BiochemistryRole of steroid 11,6-hydroxylase and steroid 18-hydroxylase in the biosynthesis of glucocorticoids and mineralocorticoids in humans (cortisol (9) recently isolated aldosterone synthase cytochrome P-450 (P-450ado), which is induced in Na+-depleted K+-replete rat adrenal cortex (10,11), and demonstrated that rat P-450ado catalyzes three successive monooxygenation reactions of DOC to form aldosterone as a final product, whereas rat P-45011p does not substantially catalyze the reaction to form aldosterone. In regard to aldosterone biosynthesis in the human adrenal cortex, it has been postulated for a long time (12)(13)(14)

A novel trypsin-like protease was purified to homogeneity from the sputum of patients with chronic airway diseases, by sequential chromatographic procedures. The enzyme migrated on SDS-polyacrylamide gel electrophoresis to a position corresponding to a molecular weight of 28 kDa under both reducing and non-reducing conditions, and showed an apparent molecular weight of 27 kDa by gel filtration, indicating that it exists as a monomer. It had an NH2-terminal sequence of Ile-Leu-Gly-Gly-Thr-Glu-Ala-Glu-Glu-Gly-Ser-Trp-Pro-Trp-Gln-Val-Ser-Leu- Arg-Leu, which differed from that of any known protease. Studies with model peptide substrates showed that the enzyme preferentially cleaves the COOH-terminal side of arginine residues at the P1 position of certain peptides, cleaving Boc-Phe-Ser-Arg-4-methylcoumaryl-7-amide most efficiently and having an optimum pH of 8.6 with this substrate. The enzyme was strongly inhibited by diisopropyl fluorophosphate, leupeptin, antipain, aprotinin, and soybean trypsin inhibitor, but hardly inhibited by secretory leukocyte protease inhibitor at 10 microM. An immunohistochemical study indicated that the enzyme is located in the cells of the submucosal serous glands of the bronchi and trachea. These results suggest that the enzyme is secreted from submucosal serous glands onto the mucous membrane in patients with chronic airway diseases.

SummaryHuman peripheral blood eosinophils released eosinophil survival-enhancing activity when stimulated with the calcium ionophore, ionomycin . The release of activity was detected as early as 3 h after stimulation and was inhibited by an immunomodulating agent, cyclosporin A . The survivalenhancing activity was completely abolished by treatment with anti-interleukin 3 (IL3) and anti-granulocyte/macrophage colony-stimulating factor (GM-CSF) monoclonal antibodies . Moreover, IL-3 and GM-CSF were measurable in ionomycin-stimulated eosinophil supernatants by immunoassay. Eosinophils produced approximately one-half as much IL3 and one-fifth as much GM-CSF as ionomycin-stimulated mononuclear cells. Neutrophils also produced 11,3 and GM-CSF, but the amounts were less than those produced by eosinophils. These observations suggest a novel role for eosinophils in pathophysiology of allergic inflammation and host defense mechanisms. E osinophils are blood leukocytes associated with helminth infections and allergic diseases, especially bronchial asthma, and may mediate immunity and tissue damage (1) . Several rytokines, including IL3, 116, and granulocyte/macrophage colony-stimulating factor (GM-CSF), induce eosinophilopoiesis and activate mature peripheral blood eosinophils in vitro (reviewed in reference 2). Recently, mRNA for these rytokines was detected in allergen-induced late-phase cutaneous reactions in atopic patients, suggesting that these rytokines play an important role in allergic inflammation (3) .Because human peripheral blood eosinophils, despite their high degree of specialization, retain the ability to synthesize rytokines such as TGF-ot and IL1 (4, 5), we investigated whether they can produce IL-3, IL-5, and GM-CSF . We found that ionomycin-stimulated eosinophils produce GM-CSF and IL-3 as shown by enhanced eosinophil survival and by immunochemical measurement .Cells and Culture Conditions. Eosinophils, neutrophils, and mononuclear cells were isolated from peripheral blood of healthy volunteers or patients with mild hay fever by discontinuous Percoll density gradient as previously described (6) . Purities of eosinophils and neutrophils were 391% and 399%, respectively. Contaminating cells in the eosinophil preparations were only neutrophils and those in the neutrophil preparations were only eosinophils . Cell viabilities were >98% . Cells were incubated (2 x 105 cells per 0 .2 ml per well) in Hybri-Care medium (American Type Culture Collection, Rockville, MD) supplemented with 50 lAg/ml gentamicin and 10% defined calf serum in 96-well flat-bottomed microtiter plates (Falcon Labware, Lincoln Park, NJ) with ionomycin (Calbiochem Corp., San Diego, CA) in the presence or absence of PMA (500 pg/ml; Calbiochem Corp.) . After 24 h at 37°C, cellfree supernatants were harvested and frozen until assayed .Eosinophil Survival Assay. To measure eosinophil survival enhancing activity (7) in supernatants, eosinophils were freshly isolated from different donors and were cultured (2 .5 x 10" cells per 0 .2 ml per well) ...

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These results indicate that tiotropium bromide can inhibit Th2 cytokine production and airway inflammation, and thus may reduce airway remodelling and AHR in a murine model of asthma.