Molecular Human Reproduction, Vol. 6, No. 12, 1085-1091,
December 2000
© 2000 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
Eosinophils in the human corpus luteum: the role of RANTES and eotaxin in eosinophil attraction into periovulatory structures
1 Institute of Anatomy, 2 Department of Gynaecology and Obstetrics, University of Leipzig, and 3 Centre for Reproductive Medicine, Goldschmidtstrasse 30, Leipzig, Germany
Abstract
We evaluated the presence and number of eosinophils at varying stages in the human corpus luteum from 27 ovaries of women at reproductive age. Eosinophils preferentially accumulated in dilated microvessels of the thecal layer transforming into septa of the corpus luteum. The granulosa layer under luteinization, the thecal layer, and haemorrhages in the former antrum each contained low, moderate and high numbers of extravasated eosinophils respectively. Eosinophils decreased rapidly during the stages of secretion and regression. Semi-quantitative reverse transcriptase–polymerase chain reaction (RT–PCR) and enzyme-linked immunosorbent assay (ELISA) systems were used to investigate the expression and regulation of the eosinophil-attracting chemokines RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxin in granulosa cells obtained from follicular aspirates from women undergoing IVF. Contaminating leukocytes were determined by CD18 mRNA quantification. Granulosa cells expressed RANTES (n = 3; 43 ± 14 pg/ml, mean ± SEM). 4ß-phorbol-12-myristate-13-acetate (PMA; 211 ± 53) and tumour necrosis factor 
(TNF) (238 ± 59), but not interleukin (IL)-1 up-regulated RANTES at significant levels. In general, higher basal and stimulated RANTES mRNA and protein were found in cultures with higher CD18 mRNA levels than in those with lower levels. We found only traces of eotaxin mRNA and no eotaxin secretion, even in stimulated granulosa cell cultures, independently of leukocyte levels. Taken together, this is the first study demonstrating the selective presence of eosinophils in human periovulatory structures. RANTES, but not eotaxin, may play an active process in the accumulation of these cells.
corpus luteum/eosinophils/eotaxin/leukocytes/RANTES
Introduction
Leukocytes are attracted into the ovary, and influence cyclically changing structures (Brännström et al., 1993; Best et al., 1996
; Machelon et al., 1997). Recently, we reported that eosinophils suddenly increase in number to ~90% of the leukocytes in the bovine corpus luteum in early development (Reibiger et al., 2000). Eosinophils have also been observed in ovine and porcine pre-ovulatory follicles (Murdoch and Steadman, 1991; Standaert et al., 1991). The presence of eosinophils has never been described in the human ovary. In rats and mice, neutrophils or macrophages are thought to take over the function of eosinophils.
The molecular trafficking of eosinophils and other immune cells is complex, involving the interactions of an entire superfamily of chemoattractant cytokines (chemokines) and their receptors. About 50 chemokines have been identified in humans; of these, RANTES (regulated on activation, normal T cell expressed and secreted) and eotaxins are favourite candidates for recruiting eosinophils (Burke-Gaffney and Hellewell, 1996; Garcia-Zepeda et al., 1996; Beck et al., 1997). The accelerated eosinophil recruitment in allergic and connective tissue diseases is caused by these chemokines (Jose et al., 1994; Ganzalo et al., 1996; Venge et al., 1996; Rajakulasingam et al., 1997). Data are very limited on the mechanisms of the cyclic migration of eosinophils in defined ovarian areas (Aust et al., 1999). Recently, we demonstrated that macrophages in the bovine ovary, located in the luteinizing theca of developing corpora lutea in early development, express RANTES mRNA. RANTES has been found in human follicular fluid at concentrations between 50–400 pg/ml. (Machelon et al., 2000). Another group (Karström-Encrantz et al., 1998) suggested that RANTES did not play a role in leukocyte attraction into the ovary, as the serum concentrations were more than 50 times as high. However, it is important to emphasize that neither plasma nor serum measurements reflect the actual circulating concentrations of RANTES, as they largely result from chemokine release from the -granules of platelets during the processing of blood samples (Polo et al., 1999). Bovine ovarian RANTES production at ovulation probably originates from local macrophages (Aust et al., 1999), whereas in the human system both leukocytes and granulosa cells release the chemokine (Machelon et al., 2000). The expression of eotaxin has not been reported in the ovary of any species.
