Ultrastructural and immunohistochemical studies on myocardial biopsies from a patient with eosinophilic endomyocarditis.

Right ventricular endomyocardial biopsy specimens from a 13-year-old boy with hypereosinophilia were studied by light and electron microscopy using the EG2 monoclonal antibody, which recognizes a common epitope of eosinophil cationic protein and eosinophil protein-X. Although the endocardial layer was of normal thickness, many eosinophils, mononuclear cells, and free eosinophil granules were observed in the endocardium and in the vicinity of degenerated myocardial cells. Under electron microscopy, many of the specific granules in and out of eosinophils had lost their crystalloid internae and displayed reversed density, and there were many degranulated eosinophils with reduced number of granules. Immunohistochemically, large amounts of eosinophil cationic protein and protein-X were observed within cardiocytes when many of them were degenerated. Deposits of the proteins were also found in some small vessels. On electron microscopy, accumulations of gold particles, which bind to eosinophil cationic protein and protein-X, were seen in association with specific granules and on the myofilaments in both degenerated and normal-appearing cardiocytes. The presence of eosinophil cationic proteins within cardiocytes may play an important role in the pathogenesis of eosinophilic endomyocardial disease.

Expression in Escherichia coli and purification of human eosinophil-derived neurotoxin with ribonuclease activity.

Eosinophil-derived neurotoxin (EDN) is a ribonuclease with neurotoxic and helminthotoxic properties. It is present in the crystalloid granules of human eosinophils. We report the expression and characterization of a functionally active recombinant human EDN using the pMAL-cRI expression system. A cDNA for mature EDN was obtained by PCR and inserted in pMAL-cRI downstream of the malE gene encoding maltose binding protein. Induction of the ptac promoter of the plasmid in Escherichia coli strain BL21(DE3) resulted in high level expression of soluble MAL-EDN fusion protein. Cleavage of affinity purified fusion protein with Factor Xa protease released recombinant EDN which comigrated with native EDN on SDS-polyacrylamide gels and cross-reacted with a polyclonal anti-EDN antiserum on Western blots. IN contrast to previous attempts at EDN expression, denatured and refolded EDN had ribonuclease activity and was prepared in microgram amounts. The availability of recombinant human EDN should facilitate studies of its structure and biological functions.

Human eosinophil major basic protein, a mediator of allergic inflammation, is expressed by alternative splicing from two promoters.

Human eosinophil major basic protein (MBP) is one of the principal mediators of injury to parasites and tissues in allergic inflammation. MBP is stored in eosinophil crystalloid granules and released with other granule constituents during eosinophil action. Previous studies have identified an MBP gene promoter that generates a 1.0 kb mRNA transcript encoding MBP preproprotein which undergoes processing to the mature storage form. To investigate how the MBP gene is regulated, we have examined the identity and levels of the MBP transcripts both in precursor cells and in blood eosinophils. It was found that the gene was expressed from two upstream promoters, a distal promoter P1 in addition to the previously described promoter P2. Evidence for the second promoter was initially provided by isolation from a human HL-60 leukaemic cell cDNA library of a novel 1.6 kb MBP cDNA that was distinct from the known 1.0 kb cDNA. The complete nucleotide sequence of the 1.6 kb cDNA was determined, and showed that the two cDNAs had identical coding and 3' untranslated regions but differed in their 5' sequences. By isolating and sequencing MBP genomic clones from an arrayed chromosome 11 library, it was demonstrated that the MBP gene is composed of nine upstream exons and five coding exons. The 1.6 and 1.0 kb cDNAs arise by differential splicing of alternate MBP transcripts from promoters P1 and P2 respectively, located 32 kb apart in the genomic DNA. Primer extension analysis identified two transcription start sites at P1, neither associated with a typical TATA box motif. Northern blotting and reverse-transcription PCR analysis showed that the 1.0 kb mRNA was present at higher levels than the 1.6 kb species in immature cells including HL-60 and bone-marrow cells. By contrast, low levels of 1.6 kb mRNA transcripts predominated in differentiated blood eosinophils. The results are compatible with differential use of P1 and P2 promoters as a mechanism for regulation of MBP expression during eosinophil maturation.

