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Regular Article |
¶
From the Departments of Stomatology,*
Obstetrics,
Gynecology and Reproductive Sciences,
Pharmaceutical Chemistry,
and
Anatomy,¶
University of California at San
Francisco, San Francisco, California; and the Division of
Cardiology,
Department of Medicine, Duke
University, Durham, North Carolina
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Limiting immune cell access to the
fetal allograft may be another mechanism involved in maternal
tolerance. Accumulating evidence supports this hypothesis. First, a
large and specific population of immune cells, termed decidual
leukocytes, infiltrates the pregnant uterus. These immune cells account
for at least 15% of all cells in the decidualized uterine wall and are
identifiable from early pregnancy through term (Yan Zhou, University of
California at San Francisco, personal communication). The
decidual leukocyte population is unique, being composed predominately
(
70%) of an unusual type of NK cell
(CD56bright/CD16-) with contributions from T
cells (15%) and monocytes (15%).4,5
These cells
express the activation markers Fas, CD69, and CD71.6-8
However, their cytolytic capacity is limited in comparison to that of
peripheral NK cells.9
Taken together, these special
features of decidual leukocytes imply that maternal tolerance relies,
at least in part, on the presence of a selected population of immune
cells at the maternal-fetal interface.
Second, by comparison to other systems,10 it is likely that the specificity of decidual leukocyte composition is governed at the level of cell trafficking. This has been demonstrated in the mouse, where microdomains of differentially expressed adhesion molecules involved in cell homing have been identified within the pregnant uterus. This expression is functionally correlated with the distinct localization of neutrophils, monocytes, and NK cells to different portions of the uterus. Accordingly, each leukocyte subset expresses adhesion molecules that interact with the endothelial counterreceptors in its respective microdomain.11
Finally, although the contribution of chemokines to the recruitment of decidual leukocytes has not previously been considered in the human, these molecules are required for effective leukocyte homing in other systems12 and likely play an analogous role during pregnancy. To date, only interleukin-8, MCP-1, and RANTES have been localized to the pregnant human uterus,13,14 and the possible effects of this expression on decidual leukocytes have not been investigated.
Therefore, this report advances the literature in three ways. First, it presents the expression patterns of a panel of 14 chemokines in the decidua. The results show that both fetal and maternal tissues are richly endowed with chemokines. Next, it examines chemokine receptor expression by decidual leukocytes. Taken together, the data identify potential ligand-receptor pairs involved in leukocyte trafficking to the pregnant uterus. Finally, the chemokine expression patterns it describes suggest the possibility of additional, nonimmune roles for these molecules in placentation and cytotrophoblast differentiation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Informed consent was obtained from all patients from whom tissue or blood was collected. Placental and decidual tissue from elective termination of pregnancy (6 to 22 weeks) or from normal term delivery (34 to 40 weeks) was collected within 1 hour of isolation, washed thoroughly in phosphate-buffered saline (PBS) with antibiotics, and placed on ice. Tissue to be used for in situ hybridization was immediately put in 10% buffered formalin. After overnight fixation, tissue was transferred to 70% ethanol and embedded in paraffin for sectioning.
Buffy coats used as a source of peripheral blood mononuclear cells (PBMCs) were obtained from Blood Centers of the Pacific (San Francisco, CA). No information was available regarding the sex, age, or pregnancy status of the blood donors.
Cell Isolation
Cytotrophoblasts
Cells were isolated from pooled first- or second-trimester human placentas by published methods.15,16 Briefly, placentas were subjected to a series of enzymatic digests that detached cytotrophoblast stem cells from the underlying stromal core of the chorionic villus. Detached cytotrophoblasts were purified over a Percoll gradient and cultured on Matrigel-coated substrates (Collaborative Biomedical Products, Bedford, MA) for various lengths of time in serum-free medium: Dulbecco’s modified Eagle’s medium, 4.5 g/L glucose (Sigma Chemical Co., St. Louis, MO) with 2% Nutridoma (Boehringer Mannheim Biochemicals, Indianapolis, IN), 1% penicillin/streptomycin, 1% sodium pyruvate, 1% Hepes, and 1% gentamicin (UCSF Cell Culture Facility).
Placental Fibroblasts
Placental fibroblasts were isolated from first-trimester placentas as previously described.15 Cells were cultured in Dulbecco’s modified Eagle’s medium H-21 with 10% fetal bovine serum, 5% glutamine, 1% penicillin/streptomycin, and 1% gentamicin.
Isolation of Human PBMCs
Human PBMCs were prepared from a buffy coat by erythrocyte sedimentation with 6% dextran T500 (Amersham Pharmacia Biotech AB, Uppsala, Sweden) followed by Histopaque-1077 (Sigma) separation. PBMCs were washed in PBS and extracted for RNA.
