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Immunocytochemical Studies of Adhesion Molecules on Mouse UNK Cells and Their Extracellular Matrix Ligands During Mouse Pregnancy.

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THE ANATOMICAL RECORD 293:1081–1088 (2010)
Immunocytochemical Studies of
Adhesion Molecules on Mouse UNK Cells
and Their Extracellular Matrix Ligands
During Mouse Pregnancy
BRUNO ZAVAN,1 ANDRÉA MOLLICA AMARANTE PAFFARO,1
PAULO PINTO JOAZEIRO,2 ÁUREO TATSUMI YAMADA,2
1
AND VALDEMAR ANTONIO PAFFARO, JR *
1
Department of Biomedical Science, UNIFAL-MG, Alfenas, Minas Gerais, Brazil
2
Department of Histology and Embryology, IB, UNICAMP, Campinas, São Paulo, Brazil
ABSTRACT
Uterine natural killer (uNK) cells are the dominant lymphocytes of
pregnant mammals’ uterus. Studies have identified four differentiation
stage of mouse uNK cells based on Dolichos biflorus lectin cytochemistry,
and their distribution showed preferential domain in the uterus through
out the pregnancy. This work was done to investigate the expression of a5,
a6, and b7 integrins on uNK cells and their ligands distribution. Section of
mouse uterus from sixth to seventeenth gestational days were submitted to
immunocytochemistry and positive reactions for a5, a6, and b7 integrins
were found on uNK from eighth to tenth gestational days but not after
twelfth gestational days. Fibronectin reactions were seemed from sixth to
tenth gestational days around uNK from the myometrium and endometrium close to the myometrium. No reaction for fibronectin was seen in the
decidualized and nondecidualized endometrium near the placenta. Laminin
reaction was seen just in the antimesometrial side. b7 integrin seems to be
the active receptor to bind with VCAM-1 or MAdCAM-1 of endothelial cells,
promoting the uNK cross through the vessels. The absence of laminin in an
uNK domain suggests these cells are not dependent of laminin and a6
integrin for their establishment. However, fibronectin seems to support
uNK migration, proliferation, differentiation, and survival in the uterus by
binding with a5 integrin. The loss of a5 integrin ligation by the down regulation of fibronectin could inhibits these events and further studies are
need to investigate whether unligated a5 can actively and initiate apoptosis, maybe in a caspase 8-dependent way that has been called integrinC 2010 Wiley-Liss, Inc.
mediated death. Anat Rec, 293:1081–1088, 2010. V
Key words: mouse pregnancy; natural killer lymphocytes;
integrin; laminin; fibronectin; decidua
Grant sponsor: Fundação de Ampáro a Pesquisa do Estado de
Minas Gerais (FAPEMIG); Grant sponsor: Fundação de Amparo
a Pesquisa do Estado de São Paulo (FAPESP); Grant sponsor:
Programa Institucional de Bolsa de Iniciação Cientı́fica (PIBIC/
CNPq).
*Correspondence to: Valdemar Antonio Paffaro, Jr, Histology
Laboratory, Department of Biological Science, Federal UniverC 2010 WILEY-LISS, INC.
V
sity of Alfenas (UNIFAL-MG), Brazil E-mail: paffaroj@unifalmg.edu.br
Received 15 July 2009; Accepted 17 December 2009
DOI 10.1002/ar.21117
Published online 3 March 2010 in Wiley InterScience (www.
interscience.wiley.com).
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ZAVAN ET AL.
INTRODUCTION
Natural killer (NK) cells represent a distinct population of circulating and tissue-resident lymphocytes that
play an important role in the early phases of immune
responses against microbial pathogens by exhibiting cytotoxic functions and secreting a number of cytokines
and chemokines. (Di Santo and Vosshenrich, 2006;
Carlino et al., 2008).
