Charles Kuhn

Fine structure of bronchiolo‐alveolar cell carcinoma

An electron microscopic study was made of 11 cases of bronchiolo‐alveolar carcinoma and the findings compared with normal lungs and with biopsies from patients with diffuse lung disease. Six of the tumors displayed bronchiolar differentiation manifested by mucin secretion, secretory granules resembling those of non‐ciliated bronchiolar cells, or by evidence of formation of ciliary basal bodies. Five other cases had atypical polymorphous cytoplasmic granules of uncertain composition. Such granules could indicate a relationship to either bronchiolar cells or to the hyperplastic alveolar epithelium common in damaged lung. Nuclear inclusions composed of membrane‐bounded vacuoles and tubules were seen in some carcinomas, in granular pneumocytes in the neighborhood of carcinomas, and in a small proportion of cases of diffuse lung disease without tumors. They are believed to form as invaginations of the inner leaflet of the nuclear membrane and may be a degenerative phenomenon.

Elastase of U-937 Monocytelike Cells: COMPARISONS WITH ELASTASES DERIVED FROM HUMAN MONOCYTES AND NEUTROPHILS AND MURINE MACROPHAGELIKE CELLS

As an approach to facilitating the understanding of proteinases associated with monocytes we have studied U-937 monocytelike cells. Elastase activity was identified in U-937 cell extracts and compared to monocyte elastase activity, neutrophil elastase, and the elastase activity from a continuous line of murine macrophagelike cells (P388D1). Serine proteinase activity which solubilized (14)C-labeled elastin accounted for >90% of the neutral proteinase activity of both U-937 cells and monocyte extracts. U-937 cell and monocyte elastase activities were similar catalytically, resembling neutrophil elastase. U-937 cells and monocytes showed other similarities: (a) both had activities reacting with [(3)H]diisopropylfluorophosphate that migrated in sodium dodecyl sulfate (SDS) polyacrylamide gels at approximately 30,000 and 60,000 daltons and (b) both contained material that cross-reacted with antiserum raised to neutrophil elastase. Preliminary characterization of U-937 cell elastase activity by affinity chromatography and ion-exchange chromatography suggested the presence of at least two distinct elastases. Minimal elastase activity was found in U-937 cell-conditioned medium, indicating that the activity is not spontaneously released by the cells. In contrast to the elastase activity associated with U-937 cells and monocytes, the elastase activity associated with P388D1 cells was a metalloproteinase and was found principally in the culture medium. These results indicate (a) U-937 cells will be useful for further investigation of proteinases associated with normal monocytes; (b) monocytes and U-937 cells contain material with catalytic and immunologic similarities to neutrophil elastase; (c) monocyte elastase activity differs from elastase activity secreted by murine macrophages and murine macrophagelike cells of the P388D1 line.

Receptor-Mediated Binding and Internalization of Leukocyte Elastase by Alveolar Macrophages In Vitro

Radioiodinated leukocyte elastase was found to bind rapidly and specifically to alveolar macrophages in vitro. In contrast to the binding of pancreatic and bacterial proteases, leukocyte elastase binding did not require the presence of alpha 2 macroglobulin. The binding was inhibited by an excess of unlabeled enzyme and was saturable by increasing elastase concentrations. Leukocyte elastase binding thus met criteria for receptor-mediated binding, with and estimated association constant of 4.97 x 10(5) M-1 and an estimated total of 640 x 10(6) binding sites/cell. It differed from the previously described binding of lysosomal glycosidases to macrophages in that it was insensitive to trypsin pretreatment, did not require calcium ions, and was not inhibited by yeast mannan. High-resolution autoradiography indicated that the cell-associated radiolabeled leukocyte elastase was rapidly incorporated into phagolysosomes. Macrophage binding may have a role in clearance of leukocyte elastase from tissue sites where alpha 2 macroglobulin is absent or present in low concentration. Thus, enzyme uptake by alveolar macrophages may be an important factor in the amelioration of lung tissue injury by extracellular leukocyte elastase.

The formation of granules in the bronchiolar Clara cells of the rat: 1. Electron microscopy

A morphologic study of bronchiolar Clara cells used rat lungs fixed by vascular perfusion or by inflation through the airways. Clara cells contained membrane-limited granules which had a crystalline structure with an 80 A periodicity when fixed by perfusion. The crystalline structure was infrequently seen after inflation fixation. The smooth endoplasmic reticulum was highly developed in the apex of Clara cells and appeared to be a major site of formation of the characteristic granules. Some nascent granules were also seen in association with the Golgi apparatus. Perfusion fixation retained a bronchiolar lining layer which was denser than the alveolar lining layer in the same sections and which resembled the matrix of Clara cells in opacity. One explanation for the difference between the bronchiolar and alveolar linings may be that the bronchiolar lining is derived in part from the secretions of Clara cells. Images suggesting exocytosis, albeit infrequent, support this suggestion.

The role of elastases in the development of emphysema.