The aim of the study was to screen human ovaries of different cyclic stages for the presence of eosinophils, and to verify the expression of both RANTES and eotaxin in granulosa cell cultures obtained from women undergoing IVF. The COV434 granulosa tumour cell line was also included. The regulation of RANTES and eotaxin by the cytokines interleukin-1 (IL-1) and tumour necrosis factor (TNF), which are highly increased in pre-ovulatory follicles and are considered important role in ovulation and corpus luteum formation, was also examined.
Materials and methods
Tissue preparations
Ovaries from 27 non-pregnant women of reproductive age, all of whom were undergoing oophorectomy for gynaecological diseases, were kindly supplied by the Institute of Pathology, University of Leipzig. The paraffin-embedded samples used here were collected over a period of 10 years. The timing of each menstrual cycle was determined by the last menstrual period and by the pathologist's report on the endometrium. The ovarian tissue had been fixed in 4% formaldehyde, and paraffin-embedded using routine procedures. Sections (7 µm thick) were mounted on slides precoated with paper glue (Cementit®; Merz and Benteli AG, Niederwangen, Germany). One slide was stained with haematoxylin and eosin (H&E) for routine histological examination. Next, samples of the cortex and the corpus luteum in early development from one ovary, which had been frozen in liquid nitrogen, were used to amplify RANTES and eotaxin mRNA.
Histology and eosinophil counting
We screened ovaries at a very early stage of corpus luteum development (n = 2), corpora lutea within development (n = 3), early (n = 6) and late (n = 8) secretory corpora lutea, and corpora lutea during regression (n = 8), according to previously described criteria (Corner, 1956; Clement, 1987). Very early stages of corpus luteum development still contained the basal membrane in some planes, clearly separating the theca layer with dilated capillaries from the granulosa layer. In developing corpora lutea, the basal membrane disappeared between luteinizing granulosa and thecal cells. The secretory corpora lutea consisted of large luteal cells and fibrocyte-like endothelial cells of the capillary bed. Regressing corpora lutea contained luteal cells with signs of degeneration, e.g. vacuolated cytoplasm.
Eosinophils were stained with Sirius red (Bayer AG, Leverkusen, Germany) as described (Reibiger et al., 2000). Sirius red (500 mg) was dissolved in 45 ml aqua bidest, 50 ml absolute ethanol and 1 ml 1% NaOH; 4ml NaCl at 20% solution was added until slight precipitation occurred. Sections were deparaffinized, stained with haematoxylin, differentiated in running tap water, treated with 70% ethanol and stained with the Sirius red solution at room temperature for 1 h.
All of the Sirius red-labelled cells were scored as positive, regardless of the intensity of staining. The numbers of positive cells/mm2 were counted by use of a light microscope evaluated by two people. Intra- and inter-observer differences were <10%. The evaluation was started at a point at the periphery of a corpus luteum and, at regular intervals, counting was continued in 10 areas, whether they happened to be luteal tissue or luteal septa. Distinct microvessels with intravascular eosinophils were included in the evaluation, whereas eosinophils in haemorrhages were disregarded. Data were reported as mean ± SEM. The Mann–Whitney test was applied to verify statistically significant differences between the groups.
To detect eosinophils in ovarian tissue, conservation in liquid nitrogen is inappropriate. Serial cryostat sections of bovine corpora lutea of very early development were stained using anti-CD18, H&E and Sirius red. In this period, nearly all of the CD18+ leukocytes contribute to the eosinophils (Reibiger et al., 2000). The CD18-stained leukocytes are mainly located in the septal area and were counted there (n = 5; mean ± SEM; mm2). In the same region of a H&E-stained section, only a small number of eosinophils could be identified. The same result was obtained from staining with Sirius red. Thus, the eosinophils were present in cryostat sections, but they could not be identified by classical H&E or Sirius red staining. These problems did not appear on paraffin sections.
