Teruko Ishizaka

Mechanisms of Reaginic Hypersensitivity and IgE Antibody Response

Immunoglobulin E was found when we were studying reaginic antibody in the serum of hay fever patients and was identified as a carrier of reaginic antibody activity (Ishizaka et al. 1966a, b, Ishizaka & Ishizaka 1967). An injection of purified IgE preparation from ragweed sensitive individuals into normal skin resulted in the sensitization of the skin site for the PrausnitzKiistner (P-K) reaction, and the removal of IgE antibody in the serum of atopic patients by anti-IgE was accompanied by loss of sensitizing activity. Subsequent studies have shown that association of reaginic activity with IgE was found not only in the ragweed system but also in the other allergenreagin systems. Antigenic analysis of the immunoglobulin (Ishizaka et al. 1967d) and identification of a imique myeloma protein ND as an E myeloma protein (Johansson & Bennich 1967, Bennich et al. 1968) established that IgE represents a unique immunoglobulin class. Since then, effort in our laboratory has been focused to study immtinological mechanisms of reaginic hypersensitivity reactions. Identification of reaginic antibody as IgE provided immunochemical approaches to solve the problems. In the past several years, we have also studied cellular events in the IgE antibody response. Although the two subjects are unrelated, we shall summarize our findings on the immunological mechanisms of IgB-mediated reactions first, followed by mechanisms of IgE antibody response.

Development of human mast cells from their progenitors.

Two types of human mast cells, which are morphologically similar to skin mast cells and lung mast cells, respectively, can be developed from pluripotent stem cells under different culture conditions. The major growth factor for mast-cell development is c-kit ligand, which induces mastocytosis in vivo. However, this cytokine is not sufficient for full maturation of the cells.

IgE formation in the rat following infection with Nippostrongylus brasiliensis. I. Proliferation and differentiation of IgE-bearing cells.

Abstract Sprague-Dawley rats were infected with Nippostrongylus brasiliensis larvae, and IgE formation was studied. Before infection, the serum IgE level was less than 0.4 μg/ml. The IgE level began to increase from the 10th day of infection, reached its maximum (50–100 μg/ml) at the 14th day and gradually declined. Reinfection of the rats resulted in an increase of the serum IgE level within 7 days. The IgE antibody response to N. brasiliensis antigens did not parallel the increase of IgE synthesis. In most animals, the antibody became detectable in the serum at the 21st day when the total IgE level already began to decrease. The animals showed a secondary IgE antibody response upon reinfection. Both mesenteric lymph nodes and spleen cell suspensions were examined for the presence of IgE-bearing cells (IgE-B cells) and IgE-forming cells by fluorescent antibody technique. The IgE-bearing lymphocytes became detectable in the mesenteric lymph nodes and spleen at the 8th day of infection. The proportion of the IgE-B cells in nonadherent cell population gradually increased and reached maximum at the 14th day; about 20% of immunoglobulin (Ig)-bearing cells in the mesenteric lymph nodes and 10% of Ig-bearing cells in spleen bore IgE on their surface. Evidence was obtained that these lymphocytes synthesized IgE. The IgE-forming cells were detected in both mesenteric lymph nodes and spleen of the infected animals. The number of IgE-forming cells was greater in the mesenteric lymph nodes than in spleen, indicating that the regional lymph nodes are the major source of serum IgE in the N. brasiliensis -infected animals.

IgE AND REAGINIC HYPERSENSITIVITY

Immunoglobulin E was detected when we studied reaginic antibodies.1 Subsequent studies of this protein have shwon that it is actually a carrier of reaginic antibody activity.2, In view of accumulated evidence that human IgE antibodies are at least the major, and probably the only, antibodies having the ability to sensitize homologous skin, the role of IgE in allergic diseases must be similar to the role of classical reaginic antibodies in the diseases. However, identification of IgE antibodies as reagin and characterization of this protein provided immunochemical approaches to study the mechanisms of reaginic hypersensitivity reactions, and the results thus obtained supported the idea that reaginic antibodies play an important role in the pathogenesis of atopic diseases. In this paper the biologic function of IgE antibodies will be summarized and the structural basis of the function will be discussed.

