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*
Departments of Biomedical Sciences and
Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, Ontario, Canada;
Institute of Cellular and Molecular Biology, University of Texas, Austin, TX 78712;
Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill, NC 27599; and
¶ Department of Immunology, Roswell Park Cancer Institute, Buffalo, NY 14263
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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4
integrin-dependent adhesion of CD56bright NK cells to
decidualizing uterus and in human PBL adhesion to lymph node
endothelium. These data support a model that attributes the dramatic
increases in human and murine uNK cells during decidualization to
precursor cell recruitment. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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45 µm diameter), acquire cytoplasmic
granules containing lytic proteins, and initiate IFN-
synthesis. In
mouse pregnancy, uNK cells are found in two specific microdomains, DB
and mesometrial lymphoid aggregate of pregnancy (MLAp, also termed
metrial gland), a zone found in the uterine wall at implantation sites
from gestation day 8 (gd8) (1, 2). The MLAp and DB are
traversed by branches of the uterine artery that will supply the
placenta beginning at gd9–10. Data from pregnancies in NK/uNK
cell-deficient mice (tg
26 and
RAG-2-/-/common -chain
(c)-/-) indicate that
DB and its spiral arteries are major targets of uNK cell actions,
mediated via release of IFN- (4, 5, 6, 7). An unresolved
question is whether adult uterus self-renews uNK cells or recruits
precursors of uNK cells (pre-uNK) from other tissues.
Generation and maintenance of specialized lymphocyte subsets occur in
some adult, nonlymphoid abdominal tissues, but have not been studied in
uterus (8, 9). To establish the site of self-renewal for
intestinal intraepithelial T lymphocytes, intestinal segment grafting
into T cell-deficient hosts was used (10). Availability of
tg26 and
RAG-2-/-/c-/-
mice which have phenotypically similar anomalous implantation sites
suggests that a uterine segment transplant approach would be possible.
However, poor outcomes have been reported following grafting of uterine
and oviductal tissues in rats, guinea pigs, rabbits, and primates
(11, 12, 13, 14). Inflammation, necrosis, and fibrosis limited the
ability of these grafts to support implantation.
NK cell progenitors (pro-NK) occur in fetal liver (15, 16) and fetal/neonatal thymus (17). In adults, bone marrow (BM) is the major source of pro-NK cells (18). Transplantable pro-NK cells are also found in spleen (19). Progenitors of uNK cells (pro-uNK) are in BM as transgene-marked adult BM injected into fetuses generates uNK cells following birth, adulthood, and pregnancy in the recipients (20). The more differentiated pre-uNK cell also occurs in BM, as pseudopregnant, irradiated mice given male rat BM differentiated some uNK cells with rat morphology (21). However, the origins of pre-uNK cells have not been studied during normal pregnancies.
Lymphocyte mobilization is induced by pregnancy; primary lymphoid
organs (BM and thymus) involute (22, 23, 24), while secondary
lymphoid organs, such as spleen and some lymph node (LN), hypertrophy
(25, 26, 27). Chemokine signaling directs cell movement toward
tissues, while egress of circulating cells from vessels requires
lymphocyte/endothelial cell interactions. Receptors for CC chemokines
are found on NK cells (CCR2 for monocyte chemoattractant protein-1,
CCR5 for macrophage-inflammatory protein-1 and RANTES) (28, 29), and these chemokines are up-regulated in pregnant uteri
(30, 31). L-selectin and 4
integrin are critical molecules involved in slowing circulating
