Molecular Human Reproduction, Vol. 6, No. 10, 899-906,
October 2000
© 2000 European Society of Human Reproduction and Embryology
Uterine physiology |
In-vitro studies of the potential role of neutrophils in the process of menstruation
Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, 3168, Victoria, Australia
Abstract
Significant numbers of neutrophils are found extravascularly within the endometrium only during the immediate premenstrual and menstrual phases of the cycle. In this study we investigated the effect of neutrophil products on the synthesis and activation of matrix metalloproteinases (MMP), enzymes considered to play a crucial role in the degradation of endometrial tissue that occurs at menstruation. Latent MMP-2, MMP-3 and MMP-9 released by endometrial stromal fibroblasts and peripheral blood neutrophils were activated when the two cell types were cultured together. Tissue inhibitors of metalloproteinases (TIMP) 1 and 2 were also degraded in this system. Neutralization studies identified a role for the serine protease, elastase, in the observed activation of MMP. Although cultured endometrial neutrophils behaved similarly to peripheral blood neutrophils in their ability to release latent MMP-9 and elastase, no active forms of MMP-2. MMP-3 and MMP-9 were detected in supernatant from co-cultures containing endometrial neutrophils and stromal fibroblasts. This appeared to be due to an alteration in the neutrophil production of elastase and inhibitors. e.g. 
1-antitrypsin, in these cultures so that active elastase was not available. Our results demonstrate that any involvement of neutrophils in the tissue destruction occurring at menstruation may be tightly regulated by the focal concentration of degradative enzymes and their respective inhibitors.
elastase/endometrium/menstruation/metalloproteinase/neutrophil
Introduction
Immunohistochemical analyses have identified leukocytes within the endometrium and have demonstrated that both the number of these cells present and the composition of this population vary during the human menstrual cycle (Salamonsen and Woolley, 1999
; Salamonsen and Lathbury, 2000). To date, the role of leukocytes at this site has not been well defined. It is likely that these cells, in addition to providing defence against invading pathogens, may play an important role in embryo implantation and pregnancy (Johnson et al., 1999; King, 2000). Leukocytes may also be involved in the tissue breakdown that occurs at menstruation through their production of various proteases, chemokines and cytokines.
Although neutrophils are barely detectable within the endometrium for most of the normal menstrual cycle, their numbers rise substantially in the immediate premenstrual phase when these cells are localized to regions of tissue degradation (Kamat and Isaacson, 1987; Poropatich et al., 1987). Neutrophils are recruited rapidly to sites of inflammation, where they respond to injurious agents by phagocytosis, by releasing preformed enzymes and proteins contained within their granules, and by the production of reactive oxygen intermediates (Weiss, 1989). Neutrophils may participate in the tissue destruction occurring at menstruation by releasing enzymes that degrade components of the extracellular matrix, by participating in the activation of enzymes released by other endometrial cells, and by regulating enzyme synthesis by these cells.
MMP are zinc-dependent enzymes that are active in the presence of Ca2+ at neutral pH, and that are capable of degrading components of both the interstitial and basement membrane extracellular matrix (Birkedal-Hansen et al., 1993). These enzymes are secreted as latent soluble or membrane-bound proteins that can be activated in vitro by a number of proteases including other MMP. MMP activity is inhibited when these proteins are bound to TIMP in a 1:1 ratio. Numerous studies have demonstrated that endometrial stromal fibroblasts and epithelial cells produce MMP, with the concentrations of some of these enzymes peaking in the immediate premenstrual and menstrual phases of the cycle (Salamonsen and Woolley, 1996, 1999). In-vitro studies using both purified endometrial cells (Salamonsen et al., 1997) and tissue explants (Marbaix et al., 1992) have demonstrated that the production of MMP in the endometrium is regulated by progesterone. Inflammatory mediators including tumour necrosis factor (TNF) (Rawdanowicz et al., 1994; Zhang et al., 1998), interleukin-1-ß (IL-1ß) (Rawdanowicz et al., 1994; Zhang et al., 1998), transforming growth factor-ß (TGF-ß) (Bruner et al., 1995) and endothelin-1 (L.A.Salamonsen, unpublished data), all of which have been identified within the endometrium, have also been shown to regulate MMP production by endometrial cells in vitro.
The aim of this study was to define the role of neutrophils in the tissue breakdown that occurs at menstruation. An in-vitro co-culture system was used to investigate the interactions between endometrial stromal fibroblasts and neutrophils purified from either peripheral blood or endometrial tissue, with particular reference to the production and activation of MMP.
Materials and methods
Source of tissue
For the first set of experiments, endometrial tissue was obtained by curettage from women with regular menstrual cycles undergoing tubal ligation or investigation of tubal patency. Late secretory and menstrual phase tissues were excluded from this study. Five samples from the proliferative phase and five samples from the early to mid-secretory phase of the cycle were used. The median age of the patients that provided this tissue was 43.5 years. For the second set of experiments, endometrial tissue was obtained from women undergoing curettage for the investigation of menorrhagia. Tissue was obtained from six patients (median age of 47.5 years), with four samples yielding sufficient neutrophils for investigation. Informed consent was obtained from patients prior to collection of tissue, and protocols were approved by the Human Ethics Committee at Monash Medical Centre.
