Affinity-dependent alterations of mouse B cell development by noninherited maternal antigen

We have examined the passage of maternal cells into the fetus during the gestation and postpartum in mice. Using enhanced green fluorescent protein (EGFP)-transgenic females, we showed that maternal cells frequently gain access to the fetus, mostly in syngeneic pregnancies, but also in allogeneic and outbred crosses. EGFP-transgenic cells, including B, T, and natural killer cells, can persist until adulthood, primarily in bone marrow and thymus. We then asked whether maternal cells, bearing antigens not inherited by the fetus, influence the development of fetal and neonatal B lymphocytes. We have used the B cell receptor 3-83 mu/delta transgenic mouse model, whose B cells recognize the major histocompatibility complex class I molecules H-2Kk and H-2Kb, with a high or moderate affinity, respectively. The fate of transgenic B cells in animals exposed to noninherited H-2Kk or H-2Kb maternal antigens (NIMA) during gestation and lactation was compared with those of nonexposed controls. In H-2Kk-exposed fetuses, NIMA-specific transgenic B cells are partially deleted during late gestation. Nondeleted cells have downmodulated their B cell receptor. In contrast, in NIMA H-2Kb-exposed neonates, transgenic B cells present an activated phenotype, including proliferation, upregulation of surface CD69, and preferential localization in the T cell zone of splenic follicles. This state of activation is still clearly detectable up to 3 wk of age. Thus, we show that fetal and neonatal B cell development is affected by maternal cells bearing antigens noninherited by the fetus and that this phenomenon is highly dependent on the affinity of the B cell receptor for the NIMA.     

Appearance of functional EGF receptor kinase during rodent embryogenesis.

Mouse and rat embryonic tissues at various stages of development were examined for epidermal growth factor (EGF) receptor kinase activity. The phosphorylated EGF receptor from embryonic tissues appeared as a band of mol. wt. 170 000 daltons on SDS gels. It was clearly demonstrable in the developing mouse fetus from 10 days of gestation onwards. The distribution of the EGF receptor kinase was studied in various tissues of 13 day mouse fetuses. The activity was apparent in the skin, developing skeletal muscles and various internal organs but was notably absent in the liver and brain. The amnion was found to be one of the richest sources of activity while the yolk sac was negative, and the placenta was weakly positive. In 16 day rat fetuses the distribution was quite similar to that of the 13 day mouse fetus. The liver acquired EGF receptor kinase activity by 18 days of gestation and had high activity in neonates. Phosphoamino acid analysis revealed that phosphotyrosine was the major labelled amino acid residue in the embryonic tissues. Thus, the EGF receptor of fetal tissues as studied by immune precipitation and phosphorylation appears to be a similar entity to that found in adult mammalian tissues. This functional EGF receptor kinase activity could first be detected at the time of onset of organogenesis.     

Uptake and metabolism of epidermal growth factor in the perfused human placenta

Uptake of 125I-labelled epidermal growth factor into trophoblast, and its subsequent fate, was studied in an isolated dually-perfused lobule of term human placenta. 125I-EGF added into the maternal circulation was rapidly taken up into the placental tissue where a portion was degraded and most of the breakdown products released back into the maternal circuit. At the end of the 2 h perfusion, radioactivity in the tissue accounted for 52% of the initial dose. 12.9% of the radioactivity remaining in the maternal circuit at the end of the perfusion, amounting to only 5.2% of the initial activity, could be identified as intact EGF by immunoaffinity chromatography. About 45 min after the start of the perfusion there was a sustained rise in the 125I activity in the fetal circulation accounting for 4.6% of the initial activity, and a small proportion of this (0.22% of the dose) could be immunologically characterised as EGF. In the presence of the acidotrophic agent chloroquine, there was a similar rapid clearance from the maternal circulation, which was not associated with breakdown. The tissue retention was slightly enhanced and there was very little transfer of activity into the fetal circulation.     

