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Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNAs overlap with
that of granzyme D–G mRNAs in the pregnant uterus. Finally, we
demonstrate that granzymes D–G are up-regulated by IL-2 and IL-15 in
primary cultures containing GMG cells. Taken together, these results
suggest that IL-2 and/or IL-15 may regulate GMG cell differentiation in
vivo, and that granzymes D–G may have different functions than
granzyme A during pregnancy. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine GMG cells belong to the NK cell lineage (3, 4, 5, 6, 7, 8), and an analogous cell type, the endometrial granulocyte, has been identified in humans (9, 10, 11). In the mouse, GMG cells are localized to the decidua and metrial gland in early to mid-gestation and are confined to the metrial gland by late gestation. GMG cell differentiation begins at about day 7 of gestation and is manifested by the accumulation of cytolytic mediators including perforin and the serine proteases, granzymes A and B, within cytoplasmic granules (7, 12, 13, 14). The murine granzymes, A and B, belong to a large family of serine proteases that includes granzymes C, D, E, F, G, and H, metase-1, and tryptase-2 (15). Cytotoxic T cells and NK cells utilize perforin and granzymes to facilitate tumor and virus-infected cell death; however, the precise role of each granzyme has yet to be determined. Recent studies of pregnant transgenic mice deficient in NK and T cells (transgenic strain, TgE26) revealed small numbers of GMG cells, placentae half the size of control mice, and high rates of fetal loss after day 10. Histological analysis of implantation sites between days 10 and 17 revealed abnormalities in the establishment of placental circulation. These results suggest that GMG cell functions may not be important in early pregnancy events such as implantation, but they may be important in placental development (3, 4).
Although the specific functions of uterine GMG cells are unclear, they may regulate trophoblast invasion into the maternal decidua. Indeed, trophoblast killing by murine and human uterine NK cells has been reported (16, 17). Other previously proposed GMG cell functions include 1) lysis of virus-infected cells present in the uterus and placenta, 2) initiation of abortion, 3) destruction of the extracellular matrix and cells at the placental/uterine interface to promote parturition, 4) nutritive functions, and 5) cytokine production (1, 18).
Several cytokines have been implicated in perforin and granzyme gene regulation in both T and NK cells, including IL-2, IL-6, IL-7, IL-12, and IL-15 (19, 20, 21, 22, 23, 24, 25). Recently, IL-15 has been shown to up-regulate perforin and granzyme A–B expression in GMG cell explants (26).
Understanding the mechanisms governing GMG cell differentiation during
pregnancy will ultimately provide a better understanding of their
function and relevance to pregnancy success. In this study, expression
of the genes coding for granzymes D, E, F, and G (granzymes D–G) was
found to be developmentally regulated in murine GMG cells during
pregnancy. Granzymes D–G were shown to be expressed in late gestation,
in contrast to the mid-gestational expression of granzyme A. This
finding suggests different roles for granzymes D–G compared with
granzyme A during pregnancy. Expression of the transcripts coding for
the IL-2Rß and IL-2R was detected in the uterus at the time when
granzymes D–G are expressed. Finally, granzymes D–G were shown to be
up-regulated by both IL-2 and IL-15 in minced primary cultures
containing GMG cells. Therefore, IL-2 and/or IL-15 may regulate GMG
cell differentiation in vivo.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Pregnant CF-1 mice were used. The morning a vaginal plug was observed was designated as day zero of pregnancy. For RNA isolation, uterine tissues removed from days 1 to 6 postcoitum also contained embryonic and placental tissue. Uterine tissues from days 7 to 19 of pregnancy were prepared with the embryos and placentae removed.
Cytokines, Abs, and cDNA clones
Recombinant human IL-15 and recombinant mouse IL-2 were
purchased from R&D Systems (Minneapolis, MN). Rabbit polyclonal
anti-mouse granzyme D–G serum was a gift from Dr. Jürg
Tschopp (Université de Lausanne, Lausanne, Switzerland). This Ab
was prepared against granzyme D but recognizes all four granzymes, D–G
(27). Horseradish peroxidase-conjugated protein A was
purchased from Sigma (St. Louis, MO). The mouse IL-2R
and IL-2R
cDNAs were gifts from Dr. Warren Leonard (National Institutes of
Health, Bethesda, MD). The mouse granzyme A cDNA was a gift from Irving
Weissman (Stanford University, Stanford, CA). The rat
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was cloned by
Fort et al. (28).
