Zvi Marom

Effects of Arachidonic Acid, Monohydroxyeicosatetraenoic Acid and Prostaglandins on the Release of Mucous Glycoproteins from Human Airways In Vitro

Human lung explants maintained in culture for 7 d incorporate [(3)H]glucosamine into mucous glycoproteins. Ethanol-precipitable, glucosamine-labeled mucous secretion was measured, and the effects of different pharmacologic agents upon this secretion were investigated. Anaphylaxed human lung generates prostaglandin (PG) synthesis and increased mucous release. Arachidonic acid (AA), PGA(2), PGD(2), and PGF(2alpha) significantly increased mucous glycoprotein release, whereas PGE(2) significantly reduced release. Evidence which suggests that lipoxygenase products of AA augment mucous release includes the following: (a) Nonsteroidal anti-inflammatory drugs (NSAID: acetylsalicylic acid and indomethacin) increase mucous release while preventing prostaglandin formation. (b) The increase in mucous release induced by AA or NSAID is additive once the agents are combined. (c) Several nonspecific lipoxygenase inhibitors (eicosa-5,8,11,14-tetraynoic acid; vitamin E; nordihydroguaiaretic acid; and alpha-naphthol) inhibit mucous release. Three additional lines of evidence directly indicate that monohydroxyeicosatetraenoic acid (HETE) causes increased mucous release: (a) the addition of a mixture of synthetic HETE (24-600 nM) increases mucous release; (b) pure 12-HETE (1-100 nM) also increases mucous release; (c) mucous release is increased synergistically by the combination of HETE and NSIAD. These data taken together demonstrate that HETE are capable of increasing mucous release and that conditions which may influence HETE production alter mucous release. Thus, although not directly demonstrating HETE production by human airways, the data strongly suggest that lipoxygenase products of AA in airways may profoundly influence mucous release; and it seems possible that lipoxygenase inhibitors may have a role in treating bronchorrhea.

Human airway monohydroxyeicosatetraenoic acid generation and mucus release.

The effects of 5-, 8-, 9-, 11-, 12-, and 15-monohydroxyeicosatetraenoic acid (HETE) (0.1-100 nM) on mucous glycoprotein release from cultured human airways were determined. Each of the HETE was an active secretagogue of mucus at concentrations greater than 1-10 nM with 12- and 15-HETE, the most active. Both 5- and 9-hydroperoxyeicosatetraenoic acid (HPETE) were also active as secretagogues at 100 nM, although of somewhat lower potency. As cultured airways were capable of responding to HETE with mucous glycoprotein release, it was of interest to identify and quantitate airway HETE formation. Accordingly, airways were incubated with tracer quantities of [14C]arachidonate for 16-48 h, and the spontaneous formation of 5-, 12- and 11- and/or 15-HETE was measured by high-pressure liquid chromatography. Indeed, sizeable quantities of 11- and/or 15- greater than 5- greater than 12-HETE were generated. This HETE generation was increased by the addition of 25 micrograms/ml of arachidonate and was reduced somewhat after 18-21 d in continuous tissue culture. Reversed anaphylaxis of human airways using anti-human IgE markedly increased the HETE formation, resulting in the production of micromolar concentrations of 5- and 11- and/or 15-HETE. Thus, human airways not only are capable of responding to the presence of HETE with mucous glycoprotein release, but also generate (both spontaneously and in response to anaphylaxis) at least three species of HETE, and do so in quantities capable of acting as mucus secretagogues.

Eosinophil cationic protein stimulates and major basic protein inhibits airway mucus secretion

Possible roles of eosinophil (EO) products in modulating the release of mucus from airway explants were investigated. Cell- and membrane-free lysates from purified human EOs (1 to 20 x 10(5)) caused a dose-dependent release of respiratory glycoconjugates (RGC) from cultured feline tracheal explants. Crude extracts from isolated EO granules also stimulated RGC release, suggesting that a granular protein might be responsible. Three proteins derived from EO granules, EO-derived neurotoxin, EO cationic protein (ECP), and major basic protein (MBP) were separated by sequential sizing and affinity chromatography. ECP (0.025 to 25 micrograms/ml) caused a dose-dependent increase in RGC release from both feline and human airway explants and also stimulated the release of the serous cell-marker, lactoferrin, from human bronchial explants. EO-derived neurotoxin (0.025 to 50 micrograms/ml) failed to affect RGC release, whereas MBP (50 micrograms/ml) significantly inhibited RGC release from feline explants. Thus, ECP stimulates RGC and lactoferrin release from airway explants, whereas MBP inhibits RGC release.

Human respiratory mucus.

