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Original Studies |
in Granulosa Cells from Human Preovulatory Follicle
INSERM, U-355 (V.M., F.N.) and U-131 (D.E.), Institut Paris-Sud sur les Cytokines, 92140 Clamart, France
Address all correspondence and requests for reprints to: Dr. Véronique Machelon, INSERM, U-355, Maturation Gamétique et Fécondation, 32 rue des Carnets, 92140 Clamart, France. veronique. machelon{at}inserm.ipsc.u-psud.fr
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Abstract |
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(TNF) and was inhibited in cultures containing a
neutralizing anti-TNF antibody. p55 TNF receptors were detected by
RT-PCR and visualized on granulosa cells by flow cytometry. RANTES
production was increased by phorbol 12-myristate 13-acetate, but not by
8-bromo-cAMP. RANTES was produced by granulosa cells from human
preovulatory follicles. This production was activated by TNF,
probably through TNF receptor p55. This suggests that RANTES may play
an active role in ovarian processes involving the local accumulation of
immune cells. |
Introduction |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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(17) have been detected in human
preovulatory follicles. Regulated on activation normal T expressed and
secreted (RANTES) is another possible candidate. RANTES belongs to the
ß-chemokine subfamily (18). It is involved in inflammatory responses
and is considered an important mediator of inflammation (19). It is a
chemoattractant for memory T lymphocytes, monocytes/macrophages, and
eosinophils (20, 21). RANTES is also a histamine-releasing factor
capable of inducing histamine release from basophils and mast cells
(22). Through these various actions this chemokine may play a key role
in the ovulatory process. In this study we measured the expression of
RANTES in cultured human preovulatory follicle granulosa cells
collected from patients undergoing in vitro fertilization,
then cultured. To investigate how RANTES production is regulated, we
tested the effect of tumor necrosis factor- (TNF), a cytokine
produced in human preovulatory follicles (23) which activates RANTES
production (24, 25). We also used pharmacological tools, such as
8-bromo-cAMP (8-Br-cAMP), a permeable analog of cAMP, and phorbol
12-myristate 13-acetate (PMA), a phorbol ester that mimics the effect
of diacylglycerol. |
Material and Methods |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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DMEM-Ham’s F-12 medium (vol/vol), trypsin-ethylenediamine
tetraacetate (1 x solution) were obtained from Life Technologies, Inc. (Cergy Pontoise, France). Percoll was
purchased from Pharmacia (St. Quentin en Yvelines, France). Human
recombinant TNF (rhTNF) and neutralizing anti-TNF were
purchased from Genzyme (Cambridge, MA). 8-Br-cAMP and the phorbol ester
PMA were obtained from Sigma-Aldrich Corp. (St. Quentin
Fallavier, France). Kits for human RANTES immunoassays (Quantikine)
were obtained from R&D Systems (Abington, UK).
R-Phycoerythrin (R-PE)-conjugated mouse anti-human
RANTES monoclonal antibody and the R-PE-conjugated mouse IgG1
isotype control, fluorescein isothiocyanate (FITC)-conjugated mouse
antihuman CD45 monoclonal antibody, and the FITC-conjugated mouse IgG1
isotype control were purchased from PharMingen (San
Diego, CA). Anti-TNF receptor p55 (htr-9) was provided by Dr. W.
Leslauer and Hoffmann-La Roche. FITC-conjugated anti-mouse IgG was
obtained from Immunotech (Marseille, France). Messenger
ribonucleic acid (mRNA) capture kit, digoxigenin (DIG) RNA labeling
mix, T3 and SP6 RNA polymerases, alkaline phosphatase-conjugated
anti-DIG, and Fast Red tablets were purchased from Roche Molecular Biochemicals (Meylan, France).
