Possible participation of a JAK2 signaling pathway in recombinant rat interleukin-5-induced prolongation of rat eosinophil survival

Recombinant rat interleukin (IL)-5-induced prolongation of rat eosinophil survival in culture was inhibited in a concentration-dependent manner by the protein synthesis inhibitor cycloheximide, the DNA-dependent RNA synthesis inhibitor actinomycin D, and the tyrosine kinase inhibitor herbimycin A when examined 96 h after incubation. The MEK-1 inhibitor PD98059 inhibited IL-5-induced phosphorylation of both p44 and p42 MAP kinases, but the IL-5-induced prolongation of eosinophil survival was not inhibited. In contrast, the JAK2 inhibitor AG490 inhibited the IL-5-induced prolongation of eosinophil survival. Treatment of eosinophils with IL-5 resulted in phosphorylation of STAT5 but not STAT1, and the IL-5-induced phosphorylation of STAT5 was inhibited by AG490. These findings suggest that the activation of JAK2 tyrosine kinase and protein synthesis are required for the prolongation of rat eosinophil survival induced by recombinant rat IL-5. STAT5 phosphorylation might also participate in the IL-5-induced survival of rat eosinophils.     

Accelerated clearance of Escherichia coli in experimental peritonitis of histamine-deficient mice

IL-5 plays a pivotal role in growth and differentiation of eosinophils. The signal transduction mechanism of IL-5Ralpha is largely unknown. We have demonstrated that IL-5 induces tyrosine phosphorylation of IL-5Ralpha in eosinophils. To identify IL-5Ralpha-associated tyrosine kinases, we have examined the expression of Src family tyrosine kinases in eosinophils. Among the Src family members, Lyn, Hck, Fgr, and Lck are present in eosinophils, and, among these four kinases, only Lyn is associated with the IL-5Ralpha under basal conditions. We also confirm the association of Janus kinase (Jak)2 with IL-5Ralpha. Lyn kinase phosphorylates both IL-5Ralpha and betacR in vitro. The importance of Lyn kinase for eosinophil differentiation was studied using antisense oligodeoxynucleotides. Lyn antisense oligodeoxynucleotide blocks eosinophil differentiation from stem cells in a dose-dependent manner. The Jak2 inhibitor tyrphostin AG490 also inhibits eosinophil differentiation. The importance of Lyn for eosinophil differentiation was further studied using Lyn knockout mice. The IL-5-stimulated eosinophil differentiation from bone marrow cells is significantly inhibited in Lyn(-/-) mice as compared with that in control mice. We conclude that both Lyn and Jak2 play an essential role in IL-5Ralpha signaling, leading to eosinophil differentiation. The effect of Lyn appears to be relatively specific for the eosinophilic lineage.     

The mechanism of IL-5 signal transduction

Cytokines are important regulators ofhematopoiesis. They exert their actions by binding to specificreceptors on the cell surface. Interleukin-5 (IL-5) is a criticalcytokine that regulates the growth, activation, and survival ofeosinophils. Because eosinophils play a seminal role in thepathogenesis of asthma and allergic diseases, an understanding of thesignal transduction mechanism of IL-5 is of paramount importance. TheIL-5 receptor is a heterodimer of - and -subunits. The-subunit is specific, whereas the -subunit is common to IL-3,IL-5, and granulocyte/macrophage colony-stimulating factor (GM-CSF)receptors and is crucial for signal transduction. It has been shownthat there are two major signaling pathways of IL-5 in eosinophils.IL-5 activates Lyn, Syk, and JAK2 and propagates signals through theRas-MAPK and JAK-STAT pathways. Studies suggest that Lyn, Syk, and JAK2tyrosine kinases and SHP-2 tyrosine phosphatase are important foreosinophil survival. In contrast to their survival-promoting activity,Lyn and JAK2 appear to have no role in eosinophil degranulation orexpression of surface adhesion molecules. Raf-1 kinase, on the otherhand, is critical for eosinophil degranulation and adhesion moleculeexpression. Btk is involved in IL-5 stimulation of B cell function.However, it does not appear to be important for eosinophil function.Thus a clear segregation of signaling molecules based on theirfunctional importance is emerging. This review describes the signaltransduction mechanism of the IL-3/GM-CSF/IL-5 receptor system andcompares and contrasts IL-5 signaling between eosinophils and B cells.