Preparation of Decidual Granulated Leukocytes
Decidua was minced with a razor blade, then incubated at 10 ml/g of tissue in RPMI-1640 (UCSF Cell Culture Facility) containing 10% fetal bovine serum, 0.1% collagenase (Sigma), 0.02% DNase I (Boehringer Mannheim), and 0.02% ethylenediaminetetraacetic acid (EDTA) (Sigma) for 1 hour at room temperature. After this digestion, the supernatant was removed and centrifuged. The cell pellet was resuspended in Hanks’ buffered saline solution, layered over Ficoll-Paque (Pharmacia Biotech AB, Uppsala, Sweden) and centrifuged for 30 minutes at 900 x g. The band of decidual leukocytes at the interface was removed, washed three times in PBS, and extracted for RNA.
In Situ Hybridization
In situ hybridization was performed using published methods.17 Tissue specimens to be used were fixed with 10% formalin in PBS at room temperature overnight, washed twice in PBS, dehydrated through a graded ethanol series, cleared in xylene, and embedded in paraffin. Sections (5 µm) were cut and mounted on poly-L-lysine-coated slides, deparaffinized, and rehydrated before use.
The EST plasmid clones with the identification numbers 490222 (BRAK),
140701 (fractalkine), 505324 (GCP-2), 1147321 (GRO
), 209366 (HCC-1),
491243 (IP-10), 1058310 (ITAC), 488534 (MCP-1), 322873 (MCP-3), 484740
(MIG), 154848 (MIP1), 183073 (SDF-1), and 503192 (SLC) were used as
templates for probe synthesis. Because these plasmids were constructed
in the same way, identical methods could be used in preparing each
probe. Plasmids were linearized on one side of the gene insert with
either EcoRI or NotI (Life Technologies, Inc.,
Rockville, MD). The linearized vectors were used as templates
for the synthesis of 35S-RNA probes using T3
(antisense) or T7 (sense) RNA polymerase.18
On day 1 of the in situ hybridization, slides were allowed
to sit at room temperature for 5 minutes. Then they were placed in PBS
for 5 minutes before incubation at room temperature in 4%
paraformaldehyde in PBS for 10 minutes, followed by a 5-minute wash in
0.5x standard saline citrate (SSC) (1x = 150 mmol/L NaCl, 15 mmol/L
sodium citrate, pH 7.4). Sections were deproteinated with 1 µg/ml
proteinase K for 10 minutes at room temperature, then washed for 10
minutes in 0.5x SSC. Then they were fixed again in 4%
paraformaldehyde for 3 minutes, followed by a final wash in 0.5x SSC
for 5 minutes. After a 3-hour prehybridization at 55 to 60°C in rHB2
buffer [50% formamide, 0.3 mol/L NaCl, 20 mmol/L Tris, pH 8.0, 5
mmol/L EDTA, 1x Denhardt solution (0.02% Ficoll, 0.02%
polyvinylpyrrolidone, 0.02% bovine serum albumin), 10% dextran
sulfate, 10 mmol/L dithiothreitol], the slides were incubated
overnight at 55 to 60°C in a humidified chamber with 200 µl of
hybridization buffer: rHB2 buffer containing 500 µg/ml yeast tRNA and
1,200,000 cpm of 35S-labeled antisense
(experimental) or sense (negative control) cRNA probes. After
hybridization, sections were washed twice, 10 minutes each, in 2x SSC
with 10 mmol/L of ß-mercaptoethanol and 1 mmol/L of EDTA. Slides were
then immersed in an RNase A solution (500 mmol/L NaCl, 10 mmol/L Tris,
pH 8.0, and 10 µg/ml RNase A) for 30 minutes at room temperature and
washed twice, 10 minutes each, in 2x SSC/ß-mercaptoethanol/EDTA.
Then the sections were subjected to a high stringency wash (0.1x SSC,
10 mmol/L ß-mercaptoethanol, 1 mmol/L EDTA) for 2 to 3 hours at
60°C before they were washed twice, 10 minutes each, in 0.5x SSC
without ß-mercaptoethanol or EDTA. Finally, the sections were
dehydrated (2 minutes/step) in a series of graded (30, 60, 80, 95, and
100%) ethanol solutions that contained 0.3 mol/L
NH4Ac. The slides were dried for 2 hours in a
fume hood before being dipped in Kodak NTB-2 liquid emulsion
(Eastman Kodak Co., Rochester, NY). Afterward they were dried overnight
in the dark, boxed, and stored at 4°C until they were developed. The
sections were stained with hematoxylin and eosin (H&E) before they were
examined in both dark and bright field using a Zeiss Axiophot
microscope. To report the data in a tabular format, results were
translated into a scoring system based on the density of white dots
that composed the signal. Designations were as follows: ++, very
intense to intense signal; +, moderately intense to weak signal; -,
extremely weak or absent signal. Various human tissues (Novagen Inc.,
Madison, WI) served as positive controls: BRAK, colon adenocarcinoma
(catalog no. 70337-3); GCP-2, endocrine tissue set (catalog no.
