Mechanism of eosinophil induced signaling in cholinergic IMR-32 cells

Eosinophils interact with nerve cells, leading to changes in neurotransmitter release, altered nerve growth, and protection from cytokine-induced apoptosis. In part, these interactions occur as a result of activation of neural nuclear factor (NF)-kappaB, which is activated by adhesion of eosinophils to neural intercellular adhesion molecule-1 (ICAM-1). The mechanism and consequence of signaling after eosinophil adhesion to nerve cells were investigated. Eosinophil membranes, which contain eosinophil adhesion molecules but not other eosinophil products, were coincubated with IMR-32 cholinergic nerve cells. The studies showed that there were two mechanisms of activation of NF-kappaB, one of which was dependent on reactive oxygen species, since it was inhibited with diphenyleneiodonium. This occurred at least 30 min after coculture of eosinophils and nerves. An earlier phase of NF-kappaB activation occurred within 2 min of eosinophil adhesion and was mediated by tyrosine kinase-dependent phosphorylation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Coimmunoprecipitation experiments showed that both extracellular signal-regulated kinase 1/2 and IRAK-1 were recruited to ICAM-1 rapidly after coculture with eosinophil membranes. This was accompanied by an induction of ICAM-1, which was mediated by an IRAK-1-dependent pathway. These data indicate that adhesion of eosinophils to IMR-32 nerves via ICAM-1 leads to important signaling events, mediated via IRAK-1, and these in turn lead to expression of adhesion molecules.     

Eosinophil adhesion to cholinergic nerves via ICAM-1 and VCAM-1 and associated eosinophil degranulation

In vivo, eosinophils localize to airway cholinergic nerves in antigen-challenged animals, and inhibition of this localization prevents antigen-induced hyperreactivity. In this study, the mechanism of eosinophil localization to nerves was investigated by examining adhesion molecule expression by cholinergic nerves. Immunohistochemical and functional studies demonstrated that primary cultures of parasympathetic nerves express vascular cell adhesion molecule-1 (VCAM-1) and after cytokine pretreatment with tumor necrosis factor-alpha and interferon-gamma intercellular adhesion molecule-1 (ICAM-1). Eosinophils adhere to these parasympathetic neurones after cytokine pretreatment via a CD11/18-dependent pathway. Immunohistochemistry and Western blotting showed that a human cholinergic nerve cell line (IMR-32) expressed VCAM-1 and ICAM-1. Inhibitory experiments using monoclonal blocking antibodies to ICAM-1, VCAM-1, or CD11/18 and with the very late antigen-4 peptide inhibitor ZD-7349 showed that eosinophils adhered to IMR-32 cells via these adhesion molecules. The protein kinase C signaling pathway is involved in this process as a specific inhibitor-attenuated adhesion. Eosinophil adhesion to IMR-32 cells was associated with the release of eosinophil peroxidase and leukotriene C(4). Thus eosinophils adhere to cholinergic nerves via specific adhesion molecules, and this leads to eosinophil activation and degranulation; this may be part of the mechanism of eosinophil-induced vagal hyperreactivity.     

Effects of eosinophils on nerve cell morphology and development: the role of reactive oxygen species and p38 MAP kinase

The adhesion of eosinophils to nerve cells and the subsequent release of eosinophil products may contribute to the pathogenesis of conditions such as asthma and inflammatory bowel disease. In this study we have separately examined the consequences of eosinophil adhesion and degranulation for nerve cell morphology and development. Eosinophils induced neurite retraction of cultured guinea pig parasympathetic nerves and differentiated IMR32 cholinergic neuroblastoma cells. Inhibition of eosinophil adhesion to IMR32 cells attenuated this retraction. Eosinophil adhesion to IMR32 cells led to tyrosine phosphorylation of a number of nerve cell proteins, activation of p38 MAP kinase, and generation of neuronal reactive oxygen species (ROS). Inhibition of tyrosine kinases with genistein prevented both the generation of ROS in the nerve cells and neurite retraction. The p38 MAP kinase inhibitor SB-239063 prevented neurite retraction but had no effect on the induction of ROS. Thus eosinophils induced neurite retraction via two distinct pathways: by generation of tyrosine kinase-dependent ROS and by p38 MAP kinase. Eosinophils also prevented neurite outgrowth during differentiation of IMR32 cells. In contrast to their effect on neurite retraction, this effect was mimicked by medium containing products released from eosinophils and by eosinophil major basic protein. These results indicate that eosinophils modify the morphology of nerve cells by distinct mechanisms that involve adhesion and released proteins.     