Preparation of endometrial stromal fibroblasts.
Endometrial stromal cells were purified as described previously (Rawdanowicz et al., 1994). Chopped tissue was digested for 45 min at 37°C with 45 IU/ml bacterial collagenase type III (Worthington Biochemical Corporation, Freehold, NJ, USA) in the presence of 3.5 µg/ml deoxyribonuclease (Boehringer Mannheim Biochimica, Mannheim, Germany) in calcium and magnesium-free phosphate-buffered saline. The cell suspension was filtered sequentially through 45 and 10 µm nylon filters to remove epithelial glands, and erythrocytes and stromal cells separated by centrifugation over Ficoll–Paque (Pharmacia, Uppsala, Sweden). Cells at the interface (predominantly stromal fibroblasts), were washed, resuspended in a 1:1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium (Trace Biosciences, Sydney, Australia) supplemented with 10% charcoal-stripped fetal calf serum (Trace Biosciences) and antibiotics (penicillin, streptomycin and fungizone), and cultured in 48-well trays (1x105 cells per well) for 4 days prior to initiation of co-cultures. For experiments with endometrial neutrophils, stromal fibroblasts were purified from the interface population using anti-fibroblast labelled magnetic microbeads (Miltenyi Biotec, Bergisch-Gladbach, Germany) and co-cultures were established on the day of isolation in 96-well trays (1 x105 fibroblasts per well).
Preparation of peripheral blood neutrophils
Peripheral blood collected from healthy donors was diluted and centrifuged over Ficoll–Paque. After erythrocytes were removed from the granulocyte-containing pellet by dextran sedimentation (0.6% in normal saline) and hypotonic water lysis, neutrophils were purified using magnetic microbeads coated with anti-CD16 monoclonal antibody (Miltenyi Biotec). Cytospin preparations of the isolated neutrophils were stained with Wright's stain (Sigma Chemical Co, St Louis, MO, USA) to determine cell purity, which was routinely >95%.
Preparation of endometrial neutrophils
Endometrial neutrophils were purified from tissue obtained from women undergoing investigation for menorrhagia, as this tissue has been found to contain increased numbers of these cells (A.J.Vincent and L.A.Salamonsen; unpublished data). Endometrial tissue was digested and filtered as described previously, and centrifuged over Ficoll–Paque. The granulocyte-containing pellet was depleted of erythrocytes, and any contaminating CD3-, CD19- and CD56-positive cells were removed using appropriately labelled magnetic microbeads (Miltenyi Biotec) before neutrophils were selected using anti-CD16 magnetic microbeads. The purity of the isolated neutrophils, which was determined as described above, was >95%. These neutrophil preparations include small numbers from within the endometrial circulation in addition to extravasated neutrophils.
Co-culture of cells
For the experiments using peripheral blood neutrophils, spent medium was first removed from the stromal fibroblasts. Peripheral blood neutrophils (2x105 per well) were then added, with the final volume in each well being 0.4 ml. In the experiments investigating endometrial neutrophils, these cells (2x104 cells per well) were combined with autologous fibroblasts, and the total volume in each well was 0.2 ml.
Cells were cultured in DMEM/F12 supplemented with insulin (10 µg/ml; human Actraprid, Novo-Nordisk Pharmaceuticals, Sydney, Australia), sodium selenite (25 ng/ml; Sigma), epidermal growth factor (50 ng/ml; Sigma), linoleic acid (10 nmol/ml; Sigma), and bovine serum albumin (0.1%; Sigma). Where indicated, oestradiol 17ß (10 nmol/l; Sigma) and natural progesterone (100 nmol/l; Sigma) were added to the culture medium.
After 24 and 48 h of incubation, supernatant was harvested, centrifuged to remove any cellular debris, divided into aliquots and stored at –20°C. Peripheral blood neutrophil viability assessed by Trypan Blue exclusion at 24 h was ~50%.
Inhibition of elastase activity in vitro
Neutrophil-conditioned medium was harvested from peripheral blood neutrophils that had been cultured for 48 h. This medium was incubated for 2 h at 4°C with a mouse monoclonal antibody against elastase (Dako, Glostrup, Denmark) at a final concentration of 1.4 µg/ml. Mock-treated neutrophil-conditioned medium was prepared by substituting an equal volume of fresh medium for the anti-elastase antibody. Equal volumes of antibody- or mock-treated neutrophil conditioned medium were then added to different wells containing stromal fibroblasts, and the volume in each well adjusted to 0.4 ml. The supernatant was harvested after 24 and 48 h of incubation, and stored as described above.
Zymographic analysis of supernatant MMP and TIMP content
Supernatants were electrophoresed under non-reducing conditions on 10% sodium dodecyl sulphate (SDS)–polyacrylamide gels containing either gelatin or casein (Sigma; 1 mg/ml). MMP (both latent and active forms) were detected as clear bands in gels that had been stained with Coomassie Blue after overnight incubation. MMP were identified by comparison with purified pro-MMP-3 (kindly provided by Dr H.Nagase, Kansas City, KS, USA), supernatant containing proMMP-2 and proMMP-9 obtained from transfected baby hamster kidney (BHK) cells (provided by Dr D.Edwards, Calgary, Canada) and molecular weight markers (Bio-Rad Laboratories Pty Ltd, Hercules, CA, USA). An equal volume of supernatant was analysed for every treatment and cell combination within each experiment.
