Pharmacologic characterization of wool dust extract in isolated guinea pig trachea.

Wool mill workers develop respiratory symptoms and lung function abnormalities associated with their work in the textile industry. As in other workplaces, which process organic materials, the dust generated in the manufacture of wool has been implicated as a cause of these respiratory problems. Pharmacologic studies of wool dust extract were performed in vitro on guinea pig tracheal (GPT) segments. A wool dust extract (WDE) was prepared from material collected from a mill previously surveyed. When the standardized WDE solution was added to an organ bath in increments of 10, 30, 100, 300, and 1000 microliters it caused a consistent, dose-dependent constriction of GPT. Pretreatment of guinea pig tracheas, prior to WDE challenge, with atropine (10(-6) M), pyrilamine (10(-6) M), indomethacin (10(-6) M), verapamil (10(-6) M), TMBS (10(-6) M), BW755C (10(-6) M) and LY171883 (10(-6) M) was studied in order to evaluate receptor-dependent and -independent characteristics of WDE-induced constriction. WDE-induced bronchoconstriction was partially inhibited by the antihistamine pyrilamine. Atropine and leukotriene inhibitors (LY171883 and BW755C) were not found to have a significant protective effect on WDE-induced constriction. Both TMBS and verapamil (intra- and extracellular calcium blocking agent) suppressed the effect of wool dust extract in the range tested. These findings suggest that in this model, WDE-induced airway constriction is only partly attributable to common mediators of bronchoconstriction (e.g., histamine). The airway effects of WDE may be modulated by calcium channel blocking agents.

Immunological findings and respiratory function in cotton textile workers.

Immunological parameters were studied in a group of 24 cotton textile workers. These were volunteers from a cohort of 106 (83 women and 23 men) previously studied textile workers. A group of 30 employees from a bottle packing plant served as a control for the immunologic studies. The subgroup of volunteers undergoing immunologic testing did not differ from the original cohort of textile workers in age, sex, smoking history, or prevalence of most chronic respiratory symptoms, nor were there any significant differences in baseline lung function or across-shift changes. The 24 cotton worker volunteers underwent skin testing with extracts of cotton dust and cotton seed. Eight of these 24 (33.3%) had positive tests, and 5 of the 8 had elevated serum immunoglobulin E (IgE) levels. Only one of the 8 skin-test-positive workers had symptoms of byssinosis. Only 1 of 30 control workers' skin tested with cotton extract reacted, and none had an increased serum IgE level (P less than 0.01). Both baseline lung function and across-shift changes did not differ between workers with positive and negative skin test reactions or between workers with normal and elevated IgE levels. Additionally, we studied the response in vitro of nonsensitized guinea pig trachea to cotton bract extract and demonstrated a dose-dependent contractile response. These data suggest that while immunological findings are frequent in textile workers, they correlate poorly with respiratory symptoms and function and may not be the basis for the airway obstruction seen in this disease.

Isolation of an inhibitor of neutrophil function from bronchial lavage of normal volunteers.

Bronchial inflammation is associated with a reduction in airway caliber. Factors that may dampen this inflammation are ill defined. We have developed a model of airway inflammation using an extract of cotton bracts (CBE). In the current study we characterize inhibitor(s) of polymorphonuclear neutrophil (PMN) function in bronchial lavage (BL) fluid and examine the relationship between inhibitor concentrations and induction of bronchial inflammation or bronchoconstriction by CBE. We report that BL obtained contains factors that inhibit PMN hydrogen peroxide production and chemotaxis to formylmethionylleucyl-phenylalanine (FMLP). In two different subject populations the relative degree of a one molecular mass inhibitor was greatest in BL from subjects that manifested little bronchoconstriction to CBE compared with other subjects. In one subject population the relative amount of this inhibitor correlated inversely with the bronchoconstricting response to CBE and a number of parameters of airway inflammation including PMN noted on bronchial biopsy after CBE instillation. Partial chemical characterization of one molecular mass BL-derived inhibitor reveals it is of low molecular mass (less than 1,000 Da), nonpolar, and sensitive to aminopeptidase digestion. The finding that the bronchial environment contains variable concentrations of an oligopeptide that inhibits PMN function has important implications for treatment of inflammatory airway diseases.

