Flow cytometric analysis of cell-surface binding elements for fibronectin on mouse lung cell isolates.

Fresh lung cell isolates from LAF1 mice were examined for the presence of fibronectin-binding elements using flow cytometric analysis. Thoroughly perfused lungs from adult male mice were dissociated using an elastase-trypsin digestion, gentle pipetting and filtering. The resulting heterogeneous cell suspension was incubated with fibronectin coated 0.5 micron fluorescent beads. Subsequent flow cytometric analysis indicated the presence of two species of specific fibronectin-binding populations; one of higher binding affinity which can be blocked with exogenous plasma fibronectin and one of lower binding affinity. We tentatively identify the lower affinity binding element with the fibronectin adhesion receptor and the higher affinity element with the putative matrix assembly receptor.

Effects of high energy particle (HZE) radiation on the distal lung.

Major sources of concern for manned space travel are the effects of high energy particle (HZE) radiation on various biological systems, and the consequences of major solar activity. To date, considerable attention has been directed toward HZE-induced alterations both on non-dividing systems, such as the retina, cornea and brain, and on dividing systems, such as the gut and testis. This paper is focused on the morphologically detectable late-occurring alterations in the distal lung, and toward a comparison of the changes with those induced by x-irradiation. Briefly, the salient alterations involve an increase in the width of the septal walls and the capillary and alveolar basal laminae, and the irregularity of the luminal surface of the capillaries, as exemplified by the presence of filipodial projections and blebbing. All alterations were focal in their localization, and no cells of any type (e.g., epithelial, endothelial or stromal) appeared to undergo damage, an observation quite unlike the cellular changes induced by x-irradiation.

Cell-cell matrix interactions in induced lung injury. IV. Quantitative alterations in pulmonary fibronectin and laminin following X irradiation.

Male LAF/1 mice were locally irradiated at doses of 5, 9, and 13 Gy and compared with untreated and sham-irradiated animals. Lungs were subsequently examined at times of 1, 4, 13, 28, 41, and 63 weeks postirradiation (PI) for alterations in pulmonary fibronectin (Fbn) and laminin (Lam) as a consequence of the irradiation. Thoroughly perfused lungs dissected clear of major airways were homogenized and fractionated by centrifugation into two fractions, soluble (supernate) and insoluble (pellet). Each fraction was analyzed by nonequilibrium competitive enzyme-linked immunoassay (ELISA) for Fbn and Lam normalized to mg protein. The results show a dose-related increase in soluble Fbn demonstrable at 1 week PI and approaching seven times control values by 28 weeks for doses of 13 Gy. Thereafter amounts decrease steadily to 63 weeks. Insoluble Fbn remains at or near control values through 13 weeks, increases in a dose-related fashion almost fivefold by 41 weeks for doses of 13 Gy, and then decreases by 63 weeks. Soluble Lam increased slightly during the duration of the study, returning to normal by 63 weeks. Insoluble Lam shows a dose-dependent increase demonstrable at 4 weeks PI which continues through 63 weeks. Interactions between these alterations in Fbn and Lam and previously reported changes in basal laminar proteoglycans may, in concert with other cellular and extracellular components, relate to the initiation and/or maintenance of radiation-induced pulmonary fibrosis.

Cell-cell matrix interactions in induced lung injury: III. Long term effects of X-irradiation on basal laminar proteoglycans.

The lungs of male LAF1 mice were locally irradiated with doses of 5, 9, and 13 Gy. The animals were killed at times corresponding to the appearance of histologically identifiable fibrosis or, for 13 Gy, at the LD50 for these doses and strain of mouse: 63, 36, and 28 weeks postirradiation (PI) respectively. Lungs were excised, incubated in buffer alone, or partially digested with enzymes for determination of relative glycosidase resistance, fixed with ruthenium red/Triton X-100 for demonstration of basal laminar anionic sites, and processed for electron microscopy. Sham-irradiated and untreated control groups (0 Gy, 0 times) were also processed. Tissue was examined ultrastructurally and alterations in both alveolar and capillary basal laminar anionic sites were quantitated. In each of the doses examined the number of anionic sites surpassed normal levels; however, the glycosidase resistance of the regenerated laminae at these late time points was not significantly altered from controls. This contrasts with the marked increase in the glycosidase resistance of laminae regenerating from radiation damage (4-12 weeks PI) reported earlier. The increased numbers of anionic sites were compared to expected values derived from models based on compensatory synthesis and continued accumulation and indicate close correlation with certain aspects of the compensatory synthesis model but not with others. The effects on basal laminar permeability, basal laminar thickening, and fibrotic induction are discussed.

Long term effects of radiation and combined modalities on mouse lung.

