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Pregnancies in diabetic women are at 4–12 more risk for pre-eclampsia, an urgent, acute onset complication of mid to late gestation, than pregnancies in normal women. Hallmarks of pre-eclampsia are hypertension, proteinuria and incomplete modification of endometrial spiral arteries. Transient, pro-angiogenic lymphocytes called uterine Natural Killer (uNK) cells are implicated in human and rodent spiral artery modification. We studied mid to late gestations in spontaneously type 1 diabetic NOD mice to ask if diabetes alters uNK cell homing and/or function.
Normoglycemic, prediabetic and diabetic NOD mice and controls were mated. Lymphocytes and endometrial endothelium and decidua were studied histologically and in functional assays.
Conception accelerated progression to overt diabetes in NOD females who had limited spiral artery development, heavier placentae and lighter fetuses displaying numerous birth defects compared with controls. UNK cell numbers were reduced in the decidua basalis of diabetic females while interferon-γ production was elevated. In diabetic NOD mice, decidual expression of the endothelial cell addressin MAdCAM-1 was aberrant in position while VCAM-1 expression was reduced. Assays of lymphocyte adhesion to tissue sections under shear forces indicated that diabetes compromises the potential homing functions of both endometrial endothelium and peripheral NK cells.
In diabetes, gestational endometrium has immune and vascular defects that likely to contribute to murine fetal loss and birth defects. Analogous problems and pre-eclampsia in diabetic women may involve similar mechanisms.
The NOD mouse is a well-characterized model of type 1 diabetes (reviewed in (1)). NOD mice develop spontaneous, T-cell mediated autoimmune insulitis, followed by diabetes that is clinically and genetically similar to human disease (reviewed in (2;3)). Disease progression is associated with a gain in islet expression of leukocyte homing molecules such as mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) and vascular cell adhesion molecule 1 (VCAM-1) (4). Immune dysfunctions in NOD mice include altered regulatory T cells (5–7) and functional as well as numerical deficits in NK and NKT cells (8;9). Despite a wealth of information on NOD mice, study of their reproductive biology is limited. Fetal consequences of maternal diabetes are described. These include death, birth defects, and potential for early development of type 1 diabetes in offspring (10–12). While normoglycemic NOD mice are excellent breeders (1), features of mid-gestation implantation sites in overtly diabetic mice are unknown.
Human pregnancies complicated by diabetes have a three to nine-fold increased risk of fetal/neonatal morbidity and mortality (13). These risks include death, intrauterine growth restriction (IUGR), neural tube defects, premature birth, and macrosomia (14). Risks are reduced but not eliminated by optimal maternal glycemic control, indicating a complex etiology. Histologically, placentae from diabetic women show evidence of hypoxia (increased nucleated fetal red blood cells), and immature villi with increased fibrinoid deposition (15). The supporting maternal vasculature is increased in length (16), but shows wall hyperplasia with lumen stenosis (16) and incomplete spiral artery modification (17). Diabetes increases the risk of pre-eclampsia (PE), a serious complication of pregnancy of unknown etiology, 12-fold (18). PE presents as hypertension and proteinuria and, without intervention, can develop into HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets) and eclampsia (convulsions). HELLP syndrome has significant maternal morbidity and mortality. Currently, the only successful treatment for PE is delivery of the placenta. PE is a multifactorial disease with immune, genetic, and environmental contributions. Histologically, hallmarks of PE are poor placentation, with hypoxic signs, and incomplete spiral artery modification (19).