Activation of the respiratory burst in eosinophil leucocytes--a transduction sequence decoupled from cytosolic Ca2+ rise.

The activation of the respiratory burst by complement factor 5a (C5a), platelet-activating factor (PAF), formyl-Met-Leu-Phe (fMLP) and neutrophil-activating peptide IL-8 was explored in eosinophils from patients with the hypereosinophilic syndrome. The amplitude of the response increased with increasing concentrations of C5a and PAF, but the time for its induction was unaffected by the amount of stimulus applied. Respiratory burst activity resulting from phorbol 12-myristate, 13-acetate (PMA)-mediated activation of protein kinase C (PKC) produced longer onset times, which shortened with increasing PMA concentrations. Total inhibition of the C5a- and PMA-mediated burst could be achieved with the PKC inhibitor staurosporine at concentrations of 100 and 5nM, respectively. Calcium depletion abolished agonist-induced rises in cytosolic free calcium ([Ca2+]i) and respiratory burst activity, but not PMA-mediated NADPH-oxidase activation. While PMA reduced elevations in [Ca2+]i, it restored the burst response to agonists in Ca(2+)-depleted eosinophils. These results agree with the agonist-induced activation of the NADPH-oxidase via PKC, but suggest a parallel, Ca(2+)-, phospholipase C- and PKC-independent signal transduction pathway. Data obtained with B. pertussis toxin showed that the respiratory burst in eosinophils is blocked by ADP-ribosylation of G(i)-proteins, but that in the presence of PMA portions of the agonist response could be recovered.

Interleukin-5 mRNA levels in blood and bone marrow mononuclear cells from patients with the idiopathic hypereosinophilic syndrome.

High blood eosinophil counts in humans are usually due to parasitic infections, allergic processes or malignant diseases. Interleukin-5 (IL-5) is thought to be the principal eosinopoietic stimulus in most of these patients. As the causes of persistent eosinophilia in patients with the idiopathic hypereosinophilic syndrome (HES) are (by definition) unknown, a semi-quantitative assay for IL-5 mRNA in eosinophils and mononuclear cells was carried out using samples from 11 patients with HES. In three patients, unstimulated peripheral blood mononuclear cells (PBMC) and bone marrow mononuclear cells (BMMC) had markedly raised levels of IL-5 mRNA. In four of five patients studied, phorbol myristate acetate (PMA) stimulation of PBMC induced them to produce more IL-5 mRNA than four healthy subjects. In five patients, bone marrow IL-5 mRNA levels were related to blood eosinophil counts. Blood eosinophils from all six patients tested, and bone marrow granulocytes from four patients had undetectable levels of IL-5 mRNA. It was concluded that HES can be separated into two groups. One has high levels of IL-5 mRNA and/or an enhanced IL-5 mRNA response to stimulation. They may well respond to treatments which inhibit the effects of this cytokine on eosinophil progenitor cells. The second group appears to have a disease which is IL-5 independent.

Expression of lacto-N-fucopentaose III (CD15)- and sialyl-Lewis X-bearing molecules and their functional properties in eosinophils from patients with the idiopathic hypereosinophilic syndrome.

As the carbohydrate lacto-N-fucopentaose III (CD15 antigen or X-determinant) and its sialylated derivative sialyl-Lewis X are involved in the adhesion of cells rolling along the surface of endothelial cells, experiments were done to study the presence of these molecules on human eosinophils from patients with the idiopathic hypereosinophilic syndrome. Normal-density eosinophils from some patients showed higher levels of expression for lacto-N-fucopentaose III than light-density eosinophils. In contrast, sialyl-Lewis X was highly expressed by light-density eosinophils. Activation of normal-density eosinophils with calcium ionophore A23187 resulted in increased expression of these molecules for a short time. Monoclonal antibodies to these carbohydrates stimulated eosinophils to secrete eosinophil cationic protein, but not eosinophil peroxidase, and acted as costimulatory signals for C3b-induced degranulation of eosinophil cationic protein. It was suggested that CD15 and sialyl-Lewis X might contribute to eosinophil-mediated tissue injury in patients with the idiopathic hypereosinophilic syndrome.