Topography of human placental receptors for epidermal growth factor

These studies were undertaken to determine whether term human placental microvillus plasma membranes, which are exposed to maternal blood, and basolateral plasma membranes, which are in close proximity to fetal blood capillaries, contain receptors for epidermal growth factor (EGF). These two highly purified membranes bound 125I-EGF with similar affinity (apparent dissociation constants, 0.07-0.12 nM, but the total number of available receptors was greater in microvillus (8.2 pmol/mg protein) compared to basolateral (4.9 pmol/mg protein) plasma membranes. Detailed characterization of 125I-EGF binding to these membranes revealed numerous similarities as well as differences. The two membranes contained two major (155 and 140 kDa) and at least three minor (115, 175, and 210 kDa) specific 125I-EGF binding proteins. The 115-kDa protein was only found in basolateral plasma membranes. The 155-kDa protein was predominantly labeled in microvillus, whereas the 140-kDa protein was labeled predominantly in basolateral plasma membranes. The addition of protease inhibitors did not alter the multiple 125I-EGF binding proteins pattern found in these membranes. EGF stimulated phosphorylation of 140- and 155-kDa proteins in both microvillus and basolateral plasma membranes. However, the 155-kDa protein was phosphorylated to a greater extent in microvillus, whereas both 140- and 155-kDa proteins were phosphorylated equally in basolateral plasma membranes. Light and electron microscope autoradiographic studies revealed that 125I-EGF preferentially associated with microvillus plasma membranes. The data demonstrates the presence of EGF receptors in outer cell membranes of syncytiotrophoblasts and suggests that maternal EGF may influence syncytiotrophoblast function by binding to receptors in microvillus plasma membranes, while fetal EGF may also influence syncytiotrophoblast function but via receptors in basolateral plasma membranes.     

Evidence for a subtype of insulin-like growth factor I receptor in brain

We examined the structure of receptors for insulin-like growth factor I (IGF-I), insulin, and epidermal growth factor (EGF) in human brain and human placenta using affinity cross-linking procedures and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In human brain, proteins specifically cross-linked to 125I-IGF-I, 125I-insulin, and 125I-EGF had apparent molecular weights of 120,000, 115,000 and 170,000, respectively. In human placenta, proteins cross-linked to 125I-IGF-I and 125I-insulin were 10 kDa larger than the corresponding subunits in brain. The receptor labeled by 125I-EGF in placenta was indistinguishable from the EGF receptor in brain. The size discrepancy of IGF-I receptors in brain and placenta was no longer apparent after removing the carbohydrate moieties of the proteins with endo-beta-N-acetylglucosaminidase F (EndoF). Furthermore, the brain IGF-I receptor was not cleaved by neuraminidase, whereas, the placental IGF-I receptor had increased mobility on SDS gels following neuraminidase treatment. The results indicate that receptors for IGF-I and insulin in human brain are structurally distinct from the corresponding receptors in human placenta, the structural heterogeneity of the receptors involves differences in N-linked glycosylation, particularly the terminal processing steps, and EGF receptors are present in human brain and human placenta but are structurally similar in these tissues. We conclude that there is a selective modification in the glycosylation of receptors for IGF-I and insulin in brain.     

Direct modulation of epidermal growth factor binding by cholecystokinin

The effects of cholecystokinin-octapeptide (CCK8), the biologically active C-terminal moiety of cholecystokinin (CCK), on the binding of epidermal growth factor (EGF) were studied in isolated rat pancreatic acini. CCK8 inhibited 125I-EGF binding in a dose-dependent manner. One-half maximal inhibition occurred at 5 X 10(-10)M, and maximal inhibition at 10(-8)M CCK8. This inhibitory effect was detectable within 5 minutes of addition of CCK8, and was not associated with enhanced degradation of 125I-EGF in incubation media. Unlabeled EGF exerted only a slightly greater inhibitory effect than CCK8 on 125I-EGF binding at equivalent molar concentrations. In contrast to CCK8, the gastrointestinal hormone vasoactive intestinal polypeptide (VIP) did not significantly alter EGF binding. CCK8 also inhibited EGF binding in mouse pancreatic acini, but did not alter binding in A-431 human carcinoma cells. These findings suggest that physiological levels of CCK may regulate EGF binding in the pancreas and other tissues with receptors for both hormones. They thus point to a previously unrecognized mechanism for hormonal interaction.     