Reverse zymography was performed on some supernatants using 15% SDS–polyacrylamide gels containing 1% gelatin and an MMP preparation from BHK-21 cells that constitutively express proMMP-2 (Hampton et al., 1999). For any one gel, an equal volume of medium from each sample was concentrated (3–10-fold, depending on the experiment) using a speedivac (Savant Co, Farmingdale, NY, USA), and the total volume of each concentrate loaded onto the gel. TIMP were visualized as dark blue bands on a cleared background in gels that had been stained with Coomassie Blue after overnight incubation. Medium from transfected BHK cells that produced TIMP-1, TIMP-2 and TIMP-3 (provided by Dr D.Edwards, Calgary, Canada) was used as a standard in these analyses.
Immunoblot analysis of supernatant enzyme and inhibitor content
Supernatants were electrophoresed under reducing conditions on 10% SDS–polyacrylamide gels. The proteins were transferred to Hybond-P membrane (Amersham Australia, Baulkham Hills, NSW, Australia) and blocked with Tris-buffered saline (TBS; pH 7.6) supplemented with 0.1% Tween 20 and 10% skimmed milk powder. Membranes were incubated overnight with either a rabbit polyclonal antibody [immunoglobulin G (IgG) fraction] against MMP-3 (kindly provided by Dr D.Woolley; University Department of Medicine, Manchester, UK) or a mouse monoclonal antibody specific for neutrophil elastase (Dako), diluted appropriately in TBS containing 0.1% Tween 20 and 5% skimmed milk. To detect 1-antitrypsin, membranes were incubated for 1 h with a rabbit polyclonal antibody (Dako) diluted in TBS containing 0.5% Tween 20 and 5% skimmed milk. After washing, the membranes were incubated with either horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham) or biotinylated horse anti-mouse IgG (Vector Laboratories Inc, Burlingame, CA, USA) followed by biotin-streptavidin–horseradish peroxidase complex (Dako), and developed by chemiluminescence (ECL system, Amersham).
Assay of elastase activity
Neutrophil elastase activity was determined spectrophotometrically at 460 nm using the specific substrate MeO-Suc-Ala-Ala-Pro-Val-7-amido-4-methyl coumarin (Sigma). The assay was performed at room temperature in 0.2 mol/l Tris–HCl, 1 mol/l NaCl (pH 8.6) with a substrate concentration of 5 µmol/l. A neutrophil lysate was prepared by adding an equal volume of 2% Triton-X100 to a neutrophil suspension (5x106 cells/ml in DMEM/F12) and stored at –80°C. A dilution series of neutrophil lysate was used to determine the elastase activity present in the test supernatants. The elastase activity released into the supernatant was expressed as nanomoles of peptide hydrolysed/s/105 neutrophils.
Results
Analysis of MMP production and activation in co-cultures of peripheral blood neutrophils and endometrial stromal fibroblasts
Endometrial stromal fibroblasts and peripheral blood neutrophils were purified and cultured either individually or together. Supernatants were harvested after 24 and 48 h of culture, and the MMP and TIMP content determined by zymography and immunoblotting. The results of a representative assay are presented in Figure 1.
|
Cultured endometrial stromal fibroblasts secreted substantial amounts of latent MMP-1 (data not shown), MMP-2 (Figure 1A
), and MMP-3 (Figure 1B), and a small amount of latent MMP-9 (Figure 1A), in agreement with earlier data from our laboratory (Rawdanowicz et al., 1994). Peripheral blood neutrophils secreted latent MMP-9 in three different molecular weight (Mr) forms (Figure 1A) of 92 000, 130 000, and 220 000 kDa. Although neutrophils also contain another MMP, neutrophil collagenase (MMP-8), this enzyme was not detected in supernatants prepared from any of the donors examined (data not shown). The specific MMP inhibitors, TIMP-1 and TIMP-2, were detected by reverse zymography in supernatant from stromal fibroblasts cultured alone, but not in supernatant from peripheral blood neutrophils (Figure 1C). Similar results were obtained from the analysis of supernatant harvested at 24 h after the initiation of culture.
The MMP and TIMP phenotype of supernatant from cultures containing both stromal fibroblasts and peripheral blood neutrophils was quite different to that of cultures containing each individual cell type. Bands representing active MMP-2, MMP-9 (Figure 1A) and MMP-3 (Figure 1B) were identified, and additional fragments with Mr of ~45 000 and 40 000 were also detected by gelatin zymography (Figure 1A). The loss of MMP-3 in the presence of neutrophils probably reflects degradation following activation. In addition to these changes, there was a substantial reduction in the amounts of TIMP-1 and TIMP-2 present in supernatant from cultures containing both cell types (Figure 1C). In four out of six studies there was an increase in the amount of active enzymes present when supernatants from 48 h of culture were compared with those of 24 h culture.
