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Background SNARE members mediate membrane fusion during intracellular trafficking underlying innate and adaptive immune responses by different cells. However, little is known about the expression and function of these proteins in human eosinophils, cells involved in allergic, inflammatory and immunoregulatory responses. Here, we investigate the expression and distribution of the Qa-SNARE syntaxin17 (STX17) within human eosinophils isolated from the peripheral blood. Methods Flow cytometry and a pre-embedding immunonanogold electron microscopy (EM) technique that combines optimal epitope preservation and secondary Fab-fragments of antibodies linked to 1.4 nm gold particles for optimal access to microdomains, were used to investigate STX17. Results STX17 was detected within unstimulated eosinophils. Immunogold EM revealed STX17 on secretory granules and on granule-derived vesiculotubular transport carriers (Eosinophil Sombrero Vesicles-EoSVs). Quantitative EM analyses showed that 77.7% of the granules were positive for STX17 with a mean±SEM of 3.9±0.2 gold particles/granule. Labeling was present on both granule outer membranes and matrices while EoSVs showed clear membrane-associated labeling. STX17 was also present in secretory granules in eosinophils stimulated with the cytokine tumor necrosis factor alpha (TNF-α) or the CC-chemokine ligand 11 CCL11 (eotaxin-1), stimuli that induce eosinophil degranulation. The number of secretory granules labeled for STX17 was significantly higher in CCL11 compared with the unstimulated group. The level of cell labeling did not change when unstimulated cells were compared with TNF-α-stimulated eosinophils. Conclusions The present study clearly shows by immunanonogold EM that STX17 is localized in eosinophil secretory granules and transport vesicles and might be involved in the transport of granule-derived cargos.

BACKGROUND: Eosinophil-associated RNases (EARs) are stored preformed in eosinophil cytoplasmic secretory granules and have a key role in eosinophil effector functions in host defense and inflammatory disorders. However, the secretion mechanisms of EARs are poorly understood. OBJECTIVE: Our study aimed to understand the involvement of cytoskeleton machinery in EAR secretion. METHODS: Fresh human and mouse eosinophils were stimulated with CCL11 and the secretion of enzymatically active EARs was detected using an RNAse activity assay. The involvement of cytoskeletal elements or microtubules was probed using specific inhibitors. RESULTS: We found that dynamic polymerization of microtubules and cytoskeletal elements, such as Rho and Rac, are required for chemokine-mediated EAR secretion from human and mouse eosinophils. However, inhibition of ROCK (Rho-associated protein kinase) increased EARs secretion in human and mouse eosinophils even in the absence of chemokine stimulation, suggesting ROCK negatively regulates EARs secretion. CONCLUSIONS: Collectively, these data suggest a cytoskeleton-dependent mechanism of EARs secretion from eosinophils, findings that are pertinent to host defense, allergy and other eosinophil-associated diseases.

CD300a is an inhibitory receptor for mast cells and eosinophils in allergic inflammation (AI); however, the spatiotemporal expression of CD300a and its potential roles in the resolution of AI are still to be determined. In this study, employing a mouse model of allergic peritonitis, we demonstrate that CD300a expression on peritoneal cells is regulated from inflammation to resolution. Allergic peritonitis–induced CD300a−/− mice had a rapid increase in their inflammatory cell infiltrates and tryptase content in the peritoneal cavity compared with wild type, and their resolution process was significantly delayed. CD300a−/− mice expressed lower levels of ALX/FPR2 receptor on peritoneal cells and had higher levels of LXA4 in the peritoneal lavage. CD300a activation on mouse bone marrow–derived mast cells regulated ALX/FPR2 expression levels following IgE-mediated activation. Together, these findings indicate a role for CD300a in AI and its resolution, in part via the specialized proresolving mediator LXA4 and ALX/FPR2 receptor pathway activation.