Granulosa cells and the COV434 cell line
Granulosa cells were taken from patients participating in oocyte retrieval after ovarian stimulation for IVF, according to routine protocols. The experimental protocol and use of the cells were submitted after obtaining the patients' consent. Follicular aspirates of multiple pre-ovulatory follicles contained mostly granulosa cells, but also leukocytes and erythrocytes. Aspirates from different follicles of one patient were pooled and centrifuged at 200 g in isotonic 45% Percoll solution (Amersham Pharmacia Biotech, Freiburg, Germany). The upper band was removed, and the cells were washed three times, seeded in 2 ml medium in a 6-well plate, and cultured in Dulbecco's modified Eagle's medium (DMEM)/Ham's F12 (Gibco BRL, Grand Island, NY, USA) with 5% fetal calf serum (FCS) for 3 days. The level of contaminating leukocytes within the granulosa cell cultures was determined by quantification of CD18 mRNA (see below). Granulosa cells of 10 patients were pooled for further experiments. The only available COV434 granulosa tumour cell line (van den Berg-Bakker et al., 1993), a kind gift of P.I.Schrier (Department of Clinical Oncology, University Hospital, Leiden, The Netherlands), was included in our study and cultured in DMEM/10% FCS.
Cell cultures
Cells (1x105) were cultured in 24-well plates for 24 h. The medium contained the desired concentration of human IL-1 (10 IU/ml; Sigma, Deisenhofen, Germany), TNF (5 ng/ml; Sigma), or 10 ng/ml 4ß-phorbol-12-myristate-13-acetate (PMA; Sigma). Triplicate cultures of each stimulator were analysed. The RANTES mRNA level reached a plateau after ~12 h, and were thus analysed at that point. The highest RANTES protein concentrations were measured after 24 h. Later, the protein concentration was reduced, probably by proteolytic degradation (data not shown). The supernatants were removed, frozen and assayed for RANTES and eotaxin by ELISA (Amersham Pharmacia). The sensitivity of the tests was 2 and 20 pg/ml respectively. The protein concentrations of cultures obtained from separate experiments (n = 3), with three replicate wells per treatment per experiment, were presented as mean ± SEM. The Mann–Whitney test was used to determine the statistical significance of the difference between basal and stimulated cultures. Lysis buffer (0.3 ml) of the Qiagen total RNA isolation kit (Qiagen GmbH, Hilden, Germany) was added to the cell culture wells, the content of three wells of every cell type was pooled and then frozen in liquid nitrogen until further mRNA analysis.
RNA isolation and cDNA synthesis
Total cellular RNA was isolated from ovarian tissues and cell cultures using the Qiagen total RNA isolation kit (Qiagen) according to the manufacturer's protocol. Genomic DNA was digested with 0.02 IU DNase/µg RNA (Roche Molecular Biochemicals, Mannheim, Germany) at 25°C for 10 min. 5 µg of total RNA was taken to synthesize cDNA using a first-strand cDNA synthesis kit from Amersham Pharmacia Biotech in a reaction volume of 15 µl.
Semi-quantitative competitive reverse transcription– polymerase chain reaction (RT–PCR)
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RANTES and CD18 cDNA were quantified using internal homologous competitors according to our published method (Aust et al., 1997). To quantify human eotaxin cDNA, a new internal homologous competitor was constructed. For this reason, cDNA from COV434 cells was used to amplify a 406 bp fragment (eotaxin mRNA; Genbank accession number D49372) with the eotaxin primer pair. The fragment was cloned into a pGEM-T vector (Promega Corporation, Madison, WI, USA); 95 bp were cut out from the cloned fragment with BalI (Roche Molecular Biochemicals), which did not cut vector sequences. The sticky ends were ligated with T4 DNA ligase (Roche Molecular Biochemicals). Amplification of the shorter eotaxin insert with the eotaxin primers resulted in the 311 bp eotaxin competitor. All competitors were quantified and stabilized at defined concentrations in the PCR tubes according to a previously described method (Köhler et al., 1997).
