Modulation of neutrophil apoptosis by plasma and peritoneal fluid from patients with advanced endometriosis

Human Reproduction, Mar 2002

BACKGROUND: The increased production of pro-inflammatory chemoattractant cytokines for neutrophils in endometriosis suggests that changes in the immune system play an important role in the pathophysiology of endometriosis. The effects of plasma and peritoneal fluid from patients with advanced endometriosis on the apoptosis of neutrophils were investigated. METHODS: Apoptotic changes of neutrophils were evaluated by morphological changes using Giemsa staining. Apoptosis was confirmed by DNA electrophoretic analysis. RESULTS: Compared with the plasma (n = 20) and peritoneal fluid (n = 5) of healthy controls, the addition of 10% plasma (n = 20) and peritoneal fluid (n = 10) from patients with endometriosis to an in-vitro culture of neutrophils from healthy subjects reduced the percentage of apoptotic cells from 65.3 ± 6.6 to 27.2 ± 4.6% (P < 0.001) and from 45.3 ± 4.8 to 10.5 ± 4.3% (P < 0.001) respectively. Neutralizing interleukin-8 antibody abrogated the delay of neutrophil apoptosis induced by peritoneal fluid, but not in the plasma of endometriosis patients. CONCLUSIONS: These findings show that interleukin-8 is one of the neutrophil survival factors in the peritoneal fluid of endometriosis patients and that an unidentified survival factor is also present in the plasma of patients with endometriosis.

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Modulation of neutrophil apoptosis by plasma and peritoneal fluid from patients with advanced endometriosis

Human Reproduction Modulation of neutrophil apoptosis by plasma and peritoneal fluid from patients with advanced endometriosis Jong-Young Kwak 1 Sang-Won Park 0 Ki-Hyung Kim 0 Yong-Jin Na 0 Kyu-Sup Lee 0 0 Department of Obstetrics and Gynecology, College of Medicine, Pusan National University , Pusan 602-739 , Korea 1 Dong-A University Institute of Medical Science , Pusan 602-103 BACKGROUND: The increased production of pro-inflammatory chemoattractant cytokines for neutrophils in endometriosis suggests that changes in the immune system play an important role in the pathophysiology of endometriosis. The effects of plasma and peritoneal fluid from patients with advanced endometriosis on the apoptosis of neutrophils were investigated. METHODS: Apoptotic changes of neutrophils were evaluated by morphological changes using Giemsa staining. Apoptosis was confirmed by DNA electrophoretic analysis. RESULTS: Compared with the plasma (n 20) and peritoneal fluid (n 5) of healthy controls, the addition of 10% plasma (n 20) and peritoneal fluid (n 10) from patients with endometriosis to an in-vitro culture of neutrophils from healthy subjects reduced the percentage of apoptotic cells from 65.3 6.6 to 27.2 4.6% (P < 0.001) and from 45.3 4.8 to 10.5 4.3% (P < 0.001) respectively. Neutralizing interleukin-8 antibody abrogated the delay of neutrophil apoptosis induced by peritoneal fluid, but not in the plasma of endometriosis patients. CONCLUSIONS: These findings show that interleukin-8 is one of the neutrophil survival factors in the peritoneal fluid of endometriosis patients and that an unidentified survival factor is also present in the plasma of patients with endometriosis. apoptosis/endometriosis/interleukin-8/neutrophils - Endometriosis is a disease in which endometrial tissue is found outside the uterus. Although retrograde menstruation is the most widely accepted theory (Sampson, 1927), the pathogenesis of the ectopic implantation of endometrial cells is poorly understood. Growth factors and cytokines that are secreted by activated immune cells have been implicated in the control of the implantation and growth of endometrial cells outside the uterus (Lebovic et al., 2001). On the other hand, it is also possible that endometrial implants activate immune responses and lead to local inflammatory changes, such as fibrous scarring and the accumulation of activated inflammatory cells (Witz, 2000; Lebovic et al., 2001). It has been reported that endometrial cell proliferation is enhanced by peripheral blood monocytes from patients with endometriosis (Braun et al., 1994) and that the plasma levels of several cytokines, including tumor necrosis factor(TNF)-, interleukin (IL)-6 (Braun et al., 1996) and monocyte chemotactic protein-1 (Akoum et al., 1996) are elevated, suggesting that endometriosis is a local and systemic immunological disease. 