The lung appears to be the major dose-limiting organ in radiation of the thorax. Early responses (less than 1 week) involve the type II pneumocyte and increased surfactant biosynthesis and secretion. Later changes, which appear to be related to the surfactant response, lead to classical radiation pneumonitis, which is often fatal. Animals which survive radiation pneumonitis develop progressive fibrosis, a late-appearing response, which reduces compliance and available air space, and is usually fatal. This study centers on the fine structural changes in the lungs of LAF1 mice, 63 weeks following various radiation exposures (5-13 Gy). Doses which are subthreshold in evoking surfactant and pneumonitic responses precipitate fibrosis and atelectasis by 63 weeks, and involve type II pneumocyte sloughing and degeneration. Of the two major deterrents to lung irradiation (pneumonitis and fibrosis), these results suggest that fibrosis always follows pneumonitis, but pneumonitis is not a necessary preliminary step to fibrosis. Bleomycin elicits several morphological alterations characteristic of radiation, and, when combined with the latter, appears to exacerbate radiation effects.

The growth and morphology of FRTL-5 thyroid epithelial cells grown as multicellular spheroids in vitro.

FRTL-5 cells, a diploid line of differentiated rat thyroid epithelial cells, have been grown as multicellular spheroids in spinner culture. Spheroids were initiated by seeding FRTL-5 cells either into Lab-Tek dishes or culture flasks with a 0.5% agar base. Thyroid stimulating hormone (TSH, greater than 1.0 mU/ml) was required for initial cell aggregation and spheroid growth. After 1 wk cellular aggregates were transferred to suspension culture in spinner flasks. As with FRTL-5 monolayer cultures, continued spheroid growth required the addition of TSH to the culture medium. The most unique characteristic of the FRTL-5 spheroids was the development of central lumina similar to thyroid follicles in vivo. Follicular structures were absent from spheroids not stimulated with TSH. In the presence of TSH epithelial cells seem metabolically active with morphological evidence of biosynthesis of thyroglobulin-like material and basal laminar-like components. In contrast, all evidence of cellular metabolic activity is absent from cells in spheroids maintained in the absence of TSH. Thus, nontransformed FRTL-5 cells grown as three-dimensional multicellular spheroids responded to hormonal manipulation in a manner comparable to follicular epithelial cells in vivo. This spheroid model might therefore prove to be a very effective tool for investigating aspects of thyroid physiology and pathology in vitro.

Cell-cell matrix interactions in induced lung injury. II. X-irradiation mediated changes in specific basal laminar glycosaminoglycans.

The thoraces of male LAF1 mice were irradiated at doses of 5, 9, or 13 Gy. The animals were killed at times of 1 hr, 1 day, 1 wk, 4 wks, and 12 wks postirradiation (PI). The lungs were removed, enzymatically or detergent digested, fixed with ruthenium red for demonstration of anionic sites, and processed for electron microscopy. Untreated (0 Gy, 0 time) and sham irradiated control groups were also processed. Sections of lungs were examined and changes in alveolar basal laminar anionic sites were quantitated. Changes in three groups of glycosaminoglycans (GAGs) were identified: chondroitin 4 and 6 sulfate-dermatan sulfate, hyaluronate, and other GAGs (principally heparan sulfate). At 1 hr PI, all groups showed a marked decrease in site number over controls, which continued to 1 wk. By 4 wks there was a marked relative increase in heparan sulfate containing sites for doses of 13 Gy and a moderate increase for the other doses. At 12 wks the level of heparan sulfate was considerably above normal for doses of 13 Gy and just above normal for the other doses. Chondroitin-dermatan sulfate had recovered by 12 wks for doses of 13 Gy, but was still subnormal for other doses; however, hyaluronate-containing sites recovered only slightly by 12 wks. The implications for this change on basal laminar permeability and the induction of fibrosis are discussed.

Topological correlation between the cell-recognition protein, R-cognin and α-bungarotoxin receptor in retinal plasma membrane.

The amount of the neural retina cell recognition protein, R-cognin, in the plasma membrane of chick embryo neural retina cells declined 43% between 10 and 17 days of embryonic development. Over this period there was a 27% increase in the plasma membrane content of the α-bungarotoxin receptor. Plasma membranes of both these ages were sonicated into vesicles and these vesicles partitioned on α-bungarotoxin agarose beads into those which contained detectable α-bungarotoxin receptor and those which did not. At 10 days, approximately 6% of the plasma membrane vesicles contained receptor. At 17 days, <2% did. At 10 days, 60% of the R-cognin was found in the α-bungarotoxin receptor-containing vesicles, at 17 days 86%. At 17 days, 6% of the retina membrane with a high concentration of both α-bungarotoxin receptor and R-cognin was of a density indicative of it being of synaptic origin. These results suggested that R-cognin and α-bungarotoxin receptor occurred close together in the plasma membrane of retina cells. However, the lack of competition between R-cognin gamma globulin and specific α-bungarotoxin binding indicated that the α-bungarotoxin receptor and R-cognin were not the same protein. Thus, R-cognin and the α-bungarotoxin receptor appear to be separate proteins which occur in close proximity on the retina plasma membrane.