Accumulation of epidermal growth factor receptors in retinoic acid-treated fetal rat lung cells is due to enhanced receptor synthesis

125I-Epidermal growth factor (EGF) binding capacity in fetal rat lung (FRL) cells is increased approximately 2 to 3-fold within 18 h of retinoic acid addition. Analysis of 125I-EGF binding assays at 0 C reveals approximately 25,000 receptors per cell, while analysis of growth factor binding to retinoic acid-treated cells demonstrates an increase in receptor levels to approximately 70,000 receptors per cell with no detectable changes in receptor affinities. We show by immunoprecipitation of 35S-methionine labeled EGF receptors that retinoic acid addition produces an increase in the accumulation of EGF receptor protein. Using brief pulses of 35S-methionine, an increase in EGF receptor synthesis can be identified within 3 h after retinoic acid addition. These results are the first to demonstrate that a retinoic acid-induced increase in 125I-EGF binding capacity is due to increased EGF receptor protein synthesis. Also, we find that a transient decrease in the rate of EGF receptor turnover occurs when retinoic acid is initially added to FRL cells. On the basis of our data, we conclude that the retinoic acid-induced accumulation of EGF receptors in FRL cells is primarily due to increased receptor synthesis. The effect of retinoic acid on EGF receptor turnover may be a secondary factor, influencing the rate at which receptors accumulate.     

Feto-maternal production and transfer of cortisol in the rhesus (Macaca mulatta)

Four pregnant rhesus monkeys cnd their fetuses. were infused constantly with ¹⁴C-cortisol and ³H-cortisol. Steady state plasma specific activities for ¹⁴C and ³H-cortisol were obtained after 80 to 90 minutes in both mother and fetus. These data and the rates of infusion of radioactivity were used to calculate the following parameters for both mother and fetus: 1) metabolic clearance rates, 2) production rates, 3) mean adrenal secretory rates, 4) transfer rates from mother to fetus and fetus to mother cnd, 5) the fraction of cortisol in each vascular compartment derived from the maternal and fetal edrenals. Plasma cortisone concentrations, as well as the fraction of cortisone derived from fetal and maternal cortisol were determined. Tetrahydrocortisol and tetrahydrocortisone concentrations were calculated. Mean cortisol secretory rates for the maternal and fetal adrenals were 60.0±11.8 and 1.82±0.42 mg/day. Fifty-eight % of the cortisol in the fetal compartment was of maternal origin. During transfer across the placenta to the fetus, cortisol was largely converted to cortisone. In fetal plasma 76% of the cortisone was of maternal origin. Cortisone concentrations in fetal plasma were higher than those of cortisol.     

Oxidative stress promotes the increase of matrix metalloproteinases-2 and -9 activities in the feto-placental unit of diabetic rats

Maternal diabetes increases the risk of congenital malformations, placental dysfunction and diseases in both the neonate and the offspring's later life. Oxidative stress has been involved in the etiology of these abnormalities. Matrix metalloproteases (MMPs), involved in multiple developmental pathways, are increased in the fetus and placenta from diabetic experimental models. As oxidants could be involved in the activation of latent MMPs, we investigated a putative relationship between MMPs activities and oxidative stress in the feto-placental unit of diabetic rats at midgestation. We found that H₂O₂ enhanced and that superoxide dismutase (SOD) reduced MMPs activities in the maternal side of the placenta and in the fetuses from control and diabetic rats. MMPs were not modified by oxidative status in the fetal side of the placenta. Lipid peroxidation was enhanced in the maternal and fetal sides of the placenta and in the fetus from diabetic rats when compared to controls, and gradually decreased from the maternal placental side to the fetus in diabetic animals. The activities of the antioxidant enzymes SOD and catalase were decreased in the maternal placental side, catalase activity was enhanced in the fetal placental side and both enzymes were increased in the fetuses from diabetic rats when compared to controls. Our data demonstrate changes in the oxidative balance and capability of oxidants to upregulate MMPs activity in the feto-placental unit from diabetic rats, a basis to elucidate links between oxidative stress and alterations in the developmental pathways in which MMPs are involved.     