In this study, as in others from our laboratory (Salamonsen et al., 1997; Hampton et al., 1999), endometrial stromal fibroblast production of latent MMP-1 and MMP-3 was inhibited in the presence of progesterone (data not shown). In contrast, peripheral blood neutrophil activation and their subsequent release of latent MMP-9 was not altered in the presence of this hormone. Similarly, stromal fibroblast production of TIMP-1 and TIMP-2 was not affected by progesterone, a finding that is also in accordance with previous studies (Salamonsen et al., 1997).
Role of neutrophil elastase in endometrial MMP activation
A number of in-vitro studies with purified proteins have described interactions between elastase, a serine protease contained within the azurophil or primary granules of neutrophils, and MMP and TIMP (Okada et al., 1988; Okada and Nakanishi, 1989; Itoh and Nagase, 1995; Rice and Banda, 1995). Therefore, analyses were performed to determine whether elastase was released by peripheral blood neutrophils under our in-vitro culture conditions, and whether this enzyme was involved in the MMP degradation and activation observed in co-cultures of neutrophils and endometrial stromal fibroblasts.
Supernatants harvested from neutrophils cultured either alone or in the presence of endometrial stromal fibroblasts were analysed by immunoblotting using a monoclonal antibody against elastase. In each case the enzyme was identified as a protein of ~Mr 30 000 (Figure 1D), demonstrating that elastase was released by neutrophils under the culture conditions utilized in this study. Slight variations in the molecular weight of the enzyme may represent different degrees of glycosylation.
To determine whether the enzyme elastase was involved in the degradation and activation of MMP in co-culture supernatants, an additional experiment was performed in which neutrophil-conditioned medium was incubated with the anti-elastase antibody before being added to stromal fibroblasts. The supernatant harvested from these cells was analysed by gelatin zymography and immunoblotting. When the neutrophil-conditioned medium was pretreated with anti-elastase antibody, active MMP-2 was still observed but there was a substantial reduction in the amount of the smaller fragments resulting from the degradation of MMP-2 (Figure 2A). The presence of anti-elastane antibody also substantially reduced and in the amount of active MMP-3 (Figure 2B) present.
|
In-vitro activity of endometrial neutrophils
Endometrial neutrophils were purified from tissue collected from women undergoing curettage for the investigation of menorrhagia, and cultured with autologous stromal fibroblasts. Culture supernatants were collected after 24 and 48 h, and the MMP and TIMP profiles determined by zymography and immunoblotting. The results from two separate experiments are presented in Figure 3
.
|
Purified endometrial stromal fibroblasts cultured immediately after isolation secreted primarily latent MMP-2 (Figure 3A
) and MMP-3 (Figure 3B), a small amount of latent MMP-9 (Figure 3A), and TIMP-1 (Figure 3C). Endometrial neutrophils cultured alone released latent MMP-9 (Figure 3A) in the same three forms as those secreted by peripheral blood neutrophils. Identical results were obtained when supernatant harvested at either 24 or 48 h after the initiation of culture was analysed. In this set of experiments, endometrial stromal fibroblast production of latent MMP-3 was reduced and endometrial neutrophil release of latent MMP-9 was unchanged in the presence of progesterone, as observed in the study utilising peripheral blood neutrophils.
The MMP and TIMP profile of supernatants harvested from co-cultures of endometrial neutrophils and stromal fibroblasts was markedly different to that of the co-cultures containing peripheral blood neutrophils. Active MMP-9 (Figure 3A) and MMP-3 (Figure 3B), and MMP-2 degradation fragments (Figure 3A) were not detected in these supernatants. In addition, TIMP-1 concentrations were similar to those present in supernatant from cultures containing stromal fibroblasts alone (Figure 3C). It is unlikely that the isolation procedure affected endometrial neutrophil function, since MMP activation and degradation were still observed in supernatant harvested from co-cultures containing peripheral blood neutrophils that had been treated with collagenase and DNase (data not shown). Furthermore, immunoblot analysis of supernatants demonstrated that elastase was released by cultured endometrial neutrophils (Figure 3D).
To test the hypothesis that MMP activation and degradation were not observed in co-cultures containing endometrial neutrophils due to an alteration in the balance between elastase and its inhibitors, two additional analyses were performed. In the first, the amount of active enzyme present in supernatants harvested from cultures containing either endometrial or peripheral blood neutrophils was determined using a specific fluorescent substrate. In the second, the amount of 1-antitrypsin (1AT), an important inhibitor of elastase (Weiss, 1989; Owen and Campbell, 1995), present in supernatant from the same experiments was determined by immunoblotting. The results of these assays are presented in Figures 4 and 5, respectively.
|
), fibroblasts and neutrophils (
), or neutrophils (
|
Active elastase was present in supernatants harvested from cultures containing peripheral blood neutrophils (Figure 4
). In contrast, the amount of active enzyme present in supernatants from cultures containing endometrial neutrophils was below the detection limit of the assay. An equal volume of supernatant from samples from each of the four donors was analysed in the experiment presented in Figure 5. Under our culture conditions on a per cell basis, substantially more 1AT was released by endometrial neutrophils (Figure 5B) than by peripheral blood neutrophils (Figure 5A). Discussion
The human endometrium is unique in that it undergoes sequential and simultaneous cycles of degradation and regeneration during the regular menstrual cycles that a woman experiences. Although numerous studies have identified leukocytes within this tissue (Salamonsen and Woolley, 1999; Salamonsen and Lathbury, 2000), very little is currently known about the way in which these cells are involved in the processes of menstruation and endometrial regeneration. In this study, we have examined the potential role of neutrophils in the tissue breakdown that occurs at menstruation.