leukocytes and facilitating rolling, tethering, and docking for vessel
egress in secondary lymphoid tissues and at extralymphoid sites of
inflammation (32). In humans, functionally active
L-selectin is expressed at extremely high levels by the numerically
minor, circulating CD56bright NK cell subset,
compared with the more prevalent CD16+,
CD56dim subset (33). The
4 integrin is expressed equivalently by
CD56bright and CD56dim NK
cells (33). Human uNK cells are
CD56bright (2), suggesting a
potential for preferential recruitment of
L-selectinbright, 4
integtrin+, CD56bright
cells into the uterus. Goals of this study were 1) to determine whether
uterus self-renews uNK cells; 2) to examine lymphoid tissues as sources
for pre-uNK cells; and 3) to explore underlying chemokine- and
adhesion-related mechanisms that contribute to mobilization of cells
into decidualizing uterus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunocompetent mice (random bred CD1; Charles River
Laboratories, St. Constant, Quebec, Canada), C57BL/6J (B6), and
C57BL/6 x 129/J F1
(F1; The Jackson Laboratory, Bar Harbor, ME))
were housed under standard husbandry in the Central Animal Facility,
University of Guelph. Immunodeficient mice (random bred
ICR-scid/scid (SCID; NK+,
T-, B-; Taconic Farms,
Germantown, NY), tg26 (H-2k/b;
NK-, T-,
B+), and
RAG-2-/-/c-/-
(H-2b; NK-,
T-, B-) were housed in
the University of Guelph’s barrier-husbandry facility. Both tg26
and
RAG-2-/-/c-/-
lack uNK cells and are referred to as NK/uNK cell deficient
(5). Mice ablated for CCR2 (H-2k/b)
and CCR5 (H-2k/b) (34, 35) were bred
at University of Texas (Austin, TX) and shipped to Guelph. Virgin
females were used at 8 wk of age, unless different ages are stated and,
if bred, were mated to males of the same strain. gd0 was the morning of
copulation plug detection. Euthanasia was by CO2,
followed by cervical dislocation.
Uterine segment transplantation
Uterine segments (10 or 5 mm), trimmed of mesentery and vessels,
were grafted from virgin donors to virgin recipients in an orthotopic
manner meant to preserve both cranial-caudal and
mesometrial-antimesometrial orientations. For autotransplantation
(n = 2), CD1 females were anesthetized (0.35 ml
xylaxine (20 mg/ml) and ketamine (100 mg/ml)), and the donor horn was
reanastomosed with simple interrupted 8-0 Vicryl (Polysorb, Norwalk,
CT) sutures. For all other grafting, donors (CD1 or SCID) were
euthanized, while recipients (tg26 or CD1) were anesthetized as
above. Recipient horns were cut at their midpoint, and donor tissue was
inserted and anastomosed. No vascular anastomosis was attempted. The
abdomen was closed surgically. After 7 days, recipients were paired for
breeding and euthanized at gd10.
Thymic engraftment
Thymuses were dissected from nonpregnant or pregnant (gd3 or 5)
adult, or neonatal (48 h) B6 mice and grafted under the renal capsule
of anesthetized gd0
RAG-2-/-/c-/-.
Adoptive transfer of BM, LN, or splenocytes
BM and spleen cell (SC) donors were nonpregnant or pregnant SCID
mice, while LN donors were B6 (gd3, 5, or 7). As pregnancy changes
cellularity of these organs, one donor tissue equivalent dose was used
for each recipient, pooling donors if several mated recipients were
available on the same day. Uteri from all gd3 donors were flushed to
confirm pregnancy by detection of preimplantation blastocysts. BM was
flushed from femurs and tibias of each donor. Twelve s.c. LN (PLN),
three iliosacral LN, and the mesenteric LN chain were harvested from
each donor. The mesenteric and iliosacral LN were pooled (MLN). LN and
spleens were dissociated mechanically. PBS (400 µl with/without
cells) was infused via tail veins into gd0 tg26 or
RAG-2-/-/c-/-
recipients, who were sacrificed on the recipients’ gd10.
