGF2
-treated rats (n = 5) and controls
(n = 4). Microfluorographs were observed just before
(baseline value) and then every 10 min after PGF2
administration.
Statistical analysis.
Results are expressed as means ± SE. The statistical significance
of differences between experimental groups was determined by one-way
analysis of variance (ANOVA) and Scheffé's multiple comparison
test. Values of P < 0.05 were considered to indicate significance.
 |
RESULTS |
PGF2
induces leukocyte accumulation in the rat
corpus luteum.
The intravital microscopic system clearly identified rhodamine
6G-labeled leukocytes in the venules of the corpus luteum. PGF2
promoted leukocyte-endothelium interactions in
venules of the corpus luteum (Fig. 1).
Intravital microscopic observations demonstrated that the number of
adherent leukocytes to venular endothelium had increased significantly
at 60 min after PGF2
administration (Fig.
2). Leukocyte adherence continued to rise throughout the entire 120-min observation period, reaching 7.9 ± 0.6 cells/100 µm of venule length at 120 min. Such an increase in
leukocyte adherence was not observed over the same period in control
rats. No significant differences in mean arterial pressure, venular
diameter, or erythrocyte velocity in venules were noted between the
control and the PGF2
groups (data not shown). PGF2
-induced neutrophil infiltration was confirmed by
means of measurement of tissue MPO activity. Functional luteolysis in this model was confirmed by serum progesterone analysis (Fig. 3). Ovarian tissue MPO activity had
increased significantly relative to the control value at 120 min of
PGF2
injection (9.6 ± 0.5 U/g) but not at 30 or 60 min. In contrast, a time-dependent decrease in serum progesterone
concentration was detected after PGF2
treatment,
reaching a value of 70.0 ± 9.9 ng/ml at 120 min, with a
significant decrease in serum progesterone demonstrated as early as 30 min after PGF2
treatment (172.6 ± 22.3 ng/ml vs.
baseline, 245.2 ± 16.7 ng/ml). These results clearly showed that
leukocyte-endothelium interactions in venules of the corpus luteum were
induced by PGF2
administration.

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Fig. 1.
Representative fluorescent microvascular images of the rat corpus
luteum before and after prostaglandin (PG)F2
administration. Leukocytes were labeled with rhodamine 6G in vivo, and
adherent leukocytes were clearly identified along the endothelium of
the venule. These representative images were recorded before
(A) and 30 (B), 60 (C), or 120 min
(D) after subcutaneous injection of PGF2 .
Note the marked increase in leukocyte adherence observed in the venule
(v) at 120 min. Bar, 50 µm.
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Fig. 2.
Time course of leukocyte adhesion in venules of the
corpus luteum after PGF2 administration. Treatment with
PGF2 (PG; 500 µg sc) significantly increased the
number of adherent leukocytes. No significant change was observed in
controls injected with vehicle. Values are expressed as means ± SE for 6 rats in each group. *P < 0.01 compared with
control value.
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Fig. 3.
Alterations of tissue-associated myeloperoxidase (MPO)
activity (A) and serum progesterone concentration
(B) after PGF2 administration. MPO activity
at 120 min was three times higher than the control value.
PGF2 treatment caused a time-dependent decrease in serum
progesterone. Values are expressed as means ± SE for 6 rats in
each group. *P < 0.05 and **P < 0.01 compared with control value; P < 0.05 compared with
60-min value.
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Anti-CD18 monoclonal antibody inhibits PGF2
-induced
leukocyte accumulation and luteolysis.
The membrane glycoprotein CD18 has been shown to act prominently in
neutrophil adherence to the endothelium and subsequent migration into
the interstitium (1, 11). To investigate whether leukocyte
adhesion to the venules of the corpus luteum was CD18 dependent, rats
were pretreated with a monoclonal antibody (WT-3) that specifically
blocks CD18-dependent adhesion (Fig. 4).