Background Eosinophils have the capacity to secrete varied cytotoxic proteins. Among the proteins are the eosinophil-associated RNases (EARs): the human eosinophil-derived neurotoxin and eosinophilic cationic protein, and their murine ortholog EARs, which have been shown to be involved in host defense, tissue remodeling, and immunity regulation. However, the signal transduction that regulates EARs secretion in response to physiological stimuli, such as chemokines, has been little studied in human and scarcely in mouse eosinophils, the foremost animal model for eosinophil-associated human diseases. Objective In this study, we aimed to understand the signal transduction involved in the secretion of enzymatically active EARs following chemokine stimulation. Methods Fresh mouse and human eosinophils were stimulated with CCL11 and CCL24, and the secretion of enzymatically active EARs was detected using an RNase activity assay. The involvement of signaling factors or integrins was probed using specific inhibitors and blocking antibodies. Adhesion was evaluated by microscopy. Results We found that secretion of mouse EARs in response to CCL11 and CCL24 was Gαi-dependent. Both mouse and human eosinophils required the activation of PI3K, ERK, and p38 MAPK. In addition, the adhesion molecules β1 and β2 integrins were found to be crucial for EAR secretion, and we suggest a mechanism in which spreading is obligatory for EAR secretion. Conclusions Collectively, these data suggest a common CCR3-mediated signaling pathway that leads to EAR secretion in both mouse and human eosinophils. These findings are applicable for eosinophil-mediated host defense and eosinophil-associated diseases.

Eosinophils are innate immune leukocytes found in relatively low numbers within the blood. Terminal effector functions of eosinophils, deriving from their capacity to release their content of tissue-destructive cationic proteins, have historically been considered primary effector mechanisms against specific parasites, and are likewise implicated in tissue damage accompanying allergic responses such as asthma. However, the past decade has seen dramatic advancements in the field of eosinophil immunobiology, revealing eosinophils to also be key participants in many other facets of innate immunity, from bridging innate and adaptive immune responses to orchestrating tissue remodeling events. Here, we review the multifaceted functions of eosinophils in innate immunity that are currently known, and discuss new avenues in this evolving story.

It is widely believed that rolling lymphocytes require successive chemokine-induced signaling for lymphocyte function-associated antigen 1 (LFA-1) to achieve a threshold avidity that will mediate lymphocyte arrest. Using an in vivo model of lymphocyte arrest, we show here that LFA-1-mediated arrest of lymphocytes rolling on high endothelial venules bearing LFA-1 ligands and chemokines was abrupt. In vitro flow chamber models showed that endothelium-presented but not soluble chemokines triggered instantaneous extension of bent LFA-1 in the absence of LFA-1 ligand engagement. To support lymphocyte adhesion, this extended LFA-1 conformation required immediate activation by its ligand, intercellular adhesion molecule 1. These data show that chemokine-triggered lymphocyte adhesiveness involves a previously unrecognized extension step that primes LFA-1 for ligand binding and firm adhesion.

Lymphocyte transendothelial migration (TEM) is promoted by fluid shear signals and apical endothelial chemokines. Studying the role of these signals in neutrophil migration across differently activated HUVEC in a flow chamber apparatus, we gained new insights into how neutrophils integrate multiple endothelial signals to promote TEM. Neutrophils crossed highly activated HUVEC in a β2 integrin-dependent manner but independently of shear. In contrast, neutrophil migration across resting or moderately activated endothelium with low-level β2 integrin ligand activity was dramatically augmented by endothelial-presented chemoattractants, conditional to application of physiological shear stresses and intact β2 integrins. Shear stress signals were found to stimulate extensive neutrophil invaginations into the apical endothelial interface both before and during TEM. A subset of invaginating neutrophils completed transcellular diapedesis through individual endothelial cells within

Leukocyte arrest on vascular endothelium under disruptive shear flow is a multistep process that requires in situ integrin activation on the leukocyte surface by endothelium-displayed chemoattractants, primarily chemokines. A genetic deficiency of leukocyte adhesion to endothelium associated with defective β2 integrin expression or function (LAD-1) has been described. We now report a novel severe genetic disorder in this multistep process associated with functional defects in multiple leukocyte integrins, reflected in recurrent infections, profound leukocytosis, and a bleeding tendency. This syndrome is associated with an impaired ability of neutrophil and lymphocyte β1 and β2 integrins to generate high avidity to their endothelial ligands and arrest cells on vascular endothelium in response to endothelial chemoattractant signals. Patient leukocytes roll normally on endothelial selectins, express intact integrins and G protein–coupled chemokine receptors (GPCR), spread on integrin ligands, and migrate normally along a chemotactic gradient. Activation of β2 integrins in response to GPCR signals and intrinsic soluble ligand binding properties of the very late activation antigen-4 (VLA-4) integrin are also retained in patient leukocytes. Nevertheless, all integrins fail to generate firm adhesion to immobilized ligands in response to in situ GPCR-mediated activation by chemokines or chemoattractants, a result of a primary defect in integrin rearrangement at ligand-bearing contacts. This syndrome is the first example of a human integrin-activation deficiency associated with defective GPCR stimulation of integrin avidity at subsecond contacts, a key step in leukocyte arrest on vascular endothelium under shear flow.

