Robert J. Mason

Differential requirement for the activation of the inflammasome for processing and release of IL-1beta in monocytes and macrophages.

The processing of pro-interleukin-1beta depends on activation of caspase-1. Controversy has arisen whether Toll-like receptor (TLR) ligands alone can activate caspase-1 for release of interleukin-1beta (IL-1beta). Here we demonstrate that human blood monocytes release processed IL-1beta after a one-time stimulation with either TLR2 or TLR4 ligands, resulting from constitutively activated caspase-1 and release of endogenous adenosine triphosphate. The constitutive activation of caspase-1 depends on the inflammasome components, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and NALP3, but in monocytes caspase-1 activation is uncoupled from pathogen-associated molecular pattern recognition. In contrast, macrophages are unable to process and release IL-1beta solely by TLR ligands and require a second adenosine triphosphate stimulation. We conclude that IL-1beta production is differentially regulated in monocytes and macrophages, and this reflects their separate functions in host defense and inflammation.

Pulmonary Alveolar Type II Cells Isolated from Rats: RELEASE OF PHOSPHATIDYLCHOLINE IN RESPONSE TO β-ADRENERGIC STIMULATION

It is unclear what factors control the secretion of pulmonary surface active material from alveolar type II cells in vivo. Other workers have suggested that cholinergic stimuli, adrenergic stimuli, and prostaglandins may all stimulate secretion. We isolated type II cells from the lungs of rats by treatment with elastase, discontinuous density centrifugation, and adherence in primary culture. beta-Adrenergic agonists, but not cholinergic agonists, caused an increase in the release of [(14)C]disaturated phosphatidylcholine, the major component of surface-active material, from type II cells in culture. The beta-adrenergic effect was stereo-selective, (-)-isoproterenol being 50 times more potent than (+)-isoproterenol. Terbutaline, 10 muM, a noncatecholamine beta-2 adrenergic agonist, caused a release of 2.0+/-0.5 (mean+/-SD) times the basal release of [(14)C]disaturated phosphatidylcholine in 3 h; the concentration of terbutaline causing half maximal stimulation was 800 nM. The terbutaline effect was blocked by propranolol, a beta-adrenergic antagonist (calculated K(d) = 6 nM), but not by phentolamine, an alpha-adrenergic antagonist. Isobutylmethylxanthine, a phosphodiesterase inhibitor, and 8-Br cyclic AMP, but not 8-Br cyclic guanosine monophosphate, also stimulated release. We conclude that type II cells secrete disaturated phosphatidylcholine in response to treatment with adrenergic stimulation.

Keratinocyte growth factor and hepatocyte growth factor/scatter factor are heparin-binding growth factors for alveolar type II cells in fibroblast-conditioned medium.

Epithelial-mesenchymal interactions mediate aspects of normal lung growth and development and are important in the restoration of normal alveolar architecture after lung injury. To determine if fibroblasts are a source of soluble growth factors for alveolar type II cells, we investigated the effect of fibroblast-conditioned medium (CM) on alveolar type II cell DNA synthesis. Serum-free CM from confluent adult human lung fibroblasts was concentrated fivefold by lyophilization. Type II cells were isolated from adult rats by elastase dissociation and incubated with [3H]thymidine and varying dilutions of concentrated CM and serum from day 1 to 3 of culture. Stimulation of type II cell DNA synthesis by fibroblast-CM was maximal after 48 h of conditioning and required the presence of serum. The activity of the CM was eliminated by boiling and by treatment with trypsin, pepsin, or dithiothreitol and was additive with saturating concentrations of acidic fibroblast growth factor, epidermal growth factor, and insulin. The growth factor activity bound to heparin-Sepharose and was eluted with 0.6 and 1.0 M NaCl. Neutralizing antibody studies demonstrated that the primary mitogens isolated in the 0.6 and 1.0 M NaCl fractions were keratinocyte growth factor (KGF, fibroblast growth factor 7) and hepatocyte growth factor/scatter factor (HGF/SF), respectively. HGF/SF was demonstrated in the crude CM and KGF was detected in the 0.6 M NaCl eluent by immunoblotting. Northern blot analysis confirmed that the lung fibroblasts expressed both KGF and HGF/SF transcripts. Human recombinant KGF and HGF/SF induced a concentration- and serum-dependent increase in rat alveolar type II cell DNA synthesis. We conclude that adult human lung fibroblasts produce at least two soluble heparin-binding growth factors, KGF and HGF/SF, which promote DNA synthesis and proliferation of rat alveolar type II cells in primary culture. KGF and HGF/SF may be important stimuli for alveolar type II cell proliferation during lung growth and after lung injury.