Cell-cell matrix interactions in induced lung injury. I. The effects of X-irradiation on basal laminar proteoglycans.

The lungs of male LAF1/J mice were locally irradiated with graded doses of radiation ranging from 5 to 13 Gy. The animals were sacrificed at 1 hr, 1 day, 1 week, 4 weeks, and 12 weeks postirradiation (PI), fixed with Ruthenium Red/Triton X-100 for demonstration of basal laminar anionic sites, and processed for electron microscopy. Sham-irradiated (0 Gy, 0 time) and untreated control groups were also processed. Sections of lungs were examined ultrastructurally and changes in both alveolar and capillary basal laminar anionic sites quantitated. A marked decrease in the number of basal laminar anionic sites was noted 1 hr PI in both alveolar and capillary basal laminae at all dose levels. The decline continued to 1 week for doses of 13 Gy and more gradually to 4 weeks following doses of 5 and 9 Gy, when the number of sites began to increase. By 12 weeks animals receiving 13 Gy were approaching normal levels while those receiving 5 or 9 Gy remained subnormal. The potential effects of the loss of proteoglycans with radiation on lung basal laminar permeability and cell-extracellular matrix interactions are discussed.

Light-microscopic immunocytochemical localization of fibronectin in the developing rat lung.

The development of the rat lung is a process of continuing morphological change. Indications from work in other mammalian systems suggest that fibronectin may be important in the control of this process. The present study has examined embryonic, neonatal, and adult lung tissue of the rat by means of the peroxidase-antiperoxidase (PAP) technique to demonstrate fibronectin at the light-microscopic level. Positive reaction was observed with anti-fibronectin serum in all stages examined. Control sections treated with pre-immune serum or no primary serum gave negative results in each case. Fibronectin in adult tissue was localized to the alveolar surface and alveolar basal lamina. Neonatal tissue showed fibronectin on pulmonary tubule walls and in basal lamina while embryonic tissue revealed localization of the protein in the basal lamina and in association with small groups of cells at the base of septal buds. These findings suggest a role for fibronectin in the control of rat lung development. The results are discussed in terms of the known functions of fibronectin as a preliminary matrix for the subsequent deposition of collagenous connective tissue, as a cellular adhesion protein, and as surface-bound material for cellular migration.

Ultrastructural immunocytochemical localization of fibronectin in the developing rat lung.

In a previous study changes in the macrodistribution of fibronectin during rat-lung development were examined. Using the peroxidase-antiperoxidase immunocytochemical technique, we have demonstrated the presence of fibronectin in embryonic, neonatal, and adult rat lung at the ultrastructural level. In the embryo, fibronectin is found both in an intra- and extracellular association with isolated pneumoblasts, and in a periodic distribution along the basal lamina. The neonate displays fibronectin in an intracellular association with early type-I cells and on their basal and luminal surfaces, but not in association with type-II cells. Neonatal basal lamina is diffusely labeled by anti-fibronectin antiserum. Fibronectin in adult tissue is found both intracellularly and on the basal and luminal surfaces of type-I cells but not in type-II cells. The basal lamina and interstitial connective tissue are slightly or non-reactive. These observations confirm and extend our initial suggestion that fibronectin is involved in rat-lung development.

Palladium chloride as a stain for elastin at the ultrastructural level.

Palladium chloride in aqueous solution stains elastic fibers in thin sections of Epon-embedded tissues. When palladium chloride is used with a lead citrate counterstain, high contrast sections with gray to black elastic fibers are obtained. The stain was tested on newborn and adult mammalian tissues and on adult tissues from lower animals. Sections were mounted on stainless steel grids, stained with 1% palladium chloride solution for 5 to 15 min, rinsed thoroughly, and counterstained with lead citrate for 7 min. Palladium chloride staining solution is stable for several months at room temperature and if the stain is filtered immediately before use, contamination of sections is not a problem. Chemical studies indicate that palladium binds directly to purified bovine ligamentum nuchae elastin and that this binding is not affected by glutaraldehyde fixation or by sodium borohydride reduction of elastin. Osmium post-fixation of glutaraldehyde-fixed elastin did significantly lower the amount of palladium bound. Palladium was shown to be chemically bound to sites on the elastin and not weakly associated. The nature of these sites is discussed.