Reactive oxygen species mediate leukocyte-endothelium interactions in prostaglandin F2alpha -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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We investigated the contribution of neutrophils to prostaglandin (PG)F2alpha -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 PGF2alpha (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 PGF2alpha -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 PGF2alpha -induced alterations. Corpora lutea preloaded with dichlorodihydrofluorescein diacetate succinimidyl ester, a fluorescent indicator for determining intracellular ROS generation, exhibited an increase in fluorescence after PGF2alpha treatment. These findings suggest that leukocyte-endothelium interactions mediated by ROS generation are important in PGF2alpha -induced luteolysis in rats.

corpus luteum; neutrophil; CD18 integrin; myeloperoxidase activity; intravital microscopy


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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)F2alpha 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 PGF2alpha (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-1beta , INF-gamma , and TNF-alpha has been described in bovine corpora lutea after PGF2alpha 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 PGF2alpha -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 PGF2alpha -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 PGF2alpha 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PGF2alpha (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 PGF2alpha 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 PGF2alpha 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 PGF2alpha 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 PGF2alpha 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 PGF2alpha administration.

Effects of anti-CD18 antibody treatment on PGF2alpha -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 PGF2alpha treatment (n = 6). Intravital microscopic observation then was performed before and 30, 60, and 120 min after PGF2alpha 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 PGF2alpha 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 PGF2alpha -induced leukocyte accumulation and luteolysis. To determine the possible role of ROS in mediating leukocyte-endothelium response to PGF2alpha , rats received subcutaneous injection of superoxide dismutase (SOD, 50,000 U/kg; Sigma) and catalase (90,000 U/kg; Sigma) 60 min before PGF2alpha 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 PGF2alpha treatment. MPO activity in the ovaries and serum progesterone concentrations were evaluated 120 min after PGF2alpha injection (n = 6).

Intravital microscopic observation of hydrogen peroxide formation after PGF2alpha treatment in the rat corpus luteum. Time course and spatial distributions of oxidative changes with or without PGF2alpha 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 PGF2alpha -treated rats (n = 5) and controls (n = 4). Microfluorographs were observed just before (baseline value) and then every 10 min after PGF2alpha 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
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

PGF2alpha 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. PGF2alpha 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 PGF2alpha 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 PGF2alpha groups (data not shown). PGF2alpha -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 PGF2alpha 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 PGF2alpha 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 PGF2alpha 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 PGF2alpha administration.


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Fig. 1.   Representative fluorescent microvascular images of the rat corpus luteum before and after prostaglandin (PG)F2alpha 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 PGF2alpha . 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 PGF2alpha administration. Treatment with PGF2alpha (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 PGF2alpha administration. MPO activity at 120 min was three times higher than the control value. PGF2alpha 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; dagger P < 0.05 compared with 60-min value.

Anti-CD18 monoclonal antibody inhibits PGF2alpha -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 PGF2alpha -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 PGF2alpha -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 PGF2alpha -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 PGF2alpha -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 PGF2alpha 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 PGF2alpha administration. The increase in MPO activity and the decrease in serum progesterone 120 min after PGF2alpha 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.

Oxygen free radical scavengers attenuate PGF2alpha -induced leukocyte accumulation and luteolysis. To investigate the possible role of ROS as mediators in PGF2alpha -induced leukocyte adhesion, pretreatment with radical-scavenging enzymes, SOD and catalase, was carried out. Pretreatment with SOD plus catalase significantly reduced PGF2alpha -induced leukocyte adhesion within 30 min after PGF2alpha 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 PGF2alpha -induced leukocyte accumulation and functional luteolysis.


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Fig. 6.   Effects of superoxide dismutase (SOD) and catalase (CAT) on PGF2alpha -induced leukocyte adhesion. Pretreatment with SOD (50,000 U/kg) plus catalase (90,000 U/kg) 60 min before PGF2alpha administration significantly inhibited PGF2alpha -induced leukocyte adhesion. Values are expressed as means ± SE for 6 rats in each group. *P < 0.01 compared with control value; dagger P < 0.01 compared with the corresponding PGF2alpha 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 PGF2alpha administration. MPO activity 120 min after PGF2alpha 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 PGF2alpha value.

Acute oxidative stress in the corpus luteum during PGF2alpha -induced luteolysis. Oxidative stress indicated by DCF fluorescence intensity was evaluated in the corpus luteum after PGF2alpha administration. Figure 8 shows the representative time course and spatial changes in DCF fluorographs in control corpus luteum and in rat corpus luteum after PGF2alpha administration. A time-dependent increase in DCF fluorescence after PGF2alpha 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 PGF2alpha administration (Fig. 9).


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Fig. 8.   Spatial and temporal distribution of oxidative changes in the corpus luteum after PGF2alpha treatment. Dichlorofluorescein microfluorographs of the corpus luteum taken before (A) and at 30 (B) and 60 min (C) after sham treatment without PGF2alpha are shown, as are others taken before (D) and at 30 (E) and 60 min (F) after PGF2alpha treatment. An increase in fluorescence intensity induced by oxygen free radical formation was apparent after PGF2alpha 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 PGF2alpha -treated corpus luteum. Fluorescence increased significantly after 30 min of PGF2alpha treatment. *P < 0.05 compared with corresponding control value.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 PGF2alpha by use of an intravital microscopy. Our experiments showed that the number of adherent leukocytes to venular endothelium increased significantly within 60 min after PGF2alpha 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 PGF2alpha -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 PGF2alpha administration. The present study demonstrates that MPO activity significantly increased in ovarian tissue exposed to 120 min of PGF2alpha 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 PGF2alpha 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 PGF2alpha treatment. The first occurrence of this functional luteolysis, within 30 min after PGF2alpha treatment, may represent a direct effect of PGF2alpha on luteal cells, which is supported by the observation that administration of PGF2alpha 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 alpha -subunit (CD11a, CD11b, and CD11c) and a common beta -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 PGF2alpha -induced neutrophil accumulation but also the decrease in serum progesterone. Thus CD18-dependent neutrophil accumulation may be closely associated with PGF2alpha -induced functional luteolysis.

Our experiments demonstrated that ROS constitute an important step in the development of the PGF2alpha -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 PGF2alpha administration by means of DCFH-dependent microfluorography. This hydrogen peroxide generation appears to precede the increase in leukocyte adhesion. Furthermore, the PGF2alpha -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 PGF2alpha -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 PGF2alpha -treated tissues is parenchymal steroidogenic cells. PGF2alpha 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 PGF2alpha -induced luteolytic models, several experiments have shown that PGF2alpha 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 PGF2alpha -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 PGF2alpha -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 PGF2alpha on leukocyte-endothelium interactions in the rat corpus luteum in vivo. The neutrophils that infiltrated tissues as a consequence contribute to functional luteolysis. PGF2alpha -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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Endocrinol Metab 283(6):E1308-E1315
0193-1849/02 $5.00 Copyright © 2002 the American Physiological Society




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