In the pregnant rodent and human uterus, a transient
accumulation of natural killer (uNK) cells are found
(Bernard et al., 1978; Stewart and Peel, 1981; King and
Loke, 1991; Croy and Kiso, 1993; Head, 1996; Paffaro
et al., 2003). In humans, NK cells are the most abundant
lymphocytes found in the mucosal tissues of maternal
uterus where their number reaches 70%–80% of the total
leukocytes in the first trimester of pregnancy, then start
to decline, and return to basal levels at the end of pregnancy (Bulmer and Lash, 2005). In rodents, these cells
appear shortly after blastocyst implantation and are restricted to the mesometrial lymphoid aggregate of pregnancy (MLAp) and decidua basalis within each
implantation site (Dickson and Bulmer, 1971; Stewart
and Peel, 1981; Stewart, 1983; Croy et al., 1997). Small
nongranular lymphocytes are recognized as uNK cell precursors (Stewart, 1983; Peel and Stewart, 1989; Parr
et al., 1991; Pollack and Linnemeyer, 1996; Van den
Heuvel et al., 1996). These precursors begin to differentiate within the uterus on fifth gestation day (Paffaro et al.,
2003). They proliferate, increase in number, size, and
granular content (Croy et al., 1997) but remain confined
to the mesometrial side of the uterus (Peel, 1989;
Kaufman, 1992, Paffaro et al., 2003). Senescence of differentiated uNK cells (i.e. not precursor) is observed before
parturition (Delgado et al., 1996) after which they are
completely absent of the uterus (Stewart and Peel, 1981;
Peel, 1989). The major roles defined to date for mouse
uNK cells are sensitization of the decidual spiral arteries
leading to the dilation of these vessels supplying the placenta and maintaining the decidual cellularity (Hunt
et al., 1997; Ashkar and Croy, 1999) through release of
Interferon-y (Ashkar et al., 2000). These findings strongly
suggest that uNK cells are pivotal in pregnancy maintenance (Koopman et al., 2003). Like mouse uNK cells,
those found in humans (CD56bright decidual cells) express
cytokines and also angiogenic factors (Li et al., 2001).
NK cells from lymphoid tissue express several surface
adhesion molecules including integrins. Expression of
these molecules appears to reflect the activation status
of the cells. NK cells bind to endothelium, extracellular
matrix (ECM), stromal cells, and target cells through adhesion receptors. The profile of expressed receptors provides the mechanism for NK cell homing, infiltration,
and recognition (Helander and Timonem, 1998).
The integrin family is a large group of receptors for
ECM proteins of immunoglobulin superfamily molecules.
Integrins are divalent cation-dependent heterodimeric
membrane glycoprotein containing noncovalently associated a and b subunits that promote cell attachment and
migration on the surrounding ECM. Each integrin subunit has an extracellular domain, a single transmembrane
region, and a short cytoplasmic region (Hynes, 2002;
Avraamides et al., 2008).
Studies suggest that the b1 and the b7 integrin have
an important hole in the homing and migration process
of the NK cells (Helander and Timonen, 1998). Longterm activated NK cells express a1b1integrin, a receptor
for laminin and collagen (Maenpaa et al., 1993). In addition, NK cells express the laminin-binding receptor a6b1
integrin that is involved in vascular-endothelial cells
interactions, exodus of the cells from the circulation, and
infiltration of the cells into tissues (Helander and Timonen, 1998). Maenpaa et al (1993) suggested the NK cell
attachment to endothelium by a4b1 integrin binding to
endothelial VCAM-1. In a in vivo melanoma study, antiVCAM-1 antibody inhibited NK cell homing to the tumor
(Fogler et al., 1996). NK cells also recognize fibronectin
through a4b1 and a5b1. Recognition of fibronectin promotes cell migration (Somersalo and Saksela et al.,
1991).
