calcimycin and methionyl-leucyl-phenylalanine

Activation of neutrophils (PMN) within the airways results in the secretion of a number of products such as reduced oxygen metabolites that could contribute to the inflammatory response associated with asthma. However, mediators of allergy, such as histamine, prostaglandin E2 (PGE2), isoproterenol, and adenosine, may serve to mitigate this inflammation through feedback inhibition of neutrophil function. To test the hypothesis that PMN activation and feedback inhibition mechanisms may be abnormal in asthmatics, we compared both superoxide production and adenosine-induced suppression of superoxide production in 12 matched pairs of asthmatics and control subjects. PMN obtained from asthmatic patients generated significantly more superoxide in response to f-met-leu-phe (fMLP) than controls (2.94 +/- 55 nmol/5 x 10(5) PMN/5 min versus 1.38 +/- 0.35 at 2 x 10(-8) M fMLP and 3.81 +/- 0.68 nmol versus 2.04 +/- 0.45 nmol at 10(-7) M; p less than 0.01 for both). In contrast, the respiratory burst generated by two receptor-independent stimuli, the calcium ionophore A23187 and phorbol myristate acetate, was equivalent between control and asthmatic subjects. At 10(-6) M, 2-chloroadenosine induced a 19.5 +/- 5.1% inhibition of fMLP-stimulated superoxide production in PMN from patients with asthma as compared to 55.6 +/- 24.6% inhibition in PMN from control subjects (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 2-Chloroadenosine; Adenosine; Asthma; Calcimycin; Cytochalasin B; Dinoprostone; Dose-Response Relationship, Drug; Humans; Isoproterenol; Lipopolysaccharides; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Superoxides

HL-60 cells differentiate into mature granulocytes in response to treatment with a variety of chemical agents. Such HL-60 cell derived granulocytes display many of the properties associated with their peripheral blood counterpart. In this study we have investigated the development of the degranulation response in dimethylsulfoxide (DMSO) or retinoic acid differentiated HL-60 cells over a six day period. The release of a number of enzymes in response to stimulation by a variety of agents was examined. Soluble aggregated IgG (SAIgG) stimulated the release primarily of elastase from HL-60 derived granulocytes with little or no release of other granule enzymes, in particular myeloperoxidase. This contrasted to what was seen when peripheral blood granulocytes were used. The lack of myeloperoxidase release was not due to the parallel release of enzyme inhibitors or failure of the stimulus to bind to the cells. Neither was it due to variations in the kinetics of enzyme release or the presence of myeloperoxidase and elastase in discrete sub-populations of HL-60 cells. When other stimuli such as fMet-Leu-Phe, A23187, or phorbol myristate acetate (PMA) were used a relatively normal degranulation response was seen. Thus, the degranulation response in granulocytes derived from HL-60 cells appears relatively normal when a range of commonly used stimuli are used but is impaired when aggregates of IgG are used.

Topics: Calcimycin; Cell Differentiation; Cell Line; Cytoplasmic Granules; Granulocytes; Humans; Immunoglobulin G; Leukemia, Promyelocytic, Acute; N-Formylmethionine Leucyl-Phenylalanine; Pancreatic Elastase; Peroxidase; Tetradecanoylphorbol Acetate

Human lung specimens were minced and treated for 30 min with collagenase (1 mg ml-1) and DNase (0.1 mg ml-1) to obtain a suspension of viable (approximately 80%) and metabolically active lung cells (5 x 10(6) cells per gram of tissue). Treatment of these mixed lung cells with bradykinin (1.25 x 10(-6) to 1 x 10(-5) M) and f-Met-Leu-Phe (f-MLP; 1 x 10(-8) to 5 x 10(-6) M) did not stimulate to a substantial extent the release of prostaglandins and thromboxanes (measured with novel Enzyme Immunoassays). The only concentration of PAF that stimulated significantly the release of icosanoids from lung cells was 5 x 10(-7) M. Phorbol myristate (PMA; 5 x 10(-8) to 2 x 10(-6) M) and ionophore a-21387 (2.5 x 10(-6) to 2 x 10(-5) M) strongly stimulated the release of prostaglandins and thromboxanes by dispersed human lung cells. These findings support previous observations showing that human lungs have the enzymes necessary for the synthesis and release of prostaglandins and thromboxanes but stimulation of the release of these mediators is not obtained with the hormonal stimuli that are active in guinea pigs. Studies in progress will purify the cell populations and characterize the cells responsible for the release of these icosanoids.

Topics: Bradykinin; Calcimycin; Cells, Cultured; Dinoprostone; Humans; Immunoenzyme Techniques; Lung; N-Formylmethionine Leucyl-Phenylalanine; Platelet Activating Factor; Prostaglandins E; Tetradecanoylphorbol Acetate; Thromboxane B2

The secretory response of cytochalasin B-treated human polymorphonuclear neutrophils to the peptide chemoattractant f-Met-Leu-Phe (FMLP), the calcium ionophore A23187 and other secretagogues was measured by assaying neutrophil supernatants for the granular enzymes beta-glucuronidase and lysozyme. The dose-dependent enzyme secretion in response to 10(-8)-10(-4) M FMLP and A23187 was unaffected by pretreatment with 10-75 microM forskolin (an activator of adenylate cyclase), but inhibited by high concentrations of prostaglandins E1 and E2. The phosphodiesterase inhibitors isobutyl-methyl-xanthine (IBMX), papaverine and Ro 20-1724 dose dependently inhibited enzyme secretion from FMLP- or A23187-treated cells, and this effect was augmented in the presence of 50-75 microM forskolin. Similar results for PGE1, forskolin and forskolin/IBMX combinations were also obtained using leukotriene B4, platelet activating factor and C5a des-Arg as secretagogues. We conclude that the adenylate cyclase system of human neutrophils is activatable by forskolin, but that the regulatory effects of adenylate cyclase stimulants in these cells are greatly attenuated unless cyclic AMP-phosphodiesterases are inhibited. Thus the phosphodiesterase activity of neutrophils may be of functional importance and is relevant to the modulation of neutrophil activity in inflammation.

Topics: Alprostadil; Calcimycin; Colforsin; Cyclic AMP; Cytoplasmic Granules; Glucuronidase; Humans; In Vitro Techniques; Muramidase; N-Formylmethionine Leucyl-Phenylalanine; Neutrophils; Phosphodiesterase Inhibitors; Radioimmunoassay; Time Factors