     

The functional role of rho and rho-associated coiled-coil forming protein kinase in eotaxin signaling of eosinophils

The CC chemokine eotaxin plays a pivotal role in local accumulation of eosinophils. Very little is known about the eotaxin signaling in eosinophils except the activation of the mitogen-activated protein (MAP) kinase family. The p21 G protein Rho and its substrate Rho-associated coiled-coil forming protein kinase (ROCK) regulate the formation of stress fibers and focal adhesions. In the present study, we studied the functional relevance of Rho and ROCK in eosinophils using the ROCK inhibitor (Y-27632) and exoenzyme C3, a specific Rho inhibitor. Eotaxin stimulates activation of Rho A and ROCK II in eosinophils. Exoenzyme C3 almost completely inhibited the ROCK activity, indicating that ROCK is downstream of Rho. We then examined the role of Rho and ROCK in eosinophil chemotaxis. The eotaxin-induced eosinophil chemotaxis was significantly inhibited by exoenzyme C3 or Y-27632. Because extracellular signal-regulated kinase (ERK)1/2 and p38 MAP kinases are activated by eotaxin and are critical for eosinophil chemotaxis, we investigated whether Rho and ROCK are upstream of these MAP kinases. C3 partially inhibited eotaxin-induced phosphorylation of ERK1/2 but not p38. In contrast, neither ERK1/2 nor p38 phosphorylation was abrogated by Y-27632. Both C3 and Y-27632 reduced reactive oxygen species production from eosinophils. We conclude that both Rho and ROCK are important for eosinophil chemotaxis and reactive oxygen species production. There is a dichotomy of downstream signaling pathways of Rho, namely, Rho-ROCK and Rho-ERK pathways. Taken together, eosinophil chemotaxis is regulated by multiple signaling pathways that involve at least ROCK, ERK, and p38 MAP kinase.     

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.     

Autocrine transforming growth factor-beta signaling mediates Smad-independent motility in human cancer cells

Transforming growth factor-beta (TGF-beta) is a pleiotropic growth factor that plays a critical role in modulating cell growth, differentiation, and plasticity. There is increasing evidence that after cells lose their sensitivity to TGF-beta-mediated growth inhibition, autocrine TGF-beta signaling may potentially promote tumor cell motility and invasiveness. To understand the molecular mechanisms by which autocrine TGF-beta may selectively contribute to tumor cell motility, we have generated MDA-MB-231 breast cancer cells stably expressing a kinase-inactive type II TGF-beta receptor (T beta RII-K277R). Our data indicate that T beta RII-K277R is expressed, can associate with the type I TGF-beta receptor, and block both Smad-dependent and -independent signaling pathways activated by TGF-beta. In addition, wound closure and transwell migration assays indicated that the basal migratory potential of T beta RII-K277R expressing cells was impaired. The impaired motility of T beta RII-K277R cells could be restored by reconstituting TGF-beta signaling with a constitutively active TGF-beta type I receptor (ALK5(TD)) but not by reconstituting Smad signaling with Smad2/4 or Smad3/4 expression. In addition, the levels of ALK5(TD) expression sufficient to restore motility in the cells expressing T beta RII-K277R were associated with an increase in phosphorylation of Akt and extracellular signal-regulated kinase 1/2 but not Smad2. These data indicate that different signaling pathways require different thresholds of TGF-beta activation and suggest that TGF-beta promotes motility through mechanisms independent of Smad signaling, possibly involving activation of the phosphatidylinositol 3-kinase/Akt and/or mitogen-activated protein kinase pathways.     