Significant numbers of neutrophils are only found in the normal endometrium during the immediate premenstrual and menstrual phases of the cycle, when they are localized to regions of tissue degradation (Kamat and Isaacson, 1987; Poropatich et al., 1987). Immunohistochemical analyses have demonstrated that these cells can express elastase, MMP-9 (Vincent et al., 1999), the membrane-type (MT) 1-MMP (Zhang et al., 2000), interferon-
(IFN-) (Yeaman et al., 1998) and activin ßA (Leung et al., 1998). Since neutrophil priming agents including TNF, granulocyte macrophage-colony stimulating factor, IFN- and interleukin-8 (IL-8) (Salamonsen and Woolley, 1996) have all been identified within premenstrual phase endometrial tissue, it is likely that neutrophils at this site respond fully to activating agents and release reactive oxygen species, their granule contents and lipid mediators.
Our analysis of supernatant from co-cultures of peripheral blood neutrophils and endometrial stromal fibroblasts has identified some of the reactions occurring between products of each cell type (Figure 1). Although latent MMP and/or TIMP were released by neutrophils and fibroblasts, MMP activation was only observed in cultures containing both cell types. If similar interactions occur in the immediate vicinity of activated neutrophils within the endometrium, one would expect active MMP-2, MMP-3 and MMP-9 to be generated, and TIMP-1 and TIMP-2 to be degraded. This alteration in the balance between MMP and their inhibitors would then allow the focal destruction of extracellular matrix.
Our observation that a proportion of the latent MMP-2 released by stromal fibroblasts was activated in the presence of peripheral blood neutrophils (Figure 1A) is in agreement with previous results (Schwartz et al., 1998a,b). We have recently demonstrated that MT1-MMP, a membrane-bound MMP essential for the activation of latent MMP-2, is expressed by endometrial neutrophils (Zhang et al., 2000). Since MT1-MMP was not the sole protein involved in the latent MMP-2 activation observed by Schwartz et al. further studies are required to determine the identity of the additional proteins involved in this process, and whether they are also involved in the activation of latent MMP-2 within the endometrium.
Our results emphasize the important role that neutrophil elastase can play in the activation cascade of MMP, and therefore in tissue degradation. Previous in-vitro studies using purified enzymes have demonstrated the involvement of this enzyme in the activation of latent MMP-2, MMP-3, and MMP-9, and in the degradation of TIMP-1 and TIMP-2 (Okada et al., 1988; Okada and Nakanishi, 1989; Itoh and Nagase, 1995; Rice and Banda, 1995; data summarized in Figure 6). The present study extends these findings to a physiologically relevant model, the human endometrium. Neutrophil elastase has also been implicated in the activation of MMP at other sites of inflammation. For example, active MMP-9 was detected in synovial fluid from a proportion of the individuals suffering rheumatoid arthritis in one study (Watanabe et al., 1997), with a direct relationship between elastase secretion and the concentration of active MMP-9 being noted.
|
Neutrophil elastase may also be involved indirectly in the degradation of endometrial tissue by other means. This enzyme has been reported to induce eosinophil degranulation, to stimulate endothelial and epithelial cell synthesis of IL-8, and to alter numerous epithelial cell functions (Hiemstra et al., 1998
). Other neutrophil products are also likely to be involved in the degradation of endometrial tissue at menstruation. For example, the serine protease, cathepsin G, which is stored in the azurophil granules of neutrophils, has been shown to digest elastin and various basement membrane components (Hiemstra et al., 1998), to activate latent MMP-3 (Okada and Nakanishi, 1989) and to influence epithelial cell activity (Hiemstra et al., 1998).
The current study has identified a role for neutrophils in the activation of endometrial MMP, but not in the regulation of MMP synthesis by endometrial stromal fibroblasts. It is likely that factors released by other endometrial cell types are responsible for this regulatory activity. Other leukocyte subsets may also be involved in the regulation of MMP synthesis within the endometrium, and in the activation of these enzymes. Indeed, we have previously defined a role for mast cells in the regulation of MMP production by endometrial cells and in the activation of these enzymes (Zhang et al., 1998).