Lipids of alveolar macrophages, polymorphonuclear leukocytes, and their phagocytic vesicles

Phagocytic vesicles were isolated from rabbit alveolar macrophages and guinea pig polymorphonuclear leukocytes that had ingested emulsified paraffin oil. Phospholipids and their fatty acids were determined in whole cells and in the phagocytic vesicle and pellet fractions separated from them. The cholesterol-to-phospholipid ratios in the vesicle fractions were distinctly higher than those of the respective whole cells or pellet fractions. The vesicle fractions also had higher phospholipid-to-protein ratios than did the whole cells. The phospholipids of the phagocytic vesicle fraction from macrophages contained relatively more sphingomyelin, lyso-(bis)phosphatidic acid, and phosphatidylserine and less lecithin, phosphatidylethanolamine, and phosphatidylinositol than did the whole cells or pellet fractions. The phospholipids of phagocytic vesicles from polymorphonuclear leukocytes contained significantly more phosphatidylinositol than did the pellet fractions. Lyso(bis)phosphatidic acid, which constituted 15% of the phospholipid in rabbit alveolar macrophages and 25% of that in their phagocytic vesicles, contained almost 60% oleic acid and 20% linoleic acid. This lipid was not detected in rabbit peritoneal macrophages or in rat alveolar macrophages.The polyunsaturated fatty acids of leukocyte phospholipids were chiefly linoleic, whereas in macrophages arachidonic accounted for almost 20% of the total fatty acids. The macrophages produced malondialdehyde when ingesting polystyrene beads or emulsified paraffin oil, from which it was inferred that peroxidation of endogenous lipid can occur during phagocytosis. Polymorphonuclear leukocytes in which less than 3% of phospholipid fatty acids were arachidonic did not produce malondialdehyde during phagocytosis of these inert particles, but did when ingesting an emulsion containing linolenate, thus providing evidence for peroxidation of ingested lipid. Isolated phagocytic vesicles from alveolar macrophages contained lipid peroxides and generated malondialdehyde when incubated with ADP, FeCl(3), and NADH.

Quantitative Studies of Phagocytosis by Polymorphonuclear Leukocytes: Use of Emulsions to Measure the Initial Rate of Phagocytosis

Polymorphonuclear leukocytes suspended in Krebs-Ringer phosphate medium ingest paraffin oil containing Oil Red O emulsified with a variety of substances. Spectrophotometric determination of Oil Red O in the cells after uningested particles have been removed by differential centrifugation provides a quantitative measure of phagocytosis. This system has been used to investigate the effects of several drugs and hormones on the initial rate of phagocytosis and to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. The rate of uptake of paraffin oil emulsified with bovine albumin was constant for 6 min and was proportional to cell concentration when saturating concentrations of paraffin oil emulsion were used. At lower concentrations of substrate, the initial rate of phagocytosis was directly proportional to paraffin oil concentration. The increment in glucose oxidation associated with phagocytosis varied directly with the initial rate of particle uptake. The rate of ingestion of the albumin emulsion was not altered by serum (2-20%, v/v), glucose (5-20 mM), or omission of potassium from the medium. The rate of phagocytosis was decreased 65% if magnesium was omitted, and was essentially zero in the absence of divalent cations. The initial rate of uptake was inhibited by inhibitors of glycolysis, by N-ethylmaleimide (0.05-1 mM), colchicine (0.001-0.1 mM), theophylline (1 and 2 mM), dibutyryl cyclic AMP (1 mM), hydrocortisone (2.1 mM), and ethanol (85 mM). Inhibitors of oxidative phosphorylation and dexamethasone (0.01 mM) were without effect, while insulin (2 mU/ml) slightly stimulated the phagocytic rate. Paraffin oil emulsified with different agents was used to approach the question of how the surface of a particle influences its acceptability as a substrate for phagocytosis. Emulsions prepared with nonionic detergents, methylated proteins, and proteins with a weak net charge at pH 7.4 were poorly ingested. On the other hand emulsions prepared with agents of strong net positive or negative charge were rapidly taken up. The effect of divalent cations on the rate of phagocytosis varied with the nature of the emulsifier, but was not related in any simple, direct fashion to the net surface charge of the particles. However, it has not been conclusively established that charge was the only variable of the emulsion particles employed.