Cell preparation
Cells were obtained from patients aged, 20–40 yr, undergoing
oocyte retrieval after ovarian stimulation for in vitro
fertilization-embryo transfer (IVF-ET) according to routine protocols
and selected at random. The experimental protocol and the use of the
cells were submitted to patient consent. Cells from aspirates of
multiple hyperstimulated preovulatory follicles were pooled and
centrifuged through a Percoll cushion [vol/vol, phosphate-buffered
saline (PBS)-Percoll] to remove red blood cells, and then cultured in
Ham’s F-12-DMEM containing 15 mmol/L HEPES, 0.365 g/liter
L-glutamine, 50 µg/ml streptomycin, and 50
IU/ml penicillin, supplemented with 10% FCS. After an initial 24-h
culture period, the medium plus floating cells were removed and
replaced by medium containing 2% FCS. This was repeated every 24
h. Granulosa luteinizing cells were identified by their
3ß-hydroxysteroid
dehydrogenase/
5-4-isomerase
(3ßHSD) activity, which metabolizes pregnenolone to form progesterone
(26). A positive reaction was observed under light microscope as
black-blue precipitate within the cells.
Detection of RANTES production
Concentrations of RANTES were measured using a RANTES sandwich enzyme-linked immunosorbent assay (Quantikine kit, R&D Systems). The sensitivity of the test was 2.5 pg/ml. Assays were run in duplicate. Absorbance was measured at 450 nm. Intra- and interassay coefficients of variation were about 5% and 8%, respectively. Amounts of RANTES in culture supernatants were expressed per µg protein to allow for changes in protein content reflecting cell number. At the end of the culture period, attached cells were dissolved in 0.5 N NaOH, the resulting solution was neutralized with 0.5 N HCl, and the protein concentration was measured using the Bio-Rad Laboratories, Inc. protein assay (Richmond, CA). Results were expressed as picograms of RANTES per µg total protein/24 h.
Immunostaining and flow cytometric analysis
Cells were fixed with 2% (wt/vol) paraformaldehyde in PBS for 20 min at 4 C, permeabilized with 0.2% saponin in PBS for 5 min at 37 C, then exposed for 1 h at room temperature to R-PE-anti-RANTES antibody diluted at 8 µg/ml in PBS containing 10% human serum AB and 0.2% saponin. R-PE-conjugated mouse IgG1 isotype was used as a control. The cells were washed repeatedly with PBS containing 0.2% saponin, then resuspended in PBS and stored at 4 C in the dark until read on the flow cytometer. Cells expressing membrane CD45 leukocyte antigen were visualized by exposure to saturated FITC-anti-CD45 antibody, which reacts with the 180-, 190-, 205-, and 220-kDa isoforms of the leukocyte antigen present on all human leukocytes. The cells were incubated with a 1:10 dilution of this antibody or with the control isotype FITC mouse IgG1 for 30 min on ice. They were then washed and resuspended in PBS until being read on the flow cytometer. TNF receptors (TNFR) were detected by incubation for 30 min at 4 C with 10 µg/ml monoclonal anti-TNFR in PBS plus 10% human serum AB. The cells were washed repeatedly with PBS and then incubated for 30 min at 4 C with a 1:40 dilution of fluorescein-conjugated F(ab')2 rabbit antimouse IgG. After successive washings in PBS, the cells were fixed with 2% (wt/vol) paraformaldehyde in PBS (20 min at 4 C) and then resuspended in PBS until they were read on the Becton Dickinson and Co. FACScan flow cytometer (Mountain View, CA). The flow cytometric data were analyzed using Becton Dickinson and Co. CellQuest software. The size and granulometry distribution of the cells were examined by simultaneous measurement of forward light scatter and side light scatter signals. Data for successive samples were acquired until 5,000 or 10,000 cells had accumulated in the gate. Fluorescence intensity histograms were generated vs. cell number. The percentage of cells that specifically bound to the test antibody was determined by subtracting the nonspecific fluorescence found for the isotype control from the total fluorescence found for the labeled antibody.