Morphometric analyses
Abdominal contents were examined grossly, then uteri were dissected, fixed in Bouin’s solution, processed routinely for paraffin embedding, serially sectioned at 7 µm (transversely for normal uteri and longitudinally for surgically manipulated uteri), and stained with H&E for routine histopathology or periodic acid Schiff’s (PAS) for uNK cell enumeration. Eleven central tissue sections from each implantation site were scored as previously described (3). One square millimeter per section was analyzed in each mesometrial microdomain, DB, and MLAp. Circular smooth muscle was used as the boundary between these. When the MLAp was absent or rudimentary, mesometrial triangle (MT) is the term used to describe the scored region, rather than MLAp. For longitudinally sectioned uteri, those containing a conceptus were scored as above. Uteri containing a deciduoma were scored in two independent 1-mm2 areas of 10 of the serial sections separated by 42 µm to avoid duplicate counting of uNK cells, which may reach 45 µm by gd10. Diameter and granularity were estimated from a total of 30 cells from 10 sections at 1000x. Means and SDs of uNK cells/mm (2) diameters, granularity, p values, and Student-Newman-Keul test for ANOVA were conducted using PC-SAS 6.12 for Windows (SAS Institute, Cary, NC).
Lymphocyte adhesion to frozen mouse tissues under shear
Human PBL were isolated from buffy coat leukocyte concentrates
of anonymous donors (American Red Cross, Buffalo, NY) by Ficoll/Hypaque
centrifugation and adherent cell depletion (36).
Information regarding donor sex and hormonal status was not available;
thus, equal numbers of nonpregnant female and male donors are probable.
Uteri and PLN from nonpregnant and pregnant (gd3, 6, or 10) B6 mice
were cryopreserved immediately upon collection. PBL were either
unstained or stained with anti-CD56 PE mAb (1/100; Coulter
Immunology, Hialeah, FL) and goat anti-mouse IgG-rhodamine
isothiocyanate (RITC) Ab to permit analysis of NK cell subset adhesion
to high endothelial venules (HEV) by fluorescence microscopy, as
described previously (36). Before assay, PBL were
incubated with or without blocking mAb (10 µg/ml) specific for
L-selectin (DREG-56; American Type Culture Collection, Manassas, VA) or
4 integrin (HP2/1; Coulter). Treated PBL
(5 x 106) were overlaid on 12-µm
cryosections of mouse tissues and rotated at 112 rpm (Labline
Instruments, Melrose Park, IL) for 30 min at 4°C. After removal of
nonadherent cells, specimens were fixed vertically in 3% formaldehyde
in PBS for 1 h. For unlabeled PBL, glutaraldehyde-fixed specimens
were permeabilized in 70% ethanol and stained with 0.5% toluidine
blue in absolute ethanol. Adhesion of PBL was scored in 300–500 HEV
per specimen. All enumeration was done in triplicate. If
CD56-prelabeled cells were used, specimens were washed in PBS and
fluorescent cells were quantified in double-blind analysis of 10 high
powered fields (equivalent to 5 mm2) at x200
magnification, while total cells were quantified in the same section by
bright field using an Olympus BH2-RFL fluorescence microscope (Olympus
Optical, Tokyo, Japan). As reported previously (33), the
accuracy of using fluorescence microscopy to visually discriminate
CD56bright and CD56dim
cells in human PBL suspensions and adherent cell populations was
verified by flow cytometry and confocal fluorescence microscopy,
respectively, to quantify the fluorescence intensity of individual
cells. Fluorescent-labeled cells were not detected in specimens labeled
with isotype-matched negative control murine IgG1 Ab (Coulter).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Feasibility of orthotopic uterine grafting was assessed in
autologously grafted CD1 mice using grafts of 10 mm (n
= 3) and 5 mm (n = 4). The longer grafts showed gross
and histological full-length necrosis, while the shorter grafts were
viable. All recipients of the 5-mm grafts were pregnant, with three
grafts containing implantation sites and the fourth a deciduoma (Table I
). Typical uNK cells (16–45 µm
diameter with 9–25, PAS-reactive cytoplasmic granules/cell) were found
in these decidualized grafts (Fig. 1, A and B). Thus, orthotopically grafted mouse
uterus supports uNK cell differentiation. Next, 5-mm uterine segments
were grafted from uNK cell-competent CD1 or SCID donors into NK/uNK
cell-deficient tg26 recipients (n = 7). Two females
mated, but were not pregnant at euthanasia, despite grossly and
histologically normal grafts. Five recipients were pregnant, and each
grafted segment contained a large deciduoma indicative of viable,
hormonally responsive graft tissue. By serial section analysis, neither
the deciduomata in donor tissues nor implantation sites in host tissues
contained uNK cells (Fig. 1, C and D). Thus, the
donor segments did not contain self-renewing pro/pre-uNK cells that
could differentiate in situ or migrate to adjacent implantation
sites.
