Paige Lacy

Eosinophils: Biological Properties and Role in Health and Disease

Eosinophils are pleiotropic multifunctional leukocytes involved in initiation and propagation of diverse inflammatory responses, as well as modulators of innate and adaptive immunity. In this review, the biology of eosinophils is summarized, focusing on transcriptional regulation of eosinophil differentiation, characterization of the growing properties of eosinophil granule proteins, surface proteins and pleiotropic mediators, and molecular mechanisms of eosinophil degranulation. New views on the role of eosinophils in homeostatic function are examined, including developmental biology and innate and adaptive immunity (as well as their interaction with mast cells and T cells) and their proposed role in disease processes including infections, asthma, and gastrointestinal disorders. Finally, strategies for targeted therapeutic intervention in eosinophil‐mediated mucosal diseases are conceptualized.

Pathways for Cytokine Secretion

Cytokine secretion is a widely studied process, although little is known regarding the specific mechanisms that regulate cytokine release. Recent findings have shed light on some of the precise molecular pathways that regulate the packaging of newly synthesized cytokines from immune cells. These findings begin to elucidate pathways and mechanisms that underpin cytokine release in all cells. In this article, we review the highlights of some of these novel discoveries.

Anaplasma phagocytophilum Utilizes Multiple Host Evasion Mechanisms To Thwart NADPH Oxidase-Mediated Killing during Neutrophil Infection

ABSTRACT Anaplasma phagocytophilum, the etiologic agent of human anaplasmosis, is a bacterial pathogen that specifically colonizes neutrophils. Neutrophils utilize the NADPH oxidase complex to generate superoxide (O2−) and initiate oxidative killing of microorganisms. A. phagocytophilums unique tropism for neutrophils, however, indicates that it subverts and/or avoids oxidative killing. We therefore examined the effects of A. phagocytophilum infection on neutrophil NADPH oxidase assembly and reactive oxygen species (ROS) production. Following neutrophil binding, Anaplasma invasion requires at least 240 min. During its prolonged association with the neutrophil plasma membrane, A. phagocytophilum stimulates NADPH oxidase assembly, as indicated by increased cytochrome b558 mobilization to the membrane, as well as colocalization of Rac and p22phox. This initial stimulation taxes the host neutrophils finite oxidase reserves, as demonstrated by time- and bacterial-dose-dependent decreases in secondary activation by N-formyl-methionyl-leucyl-phenylalanine (FMLP) or phorbol myristate acetate (PMA). This stimulation is modest, however, and does not diminish oxidase stores to nearly the extent that Escherichia coli, serum-opsonized zymosan, FMLP, or PMA do. Despite the apparent activation of NADPH oxidase, no change in ROS-dependent chemiluminescence is observed upon the addition of A. phagocytophilum to neutrophils, indicating that the bacterium may scavenge exogenous O2−. Indeed, A. phagocytophilum rapidly detoxifies O2− in a cell-free system. Once internalized, the bacterium resides within a protective vacuole that excludes p22phox and gp91phox. Thus, A. phagocytophilum employs at least two strategies to protect itself from neutrophil NADPH oxidase-mediated killing.

The influence of infections on the development and severity of allergic disorders

The frequency and severity of atopic disorders are steadily increasing, particularly in developing countries. The reason for this observation is not clear. Recent studies indicate that infections with viruses and especially with bacteria early in life may help to inhibit allergic Th2 responses by skewing the immune system towards Th1 responses. However, infections can also lead to the exacerbation of atopic disorders.

Mechanisms of Degranulation in Neutrophils

Neutrophils are critical inflammatory cells that cause tissue damage in a range of diseases and disorders. Being bone marrow-derived white blood cells, they migrate from the bloodstream to sites of tissue inflammation in response to chemotactic signals and induce inflammation by undergoing receptor-mediated respiratory burst and degranulation. Degranulation from neutrophils has been implicated as a major causative factor in pulmonary disorders, including severe asphyxic episodes of asthma. However, the mechanisms that control neutrophil degranulation are not well understood. Recent observations indicate that granule release from neutrophils depends on activation of intracellular signalling pathways, including β-arrestins, the Rho guanosine triphosphatase Rac2, soluble NSF attachment protein (SNAP) receptors, the src family of tyrosine kinases, and the tyrosine phosphatase MEG2. Some of these observations suggest that degranulation from neutrophils is selective and depends on nonredundant signalling pathways. This review focuses on new findings from the literature on the mechanisms that control the release of granule-derived mediators from neutrophils.

Human eosinophils express and release IL-13 following CD28-dependent activation

Human eosinophils produce a large number of cytokines, including immunoregulatory cytokines. Given that eosinophils store and release interleukin (IL)‐4, a key cytokine in the pathogenesis of allergic inflammation, and that IL‐4 and IL‐13 share common biological functions, we investigated the possibility that IL‐13 may be synthesized by these cells. Using flow cytometry and immunocytochemistry, we show that eosinophils synthesize and store IL‐13. Granule localization was demonstrated after subcellular fractionation, and IL‐13 immunoreactivity was localized to crystalloid, granule‐enriched fractions. Furthermore, electron microscopic analyses specifically localized IL‐13 to the dense cores of bicompartmental secondary granules. Upon CD28 ligation, IL‐13 was released by eosinophils, whereas a combination of CD28 and immunoglobulin A complexes resulted in decreased IL‐13 secretion. Furthermore, eosinophil‐derived IL‐13 exerts a biological effect, inducing CD23 expression on B cells. By having the capacity to synthesize and release IL‐13, eosinophils may participate in the development and maintenance of the T helper cell type 2 response, a prominent feature of allergic diseases.

