Regulation of alveolar epithelial cell phenotypes in fetal sheep: roles of cortisol and lung expansion

Our aim was to determine whether cortisol's effect on alveolar epithelial cell (AEC) phenotypes in the fetus is mediated via a sustained alteration in lung expansion. Chronically catheterized fetal sheep were exposed to 1) saline infusion, 2) cortisol infusion (122-131 days' gestation, 1.5-4.0 mg/day), 3) saline infusion plus reduced lung expansion, or 4) cortisol infusion plus reduced lung expansion. The proportions of type I and II AECs were determined by electron microscopy, and surfactant protein (SP)-A, -B, and -C mRNA levels were determined by Northern blot analysis. Cortisol infusions significantly increased type II AEC proportions (to 38.2 +/- 2.2%), compared with saline-infused fetuses (23.8 +/- 2.4%), and reduced type I AEC proportions (to 59.0 +/- 2.2%), compared with saline-infused fetuses (70.4 +/- 2.4%). Reduced lung expansion also increased type II AEC proportions (to 52.9 +/- 3.5%) and decreased type I AEC proportions (to 34.2 +/- 3.7%), compared with control, saline-infused fetuses. The infusion of cortisol into fetuses exposed to reduced lung expansion tended to further increase type II (to 60.3 +/- 2.1%, P = 0.066) and reduce type I AEC (to 26.6 +/- 2.3%, P = 0.07) proportions. SP-A, -B, and -C mRNA levels changed in parallel with the changes in type II AEC proportions. These results indicate that cortisol alters the proportion of type I and type II AECs via a mechanism unrelated to the degree of fetal lung expansion. However, reductions in fetal lung expansion appear to have a greater impact on the proportion of AECs than cortisol.     

Increased lung expansion alters the proportions of type I and type II alveolar epithelial cells in fetal sheep

Type I and type II alveolar epithelial cells (AECs) are derived from the same progenitor cell, but little is known about the factors that regulate their differentiation into separate phenotypes. An alteration in lung expansion alters the proportion type II AECs in the fetal lung, indicating that this may be a regulatory factor. Our aim was to quantify the changes in the proportion of type I and type II AECs caused by increased fetal lung expansion and to provide evidence for transdifferentiation of type II into type I cells. Lung tissue samples were collected from ovine fetuses exposed to increased lung expansion induced by 2, 4, or 10 days of tracheal obstruction (TO). The identities and proportions of AEC types were determined with electron microscopy. The proportion of type II cells was reduced from 28.5 +/- 2.2% in control fetuses to 9.4 +/- 2.3% at 2 days of TO and then to 1.9 +/- 0.8% at 10 days. The proportion of type I AECs was not altered at 2 days of TO (63.1 +/- 2.3%) compared with that of control cells (64.8 +/- 0.5%) but was markedly elevated (to 89.4 +/- 0.9%) at 10 days of TO. The proportion of an intermediate AEC type, which displayed characteristics of both type I and type II cells, increased from 5.7 +/- 1.3% in control fetuses to 23.8 +/- 5.1% by 2 days of TO and was similar to control values at 10 days of TO (7.7 +/- 0.9%). Our data show that increases in fetal lung expansion cause time-dependent changes in the proportion of AEC types, including a transient increase in an intermediate cell type. These data provide the first evidence to support the hypothesis that increases in fetal lung expansion induce differentiation of type II into type I AECs via an intermediate cell type.     

Increased expansion of the lung stimulates calmodulin 2 expression in fetal sheep

Obstruction of the fetal trachea causes the lungs to expand with accumulated liquid. Although this is a potent stimulus for lung growth, the mechanisms involved are unknown. Our aim was to identify genes that are differentially expressed as a result of increased fetal lung expansion. Using differential display RT-PCR, we isolated a cDNA fragment partially encoding calmodulin 2 (CALM2) and identified the remainder of the coding region by 5'-rapid amplification of cDNA ends. Differential expression of CALM2 was confirmed by Northern blot analysis; CALM2 mRNA levels were increased to 161 +/- 5% of control at 2 days of increased lung expansion, induced by tracheal obstruction (TO), and had returned to control levels at days 4 and 10. Using in situ hybridization analysis, we found that the proportion of CALM2-labeled cells increased from 10.3 +/- 1.0% to 21.4 +/- 6.8% by 2 days of TO. This increase in CALM2 expression was reflected by a tendency for calmodulin protein levels to increase from 122.7 +/- 17.3 to 156.5 +/- 17.7 at 2 days of TO. Thus increases in fetal lung expansion result in time-dependent changes in CALM2 mRNA levels, which closely parallels the changes in lung DNA synthesis rates. As calmodulin is essential for cell proliferation, increased CALM2 mRNA levels may reflect an important role for calmodulin in expansion-induced fetal lung growth.     

