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Eosinophils release their granule proteins extracellularly through exocytosis, piecemeal degranulation, or cytolytic degranulation. Findings in diverse human eosinophilic diseases of intact extracellular eosinophil granules, either free or clustered, indicate that eosinophil cytolysis occurs in vivo, but the mechanisms and consequences of lytic eosinophil degranulation are poorly understood. We demonstrate that activated human eosinophils can undergo extracellular DNA trap cell death (ETosis) that cytolytically releases free eosinophil granules. Eosinophil ETosis (EETosis), in response to immobilized immunoglobulins (IgG, IgA), cytokines with platelet activating factor, calcium ionophore, or phorbol myristate acetate, develops within 120 minutes in a reduced NADP (NADPH) oxidase-dependent manner. Initially, nuclear lobular formation is lost and some granules are released by budding off from the cell as plasma membrane-enveloped clusters. Following nuclear chromatolysis, plasma membrane lysis liberates DNA that forms weblike extracellular DNA nets and releases free intact granules. EETosis-released eosinophil granules, still retaining eosinophil cationic granule proteins, can be activated to secrete when stimulated with CC chemokine ligand 11 (eotaxin-1). Our results indicate that an active NADPH oxidase-dependent mechanism of cytolytic, nonapoptotic eosinophil death initiates nuclear chromatolysis that eventuates in the release of intact secretion-competent granules and the formation of extracellular DNA nets.

Eosinophil chemotaxis and survival within tissues are key components in the development of tissue eosinophilia and subsequent effector responses. In this study, we demonstrate a novel mechanism of eosinophil autoregulation affecting migration and survival mediated through Notch signaling. We show for the first time that human blood eosinophils express Notch receptors and Notch ligands, expressions of which are influenced by the presence of eosinophil-activating granulocyte-macrophage colony-stimulating factor (GM-CSF). Evidence of Notch receptor activation and subsequent transcription of the Notch-responsive gene HES1 were observed in GM-CSF–stimulated eosinophils, confirming functionality of eosinophil-expressed Notch-signaling components. Moreover, by inhibiting Notch signaling with γ-secretase inhibitors or Notch receptor–specific neutralizing antibodies, we demonstrate that autocrine Notch signaling enhances stimulus-mediated actin rearrangement and eosinophil chemokinesis, and impairs eosinophil viability. Taken together, these data suggest autocrine Notch signaling, enhanced in response to tissue- or inflammatory-derived signals, influences eosinophil activity and longevity, which may ultimately contribute to the development of tissue eosinophilia and exacerbation or remediation of eosinophil effector functions.

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

We used light and electron microscopy to analyze the eyelid inflammation that develops in transgenic mice that overexpress interleukin-4 (IL-4; Tepper et al, Cell 62:457, 1990). Analysis of alkaline Giemsa-stained plastic sections examined by light microscopy (Dvorak et al, J Exp Med 132:558, 1970), as well as by routine transmission electron microscopy, indicated that the mast cells in the inflammatory eyelid lesions were undergoing piecemeal degranulation, a form of secretion in which the cells' cytoplasmic granules exhibit characteristic morphologic changes that are thought to be associated with the prolonged, vesicle-mediated release of the granules' constituents. Moreover, by using a newly reported enzyme affinity-gold method, which stains histamine based on binding to diamine oxidase-gold (Dvorak et al, J Histochem Cytochem 41:787, 1993), we show that these activated mast cells had released much of their histamine content. The eyelid lesions also exhibited increased numbers of mast cells; interstitial fibrosis, particularly around cutaneous nerves and blood vessels; activated fibroblasts; focal axonal damage; venules with endothelial cells containing numerous vesiculo-vacuolar organelles; and infiltrates of neutrophils and eosinophils. Our findings illustrate that overexpression of the IL-4 gene in vivo can result in eyelid lesions associated with piecemeal degranulation of mast cells, as well as tissue fibrosis and a variety of other pathologic changes. These results also represent the first direct morphologic evidence for histamine secretion by mast cells in vivo.