VLA-4 and LFA-1 are the major vascular integrins expressed on circulating lymphocytes. Previous studies suggested that intact cholesterol rafts are required for integrin adhesiveness in different leukocytes. We found the alpha(4) integrins VLA-4 and alpha(4)beta(7) as well as the LFA-1 integrin to be excluded from rafts of human peripheral blood lymphocytes. Disruption of cholesterol rafts with the chelator methyl-beta-cyclodextrin did not affect the ability of these lymphocyte integrins to generate high avidity to their respective endothelial ligands and to promote lymphocyte rolling and arrest on inflamed endothelium under shear flow. In contrast, cholesterol extraction abrogated rapid chemokine triggering of alpha(4)-integrin-dependent peripheral blood lymphocytes adhesion, a process tightly regulated by G(i)-protein activation of G protein-coupled chemokine receptors (GPCR). Strikingly, stimulation of LFA-1 avidity to intercellular adhesion molecule 1 (ICAM-1) by the same chemokines, although G(i)-dependent, was insensitive to raft disruption. Our results suggest that alpha(4) but not LFA-1 integrin avidity stimulation by chemokines involves rapid chemokine-induced GPCR rearrangement that takes place at cholesterol raft platforms upstream to G(i) signaling. Our results provide the first evidence that a particular chemokine/GPCR pair can activate different integrins on the same cell using distinct G(i) protein-associated machineries segregated within defined membrane compartments.

Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.

The recruitment of circulating leukocytes at vascular sites in target tissue has been linked to activation of Gi-protein signaling in leukocytes by endothelial chemokines. The mechanisms by which apical and subendothelial chemokines regulate leukocyte adhesion to and migration across endothelial barriers have been elusive. We recently found that endothelial chemokines not only stimulate integrin-mediated arrest on vascular endothelial ligands but also trigger earlier very late antigen (VLA)-4 integrin-mediated capture (tethering) of lymphocytes to vascular cell adhesion molecule 1 (VCAM-1)-bearing surfaces by extremely rapid modulation of integrin clustering at adhesive contact zones. This rapid modulation of integrin avidity requires chemokine immobilization in juxtaposition with the VLA-4 ligand VCAM-1. We also observed that endothelial-bound chemokines promote massive lymphocyte transendothelial migration (TEM). It is interesting that chemokine-promoted lymphocyte TEM requires continuous exposure of lymphocytes but not of the endothelial barrier to fluid shear. It is noteworthy that lymphocyte stimulation by soluble chemokines did not promote lymphocyte TEM. Our results suggest new roles for apical endothelial chemokines both in triggering lymphocyte capture to the endothelial surface and in driving post-arrest events that promote lymphocyte transmigration across endothelial barriers under shear flow.

Highly purified eosinophils can be isolated from peripheral blood by negative selection using an antibody-based magnetic negative selection protocol. The basic protocol describes a sequential fractionation of peripheral blood in which CD16+ granulocytes are enriched first from whole blood, followed by isolation of eosinophils. This technique is easy to use, fast, and highly reproducible. Support protocols describe a staining methods that can be used to evaluate the purity of eosinophils and differentiation from other leukocyte populations.

Rapid secretion of eosinophil-associated RNases (EARs), such as the human eosinophilic cationic protein (ECP), from intracellular granules is central to the role of eosinophils in allergic diseases and host immunity. Our knowledge regarding allergic inflammation has advanced based on mouse experimental models. However, unlike human eosinophils, capacities of mouse eosinophils to secrete granule proteins have been controversial. To study mechanisms of mouse eosinophil secretion and EAR release, we combined an RNase assay of mouse EARs with ultrastructural studies. In vitro, mouse eosinophils stimulated with the chemokine eotaxin-1 (CCL11) secreted enzymatically active EARs (EC50 5 nM) by piecemeal degranulation. In vivo, in a mouse model of allergic airway inflammation, increased airway eosinophil infiltration (24-fold) correlated with secretion of active RNases (3-fold). Moreover, we found that eosinophilic inflammation in mice can involve eosinophil cytolysis and release of cell-free granules. Cell-free mouse eosinophil granules expressed functional CCR3 receptors and secreted their granule proteins, including EAR and eosinophil peroxidase in response to CCL11. Collectively, these data demonstrate chemokine-dependent secretion of EARs from both intact mouse eosinophils and their cell-free granules, findings pertinent to understanding the pathogenesis of eosinophil-associated diseases, in which EARs are key factors.—Shamri, R., Melo, R. C. N., Young, K. M., B.-B, M., Xenakis, J. J., Spencer, L. A., Weller, P. F. CCL11 elicits secretion of RNases from mouse eosinophils and their cell-free granules.