Day 11 mouse uterine-specific cDNA library construction
Polyadenylated RNA was obtained from day 11 and day 18 mouse uterine total RNA using Invitrogen Fast Track mRNA isolation reagents (Invitrogen, San Diego, CA). A subtractive library specific for day 11 uterine mRNA was constructed from day 11 and day 18 uterine poly(A)+ RNA using the PCR Select cDNA Subtraction Kit (Clontech, Palo Alto, CA) and the T-cloning vector, pT7Blue (Novagen, Madison, WI). The day 11 uterine specific cDNA library contained approximately 10,000 primary recombinants.
cDNA library screen using WSXWS oligonucleotides as probes
The day 11 mouse uterine-specific cDNA library was screened with
degenerate oligonucleotides encoding the conserved WSXWS motif of the
type I cytokine receptors (WSXWS oligo-1: (A/G)CTCCAGGN(A/G)CTCCA and
WSXWS oligo-2: (A/G)CTCCANTC(A/G)CTCCA) (29). Briefly, the
library was plated onto 150-mm Luria-Bertani (LB) agar plates
containing 50 µg/ml ampicillin and 15 µg/ml tetracycline at a
density of 15,000 colonies/plate. Colonies were replica plated onto
137-mm, 0.45-micron nitrocellulose filters (Microcon Separations,
Westboro, MA), grown at 37°C overnight and incubated for an
additional 8 h on LB agar plates containing 50 µg/ml
chloramphenicol. The bacterial colonies were then lysed and the DNA
immobilized by baking at 90°C for 90 min in a vacuum oven. The
filters were washed briefly in 6x SSC (1x SSC = 150 mM NaCl/15
mM sodium citrate, pH 7) and prehybridized at 37°C overnight in
prehybridization buffer (6x SSC, 2 mg/ml BSA, 2 mg/ml Ficoll, 2 mg/ml
polyvinyl pryrrolidone, 100 µM ATP, 10 µg/ml yeast tRNA, 2 mM
sodium pyrophosphate, 2 mg/ml salmon sperm DNA, and 0.1% Nonidet P-40
(NP-40)). The WSXWS oligonucleotides (250 pmol) were phosphorylated
with T4 polynucleotide kinase (Promega, Madison, WI) and 250 µCi
[-32P]ATP (4500 Ci/mmol) to a specific activity of
about 108 cpm/µg. Unincorporated ATP was removed using an
STE Select-D G25 spin column (5 Prime
3 Prime, Boulder, CO). The
oligonucleotides were hybridized at 37°C and 1 x
106 cpm/ml for 28 h in the prehybridization buffer
containing 0.1% SDS instead of 0.1% NP-40. The filters were washed
once for 10 min at 24°C in 6x SSC, twice for 30 min at 45°C in 6x
SSC/0.1% SDS, and twice for 20 min at 45°C in 0.5x SSC/0.1% SDS.
The filters were exposed to film for 48 to 96 h at -70°C with
intensifying screens, and then the films were developed. Colonies
chosen from the primary screen were subjected to secondary and tertiary
screens before pure clones were isolated and sequenced. Sequence
analysis was performed with the University of Wisconsin Genetics
Computer Group Sequence Analysis Software Package.