70320-3); IP-10, ovary serous tumor (catalog no. 70392-3); ITAC, lung
bronchoalveolar carcinoma (catalog no. 70367-3); MCP-1, hematal and
immune tissue set (catalog no. 70321–3); MCP-3, lung bronchoalveolar
carcinoma (catalog no. 70367-3); MIG, malignant melanoma (catalog no.
70417-3); SLC, lymph node tumor (catalog no. 70375-3); SDF-1, human
tissue set 1 (catalog no. 70312-3); and GRO, malignant melanoma
(catalog no. 70417-3).
Northern Hybridization
Total RNA was extracted from cytotrophoblasts immediately after the cells were isolated or after 12, 24, or 36 hours in culture, according to published methods.19 RNA was also collected from whole placental villi, and fibroblasts were collected as described above. Ten µg of total RNA was separated by formaldehyde-agarose gel electrophoresis, transferred to Nytran membranes (Schleicher and Schuell, Inc., Keene NH), and analyzed by Northern blot hybridization as previously described.20-22 Gels were stained with acridine orange before transfer to ensure integrity of the RNA samples and to confirm equal loading. Probes for the chemokines BRAK, GCP-2, and SLC were generated using standard methods.23 [32P]CTP, random oligonucleotide primers (Amersham Life Science, Inc., Piscataway, NJ) and the Klenow fragment of DNA polymerase I (Life Technologies, Inc.) were used to label the probes, which had a specific activity of 2 x 109 dpm/µg.
RNase Protection Assay
Total RNA from decidual granulated leukocytes was prepared as described above. RiboQuant multiprobe RNase protection assay kits were purchased from Pharmingen and used according to the manufacturer’s instructions. Briefly, three probe sets (hCR5, hCR6, and hCR8), encompassing a total of 17 receptors, were labeled with 32P and hybridized to RNA (from 2 to 20 µg) from decidual leukocytes. After RNase digestion, protected bands were resolved on sequencing gels and identified by size, using undigested probe as a reference size marker. Positive controls included probes for L32 and GAPDH transcripts, to confirm RNA integrity and loading. Yeast and PBMC RNA were additional controls for expression.
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Results |
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Figure 1
depicts the anatomy of the
region where the maternal and fetal compartments come together. With
regard to the fetal compartment, the placenta is composed of two
classes of chorionic villi—floating and anchoring—that share many of
the same histological features. Both have mesenchymal villus cores that
contain fetal blood vessels, surrounded by a layer of cytotrophoblast
stem cells that fuse to form a syncytium covering the villus surface.
But only anchoring villi form specialized structures termed
columns—aggregates of mononuclear cytotrophoblasts that bridge the gap
between the maternal and fetal compartments by attaching to, and then
invading, the uterus. Initially, this invasion is primarily
interstitial, ie, through the uterine parenchyma. Numerous fetal
cytotrophoblasts are thus distributed throughout the maternal
compartment as isolated cells or cell clusters. As invasion continues,
a subset of cytotrophoblasts targets uterine arteries, remodeling
these vessels and replacing the maternal endothelial lining.
This process happens to a lesser degree in uterine veins as well.
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. This
article focuses on chemokine expression and action in the uterine wall,
ie, decidual stroma, decidual leukocytes, uterine vessels, and invasive
fetal cytotrophoblasts. Another paper (P. Drake, K Red-Horse, MD Gunn,
SJ Fisher, in preparation) will detail chemokine expression in
the fetal compartment, ie, the placenta proper.
We first addressed the issue of chemokine expression by localizing mRNA
production in tissue sections by in situ hybridization.
Figures 2 through 6
present
representative hybridization patterns with antisense probes for the
14 chemokines assessed in this study. On the left of each figure are
bright-field micrographs of histological sections stained with H&E to
show morphology, and on the right are dark-field micrographs of the
same sections to show areas of probe hybridization, visualized by white
dots. The results are summarized in Table 1
. In general, chemokines were abundant
and intense mRNA signals were widely distributed among cell types. With
the exception of BRAK, which was regulated with gestation, expression
was remarkably stable throughout pregnancy, and in general the patterns
were highly reproducible. When not stated otherwise, the expression
pattern was found in the majority of samples examined and was
consistent throughout gestation. A few hybridization patterns were
shared by several chemokines; in these instances, the relevant figure
shows a representative example typical of all molecules within that
group. In every case, sense controls yielded no signal. For space
considerations, micrographs of these negative controls are shown in
only a portion of the figures.
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A set of chemokines shared a strong, evenly distributed expression
pattern, derived predominately from decidual stroma (Figure 2)
.
Fractalkine, HCC-1, ITAC, and GCP-2 were widely expressed throughout
the decidua. As the latter molecule was also produced by other cell
types, it is discussed below. For the remaining chemokines in this
group, decidual expression was not enhanced in areas of glandular
epithelial cells, isolated cytotrophoblasts, or leukocyte clusters,
although expression in these cell types could not be excluded. GRO
and MCP-3 (data not shown) were expressed in a similar pattern,
but with a weaker signal. Expression of MCP-3 was less consistent than
that of the other molecules (Table 1)
. Occasional samples did not fit
the dominant pattern. For example, ITAC expression specifically by
decidual leukocytes in addition to diffuse stromal production was
observed in only one of seven samples.