Plasma placental RNA allelic ratio permits noninvasive prenatal chromosomal aneuploidy detection

Current methods for prenatal diagnosis of chromosomal aneuploidies involve the invasive sampling of fetal materials using procedures such as amniocentesis or chorionic villus sampling and constitute a finite risk to the fetus. Here, we outline a strategy for fetal chromosome dosage assessment that can be performed noninvasively through analysis of placental expressed mRNA in maternal plasma. We achieved noninvasive prenatal diagnosis of fetal trisomy 21 by determining the ratio between alleles of a single-nucleotide polymorphism (SNP) in PLAC4 mRNA, which is transcribed from chromosome 21 and expressed by the placenta, in maternal plasma. PLAC4 mRNA in maternal plasma was fetal derived and cleared after delivery. The allelic ratios in maternal plasma correlated with those in the placenta. Fetal trisomy 21 was detected noninvasively in 90% of cases and excluded in 96.5% of controls.     

Expression of mRNAs for alpha-fetoprotein (AFP) and albumin and incorporation of AFP and docosahexaenoic acid in baboon fetuses.

alpha-Fetoprotein and albumin, two members of a multigene family, reversibly bind fatty acids with high affinity. The origin of alpha-fetoprotein (AFP) and albumin present in fetal tissues other than the liver and yolk sac is a subject of controversy. In this work, we have searched for the presence of the albumin and AFP mRNA molecules in different fetal organs of the baboon (Papio cinocephalus), using a highly sensitive gel-blot hybridization assay with human albumin and AFP cDNA probes. Large amounts of albumin and AFP mRNA molecules were found in the fetal liver; significant quantities were also present in the gastrointestinal tract and in the kidney. No detectable levels were found in the other tissues examined (brain, skin, spleen, pancreas, muscle, heart, thymus, placenta, and amnion). After injection of radiolabeled AFP into pregnant baboons, all fetal tissues took up the protein. White adipose tissue, kidney, intestine, lung, liver, and cerebral cortex showed a great uptake of exogenous AFP. [14C]Docosahexaenoic acid (22:6, n-3), injected at the same time, was actively transferred from the maternal compartment across the placenta and incorporated into cellular lipids by all fetal tissues and particularly by liver (around 70% of total incorporation). The levels of [14C]docosahexaenoic acid per gram of tissue increased in the order: maternal blood less than placenta less than fetal liver, indicating a selective accumulation of this fatty acid by the fetus. These results indicate that intracellular AFP in non-hepatic tissues of the developing baboon is, for the most part, of plasma origin.     

Localization and secretion of inhibins in the equine fetal ovaries

To clarify the source of inhibins in equine female fetuses, concentrations of immunoreactive (ir-) inhibin, inhibin pro-alphaC, and inhibin A in both fetal and maternal circulation and in fetal ovaries were measured. In addition, the localization of inhibin alpha and inhibin/activin beta(A), and beta(B) subunits and the expression of inhibin alpha(A) and inhibin/activin beta(A) subunit mRNA in fetal ovaries were investigated using immunohistochemistry and in situ hybridization. Concentrations of circulating ir-inhibin, inhibin pro-alphaC, and inhibin A were remarkably more elevated in the fetal than in the maternal circulation between Days 100 and 250 of gestation. Fetal ovaries contained large amounts of ir-inhibin, inhibin pro-alphaC, and inhibin A. In contrast, these inhibin forms were undetectable in both the maternal ovaries and placenta. The inhibin alpha and inhibin/activin beta(A) and beta(B) subunit proteins were localized to enlarged interstitial cells of the equine fetal ovary. Expression of inhibin alpha and inhibin/activin beta(A) subunit mRNAs were also observed in the interstitial cells. We conclude that the main source of large amounts of inhibins in fetal circulation is interstitial cells of fetal ovary and is not of maternal origin. Furthermore, these inhibins may play some important physiological roles in the development of gonads in the equine fetus.     

Inhibition of the binding of 125I-labelled epidermal growth factor to mouse cells by a mitogen in goat mammary secretions.

