Tannin-mediated secretion of a neutrophil chemotactic factor from alveolar macrophages. Potential contribution to the acute pulmonary inflammatory reaction associated with byssinosis.

Inhalation of cotton mill dust or condensed tannin, a major botanical component of cotton mill dust, induces an acute pulmonary inflammatory response characterized by a rapid influx of neutrophils into the airways. The development of neutrophil alveolitis caused by tannin inhalation is accompanied by the accumulation of low molecular weight neutrophil chemotactic factor (NCF) in the airways. To determine if the alveolar macrophage is the source of this NCF, the ability of tannin to induce the secretion of NCF from rabbit alveolar macrophages was examined in vitro. Tannin did promote the secretion of NCF from alveolar macrophages in a time- and dose-dependent manner. Secretion began immediately after challenge and was maximal after 4 to 5 h. Maximal secretion occurred at a tannin concentration of 50 micrograms/ml. Comparison with the dose response for NCF secretion by cotton dust extract indicated that tannin was the major component in the dust responsible for NCF secretion from alveolar macrophages in the time period examined. The NCF had an apparent molecular weight of greater than 800 as determined by gel chromatography. The NCF could be extracted into organic solvents, suggesting it was a lipid. Its secretion, however, could not be prevented by treatment of the macrophages with the 5-lipoxygenase inhibitor, nordihydroguaretic acid, demonstrating that the NCF was not leukotriene B4. These data indicate that the action of tannin on resident alveolar macrophages results in the secretion of a NCF that may be responsible for the acute neutrophil alveolitis associated with inhalation of cotton dust.

Changes in the levels of bioactive components of cotton bracts accompanying senescence.

A series of bracts from Deltapine 41 variety cotton were harvested weekly from October 16 through November 25, 1982, and examined for changes in biologically active components accompanying senescence. Aqueous extracts of cotton bracts (CBE) were tested for their ability to cause 5-hydroxytryptamine (5-HT) release from human platelets and contraction of isolated canine tracheal smooth muscle (TSM). In addition, the amounts of soluble tannins (the platelet-activating factor found in CBE) and endotoxin were determined for each CBE. The amount of CBE decreased significantly from October 16 through November 25, whereas endotoxin levels rose dramatically from October 23 to November 6 and declined thereafter. The amount of soluble tannin and platelet 5-HT release both decreased from highs on October 23 to unmeasurable levels on November 25. Smooth muscle contraction was attributed to two primary factors contained in CBE. The major constrictor was a 5-HT receptor agonist and its levels remained stable from October 16 through October 30, but then decreased to unmeasurable levels by November 13. In contrast, CBE also contained lesser amounts of an acetylcholine receptor agonist whose levels paralleled the changes in endotoxin levels. We conclude that several important biochemical changes occur in cotton bracts during senescence and that these changes profoundly influence their biological activity in various assays.

Protection by plasma proteins against condensed tannin-mediated platelet activation.

Condensed tannins isolated from cotton bracts are potent platelet agonists. They promote the secretion of 5-hydroxytryptamine from washed bovine platelets with an EC50 of 35 micrograms/ml. However, when platelet-rich plasma was used in lieu of washed platelets, the potency of tannin was decreased over 50-fold. Reconstitution experiments demonstrated that some component in the plasma was responsible for the diminished potency of tannin. Fractionation of platelet-poor plasma suggested that albumin may be the inhibitory plasma component. Confirmation of this hypothesis was obtained from the finding that purified serum albumin at physiologic concentrations inhibited the tannin-mediated release of platelet 5-HT by more than 70%. These data suggest that any activation of platelets which may occur in vivo by the tannins present in inhaled cotton dust would require higher tannin concentrations than would have been predicted from our previous studies using washed platelets.

Platelet 5-hydroxytryptamine release and aggregation promoted by cotton bracts tannin.

The effect of aqueous extracts of cotton bract (CBE) on platelet secretion and aggregation was examined by using washed bovine and human platelets. The CBE promoted the release of 75% to 90% of the 5-hydroxytryptamine (5-HT) stored in both human and bovine platelets in a dose-dependent manner. This release reaction occurred without the lysis of the platelets and was not inhibited by indomethacin, 2-deoxyglucose, or KCN. Fractionation of the CBE indicated that the platelet secretagogue present in the CBE was the condensed polyphenol, tannin. In addition to promoting the secretion of 5-HT, tannin also aggregated the platelets in a dose-dependent manner. We conclude that the secretion of platelet 5-HT and the aggregation of platelets by tannin could potentially contribute to the pulmonary symptoms associated with byssinosis.

Purification and substrate specificity of bovine angiotensin-converting enzyme.

Angiotensin-converting enzyme was solubilized from bovine lung with detergent and purified over 2300-fold to physical homogeneity by a combination of ammonium sulfate fractionation, molecular sieve chromatography, and ion exchange chromatography. The purified enzyme had an apparent molecular weight of 126,000 in both the denatured, and reduced, denatured forms as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 13.6 units/mg. It was inhibited by EDTA and activated by chloride ion. Chloride functioned as a nonessential activator by raising the Vmax 4.26-fold and lowering the KM 5.99-fold under saturating conditions. Under these conditions, the Vmax was 1.2 mumol/min/unit and the KM was 1.3 mM. Three series of peptides having the general structures, Hip-His-X, Hip-X-Leu, and Hip-X-His-Leu were synthesized and used to examine the binding specificity and substrate specificity of the enzyme for amino acids in the COOH-terminal (P'2), penultimate COOH-terminal (P'1), and antepenultimate COOH terminal (P1) peptide positions. These studies indicated that in terms of binding specificity, the relative importance of these three positions was P'2 > P'1 > P1, while the reverse order P1 > P'1 > P'2 was observed for the relative contribution to substrate specificity. Three peptides, Hip-His-D-Leu, Hip-D-His-Leu, and Hip-D-Phe-His-Leu, were also synthesized and used to examine the stereochemical requirements of the enzyme in terms of both peptide binding and hydrolysis. Hydrolysis was found to require an L amino acid in all three positions. In contrast, all three peptides bound to the enzyme.