Respiratory mucous glycoproteins may serve a number of protective functions for the airways; however, excessive secretions contribute to the morbidity of a variety of diseases including asthma, chronic bronchitis, and cystic fibrosis. Respiratory secretions are a mixture of cells, fluid, transudated and locally produced proteins, and mucous glycoproteins. The mucous glycoproteins give these secretions their characteristic viscosity and elasticity. While the physiologic control of mucous glycoprotein secretion is not completely understood, cholinergic, alpha-adrenergic, and beta-adrenergic stimuli may all contribute. Respiratory mucus hypersecretion seen in immediate hypersensitivity or inflammatory states may be due to reflex hypersecretion, to a variety of mediators (including histamine and cyclooxygenase or lipoxygenase pathway metabolites of arachidonic acid), or to substances released from phagocytic cells (such as macrophages, monocytes, or neutrophils). The limited number of specific approaches currently available for treating respiratory mucus hypersecretion include therapy of any underlying or intercurrent disease, improving clearance of secretions, and reducing mucus secretion with the use of glucocorticosteroids or anticholinergic drugs.

Human monocyte-derived mucus secretagogue.

Human peripheral monocytes were stimulated with opsonized zymosan or protein A-containing Staphylococcus aureus to examine whether factors might be released that were capable of stimulating mucous glycoprotein release from cultured human airways, as has recently been described with human pulmonary macrophages. While the supernatant from monocytes exposed to opsonized zymosan or protein A-containing S. aureus caused an impressive activity was found in the control samples that were cultured in parallel and exposed to nonactivated zymosan or S. aureus that was deficient in protein A. The responsible factor was termed monocyte-derived mucus secretagogue (MMS). The maximum MMS release was reached 4-8 h after stimulation, and the amount of MMS released was dependent on the dose of opsonized zymosan added. Chromatographic analyses of MMS indicate that its molecular weight was approximately 2,000 and that the isoelectric point (pI) was 5.2, with a smaller second peak of 7.4 on isoelectric focusing. MMS itself was not detected in monocyte lysates, nor was it formed by monocytes treated with the protein synthesis inhibitor, cycloheximide, before exposure to activating particles. MMS was not a prostaglandin, could not be extracted into organic solvents, and is probably not an eicosanoid. Based on these observations, we conclude that stimulated human peripheral monocytes synthesize a small, acidic molecule, termed MMS, that is capable of stimulating human airways to secrete mucus and in nearly every respect is identical to pulmonary macrophage-derived MMS.

Analysis of human nasal mucous glycoproteins

Human nasal turbinates were cultured in the presence of 3H-glucosamine, which is incorporated into nasal mucous glycoproteins. Nasal mucous glycoprotein was then characterized biochemically, and the effects of various neurohormones and immunologic stimulation on mucous glycoprotein release were analyzed. Fractionation of nasal mucous glycoprotein by gel filtration chromatography revealed a molecular size range of 2 to 200 X 10(5) (as judged by protein markers) but displayed a single, acidic charge, as reflected both in a narrow elution pattern from DEAE-cellulose and a sharp isoelectric focusing point of 2.6. Highly enriched nasal mucous glycoprotein preparations consisted of 80 per cent carbohydrate and 20 per cent protein (by weight) and included enzymatically cleavable carbohydrate side chains with molecular weights of 1,600 to 1,800. Thus, nasal mucous glycoproteins are a family of molecules that express uniform acidic charge characteristics and a wide range of molecular sizes. Cholinergic stimulation of atropine-inhibitable muscarinic receptors increased nasal mucous glycoprotein release in a dose-related manner, as did alpha-adrenergic stimulation. However, beta-adrenergic stimulation did not affect mucous glycoprotein release. Immunologic stimulation of nasal mast cells by either reversed anaphylaxis or antigen challenge after passive sensitization caused both histamine release and increased mucous glycoprotein release. Thus, nasal turbinates provide an accessible source of tissue for the analysis of nasal mucus secretion and mast cell degranulation and may provide a model for the study of pharmacologic approaches to the universally experienced discomfort of rhinorrhea.

Immunological and respiratory findings in swine farmers

The prevalence of respiratory symptoms and ventilatory capacity abnormalities in relation to immunological status was studied in 32 swine farmers and in 39 controls. A large number of swine farmers reacted to swine confinement building antigens (swine hair, 34%, swine confinement agents, 28%) but also to other extracts such as animal food (78%) and corn flour (37%). Control workers also reacted to these antigens in similar frequencies. Increased serum IgE levels were found in 3 swine farmers (9.4%) and all 3 had positive skin tests to at least one of the swine antigens. Among control workers one (2.6%) had an increased serum IgE level; this worker exhibited a positive skin reaction to swine food antigen. Swine farmers with positive skin reactions had across-shift reductions of FEF50 and FEF25 significantly larger than those with negative skin tests (P less than 0.01). Preshift measured ventilatory capacity data (FEV1, FEF50, FEF25) in swine farmers with positive skin tests were significantly lower (compared to predicted) than in those with negative skin tests. Additionally, we showed that a water-soluble swine confinement building antigen causes a dose-related contraction of nonsensitized guinea pig trachea smooth muscle studied in vitro. Our data indicate significant differences in lung function between swine workers with positive and negative skin tests. We suggest that skin testing may be helpful in identifying workers at risk for developing lung disease.

