Modulation of neutrophil apoptosis by plasma and peritoneal fluid from patients with advanced endometriosis
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.
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
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
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
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
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).
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.
The authors thank Dr Byung-Man Cho for his suggestions on statistical
analysis of the data and Dr David J.Uhlinger for his critical comments
Afford , S.C. , Pongracz , J. , Stockley , R.A. , Crocker , J. and Burnett , D. ( 1992 ) The induction by human interleukin-6 of apoptosis in the promonocytic cell line U937 and human neutrophils . J. Biol. Chem. , 267 , 21612 - 21616 .
Akoum , A. , Lemay , A. , McColl , S.R. , Paradis , I. and Maheux , R. ( 1996 ) Increased monocyte chemotactic protein-1 level and activity in the peripheral blood of women with endometriosis . Am. J. Obstet. Gynecol. , 175 , 1620 - 1625 .
American Fertility Society ( 1985 ) Revised American Fertility Society Classification of Endometriosis . Fertil. Steril., 43 , 351 - 352 .
Arici , A. , Tazuke , S.I. , Attar , E. , Kliman , H.J. and Olive , D.L. ( 1996 ) Interleukin-8 concentration in peritoneal fluid of patients with endometriosis and modulation of interleukin-8 expression in human mesothelial cells . Mol. Hum. Reprod. , 2 , 40 - 45 .
Arici , A. , Seli , E. , Senturk , L.M. , Gutierrez , L.S. , Oral , E. and Taylor , H.S. ( 1998 ) Interleukin-8 in the human endometrium . J. Clin. Endocrinol . Metab., 83 , 1783 - 1787 .
Braun , D.P. , Muriana , A. , Gebel , H. , Rotman , C. , Rana , N. and Dmowski , W.P. ( 1994 ) Monocyte-mediated enhancement of endometrial cell proliferation in women with endometriosis . Fertil. Steril., 61 , 78 - 84 .
Braun , D.P. , Gebel , H. , House , R. , Rana , N. and Dmowski , N.P. ( 1996 ) Spontaneous and induced synthesis of cytokines by peripheral blood monocytes in patients with endometriosis . Fertil. Steril., 65 , 1125 - 1129 .
Cassatella , M.A. ( 1995 ) The production of cytokines by polymorphonuclear neutrophils . Immunol. Today , 16 , 21 - 26 .
Fadok , V.A. , Bratton , D.B. , Konowal , A. , Freed , P.W. , Westcott , J.Y. and Henson , P.M. ( 1998 ) Macrophages that have ingested apoptotic cells in vitro inhibit proinflammatory cytokine production through autocrine/paracrine mechanism involving TGF-, PGE2, and PAF. J. Clin . Invest., 101 , 890 - 898 .
Garcia-Velasco , J.A. and Arici , A. ( 1999 ) Chemokines and human reproduction . Fertil. Steril., 71 , 983 - 993 .
Garzetti , G.G. , Ciavattini , A. , Provinciali , M. , Amati , M. , Muzzioli , M. and Governa , M. ( 1998 ) Decrease in peripheral blood polymorphonuclear leukocyte chemotactic index in endometriosis: role of prostaglandin E2 release . Obstet. Gynecol., 91 , 25 - 29 .
Gebel , H.M. , Braun , D.P. , Tambur , A. , Frame , D. , Rana , N. and Dmowski , W.P. ( 1998 ) Spontaneous apoptosis of endometrial tissue is impaired in women with endometriosis . Fertil. Steril., 69 , 1042 - 1047 .
Hamasaki , A. , Sendo , F. , Nakayama , K. , Ishida , N. , Negishi , I. , Nakayama , Ki. and Hatakeyama , S. ( 1998 ) Accelerated neutrophil apoptosis in mice lacking A1-a, a subtype of the bcl-2-related A1 gene . J . Exp. Med ., 188 , 1985 - 1992 .
Haslett , C. ( 1992 ) Resolution of acute inflammation and the role of apoptosis in the tissue fate of granulocytes . Clin. Sci. , 83 , 639 - 648 .
Hill , J.A. ( 1997 ) Immunology and endometriosis . Fact, artifact, or epiphenomenon? Obstet. Gynecol. Clin. North Am., 24 , 291 - 306 .
Kettritz , R. , Gaido , M.L. , Haller , H. , Luft , F.C. , Jennette , C.J. and Falk , R.J. ( 1998 ) Interleukin-8 delays spontaneous and tumor necrosis factor- - mediated apoptosis of human neutrophils . Kidney Int. , 53 , 84 - 91 .
Khorram , O. , Taylor , R.N. , Ryan , I.P. , Schall , T.J. and Landers , D.V. ( 1993 ) Peritoneal fluid concentrations of the cytokine RANTES correlate with the severity of endometriosis . Am. J. Obstet. Gynecol. , 169 , 1545 - 1549 .
Kokawa , K. , Shikone , T. and Nakano , R. ( 1996 ) Apoptosis in the human uterine endometrium during the menstrual cycle . J. Clin. Endocrinol . Metab., 81 , 4144 - 4147 .