We used light and electron microscopy to analyze the eyelid inflammation that develops in transgenic mice that overexpress interleukin-4 (IL-4; Tepper et al, Cell 62:457, 1990). Analysis of alkaline Giemsa-stained plastic sections examined by light microscopy (Dvorak et al, J Exp Med 132:558, 1970), as well as by routine transmission electron microscopy, indicated that the mast cells in the inflammatory eyelid lesions were undergoing piecemeal degranulation, a form of secretion in which the cells' cytoplasmic granules exhibit characteristic morphologic changes that are thought to be associated with the prolonged, vesicle-mediated release of the granules' constituents. Moreover, by using a newly reported enzyme affinity-gold method, which stains histamine based on binding to diamine oxidase-gold (Dvorak et al, J Histochem Cytochem 41:787, 1993), we show that these activated mast cells had released much of their histamine content. The eyelid lesions also exhibited increased numbers of mast cells; interstitial fibrosis, particularly around cutaneous nerves and blood vessels; activated fibroblasts; focal axonal damage; venules with endothelial cells containing numerous vesiculo-vacuolar organelles; and infiltrates of neutrophils and eosinophils. Our findings illustrate that overexpression of the IL-4 gene in vivo can result in eyelid lesions associated with piecemeal degranulation of mast cells, as well as tissue fibrosis and a variety of other pathologic changes. These results also represent the first direct morphologic evidence for histamine secretion by mast cells in vivo.

THE IDIOPATHIC hypereosinophilic syndrome (HES) is a leukoproliferative disorder, or more likely disorders, marked by a sustained overproduction of eosinophils. The distinctiveness of the syndrome, in addition to its eosino-philia, is its marked predilection to damage specific organs, including the heart. Such cardiac pathology is not unique to the idiopathic HES, because it may develop with eosinophilia associated with other diseases with identifiable etiologies. Conversely, yet enigmatically, not all patients with hypereo-sinophilia develop the organ damage characteristic of the HES. There are no specific tests diagnostic of the HES rather, the syndrome is defined by the combination of unexplained prolonged eosinophilia and evidence of organ involvement. We first review the more common and variable hematologic and clinical manifestations of this disorder because these are the features encountered clinically and that must be explained by investigation of the pathogenesis of HES. After considering the current understanding of the etiology and pathogenesis of the manifestations of HES, we review the therapies that may be used for HES.

The infiltration of eosinophils into tissues during pathologic responses is often associated with extracellular matrix alterations such as fibrosis. Transforming growth factor-beta 1 (TGF-beta 1) is a well-characterized multifunctional cytokine known to exert potent effects on the extracellular matrix. In this report, we showed the production of TGF-beta 1 by human eosinophils from patients with blood eosinophilia. Northern blot analysis using RNA isolated from eosinophils purified from a patient with the idiopathic hypereosinophilic syndrome (HES) detected the 2.5-kb TGF-beta 1 transcript. In situ hybridization and immunohistochemistry of leukocytes from two patients with HES and two patients with blood eosinophilia localized TGF-beta 1 messenger RNA (mRNA) and protein to eosinophils. No other cell type contained TGF-beta 1 mRNA by in situ hybridization, whereas other leukocytes contained detectable TGF-beta 1 protein by immunohistochemistry. Eosinophils from four normal donors contained little or no detectable TGF-beta 1 protein by immunohistochemistry, whereas eosinophils from two of these four normal donors labeled weakly for TGF-beta 1 mRNA by in situ hybridization. These results show that eosinophils in the peripheral blood of patients with blood eosinophilia can express TGF-beta 1, but that eosinophils in the blood of normal donors contained little or no TGF-beta 1.

Phosphatidylinositide 3-kinase (PI3K) is a key enzyme implicated in intracellular signaling of diverse cellular responses including receptor-mediated responses and neutrophil activation. Several PI3K subunits have been cloned and shown to be localized to plasma membrane receptors, the cytosol, or intracellular vesicles or caveolae. We report the localization of PI3K to a distinct intracellular site, cytoplasmic lipid bodies, in leukocytes. In U937 monocyte cells, PI3K p85 regulatory and p110β catalytic subunits were localized to lipid bodies by immunocytochemistry and/or immunoblotting and enzyme assays of subcellular fractions. In RAW murine macrophages, p55, p85, and p85β PI3K subunits were present at isolated lipid bodies. PI3K p85 was also shown to colocalize and, by co-immunoprecipitation, to be physically associated with phosphorylated Lyn kinase in lipid bodies induced to form in human polymorphonuclear leukocytes. These findings, therefore, indicate a novel site for PI3K compartmentalization and suggest that PI3K-mediated signaling is active within cytoplasmic lipid bodies in leukocytes.

