Mary E. Gray

Immunocytochemical Localization of Human Epidermal Growth Factor/Urogastrone in Several Human Tissues'

Epidermal growth factor (EGF) stimulates the growth of many tissues and inhibits stimulated gastric acid secretion. Its primary tissue of origin in man is still unknown. We used polyclonal anti-human EGF sera in the peroxidase-antiperoxidase immunocytochemical staining technique to identify immunoreactive human EGF (ihEGF) in tissue sections from 29 subjects ranging from fetuses to 63 years in age. In addition to acinar cells in the submandibular salivary glands and cells of Brunners duodenal glands, previously reported to contain ihEGF, we found ihEGF in most anterior pituitary glycopeptide hormone-secreting cells, in gastric and pyloric gland cells of the stomach, and in bone marrow cells that resembled mononuclear phagocytes in subjects of all ages. The eccrine sweat glands in the skin of adults also contained ihEGF. Cells containing ihEGF were found singly or in clusters in the trachea of the fetus only. No fetal pancreatic islet cells stained, but occasional cells in neonates and a majority of islet cells in older subjects contained ihEGF; there was no constant association with insulin, glucagon, or somatostatin. Only the lactating breast contained ihEGF. In adults, outer adrenomedullary cells contained ihEGF. Intense immunostaining was observed in the renal medulla, apparently limited to the extracellular area between the renal tubules, and increased with age; the cortex was devoid of ihEGF. No ihEGF was detected in posterior pituitary gland, thyroid gland, heart, lung, or liver at any age. An adult prostate contained ihEGF only in an area of local injury, and some primordial follicles from the ovary of a newborn appeared to contain ihEGF. Thus, many tissues appear to synthesize hEGF, which may exert exocrine, endocrine, or paracrine functions in different tissues and at different ages.

Effect of Epidermal Growth Factor on Lung Maturation in Fetal Rabbits

Summary: Injection of epidermal growth factor (EGF) into 24-day rabbit fetuses (5 μg, im or ip) induced accelerated maturation of the lung. On sacrifice at day 27, there was greater distensibility and stability on deflation associated with the appearance of a complement of type II cells approaching that of the rabbit at term. EGF treatment had no demonstrable effect on body weight or lung weight in this group of animals. Saline-injected control fetuses were not affected significantly.Speculation: EGF is capable not only of promoting epithelial cell growth but also differentiation in the fetal rabbit lung. It may be an important hormone in the maturation of the lung and capable of protecting the prematurely delivered fetus against the development of hyaline membrane disease.

Developmental expression of SP-A and SP-A mRNA in the proximal and distal respiratory epithelium in the human fetus and newborn.

We used immunolocalization and in situ hybridization to determine the distribution of SP-A and SP-A mRNA in lungs of human fetuses and normal newborn infants. Early in the second fetal trimester a few immunostained cells were observed in tracheal epithelium, often in mucosal folds near the origin of submucosal gland ducts. Non-mucous tracheal gland cells were immunostained for SP-A as they became differentiated. Expression of SP-A mRNA was similar to that of immunolocalization in the second trimester. Immunostained cells and SP-A mRNA also appeared about the same time in gestation in isolated cells of bronchial epithelium and glands. SP-A mRNA was seen in bronchiolar cells and pre-Type II cells lining terminal airways of fetuses at 19-20 weeks of gestation. Only in liveborn infants did cells of bronchioloalveolar portals and mature Type II cells contain SP-A mRNA or immunostain for SP-A. In postnatal infants, luminal material was also stained for SP-A. Although some alveolar macrophages contained immunoreactive material, SP-A mRNA was never detected. The abundance of SP-A in tracheal and bronchial glands and epithelium of conducting airways supports the importance of non-surfactant-associated functions for SP-A and may be related to a role in host defense.