Kinetics of cellular influx and histamine release in normal human bronchi.

Inhalation of certain substances can induce bronchial inflammation followed by bronchoconstriction or bronchial hyperreactivity. We have developed a model of airway inflammation and bronchoconstriction induced by an aqueous extract of cotton bracts (CBE) in which the severity of flow rate reduction correlates with the presence of bronchoalveolar neutrophils. In the current study normal human volunteers underwent local bronchial instillation of CBE. Bronchial lavage histamine concentrations and cellular populations were determined at time intervals after local challenge. In addition, bronchial biopsies were obtained after CBE instillation, and the degree of subepithelial inflammation was quantitated. We report that lavage of bronchi locally exposed to CBE contains 1) greater histamine concentrations 4 min after challenge compared with lavage from saline-challenged bronchi and 2) concentrations of eosinophils and macrophages that are greater than control lavage 8 min after exposure. Bronchial biopsies taken after CBE instillation also demonstrated a variable degree of subepithelial granulocyte infiltration that correlated with the global bronchoconstricting response to CBE assessed on a separate day. These studies suggest that inflammation can develop rapidly in normal human bronchi and may precede significant flow rate reductions by greater than 1 h.

Relationship of byssinosis to the generation of oxygen radicals by bract tissues of cotton plants.

Byssinosis is a hazardous respiratory disorder of workers in natural fiber processing industries and, in the case of cotton, is caused by exposure to respirable dust generated from leafy trash associated with raw fibers. To understand the chemical characteristics of involucral trash components that might contribute to bysinosis, we examined the human airway constricting activity and oxygen radical generating activity of dry, frost-killed cotton bracts. In response to inhalation of aerosolized bract extracts, the expiratory flow rates of human volunteers at 40% of vital capacity during partial forced expiration decreased by 3 to 32%. These values enabled us to identify two potentially byssinogenically active bract specimens, a specimen virtually inactive, and a fourth intermediately so. Using spin trapping techniques of electron spin resonance spectrometry, we found that all specimens catalyzed the generation of hydroxyl (preponderantly) and superoxide radicals from hydrogen peroxide. However, the weakest constrictor was the most potent catalyst, and vice versa. This was consistent with transition metal content of the specimens; the most potent catalyst also contained the largest amounts of those metals, suggesting a Fenton-type reaction mechanisms. Other possibilities for the inverse relationship of airway constricting (byssinogenic) activity with oxygen radical generation are discussed. We also found that neither aflatoxin nor endotoxin, contingent contaminants of bracts, catalyzed oxygen radical production from hydrogen peroxide.

Bronchial responsiveness after inhalation of cotton bract extract.

This study examined nonspecific airway responsiveness to methacholine (MC) after inhalation of cotton bract extract (CBE). In a randomized double-blind, crossover trial, 13 healthy volunteers underwent an MC inhalation challenge test prior to inhalation of CBE and normal saline solution (NSS) aerosol sham as well as 2, 8, 24, and 168 h (7 days) later. The response parameter was the concentration of MC required to induce a 25% decrement in the maximal expiratory flow at 40% of the vital capacity below total lung capacity on the partial expiratory flow-volume curve (PC25MEF40%(P]. Five of 13 subjects demonstrated a ventilatory response to CBE with a 20% or larger decrement in the MEF40%(P); no subject demonstrated such change with NSS. For the group, the maximal decrement in MEF40%(P) was to 76.5 +/- 20.3% of baseline (mean +/- SD), occurring approximately 60 to 90 min after provocation, whereas the largest decrement after normal saline was to 88 +/- 10.6% of baseline, occurring immediately after inhalation. Changes in airway responsiveness to MC were transient. For example, the PC25MEF40%(P) for the group (mean +/- SD) was 51.3 +/- 41.1 mg/ml at baseline and 25.8 +/- 30.3 and 52.2 +/- 57.3 mg/ml at 2 and 8 h. After a pre-sham baseline of 50.4 +/- 43.2 mg/ml, PC25MEF40%(P) was 57.6 +/- 83.8 and 153.8 +/- 148 mg/ml at 2 and 8 h. Repeated measures ANOVA on these acute, same-day changes (i.e., 2 and 8 h after provocation) demonstrated a statistically significant effect of CBE on airway responsiveness (p = 0.048). These data demonstrate that inhalation of CBE, in addition to bronchospasm, causes a transient increase in airway responsiveness.