Pre-colostrum and colostrum from goats cause a marked inhibition of the binding of 125I-labelled epidermal growth factor (125I-EGF) to Swiss 3T3 cells. The ability of these secretions to inhibit 125I-EGF binding is closely correlated with the ability to stimulate DNA synthesis in quiescent 3T3 cell cultures, suggesting that goat mammary secretions may contain an EGF-related mitogen. However, the material in colostrum which inhibits 125I-EGF binding to Swiss 3T3 cells is a basic protein with Mr greater than 20000 and is thus quite different from mouse and human EGF. Furthermore, the colostral-mediated inhibition of 125I-EGF binding, although rapid and apparently competitive, differs from the inhibition of binding induced by native, unlabelled EGF. Thus, the inhibitory effect of colostrum is markedly decreased when the assay temperature is shifted from 37 degrees C to 4 degrees C whereas unlabelled EGF is an effective competitive inhibitor at both 37 degrees C and 4 degrees C. Incubation of cells with EGF causes a reduction in cell surface EGF receptors whereas exposure to colostrum does not induce down-regulation of the EGF receptor. Our results suggest that the colostral factor does not bind directly to EGF receptors but inhibits 125I-EGF binding by an indirect mechanism which involves a temperature-sensitive step.     

Consumption of EGF by A431 cells: evidence for receptor recycling

We examined the extent of EGF consumption by EGFR in A431 cells. When 125I-EGF was added to A431 cell cultures at low or high density, at concentrations which corresponded to 10-fold excess of ligand over receptor on the cell surface, most of the 125I-EGF was consumed within 2 h. The amounts of 125I-EGF consumed were much greater than available EGFR on the A431 cells, by a factor of 6.5 in low-density cultures and 5.8 in high-density cultures. When the concentration of 125I-EGF was increased in low density cultures, further consumption of 125I-EGF by the A431 cells was greatly reduced, partially due to a rapid down regulation of EGFR. However, when higher concentrations of 125I-EGF were added to high density cultures, with reduced receptor down regulation, the cells continued to consume a large fraction of the EGF in the culture medium. The consumption of 125I-EGF by these cultures was in excellent agreement with the measured amount of ligand internalized into the cell. EGF consumption was far in excess of the number of EGFR down regulated or degraded. Only a minor portion of the EGFR could have been replaced during the assay period by synthesis of new EGFR or from the intracellular pool of EGFR, and the fluid-phase uptake of EGF is only temporarily increased by exposure to EGF. Our results demonstrate that EGFR in high density A431 cell cultures recycled many times. The apparent level of recycling was dependent upon the concentration of EGF and followed Michaelis-Menton kinetics for ligand concentrations as high as 215 nM. At this EGF concentration, high-density cultures consumed 45 EGF molecules per receptor over a period of 6 h.     

Oxidative stress promotes the increase of matrix metalloproteinases-2 and -9 activities in the feto-placental unit of diabetic rats

Maternal diabetes increases the risk of congenital malformations, placental dysfunction and diseases in both the neonate and the offspring's later life. Oxidative stress has been involved in the etiology of these abnormalities. Matrix metalloproteases (MMPs), involved in multiple developmental pathways, are increased in the fetus and placenta from diabetic experimental models. As oxidants could be involved in the activation of latent MMPs, we investigated a putative relationship between MMPs activities and oxidative stress in the feto-placental unit of diabetic rats at midgestation. We found that H₂O₂ enhanced and that superoxide dismutase (SOD) reduced MMPs activities in the maternal side of the placenta and in the fetuses from control and diabetic rats. MMPs were not modified by oxidative status in the fetal side of the placenta. Lipid peroxidation was enhanced in the maternal and fetal sides of the placenta and in the fetus from diabetic rats when compared to controls, and gradually decreased from the maternal placental side to the fetus in diabetic animals. The activities of the antioxidant enzymes SOD and catalase were decreased in the maternal placental side, catalase activity was enhanced in the fetal placental side and both enzymes were increased in the fetuses from diabetic rats when compared to controls. Our data demonstrate changes in the oxidative balance and capability of oxidants to upregulate MMPs activity in the feto-placental unit from diabetic rats, a basis to elucidate links between oxidative stress and alterations in the developmental pathways in which MMPs are involved.     