Increased lung expansion alters lung growth but not alveolar epithelial cell differentiation in newborn lambs

Although increased lung expansion markedly alters lung growth and epithelial cell differentiation during fetal life, the effect of increasing lung expansion after birth is unknown. We hypothesized that increased basal lung expansion, caused by ventilating newborn lambs with a positive end-expiratory pressure (PEEP), would stimulate lung growth and alter alveolar epithelial cell (AEC) proportions and decrease surfactant protein mRNA levels. Two groups of lambs were sedated and ventilated with either 0 cmH(2)O PEEP (controls, n = 5) or 10 cmH(2)O PEEP (n = 5) for 48 h beginning at 15 +/- 1 days after normal term birth. A further group of nonventilated 2-wk-old lambs was used for comparison. We determined wet and dry lung weights, DNA and protein content, a labeling index for proliferating cells, surfactant protein mRNA expression, and proportions of AECs using electron microscopy. Although ventilating lambs for 48 h with 10 cmH(2)O PEEP did not affect total lung DNA or protein, it significantly increased the proportion of proliferating cells in the lung when compared with nonventilated 2-wk-old controls and lambs ventilated with 0 cmH(2)O PEEP (control: 2.6 +/- 0.5%; 0 PEEP: 1.9 +/- 0.3%; 10 PEEP: 3.5 +/- 0.3%). In contrast, no differences were observed in AEC proportions or surfactant protein mRNA levels between either of the ventilated groups. This study demonstrates that increases in end-expiratory lung volumes, induced by the application of PEEP, lead to increased lung growth in mechanically ventilated 2-wk-old lambs but do not alter the proportions of AECs.     

Gene expression of rat alveolar type II cells during hyperoxia exposure and early recovery

Alveolar epithelial cell (AEC) injury and repair during hyperoxia exposure and recovery have been investigated for decades, but the molecular mechanisms of these processes are not clear. To identify potentially important genes involved in lung injury and repair, we studied the gene expression profiles of isolated AEC II from control, 48-h hyperoxia-exposed (>95% O(2)), and 1-7 day recovering rats using a DNA microarray containing 10,000 genes. Fifty genes showed significant differential expression between two or more time points (P<0.05, fold change >2). These genes can be classified into 8 unique gene expression patterns. Real-time PCR verified 14 selected genes in three patterns related to hyperoxia exposure and early recovery. The change in the protein level for two of the selected genes, bmp-4 and retnla, paralleled that of the mRNA level. Many of these genes were found to be involved in cell proliferation and differentiation. In an in vitro AEC trans-differentiation culture model using AEC II isolated from control and 48-h hyperoxia-exposed rats, the expressions of the cell proliferation and differentiation genes identified above were consistent with their predicted roles in the trans-differentiation of AEC. These data indicate that a coordinated mechanism may control AEC differentiation during in vivo hyperoxia exposure and recovery as well as during in vitro AEC culture.     

Changes in alveolar epithelial cell proportions during fetal and postnatal development in sheep

Basal lung expansion is an important determinant of alveolar epithelial cell (AEC) phenotype in the fetus. Because basal lung expansion increases toward term and is reduced after birth, we hypothesized that these changes would be associated with altered proportions of AECs. AEC proportions were calculated with electron microscopy in fetal and postnatal sheep. Type I AECs increased from 4.8 +/- 1.3% at 91 days to 63.0 +/- 3.6% at 111 days of gestation, remained at this level until term, and decreased to 44.8 +/- 1.8% after birth. Type II AECs increased from 4.3 +/- 1.5% at 111 days to 29.6 +/- 4.1% at 128 days of gestation, remained at this level until term, and then increased to 52.9 +/- 1.5% after birth. Surfactant protein (SP)-A, -B and -C mRNA levels increased with increasing gestational age before birth, but the changes in SP expression after birth were inconsistent. Thus before birth type I AECs predominate, whereas after birth type II AECs predominate, possibly due to the reduction in basal lung expansion associated with the entry of air into the lungs.     