cDNA library screen for genes expressed at mid-gestation but not late gestation in the uterus
The day 11 mouse uterine specific cDNA library was plated as in
the WSXWS screen. Prehybridization was carried out at 42°C overnight
in 4.8x SSC, 48% formamide, 2 mg/ml BSA, 2 mg/ml Ficoll, 2 mg/ml
polyvinyl pryrrolidone, 10% dextran sulfate, 0.1% SDS, 10 µg/ml
yeast tRNA, and 20 mM Tris, pH 7.6. Day 11 and day 18 mouse uterine
cDNAs that had been previously synthesized for subtractive library
construction were labeled with [-32P]dCTP using the
Multiprime DNA Labeling System (Amersham, Arlington Heights, IL) to a
specific activity of 2 to 4 x 109 cpm/µg. The
unincorporated nucleotides were removed using an STE Select-D G25 spin
column (5 Prime3 Prime), the probes were each boiled for 10 min with
2 mg/ml salmon sperm DNA, placed on ice for 5 min, and then hybridized
to duplicate library filters for 16 h at 42°C. The filters were
washed three times for 15 min at 24°C in 2x SSC/0.1% SDS and twice
for 20 min at 53°C in 0.2x SSC/0.1% SDS. Filters were exposed to
film for 60 h at -70°C with intensifying screens, and then the
films were developed. Clones that hybridized in duplicate to the day 11
but not the day 18 uterine cDNA probe were selected and rescreened
until pure cDNA clones were isolated. cDNA clones were sequenced and
analyzed as in the WSXWS screen.
RNA isolation and Northern blot analysis
Total RNA was isolated from frozen tissues using Trizol reagent
(GIBCO–Life Technologies, Grand Island, NY). For Northern blot
analysis, 20 to 30 µg of total RNA was separated by electrophoresis
on a 1% agarose gel containing formaldehyde and transferred to Zeta
Probe-GT cationized nylon membranes (Bio-Rad, Hercules, CA).
32P-labeled cDNA probes were synthesized using the
Multiprime DNA Labeling System (Amersham) and hybridized at 47°C for
16 to 20 h in 7% SDS, 1% polyethylene glycol (m.w.
15,000–20,000), 40% formamide, 0.1% NP-40, 0.6 M NaCl, 5 mM EDTA,
100 µg/ml yeast tRNA, 100 µg/ml salmon sperm DNA, and 40 mM sodium
phosphate, pH 7.4. Following hybridization, the membranes were washed
twice at 24°C in 0.5x SSC/0.1% SDS and twice at 47°C in 0.2x
SSC/0.1% SDS, each for 15 min. RNA transcripts were visualized by
autoradiography and quantitated by using ImageQuant software on a
PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Quantitative values
for the amount of mRNAs encoding IL-2Rß, IL-2R, granzymes D–G,
and granzyme A were normalized to that obtained for GAPDH in each
sample.
Diagnostic RT-PCR analysis of granzymes D–G mRNAs
For RT-PCR, total RNA from day 13 uterus or from the primary minced cell cultures was used. Primers of the following sequences were used: granzyme I primer, TTTAAC(T/A)CCTGTTAGAGCA; granzyme II primer, CTCT(C/T)(G/A)GAGCTGGAGCA.
Reverse transcription was performed under the following conditions: 0.175 µg/µl total RNA, 0.5 pmol/µl granzyme I primer, 1 U/µl RNasin (Promega), 0.5 mM dNTPs, 10 mM DTT, 10 U/µl Superscript II reverse transcriptase (GIBCO-Life Technologies), 75 mM KCl, 3 mM MgCl2, 50 mM Tris-HCl, pH 8.3. The reaction was allowed to proceed for 50 min at 42°C, and then the reverse transcriptase was inactivated for 15 min at 70°C. The reaction mix was stored at -20°C until needed.
PCR was performed on 15% of the reverse transcription reaction under
the following conditions: 0.8 pmol/µl each of granzyme I and granzyme
II primers, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.1% Triton X-100,
50 mM KCl, 10 mM Tris-HCl, pH 9.0. The reaction mix was heated at
94°C for 3 min, and then 0.05 U/µl Taq polymerase
(Promega) was added to commence thermal cycling; 30 cycles were
performed at 94°C for 45 sec, 56°C for 1 min, and 72°C for 1 min.
One final cycle was performed at 72°C for 7 min. In some cases, a
trace amount of [-32P]dCTP (0.1 µCi/µl) was added
to the PCR mixes to label the products.