The activity of hemolytically inactive C5b67, designated iC5b67, was evaluated as an agonist for functional responses of human polymorphonuclear leukocytes (PMN). C5b67 was formed from purified human complement components and decayed in phosphate-buffered saline (PBS) until it had no lytic activity for sheep erythrocytes in a standard assay. iC5b67, at nanomolar concentrations, stimulated PMN chemotaxis and Ca2+ fluxes, but inhibited superoxide production and failed to upregulate CR1 and CR3. There was no significant contamination of the iC5b67 with C5a to explain these results. Neither isolated C5b6 nor C7 alone exhibited the activities of iC5b67, while insolubilized anti-C7 could remove the PMN agonist activity from the iC5b67 preparation. Binding studies to define a specific receptor for iC5b67 on PMN were hampered by the very hydrophobic nature of the ligand. 125I-iC5b67, by contrast to hemolytically active 125I-C5b67, was unable to insert in erythrocytes, suggesting that iC5b67 need not insert in the PMN membrane to induce signaling. Two lines of evidence suggest that iC5b67 and C5a and FMLP share common steps in intracellular signaling (1) pretreatment of PMN with iC5b67 deactivates PMN for C5a- and FMLP-induced chemotaxis; and (2) pretreatment of PMN with pertussis toxin inhibits iC5b67-induced chemotaxis. Thus, iC5b67 has important effects on the activity of PMN and G-proteins and Ca2+ are involved in the signaling.

The infiltration of eosinophils into tissues during pathologic responses is often associated with extracellular matrix alterations such as fibrosis. Transforming growth factor-beta 1 (TGF-beta 1) is a well-characterized multifunctional cytokine known to exert potent effects on the extracellular matrix. In this report, we showed the production of TGF-beta 1 by human eosinophils from patients with blood eosinophilia. Northern blot analysis using RNA isolated from eosinophils purified from a patient with the idiopathic hypereosinophilic syndrome (HES) detected the 2.5-kb TGF-beta 1 transcript. In situ hybridization and immunohistochemistry of leukocytes from two patients with HES and two patients with blood eosinophilia localized TGF-beta 1 messenger RNA (mRNA) and protein to eosinophils. No other cell type contained TGF-beta 1 mRNA by in situ hybridization, whereas other leukocytes contained detectable TGF-beta 1 protein by immunohistochemistry. Eosinophils from four normal donors contained little or no detectable TGF-beta 1 protein by immunohistochemistry, whereas eosinophils from two of these four normal donors labeled weakly for TGF-beta 1 mRNA by in situ hybridization. These results show that eosinophils in the peripheral blood of patients with blood eosinophilia can express TGF-beta 1, but that eosinophils in the blood of normal donors contained little or no TGF-beta 1.

An international, multicenter, randomized, double-blind, placebo-controlled, parallel-group trial has evaluated the effects of mepolizumab, a humanized anti-interleukin-5 monoclonal antibody, on therapeutic prednisone dose requirements, eosinophil levels, signs and symptoms of disease in patients with hypereosinophilic syndrome (HES). The trial enrolled 85 patients (mean age 48.1 years) with HES (blood eosinophil count >1500/μl with evidence of eosinophil-related organ involvement or dysfunction and no known cause of eosinophilia), who tested negative for the FIP1L1-PDGFRα gene rearrangement and required 20–60 mg/day prednisone monotherapy to maintain blood eosinophils at Primary and secondary endpoints Endpoint Placebo (n=42) Mepolizumab (n=43) P-value (95% CI) Patients on ≤10 mg/day prednisone for ≥8 weeks (primary endpoint), % 43% 84% 30 mg/day prednisone at baseline, % 8% (n=12) 77% (n=13)

The presence of cytoplasmic lipid bodies in human eosinophils and the participation of these lipid bodies in the metabolism of arachidonic acid by human eosinophils have been studied. Lipid bodies, structures of roughly spherical shape and variable size within the cytoplasm, were identified with transmission electron microscopy by their shape and variable osmiophilia and by their lack of a limiting membrane. While generally absent from eosinophils of normal peripheral blood, lipid bodies were found in tissue eosinophils and in blood eosinophils from patients with eosinophilia. A role for lipid bodies in arachidonic acid metabolism was determined with eosinophils obtained from two eosinophilic patients. After incubation for 30 to 60 minutes with 3H- arachidonic acid, purified eosinophils took up 50% to 98% of the tritium label. By electron microscopic autoradiography, almost all tritium label was localized to lipid bodies. Only 3.6% of the cell- incorporated tritium label was free arachidonic acid, while 5.8% was neutral lipids and 66% was phospholipid. Thus, most of the tritiated arachidonic acid incorporated by human eosinophils was present in esterified form, predominantly as phospholipids, and almost all of the tritiated lipids were localized ultrastructurally to cytoplasmic lipid bodies. These results provide evidence that lipid bodies of human eosinophils have a role in the cellular metabolism of arachidonic acid.