Tissue-derived neutrophils are a heterogeneous population in respect to their activation state and hence their phenotype (Weiss, 1989). Within any tissue, such cells may undergo a progression of phenotypic changes away from that typical of peripheral blood neutrophils. Indeed, previous studies from our laboratory have shown that endometrial neutrophils are heterogeneous in respect to their expression of MMP-9 (Vincent et al., 1999), MT1-MMP (Zhang et al., 1999) and activin ßA (Leung et al., 1998). In the current study, in-vitro 1AT release by endometrial and peripheral blood neutrophils differed (Figure 5). Together these results suggest that neutrophils derived from tissue, bleeding endometrium in this case, are of a phenotype different from those in the same tissue at an earlier time or in a different physiological state, that is pre-bleeding endometrium.
The results obtained from our study of endometrial neutrophils highlight the problems associated with relating the activity of cells in vitro to their actions in tissue. Our results also emphasize that the complex interactions between proteases, such as elastase, and their specific inhibitors are critical in determining the activity of these enzymes. In-vitro cultures of endometrial neutrophils did not contain active MMP (Figure 3A,B), probably due to an alteration in the balance between elastase and its inhibitor, 1AT, in favour of the inhibitor (Figures 4 and 5). Although it is possible that a similar situation may occur within the endometrium adjacent to a proportion of the infiltrating neutrophils, it is also likely that other cells will overcome protease inhibitors and participate in extracellular matrix degradation. In support of this argument, active elastase has been detected in extracts of pre-menstrual phase endometrial tissue and tissue obtained from women undergoing investigation for menorrhagia (A.J.Vincent and L.A.Salamonsen; unpublished data).
Neutrophils, by their ability to generate chlorinated oxidants, are able to neutralize the inhibitory effect of 1AT (Zaslow et al., 1983; Ossanna, 1986; Weiss, 1989; Desrochers et al., 1992). Stimulated neutrophils can also avoid the protease inhibitors present in serum by adhering to targeted substrates and forming a sequestered microenvironment into which the entrance of the larger plasma protease inhibitors is restricted (Owen and Campbell, 1995). Since the ability of neutrophils to impair the local balance between proteases and their inhibitors has been described for rheumatoid arthritis (Abbink et al., 1993) and pulmonary disorders, e.g. bronchiectasis (Sepper et al., 1995) and emphysema (Fujita et al., 1990), these cells may perform similarly within the endometrium.
Endometrial repair begins as early as 36 h after the start of menstrual bleeding, while tissue breakdown is still in progress and at a time when substantial numbers of neutrophils are present. Therefore it is possible that neutrophils will also be involved in this process. Although the phagocytosis of wound debris is an important neutrophil role (Martin, 1997) these cells have also been shown to secrete a number of factors that may regulate the function of other endometrial cells and facilitate tissue regeneration (Cassatella, 1995). In addition, neutrophils, by their ability to oxidize 2-macroglobulin (Weiss, 1989; Wu et al., 1998), may alter the balance between tissue degradation and repair.
The results presented above support the hypothesis that neutrophils are involved in the tissue breakdown occurring at menstruation. When activated, these cells may release proMMP-9 and pro-MMP-8, enzymes that can participate in extracellular matrix degradation once activated. Other enzymes, e.g. elastase and cathepsin G, that can degrade extracellular matrix components and participate in the activation cascade of MMP may also be released by activated endometrial neutrophils. Additional in-vitro studies of purified cells and in-situ analyses of the endometrial neutrophil phenotype are required to identify other ways in which neutrophils may be involved in the tissue degradation and regeneration that occurs during the process of menstruation.
Acknowledgments
This study was supported by the following grants: NH and MRC of Australia (grant 971292), NIH (grant HD33233), WHO Human Reproduction Programme (grant 96908). We would like to thank Professor G.Kovacs, Dr J.Clarke, Dr E.Farrell and their patients for providing the endometrial tissue used in these studies, and Sr C.Canny and Sr J.McLaren for assistance in their collection. We are grateful to Ms A.Lourbakos who provided assistance with the analysis of active elastase enzyme. We thank Dr A.Vincent for sharing unpublished data. We also thank Sue Panckridge for assistance in the preparation of figures for this paper.
Notes
1 To whom correspondence should be addressed at: Prince Henry's Institute of Medical Research, PO Box 5152, Clayton, 3168, Victoria, Australia. E-mail: lois.salamonsen{at}med.monash.edu.au 
References
Abbink, J.J., Kamp, A.M., Nuijens, J.H. et al. (1993) Proteolytic inactivation of 1-antitrypsin and 1-antichymotrypsin by neutrophils in arthritic joints. Arthritis Rheum., 36, 168–180.[Web of Science][Medline]
Birkedal-Hansen, H., Moore, W.G.I., Bodden, M.K. et al. (1993) Matrix metalloproteinases: a review. Crit. Rev. Oral Biol. Med., 4, 197–250.
Bruner, K.L., Rodgers, W.H., Gold, L.I. et al. (1995) Transforming growth factor ß mediates the progesterone suppression of an epithelial metalloproteinase by adjacent stroma in the human endometrium. Proc. Natl. Acad. Sci. USA, 92, 7362–7366.
Cassatella, M.A. (1995) The production of cytokines by polymorphonuclear neutrophils. Immunol. Today, 16, 21–26.[Web of Science][Medline]
Desrochers, P.E., Mookhtiar, K., Van Wart, H.E. et al. (1992) Proteolytic inactivation of 1-proteinase inhibitor and 1-anti-chymotrypsin by oxidatively activated human neutrophil metalloproteinases. J. Biol. Chem., 267, 5005–5012.