Biology of alveolar type II cells

Abstract:  The purpose of this review is to highlight the many metabolic properties of alveolar type II cells, their production of surfactant, their role in innate immunity, and their importance in the repair process after lung injury. The review is based on the medical literature and results from our laboratory. Type II cells produce and secrete pulmonary surfactant and for that purpose they need to synthesize the lipids of surfactant. One of the regulators of lipogenesis is the transcription factor sterol regulatory element binding protein‐1c (SREBP‐1c). This is a key transcription factor regulating fatty acid synthesis. Type II cells also proliferate to restore the epithelium after lung injury, clear alveolar fluid by transporting sodium from the apical to the basolateral surface, and participate in the innate immune response to inhaled materials and organisms. The type II cell is, in many ways, the defender of the alveolus. However, the type II cells work in concert with the other cells in the gas exchange regions of the lung to keep the alveoli open and reduce inflammation due to irritants in the air we breathe.

Functional differentiation of alveolar type II epithelial cells in vitro: Effects of cell shape, cell-matrix interactions and cell-cell interactions

Alveolar type II epithelial cells rapidly lose characteristics of differentiated function when cultured on plastic dishes. We have attempted to circumvent this problem by culturing type II cells under conditions that might better reproduce their environment in vivo. Cell-matrix interactions were studied by culturing isolated adult rat type II cells on Engelbreth-Holm-Swarm (EHS) tumor basement membrane. Aggregates of type II cells formed on the surface of the matrix during 4 days in culture. Microscopic examination of these aggregates revealed cuboidal cells that retained more characteristics of differentiated type II cells than did cells cultured on plastic. Type II cells cultured on EHS matrix incorporated a higher percentage of acetate into phosphatidylcholine (PC) than did cells on plastic, and a higher percentage of this PC was saturated. Phosphatidylglycerol (PG) synthesis by these cells was no different from that seen in cells on plastic. The effects of cell-cell interactions and cell shape were evaluated by culturing type II cells on feeder layers that in turn were grown on collagen gels. The feeder layer cells included fetal rat lung fibroblasts, adult rat lung fibroblasts, fetal rat skin fibroblasts, bovine aortic endothelial cells, and rat mammary tumor epithelial cells. One-half of the gels remained attached to the culture dish and one-half of the gels were detached after 24 h and allowed to float free in the medium. Type II cells grown in association with any of the attached feeder layers became flattened and lost their differentiated phenotype. These cells incorporated no greater percentage of acetate into PC than did cells on plastic. Saturated PC synthesis was modestly increased. PG synthesis declined in parallel with that seen in cells cultured on plastic. Type II cells cultured on feeder layers that were detached assumed their native cuboidal shape and also exhibited many morphological characteristics of differentiated function. These cells incorporated a significantly greater percentage of acetate into PC compared to cells on either plastic or attached feeder layers. Saturated PC synthesis also increased markedly. These cells, however, incorporated no greater percentage of acetate into PG than did cells on plastic or attached feeder layers. These data suggest an important role for cell shape and cell-matrix interactions and maintenance of type II cell differentiation. The effects of cell-cell interactions, while beneficial, appear to be non-specific.

Surfactant protein A and surfactant protein D in health and disease

Surfactant protein (SP) A and SP-D are collagenous glycoproteins with multiple functions in the lung. Both of these proteins are calcium-dependent lectins and are structurally similar to mannose-binding protein and bovine conglutinin. Both form polyvalent multimeric structures for interactions with pathogens, cells, or other molecules. SP-A is an integral part of the surfactant system, binds phospholipids avidly, and is found in lamellar bodies and tubular myelin. Initially, most research interest focused on its role in surfactant homeostasis. Recently, more attention has been placed on the role of SP-A as a host defense molecule and its interactions with pathogens and phagocytic cells. SP-D is much less involved with the surfactant system. SP-D appears to be primarily a host defense molecule that binds surfactant phospholipids poorly and is not found in lamellar inclusion bodies or tubular myelin. Both SP-A and SP-D bind a wide spectrum of pathogens including viruses, bacteria, fungi, and pneumocystis. In addition, both molecules have been measured in the systemic circulation by immunologic methods and may be useful biomarkers of disease. The current challenges are characterization of the three-dimensional crystal structure of SP-A and SP-D, molecular cloning of their receptors, and determination of their precise physiological functions in vivo.Surfactant protein (SP) A and SP-D are collagenous glycoproteins with multiple functions in the lung. Both of these proteins are calcium-dependent lectins and are structurally similar to mannose-binding protein and bovine conglutinin. Both form polyvalent multimeric structures for interactions with pathogens, cells, or other molecules. SP-A is an integral part of the surfactant system, binds phospholipids avidly, and is found in lamellar bodies and tubular myelin. Initially, most research interest focused on its role in surfactant homeostasis. Recently, more attention has been placed on the role of SP-A as a host defense molecule and its interactions with pathogens and phagocytic cells. SP-D is much less involved with the surfactant system. SP-D appears to be primarily a host defense molecule that binds surfactant phospholipids poorly and is not found in lamellar inclusion bodies or tubular myelin. Both SP-A and SP-D bind a wide spectrum of pathogens including viruses, bacteria, fungi, and pneumocystis. In addition, both molecules have been measured in the systemic circulation by immunologic methods and may be useful biomarkers of disease. The current challenges are characterization of the three-dimensional crystal structure of SP-A and SP-D, molecular cloning of their receptors, and determination of their precise physiological functions in vivo.