RNA extraction and PCR procedures
Polyadenylated mRNA was extracted from cell lysates, then
hybridized with the biotin-labeled
oligo(deoxythymidine)20 probe and captured in
streptavidin-coated PCR tubes using the mRNA capture kit from
Roche Molecular Biochemicals according to the instructions
of the manufacturer. The immobilized mRNA was directly transcribed into
complementary DNA, then amplified using specific oligonucleotide
primers with the sequences shown in Table 1
. The RT protocol was run at 50 C for 30
min, then at 94 C for 2 min. It was followed by the PCR amplification
protocol run at 94 C for 1 min, 55 C for 1 min, and 72 C for 1 min for
30 cycles; the final extension step consisted of heating to 72 C for 10
min. The PCR products were separated on 2% agarose gel and visualized
under UV after ethidium bromide staining.
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These experiments were performed on frozen cytocentrifuged cells as previously described (27). In situ hybridization was performed with a RNA probe complementary to the coding sequence of human RANTES (28). RNA probes were labeled with digoxigenin by in vitro transcription of DNA using DIG RNA labeling mix containing T3 polymerase for antisense probe and SP6 polymerase for sense probe (negative control). After acetylation, cell spots were hybridized overnight at 50 C with DIG RNA probes in a solution containing 50% (vol/vol) deionized formamide, 0.3 mol/L NaCl, 10 mmol/L Tris (pH 8.2), 1 mmol/L ethylenediamine tetraacetate, 0.05% yeast transfer RNA, 10 mmol/L dithiothreitol, 1 x Denhardt’s solution, and 10% dextran sulfate. The slides were treated with ribonuclease A (4 µg/µl) at 37 C for 30 min and then washed in buffers containing decreasing concentrations of salts. The digoxigenin label was detected by incubating the cells for 2 h at room temperature with alkaline phosphatase-conjugated anti-DIG antibody diluted 1:500 in a solution containing 1 mol/L NaCl, 0.1 mol/L Tris, 2 mmol/LMgCl2, 0.3% Triton X-100, and 3% normal sheep serum. Alkaline phosphatase was detected using Fast Red tablets as a substrate. The cells were counterstained with hematoxylin, mounted in glycerin/PBS, and observed under a light microscope. Brownish-red staining was indicative of positive binding by the RNA probe.
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Results |
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The concentration of RANTES was measured in follicular fluid
obtained from eight women undergoing in vitro fertilization
35–36 h after hCG administration. The mean concentration of RANTES was
174 ± 12 pg/mL (n = 8; Table 2). The cells were collected from the
follicular aspirates and cultured at 105
cells/well. The supernatants were collected after 24 h, then
attached cells were dissolved to measure protein concentration. The
concentration of RANTES in culture supernatants was 29.6 ± 2.5
pg/µg total protein·24 h (n = 8; Table
2).
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The cells collected from the follicular aspirates were cultured at
105 cells/well for 24–72 h. The size and
granulometry distribution of the cells were determined by measuring the
forward light scatter and side light scatter signals. The distribution
of CD45+ cells (labeled with the anti-CD45
antibody) was examined in each experiment. Cells gated in region 1 (R1)
did not contain CD45+ cells. All cells that
stained with an anti-CD45-FITC antibody were gated in region 2 (R2).
The proportion of CD45+ cells in R2 was always
significantly lower in samples tested after 48 h in culture (Fig. 1). We looked for the cells labeled with
the RANTES antibody (RANTES+ cells) in R1 and R2.
RANTES+ cells constituted about 40–60% of the
R1-gated cells. They were always less abundant in R2, although the
proportion of CD45+ cells varied from one sample
to another one (Table 3). The percentage
of cells that stained positively for RANTES was highest during the
first 24 h of culture. After 48 h in culture, the percentage
of RANTES+ cells in R1 was slightly reduced,
whereas it had slumped to less than 1% in R2. After 72 h,
RANTES+ cells were still detected in R1, although
their number had decreased (Fig. 2).