Chemokines presented on endothelial tissues instantaneously trigger LFA-1-mediated arrest on ICAM-1 via rapid inside-out and outside-in (ligand-driven) LFA-1 activation. The GTPase RhoA was previously implicated in CCL21-triggered LFA-1 affinity triggering in murine T lymphocytes and in LFA-1-dependent adhesion strengthening to ICAM-1 on Peyer’s patch high endothelial venules stabilized over periods of at least 10 s. In this study, we show that a specific RhoA 23/40 effector region is vital for the initial LFA-1-dependent adhesions of lymphocytes on high endothelial venules lasting 1–3 s. Blocking the RhoA 23/40 region in human T lymphocytes in vitro also impaired the subsecond CXCL12-triggered LFA-1-mediated T cell arrest on ICAM-1 by eliminating the rapid induction of an extended LFA-1 conformational state. However, the inflammatory chemokine CXCL9 triggered robust LFA-1-mediated T lymphocyte adhesion to ICAM-1 at subsecond contacts independently of the RhoA 23/40 region. CXCL9 did not induce conformational changes in the LFA-1 ectodomain, suggesting that particular chemokines can activate LFA-1 through outside-in post ligand binding stabilization changes. Like CXCL9, the potent diacylglycerol-dependent protein kinase C agonist PMA was found to trigger LFA-1 adhesiveness to ICAM-1 also without inducing integrin extension or an a priori clustering and independently of the RhoA 23/40 region. Our results collectively suggest that the 23/40 region of RhoA regulates chemokine-induced inside-out LFA-1 extension before ligand binding, but is not required for a variety of chemokine and non-chemokine signals that rapidly strengthen LFA-1-ICAM-1 bonds without an a priori induction of high-affinity extended LFA-1 conformations.

Recently, we reported a rare leukocyte adhesion deficiency (LAD) associated with severe defects in integrin activation by chemokine signals, despite normal ligand binding of leukocyte integrins.1 We now report that the small GTPase, Rap1, a key regulator of inside-out integrin activation is abnormally regulated in LAD Epstein-Barr virus (EBV) lymphocyte cells. Both constitutive and chemokine-triggered activation of Rap1 were abolished in LAD lymphocytes despite normal chemokine signaling. Nevertheless, Rap1 expression and activation by phorbol esters were intact, ruling out an LAD defect in Rap1 guanosine triphosphate (GTP) loading. The very late antigen 4 (VLA-4) integrin abnormally tethered LAD EBV lymphocytes to its ligand vascular cell adhesion molecule 1 (VCAM-1) under shear flow due to impaired generation of high-avidity contacts despite normal ligand binding and intact avidity to surface-bound anti-VLA-4 monoclonal antibody (mAb). Thus, a defect in constitutive Rap1 activation results in an inability of ligand-occupied integrins to generate high-avidity binding to ligand under shear flow. This is a first report of an inherited Rap1 activation defect associated with a pathologic disorder in leukocyte integrin function, we herein term it “LAD-III.” (Blood. 2004;103:1033-1036)

Leukocyte integrins must rapidly strengthen their binding to target endothelial sites to arrest rolling adhesions under physiological shear flow. We demonstrate that the integrin-associated tetraspanin, CD81, regulates VLA-4 and VLA-5 adhesion strengthening in monocytes and primary murine B cells. CD81 strengthens multivalent VLA-4 contacts within subsecond integrin occupancy without altering intrinsic adhesive properties to low density ligand. CD81 facilitates both VLA-4-mediated leukocyte rolling and arrest on VCAM-1 under shear flow as well as VLA-5-dependent adhesion to fibronectin during short stationary contacts. CD81 also augments VLA-4 avidity enhancement induced by either chemokine-stimulated Gi proteins or by protein kinase C activation, although it is not required for Gi protein or protein kinase C signaling activities. In contrast to other proadhesive integrin-associated proteins, CD81-promoted integrin adhesiveness does not require its own ligand occupancy or ligation. These results provide the first demonstration of an integrin-associated transmembranal protein that facilitates instantaneous multivalent integrin occupancy events that promote leukocyte adhesion to an endothelial ligand under shear flow.

