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Biomedical subjects

G E Hook

Publications and source records attributed to G E Hook.

At least 19 recordsLinked to original sources
PubMed1995

Altered regulation of surfactant phospholipid and protein A during acute pulmonary inflammation.

Biochemical changes in the pulmonary surfactant system caused by exposure to toxicants are often accompanied by an influx of inflammatory cells into the lungs. We have investigated the possibility that the inflammatory and surfactant biochemical effects might be connected. Co-treatment with dexamethasone, a synthetic anti-inflammatory glucocorticoid, mitigated the increases in free cells and total intracellular surfactant phospholipid normally seen in animals given silica alone, suggesting a relationship between the free cell population of the alveoli and the surfactant system during alveolitis. Furthermore, we have investigated whether induction of the surfactant system is a universal response to alveolar inflammation. Inflammation was induced in the lungs by intratracheal injections of titanium dioxide, silica, bleomycin or lipopolysaccharide (LPS) suspended in isotonic saline. Inflammatory cell and surfactant responses were measured at 3 days and 14 days following injection. There was a distinct alveolar inflammatory cell profile following administration of each agent, at each time point, indicating a dynamic inflammatory cell population during the course of the study. Furthermore, surfactant phospholipid and protein A (SP-A) pools exhibited unique responses to the inflammatory agents. Only silica-treated lungs maintained elevated levels of surfactant phospholipids and SP-A throughout the course of the experiment. We conclude that both the surfactant components and the inflammatory cell population of the alveoli undergo dynamic changes following treatment with these inflammatory agents and that activation of the surfactant system is not a universal response to alveolar inflammation, since surfactant components were not always elevated during times of increased alveolar cellularity. The unique inflammatory cell infiltrate elicited by silica is of particular interest in that surfactant components were elevated throughout the course of the experiment in this group. Indeed, we have shown that the size of the intracellular pool of surfactant is directly proportional to the number of polymorphonuclear leukocytes but not alveolar macrophages or lymphocytes in the alveoli following silica treatment. Finally, our data suggest that the phospholipid and SP-A components of surfactant respond differentially to the pulmonary toxicants in this study.

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PubMed1993

Secretory product expression during Clara cell differentiation in the rabbit and rat.

One function of the nonciliated (Clara) cells of bronchiolar epithelium is to synthesize, store, and release small-molecular-mass (6-12 kDa) secretory proteins or Clara cell secretory protein (CCSP). This study compares the emergence of this secretory function during Clara cell differentiation in rabbits and rats. Lungs of fetal and postnatal animals were evaluated by ultrastructural morphometry and immunohistochemistry. Secretory granules were rarely seen in perinatal animals and increased to adult levels of abundance earlier in rats (1 wk postnatal) than in rabbits (3-4 wk). In contrast, rough endoplasmic reticulum was abundant in perinatal animals and decreased with age. Antibodies raised against CCSP revealed little CCSP in fetal animals; however, after birth CCSP increased to adult levels earlier in rats (1 wk postnatal) than in rabbits (3 wk). We conclude that the maturation of Clara cell secretory function 1) occurs postnatally, 2) involves a decrease in biosynthetic organelles, 3) shows close association between CCSP expression and secretory granule abundance, and 4) varies by species in timing and cellular abundance of biosynthetic machinery.

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PubMed1993

Confocal laser scanning immunofluorescence microscopy of the pulmonary surfactant system. Association of surfactant protein A with the nucleus of the alveolar type II cell.