Following the PCR, the samples were precipitated in ethanol and resuspended in water. Restriction analysis was performed with Bg1II, BstXI, or HindIII/PstI (Promega), and the DNA was resolved by electrophoresis on a 1.75% agarose gel containing 0.5 µg/ml ethidium bromide. For 32P-labeled PCR products, the gels were incubated in 10% TCA for 1 h, dried under vacuum, and exposed to PhosphorImager screens. The various DNA fragment intensities were determined using ImageQuant software on the PhosphorImager.
To determine the relative levels of granzyme D–G mRNAs, the specific DNA band intensities from the PhosphorImager were divided by the corresponding restriction fragment size to obtain relative molar equivalents for all bands. The relative molar equivalents were then divided by the sum of all molar equivalents in the gel lane to estimate the fraction of the total represented by each granzyme present.
Primary minced tissue culture
Primary cell cultures were generated similarly to Ye et al.
(26). Cultures were generated from day 8 of gestation and
contained the entire implantation site. Briefly, the implantation sites
(including the embryo, placenta, and uterus) were dissected from the
animal and minced into four to six pieces, rinsed in TBS, and cultured
in MEM (GIBCO-Life Technologies), 2% FCS, 20 mM HEPES (pH 7.4), and
10 U/ml penicillin/streptomycin. For some experiments IL-15 and/or IL-2
were added to a final concentration of 300 ng/ml, and the cultures were
maintained at 37°C for 24 h (26). After the 24-h
incubation, samples were frozen in liquid nitrogen and stored at
-70°C prior to RNA isolation.
Western blot analysis
Protein was isolated from tissues by homogenization at 4°C in 0.15 M NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml PMSF, 57 µg/ml aprotinin (Sigma), 15 mM sodium phosphate, pH 7.4. After a 30-min incubation at 4°C, the tissue debris was removed by centrifugation at 14,000 x g for 20 min at 4°C. Protein concentrations of the tissue lysates were determined using the Bradford assay. For Western blots, equal amounts of tissue lysate were resolved by SDS-PAGE on 7.5 to 15% gradient gels and transferred to nitrocellulose (NitroBind, Micron Separations). The antigranzyme DEFG serum was used at a dilution of 1:250 and was detected with horseradish peroxidase-conjugated protein A (Sigma) and ECL Western blotting detection reagents (Amersham).
Immunohistochemistry
Entire implantation sites from various days of gestation were fixed in 4% paraformaldehyde for 24 h at 4°C, embedded in paraffin, and cut into 5-µm sections. The sections were incubated with antigranzyme D–G serum (1:500), and the Abs were visualized using an anti-immunoglobulin-peroxidase stain (VectaStain ABC, Vector Laboratories, Burlingame, CA). Sections were then counter stained with periodic acid Schiff’s (PAS) stain and/or hematoxylin.
PAS reaction
Following immunohistochemistry, sections were treated with the following series of reagents: 0.5% periodic acid, 15 min at room temperature; 10 min under running water; 15 min at 4°C in Schiff’s reagent (26 mM sodium bisulfite, 15 mM basic fuschin, 0.15 M HCL); 20 sec under running water; 1 min in 2% sodium bisulfite; 5 min in water. The sections were then dehydrated to xylene, mounted with Permount (Fisher Scientific, Pittsburgh, PA) and viewed on a light microscope (Zeiss).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To identify genes expressed at mid-gestation but not late gestation in the uterus, a day 11 uterine-specific cDNA library was constructed and screened as follows. The library was probed with radiolabeled cDNAs derived from day 11 and day 18 uterine mRNA, and clones hybridizing to the day 11 uterine probe but not the day 18 probe were selected. In the primary library screen, 26 clones were selected, and 12 of these were analyzed in a secondary screen. Following the secondary screen, 4 of the 12 clones were determined to be differentially expressed in the uterus. Three of these four clones encoded partial cDNAs approximately 600 bp in length that were 98% identical to the previously identified mouse granzyme G cDNA (EMBL/GenBank accession number J02872) (31). The remaining clone encoded a full-length cDNA that was 99.7% identical to mouse granzyme D (EMBL/GenBank accession number J03255) (32).
