Ackerman, S.J.

Abstract The human eosinophil granule contains a number of cationic proteins that have been identified and purified to homogeneity, including the major basic protein (MBP), the eosinophil cationic protein (ECP), and the eosinophil-derived neurotoxin (EDN). Because of confusion in the literature regarding the distinctiveness of MBP and ECP, we investigated the immunochemical and physicochemical properties of these purified proteins by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), by specific double antibody radioimmunoassays (RIA) for MBP and ECP, and by fractionation of acid-solubilized eosinophil granules on Sephadex G-50 columns. Analysis of a mixture of the three purified proteins by SDS-PAGE showed that they migrated as three distinct bands with differing m.w. Comparison by specific RIA for MBP and ECP did not demonstrate any appreciable immunochemical cross-reactivities among the three proteins. Sephadex G-50 column fractions of acid-solubilized eosinophil granules were analyzed by RIA and by SDS-PAGE analysis of individual column fractions. MBP, ECP, and EDN eluted at different volumes from Sephadex G-50 columns as determined by RIA and SDS-PAGE. Soluble extracts of eosinophil granules from patients with the hypereosinophilic syndrome contained between six and 64 times more MBP than ECP on a weight basis. These observations demonstrate that MBP, ECP, and EDN are distinctive cationic proteins of the human eosinophil granule and that eosinophil granules from patients with eosinophilia contain considerably greater quantities of MBP than ECP.

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 Killing of bloodstream forms of Trypanosoma cruzi, the unicellular parasite that causes Chagas' disease in humans, by human eosinophils in the presence of specific antibody to the parasite was inhibited by the polyanion heparin in a dose-dependent manner. The concentration of heparin required to completely abolish eosinophil-effected killing of the flagellate increased as the eosinophil to parasite ratio increased. These results suggest that antibody-dependent eosinophil-mediated cytotoxicity is mediated by basic constituents of the eosinophil granule.

Abstract A double-antibody radioimmunoassay (RIA) was developed to measure the protein comprising the human eosinophil Charcot-Leyden crystal (CLC). Critical requirements in the development of this assay were the production of large quantities of pure CLC, the solubilization of CLC at neutral pH, and an assessment of the purity of the CLC protein. CLC were prepared from eosinophil-rich leukocyte suspensions by homogenization of cells in hypotonic-buffered saline. Centrifuged eosinophil extracts formed CLC spontaneously at 4°C, but the yield of crystals was significantly augmented by slow freezing of extracts to −20°C and thawing. Lyophilization of crystals was required to obtain material soluble at neutral pH. Soluble CLC protein produced 3 dense bands on polyacrylamide gel electrophoresis (PAGE) at pH 8.9, and these bands gave reactions of identity in immunodiffusion analyses with rabbit anti-CLC antibody. PAGE in 1% sodium dodecyl sulfate revealed a single protein band of 13,000 daltons. Isoelectric focusing of soluble CLC protein yielded 2 major and 4 minor bands with pI’s between 5.7 and 5.1, indicating the slightly acidic nature of the CLC protein. In the RIA, CLC levels were measured by inhibition of the binding of 131I-CLC reacted with specific rabbit anti-CLC antibody. Burro anti-rabbit IgG was used as the second antibody to precipitate immune complexes. The maximum precipitability of 1 ng 131I-CLC by antibody was 97%; the RIA was sensitive to 0.25 ng. CLC protein is immunologically distinct from human eosinophil major basic protein as determined by RIA and Ouchterlony analysis. Serum levels of CLC for 10 normal healthy subjects averaged 37 ± 4.7 ng/ml compared with 8507 ± 2494 ng/ml for 10 patients with diseases associated with a peripheral blood eosinophilia greater than 30%. The RIA for CLC protein provides us with a tool to investigate the biological function(s) of this protein and the role of eosinophils in certain disease states.

Abstract Eosinophilia is a common sequela of treatment in patients infected with the parasitic helminth Wuchereria bancrofti. We investigated the relationship between peripheral blood eosinophil levels and serum levels of eosinophil granule major basic protein (MBP) and Charcot-Leyden crystal (CLC) protein in 37 patients with Bancroft’s filariasis who were treated with diethylcarbamazine (DEC). Before treatment with DEC, eosinophil levels and serum levels of CLC protein, but not MBP, were found to be significantly elevated in both filariasis patients and an uninfected endemic control population. After treatment, patients with microfilaremia developed an initial eosinopenia followed by significant increases in their eosinophil levels and serum levels of both MBP and CLC protein, which peaked 1 to 2 wk after initiating treatment. Before treatment, eosinophil levels and serum levels of MBP and CLC protein were unrelated to the number of microfilariae in circulation, but after treatment, the degree of eosinophilia and the elevations in serum MBP and CLC protein were significantly correlated with the patients’ microfilarial worm burdens before treatment. In microfilaremic patients on DEC therapy, there was a statistically significant increase in serum MBP levels concomitant with the initial decrease in eosinophil levels observed on day 1 of therapy in these patients. Patients with filariasis (elephantiasis, hydrocoeles, or filarial fevers) but without microfilaremia, as well as an uninfected endemic control population, showed no change in their eosinophil levels or serum MBP or CLC protein levels during an identical course of DEC therapy. The eosinophilic response resulting from DEC therapy of patients with microfilaremia provides a unique opportunity to study the relationship between eosinophilia, eosinophil function, and possibly eosinophilopoiesis as reflected by the synthesis and/or release of eosinophil proteins in humans exposed to a quantifiable antigenic stimulus.