Dunnette, Sandra L.

Abstract The divalent cation ionophore A23187 is frequently used for studies of eosinophil degranulation. Nonetheless, the mechanism whereby A23187 induces degranulation in human eosinophils is still unclear. In the present experiments, A23187 caused human eosinophils to release a granule protein, eosinophil-derived neurotoxin (EDN) and a membrane-associated protein, Charcot-Leyden crystal (CLC) protein in a calcium and a concentration-dependent manner. However, A23187 at a concentration (1 microgram/ml) that caused 15% EDN release and 30% CLC protein release also produced release of the cytoplasmic enzyme lactic dehydrogenase (LDH) and loss of cell viability, both of which were calcium dependent. CLC protein release preceded EDN release and was detectable even at 15 min after the addition of 1 microgram/ml A23187, whereas EDN release occurred after a lag period of 30 min, and coincided with LDH release. At 1 microgram/ml A23187, neither the release of LDH nor the loss of viability occurred with purified neutrophils obtained in the same blood sample as a by-product of eosinophil purification. Electron microscopic examination demonstrated that exposure to A23187 for 15 min resulted in an increase and elongation of microridges on the cell surface, and exposure for 45 min caused cell disruption followed by extrusion of membrane-bound granules through breaks in the plasma membrane. Only once was granule exocytosis observed. These results indicate that A23187 treatment of eosinophils causes an initial release of membrane-associated CLC protein by a noncytolytic mechanism, and causes degranulation as a result of eosinophil lysis.

Abstract A double antibody radioimmunoassay (RIA) was developed to measure IgD in serum and secretions. One IgD myeloma protein was used as radiolabeled antigen and standard with antiserum to a second IgD myeloma protein. The IgD standard and normal sera yielded parallel inhibition curves in the RIA and inhibition was produced by IgD and not by any of the other immunoglobulins. The assay had a lower limit of sensitivity of 0.01 International Unit (I.U.)/ml and modifications increased the sensitivity to 0.0008 I.U./ml. Measurable IgD levels were found in all 112 normal adult sera assayed (geometric mean 13.0 I.U./ml, arithmetic mean 30.1 I.U./ml, median 14.8 I.U./ml, range 0.10 to 202 I.U./ml, 95% of values between 0.19 and 156 I.U./ml. The distribution of IgD in the 112 normal sera appeared trimodal with modes at approximately 0.25 I.U./ml, 5 I.U./ml, and 35 I.U./ml. IgD was measurable in nasal and bronchial washes and human milk, but could not be detected in parotid fluid.

Abstract Eosinophils contain four principal cationic proteins, major basic protein (MBP), eosinophil-derived neurotoxin (EDN), eosinophil cationic protein (ECP), and eosinophil peroxidase (EPO). To determine the quantities of these proteins in granulocytes and whether they are specific to eosinophils, their concentrations in ly- sates of human granulocytes were measured using specific radioimmunoassays. The effect of different methods for eosinophil lysis on the recovery of the proteins was also studied. Maximal recovery occurred at pH 2 for MBP and pH 5.6 for the other granule proteins. The proteins cosedimented with eosinophils and their concentrations (X ± SEM) in ng/106 eosinophils (and in nM/106 eosinophils) were: MBP, 8,982 ± 611 (641.6); EDN, 3,283 ± 116 (178.4); ECP, 5,269 ± 283 (250.9); and EPO, 12,174 ± 859 (171.5). Basophils from a normal person contained (in ng/106 cells) MBP, 2,374; EDN, 214; ECP, 77; and EPO, 17. Highly purified neutrophils contained (in ng/106 cells) MBP, 3 ± 0.5; EDN, 72 ± 9; and ECP, 50 ± 12. Therefore we conclude that these proteins are mainly expressed in eosinophils, but that certain ones are present in basophils and neutrophils.

SUMMARYThe participation of eosinophils in the Spanish toxic oil syndrome (TOS) was investigated. Eosinophil infiltration and degranulation in tissues from 52 patients with the TOS were examined by immunofluorescence staining for the eosinophil granule major basic protein (MBP). Serum MBP levels were determined in sera from 323 patients. Eosinophil infiltration and degranulation were found in several tissues, especially during the acute phase of the TOS, and serum MBP was significantly elevated during all phases of the disease, suggesting that eosinophils play a role in the pathogenesis of the TOS.