Adhesion-dependent interactions between eosinophils and cholinergic nerves

Eosinophils adhere to airway cholinergic nerves and influence nerve cell function by releasing granule proteins onto inhibitory neuronal M(2) muscarinic receptors. This study investigated the mechanism of eosinophil degranulation by cholinergic nerves. Eosinophils were cocultured with IMR32 cholinergic nerve cells, and eosinophil peroxidase (EPO) or leukotriene C(4) (LTC(4)) release was measured. Coculture of eosinophils with nerves significantly increased EPO and LTC(4) release compared with eosinophils alone. IMR32 cells, like parasympathetic nerves, express the adhesion molecules vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 (ICAM-1). Inhibition of these adhesion molecules alone or in combination significantly inhibited eosinophil degranulation. IMR32 cells also significantly augmented the eosinophil degranulation produced by formyl-Met-Leu-Phe. Eosinophil adhesion to IMR32 cells resulted in an ICAM-1-mediated production of reactive oxygen species via a neuronal NADPH oxidase, inhibition of which significantly inhibited eosinophil degranulation. Additionally, eosinophil adhesion increased the release of ACh from IMR32 cells. These neuroinflammatory cell interactions may be relevant in a variety of inflammatory and neurological conditions.     

Natural Killer Cells Induce Eosinophil Activation and Apoptosis

Eosinophils are potent inflammatory cells with numerous immune functions, including antigen presentation and exacerbation of inflammatory responses through their capacity to release a range of largely preformed cytokines and lipid mediators. Thus, timely regulation of eosinophil activation and apoptosis is crucial to develop beneficial immune response and to avoid tissue damage and induce resolution of inflammation. Natural Killer (NK) cells have been reported to influence innate and adaptive immune responses by multiple mechanisms including cytotoxicity against other immune cells. In this study, we analyzed the effect of the interaction between NK cells and eosinophils. Co-culture experiments revealed that human NK cells could trigger autologous eosinophil activation, as shown by up-regulation of CD69 and down-regulation of CD62L, as well as degranulation, evidenced by increased CD63 surface expression, secretion of eosinophil cationic protein (ECP) and eosinophil derived neurotoxin (EDN). Moreover, NK cells significantly and dose dependently increased eosinophil apoptosis as shown by annexin V and propidium iodide (PI) staining. Direct contact was necessary for eosinophil degranulation and apoptosis. Increased expression of phosphorylated extracellular signal-regulated kinase (ERK) in cocultured eosinophils and inhibition of eosinophil CD63 expression by pharmacologic inhibitors suggest that MAPK and PI3K pathways are involved in NK cell-induced eosinophil degranulation. Finally, we showed that NK cells increased reactive oxygen species (ROS) expression by eosinophils in co-culture and that mitochondrial inhibitors (rotenone and antimycin) partially diminished NK cell-induced eosinophil apoptosis, suggesting the implication of mitochondrial ROS in NK cell-induced eosinophil apoptosis. Pan-caspase inhibitor (ZVAD-FMK) only slightly decreased eosinophil apoptosis in coculture. Altogether, our results suggest that NK cells regulate eosinophil functions by inducing their activation and their apoptosis.     