It has been proposed that precursors of human uNK
cells present in the circulation use identical mechanisms
for movement into the human uterus. In studies that
assessed adhesion of human lymphocytes to cryostat sections of mouse uterine tissue under shear forces mimicking blood flow, it was shown that interactions involving
L-selectin and a4 integrin complexed with b1 or b7 were
promoted during pregnancy (Chantakru et al., 2002,
2003). On particular interest, functional adhesion was
demonstrated only in the central decidua basalis, where
VCAM-1 is expressed (Kruse et al., 1999), and adhesion
promoted very high enrichment (up to 25%) of CD5bright
blood lymphocytes. These data suggested that matrix
components in decidua stroma may have restricted
microdomain presentation important for homing of both
human and murine uNK precursor cells.
In b7 integrin deficient mice, uNK cells were localized
to the decidua basalis but not to the MLAp, suggesting
that b7 have a role in the uNK cell migration process
(Croy et al., 1997). However, Kruse et al (1999) showed
uNK cells in decidua basalis, surrounding VCAM-1þ,
MadCAM-1 blood vessels and suggested uNK cells endothelial transmigration through the a4b1 integrin binding in the endothelial VCAM-1.
Kiso et al (1994) reported that uNK cell precursors
(LGL-1þ), found in third to sixth gestational days, are
b1þ, a1þ, a3þ, a4þ, a5þ, and a6þ, whereas uNK cells
from eighth to fifteenth gestational days are b1þ, a4þ,
a5þ, a1, a3, and a6. These data suggested changes
in the expression of adhesion molecules as uNK cells differentiated with mature uNK cell (eighth gestational
day) retaining binding for ligands that would include
laminin, fibronectin, and VCAM-1. Additionally, fibronectin and laminin increased the survival of cultured uNK
cells that migrated from explant cultures (Croy and
Kiso, 1993).
In this study, it was undertaken a time cons study
to address the microdomain localization and possible
dynamic changes in key ECM molecules that could be
important for uterine positioning and survival of uNK
cells. With this objective, it use a high efficient uNK
staining (DBA lectin Staining) and the immunocytochemistry for b7, a6, a5 integrins, on the uNK cells,
and to the ECM molecules, fibronectin, and laminin.
The goal of this study was to purpose an unligated-a5
integrin mediated cell dead for uNK cells, associated
to the absence of fibronectin. This purpose could
explain the literature data that has showed the
increasing of in vitro uNK viability in the presence of
fibronectin.
ADHESION MOLECULES ON MOUSE UNK
1083
Fig. 1. Photomicrographs of histological sections of a pregnant
mouse uterus on tenth gestational day. (a) Area 2 of the mesometrial
region showing DBA lectin reactive uNK cells (arrows head), (b) Subtype I DBA lectin reactive uNK cell, (c) subtype III DBA lectin reactive
uNK cell, and (d) Subtype IV DBA lectin reactive uNK cell. Note the
different morphologies of the immature uNK (subtype I), mature uNK
(subtype III), and the probably degenerative form of uNK cells (subtype
IV). Blood vases (V) Diaminobenzidene and hematoxilin.
MATERIALS AND METHODS
nant mice were perfusion fixed in the same way to
collect fragments from the liver, skin, and ileum containing Peyer’s patches.
Animals
Thirty virgin females SWISS mice (from central animal facility of Unifal-MG) were mated (8–12 weeks of
age) to males of the same strain. The day a vaginal plug
was detected was called gestational day 1. All procedures and animal handling were conducted under
approved protocols.