Activation of basophils and mast cells for mediator release.

Biochemical events involved in both IgE-dependent and IgE-independent mediator release from basophils and mast cells were analyzed. The results revealed that bridging of IgE receptors activates a variety of membrane-associated enzymes, such as serine protease, phospholipase C, methyltransferases and adenylate cyclase, resulting in the stimulation of phosphatidylinositol (PI) turnover and a transient increase in both phospholipid methylation and intracellular cAMP. Mobilization of intracellular Ca2+ monitored by Quin-2 fluorescence is detected within 5 s after antigen challenge and appears to be the earliest intracellular change detectable after receptor bridging. Stimulation of PI turnover results in the generation of inositol 1,4,5-triphosphate (IP3) and of 1,2-diacylglycerol (DAG), which in turn activates protein kinase C. Evidence was obtained that the guanyl nucleotide (GTP)-binding protein Ni is not involved in the transduction of IgE-mediated triggering signals for mediator release. Although the sequence of enzyme activation following receptor bridging is not clear, the results suggest that the mobilization of intracellular Ca2+ is a crucial initial signal in the IgE-mediated activation of basophils and mast cells. In the mediator release induced by IgE-independent stimuli, enzymes involved in the mediator release are different from one stimulus to another. The results indicate the presence of multiple biochemical pathways for mediator release from basophils and mast cells.

Immunoglolulin E synthesis in parasite infection

The infection of rats with Nippostrongylus brasiliensis (Nb) larvae enhanced IgE synthesis prior to the formation of IgE antibody specific for parasite antigens. As early as the eighth to tenth day of infection, IgE-bearing lymphocytes appeared in lymph nodes and spleen. At this time, even bone marrow contained IgE-bearing blast cells. The IgE-forming plasma cells were detected in the lymph nodes and spleen at the tenth day and their number reached maximum at the fourteenth day. The proliferation of IgE-bearing lymphocytes in the lymphoid tissues was observed in neonatally thymectomized animals, indicating that T cells are not essential for the development of IgE-B cells in the infected animals. It appears, however, that T cells are involved in the differentiation of IgE-B cells to IgE-forming plasma cells. Nonspecific proliferation of IgE-B cells and their differentiation to IgE-forming plasma cells may explain potentiation of IgE antibody formation against unrelated antigens after infection with N. brasiliensis (Nb). It was also found that T cells specific for parasite antigens were primed by infection with Nb. Evidence was obtained tht Nb-specific T cells raised by the infection collaborated with hapten-specific IgE-B cells and IgG-B cells for the secondary antihapten antibody responses. By contrast, T cells primed by immunization with parasite antigen included in alum exerted the helper function for IgG antibody response but failed to collaborate with IgE-B cells. Dissociation between the helper function for IgE and IgG antibody response indicated that parasite infection generated a favorable condition for priming T cells for the IgE antibody response.

Vesicular transport of peroxidase in human eosinophilic myelocytes

We performed ultrastructural cytochemistry to detect peroxidase in developmentally arrested human eosinophilic myelocytes. Human umbilical cord blood mononuclear cells were cultured for 21 days in the presence of murine‐derived conditioned media, resulting in the development of eosinophilic myelocytes. Unlike normally developing eosinophilic myelocytes, which contain peroxidase in synthetic organelles (i.e. cisterns surrounding the nucleus and hounded by the rough endoplasmic reticulum and Golgi structures) and in immature and mature granules, the developmentally arrested cells showed ultrastructural evidence of decreased synthesis and secretor transport of peroxidase. Thus. peroxidase was generally absent in the perinuclear and rough endoplasmic cisterns, in Golgi structures, in immature granules and in the matrix‐compartment of most mature granules. Rather, bicompartmental specific granules displayed empty, peroxidase‐negative matrix and central, peroxidase‐negative core material. Peroxidase was present in perigranular vesicles, some of which were attached to granules. Such peroxidase‐loaded transport vesicles are similar to those that effect piecemeal degranulation of mature human eosinophils cultured in rhIL‐5‐containing media [1]. These findings establish vesicle‐mediated piecemeal degranulation in the secretory repertoire of immature human eosinophils and suggest the possibility that eosinophilic myelocytes may participate in vivo in important physiological and/or pathological events that require selective secretion from the specific granule matrix compartment.