Temporal expression of granzyme D–G mRNAs in the pregnant mouse uterus
To determine if the genes coding for granzymes D and G were
temporally regulated in the uterus during pregnancy, RNA isolated from
uteri at various stages throughout gestation was analyzed by Northern
blots. The granzyme D cDNA was used as a probe because it would
presumably detect both the homologous mRNA and that coding for granzyme
G, owing to the 88% identity between these sequences
(31). The mRNA transcripts were detected in the uterine
samples between days 9 and 16 of pregnancy with peak expression between
days 13 and 15 (Fig. 1
). In contrast, the
uterine expression of granzyme A was observed between days 8 and 13 and
peaked on day 9 (Fig. 1). RNA isolated from uterine samples from days 1
through 5 were also screened for granzymes A and D–G. The levels of
granzyme mRNAs in these tissues were the same or lower than those
determined for uteri isolated from day 7 of gestation.
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), even
under high stringency conditions, presumably would hybridize to
granzymes D, E, F, and G. To determine if all four of these transcripts
are expressed during pregnancy, RT-PCR was performed and combined with
a diagnostic restriction analysis. Briefly, primers were designed that
would amplify granzymes D–G but not granzymes A–C. Restriction
analysis, with BstXI, BgIII, or a combination of
HindIII/PstI, was used to distinguish the mRNAs
encoding granzymes D–G. Figure 2 shows
the results of the RT-PCR analysis on day 13 uterine RNA, and it
demonstrates that all four granzymes, D, E, F, and G, are expressed.
All four granzymes were also detected in day 8 uterine RNA samples (not
shown). Therefore, the Northern blot analysis shown in Figure 1
represents the combined gestational profile of granzymes D–G.
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Cytolytic mediators such as perforin, granzyme A, and granzyme B
have been localized to uterine GMG cell granules during pregnancy
(7, 12, 13, 14). Examination of implantation sites by
immunohistochemistry with an antigranzyme D–G serum, revealed that
granzymes D–G are also expressed in GMG cells (Fig. 3). Figure 3C shows intense
granzyme immunoreactivity on day 15 of pregnancy in a large population
of decidual cells but little staining in the placenta. The intense
staining was also observed in the metrial gland located just above the
decidua (metrial gland, not shown). Traditionally, GMG cells have been
identified by their morphology and granular PAS staining (33, 34). The granzymes D–G stain was colocalized with the PAS stain
in the sections analyzed (Fig. 3, B–D), thus
demonstrating that these proteins are expressed in the GMG cells. In
addition, the granzymes D–G protein expression pattern in the uterus,
as a function of gestation, was consistent with that obtained by
Northern blot analysis. Granzymes D–G were detected between days 9 and
17 by Western blot analysis and peaked between days 13 and 15 (Fig. 4). A similar temporal expression of
granzymes D–G as observed by Western blot analysis was observed by
immunocytochemical staining of GMG cells at different stages of
pregnancy (Fig. 3; data from day 11, 13, and 17; data not shown).
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in the uterus during gestation
A second library screen was performed to isolate cDNAs encoding
type I cytokine receptors expressed in the mid-gestational uterus. In
this screen, the day 11 uterine-specific library was probed with
oligonucleotides encoding the conserved WSXWS motif found in this
family of receptors (35). Two different oligonucleotides
were used, WSXWS-1 and WSXWS-2 (see Materials and Methods).
Using the WSXWS-2 oligonucleotide, a partial IL-2Rß (
130 bp) cDNA
was isolated. This clone was 99% identical to the previously
identified mouse cDNA sequence (EMBL/GenBank accession number
M28052).
The expression of IL-2Rß mRNA was examined in uterine tissues over
the course of gestation and determined to be temporally regulated (Fig. 5). The IL-2Rß mRNA was detected
between days 8 and 15 and reached its highest level on day 9. In
addition, the period of IL-2Rß mRNA expression overlapped with the
expression of granzyme A and granzymes D–G mRNA (Figs. 1 and 5).