Regulation of human eosinophil NADPH oxidase activity: a central role for PKCdelta.

Eosinophils play a primary role in the pathophysiology of asthma. In the lung, the activation state of the infiltrating eosinophils determines the extent of tissue damage. Interleukin-5 (IL-5) and leukotriene B4 (LTB4) are important signaling molecules involved in eosinophil recruitment and activation. However, the physiological processes that regulate these activation events are largely unknown. In this study we have examined the mechanisms of human eosinophil NADPH oxidase regulation by IL-5, LTB4, and phorbol ester (PMA). These stimuli activate a Zn2+-sensitive plasma membrane proton channel, and treatment of eosinophils with Zn2+ blocks superoxide production. We have demonstrated that eosinophil intracellular pH is not altered by IL-5 activation of NADPH oxidase. Additionally, PKCdelta inhibitors block PMA, IL-5 and LTB4 mediated superoxide formation. Interestingly, the PKCdelta-selective inhibitor, rottlerin, does not block proton channel activation by PMA indicating that the oxidase and the proton conductance are regulated at distinct phosphorylation sites. IL-5 and LTB4, but not PMA, stimulated superoxide production is also blocked by inhibitors of PI 3-kinase indicating that activation of this enzyme is an upstream event common to both receptor signaling pathways. Our results indicate that the G-protein-coupled LTB4 receptor and the IL-5 cytokine receptor converge on a common signaling pathway involving PI 3-kinase and PKCdelta to regulate NADPH oxidase activity in human eosinophils.     

IL-3 and TNFα increase Thymic Stromal Lymphopoietin Receptor (TSLPR) expression on eosinophils and enhance TSLP-stimulated degranulation

Background

Thymic stromal lymphopoietin (TSLP) and eosinophils are prominent components of allergic inflammation. Therefore, we sought to determine whether TSLP could activate eosinophils, focusing on measuring the regulation of TSLPR expression on eosinophils and degranulation in response to TSLP, as well as other eosinophil activation responses.

Methods

Eosinophil mRNA expression of TSLPR and IL-7R?? was examined by real-time quantitative PCR of human eosinophils treated with TNF?? and IL-5 family cytokines, and TSLPR surface expression on eosinophils was analyzed by flow cytometry. Eosinophils were stimulated with TSLP (with and without pre-activation with TNF?? and IL-3) and evaluated for release of eosinophil derived neurotoxin (EDN), phosphorylation of STAT5, and survival by trypan blue exclusion. A blocking antibody for TSLPR was used to confirm the specificity of TSLP mediated signaling on eosinophil degranulation.

Results

Eosinophil expression of cell surface TSLPR and TSLPR mRNA was upregulated by stimulation with TNF?? and IL-3. TSLP stimulation resulted in release of EDN, phosphorylation of STAT5 as well as promotion of viability and survival. TSLP-stimulated eosinophil degranulation was inhibited by a functional blocking antibody to TSLPR. Pre-activation of eosinophils with TNF?? and IL-3 promoted eosinophil degranulation at lower concentrations of TSLP stimulation.

Conclusions

This study demonstrates that eosinophils are activated by TSLP and that eosinophil degranulation in response to TSLP may be enhanced on exposure to cytokines present in allergic inflammation, indicating that the eosinophil has the capacity to participate in TSLP-driven allergic responses.     