Extracellular signal-regulated kinase 1/2-mediated phosphorylation of cytosolic phospholipase A2 is essential for human eosinophil adhesion to fibronectin

We examined the role of p38, p42, and p44 mitogen-activated protein kinase (MAPK) isoforms and cytosolic phospholipase A(2) (cPLA(2)) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 microg/ml FN at 30 min, and decreased after 60-90 min. Western blot analysis demonstrated that p44/42 MAPK (extracellular signal-regulated kinase (ERK)1/2) and cPLA(2) were phosphorylated during adhesion to FN, whereas p38 MAPK phosphorylation was unchanged. Preincubation of eosinophils with U0126 or PD98059, two structurally unrelated MAPK kinase inhibitors, or arachidonic trifluoromethyl ketone, a cPLA(2) inhibitor, blocked eosinophil adhesion to FN. By contrast, eosinophil adhesion was unaffected by SB203580, a p38 MAPK inhibitor. Pretreatment of eosinophils with okadaic acid, a serine/threonine phosphatase inhibitor, at the concentrations that induced ERK1/2 and cPLA(2) phosphorylation caused an increase in maximal eosinophil adhesion to FN for >60 min. MAPK kinase inhibition but not p38 inhibition also blocked FN-mediated F-actin redistribution in eosinophils and prevented cPLA(2) phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA(2) activation is essential for eosinophil adhesion to FN.     

Eosinophils increase neuron branching in human and murine skin and in vitro

Cutaneous nerves are increased in atopic dermatitis, and itch is a prominent symptom. We studied the functional interactions between eosinophils and nerves in human and mouse skin and in culture. We demonstrated that human atopic dermatitis skin has eosinophil granule proteins present in the same region as increased nerves. Transgenic mice in which interleukin-5 (IL-5) expression is driven by a keratin-14 (K14) promoter had many eosinophils in the epidermis, and the number of nerves was also significantly increased in the epidermis. In co-cultures, eosinophils dramatically increased branching of sensory neurons isolated from the dorsal root ganglia (DRG) of mice. This effect did not occur in DRG neurons co-cultured with mast cells or with dead eosinophils. Physical contact of the eosinophils with the neurons was not required, and the effect was not blocked by an antibody to nerve growth factor. DRG neurons express eotaxin-1, ICAM-1 and VCAM-1, which may be important in the recruitment, binding, and activation of eosinophils in the region of cutaneous nerves. These data indicate a pathophysiological role for eosinophils in cutaneous nerve growth in atopic dermatitis, and suggest they may present a therapeutic target in atopic dermatitis and other eosinophilic skin conditions with neuronal symptoms such as itch.     

Blockade of focal clustering and active conformation in beta 2-integrin-mediated adhesion of eosinophils to intercellular adhesion molecule-1 caused by transduction of HIV TAT-dominant negative Ras

We transduced dominant negative (dn) HIV TAT-Ras protein into mature human eosinophils to determine the signaling pathways and mechanism involved in integrin-mediated adhesion caused by cytokine, chemokine, and chemoattractant stimulation. Transduction of TAT-dnRas into nondividing eosinophils inhibited endogenous Ras activation and extracellular signal-regulated kinase (ERK) phosphorylation caused by IL-5, eotaxin-1, and fMLP. IL-5, eotaxin-1, or fMLP caused 1) change of Mac-1 to its active conformation and 2) focal clustering of Mac-1 on the eosinophil surface. TAT-dnRas or PD98059, a pharmacological mitogen-activated protein/ERK kinase inhibitor, blocked both focal surface clustering of Mac-1 and the change to active conformational structure of this integrin assessed by the mAb CBRM1/5, which binds the activation epitope. Eosinophil adhesion to the endothelial ligand ICAM-1 was correspondingly blocked by TAT-dnRas and PD98059. As a further control, we used PMA, which activates ERK phosphorylation by postmembrane receptor induction of protein kinase C, a mechanism which bypasses Ras. Neither TAT-dnRas nor PD98059 blocked eosinophil adhesion to ICAM-1, up-regulation of CBRM1/5, or focal surface clustering of Mac-1 caused by PMA. In contrast to beta(2)-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for beta(1)-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for beta(2)-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.     