Preferential Differentiation of Inflammatory Cells by Recombinant Human Interleukins

The effects of recombinant human interleukins (IL) on hematopoiesis were explored by using suspension cultures of mononuclear cells of human umbilical cord blood and bone marrow cells. The results showed that IL-5 induced the selective differentiation and proliferation of eosinophils. After 3 weeks in culture with IL-5, over 90% of nonadherent cells in both bone marrow cell and cord blood cell cultures became eosinophilic myelocytes. Culture of the same cells with IL-4 resulted in the selective growth of OKT-3+ lymphocytes. In suspension cultures of bone marrow cells and cord blood cells grown in the presence of IL-3, basophilic, eosinophilic, and neutrophilic myelocytes developed within 2 weeks. By 3 weeks, however, the majority of non-adherent cells became eosinophilic myelocytes. In contrast to mouse bone marrow cell cultures, neither IL-3 nor combination of IL-3 and IL-4 induced the differentiation of mast cells in human bone marrow or cord blood cell cultures.

Unique Features of Human Basophilic Granulocytes Developed in in vitro Culture

Human basophilic granulocytes were obtained in suspension culture of mononuclear cells from umbilical cord blood. Precursors of basophils in the cord blood are nonadherent cells and bear neither surface Ig nor T cell marker. Cultured basophils contain chondroitin-4-sulfate proteoglycan, 1.74 micrograms histamine per 10(6) cells in average, and bear 120,000-380,000 IgE receptors per cell. The IgE receptor molecule has a molecular weight of approximately 64,000 daltons. Human, rat and mouse IgE bind to the receptors with comparable high affinity and passively sensitize the cells for mediator release. Challenge of sensitized basophils with anti-IgE resulted in stimulation of phospholipid methylation, 45Ca uptake, release of both histamine and free arachidonic acid, and enhancement of phosphatidylinositol (PI) turnover. Evidence was obtained that the activation of membrane-associated proteolytic enzyme and methyltransferases is involved in subsequent IgE-mediated biochemical cascades such as PI turnover, Ca2+ uptake and mediator release.

Ultrastructural morphology of immature mast cells in sequential suspension cultures of human cord blood cells supplemented with c-kit ligand; Distinction from mature basophilic leukocytes undergoing secretion in the same cultures

The ability to culture human mast cells and, thus, to determine their ontogeny and possible relationships to other lineages has been facilitated by new studies using cocultures of cord blood cells with mouse fibroblasts and recombinant human or murine c‐kit ligand‐ supplemented suspension cultures of cord blood cells. In this study, we examined c‐kit ligand‐supplemented cord blood cell suspension cultures designed so that the effects of growth factor source, individual cord sample, and culture time (3‐17 weeks) on the developing mast cell lineage could be individually evaluated. We found that human mast cells, basophils, neutrophils, eosinophils, macrophages, megakaryocytes, and endothelial cells were present in these cultures. The numbers of mast cells and their granules increased with culture time; mature basophils, present in quantity in 3‐week cultures, decreased in number and released granule contents with increased culture times. The mast cell lineage developed similarly, regardless of which factor preparation was added to cultures, but considerable variability existed among individual donors from whom cord bloods were obtained. Unlike the mature, crystal‐containing mast cells that regularly developed in fibroblast cord blood cocultures (Furitsu et al. [1989] Proc. Natl. Acad. Sci. USA 86, 10039‐10043), human mast cells failed to attain full maturity in the suspension cultures examined here, regardless of individual cord sample, added growth factor, or culture time. Furthermore, unlike cells of the basophil lineage in which granule content release was regularly observed, morphologic evidence of secretion from human mast cells was absent. Instead, these cells were actively undergoing granule building as determined by the increasing numbers of granules and filling of these containers over culture time. Crystal granules never developed, even at the maximum culture time of 17 weeks. We conclude that fibroblasts are necessary and sufficient for the differentiation and maturation of human mast cells in vitro from their agranular precursors in cord blood but that soluble c‐kit ligand is not.