Lipid Peroxidation by Human Blood Phagocytes

Cell suspensions enriched in human blood monocytes, obtained from normal peripheral blood by sedimentation on sodium diatrizoate-Ficoll gradients or from the blood of patients with neutropenia and monocytosis, accumulated malonyldialdehyde, a labile catabolite of lipid peroxidation, during incubations with polystyrene beads or heat-killed Staphylococcus epidermidis. Mixed blood leukocytes principally composed of granulocytes or granulocytes purified by density gradient sedimentation did not accumulate malonyldialdehyde during incubations with these particles, but did when ingesting particles containing linolenate. The phospholipid fatty acid composition of monocyte-enriched and purified granulocyte preparations from the same donors were compared. The molar fraction of arachidonate (20:4) in phospholipids from monocyte-rich preparations was 62% greater than that of purified granulocytes. The findings indicate that human monocytes, possibly because of a greater content of polyunsaturated fatty acids in their membranes, peroxidize a greater quantity of endogenous lipids than granulocytes during endocytosis. Normal human granulocytes have the capacity to peroxidize ingested lipids. However, mixed leukocytes from two patients with chronic granulomatous disease produced little malonyldialdehyde when engulfing linolenate-containing particles. Therefore the capacity to peroxidize lipid is related to cellular oxygen metabolism, a function in which chronic granulomatous disease granulocytes are dificient. Malonyldialdehyde chemically prepared by hydrolysis of tetramethoxypropane, by extraction from peroxidized linolenic acid, or purified from extracts of phagocytizing rabbit alveolar macrophages had bactericidal activity against Escherichia coli and S. epidermidis. Therefore, toxic catabolites of lipid hydroperoxides may potentiate the bactericidal activity of hydrogen peroxide in mononuclear phagocytes.

Isolation and Properties of Phagocytic Vesicles from Polymorphonuclear Leukocytes

A method for the isolation of intact phagocytic vesicles from guinea pig peritoneal-exudate granulocytes and human peripheral-blood leukocytes is presented. After leukocytes ingested the particles of a stable emulsion of paraffin oil, the uningested emulsion was washed away and the cells were homogenized. The homogenate was placed in the middle of a three-step discontinuous sucrose gradient and centrifuged for 1 hr at 100,000 g. The phagocytic vesicles, containing the low density paraffin-oil particles, were simultaneously washed and collected by floatation, while the other organelles, chiefly granules, sedimented through the lower wash layer, and the particle-free supernatant remained in the middle of the gradient. Emulsion particles stained with Oil Red O were employed to assay the rate of phagocytosis and to mark the location of the particles in subcellular fractions. The dye was extracted from washed cells or cell fractions with dioxane and colorimetrically quantified. The purity of phagocytic vesicles obtained by this method was assessed by electron microscopy, chemical analysis, and assay of enzyme composition. Granule-associated enzymes, acid phosphatase, alkaline phosphatase, beta-glucuronidase, and peroxidase were present in the phagocytic vesicles and originated from the granules. Cyanide-resistant NADH (reduced form of diphosphopyridine nucleotide) oxidase was also found. Enzymes associated with the vesicles exhibited latency to Triton X-100. Uptake of particles and the transfer of total protein and phospholipid into phagocytic vesicles occurred simultaneously Accumulation of acid and alkaline phosphatase in the vesicles continued until phagocytosis ceased. Peroxidase, NADH oxidase, and beta-glucuronidase activities in the phagocytic vesicles, on the other hand, were maximal by 30 min and increased little thereafter even when phagocytosis was still going on.