After 72 h, most of the cells contained 3ßHSD activity.
Leukocytes had no 3ßHSD (our unpublished data).
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We tested the effect of TNF, a cytokine known to activate
RANTES production (24, 25). The effect of TNF on RANTES production
was tested by adding 1 ng/ml rhTNF or 20 µg/ml neutralizing
anti-TNF antibody, which blocks endogenous TNF production, to the
culture medium. We also investigated the effects of 1 mmol/L 8-Br-cAMP,
a permeable analog of cAMP, and 50 nmol/L PMA, a phorbol ester that
mimics the effect of diacylglycerol. Test reagents were added to the
culture medium for 24 h, then medium was removed, and supernatants
were collected and stored at -20 C until assayed. Cells were removed
for protein assay. RANTES accumulation in cultures was measured in
picograms of RANTES per µg total protein/h. Treated cells were
compared with untreated cells (control) in three separate experiments,
with four replicate wells per treatment per experiment. The effects of
treatments are expressed as the change in RANTES accumulation as a
percentage of the untreated control value (100%). RANTES production
was increased 5-fold by PMA and 2-fold by TNF vs. the
control value; it was decreased 2-fold by anti-TNF; 8-Br-cAMP had no
significant effect (Fig. 3). In three
other separate experiments, cultured cells were removed after
treatment, then stained with specific Ab and analyzed by flow
cytometry. The percentage of cells labeled with the RANTES antibody
(RANTES+ cells) was measured in the R1-gated
cells. rhTNF slightly increased the number of cells staining for
RANTES, whereas no cell stained for RANTES in cultures containing the
neutralizing anti-TNF antibody. PMA increased the percentage of
RANTES+ cells, whereas 8-Br-cAMP did not. Results
were similar in the three experiments. Figure 4 shows a typical experiment of the three
performed.
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TNFR were detected by RT-PCR and flow cytometry in cells cultured
for 24 h. Amplification with TNFR p55 primers produced a single
477-bp amplicon. We measured the proportion of cells expressing TNFR by
means of flow cytometry carried out after immunostaining with a p55
anti-TNFR antibody. Immunostaining for TNFR was strongly reinforced
when neutralizing anti-TNF was added to cultures. Under these
conditions more than 40% of cells in R1 labeled for TNFR (Fig. 5).
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RANTES mRNA was detected by RT-PCR. Amplification with RANTES
primers produced a single 192-bp amplicon in cells that had been
collected from follicular aspirates, suspended in culture medium for
2 h, and then cultured during 24 and 48 h (Fig. 6). The cellular expression of RANTES
mRNA was visualized in cells cultured for 24 h by in
situ hybridization. In situ hybridization was performed
using DIG-labeled human RANTES RNA probe. Red-colored cytoplasm
indicated the presence of mRNA within individual cells. No colored cell
was detected in controls using a sense RNA probe (Fig. 7). The percentage of labeled cells
actively synthesizing RANTES was determined visually; it was more than
50% of all cells in three separate experiments.
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Discussion |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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Next, we investigated how RANTES production may be regulated. The
ovulatory follicle is a site of local inflammation. The hallmark of
inflammation is the infiltration of specific leukocyte subsets
from the blood into the affected tissue. A variety of chemotactic
proteins, including RANTES, orchestrate the migration of leukocytes.
Numerous exogenous agents and endogenous proinflammatory stimulants,
including the cytokines interleukin-1ß, TNF, and interferon-
,
stimulate RANTES production in several cell types (24, 31). Therefore,
RANTES production in the ovulatory follicle may be accounted for by
many inflammatory cytokines secreted in follicular fluid. We focused
our study on TNF. TNF is found in preovulatory follicular fluid
(23); the effect of TNF on RANTES production has been reported for
endothelial cells, which require an exogenous TNF source to produce
RANTES (24, 25); binding sites for TNF have been previously
identified in swine granulo- sa cells (32). In this study, this
cytokine increased the number of cells that stained for RANTES and the
amount of RANTES in cultures. Conversely, adding neutralizing
anti-TNF antibody abolished RANTES immunostaining in granulosa cells
and decreased the amount of RANTES released into the culture
supernatants. This finding suggests that endogenous production of
TNF was required for the production of RANTES by granulosa cells.
