Person:

Weller, Peter

Eosinophils, leukocytes involved in allergic, inflammatory and immunoregulatory responses, have a distinct capacity to rapidly secrete preformed granule-stored proteins through piecemeal degranulation (PMD), a secretion process based on vesicular transport of proteins from within granules for extracellular release. Eosinophil-specific granules contain cytokines and cationic proteins, such as major basic protein (MBP). We evaluated structural mechanisms responsible for mobilizing proteins from within eosinophil granules. Human eosinophils stimulated for 30–60 min with eotaxin, regulated on activation, normal, T-cell expressed and secreted (RANTES) or platelet activating factor exhibited ultrastructural features of PMD (e.g. losses of granule contents) and extensive vesiculotubular networks within emptying granules. Brefeldin A inhibited granule emptying and collapsed intragranular vesiculotubular networks. By immunonanogold ultrastructural labelings, CD63, a tetraspanin membrane protein, was localized within granules and on vesicles outside of granules, and mobilization of MBP into vesicles within and extending from granules was demonstrated. Electron tomography with three dimension reconstructions revealed granule internal membranes to constitute an elaborate tubular network able to sequester and relocate granule products upon stimulation. We provide new insights into PMD and identify eosinophil specific granules as organelles whose internal tubulovesicular networks are important for the capacity of eosinophils to secrete, by vesicular transport, their content of preformed and granule-stored cytokines and cationic proteins.

Eosinophils are implicated prominently in allergic diseases and the host response to parasitic infections. Eosinophils may be activated in vitro by diverse classes of agonists such as immunoglobulins, lipid mediators, and cytokines. The leukocyte Ig-like receptors (LIRs) comprise a family of inhibitory and activating cell-surface receptors. Inhibitory LIRs down-regulate cellular responses through cytoplasmic immunoreceptor tyrosine-based inhibitory motifs. There are limited data on the action of the activating LIRs, which are thought to signal through the Fc receptor γ chain, which contains an immunoreceptor tyrosine-based activation motif. We now demonstrate the expression of LIR1 (inhibitory), LIR2 (inhibitory), LIR3 (inhibitory), and LIR7 (activating) on eosinophils from 4, 4, 12, and 11, respectively, of 12 healthy donors. Cross-linking of LIR7 with plate-bound antibody elicited the dose- and time-dependent release of eosinophil-derived neurotoxin and leukotriene C4. Eosinophils activated with antibodies to LIR7 embedded in gel-phase EliCell preparations showed leukotriene C4 generation at the nuclear envelope and the release of IL-12 but not IL-4 by vesicular transport. Thus, LIR7 is an activating receptor for eosinophils that elicited the release of cytotoxic granule proteins, de novo lipid mediator generation, and cytokine release through vesicular transport.

Eosinophils generate and store a battery of proteins, including classical cationic proteins, cytokines, chemokines, and growth factors. Rapid secretion of these active mediators by eosinophils is central to a range of inflammatory and immunoregulatory responses. Eosinophil products are packaged within a dominant population of cytoplasmic specific granules and generally secreted by piecemeal degranulation, a process mediated by transport vesicles. Large, pleiomorphic vesiculotubular carriers were identified recently as key players for moving eosinophil proteins from granules to the plasma membrane for extracellular release. During secretion, these specialized, morphologically distinct carriers, termed eosinophil sombrero vesicles, are actively formed and direct differential and rapid release of eosinophil proteins. This review highlights recent discoveries concerning the organization of the human eosinophil secretory pathway. These discoveries are defining a broader role for large vesiculotubular carriers in the intracellular trafficking and secretion of proteins, including selective receptor-mediated mobilization and transport of cytokines.

A distinguishing feature of eosinophils is their ability to rapidly release preformed cytokines from intracellular pools. Cytokines are delivered to the cell surface from granule stores by transport vesicles and are released in small packets at discrete locations along the cell surface through a process termed “piecemeal” degranulation. The study of this process has been hindered by lack of an assay sensitive enough to register minute protein concentrations and the inability to visualize morphology of cytokine secreting cells. These hindrances have necessitated our development of the EliCell assay, an agarose-based dual cytokine capture and detection system through which cytokine secretion and cellular morphology may be analyzed in concert. Cells are embedded within capture antibody-containing agarose and stimulated under conditions of interest. Extracellularly released cytokine is captured within the matrix at the point of release from the cell and can be labeled with a fluorochrome-conjugated antibody. Cytokine release and cellular morphology are visualized in parallel by phase contrast and fluorescence microscopy, respectively.

Infection with Fasciola hepatica, a liver trematode, is not frequently reported in the United States. We describe 2 patients, both originally from Cape Verde, who illustrate the spectrum of clinical presentations of F. hepatica as well as the means of treating infection with this parasite. Patient 1 had extensive disease and underwent multiple diagnostic procedures before the correct diagnosis was reached. Patient 2, who had few symptoms, had fascioliasis diagnosed by a noninvasive evaluation. Both patients were treated with triclabendazole without experiencing significant side effects. Fascioliasis that has been imported to the United States may elude prompt or accurate diagnosis. Obtaining a detailed travel history and recognizing the clinical presentation early in the course of infection may permit timely and noninvasive identification of infection. Triclabendazole is now the recommended drug for treating for fascioliasis because of its efficacy, safety, and ease of use.

Background:

Hypereosinophilic syndrome (HES) is a heterogeneous group of rare disorders defined by persistent blood eosinophilia ≥1.5 × 109/L, absence of a secondary cause, and evidence of eosinophil-associated pathology. With the exception of a recent multicenter trial of mepolizumab (anti–IL-5 mAb), published therapeutic experience has been restricted to case reports and small case series.