BACKGROUND: Localization of surfactant protein A (SP-A) to nucleus of type II cells isolated from the lungs of rats has been reported. Data suggested that most SP-A was located within lamellar bodies of the type II cell; however, some SP-A was found in other cytoplasmic regions of the cell and in particular in the nucleus. EXPERIMENTAL DESIGN: Type II cells and type II cell nuclei isolated from the lungs of rats were reacted with affinity-purified antibodies against SP-A. Location of SP-A was determined by using fluorescein isothiocyanate-labeled secondary antibodies and the distribution of fluorescence examined by using a laser scanning microscope fitted with a confocal aperture. Two-dimensional electrophoresis followed by Western blotting was used to separate and identify type II cell nuclear proteins. RESULTS: Nuclei were isolated from type II cells obtained from elastase-digested rat lungs and examined for the presence of SP-A. The nuclei contained both focal and diffuse deposits of SP-A. Some regions within the nuclear matrix (in particular the nucleolus) appeared to be relatively devoid of SP-A. The perinuclear membrane stained intensely for SP-A where optical sectioning showed its presence as a patchwork of punctate deposits. Analysis of the SP-A associated with the nucleus by two-dimensional electrophoresis revealed that it consisted of a family of proteins with molecular masses of 26, 32, and 36 kDa and pI 5.1. Biosynthesis of nuclear SP-A in primary cultures of type II cells was sensitive to inhibitors of glycosylation resulting in the presence of only the lowest molecular weight unprocessed form. CONCLUSIONS: These data indicate that three basic forms of SP-A are associated with the nucleus of the type II cell and are especially concentrated on the perinuclear membrane.

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PubMed1991

Application of confocal scanning laser microscopy in experimental pathology.

Confocal scanning laser microscopy (CSLM) represents an exciting new tool for scientific disciplines which focus on mechanistic studies such as experimental pathology. Enhanced resolution in the specimen plane and rejection of out-of-focus fluorescence flare allow analysis of specific nucleic acid sequences, enzymes, structural macromolecules, and cellular homeostasis utilizing fluorescent probes. Four different experimental applications are discussed which utilize CSLM to evaluate pathological processes at the subcellular, cellular, and tissue levels. Programmed cell death, or apoptosis, is a natural process of significance both during development and as a response to toxic stimuli. CSLM-imaging of nuclei of human B lymphoblastoid cells following exposure to a monofunctional alkylating agent suggests that the degradation of chromatin characteristic of apoptosis may occur in asymmetric patterns. Surfactant apoprotein-A is the major non-serum protein component of pulmonary surfactant and is essential for the extracellular function of surfactant. CSLM of alveolar type II cells suggests that apoprotein-A is present in both the cytoplasm, predominantly in lamellar bodies, and in the nucleus. The tumor promoter, phorbol myristate acetate, rapidly stimulated the formation of vacuoles in human neutrophils. CSLM using Lucifer Yellow as a probe suggests that cylindrical vacuoles are formed by fluid-phase pinocytosis. The blood-nerve barrier (BNB) in peripheral nerves may be an important target during toxin-induced neuropathies. Ricin-induced permeability of the BNB in the rat was rapidly visualized by CSLM as leakage of fluorescein isothiocynate (FITC)-dextran into the endoneurial compartment.

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PubMed1991

Alveolar proteinosis and phospholipidoses of the lungs.

Three pulmonary disease conditions result from the accumulation of phospholipids in the lung. These conditions are the human lung disease known as pulmonary alveolar proteinosis, the lipoproteinosis that arises in the lungs of rats during acute silicosis, and the phospholipidoses induced by numerous cationic amphiphilic therapeutic agents. In this paper, the status of phospholipid metabolism in the lungs during the process of each of these lung conditions has been reviewed and possible mechanisms for their establishment are discussed. Pulmonary alveolar proteinosis is characterized by the accumulation of tubular myelin-like multilamellated structures in the alveoli and distal airways of patients. These structures appear to be formed by a process of spontaneous assembly involving surfactant protein A and surfactant phospholipids. Structures similar to tubular myelin-like multilamellated structures can be seen in the alveoli of rats during acute silicosis and, as with the human condition, both surfactant protein A and surfactant phospholipids accumulate in the alveoli. Excessive accumulation of surfactant protein A and surfactant phospholipids in the alveoli could arise from their overproduction and hypersecretion by a subpopulation of Type II cells that are activated by silica, and possibly other agents. Phospholipidoses caused by cationic amphiphilic therapeutic agents arise as a result of their inhibition of phospholipid catabolism. Inhibition of phospholipases results in the accumulation of phospholipids in the cytoplasm of alveolar macrophages and other cells. While inhibition of phospholipases by these agents undoubtedly occurs, there are many anomalous features, such as the accumulation of extracellular phospholipids and surfactant protein A, that cannot be accounted for by this simplistic hypothesis.

