Gleich, Gerald J.

Abstract To study the cytotoxic reactions responsible for mediating eosinophil damage to schistosomula of Schistosoma mansoni, we have used cytoplasts (eosinophil or neutrophil vesicles devoid of granules and nuclei, with an intact oxidase in their plasma membrane) in combination with purified eosinophil cationic protein (ECP) or major basic protein (MBP) in a cytotoxicity test toward schistosomula. Suboptimal concentrations of ECP (10(-6) M) or MBP (10(-6) M) resulting in less than 10% killing were used in combination with cytoplasts. Cytoplasts alone in the presence of immune serum tested over a wide range of cytoplast:schistosomula ratios generated superoxide and hydrogen peroxide, but were unable to damage schistosomula. However, when a suboptimal ECP concentration (10(-6) M) was combined with neutroplasts or eosinoplasts, 43.9% +/- 8.5 (n = 7) and 24.7% +/- 9.8 (n = 3), respectively, of the schistosomula were killed. Oxygen metabolites were responsible for the synergism, because cytoplasts from a patient with chronic granulomatous disease were unable to act in synergy with ECP. In contrast to ECP, no synergism was found between cytoplasts and MBP (10(-6) to 2 X 10(-5)M). These results show that oxygen metabolites are important for the killing of schistosomula by lowering the concentration of ECP needed to inflict damage.

Abstract Human eosinophils contain several distinctive proteins including eosinophil granule MBP and the membrane-associated CLC protein (lysophospholipase). Human basophils also contain these proteins, indicating biochemical similarities between eosinophils and basophils. To determine whether MBP or CLC protein is present in connective tissue mast cells, we studied human lung and cutaneous mast cells by immunofluorescence by utilizing specific antibodies to CLC and MBP. Cytocentrifuge slides of enriched lung mast cells and mast cells in sections of formalin-fixed, paraffin-embedded cutaneous tissue from urticaria pigmentosa lesions were stained for CLC and MBP. Neither pulmonary nor cutaneous mast cells stained for CLC protein or MBP. In contrast, lung and cutaneous eosinophils in the same preparations showed bright staining for both proteins. The failure to find CLC protein and MBP in mast cells provides additional evidence of dissimilarity between mast cells and basophils, and an immunochemical means to distinguish between them.

Abstract Eosinophils play an important role as effector cells in allergic, parasitic, and other conditions. The mechanism(s) by which eosinophils mediate their effector functions was studied by incubation of human normodense eosinophils with Sepharose beads coupled to various Ig isotypes as targets. Controls included eosinophils incubated alone or incubated with uncoated beads, human serum albumin-, or OVA-coated beads. An eosinophil granule protein, the eosinophil-derived neurotoxin (EDN), was measured as an indicator of eosinophil degranulation. Eosinophils released eosinophil-derived neurotoxin when incubated with Sepharose beads coupled to Ig of the IgG or IgA isotypes, as well as IgA-Fc fragments. Mixtures of IgG and IgA on beads did not act synergistically. Secretory IgA (sIgA) provided the most potent signal for eosinophil degranulation and was two to three times more potent than IgG. Furthermore, 2 to 17% of the normodense eosinophils bound to IgG- or IgA-coated beads, whereas 24 to 27% of the eosinophils bound to sIgA-coated beads. Thus, sIgA may be the principal Ig mediating eosinophil effector function at mucosal surfaces in helminth infections and hypersensitivity diseases, especially bronchial asthma.

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 We previously found that sera of patients immunized with ragweed pollen extract contained a factor that interfered with the binding of IgE antibodies to solid-phase allergens in the radioallergosorbent test (RAST). We now describe an assay, RAST interference, to measure this factor, and we present evidence that the factor is IgG blocking antibody. Sera from immunized allergic patients were heated at 56 degrees C for 4 hr to destroy heat-labile Fc determinants on IgE and were tested for their ability to prevent binding of additional IgE antibody to solid-phase allergens in the RAST. Eight of 10 sera from allergic immunized patients gave RAST interference dose-response curves that did not differ from the arbitrary standard. The factor causing interference showed specificity for the immunizing antigen, was heat-stable, eluted from Sephadex G-200 in the 7S peak, was present only in sera of immunized patients, and rose after initiation of immunization. These results indicated that RAST interference can be used to measure IgG blocking antibodies with the same reagents employed for the measurement of IgE antibodies, provided the antiserum to IgE is specific for the heat-labile FC determinants on IgE.

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.

Abstract The discovery of immunoglobulin E (IgE) (1) has contributed significantly to the understanding of hypersensitivity reactions mediated by reaginic antibody (2) and has led to the development of new diagnostic procedures in the field of allergology (3, 4). Recent studies have demonstrated the production of IgE protein by peripheral blood lymphocytes in vitro and have examined the kinetics and pharmacologic control of IgE biosynthesis (5, 6). The purpose of this communication is to describe our observation of in vitro production of IgE protein and antigen-specific IgE antibodies by peripheral blood mononuclear cells obtained from atopic and nonatopic individuals. A modification of the lymphocyte culture method of Waldmann et al. (7) was used in our studies. Blood, 60 ml, was obtained from 11 patients, eight of whom are atopic (geometric mean serum IgE, 247 ng/ml; range 61 to 1079) and three of whom are nonatopic (geometric mean serum IgE, 96 ng/ml; range 73 to 143).

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 We compared the quantities and avidities of IgE and IgG antibodies from three phospholipase-A (PLA) sensitive patients during honeybee venom immunotherapy. IgG antibody was purified by affinity chromatography through a Sepharose anti-IgE Column followed by ionexchange chromatography on DEAE Sephadex. IgE antibody was obtained by elution from the Sepharose anti-IgE column with 4 M guanidinium HCl and by passage of the eluate over Sepharose anti-IgG. Absolute amounts of IgE and IgG antibody to PLA were determined by immunoabsorption and radioimmunoprecipitation, respectively. IgE antibody to PLA accounted for 25.1% to 28.7% (X̄ = 27.0%) of the total IgE protein; IgG antibody accounted for 0.073% to 0.210% (X̄ = 0.130%) of the total IgG protein. The relative avidities of IgE and IgG antibodies were assessed by the ability of equimolar amounts to bind radioiodinated PLA. In two patients, avidities of IgE and IgG antibody for PLA were comparble, whereas in the third, IgG antibody bound PLA more avidly than IgE antibody. The valence of IgE antibody was greater than 1.9, assuming that PLA behaved as a univalent ligand in extreme antigen excess. IgG antibody was able to inhibit binding of IgE antibody in the PLA radioallergosorbent test (RAST) from 10% to 40% at a molar excess of 10- to 1000-fold. The results indicate that: 1) IgE antibody to PLA accounts for a high percentage of total IgE protein; 2) the avidities of IgE and IgG antibodies are similar (two of three patients); 3) the valence of IgE antibody approaches two; and (4) IgG antibody can interfere with the measurement of IgE antibody in the PLA RAST.