A critical role for vesicle‐associated membrane protein‐7 in exocytosis from human eosinophils and neutrophils

Background:  Granulocyte exocytosis is proposed to be critically dependent on the interaction of soluble N‐ethylmaleimide‐sensitive factor attachment protein (SNAP) receptors (SNAREs) located on granules/vesicles (v‐SNAREs) and plasma membrane (t‐SNAREs). Previous studies indicated that the v‐SNARE, vesicle‐associated membrane protein (VAMP)‐2, as well as t‐SNAREs (SNAP‐23, syntaxin‐4 and ‐6) are implicated in exocytosis from human granulocytes. Vesicle‐associated membrane proteins‐7 and ‐8 have been implicated in endosome/lysosome trafficking, however, their role in granulocyte exocytosis remains obscure.

Primary granule exocytosis in human neutrophils is regulated by Rac-dependent actin remodeling

The actin cytoskeleton regulates exocytosis in all secretory cells. In neutrophils, Rac2 GTPase has been shown to control primary (azurophilic) granule exocytosis. In this report, we propose that Rac2 is required for actin cytoskeletal remodeling to promote primary granule exocytosis. Treatment of neutrophils with low doses (< or = 10 microM) of the actin-depolymerizing drugs latrunculin B (Lat B) or cytochalasin B (CB) enhanced both formyl peptide receptor- and Ca(2+) ionophore-stimulated exocytosis. Higher concentrations of CB or Lat B, or stabilization of F-actin with jasplakinolide (JP), inhibited primary granule exocytosis measured as myeloperoxidase release but did not affect secondary granule exocytosis determined by lactoferrin release. These results suggest an obligatory role for F-actin disassembly before primary granule exocytosis. However, lysates from secretagogue-stimulated neutrophils showed enhanced actin polymerization activity in vitro. Microscopic analysis showed that resting neutrophils contain significant cortical F-actin, which was redistributed to sites of primary granule translocation when stimulated. Exocytosis and actin remodeling was highly polarized when cells were primed with CB; however, polarization was reduced by Lat B preincubation, and both polarization and exocytosis were blocked when F-actin was stabilized with JP. Treatment of cells with the small molecule Rac inhibitor NSC23766 also inhibited actin remodeling and primary granule exocytosis induced by Lat B/fMLF or CB/fMLF, but not by Ca(2+) ionophore. Therefore, we propose a role for F-actin depolymerization at the cell cortex coupled with Rac-dependent F-actin polymerization in the cell cytoplasm to promote primary granule exocytosis.

Divergence of mechanisms regulating respiratory burst in blood and sputum eosinophils and neutrophils from atopic subjects.

Eosinophil respiratory burst is an important event in asthma and related inflammatory disorders. However, little is known concerning activation of the respiratory burst NADPH oxidase in human eosinophils. Conversely, neutrophils are known to assemble NADPH oxidase in intracellular and plasma membranes. We hypothesized that eosinophils and neutrophils translocate NADPH oxidase to distinct intracellular locations, consistent with their respective functions in O2−-mediated cytotoxicity. PMA-induced O2– release assayed by cytochrome c was 3.4-fold higher in atopic human eosinophils than in neutrophils, although membrane-permeable dihydrorhodamine-123 showed similar amounts of release. Eosinophil O2– release was dependent on Rac, in that it was 54% inhibited by Clostridium difficile toxin B (400–800 ng/ml). In eosinophils stimulated with PMA, a pronounced shift of cytosolic Rac to p22phox-positive plasma membrane was observed by confocal microscopy, whereas neutrophils directed Rac2 mainly to intracellular sites coexpressing p22phox. Similarly, ex vivo sputum eosinophils from asthmatic subjects exhibited predominantly plasma membrane-associated immunoreactivity for Rac, whereas sputum neutrophils exhibited cytoplasmic Rac2 staining. Thus, activated sputum eosinophils, rather than neutrophils, may contribute significantly to the pathogenesis of asthma by extracellular release of tissue-damaging O2–. Our findings suggest that the differential modes of NADPH oxidase assembly in these cells may have important implications for oxidant-mediated tissue injury.

Streptococcus pneumoniae and Staphylococcus aureus pneumonia induce distinct metabolic responses

Pneumonia is an infection of the lower respiratory tract caused by microbial pathogens. Two such pathogens, Streptococcus pneumoniae and Staphylococcus aureus, are the most common causes of community-acquired and hospital-acquired pneumonia respectively. Each expresses strains highly resistant to penicillin and other antibiotics, and a significant number of people succumb to infection by these pathogens every year. Urinary metabolite changes in a C57Bl/6 mouse model with lung infection from either S. pneumoniae or S. aureus were characterized using multivariate targeted profiling data obtained from (1)H NMR spectra. Marked changes in the urinary metabolite profile occurred within 24 h after infection with either pathogen. Specifically, significant decreases in TCA cycle intermediates, coupled with increases in fucose, creatine, and taurine were observed in the urine of S. pneumoniae-treated mice. Infection with S. aureus resulted in the decrease of a number of urinary metabolites including 1-methylnicotinamide, 3-methyl-2-oxovalerate, 2-oxoisocaproate, N-isovaleroylglycine and others. Disturbances in gut-derived microbial metabolites were also observed. Analysis of metabolic trajectory data indicated that, as the mice recovered from infection, their urinary metabolic profile became similar to that of the preinfected state. These results underline the potential of metabolomics as a tool for diagnosis, health monitoring, and drug development, and show its usefulness for understanding microbial-host interactions.