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PubMed1991

Ultrastructural immunohistochemical localization of Clara cell secretory protein in pulmonary epithelium of rabbits.

Highly purified Clara cells (93 +/- 3%) isolated from the lungs of rabbits were used to produce an antiserum against Clara cell secretory proteins. This antiserum was used to identify and study the biosynthesis and secretion of [35S]methionine-labeled proteins from isolated Clara cells. The antiserum recognized one major secretory protein with apparent molecular weight of 6 kDa and reacted weakly with a higher molecular weight protein of about 180 kDa. Biosynthesis and secretion of these proteins was not detected in preparations of isolated alveolar type II cells or alveolar macrophages. Immunocytochemical localization of the antigen with colloidal gold indicated a dual localization in bronchiolar Clara cells. Gold labeling was found over the osmiophilic secretory granules of Clara cells and smooth endoplasmic reticulum. In tracheal Clara cells, labeling was found mostly in association with secretory granules and relatively little in association with the smooth endoplasmic reticulum. Labeling was also found over the lamellar bodies of type II cells, although the reaction was weak. Labeling of ciliated cells, alveolar type I cells, capillary endothelial cells, and alveolar macrophages was not distinguishable from background. These data indicate that Clara cells of both the bronchioles and trachea of rabbits synthesize and secrete the low molecular weight protein previously called Clara cell secretory protein (CCSP). This antigen does not belong to that group of surfactant proteins whose molecular weights range from 26 to 40 kDa.

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PubMed1991

Confocal laser scanning immunofluorescence microscopy of lamellar bodies and pulmonary surfactant protein A in isolated alveolar type II cells.

We have determined the distribution of surfactant protein A (SP-A) in isolated type II cells from the lungs of rats by using immunofluorescence in conjunction with a laser scanning microscope fitted with a confocal aperture. Because of the very narrow depth of field of this microscope (less than 0.4 microns) in the confocal format, we were able to optically section type II cells and determine both the distribution of SP-A in the type II cell and the distribution of the lamellar bodies. The location of SP-A was determined by using fluorescein isothiocyanate-labeled secondary antibodies and the lamellar body distribution by using the lipid soluble fluorescent stain Phosphine 3R. SP-A was detected in the cytoplasm of type II cells as asymmetrically distributed punctate fluorescent bodies that resembled lamellar bodies in terms of size, number, and distribution within the cytoplasm of the cell. Most of the SP-A was located within bodies of the type II cell. Diffuse patches of fluorescence were seen in other cytoplasmic regions as well as the number of the cell. We conclude that SP-A is localized primarily, but not exclusively, in lamellar bodies of type II cells and that laser scanning microscopy is a much superior technique for the localization of SP-A than conventional microscopy in terms of both sensitivity and resolution.

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PubMed1990

Induction of surfactant protein (SP-A) biosynthesis and SP-A mRNA in activated type II cells during acute silicosis in rats.

The synthesis of the major surfactant protein, SP-A, was studied in activated alveolar type II cells isolated from the lungs of rats exposed to silica by intratracheal instillation. Exposure of rats to silica resulted in large increases in the levels of disaturated phosphatidylcholine and SP-A in the extracellular and intracellular surfactant compartments. Isolated type II cells were used to determine if the observed increases in SP-A were associated with increased SP-A synthesis. Type II cells were isolated by a combination of elastase digestion, centrifugal elutriation, and differential adherence on IgG-coated petri dishes. Type II cells from silica-treated lungs were separated into two populations, designated type IIA and type IIB. The type IIB, or activated population, consisted of type II cells that were larger than normal type II cells and, in addition, contained larger and more numerous lamellar bodies than normal type II cells. Type IIB cells contained 4.3-fold higher levels of SP-A compared to normal type II cells. SP-A synthesis was measured by incubating freshly isolated cells with [35S]Translabel (70% [35S]methionine, 15% [35S]cysteine) for up to 4 h in methionine-free medium, followed by immunoprecipitation of newly synthesized protein. The rate of SP-A synthesis was increased approximately 6.7-fold in the activated type II cells. Analysis of the newly synthesized protein by one-dimensional SDS-PAGE indicated three intracellular forms of SP-A with molecular weights of approximately 26,000, 30,000, and 34,000. In type II cells from control rats, the 34-kD protein accounted for approximately 93% of the newly synthesized SP-A after 4 h of incubation; only a small amount of radioactivity was associated with the lower molecular weight species. The increased biosynthesis of SP-A in the activated type II cells was associated with a 7.3-fold increase in the level of SP-A mRNA. These results indicate that the content and synthesis of SP-A are both highly elevated in activated type II cells and that these increases may be due to increased levels of SP-A mRNA.