Sample Collection
Pregnant mice at sixth, eighth, tenth, twelfth, fifteenth, and seventeenth gestational days were deeply
anesthetized with ketamine and xylazine chloridrate for
perfusion fixation with paraformaldehyde 4% in Phosphate buffer-saline (PBS) 0.05 M fixative solution. Each
animal was perfused during 15 minutes using at least
30 mL of fixative solution, and the pregnant uterus was
removed. The embryo implantation and developing sites
were dissected from the uterus and processed for conventional paraffin embedding or frozen in N2. Nonpreg-
Morphological Evaluation
Paraffin sections (7 lm) were processed for Dolichos
biflorus (DBA) lectin cytochemistry according to Paffaro
et al, (2003). Briefly, it consisted in incubation with biotinylated DBA lectin (Honen Chemical, Japan), followed
by streptavidin-peroxidase (DAKO, CA) and revealed
with 3,3,-diaminobenizidine (Sigma, St Louis) and
hydrogen peroxide reaction. The control reaction was
performed by adding 0.1 M N-acetyl-D-galactosamine
(NacGal) in the DBA lectin solution prior the incubation
with sections.
Immunocytochemistry
Cryosections or deparafined sections of pregnant
uterus (sixth to seventeenth gestational days) were
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ZAVAN ET AL.
Fig. 2. Line diagrams of histological sections from pregnant mouse uterus. (a) Seventh gestational day
implantation site and (b) tenth gestational day implantation site. Note the tree areas A1, A2, and A3 used
as references in our studies. Mesometrium (MS); Mesometrial Region (MR); Antimesometrial region
(AMR); Embryo (E); Luminal epithelium (LE); Early fetal placenta (FP).
washed in 0.05 M PBS and incubated overnight at 4 C
with the following primary antibodies: rabbit antifibronectin, rabbit anti-a5 integrin (Chemicon International),
goat anti-b7 integrin (Santa Cruz Biotechnology), or
monoclonal antibody, rat anti-a6 integrin (Chemicon
International). After washing with 0.05 M PBS the sections were incubated with one of the following Cy2 or
Cy3-conjugated secondary antibodies: anti-rabbit, antigoat, or anti-rat (Chemicon International) depending on
the primary antibody used, for 30 minutes at room temperature. The sections were washed in 0.05 M PBS and
then incubated with FITC conjugated DBA lectin,
R (Vector)
washed again, and mounted with vectaschildV
for observations under fluorescence (k ¼ 520 and 560
nm) and differential interference contrast (DIC) microscopy (Nikon Eclipse 800). The images were captured
with digital image analysis system (Cool SNAP-Image
Pro-Plus, Media Cybernetics system). Sections from
liver, skin, and ileum containing Peyer’s patches were
used as control for immunostaining.
RESULTS
DBA Lectin Cytochemistry
The DBA lectin cytochemistry selectively labeled the
cell surface and granules of uNK cells distributed in the
mesometrial side of the implantation sites of the pregnant mouse uterus (Figs. 1a–d) from sixth to seventeenth gestational days and well distinguished the sets
of their maturation (Figs. 1b,c). No positive reaction was
seen in the control reactions performed with DBA lectin
inhibited with NacGal.
Immunocytochemistry
The immunocytochemistry studies were undertaken in
three areas within the mesometrial side, where the uNK
cells were localized (Fig. 2). These areas were characterized in our previous studies by using histological sections stained by DBA lectin cytochemistry (Paffaro Jr
et al., 2003). The Area 1 was the area closed to the miometrium with predominantly immature uNK cells, the
Area 2 were the intermediate area between the Area 1
and 3 with predominantly mature uNK cells, and the
Area 3 was the area closed to the embryo with predominantly mature and degenerative forms of uNK cells.
b7 Integrin
Strong positive reactions for Integrin b7 were seen on
uNK cells surface from eighth to tenth gestational days,
mainly on those cells found near the blood vessels (Figs.
3a,b) and becomes completely negative after twelfth gestational day. The positive control developed in mouse
Peyer’s patch (from the ileum) were positives (Fig. 3c).
a6 Integrin
Week reactions for a6 integrin were also seen on cell
surface of uNK on eighth to tenth gestational days only
(Figs. 3d,e). The positive controls developed in mouse ileum were positives (Fig. 3f).
