1 Dong-A University Institute of Medical Science, Pusan 602103 and 2 Department of Obstetrics and Gynecology, College of Medicine, Pusan National University, Pusan 602739, Korea
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Abstract |
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Key words: apoptosis/endometriosis/interleukin-8/neutrophils
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Introduction |
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Apoptosis or programmed cell death, which is generally accepted as a physiological mechanism for the elimination of unwanted cells, is one of the important functions that maintains homeostasis (Stellar, 1995). Aged cells are eliminated from the functional layer of the uterine endometrium during the late secretory and menstrual phases of the menstrual cycle (Kokawa et al., 1996
). Although it has been shown that spontaneous apoptosis of endometrial tissue is impaired in women with endometriosis (Gebel et al., 1998
), the apoptosis of immune cells associated with the pathogenesis of this disease is poorly understood.
Inflammation and neovascularization have been observed in and around ectopic endometrial implants and inflammatory neutrophils were also found in these lesions (Khorram et al., 1993). Neutrophils are known to have the shortest life span among the leukocytes and their apoptotic death has also been proposed to be a critical component in the resolution of the inflammatory process (Haslett, 1992
). The increased chemotactic activity of neutrophils was demonstrated in the peritoneal fluid of patients with endometriosis (Leiva et al., 1993
). The peritoneal fluid of such patients has also been shown to contain increased concentrations of both anti-apoptotic and pro-apoptotic cytokines to neutrophils, including IL-6 and IL-8, which are known to accelerate neutrophil apoptosis (Afford et al., 1992
) or prolong their life span by inhibiting apoptosis (Kettritz et al., 1998
) respectively.
Apoptotic senescent neutrophils in tissue are recognized and phagocytosed by macrophages (Savill et al., 1989). Recently, it was proposed that the ingestion of apoptotic neutrophils triggers the production of anti-inflammatory mediators by macrophages (Fadok et al., 1998
) and conversely, that macrophages can actively induce the apoptosis of neutrophils (Meszaros et al., 2000
). Therefore, the prolonged survival and activation of neutrophils may lead to chronic inflammation and tissue damage. It has been shown that neutrophils in the endometrium produce vascular endothelial growth factor (Mueller et al., 2000
) and interferon-
(Yeaman et al., 1998
), which are known to be associated with endometrial angiogenesis (Shifren et al., 1996
) and the growth of normal uterine endometrium (Tabibzadeh, 1994
) respectively. This in turn suggests that neutrophils have more extensive roles than previously thought. In this study, we evaluated for the first time the effect of plasma and peritoneal fluid from women with endometriosis on the extent of neutrophil apoptosis.
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Materials and methods |
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Morphological assessment of neutrophil apoptosis
Neutrophils incubated in the presence or absence of plasma or peritoneal fluid were spun down on a glass slide in a cytospin (Shandon, Pittsburgh, PA, USA). Cells were fixed with methanol and stained with Giemsa staining solution (Fluka, Bushs, Switzerland). Percentages of apoptotic cells were determined by counting at least 300 cells per slide.
Detection of DNA fragmentation
Neutrophils (2x106) were harvested, washed twice with phosphate-buffered saline and incubated in cell lysis buffer (10 mmol/l Tris, pH 7.5, 1 mmol/l EDTA and 0.2% Triton X-100) containing 500 µg/ml proteinase K (Sigma) for 24 h at 55°C. Thereafter, lysates were centrifuged for 10 min at 13 000 g to separate intact chromatin from soluble low molecular weight DNA. The supernatant was separated and DNA was extracted with phenol/chloroform twice. DNA was then precipitated with isopropanol at 70°C, resuspended in distilled water, incubated with 20 µg/ml ribonuclease A (Sigma) for 1 h at 37°C, electrophoresed on a 1% agarose gel containing ethidium bromide, and photographed using a LAS 2000 luminescent analyser (Bio-Rad, Hercules, CA, USA).
Statistical analysis
Results are presented as mean ± SD. Student's t-test for unpaired samples was used to compare means. A probability value of < 0.05 was considered significant. The number of samples is represented by n; each involved independent experiments in triplicate. Analysis was carried out using Statistics Package for Social Sciences version 10.0.
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Results |
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The effects of plasma and peritoneal fluid on the apoptosis of neutrophils from endometriosis patients
We next assessed the effect of plasma and peritoneal fluid on the apoptosis of neutrophils obtained from endometriosis patients. The results are summarized in Table II. The percentage of apoptotic neutrophils from patients after 24 h of incubation in the presence of 10% plasma from control donors decreased from 65.3 ± 6.6 to 29.5 ± 4.7% (P < 0.001). In contrast to the effect of plasma from endometriosis patients on neutrophils from control donors, plasma from endometriosis patients had no further inhibitory effect on the apoptosis of neutrophils from endometriosis patients. However, the prolonged survival of neutrophils from endometriosis patients was further increased by the addition of 10% peritoneal fluid from patients when compared with the peritoneal fluid of controls (10.2 ± 3.2 versus 22.6 ± 4.7%, P < 0.001). These results show that neutrophils from women with endometriosis are relatively more resistant to spontaneous apoptosis than those from healthy female controls.