Foci of Chemokine Expression in the Decidua
As in Figure 2
, the panels in Figure 3
show second trimester decidua only.
Uterine blood vessels and leukocytes can be identified in some
sections. Antisense probes for MCP-1, MIP-1, and BRAK yielded a
strong punctate signal. This distribution pattern suggested expression
by the resident decidual leukocytes, particularly the macrophage subset
(MCP-1 and MIP-1), or by isolated patches of stromal cells (Figure 3
, BRAK, arrowhead) near clusters of decidual leukocytes (Figure 3
,
arrow). BRAK expression seemed to be gestationally regulated, as all
second trimester samples assessed were positive whereas term samples
were negative. MCP-1 and MIP-1 were also broadly expressed at a
lower level throughout the decidual stroma (data not shown).
Additionally, in one sample each, the chemokines MIG and SDF-1 were
expressed focally in the decidua.
The chemokine IP-10 displayed a distinctive expression pattern within
the maternal compartment. Figure 4
A
shows a section of first trimester decidua containing a cluster of
decidual leukocytes. These cell clusters are commonly associated with
uterine glands and were nearly always accompanied by a strong IP-10
signal, as visualized by in situ hybridization (Figure 4B
,
arrow). Although expression by leukocytes could not be excluded, at
least a portion of the signal was derived from the intercalated stromal
cells. This expression pattern was highly consistent throughout
gestation, as demonstrated by an example of hybridization signals at
term (Figure 4D)
. This sample includes numerous floating villi within
the fetal compartment (Figure 4
, left) and superficial decidua from the
maternal compartment (Figure 4
, right). Sections hybridized with the
sense probe lacked signal, as is shown on a second trimester decidual
sample (Figure 4F)
.
Cytotrophoblast Expression of Chemokines
Recently, we showed that cytotrophoblasts express
MIP-1.24
Here we report that invasive cytotrophoblasts
in the decidua also expressed the chemokines GCP-2, HCC-1, and SDF-1
(Figure 5)
. This expression pattern was
somewhat less consistent than others, in part because of the difficulty
in discriminating between cytotrophoblast- and stroma-derived signals.
Where decidual fibroblasts were replaced by deposits of extracellular
matrix, a common modification in late gestation, chemokine production
by cytotrophoblasts was unequivocal. Figure 5A
shows the morphology of
a section of second trimester decidua. In the upper part of the
section, stromal cells remain and GCP-2 expression can be seen
throughout this area (Figure 5B)
, as described above (Figure 2)
. Note
the additional production of this chemokine by cytotrophoblasts (Figure 5, A and B
, arrows). In contrast, the nearby decidual leukocytes do not
seem to be a source of GCP-2 (Figure 5, A and B
, arrowheads). HCC-1 had
a similar pattern of expression (Figure 5, C and D)
. The tissue section
focuses on an area within a second trimester decidual sample. Stromal
cells remain in the upper right side of the panel, but have been
replaced by extracellular matrix in the lower left side. Invasive
cytotrophoblasts can be seen within this matrix. Again, HCC-1
expression is apparent in the decidual stroma as described above
(Figure 2)
and in the fetal cytotrophoblast population (Figure 5D
,
arrows). Figure 5, E and F
, show a second trimester tissue section that
includes the fetal compartment (bottom). Groups of invasive
cytotrophoblasts at the decidual border are positive for the chemokine
SDF-1.
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Cells that lined a subset of vessels, which tended to be arteries,
also expressed the chemokine SDF-1. Figure 6A
shows two remodeled blood vessels
within second trimester decidua that were breached by fetal
cytotrophoblasts. The latter cells are clearly identifiable by
morphological criteria in their walls (arrows). In situ
hybridization demonstrated that these fetal cells strongly expressed
SDF-1 mRNA (Figure 6B)
; expression by resident maternal cells could not
be excluded.
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. Although expression by endovascular
cytotrophoblasts could not be excluded, clusters of these cells
within the walls of arteries did not hybridize with the antisense probe
(Figure 6D
, arrow). Sense probes yielded no signal; Figure 6F
shows a
representative example in second trimester tissue. Cultured Cytotrophoblasts Regulate Chemokine Expression with Differentiation
We confirmed the specificity of a subset of the in situ
hybridization probes by using them concurrently for Northern blot
hybridization. We compared expression by either first or second
trimester cytotrophoblasts with that observed in chorionic villi and
placental fibroblasts. For these studies, we took advantage of an
in vitro system to model cytotrophoblast differentiation in
which isolated purified cytotrophoblast stem cells recapitulate the
invasive differentiation pathway with time in culture.25
The Northern blot results dovetailed with the in situ
hybridization studies (Figure 7)
.