Eosinophil-associated RNases (EARs); the human eosinophil-derived neurotoxin and eosinophilic cationic protein and their murine orthologs, are stored in eosinophil cytoplasmic granules and secreted upon inflammation as part of innate immune responses. In eosinophil-associated diseases, such as hyper eosinophilic syndrome, allergy and asthma, EARs play crucial roles in disease pathology and can be used as biomarkers for disease severity. However, secretion mechanisms of EARs are not fully understood, especially in mouse eosinophils, the major animal model for eosinophil-associated diseases. Our study aimed to understand the mechanisms of eosinophil EAR secretion following chemokine stimulation. Both ERK and p38 MAPK activation were found to increase in mouse eosinophils in response to CCL11. CCR3-mediated EAR secretion was blocked by p38 inhibitor (SB202190), confirming previous data in human eosinophils. However, a specific inhibitor for ERK, a MEK1-derived peptide, did not inhibit CCR3-mediated secretion or migration. These results are in contrast to previous studies in human eosinophils and our studies in mouse eosinophils, showing ERK is crucial for CCR3-mediated EARs secretion and migration using PD98059, a less specific inhibitor of the ERK-activating enzyme, MEK. Collectively, these data suggest a signaling pathway for CCR3-mediated EAR secretion that includes: PI3K and p38 but not ERK. These findings are pertinent to host defense and eosinophil-associated diseases.

Eosinophils, both as end-stage effector cells and as collaborative interacting components of innate immune responses, have the capacity to secrete varied cytotoxic proteins that are stored in their cytoplasmic secretory granules, termed specific granules. Among them are the eosinophil-associated RNases (EARs): the human eosinophil-derived neurotoxin and eosinophilic cationic protein. The mouse EAR family includes at least 13 members, six of which were found to be present in specific granules. In the last decade several studies, both in vitro and in vivo, have revealed a role for eosinophils and their EARs in anti-viral defense. However, the secretion mechanism of EARs is not fully understood, especially in mouse eosinophils. Therefore, our study aimed to understand the mechanism of EAR secretion following physiological stimulation with chemokines. We found that dynamic polymerization of the actin cytoskeleton and microtubules, as well as functional integrins were critical for CCR3-mediated secretion of EARs. However, inhibition of regulatory components of the cytoskeletal machinery, such as Rac1 and ROCK-I and -II, increased EARs secretion in mouse and human eosinophils, suggesting a negative regulation of these effectors on EARs secretion. Collectively, these data suggest a mechanism by which EARs are secreted from eosinophils under physiological stimulation, findings that are pertinent to host defense against RNA viruses and eosinophil-associated diseases.

Piecemeal degranulation (PMD), in which intact eosinophils completely or partially empty granules and selectively secrete performed mediators, is the principal mechanism for human eosinophil degranulation. Unlike extensively studied human eosinophils, both the capacity and mechanisms underlying eosinophil degranulation responses of murine eosinophils have been poorly investigated and remain controversial. We applied a new methodology to detect degranulation by measuring secretion of eosinophil-associated RNases (mEARs) and found that murine eosinophils “degranulated” following stimulation with CCL11 and CCL24, as detected by an increase in RNase A and EPO activities in cell supernatants. TEM revealed signs of PMD including significant increase in the number of emptying granules. CCL11 and CCL24-mediated mouse eosinophil degranulation was Gαi and PI3K-dependent. Moreover, intact beta 1 and 2 integrins as well as dynamic cytoskeleton are required for this PMD. Unexpectedly, we found that inhibition of Rho-GTPase kinase increases PMD. Moreover, cell-free granules purified from mouse eosinophils were found to express functional CCR3 and secreted mEARs as well as EPO in response to CCL11, suggesting a unique mechanism of degranulation in cell-free context. Collectively, these data demonstrate that murine eosinophils undergo PMD in response to physiological stimulation with CCL11 and CCL24, findings that are pertinent to murine models of eosinophil-associated diseases.