calcimycin and Eosinophilia

Asthma is a multifactorial disease on genetic basis. Its development is influenced by maternal and environmental factors, i.e. allergens and adjuvants. Early identification of candidates at high risk for development of asthma will enable giving recommendations on preventive measures focussing on exposure to tobacco smoke and other pollutants, indoor and outdoor allergens and possibly viral infections during infancy.

Topics: Asthma; Bronchoalveolar Lavage Fluid; Calcimycin; Eicosanoids; Eosinophilia; Eosinophils; Histamine Release; Humans; Lymphocytes; Macrophage Activation; Macrophages, Alveolar; Mast Cells; Membrane Lipids; Oxygen; Superoxides

Use of the murine monoclonal antibodies EG1 and EG2 has been based on the assumption that EG2 recognizes activated eosinophils. We examined the reactivity of EG1 and EG2 with eosinophil cationic protein (ECP), eosinophil-derived neurotoxin (EDN), and stimulated and nonstimulated eosinophils from normal donors. By radioimmunoassay, EG1 recognized only ECP, whereas EG2 recognized both ECP and EDN. By Western blot, EG1 reacted with ECP, EG2 reacted with both ECP and EDN, but EG2 could not distinguish between lysates of stimulated and nonstimulated eosinophils. By immunofluorescence, EG1 and EG2 at 20 microg/mL stained 95-100% of nonstimulated eosinophils, regardless of fixative; EG1 and EG2 at 0.1 microg/mL stained 61-90% of acetone- and paraformaldehyde-fixed and only 5-21% of methanol-fixed nonstimulated eosinophils. Thus, the reactivity of EG1 and EG2 with eosinophils depends on the method of fixation and antibody concentration; and EG2, in contrast to previous reports, cannot reliably discriminate between resting and activated eosinophils.

Topics: Animals; Antibodies, Monoclonal; Antibody Specificity; Artifacts; Blood Proteins; Blotting, Western; Calcimycin; Calcium; Cross Reactions; Cytoplasmic Granules; Eosinophil Granule Proteins; Eosinophil-Derived Neurotoxin; Eosinophilia; Eosinophils; Epitopes; False Positive Reactions; Fluorescent Antibody Technique, Indirect; Humans; Ionophores; Mice; Proteins; Radioimmunoassay; Ribonucleases; Sensitivity and Specificity; Specimen Handling

Eosinophil degranulation induced by the calcium ionophore A23187 and opsonized zymosan particles was examined ultrastructurally in peripheral blood cells obtained from patients with the hypereosinophilic syndrome. Unstimulated hypereosinophilic syndrome eosinophils contained altered, vacuolated cytoplasmic granules and large cytoplasmic crystalloid structures not seen in normal cells. By morphometric analysis of transmission electron micrographs, the hypereosinophilic syndrome eosinophils contained a larger percentage of smaller sized cytoplasmic granules than normal cells. The hypereosinophilic syndrome eosinophils, however, were capable of undergoing noncytotoxic degranulation after stimulation with either A23187 or opsonized zymosan. Hypereosinophilic syndrome eosinophil degranulation was characterized by fusion of the perigranular membranes of adjacent cytoplasmic granules and vesiculation of the fused granules. Granule contents were released intracellularly into vacuoles after ionophore stimulation and into phagosomes containing the ingested zymosan particles. Noncytotoxic extracellular release of eosinophil peroxidase (EPO) was also observed after cell stimulation by either A23187 or zymosan. The capacity of hypereosinophilic syndrome eosinophils to degranulate after appropriate stimulation with release of toxic granule constituents such as EPO and other basic proteins may be important in the tissue injury observed in this syndrome.

Topics: Calcimycin; Cytoplasmic Granules; Eosinophilia; Eosinophils; Humans; Microscopy, Electron; Phagocytosis; Syndrome; Zymosan

The formation of arachidonic acid-derived lipoxygenase products was examined with human granulocytes obtained from eosinophilic donors. These eosinophil-enriched leukocyte populations, challenged in vitro with the ionophore of divalent cations A23187, transformed both exogenous and endogenous sources of arachidonic acid to several lipoxygenase-derived products, including 5(S), 6(R),15(S)-trihydroxy-7,9,13-trans-11-cis-eicosatetraenoic acid (lipoxin A). Lipoxin A was detected and characterized by high-pressure liquid chromatography (HPLC), ultraviolet absorbance, and gas-liquid chromatography-mass spectroscopy. Neither lipoxin B nor 6(S)-LXA was consistently detected in extracts from these incubations. The amounts of lipoxin A formed were proportional to the percentage of eosinophils present in the suspension. The results indicate that granulocytes from eosinophilic donors can generate lipoxin A.