Specificity of the probes was further indicated as bands of the
appropriate size were detected. BRAK mRNA was detected in chorionic
villi, a result in agreement with in situ analyses in this
portion of the placenta (P. Drake et al, manuscript in
preparation). In cytotrophoblasts, a very faint signal was
detected in isolated cells before culture, but at no other time points
(n = 2). In contrast, GCP-2 mRNA was not
produced by chorionic villi, but was expressed in cytotrophoblasts
after 12 hours in culture (n = 2). This
suggested that in vivo these cells increase GCP-2 production
as they invade the uterine wall, an idea supported by the in
situ hybridization data (Figure 5B)
.
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As a second step in assessing the contribution of chemokines to
decidual leukocyte recruitment, we examined the expression of chemokine
receptors by these cells at the transcriptional level. Decidual
leukocyte RNA (n = 9) was analyzed for evidence
of chemokine receptor expression, using RNase protection assays. Three
probe sets, encompassing a total of 17 receptors, were used (Figure 8)
. Each probe set is shown as a panel
with the results of a representative experiment: hCR5 (Figure 8A)
, hCR6
(Figure 8B)
, and hCR8 (Figure 8C)
. Along the left side of each panel is
a list of the chemokine receptors identified by each set. Probes for
individual receptors are distinguished from each other by size, which
decreases from top to bottom. Across the top of each panel the source
of analyzed RNA is listed: yeast, PBMCs, and decidual leukocytes from
the gestational week indicated. Bands are indicative of chemokine
receptor mRNA. Protected bands have a slightly higher mobility than the
undigested probes run as size markers in the left-hand column. Lines
connect pairs of undigested and digested probes.
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. Receptor expression was remarkably
consistent among samples. Accordingly, CCR1 and CCR5 (Figure 8A)
,
CXCR3, CXCR4, and V28/CX3CR1 (Figure 8B)
, and the
orphan receptor STRL33 (Figure 8C
, along with additional examples of
CCR5, CXCR4, and V28/CX3CR1), were highly
expressed. CCR2a, CCR2b, CCR4 (Figure 8A)
, and CCR7 (Figure 8B)
expression was consistent, although slightly weaker. Occasional
expression of CXCR1, CXCR2, CXCR5 (Figure 8B)
, and CCR8 (Figure 8A)
was
noted. A few samples containing the minimum amount of RNA (2 µg)
yielded fewer bands, providing possible clues about the relative
expression levels of these factors in decidual leukocytes.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. These include
CCR1/HCC-1, CCR2/MCP-1, CCR5/MIP-1, CCR7/SLC, CXCR3/ITAC,
CXCR4/SDF-1, and CX3CR1/fractalkine. It is
interesting to note that all of the chemokines expressed in the uterus
find a partner receptor on decidual leukocytes.
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, MCP-1, HCC-1, and SLC) (see Table 3
). Conversely,
receptors that are expressed only on cell subsets not normally found in
the pregnant uterus, such as CCR3 on eosinophils, were not identified
in our screen. CXCR1 and CXCR2, typical of neutrophils, were either not
expressed (n = 3) or present at very low levels
(n = 2). The two positive samples may have
contained neutrophils that can infiltrate the placental bed in
pathological processes.26 Through a combination of models describing cell homing by a multistep combinatorial extravasation process,12 followed by a stepwise navigation of chemokine gradients,27 one can envision a series of molecules with sequential actions that recruit decidual leukocytes from the circulation and localize them within the uterus. Because the first step in leukocyte recruitment is extravasation from the blood stream, the chemokines expressed in the lumen of uterine vessels are particularly important for identifying the initial population of immune immigrants. Interestingly, these molecules, SLC and SDF-1, were previously characterized by their involvement in the homeostatic migration of cells through secondary lymphoid organs (SLC) and the localization of immature cells during development (SDF-1). Particularly striking, considering that CD56bright NK cells comprise 70% of decidual leukocytes, is the observation that SLC preferentially attracts these cells over the CD56dim subset. Expression of SLC on cells that compose the lumen of uterine vessels may therefore serve as a selective first step, allowing CD56bright cells to dominate in the decidua.
In the context of leukocyte extravasation, it is important to note that early in pregnancy, before the utero-placental circulation is established, blood flow at the maternal-fetal interface is distinct from elsewhere in the body. Before 10 weeks of gestation, uterine arteries are occluded with aggregates of invading cytotrophoblasts, blocking direct blood flow. Thus, the maternal blood reaching the placenta is restricted to capillary flow or plasma filtrate.28 Interestingly, decidual leukocytes are thought to be recruited during this period of low shear stress, but it is not known how these unusual conditions affect their extravasation. Previously defined rolling and tethering mechanisms may be altered in response to the different requirements in this location. Conversely, blood flow at the maternal-fetal interface becomes forceful later in gestation, measured in liters per minute at term.26 In response to the gestational alterations in circulation through the intervillous space, local chemokine actions may also change. For example, SLC and SDF-1 expressed on large-bore second trimester vessels may serve to target leukocytes for emigration and return to the maternal circulation.