IL-5 and granulocyte-macrophage colony-stimulating factor activate STAT3 and STAT5 and promote Pim-1 and cyclin D3 protein expression in human eosinophils

Allergic inflammation is characterized by elevated eosinophil numbers and by the increased production of the cytokines IL-5 and GM-CSF, which control several eosinophil functions, including the suppression of apoptosis. The JAK/STAT pathway is important for several functions in hemopoietic cells, including the suppression of apoptosis. We report in this study that STAT3, STAT5a, and STAT5b are expressed in human eosinophils and that their signaling pathways are active following IL-5 or GM-CSF treatment. However, in airway eosinophils, the phosphorylation of STAT5 by IL-5 is reduced, an event that may be related to the reduced expression of the IL-5Ralpha on airway eosinophils. Furthermore, IL-5 and GM-CSF induced the protein expression of cyclin D3 and the kinase Pim-1, both of which are regulated by STAT-dependent processes in some cell systems. Pim-1 is more abundantly expressed in airway eosinophils than in blood eosinophils. Because Pim-1 reportedly has a role in the modulation of apoptosis, these results suggest that Pim-1 action is linked to the suppression of eosinophil apoptosis by these cytokines. Although cyclin D3 is known to be critical for cell cycle progression, eosinophils are terminally differentiated cells that do not proceed through the cell cycle. Thus, this apparent cytokine regulation of cyclin D3 suggests that there is an alternative role(s) for cyclin D3 in eosinophil biology.     

Dusp5 negatively regulates IL‐33‐mediated eosinophil survival and function

Mitogen-activated protein kinase (MAPK) activation controls diverse cellular functions including cellular survival, proliferation, and apoptosis. Tuning of MAPK activation is counter-regulated by a family of dual-specificity phosphatases (DUSPs). IL-33 is a recently described cytokine that initiates Th2 immune responses through binding to a heterodimeric IL-33Rα (ST2L)/IL-1α accessory protein (IL-1RAcP) receptor that coordinates activation of ERK and NF-κB pathways. We demonstrate here that DUSP5 is expressed in eosinophils, is upregulated following IL-33 stimulation and regulates IL-33 signaling. Dusp5^−/− mice have prolonged eosinophil survival and enhanced eosinophil effector functions following infection with the helminth Nippostrongylus brasiliensis. IL-33-activated Dusp5^−/− eosinophils exhibit increased cellular ERK1/2 activation and BCL-X_L expression that results in enhanced eosinophil survival. In addition, Dusp5^−/− eosinophils demonstrate enhanced IL-33-mediated activation and effector functions. Together, these data support a role for DUSP5 as a novel negative regulator of IL-33-dependent eosinophil function and survival.     

Differential regulation of human eosinophil IL-3, IL-5, and GM-CSF receptor alpha-chain expression by cytokines: IL-3, IL-5, and GM-CSF down-regulate IL-5 receptor alpha expression with loss of IL-5 responsiveness,but up-regulate IL-3 receptor alpha expression

Our recent data suggested that tissue eosinophils may be relatively insensitive to anti-IL-5 treatment. We examined cross-regulation and functional consequences of modulation of eosinophil cytokine receptor expression by IL-3, IL-5 GM-CSF, and eotaxin. Incubation of eosinophils with IL-3, IL-5, or GM-CSF led to reduced expression of IL-5R alpha, which was sustained for up to 5 days. Eosinophils incubated with IL-5 or IL-3 showed diminished respiratory burst and mitogen-activated protein kinase kinase phosphorylation in response to further IL-5 stimulation. In contrast to these findings, eosinophil expression of IL-3R alpha was increased by IL-3, IL-5, and GM-CSF, whereas GM-CSF receptor alpha was down-regulated by GM-CSF, but was not affected by IL-3 or IL-5. CCR3 expression was down-regulated by IL-3 and was transiently reduced by IL-5 and GM-CSF, but rapidly returned toward baseline. Eotaxin had no effect on receptor expression for IL-3, IL-5, or GM-CSF. Up-regulation of IL-3R alpha by cytokines was prevented by a phosphoinositol 3-kinase inhibitor, whereas this and other signaling inhibitors had no effect on IL-5R alpha down-regulation. These data suggest dynamic and differential regulation of eosinophil receptors for IL-3, IL-5, and GM-CSF by the cytokine ligands. Since these cytokines are thought to be involved in eosinophil development and mobilization from the bone marrow and are present at sites of allergic inflammation, tissue eosinophils may have reduced IL-5R expression and responsiveness, and this may explain the disappointing effect of anti-IL-5 therapy in reducing airway eosinophilia in asthma.     