Different anti-apoptotic effects of normal and asthmatic serum on normal eosinophil apoptosis depending on house dust mite-specific IgE

We investigated the effect of asthmatic serum on constitutive eosinophil apoptosis in normal subjects. Eosinophil apoptosis in normal subjects was inhibited by asthmatic serum but not normal serum. In a detailed analysis based on the presence of house dust mite (HDM) IgE, HDM IgE-positive (+) asthmatic serum was more effective for eosinophil apoptosis than that of HDM IgE-negative (?) asthmatic serum. HDM IgE+ asthmatic serum inhibited both HDM IgE? and HDM IgE+ normal eosinophil apoptosis, and HDM IgE? asthmatic serum suppressed eosinophil apoptosis of HDM IgE+ normal. HDM IgE? normal serum did not inhibit either HDM IgE? or HDM IgE+ normal eosinophil apoptosis, and HDM IgE+ normal serum inhibited HDM IgE+ normal eosinophil apoptosis. The kind of HDM IgE (Dermatophagoides pteronissinus-specific IgE and Dermatophagoides farinae-specific IgE) was not related to the effect of asthmatic serum on eosinophil apoptosis. Extracts of DP and DF, Der p1, and Der p2, were not effective for eosinophil apoptosis. HDM IgE+ asthmatic serum inhibited cleavage of procaspase 9 and procaspase 3. Asthmatic serum induced Akt and ERK phosphorylation, and ERK activation was suppressed by AKTi. Taken together, asthmatic serum inhibited normal eosinophil apoptosis via PI3K/Akt/ERK cascade. The novel approach taken in this study provided better insight into HDM-associated anti-apoptotic mechanism of eosinophils in patients with asthma.     

CCR3-active chemokines promote rapid detachment of eosinophils from VCAM-1 in vitro

Selective eosinophil recruitment is the result of orchestrated events involving cell adhesion molecules, chemokines, and their receptors. The mechanisms by which chemokines regulate eosinophil adhesion and migration via integrins are not fully understood. In our study, we examined the effect of CCR3-active chemokines on eosinophil adhesion to VCAM-1 and BSA under both static and flow conditions. When eotaxin-2 or other CCR3-active chemokines were added to adherent eosinophils, it induced rapid and sustained eosinophil detachment from VCAM-1 in a concentration-dependent manner. Adhesion was detectably reduced within 3 min and was further reduced at 10-60 min. Simultaneously, eotaxin-2 enhanced eosinophil adhesion to BSA. Preincubation of eosinophils with the CCR3-blocking mAb 7B11 completely prevented chemokine-induced changes in adhesion to VCAM-1 and BSA. Using a different protocol, pretreatment of eosinophils with chemokines for 0-30 min before their use in adhesion assays resulted in inhibition of VCAM-1 adhesion and enhancement of BSA adhesion. By flow cytometry, expression of alpha4 integrins and a beta1 integrin activation epitope on eosinophils was decreased by eotaxin-2. In a flow-based adhesion assay, eotaxin-2 reduced eosinophil accumulation and the strength of attachment to VCAM-1. These results show that eotaxin-2 rapidly reduced alpha4 integrin function while increasing beta2 integrin function. These findings suggest that chemokines facilitate migration of eosinophils by shifting usage away from beta1 integrins toward beta2 integrins.     

Dynamic change of neural cell adhesion molecule polysialylation on human neuroblastoma (IMR-32) and rat pheochromocytoma (PC-12) cells during growth and differentiation

Polysialic acid (PSA) is a regulatory epitope of neural cell adhesion molecule (NCAM) in homophilic adhesion of neural cells mediated by NCAM, is also known to be re-expressed in several human tumors, thus serves as an oncodevelopmental antigen. In this study, using a recently developed ultrasensitive chemical method in addition to immunochemical methods, growth stage-dependent and retinoic acid (RA)-induced differentiation-dependent changes of PSA expression in human neuroblastoma (IMR-32) and rat pheochromocytoma (PC-12) cells were analyzed both qualitatively and quantitatively. Both IMR-32 and PC-12 cells expressed PSA on NCAM, and the level of PSA expressed per unit weight of cells increased with post-inoculation incubation time. The most prominent feature was seen at the full confluence stage. RA induced neuronal differentiation in both IMR-32 and CP-12 cells that paralleled the change in the PSA level. Chemical analysis revealed the presence of NCAM glycoforms differing in the degree of polymerization (DP) of oligo/polysialyl chains, whose DP was smaller than 40. DP distribution of PSA was different between the cell lines and was changed by the growth stage and the RA treatment. Thus DP analysis of PSA is important in understanding both mechanism and biological significance of its regulated expression.     