Uterine Natural Killer (uNK) cells are a terminally differented NK cell subset that proliferate in the uteri of many species early in pregnancy (20). Functions of uNK cells are still under investigation, but include production of angiogenic factors (21;22). In mice, uNK cell-derived interferon (IFN)-γ is the established initiator of gestational spiral artery modification. The structural changes called, “spiral artery modification”, create large volume, low pressure vessels feeding into placentae. These changes are completed histologically in mice by midpregnancy (gestation day (gd)10 of 19–20 day term (23)). Transplantable uNK progenitor cells are not found in mouse uteri, but occur in all peripheral lymphoid tissues (24). Although hematopoeitic stem cells occur in human uteri (25), in vitro assays show that human CD56bright blood NK cells, the minor blood NK cell type, gain in functional interactions with endothelium of the decidua basalis at the pre-ovulatory menstrual cycle surge in luteinizing hormone (LH). This gain in potential for NK cell egress into decidua predicts uterine receptivity for transferred embryos (26) and is consistent with the dominance of CD56bright NK cell subset in decidua (27).
Diabetes is associated with defects in NK cell function and alterations in lymphocyte chemotaxis. We hypothesized that the incomplete spiral arterial remodeling of diabetic human pregnancies is due, at least in part, to decoy homing of uNK cells to the inflamed pancreas and/or reduced uNK cell function. The goal of this study was to characterize implantation sites in normoglycemic, prediabetic and diabetic NOD mice at mid-gestation.
NOD/LtJ, NOD/LtSz-scid.scid (NOD.scid), C56Bl/6J (B6), and FVB/NJ (FVB) male and female mice were purchased from The Jackson Laboratory (Bar Harbor, ME). C57BL/6N (B6) mice and BALB/c mice were purchased from Taconic Farms, Inc. (Germantown, NY). NOD and NOD.scid mice were housed in a barrier facility; B6 and FVB mice were housed conventionally. From nine weeks of age, NOD mice were monitored weekly for hyperglycemia using tail venipuncture and One Touch Ultra Glucose meter and strips (LifeScan, Burnaby, BC). NOD mice were classified as normoglycemic (<11 mmol/L), prediabetic (11.1–14.9 mmol/L), and diabetic (≥15 mmol/L on sequential measurements). Experimental design precluded developing a diabetes incidence curve for females in our NOD colony. The diabetes incidence for males was >50% at 30 weeks of age (n=8), consistent with published work (1).
Female NOD and NOD.scid mice with known blood glucose were paired with normoglycemic NOD males to obtain pregnancies. Copulation plug detection was named gd0. Gd-matched pregnancies in syngeneically mated B6, NOD.scid, FVB and BALB/c mice were used as controls. Mice were sacrificed at gd6 and 8 for immunohistochemistry, gd7 for adhesion assays or gd10 for morphometric analyses and quantification of IFN-γ. Peri-partum placentae and offspring were weighed.
Avertin-anaesthetized mice were euthanized by cervical dislocation. For histology, pancreas and uterus were dissected and fixed in fresh 4% neutral buffered paraformaldehyde (PFA) (Sigma-Aldrich, Oakville, ON). For adhesion assays, these tissues plus multiple peripheral lymph nodes (LN; cervical, submandibular, axial, mesenteric, iliac, superficial inguinal, and popliteal) were dissected, embedded in cryomatrix (Shandon Cryomatrix, Thermo Fisher Scientific, Inc. Waltham, MA) and snap-frozen in liquid nitrogen. All animal usage complied with protocols approved by Queen’s University’s Animal Care Committee.
PFA-fixed implantation sites were processed using an automatic tissue processor (Triangle Biomedical Sciences, Inc, Durham, NC) and paraffin embedded using standard methods (28). For each group, three implantation sites from three dams were serial sectioned at 7μm. At least 77 sections were cut at the centre of each implantation site and mounted to glass slides. Alternate slides were stained with H&E for vessel morphometry, or Periodic Acid Schiff (PAS) reagent for uNK cell ennumeration. Eleven sections, at least 42μm apart (to avoid duplicate enumeration of uNK cells), were scored per implantation site. UNK cells were enumerated within a standardized surface area in each of decidua basalis and mesometrial lymphoid aggregate of pregnancy (MLAp), a transient lymphoid structure between the circular and longitudinal smooth muscle coats of the uterus. Spiral artery wall and lumen diameters were measured and expressed as wall to lumen diameter ratios. When present in the tissue, three spiral artery cross sections were measured on each of the eleven sections from each implantation site. All analyses were performed using an AxioImager M.1 microscope and Axiovision software (Carl Zeiss, Oberkochen, Germany).