Once extravasation is completed, cell migration through the web of chemokines expressed in the decidua may occur as described in other systems.27 Here, cells retain memory of their recent environment and can prioritize a new, weaker chemotactic signal over an older, stronger one. Accordingly, the cell can follow one attractant after another, using combinations of molecules to guide it in a step-by-step manner to its destination within a tissue. For example, a possible combination of factors acting on the migration of decidual leukocytes is: SLC/CCR7 to extravasate, HCC-1/CCR1 to move within the decidual stroma, and IP-10/CXCR3 to cluster the cells near glands. The mouse provides corollary evidence, whereby specific adhesion molecules that coordinate with chemokines to effect cell targeting are reciprocally expressed in pairs on discrete immune cell subsets and vascular endothelium in particular microenvironments within the pregnant uterus.11 This expression parallels the localization of these immune subsets to these microenvironments.
In terms of receptor expression, the decidual leukocytes resemble both Th1 and Th2 cohorts, although the stronger expression is toward the Th1 group. CCR5 and CCR7, common on Th1-polarized cells, are both highly expressed, whereas CCR4 and CCR8, preferentially expressed by Th2-polarized cells, are produced at lower levels. This finding is in agreement with previous work from our laboratory that reported the production of both Th1 and Th2 cytokines at the maternal-fetal interface.29,30 In summary, the majority of chemokine receptor expression by decidual leukocytes correlates with a Th1-biased population of activated T and NK cells. However, the presence of resting or naïve cells is also suggested (eg, CCR7 and CXCR4 expression). A flow cytometry-based investigation of chemokine receptor expression on subsets of decidual leukocytes would be most informative; by analogy to other systems, it could provide clues to cell function, as well as yield information about the mechanisms of leukocyte recruitment. Recently CD56bright NK cells from the peripheral blood were found to have a chemokine receptor repertoire distinct from that of CD56dim NK cells and NK-T cells.31 These data support our results, as the former cells express high levels of CCR5, CCR7, CXCR3, and CXCR4—molecules also expressed by decidual leukocytes, which are predominately composed of CD56bright NK cells. Accordingly, these receptors interact with ligands that are expressed in the decidua.
On a teleological note, it is interesting that many of the same receptor-ligand pairs expressed at the maternal-fetal interface are also expressed in rejected allogeneic organ grafts. For instance, rejected murine cardiac tissue expresses fractalkine, IP-10, MIG, and ITAC, which are absent in nonrejected grafts. The activated T-cell infiltrate expresses the receptors for these chemokines, CX3CR1 and CXCR3. When either of these receptors is blocked with a function-perturbing antibody, graft survival is significantly prolonged.32,33 Although we do not understand the exact meaning of these data, they suggest that novel immune mechanisms are entailed in the special case of fetal allo-transplantation during pregnancy, as a subset of chemokines and/or receptors that are indicative of transplant rejection is also expressed at the maternal-fetal interface.
Given the vital nature of reproduction, it is likely that redundancies
are built into its critical components. Our data suggest that this
principle applies to chemokine function. For example, several of these
molecules shared identical expression patterns at the maternal-fetal
interface (HCC-1, ITAC, fractalkine). Additionally, some of the
molecules expressed in similar locations share the same receptors (eg,
HCC-1 and MIP-1 each bind CCR1), suggesting overlapping functions.
Finally, our results are in agreement with gene deletion studies in
mice that show that the loss of individual chemokines or receptors does
not affect leukocyte composition in the pregnant uterus.34
In addition to the recruitment of decidual leukocytes, chemokine expression patterns in the uterine wall suggest that these molecules are involved in nonimmune aspects of placentation and cytotrophoblast differentiation. One intriguing possibility is the role of chemokines in cytotrophoblast targeting to maternal spiral arterioles. Part of the invasive differentiation pathway involves the localization of a subset of these fetal cells to maternal vessels. The mechanisms underlying this homing phenomenon are not well understood. The observation that uterine veins are only rarely breached by fetal cells led to the hypothesis that oxygen gradients help target the invasive cells preferentially toward arteries.35,36 The specific expression of SDF-1 by cells that occupy uterine vessels, including cytotrophoblasts, suggests that chemokines also regulate this process. Although traditionally considered to be regulators of leukocyte trafficking, the role of chemokines in the directed migration of other cells is gaining increasing acceptance as more examples are described. For instance, metastatic tumors up-regulate the expression of chemokine receptors, and this expression is involved in the preferential seeding of particular tumor cells to specific secondary organs.37 In this context, it will be interesting to identify chemokine receptors expressed by cytotrophoblasts, and to test the effects of exogenous chemokine application on their differentiation, which includes a novel switch of their adhesion phenotype such that these epithelial cells take on vascular- and tumor-like properties.38
Finally, these data give information about chemokine networks that function at the maternal-fetal interface during normal pregnancy. The fact that expression of chemokines and their receptors tended to be constant throughout pregnancy suggests important roles from the earliest time points onward. We hypothesize that deviations are indicative of underlying pathologies, and that understanding how chemokine expression changes in specific pregnancy complications will give insights regarding their etiology. Good candidates for initial studies are complications associated with inflammatory leukocytic infiltrates, such as preterm labor with infection of the chorioamniotic membranes. Work in the mouse has already shown that the placenta produces the chemokines KC and MIP-2 in response to Listeria monocytogenes infection.39 Future investigations combining disease models with other approaches will assess at multiple levels the biology and function of chemokines at the maternal-fetal interface.
