D S Silberstein

Human eosinophils have prolonged survival, enhanced functional properties, and become hypodense when exposed to human interleukin 3.

Human eosinophils were cultured in the presence of recombinant human IL-3 for up to 14 d and their biochemical, functional, and density properties were assessed. After 3 d of culture in 10 pM IL-3, eosinophils had a viability of 70% compared with only 10% in enriched medium alone. Neither IL-1 alpha, IL-2, IL-4, tumor necrosis factor, basic fibroblast growth factor, nor platelet-derived growth factor maintained eosinophil viability. The 7- and 14-d survival of the cultured eosinophils was 55 and 53%, respectively. No other cell type, including neutrophils, was present after culture. After 7 d of culture, the normodense eosinophils were converted to hypodense cells as assessed by density centrifugation. Eosinophils exposed to 1,000 pM IL-3 for 30 min or cultured in 10 pM IL-3 for 7 d generated approximately threefold more leukotriene C4 (LTC4) in response to calcium ionophore than freshly isolated cells. Furthermore, whereas freshly isolated eosinophils killed only 14% of the antibody-coated Schistosoma mansoni larvae, these eosinophils killed 54% of the larvae when exposed to 100 pM IL-3. The enhanced helminth cytotoxicity was maintained for 7 d when eosinophils were cultured in the presence of both 10 pM IL-3 and 3T3 fibroblasts, but not when eosinophils were cultured in the presence of IL-3 alone. IL-3 thus maintains the viability of eosinophils in vitro, augments the calcium ionophore-induced generation of LTC4, enhances cytotoxicity against antibody-sensitized helminths, and induces the eosinophils to become hypodense cells. These phenotypic changes in the eosinophil may be advantageous to host defense against helminthic infections but may be disadvantageous in conditions such as allergic disease.

Human eosinophils express functional interleukin 2 receptors.

Because T cell-derived cytokines may affect the functioning of eosinophils, we have investigated the capacity of human eosinophils to respond to IL-2. IL-2 was a potent chemoattractant with ED50 of 10(-12) M with eosinophils from all normal and eosinophilic donors tested. The monoclonal antibodies anti-Tac and TU27 against p55 (Tac/CD25) and p75 receptor subunits, respectively, each inhibited IL-2-dependent eosinophil migration. The molar potency of IL-2 and the inhibitory activity of each of the above antibodies suggest that high affinity heterodimeric IL-2 receptor complexes mediated the migration responses of eosinophils to IL-2. Binding of monoclonal antibody against p75 was not detectable by flow cytometry, and high affinity binding sites for 125I-IL-2 were below the limits of quantitation on eosinophils from individuals with eosinophilia. Expression of p55 (Tac/CD25) by eosinophils, without requirement for in vitro activation, was demonstrable by flow cytometry, radioimmunoprecipitation, and Northern blotting for mRNA. Surface expression of p55 on eosinophils from normal or eosinophilic individuals increased during culture for 24-48 h with a biologic activity purified from stimulated U937 cells and to a lesser extent with granulocyte-macrophage CSF or lymphocyte chemoattractant factor but not with nine other cytokines. These studies indicate that blood eosinophils respond to IL-2 and identify one mechanism whereby activation of T lymphocytes may influence the function of eosinophils.

Trichinella spiralis: Secreted antigen of the infective L1 larva localizes to the cytoplasm and nucleoplasm of infected host cells

Antibodies were elicited against a purified antigen with an apparent molecular weight of 43K. This antibody preparation also detected a second antigen consisting of a group of closely related components of 45-50K. These antigens are stage specific for the infective first stage larva of Trichinella spiralis and are among the repertoire of secreted antigens originating from the stichosome. Antibody raised against the 43K antigen reacted with the stichosome and cuticle of the mature larva and the cytoplasm and nucleoplasm, but not nucleolus, of all nuclei of infected host cells (Nurse cells) in sections of infected tissues. Studies on sections of synchronously infected muscle tissue revealed that antigen was present only within the worm on Day 7 of the infection. On Day 9 after infection, the stichosome and cuticular surface of the larva and the cytoplasm and nucleoplasm of each nucleus of the Nurse cell reacted with antibody. Nurse cell cytoplasmic and nuclear reactivity increased in intensity until Day 18 after infection. These results suggest that stichocyte-specific antigens are synthesized during the early phase of infection in the muscle, and that as the Nurse-parasite complex develops, some of the antigen is secreted into the milieu of the Nurse cell. The presence of antigen in the cytoplasm and nucleoplasm of the infected host cell is discussed in relation to Nurse cell formation and maintenance.

IMMUNOCYTOLOCALIZATION OF TWO PROTECTION-INDUCING ANTIGENS OF TRICHINELLA SPIRALIS DURING ITS ENTERAL PHASE IN IMMUNE AND NON-IMMUNE MICE

Monoclonal antibodies (mAb) recognizing epitopes on the 48K (beta stichocyte specific) and the 50/55K antigen (alpha stichocyte specific) were used as first ligands for immunocytolocalization on de-paraffinized sections of infected gut tissue of non-immune and immune CFW strain mice. The enteral phase was studied at 6, 14, 23, 30 hr and 7 days after initiation of infection via the oral route, times corresponding in worm development to the first (L1), second (L2), and third (L3) stage larva and adult. No change in the intensity of the immune reaction with either mAb was noted in parasites developing within immune or non-immune mice for any of the time-points studied. The 48K and the 50/55K antigens were present within the stichocytes at 6 hr. Enterocytes adjacent to some worms also stained positive for both epitopes at this time. Throughout worm development, the amount of each antigen within the worm diminished, until almost none was left at 30 hr. At day 7, the 48K antigen was present within a few stichocyte cells, the canalicular tree, and within the lumen of the midgut. The 50/55K antigen at this time point was localized within only a few stichocyte granules and on the lining of the worms gut. Embryo stages did not possess either the 48K or 50/55K epitopes. A marked increase in cells bearing IgG in the lamina propria was noted in immune mice when compared with their non-immune counterparts.

Immunization with purified antigens protects mice from lethal infection with Trichinella spiralis.

The use of monoclonal antibodies with affinity chromatography has allowed the purification of 2 antigens, the 48K antigen and the 50/55K antigen, from the infective L, larva of Trichinella spiralis. Immunization of mice with either ofthese antigens induced strong resistance to the development of muscle larvae following a challenge infection of 360 infective L, larvae (Silberstein and Despommier, 1984, Journal of Immunology 132: 898-904). This infective dose was too low to cause severe disease or death in either test or control mice. Use of a low infective dose facilitates quantitation of parasite development, but it does not allow investigation of several interesting possibilities: 1) that immunization with single purified T. spiralis antigens may protect the host from severe disease or death; 2) that a high dose of infective larvae may suppress the effects of immunity; or 3) that immunization with certain antigens may exacerbate disease even though it inhibits parasite development. A preliminary trial of the effect of 48K and 50/55K immunization on lethal infections of mice was conducted. Three-mo-old male CFW strain mice (Charles River Breeding Laboratories, Wilmington, Massachusetts) were immunized by intraperitoneal injection of antigen in pH 7.0, 0.01 M sodium phosphate buffer emul-