Indirect antibody staining was performed for peripheral lymph node addressin (PNAd), MAdCAM-1 and VCAM-1 on paraffin-embedded sections of pancreas and uterus from virgin, gd6, or gd8 normoglycemic NOD mice, diabetic NOD mice and B6 mice. Three viable implantation sites and six representative sections per antibody were used, plus isotype controls. Sections were deparaffinized, followed by antigen retrieval for MAdCAM-1 and VCAM-1 (citrate buffer and Proteinase K methods, respectively). Endogenous peroxidase was quenched with 0.3% H202 (30 min), followed by blocking with 10% normal rabbit serum in 1% BSA (1h). Primary antibodies were incubated overnight at 4°C (biotinylated rat anti-mouse PNAd, 1:100, MECA-79, Biolegend, San Diego, CA; LEAF™-purified rat anti-mouse MAdCAM-1, 1:100, MECA-367, Biolegend; rat anti-mouse VCAM-1, 1:20, MK-2, Antigenix America, Huntington Station, NY). MAdCAM-1 and VCAM-1 sections were incubated with rabbit anti-rat IgG biotinylated secondary antibody (1:100, Vector Labs, Burlingame, CA) (30 min). All sections were subjected to amplification with Vectastain® Elite ABC kit (Vector Labs), and DAB substrate for visualization.
12μm cryostat sections of substrate tissue (mouse LN, pancreas, and gd7 uterus) were cut immediately before use in the modified Stamper-Woodruff adhesion assay. Two sources of indicator lymphocytes were used; human peripheral blood lymphocytes (PBL) or mouse splenocytes. Human blood in ACD anticoagulant (BD Biosciences, San Jose, CA) was provided by a healthy, nulliparous, non-cycling adult female using depot-medroxyprogesterone acetate contraception. This donor had normal random and fasting blood glucose, insulin, and lipid profiles. PBL were isolated as previously described (24;29), adjusted to 5×106 cells/μL in serum-free RPMI, and incubated with anti-CD56 mAb (1:100, Coulter Immunology, Hialeah, FL).
Mouse splenocytes were dissociated mechanically, erythrocytes were lysed and lymphocytes were harvested following centrifugation over Lympholyte-M® (Cedarlane Laboratories, Burlington, ON). Aliquots of 5×106 cells/μL in RPMI, and labeled with either anti-CD49b mAb (DX5, 1:100, eBioscience, San Diego, CA) or CellTracker Blue CMAC (Molecular Probes from Invitrogen, Burlington, ON), a fluorescent intravital dye without effects on adhesive function (30).
To assess changes in endothelial cell function induced by diabetes, aliquots of PE-Cy5-CD56 labeled human PBL were overlaid onto sections of normoglycemic or diabetic NOD pancreas, LN, or gd7 uterus or matched B6 tissues on a rotating table (112 rpm) and incubated (30 min, 4°C). Non-adherent cells were removed by dipping the slides into PBS; slides were then fixed (4% PFA, 30 min), rinsed (PBS then dH2O) and mounted. Fluorescent adherent cells were scored in 25 high power fields (HPF)/specimen at 400× magnification.
To assess changes in lymphocyte function with progression to diabetes, B6 substrate tissues were incubated with splenocytes from normoglycemic or diabetic NOD mice, or B6 mice. The experiment was conducted as above.
Uteri were collected from virgin or gd10 normoglycemic, prediabetic or diabetic NOD mice and from a gd10 BALB/c mouse. The MLAp and decidua basalis were dissected from healthy implantation sites; each was pooled by uterus in a microcentrifuge tube containing 100μL RPMI, and homogenized by Kontes micropestle (Fisher Scientific Inc. Waltham, MA). Mesometrial tissue was dissected from virgin mice and handled similarly. Following centrifugation (800g; 5 min), supernatants were collected and stored at −20°C until assayed for IFN-γ by ELISA as previously described (31). The limit of detection of this assay was 0.01 IU IFN-γ.