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The effects of actinomycin D on delayed apoptosis induced by plasma and peritoneal fluid
The inhibition of protein expression by actinomycin D (Stringer et al., 1996) or cycloheximide (Whyte et al., 1997
) triggers neutrophil apoptosis, which suggests that protein synthesis is important in the regulation of apoptosis. Therefore, we examined the effect of actinomycin D upon the inhibitory apoptotic effects of the plasma and the peritoneal fluid of patients. Neutrophils of controls were treated and incubated with or without actinomycin D for 12 h, because prolonged incubations cause cell necrosis. Plasma from patients decreased the percentage of apoptosis in neutrophils triggered by 0.1 µmol/l actinomycin D compared with plasma from controls (42.5 ± 3.5 versus 22.6 ± 6.3; P < 0.001, n = 10) (Figure 3
). In the presence of 1 µmol/l of actinomycin D, neutrophils showed a high percentage of apoptosis, up to 90% after 12 h of incubation. Although the inhibitory effect of plasma from endometriosis patients on actinomycin D-triggered apoptosis was greater than that of the controls, neutrophil apoptosis was not completely inhibited. A similar result was observed with peritoneal fluid in this experiment.
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Discussion |
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We also considered the non-specific or hormonal effect of plasma and peritoneal fluid on neutrophil apoptosis. When autologous instead of heterologous plasma was used as a control, similar results were obtained, which indicates that the possibility of non-specific immune reactions can be ruled out. Neutrophil apoptosis is also known to be associated with hormonal changes (Liles et al., 1995). Hydrocortisone reduces neutrophil apoptosis, but progesterone (Liles et al., 1995
) and estrogen (unpublished results), which are the major contributory hormonal factors in the aetiology of endometriosis, have no effect. Therefore, the possibility that reduced apoptosis may be caused by different levels of estrogen and progesterone may be eliminated.
In this study, although the spontaneous apoptosis of neutrophils was delayed by plasma from endometriosis patients, the peripheral blood neutrophil count was within the normal range. Neutrophils harvested from certain inflammatory sites have been shown to have an enhanced longevity in culture, but delayed apoptosis is not always associated with increased cellularity because neutrophil survival in inflammation may be highly transient (Watson et al., 1997; Hamasaki et al., 1998
). Therefore, it is important to consider the derangement of immune response by aged neutrophils, rather than rely on the count of cells in the peripheral blood, endometrium or peritoneal fluid.
Gebel et al. reported that the spontaneous apoptosis of endometrial cells obtained from patients with endometriosis was lower than that found in healthy controls (Gebel et al., 1998). These workers suggested that the decreased susceptibility of endometrial tissue to a certain factor might contribute to the aetiology of endometriosis. However, it is not known whether the spontaneous apoptosis of ectopic endometrial cells can be triggered by peritoneal fluid containing higher levels of cytokines in endometriosis patients. Although the biological significance of delayed apoptosis of neutrophils is still obscure, the following possibilities can be considered. First, aged neutrophils or cytokine(s) that inhibit neutrophil apoptosis may affect the functions and spontaneous apoptosis of endometrial cells. Second, the secretory products of monocytes and macrophages can suppress the activities and survival time of other immune cells, such as neutrophils.
One remarkable finding in the peritoneal fluid of patients was the increased concentration of IL-8 (Ryan et al., 1995; Arici et al., 1996
), which exerts a chemotactic activity primarily on neutrophils (Oppenheim et al., 1991
) and inhibits the apoptosis of neutrophils even in the presence of Fas engagement (Leuenroth et al., 1998
). These findings suggest that neutrophils may be related to the pathogenesis of endometriosis. IL-8 was found to be expressed in the human endometrium (Arici et al., 1998
); moreover, the peripheral blood monocytes of women with endometriosis were found to secrete greater amounts of IL-8 than controls (Braun et al., 1996
). Although neutrophil apoptosis was delayed by IL-8, IL-8 had no noticeable effect on the apoptosis of neutrophils from endometriosis patients. These results suggest that some other survival factor(s) in the plasma of patients with endometriosis may delay neutrophil apoptosis. In contrast to observations on plasma, anti-IL-8 antibody abrogated the effect of the peritoneal fluid of patients on the spontaneous apoptosis of control neutrophils. This result suggests that IL-8 is one of the survival factors in the peritoneal fluid of endometriosis patients that delays neutrophil apoptosis. It is also possible that other factor(s) in the peritoneal fluid may stimulate the production of IL-8 or enhance the effect of IL-8 on apoptosis. It has been shown that TNF-
induces the production of IL-8 in neutrophils (Cassatella, 1995
), which suggests that TNF-
in peritoneal fluid induces apoptotic delay by mediating the secretion of IL-8. In our experiments, neutralizing TNF-
antibody did not abolish the apoptotic delay induced by the peritoneal fluid of endometriosis patients (data not shown), indicating that the effect of these patients' peritoneal fluid was not mediated by this cytokine.
Actinomycin D, a transcription inhibitor, accelerates neutrophil apoptosis in vitro, as shown here and by others (Stringer et al., 1996). The delayed apoptosis of neutrophils by the plasma or peritoneal fluid of endometriosis patients was partially inhibited by actinomycin D, suggesting that the plasma or peritoneal fluid of these patients may not only affect the transient expression of anti-apoptotic proteins, but also alter the activities of pre-existing proteins. However, the anti-apoptotic effect of both the endometriosis patients' plasma and peritoneal fluid appeared to be independent of changes in the expressions of the anti-apoptotic proteins of the Bcl-2 family, such as Mcl-1 (data not shown).
In this study, we show that the spontaneous apoptosis of neutrophils is reduced by the plasma and by the peritoneal fluid of patients with endometriosis, which suggests that the impaired clearance of cells responsible for innate immunity is associated with endometriosis.
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Acknowledgements |
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Notes |
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Submitted on May 1, 2001; resubmitted on September 6, 2001
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References |
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Submitted on May 1, 2001; resubmitted on September 6, 2001; accepted on November 14, 2001.