Variations across different cDNA preparations were corrected. All samples were first adjusted to contain equal-input GAPDH cDNA concentrations in a semi-quantitative RT–PCR (Aust et al., 1997). We then estimated RANTES, eotaxin and CD18 cDNA in these adjusted samples. cDNA samples were titrated into RT–PCR amplification solutions containing known copies of the competitor. Both the sample cDNA and the competitor were co-amplified using the same primer pair. With this approach, two products were generated. One was derived from the cDNA and the other, smaller in size, from the competitor (Table I). Based on the length difference, the sample cDNA and competitor PCR products were resolved by gel electrophoresis. The sample cDNA and competitor were quantified by measuring the intensity of ethidium fluorescence with a cooled CCD 8-bit image sensor, and data were analysed by Phoretix 1 D plus software (Phoretix International, Newcastle upon Tyne, UK). The sequences of the RANTES, eotaxin, CD18 and cytokine receptor primer pairs are shown in Table I; the GAPDH primers were published previously (Aust et al., 1997). The target copies equation was used to determine the ratio of sample cDNA copies/PCR to the number of competitor copies added, multiplied by the quotient of the cDNA signal and divided by the competitor signal.
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The intra- and inter-assay coefficients of variation (CVs) for mRNA quantification were <15%. This is in agreement with other studies using quantitative competitive RT–PCR (Hockett et al., 1995
; Vats et al., 1997; Uusi-Oukari et al., 2000). Each 25 µl amplification reaction contained 2.5 µl 10x concentrated PCR buffer (15 mmol/l MgCl2), 0.3 IU Taq DNA polymerase (Roche Molecular Biochemicals), 100 µmol/l dNTPs, 0.1 µmol/l of each primer, 1 µl sample cDNA and the stabilized competitor in adjusted dilutions. PCR reactions consisted of n cycles for 15 s at 94°C, 30 s at 64°C (GAPDH, RANTES) or at 58°C (eotaxin) and 35 s at 72°C.
Results
Eosinophils in corpora lutea
The freshly ruptured mature follicles showed strikingly dilated capillaries and venules in the thecal layer forming septa between the infoldings of the luteinizing granulosa layer. Vessel dilations were also seen in the corpora lutea at the early stage of development, but not in subsequent stages. Vessel dilations were accompanied by the presence of eosinophils. They were frequently seen clustered in blood vessels, often found adhering to the endothelial cells, and less often distributed in the thecal layer of ruptured follicles and in the luteinizing thecal layer of developing corpora lutea (Figure 1). In comparison with the former thecal layer, the granulosa layer under luteinization displayed a lower number of extravasated eosinophils (Figures 1 and 2). When haemorrhages into the former antrum were present, they were rich in extravasated eosinophils. The visual impression of high numbers of eosinophils in freshly ruptured follicles and developing corpora lutea and the lower-to-negligible number of eosinophils in secretory and in regressing corpora lutea was confirmed by our data from leukocyte counting (Figure 2).
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RANTES mRNA and protein expression
Samples of the cortex and the corpus luteum in early development were positive for RANTES mRNA in a simple RT–PCR (data not shown). In order to define the cellular source of RANTES, we investigated follicle-derived granulosa cell cultures and the COV434 granulosa tumour cell line. Both cell types were positive for IL-1 receptor (p80 and p68) and TNF
receptor (p75 and p55) mRNA (Figure 3). Granulosa cells expressed RANTES mRNA (n = 3, mean ± SEM, 121 ± 91x10–20 mol RANTES mRNA/PCR) as well as protein (43 ± 14 pg/ml) under basal conditions. PMA (cDNA: 825 ± 432x10–20 mol RANTES mRNA/PCR; protein: 211 ± 53 pg/ml) and TNF (mRNA: 1271 ± 519x10–20 mol RANTES mRNA/PCR; protein: 238 ± 59pg/ml), but not IL-1, significantly up-regulated RANTES (P < 0.02). The high SEM indicates that different amounts of leukocytes may influence the basal and stimulated RANTES levels. Thus, we quantified CD18 mRNA in these cultures as a marker for leukocytic contamination. In general, higher basal and stimulated RANTES mRNA and protein were found in cultures with higher CD18 mRNA levels than in those with low levels. Figure 4 shows two typical experiments with very low and high leukocyte contamination. Under basal conditions, cultures with higher CD18 mRNA level showed a RANTES mRNA expression level of up to 20-fold higher, compared with cultures with very low CD18 mRNA (Figure 3). However, as well as leukocytes, stimulated granulosa cells themselves expressed RANTES mRNA. Only traces of RANTES mRNA and no RANTES protein expression were found in the granulosa tumour cell line COV 434 cells (data not shown).