Kwak , J.Y. , Lopez, I. , Uhlinger , D.J. , Ryu , S.H. and Lambeth , J.D. ( 1995 ) RhoA and a cytosolic 50-kDa factor reconstitute GTPS-dependent phospholipase D activity in human neutrophil subcellular fractions . J. Biol. Chem. , 270 , 27093 - 27098 .
Lebovic , D.I. , Mueller , M.D. and Taylor , R.N. ( 2001 ) Immunobiology of endometriosis . Fertil. Steril., 75 , 1 - 10 .
Leiva , M.C. , Hasty , L.A. , Pfeifer , S. , Mastroianni , L. Jr and Lyttle , C.R. ( 1993 ) Increased chemotactic activity of peritoneal fluid in patients with endometriosis . Am. J. Obstet. Gynecol. , 168 , 592 - 598 .
Leuenroth , S. , Lee , C. , Grutkoski , P. , Keeping , H. and Simms , H.H. ( 1998 ) Interleukine-8-induced suppression of polymorphonuclear leukocyte apoptosis is mediated by suppressing CD95 (Fas/Apo-1) Fas-1 interactions . Surgery, 124 , 409 - 417 .
Liles , W.C. , Dale , D.C. and Klebanoff , S.J. ( 1995 ) Glucocorticoids inhibit apoptosis of human neutrophils . Blood , 86 , 3181 - 3188 .
Meszaros , A.J. , Reichner , J.S. and Albina , J.E. ( 2000 ) Macrophage-induced neutrophil apoptosis . J. Immunol. , 165 , 435 - 441 .
Mueller , M.D. , Lebovic , D.I. , Garrett , E. and Taylor , R.N. ( 2000 ) Neutrophils infiltrating the endometrium express vascular endothelial growth factor: potential role in endometrial angiogenesis . Fertil. Steril., 74 , 107 - 112 .
Oppenheim , J.J. , Zachariae , C.O. , Mukaida , N. and Matsushima , K. ( 1991 ) Properties of the novel proinflammatory supergene 'intercrine' cytokine family . Ann. Rev. Immunol. , 9 , 617 - 648 .
Ryan , I.P. , Tseng , J.F. , Schriock , E.D. , Khorram , O. , Landers , D.V. and Taylor , R.N. ( 1995 ) Interleukin-8 concentrations are elevated in peritoneal fluid of women with endometriosis . Fertil. Steril., 63 , 929 - 932 .
Sampson , J.A. ( 1927 ) Peritoneal endometriosis due to menstrual dissemination of endometrial tissue into the peritoneal cavity . Am. J. Obstet. Gynecol. , 14 , 422 - 429 .
Savill , J.S. , Henson , P.M. and Haslett , C. ( 1989 ) Phagocytosis of aged human neutrophils by macrophages is mediated by a novel 'charge-sensitive' recognition mechanism . J. Clin. Invest ., 84 , 1518 - 1527 .
Shifren , J.L. , Tseng , J.F. , Zaloudek , C.J. , Ryan , I.P. , Meng , Y.G. , Ferrara , N. , Jaffe , R.B. and Taylor , R.N. ( 1996 ) Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis . J. Clin. Endocrinol . Metab., 81 , 3112 - 3118 .
Stellar , H. ( 1995 ) Mechansims and genes of cellular suicide . Science , 267 , 1445 - 1449 .
Stringer , R.E. , Hart , C.A. and Edwards , S.W. ( 1996 ) Sodium butyrate delays neutrophil apoptosis: role of protein biosynthesis in neutrophil survival . Br. J. Haematol. , 92 , 169 - 175 .
Tabibzadeh , S. ( 1994 ) Regulatory roles of IFN- in human endometrium . Ann. NY Acad. Sci. , 734 , 1 - 6 .
Watson , R.W. , Rotstein , O.D. , Nathens , A.B. , Parodo , J. and Marshall , J.C. ( 1997 ) Neutrophil apoptosis is modulated by endothelial transmigration and adhesion molecule engagement . J. Immunol. , 158 , 945 - 953 .
Whyte , M.K. , Savill , J. , Meagher , L.C. , Lee , A. and Haslett , C. ( 1997 ) Coupling of neutrophil apoptosis to recognition by macrophages: coordinated acceleration by protein synthesis inhibitors . J. Leukoc. Biol ., 62 , 195 - 202 .
Witz , C.A. ( 2000 ) Interleukin-6: another piece of the endometriosis-cytokine puzzle . Fertil. Steril., 73 , 212 - 214 .
Yeaman , G.R. , Collins , J.E. , Currie , J.K. , Guyre , P.M. , Wira , C.R. and Fanger , M.W. ( 1998 ) IFN- is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils . J. Immunol. , 160 , 5145 - 5153 .
Submitted on May 1 , 2001 ; resubmitted on September 6, 2001 ; accepted on November 14 , 2001