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At gd10 in normal and SCID mice, the range in uNK cell frequency
is 27–53 cells/mm2 in DB and 72–129
cells/mm2 in MLAp (Table II). Uterine segment transplantation
suggested that migration of pre-uNK cells accounts for filling of these
microdomains. Lymphoid tissues were assessed for pre-uNK cells by
grafting to mated, NK/uNK cell-deficient mice. Thymic engraftment
generated limited numbers of uNK cells at gd10 (Table II). There were
no statistical differences in reconstitution of DB or MT by thymuses of
different ages or from different donor pregnancy states
(p > 0.05). BM from nonpregnant donors or
donors at three early times of pregnancy also gave low uNK cell
reconstitution in all recipients (Table II). No significant differences
were found in uNK cells/mm2 in DB or MT between
the BM donor groups (p > 0.05). MLN gave low
uNK cell restoration, while implantation sites in recipients of PLN
showed MLAp development. Both MLAp and DB of PLN-grafted mice contained
mature uNK cells. Implantation sites in NK/uNK cell-deficient mice
receiving SC from pregnant donors also showed development of MLAp and
high levels of uNK cells in both MLAp and DB. However, if the SC donors
were not pregnant, uNK cell reconstitution was much lower
(p < 0.001) in both areas. As shown in Fig. 1
, E and F, levels of engraftment resulting from
inoculation of SC from pregnant donors were sufficient to modify the
decidual spiral arteries. In sharp contrast, host arterial vasculopathy
persisted in recipients of SC from nonpregnant donors. For all SC
donors, uNK cells were present at higher frequencies in MLAp than in DB
(p < 0.01), the typical gd10 pattern in normal
mice. Morphological assessment of graft-derived uNK cells showed that
uNK cells derived from thymus, BM, LN, and SC were similar in diameter
(means ± SD of 14.5 ± 4, 15.6 ± 4.5, 18.7 ±
7.5, and 13.9 ± 4.7 µm, respectively) and in numbers of
granules/cell (12.8 ± 5.8, 15.6 ± 5.5, 20.5 ± 11.9,
and 17.8 ± 7.7 granules/cell, respectively). These properties are
identical with uNK cells in unmanipulated B6 (14.3 ± 2.9 µm
diameter and 18 ± 8.2 granules/cell) and SCID (15.9 ± 4
µm diameter, 19.4 ± 9 granules/cell) mice, implying equivalent
maturity.
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c-/-
females were infused with SC from pregnant (gd3 or 5)
CCR2-/- or CCR5-/-
mice. High levels of uNK cells were found in all recipients that did
not differ numerically (Table II) or morphometrically from uNK cells in
gd-matched, F1 controls. These data indicate that
the chemokines macrophage-inflammatory protein-1, monocyte
chemoattractant protein-1, and RANTES are not essential for uterine
recruitment of pre-uNK cells. Human PBL adhesion under shear to murine tissue sections
Human anti-CD56 mAb-labeled cells were assessed for adhesion
under shear to frozen uterine sections from nonpregnant or pregnant B6
mice (gd3, 6, 10). Consistent with previous reports (33, 47), the proportion of labeled cells in test PBL
suspensions was 10–12% CD56+, with <1.5%
being CD56bright. Prelabeling with anti-CD56
(murine IgG1) did not affect the total number of cells bound to uterine
tissues at any of the time points (data not shown).