The presence of cytoplasmic lipid bodies in human eosinophils and the participation of these lipid bodies in the metabolism of arachidonic acid by human eosinophils have been studied. Lipid bodies, structures of roughly spherical shape and variable size within the cytoplasm, were identified with transmission electron microscopy by their shape and variable osmiophilia and by their lack of a limiting membrane. While generally absent from eosinophils of normal peripheral blood, lipid bodies were found in tissue eosinophils and in blood eosinophils from patients with eosinophilia. A role for lipid bodies in arachidonic acid metabolism was determined with eosinophils obtained from two eosinophilic patients. After incubation for 30 to 60 minutes with 3H- arachidonic acid, purified eosinophils took up 50% to 98% of the tritium label. By electron microscopic autoradiography, almost all tritium label was localized to lipid bodies. Only 3.6% of the cell- incorporated tritium label was free arachidonic acid, while 5.8% was neutral lipids and 66% was phospholipid. Thus, most of the tritiated arachidonic acid incorporated by human eosinophils was present in esterified form, predominantly as phospholipids, and almost all of the tritiated lipids were localized ultrastructurally to cytoplasmic lipid bodies. These results provide evidence that lipid bodies of human eosinophils have a role in the cellular metabolism of arachidonic acid.

The Charcot-Leyden crystal (CLC) protein is a unique constituent of eosinophils and basophils. This protein forms the hexagonal bipyramidal crystals observed in tissues at sites of eosinophil accumulations, possesses lysophospholipase activity (lysolecithin acylhydrolase E.C.3.1.1.5), and comprises an estimated 7% to 10% of total eosinophil protein. The ultrastructural localization of CLC protein was studied in mature peripheral blood eosinophils from normal donors and from patients with the idiopathic hypereosinophilic syndrome. Subcellular localization was evaluated by immunoelectron microscopy using eosinophils, both from buffy coat and purified cell suspensions, that were fixed by a variety of methods. Immunochemical detection of CLC protein employed rabbit antiserum to eosinophil CLC protein, affinity chromatography-purified monospecific IgG antibodies, and postembedding immunogold techniques. Controls for specificity included (1) omission of the primary antibody to CLC protein and (2) substitution of primary antibody with a nonimmune preimmunization serum, a protein A-purified nonimmune IgG, or a protein A-purified nonreactive IgG prepared from solid-phase CLC protein-Sepharose-absorbed anti-CLC antiserum. CLC protein was localized to a minor (approximately 5%) subpopulation of eosinophil granules. These membrane-bound cytoplasmic granules were large (greater than 0.5 mu), were devoid of crystalloid inclusions, and were morphologically compatible with persisting eosinophil primary granules. The crystalloid-containing, large, specific granules did not stain for CLC protein. Insufficient numbers of small dense granules, lipid bodies, and vesiculotubular structures were present to adequately evaluate their potential as additional sites for the subcellular localization of CLC protein. The cellular specificity of the immunogold localization of CLC protein in the eosinophil was affirmed by the absence of staining in neutrophils and lymphocytes present in the same sections. The ultrastructural immunogold localization of CLC protein (lysophospholipase) to a large, crystalloid-free granule in mature circulating eosinophils supports the persistence of a distinct “primary” granule population that serves as a major intracytoplasmic repository for this enzyme.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are abnormally sensitive to complement. Two membrane proteins, the C8 binding protein (C8bp) and the decay accelerating factor (DAF), which are expressed on normal cells, function to restrict lysis by homologous complement, and both of these proteins are absent from PNH erythrocytes. DAF is anchored to the plasma membrane on normal cells by a phosphatidylinositol linkage. The investigators found that a purified phosphatidylinositol-specific phospholipase C cleaved C8bp from the surface of normal lymphocytes and monocytes. This finding indicates that the abnormal complement sensitivity of PNH erythrocytes arises from a common defect, the inability to attach the phosphatidylinositol- containing anchor that is necessary for the membrane expression of both membrane complement regulatory proteins, the C8bp, and DAF.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) are abnormally sensitive to complement. Two membrane proteins, the C8 binding protein (C8bp) and the decay accelerating factor (DAF), which are expressed on normal cells, function to restrict lysis by homologous complement, and both of these proteins are absent from PNH erythrocytes. DAF is anchored to the plasma membrane on normal cells by a phosphatidylinositol linkage. The investigators found that a purified phosphatidylinositol-specific phospholipase C cleaved C8bp from the surface of normal lymphocytes and monocytes. This finding indicates that the abnormal complement sensitivity of PNH erythrocytes arises from a common defect, the inability to attach the phosphatidylinositol-containing anchor that is necessary for the membrane expression of both membrane complement regulatory proteins, the C8bp, and DAF.