TGF-β1 perturbs vascular development and inhibits epithelial differentiation in fetal lung in vivo

Members of the transforming growth factor β (TGF‐β) family of polypeptides have been implicated in morphogenesis and differentiation in numerous tissues, including the lung. In order to further define effects of TGF‐β signaling in lung morphogenesis, a constitutively active form of TGF‐β1 was expressed in respiratory epithelial cells of the fetal mouse lung in vivo. Expression of TGF‐β1 arrested lung morphogenesis in the pseudoglandular stage of development, inhibiting synthesis of differentiation‐dependent proteins, SP‐B, SP‐C, and CCSP, and maintaining embryonic patterns of staining for thyroid transcription factor‐1 (TTF‐1) and hepatocyte nuclear factor‐3β (HNF‐3β). The pulmonary mesenchyme was thickened and vascular density was increased by TGF‐β1. TGF‐β1 decreased expression of vascular endothelial growth factor‐A (VEGF‐A) mRNA and protein, and the abundance of Flk‐1 mRNA in the lung mesenchyme. Distribution of platelet‐endothelial cell adhesion molecule (PECAM)‐1, a marker of pulmonary blood vessels, was altered, and ultrastructural studies demonstrated that TGF‐β1 inhibited vascular development in the fetal lung. TGF‐β1 perturbed both epithelial cell differentiation and formation of the pulmonary vasculature, supporting the concept that precise control of signaling via the TGF‐β receptor pathway is critical for normal lung morphogenesis.

Pulmonary hemodynamic and ultrastructural changes associated with Group B streptococcal toxemia in adult sheep and newborn lambs.

Summary: A toxin isolated from Group B β-hemolytic streptococci, Type III was infused into adult sheep and newborn lambs. A twophased reaction was observed. There was an initial phase of pulmonary hypertension and high flow of protein-poor lymph. This was followed by a second phase when pressures returned to baseline but lymph flow remained twice the baseline values and protein concentration in lymph increased. During the second phase there was a significant increase in lymph protein clearance, suggestive of increased microvascular permeability to protein. The absolute granulocyte count decreased to 10% of baseline values by 60 min after the infusion, and was followed by a variable return to baseline. The sheep with the largest changes in protein clearance were those who had the slowest return to baseline values. Pathologic examination of lung tissue revealed there was capillary dilation, interstitial edema, and large numbers of granulocytes in the lungs. The basement membranes of both capillaries and arterioles showed disruption and widening, along with fragmentation of the internal elastic membrane. This study provides morphologic and physiologic evidence of increased pulmonary vascular permeability after injection of streptococcal toxin associated with granulocyte trapping in the lung. We postulate that granulocytes may be involved as mediators of the pulmonary vascular injury.

Temporal-Spatial Distribution of Hepatocyte Nuclear Factor-3β in Developing Human Lung and Other Foregut Derivatives

SUMMARY We assessed the temporal-spatial distribution of hepatocyte nuclear factor-3β (HNF-3β) in developing human lung and other foregut derivatives. Tissue from 31 fetuses (10-40 weeks) and 24 infants with hyaline membrane disease (HMD) or bronchopulmonary dysplasia (BPD) (2 days to 7 months) was studied. HNF-3β was detected in nuclei of epithelial cells of trachea and of conducting and terminal airways at 10 weeks. Thereafter, epithelial nuclei were immunolabeled more widely in peripheral than proximal airways. HNF-3β was confined to bronchiolo-alveolar portals and Type II cells in nonfetal lung. In infants with BPD, HNF-3 β was expressed abundantly in regenerating epithelial cells at the periphery of lung lobules. HNF-3 β was also detected in fetal esophagus, pancreas, duodenum, stomach, and gallbladder, suggesting that it is a marker for progenitor cells in foregut derivatives. The pattern of expression of HNF-3 β in the lung was similar to that of thyroid transcription factor-1 (TTF-1) at all ages. The temporal-spatial patterns of HNF-3 β and TTF-1 in the developing and regenerating lung are consistent with their proposed role in epithelial cell differentiation, regeneration, and surfactant protein gene expression.

The lipid composition of isolated rat spermatids and spermatocytes.

The lipids composition of enriched fractions of spermatids and spermatocytes, isolated from rat testicular tissue, has been investigated. More than 20% of the total fatty acids of spermatids but only 10% of those of spermatocytes, isolated from testes of mature rats, was 4,7,10,13,16-docosapentaenoic acid. Spermatocyte-enriched fractions isolated from testes of immature rats had fatty acid compositions similar to those isolated from testes of mature rats. On the other hand, spermatids isolated from immature rats had a level of docosapentaenoic acid which was intermediate between the level found in spermatocytes and that of spermatids from mature rats. Major phospholipid classes and the triacylglycerols of spermatids contained much more of the docosapentaenoic acid than the corresponding lipid types from spermatocytes. Differences in content of total phospholipids, individual classes of phospholipids and triacylglycerols among spermatocytes, spermatids and late spermatids were also observed.