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, IL-2Rß, and IL-2R) and an intermediate
affinity dimeric receptor (IL-2Rß and IL-2R). The IL-15 receptor
consists of IL-2Rß and IL-2R with an additional IL-15 subunit
that has been demonstrated to exist in the uterus (26).
Therefore, uterine tissues were analyzed by Northern blot to determine
the expression patterns of the IL-2R and - subunits during
pregnancy. The IL-2R mRNA was undetectable in the uterus during
gestation (not shown). However, the IL-2R subunit mRNA was present
throughout gestation and increased between days 7 and 13 similarly to
the IL-2Rß subunit (Fig. 5). IL-2 and IL-15 induction of granzyme D–G expression in primary cultures containing GMG cells
The fact that the receptor subunits utilized in IL-2 and IL-15
signaling are present in the uterus at the time when granzymes D–G are
expressed suggested that IL-2 and/or IL-15 may regulate granzyme D–G
expression in GMG cells. To examine this possibility, primary minced
implantation sites from day 8 of gestation were cultured with IL-2
and/or IL-15, and the amount of granzyme D–G mRNAs present after
24 h was determined. A 2- to 5-fold increase in granzyme D–G
mRNAs was observed in the primary cultures incubated with IL-15,
whereas cultures treated with IL-2 showed a 3- to 6-fold increase (Fig. 6). The combination of both cytokines
resulted in a stimulation that was approximately equal to that obtained
with either cytokine alone (not shown). To eliminate possible
influences of cells in the cultures other than the decidual cells, the
effects of IL-2 and IL-15 were also tested in cultures containing the
decidua alone. The effects of IL-2 and IL-15 on granzyme D–G mRNAs
were similar in the cultures containing only decidual cells compared to
those containing entire implantation sites. In an experiment in which
the response to IL-15 of complete implantation sites and isolated
deciduae were tested in triplicates, the former showed a 3.6-fold
stimulation over the control and the latter showed a 5-fold stimulation
over the control value.
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. When the
relative amounts of the various granzymes in each sample were compared
between stimulated and unstimulated samples, there was no significant
difference in the relative amounts of any of the granzyme mRNAs in
response to either IL-2 or IL-15 (Fig. 7). Thus, we conclude that IL-2 and IL-15
equally increase the expression of granzyme D–G mRNAs.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Many investigators have proposed that GMG cells may lyse placental trophoblasts invading the maternal decidua. Controlling the extent of trophoblast invasion is extremely important to the maintenance of pregnancy, because in humans diminished invasion may result in problems such as pre-eclampsia and still birth, whereas extensive invasion may cause maternal death due to uterine rupture (37). The granzyme A and B expression patterns are well timed with respect to the period of trophoblast invasion, and perhaps they play a role in these events. GMG cells only have lytic activity between days 6.5 and 9.5 of gestation (38), and this lytic activity is coincident with granzyme A and B expression and with the period of trophoblast invasion.
The late gestational expression of granzymes D–G suggests that these serine proteases may not be regulators of trophoblast invasion. Parturition, which requires considerable tissue remodeling, occurs between 19 and 21 days of gestation in the mouse. Although there is no experimental evidence supporting regulated granzyme secretion from GMG cells, Croy et al. (39) proposed that granzymes may be released in late gestation to eliminate the extracellular matrix and cells at the uterine/placental interface to promote parturition. Experimental evidence in support of this hypothesis was recently demonstrated. Delgado et al. (40) showed that GMG cells began to undergo degenerative changes from day 12 through parturition. Furthermore, degenerating GMG cells rupture and release their granule contents in late gestation (39, 40).
In this study, granzyme D–G proteins were detected between days 9 and 17 in the uterus. Mesometrial decidual regression begins at about day 10 and continues until birth (2, 41). Although they are generally believed to be involved in cell-mediated cytotoxicity, there is some evidence that granzymes can also participate in other events (42). It is intriguing to consider that granzymes D–G released from degenerating GMG cells in late gestation may prepare the feto-maternal interface for parturition.
