Margaret M. Grant

Alterations in lung basement membrane during fetal growth and type 2 cell development.

We studied basement membrane development in the late fetal and in the neonatal rat lung, from the 18th day of gestation (term = 22 days) through the 8th postnatal day, with particular emphasis on the gas-exchange region of the lung. In the periphery of the lung, as type 2 cells differentiate, the continuous basement membrane develops openings beneath these cells. Basal cytoplasmic foot processes extend through these discontinuities into the underlying interstitium, often approaching interstitial cells closely. These discontinuities and extensive foot processes are associated only with type 2 epithelial cells and not with either differentiated airway cells or with the type 1 alveolar lining cells derived from type 2 cells. The type 2 cell basement membrane discontinuities and penetrating foot processes are maximal in the perinatal period and decrease in the week after birth. The appearance of openings in type 2 cell basement membrane and changes in distribution, linear density, and ruthenium red staining of anionic sites suggest that the epithelial basement membrane undergoes continuous remodeling throughout development, particularly in association with type 2 cell differentiation and growth of lung surface area. Epithelial cell foot processes may interact with underlying interstitial cells and affect the coordination of lung surface growth with the development of its connective tissue framework.

Development of Neuroepithelial Bodies in Fetal Rabbit Lungs.: I. Appearance and Functional Maturation as Demonstrated by High-Resolution Light Microscopy and Formaldehyde-Induced Fluorescence

Developing lungs of fetal rabbits aged 13 days through early postnatal stages were examined for periodic acid-Schiff (PAS)-lead hematoxylin staining, serotonin fluorescence, and argyrophilia, methods selective for small-granule (neuro) endocrine cells. Later stages were also studied by electron microscopy. These cells arise from precursors that first appear around days 18-19 in the endodermal epithelium; the latter stand out as clear cells from their undifferentiated, glycogen laden neighbors. The cells first form in the main and lobar bronchi, then continue to arise in the lining of newly laid down branches of the extending bronchial tree, tending to concentrate near points of branching. Some cells have been seen to divide, and clusters of two, four, or more soon appear. Morphologically mature neuroepithelial bodies begin to appear in larger bronchi around 23 days, although the largest examples occur in 25-day and older fetuses. After 23 days, additional bodies mature from clear-cell precursors in more recently laid down distal lung, so that by the terminal sac period (around days 28-29) all conducting airways contain them, and the clear cells and clear-cell clusters have virtually disappeared. A pinkish staining by PAS-lead hematoxylin and dense-core secretory granules, which begin to appear in the clear cells, are present in mature small-granule cells whether solitary or aggregated. Argyrophilia is less consistently demonstrable. Fluorescence for serotonin is first evident in mature neuroepithelial bodies at 23 days, increases considerably by 26 days, and reaches a prenatal peak around days 28-29, when neuroepithelial bodies and solitary small-granule cells along the whole conducting bronchial tree may participate. Fluorescence appears to decrease during days 30-31 but strengthens again after birth. Composed of these solitary small-granule cells and neuroepithelial bodies, the pulmonary APUD system of rabbits thus appears substantially functional during the final quarter of gestation.

Influence of maternal diabetes on basement membranes, type 2 cells and capillaries in the developing rat lung

To determine the effect of maternal diabetes on rat lung development, we studied the ultrastructure of the alveolar wall from the ninteenth day of gestation (term = 22 days) through the eighth postnatal day in fetal and neonatal rats of mothers with streptozotocin-induced diabetes. In normal fetal lung development, epithelial basement membranes develop large discontinuities beneath type 2 cells, through which cytoplasmic foot processes extend into the interstitium. Maternal diabetes delays the appearances of these epithelial basement membrane discontinuities and reduces the number of type 2 cell processes that penetrate it. These alterations in epithelial basement membrane are reversed after birth. There is no ultrastructural evidence of a delay in type 2 cell maturation as assessed by lamellar body volume density morphometry. Endothelial basement membranes, which are not present around the growing pulmonary capillary bed in the pseudoglandular lung, are seen late in normal gestation, primarily around capillaries forming the mature air-blood barrier. This development of endothelial basement membrane may be delayed in the fetuses of diabetic mothers and reflects a significant delay in the expansion of the pulmonary capillary network in these animals as assessed by morphometric volume density measurements. This effect on capillary growth is not reversed in the newborn animals through 8 days after birth. The summation of these effects indicates a generalized slowing of fetal lung development by maternal diabetes, some of which effects persist after birth and may continue to influence lung development during the period of postnatal alveolar septal growth.

Production of Lymphocyte Chemokinetic Activity by Stimulated Alveolar Macrophages

While the presence of a lymphocytic parenchymal infiltrate is characteristic of several lung diseases, the mechanisms responsible for the focal accumulation of lymphocytes within the lungs remain unclear. Since alveolar macrophages secrete several substances that affect lymphocyte function, we examined supernatants of stimulated, cultured guinea pig alveolar macrophages for their ability to alter lymphocyte motility. Guinea pigs were immunized by footpad injection of ovalbumin (OVA) emulsified in complete Freunds adjuvant. Fourteen days later, alveolar macrophages were obtained by bronchial lavage or teasing the lung parenchyma, enriched by adherence to plastic, and incubated for 3 and 24 hours in culture medium alone or medium containing either latex beads, OVA, or human serum albumin (HSA). Conditioned medium was harvested and assayed for chemoattractant activity against rat splenic lymphocytes in modified Boyden chambers. Regardless of stimulus, there was no evidence of enhanced lymphocyte motility above control values in supernatants harvested at 3 hours. At 24 hours, alveolar macrophages from OVA-sensitized guinea pigs stimulated with latex or OVA generated significant amounts of lymphocyte migration stimulating activity (LCA) (250 +/- 25 and 247 +/- 24 percent of control migration, respectively) compared to cells incubated in medium alone or with HSA (162 +/- 23 and 147 +/- 14 percent, respectively). Antigen recognition appears to be related to the presence of cytophilic anti-OVA antibody on the surfaces of alveolar macrophages of sensitized guinea pigs. LCA is resistant to neuraminidase, chymotrypsin, and heating to 56 degrees C, and was chemokinetic for T-lymphocytes. it elutes from Sephadex G-100 in two regions: one at approximately 67,000 d, and a second at approximately 15,000 d. These studies indicate that following systemic immunization, the guinea pig alveolar macrophage can react to specific antigen or phagocytosis of inert particulates by secreting a chemokinetic factor for T-lymphocytes, and may play a role in the pathogenesis of some types of antigen-induced lung disease.