Influence of growth hormone on the lung growth response to tracheal obstruction in fetal sheep

Obstructing the fetal trachea is a potent stimulus for fetal lung growth, but little is known about the factors that regulate this process. Our aim was to determine the role of growth hormone (GH) in regulating the increase in lung growth induced by obstruction of the trachea in fetal sheep. Twenty chronically catheterized fetal sheep, nine of which were hypophysectomized, were divided into four experimental groups: 1) control group (n = 4), 2) a group in which the fetal trachea was obstructed for 3 days (3-day obstructed; n = 6), 3) a 3-day obstructed group in which the pituitary was removed [hypophysectomized (HX)] and the fetus was given maintenance infusions of ACTH, thyroxine, and human GH (hGH; HX hGH 3-day obstructed; n = 5), and 4) a HX 3-day obstructed group in which the fetus was given maintenance infusions of ACTH and thyroxine (n = 5). Tracheal obstruction significantly increased fetal lung liquid volumes from 37.2 +/- 3.2 ml/kg in control fetuses to 75.6 +/- 9.0 ml/kg in 3-day obstructed fetuses, and the presence or absence of GH did not affect this increase. Similarly, the presence or absence of GH did not affect the increase in lung weight or protein content induced by 3 days of tracheal obstruction. However, in the absence of GH, 3 days of tracheal obstruction failed to increase total lung DNA content above unobstructed control values (107.9 +/- 5.3 and 94. 1 +/- 7.0 mg/kg for control and HX 3-day obstructed groups, respectively). In contrast, 3 days of tracheal obstruction increased total lung DNA content to a similar extent in fetuses with an intact pituitary and HX fetuses that received GH replacement (126.0 +/- 4.4 and 126.7 +/- 4.0 mg/kg for 3-day obstructed and HX hGH 3-day obstructed groups, respectively). These data indicate that the absence of GH either abolishes or delays the acceleration in cell division caused by an increase in fetal lung expansion.     

Effects of cortisol on cardiac myocytes and on expression of cardiac genes in fetal sheep

In 17 fetal sheep aged 129 days, the effects of large-dose infusions of cortisol (72.1 mg/day for 2-3 days) on proliferation, binucleation, and hypertrophy of cardiac myocytes, cardiac expression of angiotensinogen, angiotensin receptor subtypes 1 and 2, Glut-1, glucocorticoid and mineralocorticoid receptors, proteins of the MAPK pathways and calcineurin were studied. Cortisol levels were 8.7 +/- 2.3 nM (SE) in 8 control and 1,028 +/- 189 nM in 9 treated fetuses (P < 0.001). Cortisol had no effect on myocyte binucleation. Left ventricular free wall (LVFW) uni- and binucleated myocytes were larger in cortisol-treated fetuses (P < 0.001, P < 0.05). Cortisol-treated fetuses had higher right ventricular free wall (RVFW) and LVFW angiotensinogen (Aogen) mRNA levels (treated: 2.30 +/- 0.37, n = 8 and 2.05 +/- 0.45, n = 7 vs. control: 0.94 +/- 0.12, n = 8 and 0.67 +/- 0.09, n = 7, P < 0.02). Levels of the glucose transporter Glut-1 mRNA were lower in the LVFW of treated fetuses (0.83 +/- 0.23 vs. 1.47 +/- 0.30 in control, P < 0.05, n = 7, 8). The higher the cortisol level, the greater the Aogen mRNA level (RVFW, r = 0.61, P < 0.01, n = 16; LVFW, r = 0.83, P < 0.0003, n = 14). There were no other changes in mRNA levels nor in levels of extracellular kinase, JNK, p38, their phosphorylated forms, and calcineurin. Thus high levels of cortisol such as occur after birth do not affect fetal cardiac myocyte binucleation or number but are associated with higher levels of ventricular Aogen mRNA, lower levels of Glut-1 mRNA, and hypertrophy of LVFW myocytes. These effects could impact on postnatal cardiac development.     

