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Departments of Molecular and Integrative Physiology (H.M., B.L., G.D., M.J.S.) and Anatomy and Cell Biology (J.S.H.), University of Kansas Medical Center, Kansas City, Kansas 66160; the Department of Biomedical Sciences, Ontario Veterinary College, University of Guelph (B.A.C.), Guelph, Ontario, Canada N1G 2W1; and the Green Center for Reproductive Biology Sciences, University of Texas Southwestern Medical Center (J.R.H.), Dallas, Texas 75235-9051
Address all correspondence and requests for reprints to: Michael J. Soares, Ph.D., Department of Molecular and Integrative Physiology, University of Kansas Medical Center, 39th and Rainbow Boulevard, Kansas City, Kansas 66160. E-mail: msoares{at}kumc.edu
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26 mice, and the specific interaction of PLP-A
with a rat NK cell line, RNK-16. We have further demonstrated that
PLP-A effectively suppresses RNK-16 cell cytolytic activities. Our
results provide evidence for a new paradigm of embryonic-maternal
communication involving a PLP-A signaling pathway between trophoblast
cells and uterine NK lymphocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have implemented a strategy of tagging members of the uteroplacental PRL family with alkaline phosphatase (AP). These AP-cytokine fusion proteins have facilitated the identification of target tissues for members of the uteroplacental PRL family (3). In the present report we begin to explore the biology of one member of the PRL family referred to as PRL-like protein A (PLP-A). PLP-A is expressed by trophoblast cells at the ontogeny of chorioallantoic placenta formation (4, 5, 6, 7, 8). PLP-A is considered a nonclassical PRL family member, in that it does not use the PRL receptor (1, 9). At present, the target cells and actions of PLP-A are unknown.
Using the AP-tagging strategy, we show that PLP-A specifically interacts with a population of natural killer (NK) lymphocytes within the mesometrial compartment of uteri from pregnant rats and mice. NK cells participate in immune surveillance, where they effectively recognize and kill virally infected and transformed cells and secrete an array of bioeffector molecules (10, 11, 12). NK cells are conspicuous residents of the uterus of rodents and humans during pregnancy (11, 13, 14, 15). The distribution and phenotype of uterine NK cells change from implantation to midgestation (11, 13). At mid-gestation in the mouse and rat, the mesometrial decidual region is characterized by the presence of a prominent population of NK cells (11, 13, 16). These uterine NK cells are juxtaposed to trophoblast cells of the developing chorioallantoic placenta and are distinct in their cytokine and bioeffector secretory profiles and their relative absence of cytolytic activities (17, 18, 19, 20, 21). Our data indicate that PLP-A is a candidate regulator of the pregnancy-associated uterine NK cell phenotype.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Animals and tissue preparation
Holtzman rats were obtained from Harlan Sprague Dawley, Inc. (Indianapolis, IN). A breeding colony of transgenic tg26
mice (22) was maintained in the barrier-sustained environment of the
isolation unit at the University of Guelph. Timed pregnancies and
pseudopregnancies and tissue dissections were performed as previously
described (23, 24, 25, 26). The presence of a copulatory plug or sperm in the
vaginal smear was designated day 0 of pregnancy. The first day of
pseudopregnancy was defined as the first day of leukocytic vaginal
smears after stimulation. Deciduomal responses were induced in
pseudopregnant rats by the injection of 50–100 µl sesame oil/uterine
horn on day 4 of pseudopregnancy (25). Protocols for the care and use
of animals were approved by the University of Kansas animal care and
use committee.
Cell culture
Human fetal kidney 293 cells were used as hosts for the
expression of AP fusion proteins. 293 cells were routinely maintained
in MEM supplemented with 20 mM HEPES, 100 U/ml penicillin,
100 µg/ml streptomycin, and 10% FBS. Some aspects of NK cell biology
were evaluated in the rat NK cell line, RNK-16 (27, 28). RNK-16 cells
were maintained in RPMI 1640 culture medium supplemented with HEPES (10
mM), L-glutamine (2 mM), sodium
pyruvate (1 mM), nonessential amino acids, 50
µM 2-mercaptoethanol, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 10% FBS. YAC-1 cells were used as targets for
cytotoxicity assays and were maintained in RPMI 1640 culture medium
supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and
10% FBS. CHO cells were used as hosts for the PRL receptor expression
plasmid and were routinely maintained in DMEM/MCDB-302 culture medium
containing 1 mM proline, 100 U/ml penicillin, 100 µg/ml
streptomycin, and 10% FBS.