Bronchoalveolar cell activation after inhalation of a bronchoconstricting agent.

Airway inflammation is thought to be an important determinant of bronchoconstriction and bronchial hyperreactivity. We have recently demonstrated that bronchoconstriction induced by an aqueous extract of cotton bracts (CBE) is associated with bronchoalveolar complement activation, release of polymorphonuclear neutrophil (PMN) chemoattractants by pulmonary cells, and increased numbers of bronchoalveolar lavage PMN's. In the present study we performed bronchoalveolar lavage (BAL) on subjects after CBE or control (saline) challenge and examined whether BAL cells were activated in vitro to produce other inflammatory agonists. After CBE administration, cultured BAL cells released increased amounts of the reactive O2 species, superoxide (O2-.), and the cyclooxygenase products prostaglandin E2 and thromboxane B2. Although none of these in vitro parameters of BAL cell activation appeared to correlate with the degree of bronchoconstriction induced by CBE, BAL fluid levels of thromboxane B2 were also increased after CBE administration and in vivo amounts of this eicasanoid did correlate with the degree of bronchoconstriction induced by CBE (r = 0.50, P less than 0.04). Finally, although cell culture supernatants were highly chemotactic for PMN's, concentrations of leukotriene B4 were not increased, suggesting other chemotaxins were released by BAL cells in this setting. We conclude that CBE administration activates bronchoalveolar cells to release reactive O2 species and cyclooxygenase products that may be important in the bronchoconstricting response to CBE.

Acute pulmonary response to cotton bract extract in monkeys: lung function and effects of mediator modifying compounds.

It is well established that cotton dust inhalation can compromise lung function in textile workers. Challenges with a water-soluble extract of cotton bract (CBE) can also induce reversible airway obstruction in healthy volunteers. We have examined the effect of inhaled CBE in nonhuman primates and have attempted to inhibit the bronchoconstrictive response with mediator modifying compounds. CBE (34 mg/ml or 100 mg/ml) was administered via IPPB for 15 minutes (15 breaths/min) in 12 intubated, anesthetized, adult male monkeys (Macaca fascicularis). Breath-by-breath determinations of pulmonary resistance, dynamic compliance (Cdyn), tidal volume, and breathing frequency were calculated from the transpulmonary pressure (esophageal balloon) and airflow signals and monitored for 2 hr postchallenge. Control challenges with distilled water were also performed in 3 monkeys with the greatest response from CBE. Five animals (42%) were found to respond to CBE with peak % changes in Cdyn greater than 45%. In 3 of these animals, we attempted to blunt the CBE response with chlorpheniramine (0.1 mg/kg i.v.) and a mast cell stabilizer lodoxamide (0.1 mg/ml aerosol). In these 3 animals the mean (+/- SD) peak % changes in Cdyn to CBE alone was -47.4 +/- 1.8. The CBE response following chlorpheneramine was -49 +/- 15.7 and following lodoxamide was -47.0 +/- 5.4. These data suggest that monkeys, like humans, can develop reproducible bronchoconstriction following an aerosol challenge with CBE. Furthermore, this bronchoconstriction in the monkey is probably not explained by the action of histamine or mediator release alone and an acute inflammatory reaction may be involved.

The relationship between bronchoalveolar neutrophil recruitment and bronchoconstriction induced by a soluble extract of cotton bracts.