Enzymes which degrade elastin can disorganize the network of elastic fibers in the lungs of experimental animals and produce emphysema. Two sources of endogenous elastases in the lung are neutrophils and alveolar macrophages. The neutrophil elastase is an intracellular, granule-associated enzyme which is inhibited by α1-antitrypsin and has the capacity to produce emphysema in experimental animals. The recently identified macrophage elastase appears to be a secretory enzyme, not associated with granules and less effectively inhibited by α1-antitrypsin. The demonstration that macrophages from cigarette smokers release elastase in culture, and that cigarette smoke interferes with the action of inhibitors of elastase, suggests that elastases may be involved in the pathogenesis of emphysema in man. Further research is needed to establish whether degradation of elastin occurs in humans developing emphysema.

The topography of the pulmonary alveolus: scanning electron microscopy using different fixations.

Scanning electron microscopy combines great depth of field with relatively high resolution, permitting a three-dimensional appreciation of lung structure. This study compares the topography of pulmonary alveoli by scanning electron microscopy using several different fixation procedures and correlating scanning with transmission electron microscopy. When lungs are distended with fixative through the airways, the alveoli tend to be polyhedral and considerable structural detail can be seen with the SEM. Granular pneumocytes, membranous pneumocytes, and macrophages all present recognizable surface specializations. When the lung is inflated with air at 20 cm of water pressure and fixation is achieved with either rapid freezing or intravascular perfusion of fixative, the alveolar contour is round and the alveolar surface is smooth. Collapse of capillaries due to intra-alveolar pressure, preservation of the extracellular alveolar lining layer, and surface forces all contribute to this appearance. If fixation is carried out under conditions in which capillary pressure exceeds transpulmonary pressure, the capillary bed is visible in relief as a reticular pattern of ridges.

The fine structure of so-called minute pulmonary chemodectomas

Electron microscopy was performed on several minute tumors of the type called chemodectomas, all from the lung of a single patient. The cells had a whorling pattern with extensive interdigitating cytoplasmic processes joined by desmosomes. Except for tangles of cytoplasmic fibrils, the tumor cells had few distinctive organelles. They had no endocrine-like granules and were not associated with nerves or basement membranes. The tumors had little resemblance to paragangliomas, but displayed a puzzling similarity to meningiomas. Our observations permit no definite conclusions as to the histogenesis of these lung tumors. Viewed in the light of recent physiologic studies, they cast doubt on the presence of special chemoreceptive paraganglia in the lung.

Extramembranous particles in tubular myelin from rat lung.

Tubular myelin is one morphologic form taken by extracellular pulmonary surfactant. We used electron microscopy after fixation in a mixture of tannic acid and glutaraldehyde followed by osmication to examine tubular myelin fixed in situ , isolated from lavage fluid by isopyknic centrifugation in sucrose gradients, and prepared in vitro from isolated lamellar bodies by the addition of 5 m M Ca 2+ . In each case, the bilayered membranes of tubular myelin were seen as parallel arrays or as a lattice with a spacing of approximately 5 nm. The use of tannic acid produced good visualization of rows of rod-like particles 5 nm in diamter and 14–16 nm in length. The particles were regularly spaced at intervals of 14–16 nm in rows along the angels formed by intersecting membranes. Particles on opposite sides of the bilayer were in register. Since these particles were observed on tubular myelin formed by the addition of calcium to purified lamellar bodies as well as on tubular myelin formed in vivo , they appear to form from material prepackaged in the lamellar bodies of the type II cells. We suggest that these particles are protein and are involved in the formation or maintenance of the structure of tubular myelin.

The formation of granules in the bronchiolar clara cells of the rat. II. Enzyme cytochemistry

The Clara cells of the rat contain characteristic round and rod-shaped electron-opaque granules in the apical cytoplasm. Small membranous sacs containing material resembling the contents of the granules were found among cisterns of smooth endoplasmic reticulum in the apex of the cell and in the region of the Golgi apparatus. These sacs were thought to be precursors of mature granules. To investigate the origin of these sacs, the localization of four phosphatases was studied cytochemically. Alkaline phosphatase was found in the apical plasma membrane; acid phosphatase was found in basilar lysosomes, the Golgi apparatus, and infrequently in mature granules; glucose-6-phosphatase (G-6-Pase) was found in the endoplasmic reticulum and thiamin pyrophosphatase (TPPase) in the Golgi apparatus. Mature granules and the majority of granule precursors lacked both G-6-Pase and TPPase. Rare granule precursors near the Golgi contained TPPase while a few in the apex contained G-6-Pase. The cytochemical results support a dual origin for the granules: Some form in the Golgi apparatus while others separate directly from smooth endoplasmic reticulum.

Particles resembling microbodies in normal and neoplastic perianal glands of dogs

SummaryBy electron microscopy, the parenchymal cells of the perianal glands of dogs contain granules which have the morphological features of microbodies (peroxisomes) including marginal plates and, occasionally, dense nucleoids. Like microbodies, they are occasionally attached to the endoplasmic reticulum. Histochemical evidence is presented suggesting that they contain at least one of the peroxisomal enzymes, L-α-hydroxy acid oxidase. The granules of a perianal gland adenoma showed abnormal morphologic variations.