Eosinophils 1992.

This report from a recent meeting* highlights some of the key developments in eosinophil research and assesses their impact on current views of the role of eosinophils in inflammation. Considerable progress has been made in defining the structure, constituents and properties of eosinophils in vitro and these new insights into eosinophil biology are summarized. A new era is beginning in which the roles of eosinophils can be assessed and defined in vivo.

Human immunodeficiency virus infection of eosinophils in human bone marrow cultures.

Normal human bone marrow, cultured in vitro with interleukin 5 to promote eosinophil production and maturation, was inoculated with cell-free isolates of human immunodeficiency virus type 1 (HIV-1). CD4 expression by eosinophil precursors, determined by immunocytochemistry, was found to be greatest early in their maturation with a rapid decline after 28 d in culture. Productive HIV infection of eosinophil precursors was detected 14 d after inoculation, by a combination of immunostaining for HIV-1 p24 and gp41/160 and in situ hybridization for viral RNA, together with assay of culture supernatants for p24 antigen and reverse transcriptase activity. Thus, eosinophils are susceptible to productive HIV-1 infection in vitro and may be an important reservoir for the virus in vivo.

Dilated cardiomyopathy and myocarditis: monoclonal antibodies to diseased heart tissues.

This study looked for the presence of abnormal contractile protein antigens and alterations in contractile protein expression in dilated cardiomyopathy (DCM). Monoclonal antibodies were raised to extracts from hearts removed at cardiac transplantation from two patients with dilated cardiomyopathy (DCM), and one with myocarditis. The specificities of the antibodies were assessed on cryostat sections from eight hearts with DCM. Although an extensive search was made for DCM-specific antibodies among over 1500 clones, none were found. However, a panel of antibodies was prepared and characterized, including antibodies to human adult myosin heavy chain beta, actin and troponin-I, which were selected for their value as reagents for immunocytochemical studies on cardiac, skeletal and smooth muscle. No significant alteration in the distribution of the epitopes recognized by these antibodies was found in DCM although more atrial myocytes in patients with DCM contained myosin adult heavy chain-beta. As a similar increase was found in atria from patients with other diseases who had a normal filling pressure, it was concluded that this alteration was unrelated to filling pressure, and was not specific for DCM. Further work with well defined monoclonal antibodies to other cardiac components in DCM could be useful in defining the alterations which lead to the functional defects in DCM and other cardiac diseases of unknown cause.

Effects of IL-5, granulocyte/macrophage colony-stimulating factor (GM-CSF) and IL-3 on the survival of human blood eosinophils in vitro.

The mechanisms that could affect the lifespan of eosinophils after they have left the bone marrow, and their capacity to respond to activation factors were studied by examining the effects of IL-5, GM-CSF and IL-3 on purified human blood eosinophils in culture. All three cytokines prolonged the lifespan of the majority of blood eosinophils. This effect was dose-dependent: IL-5 greater than GM-CSF greater than IL-3. Light density eosinophils from most patients had a longer lifespan in culture than did normal density eosinophils, with or without the three cytokines. Eosinophil death in the absence of these cytokines occurred by apoptosis. Eosinophils from two patients did not survive when cultured with IL-5, although they survived in the presence of IL-3 or GM-CSF. IL-5, GM-CSF and IL-3 induced the expression of the activation epitope on the eosinophil ribonucleases recognized by monoclonal antibody EG2. We conclude that small amounts of IL-5, GM-CSF and IL-3 prevented programmed cell death in human blood eosinophils and induced the expression of the activation forms of eosinophil ribonucleases. We suggest that differences in the capacity of normal and light density eosinophils to survive in culture, and in the ability of eosinophils from some patients to respond to IL-5 could account for variations in the severity of disease seen in patients with persistent eosinophilia.