CD56+ cells adhered to nonpregnant endometrial
stroma in a randomly dispersed, low frequency manner (Fig. 2). Numbers of
CD56+ cells adhering to nondecidualized uterine
stroma markedly increased in gd3 tissue, and the adhering cells
remained randomly distributed. At gd6 and 10, cell adhesion was
significantly elevated, compared with nonpregnant uterus
(p < 0.001), and the cells were restricted to
the mesometrial side of the uterus and localized exclusively to DB. The
adhering CD56+ cells were predominantly small
sized (6.8 ± 0.4 µm, measured from 12 adherent cells). A minor
adhering population (1–10%) of larger, CD56+
cells (8.01–9.01 µm, measured from three adherent cells) was also
detected (Fig. 2B). In comparison with the numbers of
CD56bright cells in the initial overlay cell
suspension, adhesion to uterine tissue significantly enriched the
CD56bright cells (Fig. 2). Pregnancy-induced
adhesion was dependent on both L-selectin and
4 integrin homing receptors (Fig. 2) because
it was inhibited by specific function-blocking Abs, i.e., DREG-56 and
HP2/1, respectively, but not by an IgG1 isotype-matched control Ab.
Pregnancy induced dramatic and unexpected gains in lymphocyte adhesion
in HEV of PLN from gd6 in C56/BL6 mice (Fig. 2). As predicted from
earlier reports, lymphocyte adhesion to LN HEV from nonpregnant and
pregnant mice was L-selectin dependent and did not involve
4 integrin (32). Only uniformly
sized small lymphocytes bound to PLN HEV.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To explain the dramatic rise in human uNK cells during decidualization,
some authors suggest that uNK cells self-renew in the uterus
(38), while others suggest direct trafficking from BM is
the major supply mechanism (39). Our studies indicate that
both ideas may be incorrect. In our adoptive transfers, BM made only a
minor contribution to uNK cell numbers during early to mid gestation.
Despite the known and observed pregnancy-induced involution of BM
(Table II), there was no loss in BM-derived progenitors able to
populate uterus with uNK cells, during the first trimester of
pregnancy. Levels of uNK cell generation from thymus were also low and
independent of donor age or pregnancy status. However, our results show
for the first time that thymus retains its capacity for NK cell
generation into adulthood.
LN was shown to be a source of pre-uNK cells, but not all LN had transplantable pre-uNK. Hypertrophy was anticipated in the iliosacral LN draining the uterus (26, 27), but was not measurable when these smaller nodes were pooled with the much larger mesenteric LN chain. This pool, which includes the LN draining the pelvic region, hind limb, and uterus, had limited pre-uNK cells. Transplantable pre-uNK cells were present in PLN. However, further study would be necessary to precisely define which LN in the PLN pool contained transplantable pre-uNK. Development of the LN in the PLN that were pooled is heterogeneously regulated with some nodes (i.e., cervical) differentiating analogously to MLN (40). These nodes would perhaps lack transplantable pre-uNK cells.
Spleen contained pre-uNK cells that were mobilized to the uterus by
pregnancy. In comparison with nonpregnant donors, SC numbers doubled in
pregnant donors, while numbers of uNK cell progeny increased 4–47
times (Table II). This suggests that numerical alterations are not the
sole pregnancy-induced changes in SC accounting for uNK cell
reconstitution. The developmental stages of hemopoietic cells that move
into the uterus are not yet known. Because uNK cells differentiating
from thymus, BM, LN, and SC are identical morphologically and
morphometrically and match those in gd10 unmanipulated, normal mice,
the cells that moved into the uterus from these tissues were probably
at relatively similar stages of differentiation. Alternatively, uNK
cells may differentiate rapidly, and cells at various pro/pre-uNK
stages may have had sufficient time to complete differentiation under
our experimental conditions. The heterogeneity in size of human
lymphocytes adhering to murine uterus suggests that circulating cells
at more than one stage of differentiation/activation may have uterine
homing potential. Despite the fact that splenic NK cells express the
genes for chemokine receptors CCR2 and CCR5 (Refs. 28, 29 ; W. A. Kuziel, unpublished data) and that pregnant mouse and
human uteri express high levels of the ligands for these receptors
(30, 31), we found that the targeted absence of either
CCR2 or CCR5 on pre-uNK cells did not reduce homing from spleen to
uterus. These observations raise the possibility that the CCR2 and CCR5
chemokine receptors are functionally redundant, or that pre-uNK cells
use alternative receptors for their recruitment.