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Acknowledgements |
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Footnotes |
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Supported by a grant from the National Institutes of Health (HL64597, to S. J. F.), The Sandler Family Supporting Foundation (to P. D.), a predoctoral fellowship from The University of California Tobacco-Related Disease Program (8DT-0176, to P. D.), and a National Institutes of Health National Institute of General Medical Sciences Minority Biomedical Research Support Research Initiative for Scientifc Enhancement graduate training fellowship (R25GM59298-03) through the San Francisco State University (to K. R.-H.).
Accepted for publication September 9, 2001.
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  M. S. M. van Mourik, N. S. Macklon, and C. J. Heijnen Embryonic implantation: cytokines, adhesion molecules, and immune cells in establishing an implantation environment J. Leukoc. Biol., January 1, 2009; 85(1): 4 - 19. [Abstract] [Full Text] [PDF]
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C. G. Peralta, V. K. Han, J. Horrocks, B. A. Croy, and M. J. van den Heuvel CD56bright cells increase expression of {alpha}4 integrin at ovulation in fertile cycles J. Leukoc. Biol., October 1, 2008; 84(4): 1065 - 1074. [Abstract] [Full Text] [PDF]
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 C.A. Gifford, A.M. Assiri, M.C. Satterfield, T.E. Spencer, and T.L. Ott Receptor Transporter Protein 4 (RTP4) in Endometrium, Ovary, and Peripheral Blood Leukocytes of Pregnant and Cyclic Ewes Biol Reprod, September 1, 2008; 79(3): 518 - 524. [Abstract] [Full Text] [PDF]
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N. J Hannan and L. A Salamonsen CX3CL1 and CCL14 Regulate Extracellular Matrix and Adhesion Molecules in the Trophoblast: Potential Roles in Human Embryo Implantation Biol Reprod, July 1, 2008; 79(1): 58 - 65. [Abstract] [Full Text] [PDF]
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 Y. Huang, X.-Y. Zhu, M.-R. Du, and D.-J. Li Human Trophoblasts Recruited T Lymphocytes and Monocytes into Decidua by Secretion of Chemokine CXCL16 and Interaction with CXCR6 in the First-Trimester Pregnancy J. Immunol., February 15, 2008; 180(4): 2367 - 2375. [Abstract] [Full Text] [PDF]
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 B. W. Whitcomb, E. F. Schisterman, M. A. Klebanoff, M. Baumgarten, A. Rhoton-Vlasak, X. Luo, and N. Chegini Circulating Chemokine Levels and Miscarriage Am. J. Epidemiol., August 1, 2007; 166(3): 323 - 331. [Abstract] [Full Text] [PDF]
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 M. Kallikourdis, K. G. Andersen, K. A. Welch, and A. G. Betz Alloantigen-enhanced accumulation of CCR5+ 'effector' regulatory T cells in the gravid uterus PNAS, January 9, 2007; 104(2): 594 - 599. [Abstract] [Full Text] [PDF]
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A.P. Hess, A.E. Hamilton, S. Talbi, C. Dosiou, M. Nyegaard, N. Nayak, O. Genbecev-Krtolica, P. Mavrogianis, K. Ferrer, J. Kruessel, et al. Decidual Stromal Cell Response to Paracrine Signals from the Trophoblast: Amplification of Immune and Angiogenic Modulators Biol Reprod, January 1, 2007; 76(1): 102 - 117. [Abstract] [Full Text] [PDF]
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Y. Hirota, Y. Osuga, K. Koga, O. Yoshino, T. Hirata, C. Morimoto, M. Harada, Y. Takemura, E. Nose, T. Yano, et al. The Expression and Possible Roles of Chemokine CXCL11 and Its Receptor CXCR3 in the Human Endometrium J. Immunol., December 15, 2006; 177(12): 8813 - 8821. [Abstract] [Full Text] [PDF]
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N. J. Hannan, R. L. Jones, C. A. White, and L. A. Salamonsen The Chemokines, CX3CL1, CCL14, and CCL4, Promote Human Trophoblast Migration at the Feto-Maternal Interface Biol Reprod, May 1, 2006; 74(5): 896 - 904. [Abstract] [Full Text] [PDF]