Data were analyzed using Prism 4.03 Statistical Software package (GraphPad, Inc., San Diego, CA), and are presented as mean ± SEM. Blood glucose values were analyzed by repeated measures ANOVA, followed by both linear trend and Bonferroni’s Test. Fetal and placental weights were analyzed by independent 1-tailed t-tests. Resorption rates, MLAp uNK counts, spiral artery lumen diameters and adhesion assays were analyzed using ANOVA with Bonferroni’s post-test. Kruskal-Wallis tests were used to analyze uNK counts within the decidua basalis, IFN-γ quantification and spiral artery wall:lumen ratios, followed by Dunn’s post-test. A p-value of <0.05 was considered statistically significant.
Pregnant mammals develop mild glucose intolerance at mid-gestation to elevate fetal glucose availability. In normal mice, blood glucose increases at gd4.5 (32). All pregnant NOD mice (normoglycemic, n=5; prediabetic, n=7; diabetic, n=7) displayed significant increases in blood glucose from their individual baselines (three prior measurements; Figure 1), p<0.005. Mean gd10 blood glucose values were significantly increased only in mice overtly diabetic (19.30±4.352, vs. normoglycemic, 6.885±0.8947 and prediabetic, 7.950±1.698) prior to mating (p<0.005).
The impact of diabetes on NOD fetuses was scored grossly (Table 1). Diabetic mice (n=4) had significantly more resorption sites than normoglycemic (n=3), prediabetic (n=6), and NOD.scid (n=3) mice at gd10 (p=0.021). However, gross study underestimated losses in diabetic NOD mice since additional resorptions were found histologically. Weights of gd18 fetuses and placentas from one overtly diabetic NOD dam (n=1) were compared to fetal weights from FVB dams (n=3). NOD placental weight was significantly greater than FVB placental weight (0.13±0.0080g vs. 0.086±0.0031g, p<0.0001). Although NOD placental weights were greater, morphometry revealed a smaller, more compact placental area than FVB placentae (data not shown).
Fetuses from the diabetic NOD dam were smaller than those from FVB dams (0.98±0.065g vs.1.22±0.068g, p<0.05). FVB fetuses included one that was very underweight fetus but had no gross abnormalities and another fetus with agnathia. In contrast, NOD fetuses had gross neural tube defects (spina bifida and exencephaly, n=3), and growth restriction (Figure 2). Further necropsy revealed additional defects including situs inversus, diaphragmatic hernia, agnathia, anopthalmia, cardiac hypertrophy, pancreatic hyperplasia, asplenia and an occult spina bifida. Each NOD fetus had at least one defect, while most, as reported by others (10;11), had multiple defects.
In NOD.scid (n=3) and normoglycemic NOD mice (n=3), uNK cell numbers in the MLAp (site of most uNK cell proliferation) and decidua basalis (site dominated by post-mitotic uNK cells) were consistent with published numbers in normal mice (33). Prediabetic (n=3) and diabetic NOD mice (n=3) had reduced uNK cell numbers in the MLAp (81.9±1.18; 80.8±1.02) compared to NOD.scid mice (87.5±2.22), p<0.05, Figure 3. Diabetic NOD mice had drastically reduced decidual uNK numbers compared to all other groups (26.1±0.67), p<0.001. Decidual uNK cells were small and had with limited cytoplasmic granularity, suggesting immaturity.
To further assess uNK cell function in NOD mouse implantation sites, IFN-γ was quantified in the MLAp and decidua basalis. Gd10 BALB/c (n=1) MLAp and decidua basalis were positive controls, and IFN-γ concentrations matched a previous report for immune-competent mice (9.04 and 12.92, respectively) (31;31). Mesometrial uterus from virgin, normoglycemic NOD (n=1) mouse lacked IFN-γ, a finding typical of virgin uteri from normal mice. IFN-γ concentrations in decidua basalis and MLAp from normoglycemic (n=3) and prediabetic NOD (n=3) mice did not differ. However, IFN-γ concentration within the decidua basalis of diabetic NOD mice (n=3) was significantly higher than in decidua basalis from normoglycemic NOD mice (34.01±4.47 vs. 9.35±3.74, p<0.05). No difference in IFN-γ concentration within the MLAp was found between normoglycemic NOD, prediabetic NOD, and diabetic NOD mice.