Functional assays of CD56+ lymphocyte (male or
female) adhesion to frozen tissue sections under shear showed that all
uteri (nonpregnant and gestational) bound labeled cells, including a
rare population of larger cells. Adherent cell localization was
dramatically altered by development of decidua, changing from random to
mesometrial indistribution. At gd6, adhering cells were absent over
fetal trophoblasts and maternal MT/MLAp regions and localized
exclusively over DB. In gd10 mice, 10% of the large, mature uNK
cell population is intravascular in DB, while intravascular uNK cells
are rare in MLAp, a domain containing uNK cells with less mature
morphology (41). Thus, adherence of
CD56+ cells in DB could indicate a specialized
endothelium participating in pre-uNK cell recruitment and/or serving as
a target of uNK cell functions within implantation sites.
Selective recruitment of NK cells to uterus most likely involves dual
control of adhesion molecules on both trafficking NK cells and target
uterine vascular endothelial cells. CD56+ cell
adhesion to uterine tissues was dependent on both L-selectin and
4 integrins. Coordinate expression of these
molecules has been previously documented on circulating
CD56bright cells, with L-selectin expressed at
very high density (33). Both L-selectin and
4 integrins (i.e.,
4
7 and
41) function as
gatekeepers, controlling lymphocyte extravasation through their ability
to bind to vascular addressins under shear (32, 43). Thus,
it is tempting to speculate that pregnancy induces differential display
of vascular adhesion molecules on uterine microvessels. The almost
complete blocking of CD56bright cell adhesion by
mAb to both L-selectin and 4 integrin
suggested that these adhesion molecules are used by the same
CD56bright subset. The data are consistent with
the coutilization of a common endothelial ligand for both L-selectin
and 4 integrin, analogous to the situation in
Peyer’s patches (PP) in which trafficking of naive lymphocytes
is mediated by cooperative interactions of L-selectin and
47 integrins with
spatially distinct domains within the mucosal addressin cell adhesion
molecule (MAdCAM-1) (44, 45). Blockade of L-selectin and
4 integrins under the same conditions as used
in the present study results in complete inhibition of human PBL
binding to PP HEV (46). Alternatively, if independent
ligands are involved in mediating CD56+ NK cell
adhesion to uterine tissues, partial inhibition by
L-selectin and 4 integrin-blocking mAb might
be expected in frozen-section adhesion assays, as reported for
mesenteric LN that dually express peripheral LN addressins (PNAd; an
L-selectin ligand) and MAdCAM-1 (a ligand for
4 integrins and L-selectin)
(44, 45).
The ligands for 4 integrins and L-selectin in
uterine stroma remain to be defined. Recent studies by Kruse et al.
(42) showed VCAM-1 is restricted to endothelium of the
central DB in gd9 mice. While this could account for the
4 integrin-mediated binding observed in the
present study, caution must be used in this interpretation because
extracellular matrix is also exposed in the assayed tissue and
4 integrins bind to fibronectin, a molecule
prevalent in mesometrial decidua (47). High level MAdCAM-1
expression has also been reported on mesometrial endothelium in the
vascular zone, lateral to the DB in gd9 mice (42). This
counterreceptor for L-selectin and
47 in PP
(32) may have the same function in decidualized uterus.
Message for glycosylation-dependent cell adhesion molecule-1, an
L-selectin ligand (48), is up-regulated mesometrially
between gd6 and 10 in B6 uterus, while P-selectin glycoprotein
ligand-1, a P-selectin ligand (49) used by NK cells, is
constitutively expressed at these times (S. Chantakru and B. A.