Laminin
In all analyzed gestation days, any positive reaction to
the laminin in the mesometrial side of the pregnant
uterus where the uNK cells were localized was not
1085
ADHESION MOLECULES ON MOUSE UNK
Fig. 3. Photomicrographs of histological sections submitted to the
immunocytochemistry. (a) Area 2 of the mesometrial region of a pregnant mouse uterus on tenth gestational day showing uNK cells
(arrows) reactive to Immunofluorescent (Cy3) anti-b7 integrin, (b) the
same area under differential interference contrast (DIC) showing the
typical morphology of these uNK cells (arrows), (c) positive control
developed in Payer’s patches showing Immunofluorescent (Cy3) antib7 integrin reactive cell, (d) Area 2 of the mesometrial region of a
pregnant mouse uterus on tenth gestational day showing uNK cells
(arrows) reactive to Immunofluorescent (Cy3) anti-b6 integrin, (e) the
same area under differential interference contrast (DIC) showing the
typical morphology of these uNK cells (arrows), and (f) positive control
developed in ileum showing Immunofluorescents (Cy3) anti-b6 integrin
reactive epithelial cells that are known to be attached to the laminin in
the basal membrane through this integrin. Blood vases (v).
detected (Fig. 4a). Positive reactions to this ECM molecule were found only in the antimesometrial side of the
pregnant uterus, in the blood vessel wall, around the
muscle fibers of the miometrium, in the decidual cell
cytoplasm, and in the decidualized ECM (Fig. 4b). The
positive controls developed in mouse ileum were positive
(Fig. 4c).
a5 Integrin
Fibronectin 1 DBA Lectin
It was observed positive reaction to the antifibronectin
antibody in the miometrium, in the endometrium localized in the mesometrial and antimesometrial region, and
in the mesometrium. In the mesometrial side, where the
uNK cells were localized, positive reaction was localized
around the uNK cells in the Area 1 (Fig. 4d) and 2 (Fig.
4e) at sixth to tenth gestational days. Particularly, the
strong positive reactions to the fibronectin around the
uNK cells were found in the Area 2 at eighth to tenth
gestational days (Fig. 4e). There were not fibronectin
positive reactions in the high-decidualized area in the
Area 3 (Fig. 4f). However in twelfth, fifteenth, and seventeenth gestational days the fibronectin reaction was
very weak in the mesometrial side of the uterus, and
there was nonpositive reaction around the uNK cells
(Fig. 4g). The positive controls developed in mouse liver
were positives (Fig. 4h).
In the mesometrial side of the implantation sites it
was found a5 integrin positive reactions at sixth and
seventh gestational days only on stromal cells in the
Area 1, 2, and 3, but the uNK cells were totally a5 integrin negative at these gestation days. In the antimesometrial side, it was observed positive reaction on the
decidual cells. This reaction pattern was also observed
until the ninth gestational day. However, at eighth to
tenth gestational days a5 integrin positive uNK cell
were observed in the Area 2 and 3 but not in the Area 1
(Figs. 4i,j). At twelfth to seventeenth gestational days,
there were nonpositive a5 integrin reaction on the uNK
cells in any analyzed area, whereas the reaction for this
integrin on the stromal cell from the mesometrial and
antimesometrial side was still observed. Between twelfth
to seventeenth gestational days, it was also observed
positive reaction on the trophoblast giant cells, spongiotrophoblast, and labyrinth trophoblast cells plasma
surfaces. The positive controls performed in mouse skin
were positive (data not showed).
DISCUSSION
In our previous studies, we have characterized four
uNK maturation stages and also their distribution within
the implantation sites during the mouse pregnancy
through morphological, ultrastructural, citochemical, and
1086
ZAVAN ET AL.