Placental restriction of fetal growth decreases IGF1 and leptin mRNA expression in the perirenal adipose tissue of late gestation fetal sheep

Placental restriction (PR) of fetal growth results in a low birth weight and an increased visceral fat mass in postnatal life. We investigated whether PR alters expression of genes that regulate adipogenesis [IGF1, IGF1 receptor (IGF1R), IGF2, IGF2R, proliferator-activated receptor-gamma, retinoid-X-receptor-alpha], adipocyte metabolism (lipoprotein lipase, G3PDH, GAPDH) and adipokine signaling (leptin, adiponectin) in visceral adipose tissue before birth. PR was induced by removal of the majority of endometrial caruncles in nonpregnant ewes before mating. Fetal blood samples were collected from 116 days gestation, and perirenal visceral adipose tissue (PAT) was collected from PR and control fetuses at 145 days. PAT gene expression was measured by quantitative RT-PCR. PR fetuses had a lower weight (PR 2.90 +/- 0.32 kg; control, 5.12 +/- 0.24 kg; P < 0.0001), mean gestational arterial Po(2) (P < 0.0001), plasma glucose (P < 0.01), and insulin concentrations (P < 0.02), than controls. The expression of IGF1 mRNA in PAT was lower in the PR fetuses (PR, 0.332 +/- 0.063; control, 0.741 +/- 0.083; P < 0.01). Leptin mRNA expression in PAT was also lower in PR fetuses (PR, 0.077 +/- 0.009; control, 0.115 +/- 0.013; P < 0.05), although there was no difference in the expression of other adipokine or adipogenic genes in PAT between PR and control fetuses. Thus, restriction of placental and hence, fetal substrate supply results in decreased IGF1 and leptin expression in fetal visceral adipose tissue, which may alter the functional development of the perirenal fat depot and contribute to altered leptin signaling in the growth-restricted newborn and the subsequent emergence of an increased visceral adiposity.     

Chronic umbilical cord compression results in accelerated maturation of lung and brown adipose tissue in the sheep fetus during late gestation

Umbilical cord compression (UCC) sufficient to reduce umbilical blood flow by 30% for 3 days, results in increased fetal plasma cortisol and catecholamines that are likely to promote maturation of the fetal lung and brown adipose tissue (BAT). We determined the effect of UCC on the abundance of uncoupling protein (UCP)1 (BAT only) and -2, glucocorticoid receptor (GR), and 11beta-hydroxysteroid dehydrogenase (11beta-HSD)1 and -2 mRNA, and mitochondrial protein voltage-dependent anion channel (VDAC) and cytochrome c in these tissues. At 118 +/- 2 days of gestation (dGA; term approximately 145 days), 14 fetuses were chronically instrumented. Eight fetuses were then subjected to 3 days of UCC from 125 dGA, and the remaining fetuses were sham operated. All fetuses were then exposed to two 1-h episodes of hypoxemia at 130 +/- 1 and 134 +/- 1 dGA before tissue sampling at 137 +/- 2 dGA. In both tissues, UCC upregulated UCP2 and GR mRNA, plus VDAC and cytochrome c mitochondrial proteins. In lung, UCC increased 11beta-HSD1 mRNA but decreased 11beta-HSD2 mRNA abundance, a pattern reversed for BAT. UCC increased UCP1 mRNA and its translated protein in BAT. UCP2, GR, 11beta-HSD1 and -2 mRNA, plus VDAC and cytochrome c protein abundance were all significantly correlated with fetal plasma cortisol and catecholamine levels, but not thyroid hormone concentrations, in the lung and BAT of UCC fetuses. In conclusion, chronic UCC results in precocious maturation of the fetal lung and BAT mitochondria, an adaptation largely mediated by the surge in fetal plasma cortisol and catecholamines that accompanies UCC.     

Long-term hypoxia increases leptin receptors and plasma leptin concentrations in the late-gestation ovine fetus

This study was designed to test the hypothesis that long-term hypoxia (LTH) increases fetal plasma leptin and fetal adipose or placental leptin expression and alters hypothalamic and adrenocortical leptin receptor (OB-R) expression. Pregnant ewes were maintained at high altitude (3,820 m) from day 30 to approximately 130 days of gestation. Reduced Po2 was maintained in the laboratory by nitrogen infusion through a maternal tracheal catheter. On day 132, normoxic control and LTH fetuses underwent surgical implantation of vascular catheters (n=6 for each group). Five days after surgery, maternal and fetal arterial blood samples were collected for leptin, insulin, and glucose analysis. Placental tissue, periadrenal fat, and fetal hypothalami and adrenal glands were collected from additional control (n=7) and LTH (n=8) fetuses for analysis of leptin mRNA by quantitative, real-time, RT-PCR (qRT-PCR). There was a significant (P<0.03) elevation in fetal plasma leptin in the LTH fetuses (3.5+/-0.7 ng/ml) vs. control (1.1+/-0.1 ng/ml). There were no differences in either glucose or insulin concentrations between the two groups. Periadrenal adipose leptin mRNA was significantly higher in the LTH group compared with control, as was placental leptin expression. The levels of leptin mRNA in adipose were approximately 70 times higher vs. placenta. LTH significantly reduced expression of OB-Ra (short-isoform) in the hypothalamus (P=0.0156), while resulting in a significant increase in adrenal OB-Rb (long-form) expression (P<0.03). Our data suggest that leptin is a hypoxia-inducible gene in the ovine fetus and OB-R expression is altered by LTH. These changes may be responsible in part, for our previously observed alterations in fetal hypothalamic-pituitary-adrenal function following LTH.     