Objective:

The purpose of the study was to collect and summarize baseline demographic, clinical, and laboratory characteristics in a large, diverse cohort of patients with HES and to review responses to treatment with conventional and novel therapies.

Methods:

Clinical and laboratory data from 188 patients with HES, seen between January 2001 and December 2006 at 11 institutions in the United States and Europe, were collected retrospectively by chart review.

Results:

Eighteen of 161 patients (11%) tested were Fip1-like 1–platelet-derived growth factor receptor α (FIP1L1-PDGFRA) mutation—positive, and 29 of 168 patients tested (17%) had a demonstrable aberrant or clonal T-cell population. Corticosteroid monotherapy induced complete or partial responses at 1 month in 85% (120/141) of patients with most remaining on maintenance doses (median, 10 mg prednisone equivalent daily for 2 months to 20 years). Hydroxyurea and IFN-α (used in 64 and 46 patients, respectively) were also effective, but their use was limited by toxicity. Imatinib (used in 68 patients) was more effective in patients with the FIP1L1-PDGFRA mutation (88%) than in those without (23%; P < .001).

Conclusion:

This study, the largest clinical analysis of patients with HES to date, not only provides useful information for clinicians but also should stimulate prospective trials to optimize treatment of HES.

Although leukocytes of the innate immune system, including eosinophils, contain within their granules preformed stores of cytokines available for selective and rapid release, little is known about the mechanisms governing the mobilization and secretion of these cytokines. Here we show that a cytokine receptor, the IL-4 receptor α chain, mediates eotaxin-stimulated mobilization of preformed IL-4 from eosinophil granules into secretory vesicles. Eosinophils contain substantial intracellular quantities of several granule- and vesicle-associated cytokine receptors, including IL-4, IL-6, and IL-13 receptors as well as CCR3. Both IL-4 and IL-4 receptor α chain colocalized in eosinophil granules; and after eotaxin-stimulation, IL-4 receptor α chain, bearing bound IL-4, was mobilized into secretory vesicles. These findings indicate that intracellular cytokine receptors within secretory vesicles transport their cognate cytokines requisite for the secretion of cytokines preformed in innate immune leukocytes.

Purpose of review — Hypereosinophilic syndromes (HESs) are disorders characterized by sustained blood or tissue hypereosinophilia or both with subsequent damage to various organs due to eosinophilic infiltration and release of mediators. HES are now recognized to include varied eosinophilic disorders for some of which there are recent insights into their pathogenesis and targeted treatment. Recent findings—Studies have helped delineate two subtypes of HES: the myeloproliferative variants of HES and the lymphocytic variants of HES. Many, but not all, myeloproliferative-HES patients have interstitial deletions on chromosome 4q12 that lead to fusion of the FIP1-like 1 and platelet-derived growth factor receptor α genes, with the fusion product encoding a protein that has constitutive tyrosine kinase activity. Lymphocytic-HES is a primary lymphoid disorder characterized by nonmalignant expansion of a T-cell population able to produce eosinophilopoietic cytokines, with the T-cell population being identified by flow cytometry or reverse transcriptase-PCR for T-cell receptor usage or both. Other HES subtypes are of uncertain causes and are included in recent diagnostic algorithms for the spectrum of HES. Summary—The contemporary definition of the hypereosinophilic syndromes encompasses a range of eosinophilic disorders characterized by chronic blood hypereosinophilia often with eosinophil-mediated damage to various organs.

Human eosinophils contain within their cytoplasmic granules multiple preformed proteins, including over three dozen cytokines with nominal Th1, Th2 and immunoregulatory capabilities, and four distinctive cationic proteins. The secretion of these granule-derived proteins within eosinophils occurs principally by a mechanism whereby selected proteins are mobilized into vesicles for transport to and release at the cell surface. In contrast, the enigmatic presence of membrane-bound cell-free granules extruded from eosinophils has been long recognized in tissues associated with eosinophilia, including allergic diseases and responses to helminths. Functional capabilities for extracellular granules have recently been demonstrated. Eosinophil granules express cytokine receptors on their membranes and function, upon extrusion from eosinophils, as independent secretory organelles releasing granule constituents in response to activating cytokines and chemokines. We provide an update on the processes that mediate selective protein secretion from within eosinophil granules both as intracellular organelles and, as novelly demonstrated, as cell-free extracellular structures.

Lipid body accumulation within leukocytes is a common feature in both clinical and experimental infectious, neoplasic and other inflammatory conditions. Here, we will review the contemporary evidence related to the biogenesis and structure of leukocyte lipid bodies (also known as lipid droplets) as inflammatory organelles. Studies of leukocyte lipid bodies are providing functional, ultrastructural and protein compositional evidences that lipid bodies are not solely storage depots of neutral lipid. Over the past years substantial progresses have been made to demonstrate that lipid body biogenesis is a highly regulated process, that culminate in the compartmentalization of a specific set of proteins and lipids, that place leukocyte lipid bodies as inducible cytoplasmic organelles with roles in cell signaling and activation, regulation of lipid metabolism, membrane trafficking and control of the synthesis and secretion of inflammatory mediators. Pertinent to the roles of lipid bodies in inflammation and cell signaling, enzymes involved in eicosanoid synthesis are localized at lipid bodies and lipid bodies are sites for eicosanoid generation. Collectively, lipid bodies in leukocytes are emerging as critical regulators of different inflammatory diseases, key markers of leukocyte activation and attractive targets for novel anti-inflammatory therapies.