The mechanisms governing GMG cell differentiation during pregnancy are
not well understood, and determining the signaling pathways required
for their differentiation may ultimately lead to a better functional
understanding of these cells. In this study, Northern blot analysis of
uterine tissues revealed that the IL-2Rß mRNA is temporally regulated
during pregnancy. Although mouse uterine IL-2Rß expression has been
reported in the past (26, 43), its quantitative mRNA
profile over the course of gestation has not previously been reported.
The IL-2Rß subunit was shown to be expressed between days 8 and 16,
with peak expression on day 9. This expression pattern, overlapping
with that of granzymes D–G, prompted us to investigate whether IL-2
and/or IL-15, the only cytokines known to bind the IL-2Rß, regulate
granzyme D–G expression and therefore GMG cell differentiation.
Because IL-2 and IL-15 signaling occurs through several heteromeric
receptors, uterine tissues from various days of gestation were analyzed
for the additional receptors that might be involved, the IL-2R and
IL-2R. By Northern blot analysis, the IL-2R mRNA was undetectable
throughout gestation. In agreement with this finding, Ye et al.
(26) also did not detect the IL-2R mRNA by RT-PCR.
However, low levels of IL-2R have been reported in the murine and
human uterus by other investigators (43, 44, 45). The IL-2R
mRNA was present throughout gestation and increased at mid-gestation
following a similar pattern to what was observed for the IL-2Rß mRNA.
Together, these experiments provide evidence that intermediate affinity
IL-2 receptors (IL-2Rß/IL-2R) are present in the mouse uterus at
mid-gestation.
Ye et al. (26) reported mid-gestational expression of
mouse IL-15R in the uterus, which they detected by RT-PCR analysis.
Combined with our results of the IL-2Rß and IL-2R mRNA expression,
these results suggest that an IL-15R composed of the IL-15R,
IL-2Rß, and IL-2R may exist in the uterus at mid-gestation.
To determine whether IL-15 and/or IL-2 regulate granzyme D–G expression in GMG cells, primary minced tissue cultures that contained GMG cells were treated with both of these cytokines individually or together. GMG cells represent the predominant immune effector cell population in the rodent uterus during pregnancy (38). Therefore, any effect on granzyme D–G expression in these primary minced cultures are likely to be a response of GMG cells. IL-15 and IL-2 stimulated granzyme D–G expression to similar levels in day 8 primary implantation cultures. The combination of IL-2 and IL-15 was not synergistic, suggesting that IL-2 and IL-15 may use the same signal transduction pathways in GMG cells.
In addition to Northern blot analysis, diagnostic RT-PCR was performed on RNA isolated from the primary cultures to determine if granzymes D–G were differentially regulated by IL-2 and IL-15. The analysis showed no change in the relative proportions of individual granzyme mRNAs after addition of IL-15 or IL-2. This suggests that the 2- to 6-fold increase in granzyme D–G mRNAs observed on Northern blots was due to the cumulative increase in the mRNAs encoding all four granzymes.
Ultimately, the in vivo regulation of perforin, granzymes A and B, and granzymes D–G gene expression will depend on the presence of IL-2 and IL-15 in the local GMG cell environment. Expression of IL-2 mRNA has been reported in the human and murine placenta (46, 47, 48) and in human decidual and T cells (49, 50). Interestingly, the placentae of mice that are prone to high rates of spontaneous fetal resorptions contain elevated IL-2 mRNA (47). This is in agreement with other reports showing that elevated IL-2 levels have deleterious effects on pregnancy (51). Our results suggest that uterine GMG cells may be IL-2 targets in vivo. IL-2 is widely recognized as a regulator of T and NK cell activation and most activated T and NK cells express cytolytic mediators. In analogy, elevated IL-2 expression may aberrantly activate GMG cells to a highly lytic state resulting in extensive cell lysis and abortion.