Enhanced antiviral and antiproliferative properties of a STAT1 mutant unable to interact with the protein kinase PKR

We have previously reported a physical association between STAT1 and the protein kinase double-stranded RNA-activated protein kinase (PKR). PKR inhibited STAT1 function in a manner independent of PKR kinase activity. In this report, we have further characterized the properties of both molecules by mapping the sites of their interaction. A STAT1 mutant unable to interact with PKR displays enhanced interferon gamma (IFN-gamma)-induced transactivation capacity compared with STAT1. This effect appears to be mediated by the higher capacity of STAT1 mutant to heterodimerize with STAT3. Furthermore, expression of STAT1 mutant in STAT1(-/-) cells enhances both the antiviral and antiproliferative effects of IFNs as opposed to STAT1. We also provide evidence that STAT1 functions as an inhibitor of PKR in vitro and in vivo. That is, phosphorylation of eIF-2alpha is enhanced in STAT1(-/-) than STAT1(+/+) cells in vivo, and this correlates with higher activation capacity of PKR in STAT1(-/-) cells. Genetic experiments in yeast demonstrate the inhibition of PKR activation and eIF-2alpha phosphorylation by STAT1 but not by STAT1 mutant. These data substantiate our previous findings on the inhibitory effects of PKR on STAT1 and implicate STAT1 in translational control through the modulation of PKR activation and eIF-2alpha phosphorylation.     

Blockade of JAK2 by tyrphostin AG-490 inhibits antigen-induced eosinophil recruitment into the mouse airways

We studied the effect of tyrphostin AG-490, a specific Janus kinase 2 (JAK2) inhibitor, on antigen-induced eosinophil recruitment into the airways of sensitized mice and on IL-5-induced chemokinesis and adhesiveness of eosinophils. The in vivo administration of AG-490 prevented antigen-induced eosinophil infiltration in the airways of sensitized mice in a dose-dependent manner. However, the administration of AG-490 did not affect antigen-induced IL-5 production in the airways nor in vitro antigen-induced IL-5 production and T cell proliferation of spleen cells. Furthermore, AG-490 inhibited IL-5-induced chemokinesis and beta1-integrin adhesiveness of eosinophils in vitro. Because antigen-induced eosinophil recruitment into the airways is mediated by IL-5, these results indicate that JAK2 activation is critical for antigen-induced, IL-5-dependent mobilization of eosinophils into the tissue.     

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.     

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.     

A novel Lyn-binding peptide inhibitor blocks eosinophil differentiation, survival, and airway eosinophilic inflammation.

Receptor antagonists block all receptor-coupled signaling pathways indiscriminately. We introduce a novel class of peptide inhibitors that is designed to block a specific signal from a receptor while keeping other signals intact. This concept was tested in the model of IL-5 signaling via Lyn kinase. We have previously mapped the Lyn-binding site of the IL-5/GM-CSF receptor common beta (beta c) subunit. In the present study, we designed a peptide inhibitor using the Lyn-binding sequence. The peptide was N-stearated to enable cellular internalization. The stearated peptide blocked the binding of Lyn to the beta c receptor and the activation of Lyn. The lipopeptide did not affect the activation of Janus kinase 2 or its association with beta c. The inhibitor blocked the Lyn-dependent functions of IL-5 in vitro (e.g., eosinophil differentiation from stem cells and eosinophil survival). It did not affect eosinophil degranulation. When applied in vivo, the Lyn-binding peptide significantly inhibited airway eosinophil influx in a mouse model of asthma. The lipopeptide had no effect on basophil histamine release or on the proliferation of B cells and T cells. To our knowledge, this is the first report on an inhibitor of IL-5 that blocks eosinophil differentiation, survival, and airway eosinophilic inflammation. This novel strategy to develop peptide inhibitors can be applied to other receptors.