Human respiratory mucous glycoproteins.

Biochemical characterization of human respiratory mucus has generally utilized expectorated specimens. In order to exclude extraneous contaminants in the analysis of airway glycoproteins, human airways were cultured and the mucous glycoprotein released into the supernatant analyzed. By incorporating 3H-labeled glucosamine or 14C-threonine into the media, the airways biosynthetically labeled the mucous glycoproteins (MGP), facilitating their analysis. The MGP chromatograph by gel filtration on Sepharose 2B in two fractions: one excluded from the column and one that enters the column. However, employing a gel filtration column with the ability to fractionate larger molecules, Sephacryl S-1000, it was found that MGP fractionate over a large range in molecular sizes and do not segregate into distinct fractions. The diffuse, broad peak of MGP fractionation on Sephacryl S-1000 is not affected by reduction and alkylation or by chromatography in 1 M NaCl. The fractionated MGP from Sepharose 2B were divided into larger and smaller molecular species, and their charge characteristics were determined by DEAE chromatography and preparative isoelectric focussing. MGP exhibit strong acidic charge characteristics that are uniform, as reflected in elution from DEAE and a single, sharp isoelectric focussing point. Enzymatic cleavage of the oligosaccharide side chains from MGP liberates more than 70% of the radiolabeled side chains. The side chains enzymatically cleaved from the larger and smaller molecular species of MGP are similar in size. Highly purified MGP were found to be 73% carbohydrate and 27% protein. Thus, human airways release a family of MGP that express marked heterogeneity in size but a uniform, strong acid charge and include side chains of similar size.

Mucus secretagogue production by a human macrophage hybridoma

A pulmonary macrophage-monocyte-derived mucus secretagogue (MMS) oligopeptide has been previously reported to induce mucus secretion in an in vitro model system with human airway explants and secretory epithelial cells. To understand the possible role of macrophages in the regulation of secretion of mucus, our laboratory has used a series of human macrophage hybridomas that were generated by fusing an hypoxanthine guanine phosphoribosyl transferase-deficient promonocytic line, U937, with macrophages obtaining by maturing monocytes in Teflon bags. The cell lines were proven to be true hybridomas by acquisition of donor class I antigens, additional chromosomes, as well as macrophage specific (maximum velocity) not present on the U937 parent line. One clone, clone 63, produced large amounts of an oligopeptide with an approximate molecular weight of 2000, which was identified from culture supernatants by ultrafiltration, chromatography, isoelectric focusing, and Western blot. Processed clone 63 supernatant had biologic activity causing increased secretion of radiolabeled glycoconjugate in both cultured airways and secretory epithelial cells. Immunoblot analysis with a polyclonal rabbit antisera generated against MMS was positive, and Western blot analysis produced a band at approximately 2000 daltons, consistent with the previously described MMS. MMS secretion could be stimulated by zymosan and lipopolysaccharide and inhibited by both cycloheximide and erythromycin. Dexamethasone had a different effect, appearing to stimulate MMS production intracellularly but inhibiting its release once it was synthesized. The availability of cloned hybridomas allows for study of the regulation of mucus secretagogue production as well as purification of molecular species and provides a valuable tool for the study of mucus secretion.

Isolation and characterization of a macrophage-derived high molecular weight protein involved in the regulation of mucus-like glycoconjugate secretion

Pulmonary macrophages release a variety of mediators that are involved in inflammatory processes and probably are involved in respiratory mucus secretion. Conditioned media obtained from activated pulmonary macrophages were found to contain a protein that functioned as a secretagogue for mucus-like glycoconjugate (MLGC) in an in vitro bioassay. A human macrophage-derived hybridoma cell line, HB-63, exhibited the same properties and was very useful in obtaining large amounts of the protein for purification and characterization. With ultrafiltration membranes and gel electrophoresis, the protein isolated from the conditioned media of zymosan- or lipopolysaccharide-treated cells was found to have a molecular weight of approximately 68 kd. The purified protein obtained from hybridoma cells and from pulmonary macrophages exhibited strong biologic activity when it was used to stimulate MLGC secretion, both in human airway explants and in an in vitro human secretory epithelial cancer cell line. The proteins from both sources were found to have similar amino acid compositions. Preliminary results indicate the presence of the 68 kd protein in the bronchoalveolar lavage fluid of a patient with severe chronic bronchitis and mucus hypersecretion. The role of this novel protein in the lungs is, so far, speculative. The 68 kd protein may be a useful tool for studying the biosynthesis and regulation of MLGC secretion and hypersecretion.