IL-15 mRNA expression has also been reported in the placenta and uterus during pregnancy (26, 52). While IL-2 is expressed mainly in T cells, IL-15 has been detected in a wide variety of cells and tissues (52). This recently identified cytokine has been shown to function similarly to IL-2 in many instances (53, 54), whereas it is expressed in many different cell types and probably has functions outside the immune system. For example, IL-15 can act as an anabolic agent in muscle cells (55). Therefore, although our studies and the results of others show that IL-15 may regulate GMG cell differentiation (26), it may have additional effects on GMG cells during pregnancy as well as distinct effects on specific granzymes.
Although the in vivo regulators of GMG cell differentiation remain to be elucidated, the primary cultures in this study provide strong evidence that IL-2 and/or IL-15 may be involved. In addition, the distinct expression patterns of granzyme A and granzymes D–G provide further evidence that granzymes may have several functions throughout pregnancy. Ultimately, determining granzyme functions during pregnancy may provide a better understanding of normal placentation, uterine tissue remodeling, and fetal/maternal interactions in general.
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Acknowledgments |
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Footnotes |
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2 Present address: Department of Medicine, Division of Endocrinology, University of Colorado Health Sciences Center, 4200 East 9th Ave., Denver, CO 80262.
3 Address correspondence and reprint requests to Dr. Marit Nilsen-Hamilton, 3206 Molecular Biology Building, Department of Biochemistry and Biophysics, Iowa State University, Ames, IA 50011–3260. E-mail address:
4 Abbreviations used in this paper: GMG, granulated metrial gland; PAS, periodic acid Schiff’s stain; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NP-40, Nonident P-40.
Received for publication December 1, 1997. Accepted for publication May 19, 1998.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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 A. Mallakin, L. W. Kutcher, S. A. McDowell, S. Kong, R. Schuster, A. B. Lentsch, B. J. Aronow, G. D. Leikauf, and S. E. Waltz Gene Expression Profiles of Mst1r-Deficient Mice during Nickel-Induced Acute Lung Injury Am. J. Respir. Cell Mol. Biol., January 1, 2006; 34(1): 15 - 27. [Abstract] [Full Text] [PDF]
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 C. Tayade, Y. Fang, G. P. Black, P. VA Jr, A. Erlebacher, and B. A. Croy Differential transcription of Eomes and T-bet during maturation of mouse uterine natural killer cells J. Leukoc. Biol., December 1, 2005; 78(6): 1347 - 1355. [Abstract] [Full Text] [PDF]
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N. Takano, H. Matusi, and T. Takahashi Granzyme N, a Novel Granzyme, Is Expressed in Spermatocytes and Spermatids of the Mouse Testis Biol Reprod, December 1, 2004; 71(6): 1785 - 1795. [Abstract] [Full Text] [PDF]
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 J. T. Fassett and M. Nilsen-Hamilton Mrp3, a Mitogen-Regulated Protein/Proliferin Gene Expressed in Wound Healing and in Hair Follicles Endocrinology, May 1, 2001; 142(5): 2129 - 2137. [Abstract] [Full Text]
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 T. A. Fehniger and M. A. Caligiuri Interleukin 15: biology and relevance to human disease Blood, January 1, 2001; 97(1): 14 - 32. [Full Text] [PDF]
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 H. Okada, T. Nakajima, M. Sanezumi, A. Ikuta, K. Yasuda, and H. Kanzaki Progesterone Enhances Interleukin-15 Production in Human Endometrial Stromal Cells in Vitro J. Clin. Endocrinol. Metab., December 1, 2000; 85(12): 4765 - 4770. [Abstract] [Full Text]
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J. T. FASSETT, R. T. HAMILTON, and M. NILSEN-HAMILTON Mrp4, A New Mitogen-Regulated Protein/Proliferin Gene; Unique in this Gene Family for its Expression in the Adult Mouse Tail and Ear Endocrinology, May 1, 2000; 141(5): 1863 - 1871. [Abstract] [Full Text] [PDF]
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 S. Okada, H. Okada, M. Sanezumi, T. Nakajima, K. Yasuda, and H. Kanzaki Expression of interleukin-15 in human endometrium and decidua Mol. Hum. Reprod., January 1, 2000; 6(1): 75 - 80. [Abstract] [Full Text] [PDF]
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0.05; IL-15
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