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PubMed1990

Pathways of differentiation of airway epithelial cells.

The question being examined is whether one or more morphologically distinct cell types can be identified in the conducting airways of adult rabbits possessing stem cell functions. The term "stem cell" is used to denote cells with extensive self-replicating potential and the ability to produce differentiated progeny. According to various models of cell renewal in the conducting airways that have been proposed over the years, two different cell types have to be regarded as primary candidates for the stem cell: basal cells and some type of secretory cells. The question is complicated by the fact that significant differences exist between species in the distribution and morphological characteristics of airway cell types. In addition, different airway segments may or may not be occupied by different populations of stem cells. Previously, investigators have addressed the problem by studying normal cell regeneration or injury induced cell regeneration in vivo in the whole animal. We decided to attempt a different approach, namely, to separate specific cell types and to study the proliferation and differentiation capacity of such cell isolates using in vitro and in vivo cell culture techniques. Our studies lead us to conclude that the conducting airways of adult rabbits contain at least two distinct cell populations endowed with stem cell potential, namely basal cells and bronchiolar Clara cells. From that it follows that the trachea and bronchi, on one hand, and the bronchioles, on the other hand, are occupied by two different stem cell populations governing renewal of the epithelial lining.

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PubMed1990

Hypertrophy and hyperplasia of alveolar type II cells in response to silica and other pulmonary toxicants.

Alveolar Type II cells serve two major functions in the lung, both of which are essential for the preservation of normal lung function. First, Type II cells synthesize and secrete pulmonary surfactant, and second, they function as progenitor cells for maintaining the alveolar epithelium. The Type II cell population of the lung is quite sensitive to the deposition of toxicants in the distal lung, responding in two principal ways. Damage to the Type I epithelium stimulates Type II cells to proliferate and subsequently differentiate to replace the injured Type I cells. Second, a portion of the Type II cell population may become hypertrophic. Both of these events are frequent findings in the diseased or damaged lung. The Type II cell changes are often associated with increases in surfactant pools. In those cases where ultrastructural characteristics of hypertrophic Type II cells were examined, the appearance of these cells was consistent with that of an activated cell type. Alterations in the lamellar body compartment are a common finding in hypertrophic Type II cells, with increases in both lamellar body size and number. It is likely that the hypertrophic, or activated, Type II cells account for the increased levels of surfactant found in the lungs after exposure to a variety of toxic agents. We examined, in detail, Type II cell hyperplasia and hypertrophy induced by silica deposition. Both Type II cell hyperplasia and hypertrophy were prominent responses. The proliferative response led to an approximate doubling of the number of Type II cells in the lung.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1989

Kinetics of pulmonary surfactant phosphatidylcholine metabolism in the lungs of silica-treated rats.