Shape changes, exocytosis, and cytosolic free calcium changes in stimulated human eosinophils.

Essentially pure preparations of normal density eosinophils obtained from patients with hypereosinophilic syndrome (HES) were stimulated with complement factor 5a (C5a), platelet-activating factor (PAF), FMLP and neutrophil-activating peptide (NAP-1/IL-8). Three responses were studied, the transient rise in cytosolic free calcium concentration ([Ca2+]i) (derived from indo-1 fluorescence), shape changes (measured by laser turbidimetry), and exocytosis of eosinophil peroxidase (EPO) (assessed by H2O2/luminol-dependent chemiluminescence). Responses were obtained with all four agonists, but C5a and PAF were by far more potent than FMLP and NAP-1/IL-8, which induced only minor effects. Pretreatment of the cells with pertussis toxin attenuated [Ca2+]i changes, EPO release and, to a lesser extent, shape changes, indicating that GTP-binding proteins of Gi-type are involved in receptor-dependent signal transduction processes leading to these responses. A clear dissociation was observed in the control of the shape change response and EPO exocytosis. The shape change was not affected by Ca2+ depletion or treatment with the protein kinase inhibitor staurosporine, but exocytosis was prevented by Ca2+ depletion and markedly enhanced by staurosporine. The activation of the contractile system, leading to shape changes and motility, thus appears to be independent of the classical signal transduction pathway involving phospholipase C, a [Ca2+]i rise and protein kinase C activation. Exocytosis is, as expected, Ca2+ dependent and appears to be under a negative control involving protein phosphorylations.

The effects of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 on the secretory capacity of human blood eosinophils.

The effects of recombinant human GM-CSF and interleukin-3 (IL-3) on human blood eosinophil survival, activation, and secretion were studied. Purified normal density eosinophils from patients with the idiopathic hypereosinophilic syndrome (HES) survived in culture for 7 days (50% viable) in the presence of 50 nM GM-CSF or 50 nM IL-3. Neutrophils did not survive after 4 days. No eosinophils survived in the absence of GM-CSF or IL-3. In two out of five patients studied, the cultured eosinophils became elongated with numerous processes. In all five patients the cells became adherent, but there were no morphological signs of degranulation. Both GM-CSF and IL-3 activated eosinophils, transforming the storage form of eosinophil cationic protein (ECP) into the secreted form. The proportion of activated cells increased from less than 20% to over 50% after 4 days in culture. However, GM-CSF and IL-3 did not induce secretion on their own. On the other hand, when GM-CSF/IL-3-activated eosinophils were exposed to known secretory stimuli, there was a six-fold increase in the amount of ECP released when the cells were stimulated with sepharose coated with C3b, and a two-fold increase when they were stimulated with sepharose-activated whole autologous serum. Eosinophils from patients taking steroids were unable to secrete their granule contents, even though they became activated by GM-CSF and IL-3. A novel finding was that sepharose-activated whole serum was an extremely potent secretory signal for ECP, releasing up to 50% of the total ECP content. These studies showed that GM-CSF and IL-3 prime eosinophil effector function by initiating granule solubilization which is the first step in the secretory event, without affecting the subsequent extracellular release of granule proteins.

Aquagenic pruritus associated with the idiopathic hypereosinophilic syndrome.

Patients with the idiopathic hypereosinophilic syndrome (HES) may develop associated skin disorders. We describe a patient who had xerosis since birth, but who first developed symptoms of aquagenic pruritus soon after he presented with HES. Photochemotherapy with psoralen and UVA treatment reduced his peripheral blood eosinophil count. The good response to treatment suggests that there was a close relationship between the dermatosis and the blood disorder.

Activation of eosinophils in cancer patients treated with IL-2 and IL-2-generated lymphokine-activated killer cells.