Fewer spiral arteries were found in implantation sites from prediabetic (n=3) and diabetic NOD (n=3) mice than in normoglycemic NOD (n=3) or NOD.scid mice (n=3). Spiral artery lumen diameters shortened as blood glucose increased. For arteries in prediabetic (26.45±0.62μm) and diabetic dams (23.36±0.57μm), values were statistically different compared with normoglycemic NOD (36.82±1.04μm) or NOD.scid mice (34.43±0.81μm), p<0.001 (Figure 4A). Spiral arterial wall:lumen diameter ratios for normoglycemic NOD mice and NOD.scid mice were greater than reported for B6 mice (34). This may indicate a basal arterial defect in the NOD strain. Spiral arteries from prediabetic (3.33±0.092) and diabetic NOD mice (3.82±0.11) had even greater wall:lumen ratios than normoglycemic NOD (2.39±0.058) and NOD.scid mice (1.82±0.044), suggesting progressive loss of capacity for spiral artery modification with increasing hyperglycemia, p<0.01 (Figure 4B).
To determine if decidua basalis of diabetic NOD mice expressed addressins aberrantly, PNAd, MAdCAM-1, and VCAM-1 were localized at gd6 and gd8. PNAd was not significantly detected in any implantation sites. MAdCAM-1 was localized to the lateral sinusoids in B6 (n=3) while, in NOD mice (n=3), reactivity was found in anti-mesometrial decidua. VCAM-1 was strongly expressed in the central decidua, mesometrial and anti-mesometrial decidua of B6 mice (Figure 5A). VCAM-1 was similarly localized in NOD mice, but staining intensity was weaker (Figure 5B).
PNAd, MAdCAM-1, and VCAM-1 expression was absent from pancreatic islets from pregnant B6 mice (Figure 5C). Moderate expression of PNAd, MAdCAM-1, and VCAM-1 was observed in islets from pregnant normoglycemic NOD mice. These molecules were strongly expressed in some of the islets remaining in pregnant diabetic NOD mice (Figure 5D).
To assess endothelial homing receptor function, CD56-tagged indicator lymphocytes were evaluated for adhesion to various tissues. Adhesion to LN was used as a positive control and was similar for normoglycemic NOD (n=8), diabetic NOD (n=10) and B6 (n=6) substrates (Figure 6). Adhesion to diabetic NOD pancreas (5.28±1.03 cells/HPF) was greater than to B6 pancreas (1.58±0.19 cells/HPF), p<0.05. Low adhesion to B6 pancreas is consistent with published data. Adhesion to B6 uterus was localized to decidua basalis and low, concurring with previous reports for donors not at the ovulatory stage of their menstrual cycle (35). Adhesion to normoglycemic NOD uterus (13.9±3.3 cells/HPF) was greater than to diabetic NOD (6.7±0.50 cells/HPF) or B6 uterus (3.46±0.54 cells/HPF), p<0.05. CD56+ cells showed less localized binding to decidua basalis in decidual sections from diabetic NOD mice; cells bound to uterine glands, antimesometrial decidua, and myometrium. Comparisons to controls used only cells bound to decidua basalis.
Fluorescently tagged splenic NK cells from diabetic NOD mice (n=2) showed significantly more adhesion to normal B6 (n=2) peripheral LN (6.28±0.24 vs. 3.46±0.38 cells/HPF), p<0.05 (Figure 7A). Adhesion of splenic NK cells from NOD mice (diabetic NOD, 4.80±0.0050; normoglycemic NOD, 3.39±0.43 cells/HPF) to B6 pancreatic endothelium, regardless of blood glucose, was increased over NK cells from B6 mice (1.84±0.0040 cells/HPF), p<0.05. There were no differences between the three splenic NK cell populations in adhesion to gd7 B6 uterus.