Eosinophil resistance to glucocorticoid-induced apoptosis is mediated by the transcription factor NFIL3

The mainstay of asthma therapy, glucocorticoids (GCs) exert their therapeutic effects through the inhibition of inflammatory signaling and induction of eosinophil apoptosis. However, laboratory and clinical observations of GC-resistant asthma suggest that GCs’ effects on eosinophil viability may depend on the state of eosinophil activation. In the present study we demonstrate that eosinophils stimulated with IL-5 show impaired pro-apoptotic response to GCs. We sought to determine the contribution of GC-mediated transactivating (TA) and transrepressing (TR) pathways in modulation of activated eosinophils’ response to GC by comparing their response to the selective GC receptor (GR) agonist Compound A (CpdA) devoid of TA activity to that upon treatment with Dexamethasone (Dex). IL-5-activated eosinophils showed contrasting responses to CpdA and Dex, as IL-5-treated eosinophils showed no increase in apoptosis compared to cells treated with Dex alone, while CpdA elicited an apoptotic response regardless of IL-5 stimulation. Proteomic analysis revealed that both Nuclear Factor IL-3 (NFIL3) and Map Kinase Phosphatase 1 (MKP1) were inducible by IL-5 and enhanced by Dex; however, CpdA had no effect on NFIL3 and MKP1 expression. We found that inhibiting NFIL3 with specific siRNA or by blocking the IL-5-inducible Pim-1 kinase abrogated the protective effect of IL-5 on Dex-induced apoptosis, indicating crosstalk between IL-5 anti-apoptotic pathways and GR-mediated TA signaling occurring via the NFIL3 molecule. Collectively, these results indicate that (1) GCs’ TA pathway may support eosinophil viability in IL-5-stimulated cells through synergistic upregulation of NFIL3; and (2) functional inhibition of IL-5 signaling (anti-Pim1) or the use of selective GR agonists that don’t upregulate NFIL3 may be effective strategies for the restoring pro-apoptotic effect of GCs on IL-5-activated eosinophils.     

Ligation of Fc gamma RII (CD32) pivotally regulates survival of human eosinophils

The low-affinity IgG Fc receptor, FcgammaRII (CD32), mediates various effector functions of lymphoid and myeloid cells and is the major IgG Fc receptor expressed by human eosinophils. We investigated whether FcgammaRII regulates both cell survival and death of human eosinophils. When cultured in vitro without growth factors, most eosinophils undergo apoptosis within 96 h. Ligation of FcgammaRII by anti-CD32 mAb in solution inhibited eosinophil apoptosis and prolonged survival in the absence of growth factors. Cross-linking of human IgG bound to FcgammaRII by anti-human IgG Ab or of unoccupied FcgammaRII by aggregated human IgG also prolonged eosinophil survival. The enhanced survival with anti-CD32 mAb was inhibited by anti-granulocyte-macrophage-CSF (GM-CSF) mAb, suggesting that autocrine production of GM-CSF by eosinophils mediated survival. In fact, mRNA for GM-CSF was detected in eosinophils cultured with anti-CD32 mAb. In contrast to mAb or ligands in solution, anti-CD32 mAb or human IgG, when immobilized onto tissue culture plates, facilitated eosinophil cell death even in the presence of IL-5. Cell death induced by these immobilized ligands was accompanied by DNA fragmentation and was inhibited when eosinophil beta2 integrin was blocked by anti-CD18 mAb, suggesting that beta2 integrins play a key role in initiating eosinophil apoptosis. Thus, FcgammaRII may pivotally regulate both survival and death of eosinophils, depending on the manner of receptor ligation and beta2 integrin involvement. Moreover, the FcgammaRII could provide a novel mechanism to control the number of eosinophils at inflammation sites in human diseases.     