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. Materials and methods The endometriosis patients (n 20) included women with stage III and IV endometriosis, which was staged according to the revised American Fertility Society scoring system (American Fertility Society, 1985). The presence of endometriosis was assessed at the time of operation and was later confirmed by pathology. Women taking oral contraceptives and GnRH analogues were excluded. Venous blood (n 20) and peritoneal fluid (n 10) samples from the endometriosis group were collected in a sterile manner before any operative manipulations. Control peritoneal fluids (n 5) were obtained from fertile women during laparoscopic operation for various gynaecological indications other than endometriosis. Sampling of peritoneal fluid was performed in the follicular phase of the menstrual cycle, because the numbers of neutrophils and macrophages are associated with the cyclic phase (Mueller et al., 2000). These fluids were centrifuged at 800 g for 10 min and the supernatants were stored at 70C. Healthy young blood donors (n 20) who did not have subfertility, dysmenorrhoea, pelvic pain or dyspareunia were enrolled as controls. Control donors and patients were confirmed not to have taken anti-inflammatory drugs for at least 3 weeks before operation. Menstrual cycle dating was determined according to the regularity of the cycle and the date of the previous menses and measurement of 17-estradiol and progesterone levels (Table I). Informed consent was obtained from all subjects. Peripheral blood neutrophils were isolated from patients and controls using a method involving dextran sedimentation and differential centrifugation through a FicollHypaque density gradient (Kwak et al., 1995). Venous blood was collected on sodium citrate solution (3.8%). The cellular part of the blood was mixed with a solution of 3% dextran (Amersham Pharmacia Biotech, Uppsala, Sweden) in 0.9% NaCl solution and kept for 45 min at 25C. The neutrophilrich upper layer of the suspension was then collected and centrifuged (250 g for 10 min). Residual erythrocytes were removed by hypotonic lysis and the pellet obtained was suspended in HEPES-buffered saline (25 mmol/l HEPES, pH 7.4, 125 mmol/l NaCl, 0.7 mmol/l MgCl2 and 0.5 mmol/l EDTA). The suspension was then centrifuged (250 g for 30 min) on Histopaque solution (Sigma Chemical Co., St Louis, MO, USA) at 4C. Isolated neutrophils (2 105/100 l) were maintained in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco-BRL, Rockville, MD, USA) supplemented with 1% glutamine, 100 IU/ml penicillin and 100 mg/ml streptomycin in 96-well flat bottomed plates at 37C in a humidified atmosphere containing 5% CO2. The neutrophils obtained were shown by microscopy to be morphologically 95% pure. 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 (2 106) were harvested, washed twice with phosphatebuffered 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 g/ml proteinase K (Sigma) for 24 h at 55C. 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 70C, resuspended in distilled water, incubated with 20 g/ml ribonuclease A (Sigma) for 1 h at 37C, electrophoresed on a 1% agarose gel containing ethidium bromide, and photographed using a LAS 2000 luminescent analyser (Bio-Rad, Hercules, CA, USA). Results are presented as mean SD. Students 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. The effects of plasma and peritoneal fluid from patients with endometriosis on neutrophils obtained from controls Neutrophils cultured in vitro undergo constitutive apoptosis. The percentage of apoptotic cells was monitored using Giemsastained cytocentrifuge preparations. Apoptotic cells, showing diminished cell volume and nuclear condensation, were readily observed (Figure 1A). Neutrophils from control donors were incubated for 24 h in RPMI medium without serum or plasma to avoid growth factor effects. The mean percentage of neutrophils showing apoptotic morphology after this period was 75 6.8% (n 20). Neutrophils from control donors were then incubated for 24 h in serial dilutions (range 120% on the culture medium) of plasma or peritoneal fluid from patients . After 24 h in-vitro incubation, the addition of 10% plasma from patients with endometriosis gave rise to a more striking inhibition of neutrophil apoptosis than that of the controls (27.2 4.6 versus 65.3 6.6%, P 0.001). Although peritoneal fluid from the controls reduced the apoptosis of control neutrophils (45.3 4.3%), the amount of apoptotic neutrophils was reduced to 10.5 4.3% (P 0.001) after incubating the neutrophils in the presence of 10% peritoneal fluid from endometriosis patients. The percentage of apoptotic cells decreased at a concentration of 5% of control plasma or peritoneal fluid, but increased at higher concentrations. In contrast, the percentage of neutrophil apoptosis decreased with increasing concentrations of endometriosis patients plasma and peritoneal fluid (Figure 2A). In this case, the effect of peritoneal fluid on the delay of neutrophil apoptosis was more prominent than that of plasma when the same concentrations of samples were used. To confirm that this delay of apoptosis of neutrophils was associated with decreased DNA fragmentationwhich is regarded