Calcitonin gene-related peptide in human fetal lung and in neonatal lung disease.

The ontogeny of calcitonin gene-related peptide immunoreactivity (CGRP-IR) was evaluated immunohistochemically in 67 human fetal or newborn lungs previously analyzed for calcitonin immunoreactivity (CT-IR). CGRP-IR was present by 10 weeks of gestation in rare, solitary neuroendocrine (NE) cells of developing conducting airways in two of eight first-trimester lungs. During the second trimester, cells with CGRP-IR were found consistently (21/23 fetuses). However, the numbers of positively staining cells did not appear to increase in these fetuses or in third-trimester infants dying of non-pulmonary causes. The highest concentrations of CGRP-IR cells were seen in lungs of premature infants with advancing chronic lung disease associated with bronchopulmonary dysplasia (BPD). CGRP-IR was seen earlier in gestation and in greater numbers of NE cells than was calcitonin immunoreactivity (CT-IR) reported previously in these same fetal lungs (Lab Invest 52:52, 1985). Its presence paralleled that of CT-IR in postnatal chronic lung disease.

Ontogeny of epidermal growth factor receptor/kinase and of lipocortin-1 in the ovine lung

ABSTRACT: We have examined the ontogeny and distribution of the epidermal growth factor receptor/kinase (EGF receptor) and of lipocortin-1, a major cellular substrate of the EGF receptor, in a developmental series of 13 normal ovine fetal lungs (44-145 d of gestation) using the peroxidase anti-peroxidase technique with two extensively characterized polyclonal antibodies recognizing the EFG receptor and one polyclonal antibody recognizing lipocortin-1. Immunoreactive EGF receptor/kinase and lipocortin-1 were detected in conducting airway epithelium by the end of the first trimester of pregnancy before bronchial glands could be identified. This was followed at two-thirds of gestation by immunoreactivity in bronchial glands and large bronchioles adjacent to postive bronchi. By seveneighths of gestation conducting airway epithelium no longer contained consistently detectable immunostaining for EGF receptor, although lipocortin-1 was identified until term in all levels of conducting airways. In contrast, neither EGF receptor nor lipocortin-1 immunoreactivity was detected in alveolar type I or type II epithelial cells, fibrocytes, chondrocytes, smooth muscle, or endothelial cells at any gestational age. These findings suggest that EGF receptor and lipocortin-1 may participate in early airway development.

Human Surfactant Protein-A Contains Blood Group A Antigenic Determinants

ABSTRACT: A major blood group antigenic epitope was identified on human pulmonary surfactant protein A (SPA). MAb and polyclonal antibodies generated against purified human SP-A aggregated blood group A human erythrocytes and immunostaine epithelial cells in a variety of human tissues, consistent with the tissue distribution of major blood group antigens. SP-A MAb (MAb-8) agglutinated red cells and immunostained tissues from A or AB blood groups, but did not react with cells or tissues from O or B individuals. MAb-8 immunostaining of tissue from blood group A individuals was ablated by incubation with blood group A red cells. MAb and polyclonal antibodies directed against A blood group antigens reacted strongly with purified SP-A obtained from a blood group A individual with alveolar proteinosis. MAb and polyclonal antibodies specific for B blood group antigen failed to react with SP-A from this patient or from patients who were in blood group B. Reactivity of anti-blood group MAb was lost after treatment of SP-A with endoglycosidase-F, demonstrating its reactivity with an epitope dependent on the asparagine-linked oligosaccharide at asparagine 187. Reactivity of MAb-8 with SP-A persisted after endoglycosidase-F treatment, but was lost after digestion with collegenase as assessed by Western blot after SDS-PAGE. Reactivity of MAb to SP-A was sensitive to β-elimination, supporting the presence of another blood group antigenic site distinct from the epitope dependent on the asparagine-linked carbohydrate. The finding that the SP-A molecule contains a major blood group epitope has implication for the clinical use of surfactant replacement preparations and diagnostic reagents based on this protein.