Pretreatment with WT-3 significantly reduced the
PGF2
-induced recruitment of adherent leukocytes at 120 min (4.3 ± 0.4 cells/100 µm of venule length). Treatment with
nonspecific mouse IgG1 did not significantly affect
PGF2
-induced leukocyte adhesion. No significant
differences in mean arterial pressure, venular diameter, or erythrocyte
velocity in venules were noted between control and treatment groups
(data not shown). Pretreatment with WT-3 significantly attenuated the PGF2
-induced elevation in MPO activity (7.6 ± 0.5 U/g) and the decrease in progesterone level (128.0 ± 17.3 ng/ml;
Fig. 5). Thus PGF2
-induced
leukocyte accumulation and luteolysis are CD18 dependent.

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Fig. 4.
Effects of anti-CD18 monoclonal antibody on leukocyte
adhesion in the venules of the corpus luteum. Anti-CD18 (WT-3; 0.8 mg/kg) or nonspecific mouse immunoglobulin G1 (IgG1; 0.8 mg/kg) was
injected intravenously 30 min before PGF2
administration. The number of adherent leukocytes was significantly
decreased in the WT-3-treated group. Values are expressed as means ± SE for 6 rats in each group. *P < 0.01 compared
with corresponding nonspecific IgG1 value.
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Fig. 5.
Effects of anti-CD18 monoclonal antibody on
tissue-associated MPO activity (A) and serum progesterone
concentration (B) 120 min after PGF2
administration. The increase in MPO activity and the decrease in serum
progesterone 120 min after PGF2 administration were
significantly inhibited by pretreatment with WT-3. Values are expressed
as means ± SE for 10 rats in each group. *P < 0.05 and **P < 0.01 compared with the nonspecific IgG1
value.
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Oxygen free radical scavengers attenuate
PGF2
-induced leukocyte accumulation and luteolysis.
To investigate the possible role of ROS as mediators in
PGF2
-induced leukocyte adhesion, pretreatment with
radical-scavenging enzymes, SOD and catalase, was carried out.
Pretreatment with SOD plus catalase significantly reduced
PGF2
-induced leukocyte adhesion within 30 min after
PGF2
injection (2.2 ± 0.1 cells/100 µm of venule
length), continuing for 120 min (Fig. 6).
No significant differences in mean arterial pressure, venular diameter,
or erythrocyte velocity in venules were noted between control and
treatment groups (data not shown). Simultaneous administration of SOD
and catalase also blocked the increase in MPO activity at 120 min
(6.0 ± 0.5 U/g) and significantly attenuated the decrease in
serum progesterone concentration (124.0 ± 7.4 ng/ml; Fig.
7). Oxygen-derived free radicals
therefore contribute significantly to PGF2
-induced leukocyte accumulation and functional luteolysis.

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Fig. 6.
Effects of superoxide dismutase (SOD) and catalase
(CAT) on PGF2 -induced leukocyte adhesion. Pretreatment
with SOD (50,000 U/kg) plus catalase (90,000 U/kg) 60 min before
PGF2 administration significantly inhibited
PGF2 -induced leukocyte adhesion. Values are expressed as
means ± SE for 6 rats in each group. *P < 0.01 compared with control value; P < 0.01 compared with the
corresponding PGF2 value.
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Fig. 7.
Effects of oxygen free radical scavengers on
tissue-associated MPO activity (A) and serum progesterone
concentration (B) after PGF2 administration.
MPO activity 120 min after PGF2 administration and the
serum progesterone were significantly inhibited by pretreatment with
SOD plus catalase. Values are expressed as means ± SE for 6 rats
in each group. *P < 0.01 compared with the
PGF2 value.
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Acute oxidative stress in the corpus luteum during
PGF2
-induced luteolysis.
Oxidative stress indicated by DCF fluorescence intensity was evaluated
in the corpus luteum after PGF2
administration. Figure
8 shows the representative time course
and spatial changes in DCF fluorographs in control corpus luteum and in
rat corpus luteum after PGF2
administration. A
time-dependent increase in DCF fluorescence after PGF2
treatment was observed within the entire corpus luteum except for large
vessels. DCF fluorescence intensity was significantly increased
compared with the control at 30, 40, 50, and 60 min after
PGF2
administration (Fig. 9).