as another indicator of apoptosis agarose gel electrophoresis was performed on neutrophils. The DNA of neutrophils incubated in vitro for 24 h was markedly fragmented, and showed a distinctive ladder pattern of multiple ca. 200 base pair fragments (Figure 1B). Treatment of the cells with plasma or peritoneal fluid from patients with endometriosis led to a decrease in the amount of low molecular weight DNA. We also measured the proportion of apoptotic neutrophils after prolonged incubation of the control cells in endometriosis patients plasma or peritoneal fluid. As shown in Figure 2B, extended incubation showed that 70% of the neutrophils underwent apoptosis spontaneously within 48 h in the presence of control plasma or peritoneal fluid, but that the inhibition of apoptosis was still evident 48 h after treatment with the plasma or peritoneal fluid of endometriosis patients (38.0 7.0 for plasma and 15.0 10.0% for peritoneal fluid). aP 0.001 with versus without anti-IL-8 antibody. bP 0.001 cells from endometriosis patients versus cells from control donors. cP 0.001 peritoneal fluid from patients with endometriosis versus peritoneal fluid from control donors. dP 0.001 with versus without anti-IL-8 antibody. Neutrophils were prepared from patients (n 20) and healthy controls (n 20). Cells (2 105/100 l) were cultured for 24 h with 10% plasma or peritoneal fluid from controls and patients with endometriosis in the presence or absence of IL-8 (100 ng/ml) and anti-IL-8 antibody (5 g/ml IgG). Data are reported as the percentage of cells showing morphologic features of apoptosis. Results represent the means SD. 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. It has been reported that IL-8 can modulate the apoptosis of neutrophils (Kettritz et al., 1998; Leuenroth et al., 1998) and that its concentration is elevated in the peritoneal fluid of endometriosis patients (Ryan et al., 1995; Arici et al., 1996). Therefore, we examined whether neutrophil apoptosis is influenced by IL-8 and its neutralizing anti-human IL-8 antibody. As shown in Table II, apoptosis was clearly decreased when neutrophils from the control group were incubated with 100 ng/ml of IL-8 (R&D Systems, Minneapolis, MN, USA) in the absence of plasma and peritoneal fluid, which is in keeping with previous reports (Kettritz et al., 1998; Leuenroth et al., 1998). Furthermore, the effect of IL-8 on the apoptosis of neutrophils from the control group was independent of plasma or peritoneal fluid from both the controls and the endometriosis patients. However, the capacity of the plasma from endometriosis patients to delay neutrophil apoptosis was unaffected by anti-IL-8 antibody. In contrast, IL-8 had no inhibitory effect on the apoptosis of neutrophils obtained from endometriosis patients. To examine whether the delayed apoptosis of neutrophils by the peritoneal fluid of endometriosis patients was elicited by IL-8, cells were preincubated with the anti-IL-8 antibody (R&D Systems) for 1 h and then treated with the peritoneal fluid of patients. This preincubation with anti-IL-8 antibody effectively blocked the apoptotic delay induced by the peritoneal fluid of endometriosis patients. 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 mol/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 mol/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. Endometriosis is associated with changes in the immune system involving alterations in both cell-mediated and humoral immunity (Hill, 1997). A local sterile inflammation occurs in the peritoneal cavity of women with endometriosis. This finding suggests that fewer neutrophils are present in the peritoneal fluid of those with endometriosis, because of an impaired response to chemokines or the accelerated clearance of cells (Garzetti et al., 1998). Garcia-Velasco and Arici suggested that growth factors secreted from peritoneal granulocytes are involved in the pathogenesis of endometriosis (GarciaVelasco and Arici, 1999). However, neutrophil apoptosis has not previously been studied as a potential contributory factor or epiphenomenon of endometriosis. Peripheral blood and peritoneal fluid from endometrial patients contain various cytokines and growth factors that are both pro- and antiapoptotic, depending on the cell type. In the present study, the delay of spontaneous neutrophil apoptosis was more prominent in the plasma and peritoneal fluid of endometriosis patients than in the controls. 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 antiapoptotic 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). 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Jong-Young Kwak, Sang-Won Park, Ki-Hyung Kim, Yong-Jin Na, Kyu-Sup Lee. Modulation of neutrophil apoptosis by plasma and peritoneal fluid from patients with advanced endometriosis, Human Reproduction, 2002, 595-600, DOI: 10.1093/humrep/17.3.595