The oncogene Trop2 regulates fetal lung cell proliferation

The factors regulating growth of the developing lung are poorly understood, although the degree of fetal lung expansion is critical. The oncogene Trop2 (trophoblast antigen 2) is upregulated during accelerated fetal lung growth, and we hypothesized that it may regulate normal fetal lung growth. We investigated Trop2 expression in the fetal and neonatal sheep lung during accelerated and delayed lung growth induced by alterations in fetal lung expansion, as well as in response to glucocorticoids. Trop2 expression was measured using real-time PCR and localized spatially using in situ hybridization and immunofluorescence. During normal lung development, Trop2 expression was higher at 90 days gestational age (GA; 4.0 ± 0.8) than at 128 days GA (1.0 ± 0.1), decreased to 0.5 ± 0.1 at 142 days GA (full term ～147 days GA), and was positively correlated to lung cell proliferation rates (r = 0.953, P < 0.005). Trop2 expression was regulated by fetal lung expansion, but not by glucocorticoids. It was increased nearly threefold by 36 h of increased fetal lung expansion (P < 0.05) and was reduced to ～55% of control levels by reduced fetal lung expansion (P < 0.05). Trop2 expression was associated with lung cell proliferation during normal and altered lung growth, and the TROP2 protein colocalized with Ki-67-positive cells in the fetal lung. TROP2 was predominantly localized to fibroblasts and type II alveolar epithelial cells. Trop2 small interfering RNA decreased Trop2 expression by ～75% in cultured fetal rat lung fibroblasts and decreased their proliferation by ～50%. Cell viability was not affected. This study demonstrates that TROP2 regulates lung cell proliferation during development.     

Cell proliferation and differentiation in the fetal and early postnatal mouse thymus

The relationships between cell proliferation and cell differentiation during thymus ontogeny were studied by labeling DNA-synthesizing thymocytes with bromodeoxyuridine and staining with antibodies against CD4, CD8, J11d, phagocytic glycoprotein 1, TCR V beta 8 chain, Thy-1, and IL-2R surface proteins. The development of the thymus was discontinuous, with two well defined growth periods from 13 days to 18 days of fetal life and from 3 days to 6 days after birth, and more progressive growth from day 8 to 2 wk. Cell proliferation started on fetal day 12, 1 day after the arrival of hemopoietic stem cells in the third branchial pouch. These cells were phagocytic glycoprotein 1-positive but IL-2R and Thy-1 negative. Thus, cell proliferation preceded IL-2R expression. Until day 15, CD4-8- thymocytes expanded without differentiation. Then CD4-8+ and CD4+8+ cells appeared; this induction was proliferation dependent and occurred on cells which had already lost IL-2R, but just after maximum expression of this receptor. During several days, the thymus remained of constant size (around 10(7) cells) and behaved like the steady state thymus. On day 3 after birth, expansion started again and was correlated with an increase in CD4-8- proliferation index and IL-2R expression. At the same time, the thymic subset capable of expansion without differentiation was again, transiently, detectable. These results suggest that the inflow of precursor cells into the thymus is permanent but transiently increased at several times during ontogeny. Moreover, the behavior of fetal CD4-8- cells does not appear radically different from that of adult precursors, but the actual difference resides in the variation of the relative proportion of CD4-8- cells at different maturation stages, as revealed by striking variations of IL-2R expression by cycling cells.     