Fig. 4. Photomicrographs of histological sections submitted to the
immunocytochemistry. (a) Mesometrial region of a pregnant mouse
uterus on tenth gestational day showing nonreactivity to the Immunofluorescent (Cy3) anti-laminin, (b) the positive reactions were only in
the antimesometrial side of the uterus, where no uNK cells are
observed, (c) positive control developed in ileum showing positive
reaction to the Immunofluorescent (Cy3) antilaminin antibody in the
basal membrane where the intestinal epithelial are attached, (d) Area 1
of the mesometrial region of a pregnant mouse uterus on tenth gestational day showing fluorescent (FITC) DBA lectin reactive uNK cells
(green) and the fluorescent (Cy3) antifibronectin positive reaction (red)
around the uNK cells. (e) Area 2 of the mesometrial region of a pregnant mouse uterus on tenth gestational day showing fluorescent
(FITC) DBA lectin reactive uNK cells (green) and the fluorescent (Cy3)
anti-fibronectin positive reaction (red) around the uNK cells, (f) Area 3
of the mesometrial region of a pregnant mouse uterus on tenth gestational day showing fluorescent (FITC) DBA lectin reactive uNK cells
(green) and the nonreactivity to the fluorescent (Cy3) antifibronectin
antibody around the uNK cells, (g) Area 2 of the mesometrial region of
a pregnant mouse uterus on twelfth gestational day showing fluorescent (FITC) DBA lectin reactive uNK cells (green) and the nonreactivity
to the fluorescent (Cy3) antifibronectin antibody around the uNK cells,
(h) positive control developed in liver showing the positive reaction to
the fluorescent (Cy3) antifibronectin antibody in the extra cellular matrix of this organ, (i) Area 2 of the Mesometrial region of a pregnant
mouse uterus on tenth gestational day showing uNK cells (arrows) reactive to Immunofluorescent (Cy3) anti-a5 integrin, and (j) the same
area under differential interference contrast (DIC) showing the typical
morphology of these uNK cells (arrows).
stereological techniques. These subsets differ in numbers
of cytoplasmic granules, cell diameter, and chromatin
ultrastructure. These previous data also suggested that
the uNK cells immature forms were mainly distributed in
a area closed to the miometrium in the mesometrial side,
whereas the mature forms were found preferentially in
the Area 2 and 3 (Paffaro et al., 2003).
In this study, it was analyzed the presence of b7, a6,
a5 integrins, on the uNK cells, and ECM compounds as
fibronectin and laminin.
Integrin ligation promotes integrin clustering and subsequent integrin-mediated intracellular signal transduction (Mitra et al., 2005; Mitra and Schlaepfer, 2006;
Avraamides et al., 2008). In this work, it was observed
the b7 integrin positive reaction on the uNK cell surface.
The reaction pattern observed that the positive b7 reactions were predominantly concentrated in a side of the
uNK cells (clustering) closed to the uterine blood vessels,
suggested the uNK cell can bind to the blood vessels
through their b7 integrin molecules probably associated
ADHESION MOLECULES ON MOUSE UNK
to the a4 subunit forming the a4b7 molecule. Therefore,
this data suggest the uNK cell could use the a4b7 to
binding in the endothelial VCAM-1 or MAdCAM-1 to
infiltrate in the uterus and to bind in the fibronectin to
migrate in the uterus as the NK cells found in other
sites (Berlin et al., 1993; Erle et al., 1994; Helander and
Timonen., 1998; Cortijo et al., 2006). This also supports
Croy, et al (1997) who identified the alterations of the
uNK cells distribution in b7 integrin deficient mice.
Not expected, despite the a6 integrin positive reaction
on the uNK cells surface, no laminin positive reactions
were observed around these cells and in all extension of
the mesometrial side of the uterus. This data suggest
two possible functional roles to the a6 integrin on uNK
cell. Therefore, the uNK cells could use their a6 integrin,
forming with b1 or b4 integrin subunit, the VLA-6, or
the a6b4 integrin molecule, in other sites, as secondary
lymphoid organs before they migrate to the uterus, or
the a6 integrin in uNK could bind to the other matrix
compound of the uterus but not to the laminin.