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Eotaxin mRNA and protein expression
Samples of the cortex and the corpus luteum in early development were positive for eotaxin mRNA in a simple RT–PCR (data not shown). However, granulosa cells showed minimal eotaxin mRNA levels near the detection limit of the method. IL-1 and TNF
slightly stimulated the eotaxin mRNA independently of the CD18 mRNA level (data not shown). Eotaxin protein expression was not detectable in our ELISA system (detection limit = 20 pg/ml). COV434 cells showed a higher basal and stimulated eotaxin expression compared with granulosa cell cultures (n = 3, mean ± SEM, pg/ml; basal: 28 ± 20, PMA: 56 ± 37, IL-1: 143 ± 46, TNF: 80 ± 49). However, the concentrations were near the detection limit of the assay or very low, and did not reflect physiological relevance. Discussion
This is the first study that demonstrates the presence of eosinophils in developing corpora lutea from human ovaries. Other comparable studies did not detect a sudden increase in the number of eosinophils. Another group (Karström-Encrantz et al., 1998) mentioned that eosinophils are rarely detected inside the ovary at any stage of the cycle. In contrast, macrophages/monocytes, T-lymphocytes and natural killer cells have been located in human corpora lutea (Wang et al., 1992; Katabuchi et al., 1997; Gaytan et al., 1998; Suzuki et al., 1998). Perhaps the rarity of ovaries with developing corpora lutea at the very early stage among the few available intact ovaries of women at reproductive age or the fact that Sirius red staining has not been carried out on histological sections by others may explain the failure in eosinophil detection (Brännstrom et al., 1994). Moreover, in half of the bovine ovary samples, we have found that the number of detectable Sirius red-stained eosinophils in cryostat sections is significantly lower compared with paraffin-embedded samples. In part, this is likely to be due to spontaneous degranulation of eosinophils during shock freezing. Similarly, the density of macrophages in pre-ovulatory follicles has been controversially reported. It is either dramatically increased, leading to the hypothesis that macrophages regulate ovulation (Brännstrom et al., 1994), or in other studies, only a few macrophages are detected in human pre-ovulatory follicles and no functional role was indicated (Best et al., 1996; Takaya et al., 1997). Here, we emphasize that only the very early stage of corpus luteum development shows a dense eosinophil presence. Thus, the time frame for eosinophil recruitment is very short.
The function of eosinophils in the corpus luteum remains speculative. In sheep, eosinophils are assumed to influence angiogenesis because cortisol-induced eosinopenia causes small-sized corpora lutea with an underdeveloped vascular bed, and thus insufficient endocrine function (Murdoch and Steadman, 1991). In the human corpus luteum, eosinophil occurrence runs parallel to the period of angiogenesis and luteinization. Blood vessels grow from the former theca between the luteinizing granulosa cells towards the centre of the former antrum. Eosinophils which have accumulated in the blood vessels and outside of those of the forming septa may influence the architecture of vessel sprouting in three dimensions, and thus mediate the orientation between endocrine and endothelial cells. The conspicuously high number of eosinophils in the haemorrhages of the freshly ruptured follicles and developing corpora lutea may be involved in such a controlled three-dimensional blood vessel growth.
The marked increase of eosinophils in periovulatory structures suggests the expression of specific eosinophil-attracting chemokines. Tissue eosinophilia under allergic conditions is associated with high expression levels of RANTES and/or eotaxin (Beck et al., 1997; Rajakulasingam et al., 1997; Ying et al., 1997). Here, granulosa cells secreted RANTES, but no eotaxin, under basal conditions in the range 10–300 pg/ml, depending on the CD18 mRNA level in the cultures. This result confirms our own previous data on RANTES mRNA expression in isolated and in-situ macrophages detected by mRNA quantification and in-situ hybridization in the bovine ovary (Aust et al., 1999). Human granulosa cells indeed produce RANTES. In agreement with a recently published study (Machelon et al., 2000), we found basal and stimulated RANTES secretion up to 100 pg/ml in granulosa cell cultures nearly free of contaminating leukocytes. However, these RANTES levels are still low compared with other studies quantifying the chemokine in stimulated cultures of fibroblasts (Teran et al., 1999), or thyroid-infiltrating leukocytes (Simchen et al., 2000). RANTES induces optimal chemotactic activity in the concentration range 1–100 ng/ml. To confirm the significance of RANTES expression by granulosa cells, we examined the COV434 granulosa tumour cell line, but found only faint RANTES mRNA signals not indicating any physiological significance. In contrast, physiological relevant levels of up to 5 µg/ml of constitutive RANTES production have been shown in two breast, and four out of eight melanoma tumour cell lines respectively (Luboshits et al., 1999; Mrowietz et al., 1999).