In these experiments, total (CMAC-stained) splenocyte binding was enumerated on adjacent tissue sections to enable calculation of NK cell enrichment (Figure 7B). About 10% of the splenocytes from B6 and normoglycemic NOD mice adhering to B6 LN and pancreas were DX5+ NK cells. In diabetic NOD mice, the proportional binding of NK cells increased to B6 LN (30.7±4.27%), and was significant in pancreas (51.5±18.1%), p<0.05. The proportion of DX5+ B6 splenocytes binding to B6 uterine endothelium was high (67.0±0.88%). This proportion was lower for splenocytes from normoglycemic NOD mice (33.4±9.12%) and was significantly different for diabetic NOD mice (27.1±2.81%), (p<0.05). This suggests that NK cells from diabetic mice are reduced in ability to engage with uterine endothelium for extravasation and that other cell types compete for these receptors. This may skew the immune cell populations at a diabetic maternal-fetal interface.
This study addressed whether reduced uterine homing and/or function of NK cells occurs in diabetic pregnancy that would impair pregnancy-associated spiral arterial modification and link endometrial lymphocytes to the pregnancy complications of diabetic women. Spontaneous disease in type 1 diabetic NOD mice was used as a model for gradual disease progression. Unexpectedly, defective spiral arterial development and modification were found in normoglycemic NOD and NOD.scid mice. This pathology is absent in implantation sites of pregnant scid mice, which lack T and B cells (36). NOD.scid mice have similar NK cell defects to NOD mice, but do not develop insulitis or diabetes. Of particular note was the paucity of spiral arteries, which are major vessels, in NOD implantation sites. This has not been reported in other strains. Reduced neovascularization has been reported in NOD mice subjected to femoral artery ischemia. This was attributed to lower induction of vascular endothelial growth factor (VEGF) in ischemic NOD tissue (37). VEFG (38) and the VEGF family member placental growth factor (PlGF) (39) are major uNK cells products. In other tissues, VEGF regulates the expression of chemokine ligands that position recruited bone marrow-derived circulating cells (40) and promote flexible (i.e. not developmentally hard wired) angiogenesis. Endometrium requires flexible angiogenesis since the number of arriving blastocysts is variable between reproductive cycles. The vascular histopathology was more severe in pre-diabetic and diabetic NOD mice (5;41).
Progesterone, the essential hormone of pregnancy, and IFN-γ are among the regulators of VEGF transcription (42;43). Progesterone is absolutely required for terminal uNK cell differentiation and uNK cells are the major source of mouse implantation site IFN-γ during the first half of gestation. Since spiral artery modification in mice is normally initiated by IFN-γ released from uNK cells (36;44), this cytokine was quantified in implantation sites. As in other strains, IFN-γ was not detected in virgin uterus but was present between gd6-10 in NOD implantation sites. Levels were comparable to those in a normal mouse and consistent with our earlier published data (31), except in the decidua basalis of overtly diabetic gd10 females. This tissue, in which uNK cells are reduced, unexpectedly showed elevated IFN-γ. This finding suggests that the primary deficit in spiral artery development in NOD mice that is not overcome by the influx of IFN-γ producing uNK cells. The elevated IFN-γ we saw may relate to the disease stage we studied. Rodacki et al. (45) showed in type 1 diabetic patients, numbers and activation status of blood NK cells vary with disease stage. At onset, NK cells are reduced but unusually activated producers of IFN-γ. With longer standing disease, both patients and NOD mice lose NK cell activation receptors (45;46). Because spontaneously diabetic NOD mice are fragile, usually older than mice used for first pregnancy studies and prescribed for removal from breeding colonies, we bred our mice as soon as they became diabetic. This may have been in the interval of elevated NK cell activation. We attempted support of diabetic females by subcutaneously implanted slow release insulin rods before mating. This normalized blood glucose only until mating. By gd10, all insulin-treated females had reverted to hyperglycemia (n=6; unpublished).