The immunocytochemistry to the fibronectin showed a
heterogeneous distribution of this molecule in the pregnant uterus. It were not found to be fibronectin positive
reactions in the most decidualized areas of the Area 3,
and these could be explained by the occurrence of
decidualizing process in the pregnant uterus (Rider
et al., 1992). Therefore, the decidualization phenomenon,
which is characterized by the uterine fibroblast-like
transformation in decidual cells (Abrahamson and Zorn,
1993), followed to the ECM contend reduction (Rider
et al., 1992), suggest a fibronectin decrease in the decidualized areas as in the Area 3.
The fibronectin positive reactions around the uNK
cells in the Area 1 and 2 from the sixth to tenth gestational days, and the a5 integrin positive uNK cells
appear lately at eighth gestational day, agree to the
finds that cells tend to express integrins that are
matched to the ECM ligands present within their local
microenvironment (Stupack and Cheresh, 2002a) This
matched expression of adhesion receptor and ECM
ligand is also observed during the developmental (Drake
et al., 1992; Rupp and Little, 2001) and other tissue
remodeling events (Brooks et al., 1994). In these cases,
the repertoire of specific integrins expressed on a given
cell type is altered to adjust to concurrent changes
within the local ECM (Stupack and Cheresh, 2002b). In
addition, the nonexpression of fibronectin in all mesometrial side of implantation sites from twelfth to seventeenth gestational days also match to the a5 integrin
nonexpression in this period. These data suggested that
the uNK cells use the a5 integrin together with the b1
subunit constitutes the VLA-5 molecule to bind with fibronectin and migrate in the uterus. This affirmation is
supported to the data that have showed the uNK cells
viability in the fibronectin environment ‘‘in vitro’’ (Croy
and Kiso., 1993). These data also corroborates to our
findings (Paffaro et al., 2003), which suggested that the
uNK cell precursors migrate from the Area 1
(fibronectinþ), through the Area 2 (fibronectinþ), and
getting differentiated in the Area 3 (fibronectin).
It has been known that the number of uNK cells
decrease from the tenth gestational day to the end of
pregnancy (Stewart and Peel., 1981; Peel, 1989; Delgado
et al., 1996; Paffaro et al., 2003), and in the areas closed
to the embryo (Area 3) the senescent subtype of uNK
1087
cell are large in number. When the pregnancy develops,
the senescent uNK cell number increase in all mesometrial region including the Area 1 and 2 (Paffaro et al.,
2003). These previous findings match to the fibronectin
expression data of this study in which it was observed
the nonexpression of these molecule in the Area 3 (rich
on senescent uNK cells) and in all areas of mesometrial
side from twelfth to the seventeenth gestational days.
Notably, the a5 integrin expression was seen to be
totally negative fibronectin in Area 3, and this region
was described as a senescent uNK cell rich area (Paffaro
et al., 2003). This result agree to the findings that
showed over expression of unligated integrin a5, which
has been associated with apoptosis and reduced tumor
cell growth in vitro and in vivo (Giancotti and Ruoslahti,
1990; Varner et al., 1995; Kim et al., 2000; Plath et al.,
2000; Stupack et al., 2001). These authors suggest that
the expression of specific integrin complexes, in the absence of a suitable ligand, may promote cell death in a
caspase 8-dependent way (Stupack, 2005; Avraamides
et al., 2008).
Our studies showed trough the immunocytochemical
and a high selective DBA lectin cytochemical method
that fibronectin and a5 integrins are apparently the key
molecules for the establishment, proliferation, differentiation, migration, and survival of uNK cells during
pregnancy of mice. Further studies should be undertaken to show whether the loss of a5 integrin ligation
could inhibits these events and unligated a5 integrins
could actively and initiate apoptosis, which could characterizes the uNK integrin-mediated death.
ACKNOWLEDGMENTS
The authors are grateful to Dr. B. Anne Croy for
reviewing the manuscript and for valuable suggestions.
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