Generation and characterization of the AP-PLP-A fusion
protein
A fusion protein consisting of a modified human placental AP
(PLAP) and rat PLP-A was generated and used to monitor PLP-A target
cell interactions. Procedures for generating the fusion protein were
similar to those previously reported for the generation of an AP-PL-I
fusion protein (3). A vector containing ampicillin and neomycin
resistance genes and a secreted version of PLAP (SEAP) (29) situated
downstream of a cytomegalovirus (CMV) promoter (pCMV/SEAP) was
commercially obtained (Tropix). A nucleotide region representing the
mature rat PLP-A protein was then amplified and ligated into the
pCMV/SEAP vector. Ligation with the PLP-A insert resulted in a CMV
promoter-driven vector containing the ligated cDNAs encoding a
SEAP-PLP-A fusion protein (AP-PLP-A; see Fig. 1
). DNA sequencing of the insert was
performed to verify the accuracy of the PCR amplification. After
linearization with BglII, the AP-PLP-A construct was
electroporated into 293 human fetal kidney cells. After a 2-week
selection with 500 µg/ml G418, single clones were isolated by
limiting dilution and were screened for AP expression. An unmodified
pCMV-SEAP vector (AP) was similarly transfected, selected, and served
as a negative control. Transfected 293 cells were cultured in MEM
supplemented with 20 mM HEPES, 100 U/ml penicillin, 100
µg/ml streptomycin, and 10% FBS in an atmosphere of 5%
CO2-95% air at 37 C in a humidified incubator. After the
cells reached confluence, the medium was changed to serum-free MEM and
HEPES, further conditioned for 72 h, collected and clarified by
centrifugation, sterile filtered (0.22 µm), and stored at -20 C
until used. AP activity was measured from conditioned medium via a
colorimetric assay (3). Western blot analysis for PLP-A was performed
as previously described (9). AP-PLP-A preparations were isolated from
conditioned medium using immunoprecipitation with monoclonal antibodies
to AP conjugated to agarose (Sigma Chemical Co.). Native
PLP-A isolated from junctional zone conditioned medium and recombinant
PLP-A isolated from 293 cells were used as controls for the Western
blotting (8, 9). PRL-like biological activities were assessed through
the use of the rat Nb2 lymphoma cell proliferation assay (30, 31) and
through incubation with CHO cells transiently transfected with the long
form of the rat PRL receptor (32).
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Identification of PLP-A in the midgestation rat conceptus
PLP-A messenger RNA was detected in frozen tissue sections from
the rat day 9 conceptus using previously described procedures (33). The
full-length rat PLP-A cDNA (4) was linearized and used as a template
for the synthesis of 35S-labeled sense and antisense RNA
probes. PLP-A protein was identified by immunocytochemistry in rat day
9 conceptus tissue sections as previously described (8).
Immunocytochemical localization of gp42
The protein, gp42, is expressed on the surface of
interleukin-2-activated rat NK cells and can be specifically recognized
with the monoclonal antibody, 3G7-E (34, 35). The gp42 protein was
localized in frozen tissue sections of uteri from pregnant and
pseudopregnant/decidualized rats using the 3G7-E monoclonal antibody
and a streptavidin-biotin immunoperoxidase kit for mouse IgG (33, 34).
The specificity of the immunoreactions was demonstrated using nonimmune
mouse IgG.
Immunomagnetic isolation of rat uterine NK cells
The 3G7-E monoclonal antibody to gp42 and magnetic cell sorting
with antimouse IgG MicroBeads (Miltenyi Biotech, Auburn,
CA) were used to isolate NK cells from uteri of pregnant and
pseudopregnant/decidualized rats. Decidual cells were dissociated from
mesometrial decidua isolated on day 10 of gestation or from whole
deciduoma isolated on day 10 of pseudopregnancy. Decidual tissue was
minced with fine scissors and dispersed in Dispase (2.4 U/ml)
containing deoxyribonuclease I (80 U/ml) for 1 h at 37 C.