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Fig. 8.
Spatial and temporal distribution of oxidative changes in the
corpus luteum after PGF2 treatment. Dichlorofluorescein
microfluorographs of the corpus luteum taken before (A) and
at 30 (B) and 60 min (C) after sham treatment
without PGF2 are shown, as are others taken before
(D) and at 30 (E) and 60 min (F) after
PGF2 treatment. An increase in fluorescence intensity
induced by oxygen free radical formation was apparent after
PGF2 treatment throughout the corpus luteum except for
large vessels. Bar, 500 µm.
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Fig. 9.
Time course of fluorescence intensity in the
PGF2 -treated corpus luteum. Fluorescence increased
significantly after 30 min of PGF2 treatment.
*P < 0.05 compared with corresponding control value.
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 |
DISCUSSION |
The present study provides the first in vivo demonstration of
leukocyte-endothelium interactions in the venules of the corpus luteum
during functional luteolysis in response to PGF2
by use
of an intravital microscopy. Our experiments showed that the number of
adherent leukocytes to venular endothelium increased significantly
within 60 min after PGF2
treatment, continuing to
increase through the 120-min observation. Most previous in vivo studies
on leukocyte accumulation during luteolysis have been shown by static
histological observations (3). Some uncertainty remains as
to whether accumulated leukocytes actually influence the corpus luteum
function in vivo. Intravital microscopy, which delineates temporal and
quantitative alterations at the microcirculatory level, is a useful in
vivo system for investigating factors associated with leukocyte
accumulation that causes luteolysis. The results of our experiments
lend further support to evidence of leukocyte involvement in
PGF2
-induced functional luteolysis.
Our observations of leukocyte-endothelium interactions in the venules
of the corpus luteum are confirmed by measurement of ovarian tissue MPO
activity after PGF2
administration. The present study
demonstrates that MPO activity significantly increased in ovarian
tissue exposed to 120 min of PGF2
treatment.
Tissue-associated MPO activity reflects the presence of adhered and
migrated leukocytes (i.e., neutrophils, eosinophils, monocytes).
Because the circulating numbers of eosinophils and monocytes are small
compared with neutrophils, tissue MPO activity is considered an
indirect index of neutrophil infiltration (10). Therefore,
the increased MPO activity in our model indicates that neutrophils
infiltrate ovarian tissue 120 min after PGF2
administration. The distribution of individual leukocyte subsets has
been demonstrated within the regressing corpus luteum (3).
Neutrophils are observed in great numbers in the initial phase of the
regression in pseudopregnant rats, whereas monocytes, macrophages, and
lymphocytes are scarce (4). Thus leukocytes, particularly
neutrophils, may contribute to corpus luteum function during luteolysis.
The present study clearly demonstrates two phases of decrease in serum
progesterone after PGF2
treatment. The first occurrence of this functional luteolysis, within 30 min after PGF2
treatment, may represent a direct effect of PGF2
on
luteal cells, which is supported by the observation that administration
of PGF2
to rats causes a significant fall in blood
progesterone within 20-40 min (16, 29). The second
phase of progesterone decrease, within 120 min, corresponds to the time
of neutrophil infiltration into ovarian tissue. Pepperell et al.