Topics: Arachidonate 5-Lipoxygenase; Arachidonic Acid; Arachidonic Acids; Calcimycin; Eosinophilia; Granulocytes; Humans; Hydroxyeicosatetraenoic Acids; Lipoxins; SRS-A

The formation of 5- and 15-lipoxygenase products of arachidonic acid metabolism was examined in human peripheral blood eosinophils obtained from five normal individuals and five patients with the hypereosinophilic syndrome (HES). Normal and HES eosinophils after stimulation with the calcium ionophore A23187 produced in comparable amounts leukotriene (LT)C4, LTD4, LTB4 and two of its isomers (5-(S),12-(R)-6-trans-LTB4 and 5-(S), 12-(S)-6-trans-LTB4), 15-hydroxy-eicosatetraenoic acid (HETE), 5,15-di-HETE and 8,15-di-HETE. No single lipoxygenase product predominated in the absence of added arachidonic acid whereas 15-HETE was the major product formed when either normal or HES eosinophils were stimulated with A23187 in the presence of added arachidonic acid (10(-4) M).

Topics: Arachidonate Lipoxygenases; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Eosinophilia; Eosinophils; Humans; Leukotriene B4; Lipoxygenase; SRS-A; Syndrome

Blood samples were obtained from a group of 20 patients with hypereosinophilia (greater than or equal to 1500 eosinophils/mm3). The polymorphonuclear leukocytes (PMNLs) were prepared from blood treated with ethylenediaminetetra-acetic acid by successive dextran sedimentation of the red blood cells, separation of mononuclear leukocytes and PMNLs on Ficoll-Paque, and ammonium chloride treatment of the PMNL fraction. The eosinophil content of the final PMNL preparations ranged from 15% to 75%, as assessed by Wright-stained smears, and the remaining leukocytes were predominantly neutrophils with only 3% to 5% mononuclear cells. The eosinophil-rich PMNL preparations as well as PMNL preparations from normal volunteers were incubated under various conditions and the arachidonic acid metabolites were analyzed by reverse-phase high-performance liquid chromatography. The synthesis of 5-lipoxygenase products was strongly stimulated by the ionophore A23187 in both normal and eosinophil-rich PMNL preparations. Whereas the normal PMNL preparations, which were eosinophil poor, produced 10 to 25 times more leukotriene B4 than leukotriene C4, the eosinophil-rich PMNL preparations characteristically released leukotriene C4 in equal or up to 20 times greater amounts than leukotriene B4.

Topics: Arachidonate Lipoxygenases; Arachidonic Acid; Arachidonic Acids; Calcimycin; Chromatography, High Pressure Liquid; Eosinophilia; Eosinophils; Humans; Lipoxygenase; Neutrophils; Reference Values; SRS-A

Considerable variations in the distribution of eosinophil populations were revealed by density gradient centrifugation of peripheral blood leukocytes from 14 normal subjects and 31 patients with a transient or persistent hypereosinophilia. Such a purification procedure enabled us to collect in healthy controls, eosinophils (77.1 +/- 15.9% SD purity) with an appreciable cell recovery (21.7 +/- 14.0% SD) in the densest gradients ('normodense' cells). A high degree of purity was obtained in the same gradients, with leukocytes from patients with hypereosinophilia (84.4 +/- 11.9% SD pure eosinophils) but the cell recovery was significantly decreased (4.1 +/- 3.4% SD; p less than 0.001). A study of the various fractions found to contain eosinophils showed cells with an altered cellular density ('hypodense' cells) especially in marked hypereosinophilia. Furthermore, studies on leukocytes from patients with a very high hypereosinophilia (differential cell count greater than 70%) led to the recovery of almost pure fractions of eosinophils in the low density gradients. Functional studies were performed on each distinct cell fraction collected. They included investigations of their cytotoxic ability in a heterologous antibody-dependent cell-mediated cytotoxicity assay and their biochemical activity by using a previously described technique evaluating the membrane hexose transport. Variability in the functional potentialities was observed in relation to the cellular density: higher cytotoxic eosinophil abilities were noted in the case of the 'hypodense' cell population. According to biochemical criteria, these latter cells appear to be more activated and capable of responding to stimulation.

Topics: Antibody-Dependent Cell Cytotoxicity; Biological Transport; Calcimycin; Cell Separation; Centrifugation, Density Gradient; Deoxyglucose; Eosinophilia; Eosinophils; Heparin; Humans; Leukocyte Count