Decline in lung liquid volume and secretion rate during oligohydramnios in fetal sheep

Reduced amniotic fluid volume often results in fetal lung hypoplasia. Our aim was to examine the effects of prolonged drainage of amniotic and allantoic fluids on lung liquid volume (Vl), secretion rate (Vs), and tracheal flow rate (Vtr) in fetal sheep. In five experimental animals, amniotic and allantoic fluids were drained from 107 to 135 days of gestation. The volume of fluid drained from the experimental animals was 411.8 +/- 24.4 ml/day (n = 140). In six control animals, amniotic fluid volume was 747.7 +/- 89.7 ml (n = 15). Wet and dry lung weights were 20-25% lower in experimental fetuses than in control fetuses. Fetal hemoglobin, O2 saturation, arterial PO2, pH, and hematocrit were unchanged by drainage. During the drainage period, Vl was up to 65% lower, Vs was up to 35% lower, and Vtr was up to 40% lower in experimental fetuses than in control fetuses. We conclude that prolonged drainage of amniotic and allantoic fluids decreases Vl, Vs, and Vtr in fetal sheep. These findings indicate that fetal lung hypoplasia associated with oligohydramnios may be the result of a prolonged reduction in Vl.     

Increases in lung expansion alter pulmonary hemodynamics in fetal sheep.

Prolonged increases in fetal lung expansion stimulate fetal lung growth and development, but the effects on pulmonary hemodynamics are unknown. Our aim was to determine the effect of increased fetal lung expansion, induced by tracheal obstruction (TO), on pulmonary blood flow (PBF) and vascular resistance (PVR). Chronically catheterized fetal sheep (n = 6) underwent TO from 120 to 127 days of gestational age (term approximately 147 days); tracheas were not obstructed in control fetuses (n = 6). PBF, PVR, and changes to the PBF waveform were determined. TO significantly increased lung wet weight compared with control (166.3 +/- 20.2 vs. 102.0 +/- 18.8 g; P < 0.05). Despite the increase in intraluminal pressure caused by TO (5.0 +/- 0.9 vs. 2.4 +/- 1.0 mmHg; P < 0.001), PBF and PVR were similar between groups after 7 days (TO 28.1 +/- 3.2 vs. control 34.1 +/- 10.0 ml.min(-1).100 g lung wt(-1)). However, TO markedly altered pulmonary hemodynamics associated with accentuated fetal breathing movements, causing a reduction rather than an increase in PBF at 7 days of TO. To account for the increase in intraluminal pressure, the pressure was equalized by draining the lungs of liquid on day 7 of TO. Pressure equalization increased PBF from 36.8 +/- 5.2 to 112.4 +/- 22.8 ml/min (P = 0.01) and markedly altered the PBF waveform. These studies provide further evidence to indicate that intraluminal pressure is an important determinant of PBF and PVR in the fetus. We suggest that the increase in PBF associated with pressure equalization following TO reflects an increase in growth of the pulmonary vascular bed, leading to an increase in its cross-sectional area.     

Sarcoidosis is not associated with a generalized defect in T cell suppressor function

In pulmonary sarcoidosis, the marked expansion of CD4+ (helper/inducer) T cells in the alveolar structures of the lung is maintained by local IL-2 release by activated CD4+ HLA-DR+ T cells without concomitant expansion and activation of CD8+ (suppressor/cytotoxic) T cells, suggesting that sarcoid may be associated with a generalized abnormality of CD8+ T cells. Consistent with this concept, evaluation of the expression of the IL-2R on fresh lung T cells from individuals with active sarcoidosis demonstrated that 7 +/- 1% of sarcoid lung CD4+ T cells are spontaneously expressing the IL-2R compared with only 1 +/- 1% lung CD8+ T cells (p less than 0.01). However, stimulation of purified sarcoid blood CD8+ T cells with the anti-T3/TCR complex mAb OKT3 was followed by the normal expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). In addition, lung sarcoid CD8+ T cells responded to OKT3 similarly to normal lung CD8+ T cells and to autologous blood CD8+ T cells as regards expression of IL-2R (p greater than 0.1) and proliferation (p greater than 0.1). Finally, using CD4+ cells activated with allogenic Ag to induce, in coculture, fresh autologous CD8+ cells to suppress proliferation of fresh autologous CD4+ cells to the same Ag, sarcoid CD8+ T cells suppressed CD4+ cell proliferation in a normal fashion (p greater than 0.1). These results demonstrate that sarcoid CD8+ (suppressor/cytotoxic) T cells are competent to respond to a proliferation signal normally and can be induced to normally suppress CD4+ T cell proliferation to Ag, suggesting that the expansion of activated CD4+ T cells in pulmonary sarcoidosis is not due to a generalized abnormality of CD8+ T cells or of their suppressor T cell function.     