Dispersed cells were recovered by centrifugation and washed with HBSS
to remove residual Dispase. Red blood cells were lysed via incubation
of the cell suspensions with 10 vol lysis buffer (155 mM
NH4Cl, 10 mM NaHCO3, and 0.1
mM EDTA) for 5 min at room temperature. Cells were
recovered by centrifugation and washed twice in 10 vol PBS containing 2
mM EDTA and 0.5% BSA (isolation buffer). The cell
preparation was adjusted to 107 cells/100 µl and
incubated with anti-gp42 (34) for 10 min at 5 C with continual mixing.
Cells were then washed twice with 10 vol isolation buffer, recovered by
centrifugation, resuspended at a final concentration of 107
cells/100 µl, and incubated with antimouse IgG MicroBeads
(Miltenyi Biotech) for 15 min at 5 C. After incubation,
the cells were washed, resuspended in a final volume of 500 µl
isolation buffer, and loaded onto a Magnetic Separation Plus column
(Miltenyi Biotech) affixed to a magnet. The column was
washed three times with isolation buffer (500 µl each), and the
eluting cells were collected (gp42-immunonegative fraction). The column
was then removed from the magnet, and cells were eluted in 1 ml
isolation buffer (gp42-immunopositive fraction). Aliquots of cells from
both fractions were adhered to glass slides and processed for PLP-A
binding.
Cytotoxicity assays
NK cell cytolytic activities were monitored by 51Cr
release assay (36). YAC-1 target cells were incubated for 1 h at
37 C with 200 µCi 51Cr. Radiolabeled YAC-1 target cells
were washed three times and then incubated with RNK-16 cells to
evaluate killing activity. The two populations of cells were
coincubated at various ratios at 37 C, and after 4 h the amount of
51Cr released into the culture medium was measured with a
-counter. Cells were exposed to medium conditioned by 293 cells mock
transfected or transfected with a PLP-A expression vector, or they were
exposed to an enriched preparation of recombinant PLP-A (purity, >90%
by PAGE). Target cells in medium alone served as spontaneous release
controls, and target cells in Triton X-100 served as maximal release
controls. Spontaneous release values were subtracted from the test and
maximal release values to calculate specific release. Data are
presented as the mean ± SEM percentage of maximal
killing.
Statistical analysis
Data were analyzed by ANOVA. The source of variation from
significant F ratios was determined with Student’s two-tailed
t test (37).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). The AP-PLP-A construct
and an unmodified AP control vector were transfected via
electroporation into 293 cells. The presence of AP activity was
evaluated in conditioned medium from transfected cells and
nontransfected cells using a colorimetric assay. Anti-PLP-A antibodies
specifically recognized the AP-PLP-A fusion protein. AP and the
AP-PLP-A fusion protein were initially enriched by immunoprecipitation
with a monoclonal antibody to human PLAP conjugated to agarose. PLP-A
antibodies recognized an AP-PLP-A protein species approximating 110 kDa
and native PLP-A protein species of 29 and 33 kDa (Fig. 1B). The
110-kDa Mr of the AP-PLP-A fusion protein was consistent
with the predicted Mr of its AP and PLP-A components. The
AP control preparation was not recognized by the PLP-A antibodies. The AP-PLP-A fusion protein was also evaluated for its ability to activate the PRL receptor signaling system. Both AP-PLP-A and AP failed to stimulate the proliferation of rat Nb2 lymphoma cells (data not shown) and failed to bind to CHO cells transiently transfected with the long form of the rat PRL receptor (data not shown). In contrast, AP-PL-I controls effectively stimulated rat Nb2 lymphoma cell proliferation and interacted with the PRL receptor (data not shown). These observations are consistent with earlier reports that PLP-A does not activate the PRL receptor signaling pathway (9).
Identification of PLP-A targets with the AP-PLP-A fusion
protein
We next examined the binding of AP-PLP-A to uteroplacental
tissues during various stages of gestation. AP-PLP-A specifically bound
to a population of cells situated in the midgestation mesometrial
compartment of the deciduum immediately overlying the developing
chorioallantoic placenta (Fig. 2, A–C).
This distinct binding pattern was first observed on day 9 of gestation
in the rat, earlier than reported for PLP-A expression (4, 5, 6).