Croy, unpublished data). Regardless of the mechanisms involved,
exposure of indicator PBL to pregnant uterine tissue results in
preferential adhesion and the enrichment of the
CD56bright NK cell subset. Further studies are
required to determine whether pregnancy alters human lymphocyte
function in the binding assay. This outcome is anticipated because
pregnancy conferred increased potential for reconstitution of uNK cells
in the splenocyte adoptive transfer experiments and from recent studies
in our laboratories showing that pregnancy promotes mouse splenocyte
adhesion to HEV in LN from virgin mice (S. Chantakru, W. C. Wang,
B. A. Croy, and S. S. Evans, manuscript in preparation).
Pregnancy, unexpectedly, altered lymphocyte/endothelial interactions in nonuterine sites. Thus, elevated adhesion to LN HEV was observed by gd3, whereas maximal adhesion occurred by gd6 that was sustained throughout pregnancy (shown in this study, S. Chantakru, W. C. Wang, B. A. Croy, and S. S. Evans, manuscript in preparation). The physiological roles of these changes in endothelial cells in peripheral lymphoid tissues are undefined, but they may contribute to cell recruitment to the uterus or in immune protection by surveillance for fetal Ag-sensitized cells. This study has shown that pregnancy induces functional changes in lymphocytes in vivo and adhesion molecules on endothelial cells, and it provides novel transplantation and adhesion assays that will facilitate further studies on the mechanisms regulating pre-uNK cell recruitment to the uterus in both mice and women.
|
Acknowledgments |
|---|
26 and
RAG-2-/-/c-/- and for
discussions of unpublished transplantation protocols. We are indebted
to the Ontario Ministry of Agriculture, Food, and Rural Affairs
Isolation Unit staff for the outstanding level of husbandry provided to
our immunodeficient mice. |
Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Sirirak Chantakru, Department of Biomedical Sciences, Building No. 40, University of Guelph, Guelph, Ontario, Canada, N1G 2W1. E-mail address: schantak{at}uoguelph.ca
3 Abbreviations used in this paper: uNK, uterine NK; BM, bone marrow; DB, decidua basalis; gd, gestation day; HEV, high endothelial venule; LN, lymph node; MAdCAM-1, mucosal addressin cell adhesion molecule-1; MLAp, mesometrial lymphoid aggregate of pregnancy; MLN, pools of mesenteric and iliosacral LN; MT, mesometrial triangle; PAS, periodic acid Schiff’s; PLN, pools of s.c. LN; PP, Peyer’s patch; pre-NK, precursors of NK cell; pro-NK/uNK, progenitors of NK/uNK cell; RITC, rhodamine isothiocyanate; SC, spleen cell; c, common -chain.
Received for publication July 9, 2001. Accepted for publication October 22, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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26 mice. J. Exp. Med. 187:217. contributes to initiation of uterine vascular modification, decidual integrity, and uterine natural killer cell maturation. J. Exp. Med. 192:259. and revealed in lymphotoxin deficient mice. Immunity 6:491.[Medline]
4 integrins in lymphocyte homing to mucosal tissues in vivo. J. Immunol. 152:3282.[Abstract]
47 integrin dependent lymphocyte-endothelial adhesion. Int. J. Hyperthermia 16:45.[Medline]
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A. M. Sharkey, L. Gardner, S. Hiby, L. Farrell, R. Apps, L. Masters, J. Goodridge, L. Lathbury, C. A. Stewart, S. Verma, et al. Killer Ig-Like Receptor Expression in Uterine NK Cells Is Biased toward Recognition of HLA-C and Alters with Gestational Age J. Immunol., July 1, 2008; 181(1): 39 - 46. [Abstract] [Full Text] [PDF]
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J. L. Herington and B. M. Bany Effect of the Conceptus on Uterine Natural Killer Cell Numbers and Function in the Mouse Uterus During Decidualization Biol Reprod, April 1, 2007; 76(4): 579 - 588. [Abstract] [Full Text] [PDF]