Exposure of rats to silica by intratracheal injection increased the intra- and extracellular compartments of pulmonary surfactant phospholipid. These changes were dose and time dependent, but both pools were not affected equally. Seven days after the instillation of 10 mg of silica, the intracellular pool increased 13.3-fold, from 1.49 +/- 0.30 to 19.86 +/- 0.77 mg of surfactant phospholipid per pair of lungs, and the extracellular pool increased 7.4-fold, from 1.87 +/- 0.79 to 13.79 +/- 0.72 mg of surfactant phospholipid per pair of lungs. To investigate the physiologic processes responsible for these massive accumulations of surfactant. [14C]choline was injected into the tail veins of control and silica-treated rats and the specific activity of surfactant phospholipids within the intracellular and extracellular pools was determined at various times between 0 and 26 hr after injection. All of the processes measured were increased in response to silica exposure, but not to the same extent. At 1 hr, incorporation of [14C]choline into the intracellular surfactant pool was increased 12.6-fold above controls, from 4.8 +/- 1.1 x 10(3) to 60.6 +/- 26.6 x 10(3) dpm. The flux of [14C]choline-labeled phospholipid from the intracellular to the extracellular pool was increased 7.3-fold, from 102 +/- 10 to 749 +/- 39 micrograms/hr in silica-treated animals, but its disappearance from the extracellular pool was increased only 5.0-fold, from 87 +/- 8 to 434 +/- 21 micrograms/hr. The half-life of [14C]choline-labeled phospholipids in the intracellular pool of surfactant was increased from 10.1 +/- 1.0 to 18.3 +/- 5.3 hr and that in the extracellular surfactant pool from 14.8 +/- 1.4 to 21.9 +/- 4.9 hr. Expansion of the intra- and extracellular pools of surfactant phospholipids may be explained on the basis of a metabolic imbalance in which the intracellular production of surfactant is increased above its secretion rate into the extracellular compartment, and the secretion rate is elevated above the rate at which surfactant phospholipids are cleared from the alveoli.

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PubMed1989

In vitro and in vivo growth and differentiation of clones of tracheal basal cells.

Studies were performed to examine the hypothesis that the basal cells in the large conducting airways function as stem cells. Basal cells were isolated from the tracheas of adult rabbits using centrifugal elutriation procedures. Such cell isolates that contained greater than 90% basal cells were plated in culture at low cell density to produce individual basal cell clones. Clones were similarly obtained from unfractionated tracheal cells (mixed tracheal cells). Basal cell and mixed cell clones were tested to compare their in vitro growth capacity and their ability to reepithelialize denuded tracheal grafts with a mucociliary epithelium. Both basal cell and mixed cell clones underwent 15 to 20 population doublings in culture. The sizes of their clonogenic cell compartments were roughly similar. When inoculated into denuded tracheal grafts, both types of clones were equally efficient in establishing a mucociliary epithelium. The major secretory cell types observed in tracheas repopulated with the two types of cell clones were cells containing small secretory granules and either rough or smooth endoplasmic reticulum. Goblet cells were seen in approximately 40% of tracheas repopulated with either of the two types of cell clones. It is concluded that in rabbits tracheal basal cells can generate secretory cells with varying morphologic characteristics as well as ciliated cells and may be regarded as epithelial stem cells.

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PubMed1988

Stimulation of surfactant phospholipid biosynthesis in the lungs of rats treated with silica.

The effects of intratracheally instilled silica (10 mg/rat) on the biosynthesis of surfactant phospholipids was investigated in the lungs of rats. The sizes of the intracellular and extracellular pools of surfactant phospholipids were measured 7, 14 and 28 days after silica exposure. The ability of lung slices to incorporate [14C]choline and [3H]palmitate into surfactant phosphatidylcholine (PC) and disaturated phosphatidylcholine (DSPC) was also investigated. Both intra- and extra-cellular pools of surfactant phospholipids were increased by silica treatment. The intracellular pool increased linearly over the 28-day time period, ultimately reaching a size 62-fold greater than controls. The extracellular pool also increased, but showed a pattern different from that of the intracellular pool. The extracellular pool increased non-linearly up to 14 days, and then declined. At its maximum, the extracellular pool was increased 16-fold over the control. The ability of lung slices to incorporate phospholipid precursors into surfactant-associated PC and DSPC was elevated at all time periods. The rate of incorporation of [14C]choline into surfactant PC and DSPC was maximal at 14 days and was nearly 3-fold greater than the rate in controls. The rate of incorporation of [3H]palmitate was also maximal at 14 days, approx. 5-fold above controls for PC and 3-fold for DSPC. At this same time point, the microsomal activity of cholinephosphate cytidylyltransferase was increased 4.5-fold above controls, but cytosolic activity was not significantly affected by silica treatment. These data indicate that biosynthesis of surfactant PC is elevated after treatment of lungs with silica and that this increased biosynthesis probably underlies the expansion of the intra- and extra-cellular pools of surfactant phospholipids.

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