Decreased epidermal growth factor binding in cells growth arrested in G1 by nutrient deficiency

Previous studies have shown that the nontransformed AKR-2B mouse embryo derived cell line may growth arrest by two separate mechanisms in the G₁ phase of the cell cycle-growth factor deficiency arrest (G₀) and low molecular weight nutrient deficiency arrest. An examination of epidermal growth factor (EGF) receptors under the different resting or growth conditions has shown that rapidly growing cells or cells arrested due to growth factor deficiency have the expected amount of ¹²⁵I-EGF binding with approximately 10⁵ receptors per cell being present in G₀ arrested cells. In contrast, cells arrested due to nutrient deficiency show a reduction in ¹²⁵I-EGF binding to 10--20% of that observed under the other conditions. This effect appears to be due to decreased receptor number and not to a change in the affinity of the receptor. Stimulation of DNA synthesis by nutrient replenishment causes a tenfold increase in EGF binding 20 hours later, with some increase in binding being detectable as early as six hours. The increase in binding is inhibited by cycloheximide and actinomycin D. This suggests that new mRNA synthesis as well as increased protein synthesis is required for the increase in EGF binding.     

Pathogenesis of in utero infections with abortogenic and non-abortogenic alphaviruses in mice.

The initial stages of infection of pregnant mice at gestation day 11 with either the T48 strain of Ross River virus or avirulent Semliki Forest virus are similar. With both infections, a hematogenous spread of virus to the placenta occurs. The viruses subsequently replicate to high titer in all placentas and are able to persist in the presence of specific maternal antiviral antibodies. There is a delay of at least 1 to 2 days between the initial detection of virus in the placenta and the onset of fetal infection. With Semliki Forest virus, abortion occurred in all mothers and appeared to be preceded by infection of all fetuses. However, when Semliki Forest virus was given at other stages of pregnancy, abortion was less common, and in all non-aborted pregnancies at least one uninfected fetus was observed. This situation was similar to that with Ross River virus, in which abortion was not observed and fetal infection and death were only seen in a proportion of fetuses. Within each pregnancy, the outcome of the two in utero infections appeared to result from similar mechanisms, with the fate of an individual fetus depending upon the timing of the passive transfer of anti-viral immunoglobulin G from the mother relative to the timing of fetal infection by virus from the placenta. Although the passive maternal immunoglobulin G protected susceptible fetuses against infection, antibody did not mediate in utero recovery of infected fetuses or clear placental infection.     

Lack of maternal to fetal transfer of 125I-labelled erythropoietin in sheep.

We administered tracer quantities of biologically active 125I-labelled recombinant human erythropoietin by intravenous bolus injection to seven late gestation pregnant ewes. Maternal and fetal blood was sampled over the subsequent six hours and assayed for erythropoietin-specific radioactivity. Despite the expected increase in maternal plasma immunoprecipitable 125I-labelled erythropoietin radioactivity, fetal plasma levels remained unchanged throughout the study. In addition, erythropoietin receptors were not detected in ovine and human placental tissue. We conclude that biologically active 125I-labelled erythropoietin does not cross the placenta from mother to fetus in measurable quantities in sheep, and likely in humans. Thus, these data indicate the levels of erythropoietin measured in fetal plasma are reflective of fetal, and not maternal, erythropoietin production and elimination.     

Preparation and properties of monoclonal and polyclonal antibodies to mouse epidermal growth factor (EGF) receptors: evidence for cryptic EGF receptors in embryonal carcinoma cells

Monoclonal antibodies to mouse epidermal growth factor (EGF) receptor were prepared by the immunization of rats with receptor glycoprotein purified from mouse liver by affinity chromatography on immobilized EGF. Purified mouse EGF receptor retained EGF-inducible autophosphorylating activity and was antigenic in rats and rabbits. The monoclonal antibodies cross react very poorly with human EGF receptor, while polyclonal rabbit antibodies immunoprecipitate human, rat and mouse EGF receptor equally well. The rabbit antibody blocks EGF binding to mouse fibroblast cells and, at 20-fold higher concentrations, stimulates uptake of tritiated thymidine into DNA. This indicates that antibodies bind at or close to the EGF-binding site and can mimic the effects of the growth factor. None of the monoclonals bind at the EGF site of the receptor. Immunoprecipitation, immunoblotting, 125I-EGF cross linking, 125I-surface labelling, immunohistochemistry and autophosphorylation techniques were used to delineate the basis for the induction of EGF receptors when OC15 embryonal carcinoma (EC) cells differentiate into endodermal derivatives (END). EGF-stimulated autophosphorylation of a 170 X 10(3) Mr protein in solubilized OC15 EC cells is readily detectable, although intact EC cells do not bind or respond to EGF by all other tests. The results suggest that cryptic EGF receptors are present in EC stem cells, a finding with implications in development.