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T. Yamaguchi, K. Kitaya, N. Daikoku, T. Yasuo, S. Fushiki, and H. Honjo Potential Selectin L Ligands Involved in Selective Recruitment of Peripheral Blood CD16(-) Natural Killer Cells into Human Endometrium Biol Reprod, January 1, 2006; 74(1): 35 - 40. [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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X. Xie, H. He, M. Colonna, T. Seya, T. Takai, and B. A. Croy Pathways Participating in Activation of Mouse Uterine Natural Killer Cells During Pregnancy Biol Reprod, September 1, 2005; 73(3): 510 - 518. [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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M. J. van den Heuvel, J. Horrocks, S. Bashar, S. Taylor, S. Burke, K. Hatta, J. E. Lewis, and B. A. Croy Menstrual Cycle Hormones Induce Changes in Functional Interactions between Lymphocytes and Decidual Vascular Endothelial Cells J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2835 - 2842. [Abstract] [Full Text] [PDF]
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G. Schofield and S. J. Kimber Leukocyte Subpopulations in the Uteri of Leukemia Inhibitory Factor Knockout Mice During Early Pregnancy Biol Reprod, April 1, 2005; 72(4): 872 - 878. [Abstract] [Full Text] [PDF]
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C. L. Sentman, S. K. Meadows, C. R. Wira, and M. Eriksson Recruitment of Uterine NK Cells: Induction of CXC Chemokine Ligands 10 and 11 in Human Endometrium by Estradiol and Progesterone J. Immunol., December 1, 2004; 173(11): 6760 - 6766. [Abstract] [Full Text] [PDF]
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K. Deb, M. M Chaturvedi, and Y. K Jaiswal A 'minimum dose' of lipopolysaccharide required for implantation failure: assessment of its effect on the maternal reproductive organs and interleukin-1{alpha} expression in the mouse Reproduction, July 1, 2004; 128(1): 87 - 97. [Abstract] [Full Text] [PDF]
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S. Chantakru, W.-C. Wang, M. van den Heuvel, S. Bashar, A. Simpson, Q. Chen, B. A. Croy, and S. S. Evans Coordinate Regulation of Lymphocyte-Endothelial Interactions by Pregnancy-Associated Hormones J. Immunol., October 15, 2003; 171(8): 4011 - 4019. [Abstract] [Full Text] [PDF]
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A. A. Ashkar, G. P. Black, Q. Wei, H. He, L. Liang, J. R. Head, and B. A. Croy Assessment of Requirements for IL-15 and IFN Regulatory Factors in Uterine NK Cell Differentiation and Function During Pregnancy J. Immunol., September 15, 2003; 171(6): 2937 - 2944. [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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J. P. Goodridge, C. S. Witt, F. T. Christiansen, and H. S. Warren KIR2DL4 (CD158d) Genotype Influences Expression and Function in NK Cells J. Immunol., August 15, 2003; 171(4): 1768 - 1774. [Abstract] [Full Text] [PDF]
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), 
), or an
isotype-matched control Ab (mouse IgG1; 
). To permit discrimination
of NK cells bound to uterine tissues, PBL were initially stained with
anti-CD56 mAb and goat anti-mouse IgG-RITC secondary Ab. The
initial PBL sample was comprised of 11% CD56+ cells and
<1.5% CD56bright cells (not shown). The relative
proportion of CD56bright cells within the total lymphocyte
adherent populations was determined by direct analysis of 10 high
powered fields (5-mm2 area) under fluorescence and light
microscopy. *, Denotes statistical differences calculated using an
unpaired two-tailed Student’s t test for the increase
in adhesion detected in response to pregnancy, p <
0.001. Data are the mean ± SD of triplicate specimens prepared
using a single lymphocyte pool. Results are representative of three
independent studies, each using different human donors and a different
series of gestationally timed murine tissues. B,
Photomicrographs showing adhesion of CD56bright cells to
uterine tissues (left panels) or toluidine-stained PBL
bound to PLN HEV (right panels). Fluorescently labeled
lymphocytes were not detected in uterine tissues when PBL were stained
with isotype-matched control Ab and goat anti-mouse IgG-RITC
secondary Ab (not shown). Bar, 25 µm.