Fujita, J., Nelson, N.L., Daughton, D.M. et al. (1990) Evaluation of elastase and antielastase balance in patients with chronic bronchitis and pulmonary emphysema. Am. Rev. Respir. Dis., 142, 57–62.[Web of Science][Medline]
Hampton, A.L., Nie, G.Y., Salamonsen, L.A. (1999) Progesterone analogues similarly modulate endometrial matrix metalloproteinase-1 and matrix metalloproteinase-3 and their inhibitor in a model for long-term contraceptive effects. Mol. Hum. Reprod., 5, 365–371.
Hiemstra, P.S., van Wetering, S. and Stolk, J. (1998) Neutrophil serine proteinases and defensins in chronic obstructive pulmonary disease: effects on pulmonary epithelium. Eur. Respir. J., 12, 1200–1208.[Abstract]
Itoh, Y. and Nagase, H. (1995) Preferential inactivation of tissue inhibitor of metalloproteinases-1 that is bound to the precursor of matrix metalloproteinase 9 (progelatinase B) by human neutrophil elastase. J. Biol. Chem., 270, 16518–16521.
Johnson, P.M., Christmas, S.E. and Vince, G.S. (1999) Immunological aspects of implantation and implantation failure. Hum. Reprod., 14 (Suppl. 2), 26–36.
Kamat, B.R. and Isaacson, P.G. (1987) The immunocytochemical distribution of leukocyte subpopulations in human endometrium. Am. J. Pathol., 127, 66–73.[Abstract]
King, A. (2000) Uterine leukocytes and decidualization. Hum. Reprod. Update, 6, 28–36.
Leung, P.H.Y., Salamonsen, L.A. and Findlay, J.K. (1998) Immunolocalization of inhibin and activin subunits in human endometrium across the menstrual cycle. Hum. Reprod., 13, 3469–3477.
Marbaix, E., Donnez, J., Courtoy, P.J. et al. (1992) Progesterone regulates the activity of collagenase and related gelatinases A and B in human endometrial explants. Proc. Natl. Acad. Sci. USA, 89, 11789–11793.
Martin, P. (1997) Wound healing- aiming for perfect skin regeneration. Science, 276, 75–81.
Okada, Y. and Nakanishi, I. (1989) Activation of matrix metalloproteinase 3 (stromelysin) and matrix metalloproteinase 2 (`gelatinase') by human neutrophil elastase and cathepsin G. FEBS Letts., 249, 353–356.[Web of Science][Medline]
Okada, Y., Watanabe, S., Nakanishi, I. et al. (1988) Inactivation of tissue inhibitor of metalloproteinases by neutrophil elastase and other serine proteinases. FEBS Letts., 229, 157–160.[Web of Science][Medline]
Ossanna, P.J., Test, S.T., Matheson, N.R. et al. (1986) Oxidative regulation of neutrophil elastase-alpha-1-proteinase inhibitor interactions. J. Clin. Invest., 77, 1939–1951.
Owen, C.A. and Campbell, E.J. (1995) Neutrophil proteinases and matrix degradation. The cell biology of pericellular proteolysis. Semin. Cell. Biol., 6, 367–376.[Web of Science][Medline]
Poropatich, C., Rojas, M. and Silverberg, S.G. (1987) Polymorphonuclear leukocytes in the endometrium during the normal menstrual cycle. Int. J. Gynecol. Pathol., 6, 230–234.[Web of Science][Medline]
Rawdanowicz, T.J., Hampton, A.L., Nagase, H. et al. (1994) Matrix metalloproteinase secretion by cultured human endometrial stromal cells: identification of interstitial collagenase, gelatinase A, gelatinase B, and stromelysin 1 and their differential regulation by interleukin 1- and tumor necrosis factor-. J. Clin. Endocrinol. Metab., 79, 530–536.[Abstract]
Rice, A. and Banda, M.J. (1995) Neutrophil elastase processing of gelatinase A is mediated by extracellular matrix. Biochemistry, 34, 9249–9256.[Medline]
Salamonsen, L.A., Butt, A.R., Hammond, F.R. et al. (1997) Production of endometrial matrix metalloproteinases, but not their tissue inhibitors, is modulated by progesterone withdrawal in an in vitro model for menstruation. J. Clin. Endocrinol. Metab., 82, 1409–1415.
Salamonsen, L.A. and Lathbury, L.J. (2000) Endometrial leukocytes and menstruation. Hum. Reprod. Update, 6, 16–27.
Salamonsen, L.A. and Woolley, D.E. (1996) Matrix metalloproteinases and their tissue inhibitors in endometrial remodelling and menstruation. Reprod. Med. Rev., 5, 185–203.