Changes in patterns of vascular addressins are found in diabetic NOD mice (4;47) but effects of pregnancy on these molecules have not been addressed nor have comparisons of expression been made between gestational endometrium and pancreas. We found alterations in the normal pattern of MAdCAM-1 and VCAM-1 expression within the early gestational uterus of diabetic NOD mice. MAdCAM-1 was expressed in an aberrant location, and VCAM-1 expression was less intense. VCAM-1 is thought to be the key addressin in early uNK cell recruitment to deciduas basalis (48). The successful localization of many uNK cells in diabetic NOD pregnancy supports this postulate (48). Homing receptor elevation underlies insulitis, which is present in all adult NOD mice, regardless of diabetic state. Assays of lymphocyte adhesion using NOD pancreas showed that, in pregnancy as in non pregnant NOD mice, hyperglycemia elevates islet adhesion (49). The pattern of lymphocyte adhesion to decidualized uterus was less straightforward. There was a striking increase in the level of adhesion to uterus from normoglycemic NOD mice relative to control. One interpretation of these data is that endometrial endothelium in gestational uterus compensates against pancreatic endothelial cell recruitment signals early in disease, but cannot sustain this state as disease progresses. Adhesion assays using NOD splenocytes on B6 tissue, showed diabetes-promoted gains in the ability of lymphocytes to adhere to pancreas and peripheral LN but not to uterus. This is consistent with the relatively normal numbers of uNK cells quantified within gd6-8 implantation sites. It also suggests there is a large reserve of circulating NK cells with tissue homing potential enabling NK cell elevation in one tissue without decoy homing from another tissue. Previous studies of normal lymphocytes reported NK cell enrichment among cells adhering to gd7 uterus (26). Similar enrichment was not observed in assays using splenocytes from diabetic NOD mice. Splenic NK cells from diabetic NOD mice were enriched, however, by adhesion to pancreas, suggesting preferential homing to this organ over pregnant uterus.
In human diabetic pregnancy, placental and neonatal sizes are usually correlated; diabetic women frequently having abnormally large placentae with macrosomic infants. The diabetic NOD placentae were abnormally heavy, but fetal trophoblastic areas were greatly reduced relative to maternal decidua basalis, in all pregnant NOD and NOD.scid mice. Human pregnancies complicated by both diabetes and IUGR or PE often display hypercellular placental histopathology accompanying small size. Human pregnancies complicated by diabetes have various vasculopathies, which tend to perturb spiral artery modification. Most frequently, long, non-spiraling arteries persist that remain responsive to vasoactive substances. In pathological diabetic pregnancies (i.e. co-morbid PE or IUGR), spiral arteries are incompletely modified resulting in placenta hypoxia. Depending on the timing of the insult, placental insufficiency and/or hypoxia can result in growth restriction, or abnormal organ development, intrauterine death or fetally-programmed risk for post-natal health impairment. Fetuses of overtly diabetic NOD dams showed early and late resorption, growth restriction, and a high rate of developmental defects. Our study shows there are major endometrial contributions associated with pathogenesis that include poor vascular bed development, and altered endometrial lymphocyte recruitment and function, particularly of proangiogenic uNK cells. Thus, gestation in spontaneously diabetic NOD females appears to model IUGR and PE in human diabetic pregnancy. Links between NK cells and autoimmunity are beginning to be understood. Their relationships during physiological stress such as pregnancy require further study.
We thank Drs. E. Leiter (Jackson Laboratory, Bar Harbor, ME), M. van den Heuvel (University of Western Ontario, London, ON), C. McKerlie and Mr. C. Fleming (Mount Sinai Hospital, Toronto, ON) for invaluable advice, and M. Bilinski and R. Watering for technical support. This work is supported by the Canadian Institutes for Health Research. B.A.C. is a Tier 1 Canada Research Chair.