Of 16 patients, a total of 13 who received IL-2 and autologous IL-2-generated lymphokine-activated killer LAK cells developed eosinophilia late during the course of treatment. To understand the direct or indirect effects of IL-2 on eosinophils, the physical and functional characteristics of the late-treatment eosinophils were compared to those of early-treatment and control eosinophils. Late-treatment eosinophils differed from early-treatment and control eosinophils in the following respects: they had somewhat reduced density, hypersegmented nuclei, eosinophil cationic protein converted from the storage form to the secretory form, and a greater than 200% increased ability to kill larvae of Schistosoma mansoni by an antibody-dependent mechanism (cytotoxic function). In vitro, IL-2 (1000 U/ml in medium as used to culture LAK cells) did not affect the cytotoxic function of eosinophils from cancer patients or from control subjects. However, LAK cell-conditioned medium enhanced the cytotoxic function of eosinophils from early-treatment cancer patients and from normal subjects by greater than 150%. Thus, eosinophils late in the course of IL-2/LAK cell treatment undergo physical changes and become functionally activated. The involvement of IL-2 in these changes is probably indirect, as an inducer of factors that enhance eosinophil function.

Active participation of eosinophils in patch test reactions to inhalant allergens in patients with atopic dermatitis.

Intracutaneous testing and patch tests with house dust mite and grass pollen allergens were performed in patients with atopic dermatitis. Only patients with an immediate type skin reaction to house dust mite or grass pollen allergens showed a positive patch test reaction to these allergens 24-48 h after testing. Occasionally positive patch test reactions at 20 min, 2 h and 6 h were also observed. Patch test reactions were not found in normal controls or atopic patients without atopic dermatitis. Analysis of the cellular infiltrate demonstrated an influx of eosinophils into the dermis, starting from 2-6 h after patch testing. Immunostaining with antibodies against granular constituents of the eosinophils revealed that the infiltrating eosinophils were in an activated state and had lost part of their granular contents. At 24 h eosinophils also appeared in the epidermis. Electron microscopy showed that in the epidermis, some eosinophils were in close contact with Langerhans cells, suggesting a cell-cell interaction. Taken together, these results strongly suggest an active role for eosinophils in patch test reactions to inhalant allergens in atopic dermatitis patients.

Lymphomatoid papulosis and its relationship to "idiopathic" hypereosinophilic syndrome.

Persistent hypereosinophilia, endomyocardial fibrosis, and a recurrent self-healing papulonodular eruption with the histologic features of lymphomatoid papulosis are described in three patients. One patient died after developing an acute myeloblastic transformation in the eosinophil series. Immunocytochemical studies of cutaneous lesions in two of the patients suggested a mature T-cell phenotype with a predominant population of CD4-positive cells. Immunostaining of cutaneous tissue with monoclonal antibodies BE1 and BE2 yielded negative findings. Because it is now known from in vitro studies that T lymphocytes secrete the eosinopoietic factor, interleukin 5, it is possible that the cutaneous lesions, hypereosinophilia, and associated endomyocardial fibrosis were induced by transformed helper T lymphocytes in these three patients.

Synergism between eosinophil cationic protein and oxygen metabolites in killing of schistosomula of Schistosoma mansoni.

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.

Killing of Plasmodium falciparum by eosinophil secretory products.

The multiplication of two strains of Plasmodium falciparum in culture, as measured by [3H]hypoxanthine incorporation, was inhibited in a dose-dependent manner by granule proteins secreted by purified eosinophils obtained from patients with the hypereosinophilic syndrome. Morphological examination revealed the presence of abnormal parasites inside erythrocytes, indicating that they were killed in situ, and the later stages of the developmental cycle were found to be most susceptible to these toxic effects. A monoclonal antibody against eosinophil cationic protein partially blocked the inhibitory effect, suggesting that it was caused by more than one of the eosinophil granule proteins. Thus some of the antimalarial effects of molecules such as the tumor necrosis factor, which activates eosinophils, may be mediated through the enhanced production of eosinophil secretion products.