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 Y. Huang, X.-Y. Zhu, M.-R. Du, X. Wu, M.-Y. Wang, and D.-J. Li Chemokine CXCL16, a scavenger receptor, induces proliferation and invasion of first-trimester human trophoblast cells in an autocrine manner Hum. Reprod., April 1, 2006; 21(4): 1083 - 1091. [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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 E. Dimitriadis, C.A. White, R.L. Jones, and L.A. Salamonsen Cytokines, chemokines and growth factors in endometrium related to implantation Hum. Reprod. Update, November 1, 2005; 11(6): 613 - 630. [Abstract] [Full Text] [PDF]
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 K. Red-Horse, M. Kapidzic, Y. Zhou, K.-T. Feng, H. Singh, and S. J. Fisher EPHB4 regulates chemokine-evoked trophoblast responses: a mechanism for incorporating the human placenta into the maternal circulation Development, September 15, 2005; 132(18): 4097 - 4106. [Abstract] [Full Text] [PDF]
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X. Wu, L.-P. Jin, M.-M. Yuan, Y. Zhu, M.-Y. Wang, and D.-J. Li Human First-Trimester Trophoblast Cells Recruit CD56brightCD16- NK Cells into Decidua by Way of Expressing and Secreting of CXCL12/Stromal Cell-Derived Factor 1 J. Immunol., July 1, 2005; 175(1): 61 - 68. [Abstract] [Full Text] [PDF]
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 C. Dosiou and L. C. Giudice Natural Killer Cells in Pregnancy and Recurrent Pregnancy Loss: Endocrine and Immunologic Perspectives Endocr. Rev., February 1, 2005; 26(1): 44 - 62. [Abstract] [Full Text] [PDF]
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 M. A. Crane-Godreau and C. R. Wira CCL20/Macrophage Inflammatory Protein 3{alpha} and Tumor Necrosis Factor Alpha Production by Primary Uterine Epithelial Cells in Response to Treatment with Lipopolysaccharide or Pam3Cys Infect. Immun., January 1, 2005; 73(1): 476 - 484. [Abstract] [Full Text] [PDF]
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 N. J. Hannan, R. L. Jones, H. O. D. Critchley, G. J. Kovacs, P. A. W. Rogers, B. Affandi, and L. A. Salamonsen Coexpression of Fractalkine and Its Receptor in Normal Human Endometrium and in Endometrium from Users of Progestin-Only Contraception Supports a Role for Fractalkine in Leukocyte Recruitment and Endometrial Remodeling J. Clin. Endocrinol. Metab., December 1, 2004; 89(12): 6119 - 6129. [Abstract] [Full Text] [PDF]
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K. Kitaya, T. Nakayama, N. Daikoku, S. Fushiki, and H. Honjo Spatial and Temporal Expression of Ligands for CXCR3 and CXCR4 in Human Endometrium J. Clin. Endocrinol. Metab., May 1, 2004; 89(5): 2470 - 2476. [Abstract] [Full Text] [PDF]
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 L. Pereira, E. Maidji, S. McDonagh, O. Genbacev, and S. Fisher Human Cytomegalovirus Transmission from the Uterus to the Placenta Correlates with the Presence of Pathogenic Bacteria and Maternal Immunity J. Virol., December 15, 2003; 77(24): 13301 - 13314. [Abstract] [Full Text] [PDF]
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Y. Sato, T. Higuchi, S. Yoshioka, K. Tatsumi, H. Fujiwara, and S. Fujii Trophoblasts acquire a chemokine receptor, CCR1, as they differentiate towards invasive phenotype Development, November 15, 2003; 130(22): 5519 - 5532. [Abstract] [Full Text] [PDF]
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 J. Hanna, O. Wald, D. Goldman-Wohl, D. Prus, G. Markel, R. Gazit, G. Katz, R. Haimov-Kochman, N. Fujii, S. Yagel, et al. CXCL12 expression by invasive trophoblasts induces the specific migration of CD16- human natural killer cells Blood, September 1, 2003; 102(5): 1569 - 1577. [Abstract] [Full Text] [PDF]
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 K. Nagaoka, H. Nojima, F. Watanabe, K.-T. Chang, R. K. Christenson, S. Sakai, and K. Imakawa Regulation of Blastocyst Migration, Apposition, and Initial Adhesion by a Chemokine, Interferon {gamma}-inducible Protein 10 kDa (IP-10), during Early Gestation J. Biol. Chem., August 1, 2003; 278(31): 29048 - 29056. [Abstract] [Full Text] [PDF]
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K. Kitaya, T. Nakayama, T. Okubo, H. Kuroboshi, S. Fushiki, and H. Honjo Expression of Macrophage Inflammatory Protein-1{beta} in Human Endometrium: Its Role in Endometrial Recruitment of Natural Killer Cells J. Clin. Endocrinol. Metab., April 1, 2003; 88(4): 1809 - 1814. [Abstract] [Full Text] [PDF]
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