Byssinosis is characterized by an obstructive bronchitis in some workers exposed to cotton dust. Mechanisms of airway alterations induced by cotton dust are unknown. To study mechanisms of these alterations, we administered an aqueous extract of cotton bract (CBE) to normal human subjects. After CBE inhalation, we demonstrated reproducible reductions in flow rates of variable magnitude. The percentage of polymorphonuclear leukocytes (PMN) recovered by bronchoalveolar lavage (BAL) and total PMN per milliliter BAL fluid were increased after challenge with CBE, and these parameters were correlated (r = 0.556 and r = 0.553, respectively) with the degree of bronchoconstriction induced by CBE. In addition, chemotactic factors for PMN were noted in BAL fluid and in supernatant from BAL cell cultures. The degree of BAL fluid chemotactic activity correlated with the degree of bronchoconstriction induced by CBE. Assessment of molecular sieve column effluent demonstrated BAL fluid to have 3 distinct peaks of chemotactic activity (approximate molecular weight, greater than 25,000, 10,000, and less than 1,000 daltons) and cell culture supernatant to contain 1 peak (approximate MW, 10,000 daltons). Complement activation as one source of BAL chemotactic activity was suggested by almost complete inhibition of BAL chemotactic activity after incubation with antibody to the fifth component of complement (C5) and detection of C5a des Arg in BAL fluid from some subjects challenged with CBE. These findings suggest that exposure of volunteer subjects to CBE results in a bronchoalveolar inflammatory response related to both complement activation and chemotactic factor synthesis by luminal cells.

Biologic activity of purified cotton bract extracts in man and guinea pig.

Purified aqueous extracts of cotton bract induce acute airway constriction in healthy volunteers never before exposed to cotton bract. The response is similar to that of textile workers who inhale cotton dust. Approximately 60% of volunteers respond to bract extract with significant decreases in lung function, and these volunteers show an increased number of lymphocytes present in their lungs. Following inhalation of bract, the percent of polymorphonuclear leukocytes increases. Macrophages obtained by bronchoalveolar lavage from volunteers pre-challenged with bract extract release increased amounts of chemotactic factor and superoxide anion. Efforts to detect release of histamine and leukotrienes in volunteers following challenge with bract show no increase in urinary histamine and no significant release of leukotrienes in lung lavage fluid. Purified extracts exhibit chemotactic activity in vitro. They also contract guinea pig ileal longitudinal muscle in vitro. This preparation contains mast cells but no basophils, and the H-1 blocker, mepyramine blocks the contraction. Purified bract extracts contain no histamine or endotoxin but other contractors of smooth muscle may be present. The purified extract exhibits spectral, fluorescent, and radioimmune assay properties similar to a leukotriene B-like component. Cotton bract appears to have direct as well as cell-mediated activities.

Vegetable dust and airway disease: inflammatory mechanisms.

Exposure to cotton or grain dust causes an obstructive bronchitis in certain subjects, mechanisms of which are poorly understood. A difficulty encountered in discerning mechanisms of this airway disease is the lack of knowledge of the active components of these dusts. Clinical features suggest common but not exact mechanisms of the airway disease associated with these vegetable dusts. Human and animal studies show evidence of acellular and cellular inflammatory mechanisms of the bronchoconstriction and inflammation associated with these disorders. Potential cellular sources include alveolar macrophages, polymorphonuclear leukocytes, mast cells, basophils, eosinophils and lymphocytes. Acellular origins include the complement and humoral antibody systems, both of which have been implicated, although their pathogenic role in grain or cotton dust disorders is uncertain. In this review we critically address potential inflammatory mechanisms of airway alterations resulting from cotton or grain dust exposure. General mechanisms of bronchoconstriction are first presented, then specific studies dealing with either of the two dusts are discussed. We believe this area of research may be fruitful in dissecting mechanisms of bronchoconstriction and airway inflammation, especially as more human studies are undertaken.

Airway constrictor response to cotton bract extracts in the absence of endotoxin.

Crude and purified aqueous extracts of cotton bracts shown to cause airway constriction in naive subjects were assayed for endotoxin content. Pulmonary function measured by flow changes on partial expiratory flow volume curves was used to assess airway responses to the bract extracts after their inhalation by a panel of volunteers. These responses are similar to the acute responses experienced by cotton textile workers. Crude aqueous extracts from various bracts harvested before and after senescence of the cotton plant displayed endotoxin concentrations ranging widely from 0.086 to 50 micrograms/ml. No correlation was found between these differences in endotoxin content and the severity of the airway constrictor response. Purifying the bract extract by a series of procedures that included precipitation of polymeric material by addition of methanol and chromatography on an anion exchange column of DEAE-Sephacel resulted in the elimination of almost all the endotoxin. The partially purified extract contained less than 1 ng/ml of endotoxin. The panel of volunteers responded to this purified bract extract, however, with a decrease in pulmonary function which was more than 60% of that seen with the crude extract of bracts. It is concluded that aqueous extracts of cotton bract contain an agent(s) other than endotoxin that causes acute airway constriction in people.