Consequently, we examined the expression of PLP-A messenger RNA by the
rat day 9 conceptus and observed strong hybridization in developing
trophoblast (Fig. 2, D and E). Similar localization results were
obtained with PLP-A immunocytochemistry. Thus, the expression of the
PLP-A ligand coincided with the appearance of cells capable of binding
PLP-A.
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), and the binding of
AP-PLP-A was specifically competed by the addition of PLP-A (Fig. 2C),
but not by coincubation with ovine PRL or bovine GH (data not
shown).
The distribution of PLP-A binding in the midgestation rat conceptus was
reminiscent of the distribution of NK cells in rat and mouse
mesometrial decidua (Fig. 2F) (13, 14, 16, 38). To clarify this
relationship, we compared PLP-A binding with the localization of rat NK
cells using a monoclonal antibody to gp42, a protein expressed on the
surface of activated rat NK cells (34, 35). gp42 has been previously
shown to be expressed on differentiated rat uterine NK cells (13).
Similar distributions of PLP-A binding and gp42 immunoreactivity were
observed in decidual tissues from days 9–11 of pseudopregnancy or
pregnancy and in the metrial gland from day 14 of pregnancy (Fig. 3). The number of gp42-positive cells
always outnumbered the number of AP-PLP-A-binding cells, especially on
days 9–11 of pregnancy or pseudopregnancy. Of particular interest was
the association of gp42-positive cells and PLP-A-binding cells with the
vasculature of the mesometrium and the developing metrial gland (Fig. 3). In specimens from pregnant females, uterine NK cells radiated
dramatically from developing trophoblast cells and were prominently
associated with mesometrial vasculature, whereas NK cells developing in
the absence of trophoblast were more randomly dispersed throughout the
mesometrial compartment.
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). Pseudopregnant/decidualized
uteri proved to be a richer source of gp42-positive cells than
mesometrial decidua from conceptuses.
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26 mice (22) and a rat NK cell
line, RNK-16 (27, 28). AP-PLP-A specifically bound to cells in the
mesometrial compartment of controls, but not in the NK cell-deficient
conceptuses (Fig. 5). Finally, AP-PLP-A
specifically bound to cells of the rat NK cell line, RNK-16 (Fig. 5).
The extent of binding to RNK-16 cells was variable and may relate to
their differentiation state.
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Effects of PLP-A on NK cell cytotoxicity
NK cells are components of natural/innate immunity and participate
in immune surveillance through their targeting of virus-infected cells,
tumor cells, and potentially other cells for destruction without prior
sensitization (10, 11, 12). The phenotype of uterine NK cells during
pregnancy is characterized by their reduced capacity for killing (11, 17, 18, 19, 39). Thus, we examined the effects of PLP-A on killing activity
of RNK-16 cells toward YAC-1 cells. PLP-A significantly inhibited
RNK-16 cell killing in a dose-dependent manner (Fig. 6) without directly affecting the
viability or proliferation of either RNK-16 or YAC-1 cells. The data
are consistent with the participation of PLP-A in the regulation of the
pregnancy-specific uterine NK cell phenotype.
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Discussion |
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Uterine NK cells have been proposed to participate in immunological adjustments to pregnancy and in the establishment of the chorioallantoic placenta (13, 14, 38). During pregnancy, NK cells undergo considerable morphological and functional changes, creating, in effect, a NK cell with a unique phenotype. Midgestation uterine NK cells are distinct in their cytokine and bioeffector secretory profile and their relative absence of cytolytic activities (17, 18, 19, 20, 21). Further evidence from genetically mutant mice possessing defective cytolytic activities clearly indicate that NK cell cytolytic activities are not required for a successful pregnancy (41, 42). However, there is some evidence suggesting that aberrant uterine NK cell cytolytic activity is associated with pregnancy termination (43, 44, 45, 46, 47). Presumably, dysregulated maternal uterine NK cells target genetically foreign extraembryonic and embryonic cells for destruction. Specific cause and effect relationships between uterine NK cells and pregnancy termination have not been established. Although NK cell cytolytic activities are not required for normal progression of pregnancy, it is apparent from studies on NK cell-deficient mice that noncytolytic actions of NK cells are essential for normal uterine vasculature and chorioallantoic placental development (48, 49, 50). Thus, it is evident that uterine NK cell transformation into a pregnancy-dependent phenotype is vital for normal progression of the gestational state. Some of the maturational changes accompanying the pregnancy-dependent uterine NK cell phenotype occur in the absence of extraembryonic and embryonic tissues (51, 52, 53), whereas other changes are probably dependent upon modulators produced by extraembryonic sources. The nature of the modulators controlling the pregnancy-dependent uterine NK cell phenotype is yet to be resolved.