TNF p55 receptors were detected by RT-PCR and visualized in granulosa
cells by flow cytometry. These receptors may mediate the effects of
TNF. The possibility that RANTES production is mediated by adenylate
cyclase/protein kinase A signal transduction was tested by incubating
cells with 8-Br-cAMP. Results suggest that the gonadotropins,
which are released in high amounts just before ovulation and act on
granulosa cells via an increase in cAMP, probably do not induce RANTES
production. On the other hand, PMA increased the number of granulosa
cells staining for RANTES. The effect of PMA in granulosa cells is
rather complex (33). PMA is known to activate protein kinase C (PKC)
(34). In contrast, prolonged treatment may also down-regulate
phorbol-sensitive isozyme (35). We previously tested the effect of PMA
in human granulosa luteinizing cell cultures; PMA treatment decreased
progesterone release by 2- to 3-fold, and this inhibitory effect was
antagonized by staurosporine (36). This finding suggested that PMA
activates PKC under our experimental conditions. Signal transduction
after TNFR activation may involve PKC (37, 38). The PKC pathway has
been implicated in TNF-mediated effects in Leydig cells (39) and
ovarian thecal cells (40). It may also be a critical component in TNF
activation of nuclear factor-B (NF-B) (41). Like PMA, TNF may
activate NF-B through PKC (42, 43). B-like sequences have been
identified within RANTES promoter (44). All of these considerations
suggest that TNF may mediate RANTES production in granulosa cells
via the activation of PKC and NF-B, although this remains to be
demonstrated. Indeed, further experiments quantifying PKC by Western
block should be conducted to confirm these data.
TNF is found in preovulatory follicular fluid at concentrations
similar to that used in the present study (23), and this cytokine
presumably accounts for RANTES production in the human preovulatory
follicle. TNF might directly activate the production of RANTES by
granulosa cells via TNFR. In addition, it might induce the production
of factors activating RANTES production. Preovulatory events prompt
massive ovarian infiltration by white blood cells. Paving the way are
the mast cells, which play a critical role in initiating the ovulatory
process. The preovulatory ovary contains numerous mast cells, and the
release of granules from these cells probably initiates the chemotactic
response (4). Mast cells alone contain TNF in their granules in a
form that they can release immediately (45, 46). The preovulatory LH
surge causes ovarian mast cells to granulate and release histamine and
TNF. This cytokine may activate RANTES production by granulosa
cells. In addition, RANTES is a very potent activator of mast cells and
leads to their degranulation (22). This suggests an autoamplified
mechanism between RANTES production and the progression of the
ovulatory process. Next to arrive are eosinophils and T lymphocytes,
followed in quick succession by the phagocytic monocytes. RANTES is a
chemoattractant for these three types of immune cells. It is likely to
contribute to their local recruitment. By producing RANTES, granulosa
cells may contribute to the influx of immune cells from vessels into
the ovulatory follicle, where they accumulate and contribute to the
ovulation process and to the formation of the corpus luteum. Further
studies identifying RANTES in human ovarian tissues would support these
hypotheses.
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Acknowledgments |
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Received February 19, 1999.
Revised May 24, 1999.
Revised September 10, 1999.
Accepted October 3, 1999.
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References |
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TopAbstractIntroductionMaterial and MethodsResultsDiscussionReferences |
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untransformed swine granulosa cells. Endocrinology. 129:641–648. induces interleukin-6 mRNA and
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inhibition of 17-hydroxylase/C17–20 lyase gene
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