(24) reported that in vitro activation of neutrophils
leads to passage of ROS into neighboring rat luteal cells and
interferes with lutenizing hormone stimulation of cAMP and progesterone
production. In our observations, one possible explanation for the
correspondence of progesterone decrease to neutrophil infiltration may
be a contribution of neutrophils to inhibition of progesterone
synthesis by producing ROS. To confirm this interpretation, we carried
out the experiments in the presence of a common leukocyte antibody for
lack of a specific antibody against neutrophils. Leukocyte integrin
CD11/CD18 has an essential role in establishing the leukocyte adherence
to endothelium. This adhesive CD11/CD18 glycoprotein complex is
composed of a family of three heterodimers, each consisting of a
variable
-subunit (CD11a, CD11b, and CD11c) and a common
-subunit
(CD18). Administration of a monoclonal antibody to CD18 (WT-3) has been
shown to attenuate local neutrophil accumulation and tissue injury in
other organs (19, 27, 34). In the present study,
pretreatment with WT-3 significantly inhibited not only
PGF2
-induced neutrophil accumulation but also the
decrease in serum progesterone. Thus CD18-dependent neutrophil
accumulation may be closely associated with PGF2
-induced
functional luteolysis.
Our experiments demonstrated that ROS constitute an important step in
the development of the PGF2
-induced increase in neutrophil adhesion and infiltration observed in vivo. The present study first showed hydrogen peroxide formation in the corpus luteum within 30 min after PGF2
administration by means of
DCFH-dependent microfluorography. This hydrogen peroxide generation
appears to precede the increase in leukocyte adhesion. Furthermore, the
PGF2
-induced increase in leukocyte adhesion and MPO
activity was attenuated by pretreatment with SOD and catalase. A
previous in vivo observation in rat intestinal mucosa (39)
demonstrated that treatment with either SOD or catalase significantly
inhibited the increase in the MPO activity induced by
ischemia-reperfusion injury, including microvascular
dysfunction and postischemic tissue damage. In other organs,
recent studies have shown that exogenous hydrogen peroxide promotes
neutrophil interaction with venular endothelial cells by mechanisms
that involve CD11/CD18 on neutrophils and intercellular adhesion
molecule-1, a ligand for CD11/CD18, on venular endothelial cells
(7, 17, 33). Our results suggest that ROS-mediated neutrophil accumulation by activation of CD18 may occur in the course
of PGF2
-induced luteolysis.
The precise source of ROS that mediate neutrophil activation and
invasion in our model remains to be elucidated. However, at least two
sources are possible. The primary source of ROS in PGF2
-treated tissues is parenchymal steroidogenic cells. PGF2
has been demonstrated to stimulate production of
ROS by rat luteal cells in vitro (36) and cause an
increase in hydrogen peroxide in regressing rat corpora lutea in vivo
(26). Endothelial cells of the microvasculature within the
corpus luteum are another possible source. ROS generation by
endothelial cells can be induced by ischemia-reperfusion
through activation of the hypoxanthine-xanthine oxidase system,
resulting in neutrophil activation and adhesion (8). In
PGF2
-induced luteolytic models, several experiments have
shown that PGF2
did not affect luteal hemodynamics in the early stage of luteolysis (5, 14, 22) and that
xanthine oxidase activity in the rat corpus luteum did not change
during PGF2
-induced luteolysis (18). In our
model, no significant microvascular hemodynamic alteration was
noted during the 120-min observation period. These findings indicate
that endothelial cells are not necessarily the major source of ROS in
this organ. Pretreatment with SOD and catalase, large molecules that do
not easily penetrate cell membranes, prevented
PGF2
-induced functional luteolysis; this favors the
possibility that neutrophils may be the major source of ROS in
functional luteolysis.
In conclusion, the present study characterizes the stimulatory effect
of PGF2
on leukocyte-endothelium interactions in the rat
corpus luteum in vivo. The neutrophils that infiltrated tissues as a
consequence contribute to functional luteolysis. PGF2
-induced ROS generation may be a key mediator of
neutrophil accumulation within the regressing corpus luteum.
 |
ACKNOWLEDGEMENTS |
This work was supported in part by a Grant-in-Aid (no. 10770855)
for Scientific Research from the Ministry of Education, Science, and
Culture of Japan.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: K. Minegishi, Dept. of Obstetrics and Gynecology, Keio Univ. School of
Medicine, Shinjuku-ku 160-8582, Tokyo, Japan.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
July 30, 2002;10.1152/ajpendo.00240.2002
Received 3 June 2002; accepted in final form 23 July 2002.
 |
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