Salamonsen, L.A. and Woolley, D.E. (1999) Menstruation: induction by matrix metalloproteinases and inflammatory cells. J. Reprod. Immunol., 44, 1–27.[Web of Science][Medline]
Schwartz, J.D., Shamamian, P., Monea, S. et al. (1998a) Activation of tumor cell matrix metalloproteinase-2 by neutrophil proteinases requires expression of membrane-type 1 matrix metalloproteinase. Surgery, 124, 232–238.[Web of Science][Medline]
Schwartz, J.D., Monea, S., Marcus, S.G. et al. (1998b) Soluble factor(s) released from neutrophils activates endothelial cell matrix metalloproteinase-2. J. Surg. Res. 76, 79–85.[Web of Science][Medline]
Sepper, R., Konttinen, Y.T., Ingman, T. et al. (1995) Presence, activities, and molecular forms of cathepsin G, elastase, 1-antitrypsin, and 1-antichymotrypsin in bronchiectasis. J. Clin. Immunol., 15, 27–34.[Web of Science][Medline]
Vincent, A.J., Malakooti, N., Zhang, J. et al. (1999) Endometrial breakdown in women using Norplant is associated with migratory cells expressing matrix metalloproteinase-9 (gelatinase B). Hum. Reprod., 14, 807–815.
Watanabe, Y., Shimamori, Y., Fujii, N. et al. (1997) Correlation between the appearance of gelatinases in the synovial fluid of patients with rheumatoid arthritis and polymorphonuclear elastase, stromelysin-1, and the tissue inhibitor of metalloproteinase-1. Clin. Exp. Rheumatol. 15:255–261.[Web of Science][Medline]
Weiss, S.J. (1989) Tissue destruction by neutrophils. N. Eng. J. Med., 320, 365–376.[Web of Science][Medline]
Wu, S.M., Patel, D.D. and Pizzo, S.V. (1998) Oxidised 2-macroglobulin (2M) differentially regulates receptor binding by cytokines/growth factors: implications for tissue injury and repair mechanisms in inflammation. J. Immunol., 161, 4356–4365.
Yeaman, G.R., Collins, J.E., Currie, J.K. et al. (1998) IFN- is produced by polymorphonuclear neutrophils in human uterine endometrium and by cultured peripheral blood polymorphonuclear neutrophils. J. Immunol., 160, 5145–5153.
Zaslow, M.C., Clark, R.A., Stone, P.J. et al. (1983) Human neutrophil elastase does not bind to alpha1-protease inhibitor that has been exposed to activated human neutrophils. Am. Rev. Respir. Dis., 128, 434–439.[Web of Science][Medline]
Zhang, J., Hampton, A.L., Nie, G.Y. et al. (2000) Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium. Biol. Reprod., 62, 85–94.
Zhang, J., Nie, G.Y., Wang, J. et al. (1998) Mast cell regulation of endometrial stromal cell matrix metalloproteinases: a mechanism underlying menstruation. Biol. Reprod., 59, 693–703.
Submitted on March 20, 2000; accepted on July 20, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Deng, X. Pan, Y. Wang, L. Wang, X. E Zhou, M. Li, Y. Feng, Q. Wu, B. Wang, and N. Huang Hemoglobin and its derived peptides may play a role in the antibacterial mechanism of the vagina Hum. Reprod., January 1, 2009; 24(1): 211 - 218. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C.-w. Cheng, H. Bielby, D. Licence, S. K. Smith, C. G. Print, and D. S. Charnock-Jones Quantitative Cellular and Molecular Analysis of the Effect of Progesterone Withdrawal in a Murine Model of Decidualization Biol Reprod, May 1, 2007; 76(5): 871 - 883. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. J. Lockwood, P. Toti, F. Arcuri, M. Paidas, L. Buchwalder, G. Krikun, and F. Schatz Mechanisms of Abruption-Induced Premature Rupture of the Fetal Membranes: Thrombin-Enhanced Interleukin-8 Expression in Term Decidua Am. J. Pathol., November 1, 2005; 167(5): 1443 - 1449. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Jones, N. J. Hannan, T. J. Kaitu'u, J. Zhang, and L. A. Salamonsen Identification of Chemokines Important for Leukocyte Recruitment to the Human Endometrium at the Times of Embryo Implantation and Menstruation J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6155 - 6167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. E. King, H. O. D. Critchley, J.-M. Sallenave, and R. W. Kelly Elafin in Human Endometrium: An Antiprotease and Antimicrobial Molecule Expressed during Menstruation J. Clin. Endocrinol. Metab., September 1, 2003; 88(9): 4426 - 4431. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. E. Curry Jr. and K. G. Osteen The Matrix Metalloproteinase System: Changes, Regulation, and Impact throughout the Ovarian and Uterine Reproductive Cycle Endocr. Rev., August 1, 2003; 24(4): 428 - 465. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang and L. A. Salamonsen In Vivo Evidence for Active Matrix Metalloproteinases in Human Endometrium Supports their Role in Tissue Breakdown at Menstruation J. Clin. Endocrinol. Metab., May 1, 2002; 87(5): 2346 - 2351. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Zhang and L.A. Salamonsen Expression of hypoxia-inducible factors in human endometrium and suppression of matrix metalloproteinases under hypoxic conditions do not support a major role for hypoxia in regulating tissue breakdown at menstruation Hum. Reprod., February 1, 2002; 17(2): 265 - 274. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||