Eosinophils and airway nerves in asthma

In the lungs, neuronal M2 muscarinic receptors limit the release of acetylcholine from postganglionic cholinergic nerves. However, these receptors are not functional under certain circumstances in animal models of hyperreactivity such as occurs after exposure of sensitised animals to an allergen or during a respiratory tract virus infection. This loss of M2 receptor function leads to an increase in acetylcholine release from cholinergic nerves and thus is a mechanism for the vagally mediated hyperreactivity seen in these animals. Studies in animal models of hyperreactivity have shown that eosinophils localise to the airway nerves of sensitised animals after antigen challenge. Inhibiting this localisation of eosinophils either with an antibody to the eosinophil survival cytokine IL-5 or the eosinophil adhesion molecule VLA-4 prevents loss of M2 muscarinic receptor function. It is likely that eosinophil MBP is responsible for the loss of M2 receptor function, since inhibiting eosinophil MBP with an antibody or neutralising MBP with heparin prevents this loss of function. These data are also supported by ligand binding studies where it has been shown that eosinophil MBP is an allosteric antagonist at neuronal M2 muscarinic receptors. Loss of function of lung neuronal M2 muscarinic receptors may also occur under certain circumstances in patients with asthma, although the mechanisms are not yet established.     

Carnosic acid, a rosemary phenolic compound, induces apoptosis through reactive oxygen species-mediated p38 activation in human neuroblastoma IMR-32 cells

Carnosic acid (CA), a rosemary phenolic compound, has been shown to display anti-cancer activity. We examined the apoptotic effect of CA in human neuroblastoma IMR-32 cells and elucidated the role of the reactive oxygen species (ROS) and mitogen-activated protein kinase (MAPK) associated with carcinogenesis. The result indicated that CA decreased the cell viability in a dose-dependent manner. Further investigation in IMR-32 cells revealed that cell apoptosis following CA treatment is the mechanism as confirmed by flow cytometry, hoechst 33258, and caspase-3/-9 and poly(ADP-ribose) polymerase (PARP) activation. Immunoblotting suggested a down-regulation of anti-apoptotic Bcl-2 protein in the CA-treated cells. In flow cytometric analysis, CA caused the generation of reactive oxygen species (ROS); however, pretreatment with the antioxidant N-acetylcysteine (NAC) attenuated the CA-induced generation of ROS and apoptosis. This effect was accompanied by increased activation of p38 and by decreased activation of extracellular signal-regulated kinase (ERK) as well as activation of c-Jun NH₂-terminal kinase (JNK). Moreover, NAC attenuated the CA-induced phosphorylation of p38. Silencing of p38 by siRNA gene knockdown reduced the CA-induced activation of caspase-3. In conclusion, ROS-mediated p38 MAPK activation plays a critical role in CA-induced apoptosis in IMR-32 cells.     

ASK1 resistant neuroblastoma is deficient in activation of p38 kinase

Apoptosis Signal-regulating Kinase 1 (ASK1) is known to either induce apoptosis or differentiation in various cell lines of neuronal origin. We analyzed the effect of the constitutively active mutant of ASK1 (ASK1-Delta N) in an adenoviral vector in four neuroblastoma cell lines, two murine, C1300 and NXS2, and two human, SH-SY5Y and IMR-32. Already after 24 h upon infection, C1300 and SH-SY5Y cells arrested in growth when judged by [(3)H]thymidine incorporation, and the majority of the cells demonstrated apoptotic appearance, which was confirmed by DNA-laddering in gel electrophoresis. In contrast, NXS2 and IMR-32 cell lines remained unaffected. Immunoblotting revealed strongly phosphorylated p38 MAPK accompanied by weakly phosphorylated JNK in C1300 and SH-SY5Y, whereas none of these kinases were activated by adenoviruses expressing the kinase negative ASK1 mutant or beta-galactosidase. There was no expression of phosphorylated kinases in IMR-32 cells, but NXS2 showed a faint band of phosphorylated p38 MAPK. Addition of the p38 MAPK specific inhibitor, SB203580, protected C1300 and SH-SY5Y cells from apoptosis induced by ASK1-Delta N. The anti-neoplastic agent, paclitaxel, activates ASK1 and JNK, and promotes the in vitro assembly of stable microtubules. Addition of 10 nM paclitaxel sensitised the NXS2 cell line to ASK1-induced cell death. Our results indicate that ASK1 induces apoptosis in neuroblastoma cells mainly via the p38 MAPK pathway, and resistant neuroblastoma cells can be sensitised to ASK1 by paclitaxel.