ANG II type 2 receptor regulates smooth muscle growth and force generation in late fetal mouse development

Although evidence from culture studies implicates the angiotensin II (ANG II) type 2 receptor (AT(2)R) in the regulation of growth and differentiation of arterial smooth muscle (SM) cells (SMC), the lack of its expression in adult arteries has precluded direct investigation of its role in vivo. The goal of the present study was to determine the role of AT(2)R in the control of fetal SMC growth, contractility, and differentiation during vascular development. Determination of isometric tension in fetal aortas showed potentiated ANG II-induced contraction by treatment with the selective AT(2)R antagonist PD-123319, demonstrating the presence of functional AT(2)Rs that mediate reduced force development in vascular SMC. In direct contrast to numerous cell culture studies, proliferation indexes were decreased rather than increased in aortic SMC of fetal homozygous AT(2)R knockout compared with wild-type or heterozygous knockout mice. Experiments using SMC tissues from heterozygous female AT(2)R knockout mice, which are naturally occurring chimeras for AT(2)R expression, showed that AT(2)R mRNA expression was exactly 50% of that of wild type. This indicated that loss of AT(2)R expression did not confer a selective advantage or disadvantage for SMC lineage determination and expansion. Real time RT-PCR analyses showed no significant difference in expression of SM-alpha-actin, SM myosin heavy chain, and myocardin in various SM tissues from all three genotypes, suggesting that knockout of AT(2)R had no effect on subsequent SMC differentiation. Taken together, results indicate that functional AT(2)R are expressed in fetal aorta and mediate reduced force development but do not significantly contribute to regulation of SMC differentiation.     

Intrathymic radioresistant stem cells follow an IL-2/IL-2R pathway during thymic regeneration after sublethal irradiation

Sublethally irradiated mice undergo thymic regeneration which follows a phenotypic pattern of events similar to that observed during normal fetal development. Thymic regeneration after irradiation is the product of a limited pool of intrathymic radioresistant stem cells undergoing simultaneous differentiation. We show that in this model of T cell development, thymic regeneration follows a pathway in which the IL-2R is transiently expressed on CD4-/CD8- cells. IL-2R expression occurred during the exponential growth period of thymic regeneration, and IL-2R blocking prevented this explosive growth. Flow cytometry analysis revealed that the IL-2R blockade affected primarily the development of the immature CD3-/CD4-/CD8- (triple negative) cells and their ability to generate CD3+/CD4+/CD8+ or CD3+/CD4+/CD8- and CD3+/CD4-/CD8+ thymocytes. Thus, our findings demonstrate that blocking of the IL-2R resulted in an arrest in proliferation and differentiation by intrathymic radioresistant stem cells, indicating that the IL-2/IL-2R pathway is necessary for the expansion of immature triple negative T cells.     

A premature increase in circulating cortisol suppresses expression of 11beta hydroxysteroid dehydrogenase type 2 messenger ribonucleic acid in the adrenal of the fetal sheep

We have investigated the effect of intrafetal cortisol administration, before the normal prepartum cortisol surge, on the expression of 11beta hydroxysteroid dehydrogenase (11betaHSD) type 2 mRNA in the fetal adrenal. We also determined whether increased fetal cortisol concentrations can stimulate growth of the fetal adrenal gland or increase expression of adrenal steroidogenic enzymes. Cortisol (hydrocortisone succinate: 2.0-3.0 mg in 4.4 ml/24 h) was infused into fetal sheep between 109 and 116 days of gestation (cortisol infused; n = 12), and saline was administered to control fetuses (saline infused; n = 13) at the same age. There was no effect of cortisol infusion on the fetal adrenal:body weight ratio (cortisol: 101.7 +/- 5.3 mg/kg; saline: 108.2 +/- 4.3 mg/kg). The ratio of adrenal 11betaHSD-2 mRNA to 18S rRNA expression was significantly lower, however, in the cortisol-infused group (0.75 +/- 0.02) compared with the group receiving saline (1.65 +/- 0.14). There was no significant effect of intrafetal cortisol on the relative abundance of adrenal CYP11A1, CYP17, CYP21A1, and 3betaHSD mRNA. A premature elevation in fetal cortisol therefore resulted in a suppression of adrenal 11betaHSD-2. Increased intra-adrenal exposure to cortisol at this stage of gestation is, however, not sufficient to promote adrenal growth or steroidogenic enzyme gene expression.