The low RANTES and the missing eotaxin expression reflect several problems. First, other cell populations, e.g. fibroblasts, which are known to be potential RANTES and eotaxin producers (Rathanaswami et al., 1993; Teran et al., 1999), may additionally be responsible for chemokine expression. Our preliminary positive eotaxin and RANTES mRNA results obtained from the cortex region of the human ovary support this assumption. Secondly, there may be different chemokine patterns in attracting eosinophils into the normal cycling ovary than into inflamed or allergy affected tissues. This is supported by the striking accumulation of eosinophils within the dilated vessels of the thecal layer under reorganization. Thirdly, additional chemokines or chemokine receptors may be involved in eosinophil attraction. Eotaxin-2 (Forssmann et al., 1997) and the recently identified eotaxin-3, which is constitutively expressed in the ovary (Kitaura et al., 1999), also control eosinophil trafficking via CCR3. Recently, two groups (Jinquan et al., 2000; Nagase et al., 2000) reported that eosinophils express CXC chemokine receptors. The CXC chemokines, IP-10 and Mig, induce eosinophil chemotaxis via CXCR3; whereas stromal-derived factor-1 (SDF-1), the natural ligand of CXCR4, elicites an apparent Ca2+ influx in eosinophils and induces a strong migratory response comparable with that by eotaxin. Moreover, the CXC chemokines, IL-8, chemokine growth-regulated oncogene (GRO)-, and monocyte chemotactic peptide-1 (MCP-1) are directly or indirectly implicated in the development and ovulation of follicles, in corpus luteum stages such as development, secretion and regression, and in steroidogenesis (Runesson et al., 1996; Arici et al., 1997; Karström-Encrantz et al., 1998). IL-8, GRO- and MCP-1 mRNA and protein have been demonstrated in the thecal and granulosal regions of dominant follicles and/or in ovarian stromal and granulosa–lutein cells (Arici et al., 1997; Karström-Encrantz et al., 1998). Thus, the chemokine–chemokine receptor system involved in eosinophil attraction has not yet been completely defined.
As well as basal RANTES and eotaxin expression, we have investigated how their expression may be regulated. Numerous pro-inflammatory stimulants, including the cytokines IL-1 and TNF, stimulate RANTES and eotaxin in several cell types (Rathanaswami et al., 1993; Sticherling et al., 1995; Bartels et al., 1996; Lilly et al., 1997; Teran et al., 1999). Therefore, RANTES and eotaxin production of the periovulatory follicle may be dependent on the local IL-1 and TNF secretion in the follicular fluid. Firstly, we demonstrated the expression of IL-1 receptor mRNAs for both known IL-1 receptors on granulosa cells and the cell line COV434 and confirmed the results for the expression of TNF receptors shown previously (Machelon et al., 2000). TNF and PMA showed the highest capacity to stimulate granulosa cell cultures for RANTES production, whereas IL-1 only slightly increased the chemokine.
In summary, eosinophils are abundant in the human corpus luteum at the very early development stage, pointing to a role in the regulation of corpus luteum growth. RANTES is expressed in granulosa cells as well as leukocytes and may be involved in eosinophil attraction.
Acknowledgments
We thank Silke Kiessling and Doreen Sittig for their excellent technical assistance. The study was financially supported by the Interdisciplinary Center of Clinical Research (IZKF, project B6), University of Leipzig.
Notes
4 To whom correspondence should be addressed at: Institute of Anatomy, Liebigstrasse 13, Leipzig, 04103, Germany. E-mail: ausg{at}medizin.uni-leipzig.de 
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Submitted on May 22, 2000; accepted on September 14, 2000.
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