Several years ago, Parr and co-workers postulated the idea of PRL or PRL-related proteins arising from decidual or placental tissues as regulators of uterine NK cells (54). They proposed that uteroplacental PRL family members participated in transforming uterine NK cells into their pregnancy-specific phenotype. Indeed, ligands for the PRL receptor are secreted throughout gestation via either decidual (decidual luteotrophin) or trophoblast (PL-I, PL-II, and PL-I variant) tissues (1, 55). Furthermore, PRL is known to interact with and influence an array of different immune cells, including NK cells (56, 57). The surfaces of NK cells possess PRL receptors (58), and PRL appears to synergize with interleukin-2 to promote NK cell activation, including killing activities (59). Such actions are not entirely consistent with the midgestation uterine NK cell phenotype, which exhibits limited cytolytic activities (17, 18, 19). Another PRL family member investigated in the present report, PLP-A, is a new candidate regulator of uterine NK cell function during pregnancy. PLP-A specifically binds to uterine and some nonuterine NK cells and can inhibit the cytolytic activities of rat NK cells (present study). PLP-A binding appears to be best associated with NK cell activation, as demonstrated by rat uterine NK cells expressing the activation surface protein, gp42. In further support of a regulatory role for PLP-A during pregnancy, trophoblast cell expression of PLP-A coincides with the appearance of proximally located NK cells with diminished cytolytic activities (Refs. 4, 5, 6, 7, 8, 19 and the present study). Whether PLP-A participates in the modulation of uterine NK cell killing or other aspects of the pregnancy-dependent uterine NK cell phenotype remains to be determined.
The nature of PLP-A’s actions on NK cells is not known. PLP-A does not interact with the PRL receptor (Ref. 9 and the present study). Consequently, PLP-A actions are probably mediated by alternative signaling pathways. It is not entirely clear whether specific and unique receptor signaling systems have coevolved with each PRL family member, whether PRL family members use signaling pathways for other known ligands, or whether PRL family members act through receptor-independent mechanisms (e.g. transport proteins, binding proteins, etc.). PGs have been implicated in mediating pregnancy-associated changes in the uterine NK cell phenotype (47, 60, 61) and may represent an intracellular mediator of PLP-A action.
In summary, using the AP-tagging strategy, PLP-A is shown to specifically interact with a population of NK cells within the mesometrial compartment of the pregnant uterus. NK cells comprise part of the maternal immune surveillance with potent cytolytic activities and represent a rich source of bioeffector molecules. Appropriate modulation of uterine NK cells is critical for the establishment of vascular connectivity between mother and embryo without immunological rejection of the embryo. We propose that PLP-A participates in the regulation of uterine NK cells during pregnancy. Our results provide evidence for a new paradigm of embryonic-maternal communication involving a PLP-A signaling pathway between trophoblast cells and uterine NK cells.
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Acknowledgments |
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Footnotes |
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2 Supported by a fellowship from the Deutsche Forschungsgemeinschaft
of Germany (Mu 1183/1–1). Present address: Department of Obstetrics
and Gynecology, University of Rostock, Doberaner Strasse 142, 18057
Rostock, Germany.
Received December 8, 1998.
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1
µg/ml; purity, >90% by PAGE). Note that control cells killed to a
level of 36%, whereas NK cells exposed to PLP-A showed diminished
killing. NK cells exposed to 100% 293-PLP-A conditioned medium
(293-PLP-A CM) significantly decreased killing (P
< 0.05), whereas exposure to the recombinant PLP-A preparation
diminished killing even more (P < 0.01). PLP-A did
not significantly affect the viability or proliferation rates of RNK-16
or YAC-1 cells (data not shown). CM, Conditioned medium.