Immunocytochemical Studies of Adhesion Molecules on Mouse UNK Cells and Their Extracellular Matrix Ligands During Mouse Pregnancy.код для вставкиСкачать
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: firstname.lastname@example.org 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). 1082 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 1084 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. 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