The increase in airway responsiveness induced by O3 exposure in dogs is associated with airway epithelial inflammation, as evidenced by an increase in the number of neutrophils (polymorphonuclear leukocytes) found in epithelial biopsies and in bronchoalveolar lavage fluid. We investigated in 10 healthy, human subjects whether O3-induced hyperresponsiveness was similarly associated with airway inflammation by examining changes in the types of cells recovered in bronchoalveolar lavage fluid obtained after exposure to air or to O3 (0.4 or 0.6 ppm). We also measured the concentrations of cyclooxygenase and lipoxygenase metabolites of arachidonic acid in lavage fluid. We measured airway responsiveness to inhaled methacholine aerosol before and after each exposure and performed bronchoalveolar lavage 3 h later. We found more neutrophils in the lavage fluid from O3-exposed subjects, especially in those in whom O3 exposure produced an increase in airway responsiveness. We also found significant increases in the concentrations of prostaglandins E2, F2 alpha, and thromboxane B2 in lavage fluid from O3-exposed subjects. These results show that in human subjects O3-induced hyperresponsiveness to methacholine is associated with an influx of neutrophils into the airways and with changes in the levels of some cyclooxygenase metabolites of arachidonic acid.

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Since the initial descriptions of Charcot-Leyden crystals more than 100 years ago, the presence of these slender, dipyramidal crystals in human tissues and biologic fluids has become a hallmark of eosinophilic leukocyte infiltration, especially in association with allergic and helminthic diseases. The formation of these crystals in vitro after disruption of human eosinophils, but not of other cell types, in hypotonic saline or detergent established the eosinophil as the unique cellular source of the crystalline protein. Charcot-Leyden crystals have now been found to express lysophospholipase activity (lysolecithin acylhydrolase, EC 3.1.1.5), and the solubilized Charcot-Leyden crystal protein presents a single stained protein band that is coincident with the lysophospholipase activity eluted from replicate gels on alkaline polyacrylamide gel electrophoresis. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the solubilized Charcot-Leyden crystal protein migrates with a molecular weight of 17,400, which is comparable to that of eosinophil lysophospholipase purified chromatographically to homogeneity; further, on combination, the two proteins comigrate as a single staining band. Finally, the chromatographically purified eosinophil lysophospholipase in hypotonic buffer forms dipyramidal crystals morphologically identical to Charcot-Leyden crystals. The findings that chromatographically purified, homogeneous eosinophil lysophospholipase and Charcot-Leyden crystal protein express the same enzymatic activity, are of the same size and charge, and form crystals of identical morphology indicate that human eosinophil lysophospholipase is the constituent of Charcot-Leyden crystals.

The results of recent studies of the mechanism of leukotriene B4-induced hyperalgesia suggest a dependence on polymorphonuclear leukocytes (PMNLs). In this study, we addressed the contribution of PMNLs to hyperalgesia evoked by the peptide chemotactic factors N-formyl-methionyl-leucyl-phenylalanine (fMLP) and the anaphylatoxin fragment of the fifth component of the complement pathway (C5a). Local injection of glycogen, which attracts but does not activate PMNLs, produced a marked shift to the left (toward lower concentrations) in the concentration dependence curve of fMLP-induced hyperalgesia. In addition, PMNL repletion by transfusion with syngeneic PMNLs reestablished fMLP-induced hyperalgesia in PMNL-depleted rats. Finally, supernatants from rat and human PMNLs, that had been stimulated with fMLP in vitro, produced hyperalgesia in PMNL-depleted rats. Preliminary characterization of the hyperalgesia-inducing activity released by stimulated PMNLs indicated that it is lipid in nature. The nonsteroidal anti-inflammatory indomethacin did not attenuate C5a and fMLP-induced hyperalgesia. Thus, the hyperalgesia produced by fMLP and C5a is similar to that produced by leukotriene B4 in that it is dependent on PMNLs and independent of the cyclo-oxygenation of arachidonic acid. Taken together, these data suggest that structurally diverse PMNL-chemotactic factors produce hyperalgesia by a novel mechanism, involving PMNL-derived factors.

Preferential eosinophil chemotactic activity exhibiting a molecular weight comparable to that released from sensitized human lung fragments challenged with specific antigen and designated eosinophil chemotactic factor of anaphylaxis has been isolated from extracts of human lung fragments by sequential purification on Sephadex G-25, Dowex-1, Sephadex G-10, and paper chromatography. Two eosinophilotactic tetrapeptides of amino acid sequence ValGly-Ser-Glu and Ala-Gly-Ser-Glu were recovered from the extracts in 4-12% overall yield of the low molecular weight peak from Sephadex G-25. Purified eosinophil chemotactic factor of anaphylaxis and the synthetic tetrapeptides were maximally active in amounts from 0.1 to 1.0 nmo per chemotactic chamber, and the activity was dependent on both the NHrterminal and the COOH-terminal residues. Both natural and synthetic peptides were preferentially chemotactic for eosinophils and rendered them unresponsive to a subsequent stimulus.The eosinophil chemotactic factor of anaphylaxis (ECF-A) was discovered in 1971 as a mediator released during immediate hypersensitivity reactions in guinea pig (1) and human (2) lung slices. ECF-A was subsequently recognized to be present totally preformed in rat mast cells in association with the granules (3), human leukemic basophils (4), and mast cell-rich tissues such as human lung and nasal polyps (3, 5). The release of ECF-A from human tissue by IgE-dependent mechanisms has biochemical requirements and is modulated by the intracellular levels of cyclic nucleotides in a manner comparable to histamine release from the same tissues (5, 6). ., N.Y.), t-butoxycarbonyl (BOC)-amino acids (Beckman Instruments, Inc., Palo Alto, Calif.), 3,5-dinitrophenyl-glutamic acid (Cyclo Chemical Co., Los Angeles, Calif.), and Rhodamine 6-G and N, N'-diisopropylethylamine (Matheson, Coleman and Bell, East Rutherford, N.J.) were obtained as specified. All other solvents were either reagent grade from Eastman Kodak Co., or Fisher-certified (Fisher Scientific Co., Medford, Mass.) and were redistilled before use. Methylene chloride was further purified by chromatography on a column of alumina (alumina adsorption, Fisher Scientific Co.). N, N'-Dimethylformamide was passed through a column of 4A molecular sieves (Matheson, Coleman and Bell, East Rutherford, N.J.) and stored over fresh sieves for 72 hr before use (11).Chemotaxis. Blood from normal subjects or patients with peripheral blood hypereosinophilia of 20-95% was incubated for 45 min at 370 with citrate anticoagulant and dextran to sediment the erythrocytes (12). The leukocyte-rich supernatant plasma was removed and centrifuged at 100 X g for 10 min at room temperature. The leukocyte pellet was either washed and suspended in Medium 199 made 0.4% in ovalbumin and 0.01 M in Tris-HCl pH 7.4 (Medium 199-ovalbumin) and used directly in the chemotactic assays, or was used as a source of a specific leukocyte population. Eosinophils were enriched by centrifugation on metrizoate cushions (13), and neutrophils and...