Skin testing with an aqueous extract of cotton bract.

Airway challenge with cotton bract extract (CBE) causes reversible bronchospasm in most volunteers never before exposed to CBE or the textile industry. Lung function abnormalities develop slowly after inhalation with a maximum effect reached within 2 hours and lung function slowly improving thereafter. When CBE was injected intradermally in three naive subjects with negligible airway response to CBE, we observed an initial wheal-and-flare reaction within minutes followed by erythema and subsequent induration that persisted for 24 to 48 hours. Microscopic examination of skin biopsy specimens obtained during the course of these reactions revealed edema in the early phase followed by perivascular infiltration of a mixed cellular nature in the subsequent stages of the reaction. Degranulation of mast cells was noted throughout the course of the reaction. These findings indirectly suggest that a nonspecific inflammatory reaction initiated by mast cell-derived mediators and sustained by the presence of infiltrating cells may be responsible for some of the airway effects observed in byssinosis.

Airway reactivity and cotton bract-induced bronchial obstruction.

Most seemingly healthy persons challenged with an aerosol of cotton bract extract develop some degree of bronchospasm. The role of nonspecific reactivity of the airways in this reaction to cotton bract extract is undefined. We examined the relationship between airway responses to cotton bract extract and to methacholine, as well as between airway responses to cotton bract extract and to a bronchodilator. Twenty-two healthy subjects were screened for sensitivity to inhaled cotton bract extract. Pulmonary function was measured using partial expiratory flow-volume curves on which flow at 60 percent of the control vital capacity below total lung capacity was measured (MEF40%[P]) following ten minutes of inhalation of cotton bract extract. In the group screened, 12 were found to be responders to the extract, with drops in MEF40%(P) of 20 percent or more, and ten were found to be nonresponders. On separate days, we measured the responses of our subjects' airways to inhaled metaproterenol and methacholine. The mean threshold dose for methacholine in the responding group was 26.8 mg/ml, as compared to 55.6 mg/ml for the nonresponders (t = 2.52; p less than 0.05). Furthermore, the mean percent increase in MEF40%(P) following inhalation of metaproterenol was 41 percent in responders and 24 percent in nonresponders (t = 2.19; p less than 0.05). We conclude that some responders to cotton bract extract exhibit greater reactivity of the airways than nonresponders.

Stimulation of ion transport by ascorbic acid through inhibition of 3':5'-cyclic-AMP phosphodiesterase in the corneal epithelium and other tissues.

Ascorbic acid stimulates active transport of Cl-minus by the isolated intact cornea. The effect is not present in corneas previously stimulated by the theophylline, an inhibitor of 3':5"-cyclic-AMP phosphodiesterase (EC 3.1.4.17), and vice versa, theophylline has no action after stimulation with ascorbic acid. This indicated inhibition of 3':5'-cyclic-AMP phosphodiesterase by ascorbic acid. Assay of phosphodiesterase using 3-H-labeled cyclid AMP of frog and rabbit corneal epithelial homogenates showed an inhibitory effect of ascorbic acid. Concentration of 5 mM produced 16% inhibition with 20 mM producing 46%. This compares with 58% inhibition by theophylline at 5 mM. Phosphodiesterase activity is mostly soluble in frog corneal epithelium but in rabbit 45% is particulate. Soluble and particulate fractions are inhibited by ascorbate, but in rabbits greater inhibition (50%) was observed in the particulate fraction than in the soluble fraction. Other tissues showed inhibition also: frog retina 12%, rat brain (caudate nucleus) 48%, rabbit brain 14%, rabbit liver 16%. It is concluded that ascorbate produces an increase in cyclic AMP content of corneal epithelium and other tissues by inhibition of 3':5'-cyclic-AMP phosphodiesterase. This action may be one of the main functions of the high ascorbic acid content of ocular tissues and explain some of the effects of high dosis of ascorbate in other systems.