The role of STAT1 in activation of IL-3- and IL-5-induced eosinophils by interferon gamma

Tyrosine phosphorylation of STAT1alpha in eosinophils after IFN-gamma stimulation has been shown, but the biological significance of eosinophil STAT1alpha activation in transmitting the signals through the IFN-gamma receptor remains unknown. The purpose of this study is to determine whether STAT1 is involved in the regulation of eosinophils by IFN-gamma-IFN-gamma receptor interaction. rhIL-3- and rhIL-5-induced eosinophils from CD34+ cells of cord blood on day 28 of culture were used. The cells were washed and further incubated in IL-3- and IL-5-free medium for 48 h. The induced eosinophils constitutively expressed CD69 and lost this expression after a further 48-hour incubation without the cytokines. IFN-gamma significantly upregulated CD69 expression on the 48-hour incubated cells. In inhibitory experiments on STAT1, a phosphorothioate oligo antisense DNA against STAT1alpha was added to IL-3- and IL-5-containing medium from day 15 to day 28 of culture. The oligo DNAs altered neither the expressions of myeloid cell marker CD9 and 13 nor the expression of IFN-gamma receptor on the cells. The added STAT1alpha antisense, but not sense, DNA significantly reduced STAT1alpha mRNA expression in the cells. The STAT1 antisense also significantly inhibited IFN-gamma-induced CD69 expression on the 48-hour incubated eosinophils. In conclusion, these results indicate that IFN-gamma induces CD69 expression in the induced eosinophils through STAT1alpha, suggesting that STAT1alpha may play a significant role in eosinophil regulation by IFN-gamma.     

Regulation of interleukin-5-induced beta2-integrin adhesion of human eosinophils by phosphoinositide 3-kinase

We examined the role of phosphoinositide 3-kinase (PI3K) in integrin-mediated eosinophil adhesion. Deltap85, a dominant-negative form of the class IA PI3K adaptor subunit, was fused to an HIV-TAT protein transduction domain (TAT-Deltap85). Recombinant TAT-Deltap85 inhibited interleukin (IL)-5-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. beta(2)-Integrin-dependent adhesion caused by IL-5 to the plated intracellular adhesion molecule-1 surrogate, bovine serum albumin, was inhibited by TAT-Deltap85 in a concentration-dependent manner. Similarly, two PI3K inhibitors, wortmannin and LY294002, blocked eosinophil adhesion to plated bovine serum albumin. By contrast, beta(1)-integrin-mediated eosinophil adhesion to vascular cell adhesion moelcule-1 was not blocked by TAT-Deltap85, wortmannin, or LY294002. Rottlerin, a protein kinase C (PKC)-delta inhibitor, also blocked beta(2)-integrin adhesion of eosinophils caused by IL-5, whereas beta(1) adhesion to vascular cell adhesion molecule-1 was not affected. IL-5 caused translocation of PKCdelta from the cytosol to cell membrane; inhibition of PI3K by wortmannin blocked translocation of PKCdelta. Western blot analysis demonstrated that extracellular signal-regulated kinase phosphorylation, a critical intermediary in adhesion elicited by IL-5, was blocked by inhibition of either PI3K or PKC-delta. These data suggest that extracellular signal-regulated kinase-mediated adhesion of beta(2)-integrin caused by IL-5 is mediated in human eosinophils by a class IA PI3K through activation of a PKCdelta pathway.     

Beta 1/beta 3 integrin ligation is uncoupled from ERK1/ERK2 activation in cytotoxic T lymphocytes

beta 3 integrins mediate fibronectin binding and enhanced activation of cytotoxic T lymphocytes (CTL). The intracellular signals initiated by beta 3 integrins in lymphocytes are not well characterized, but in many cell types, beta 1 integrin ligation activates mitogen-activated protein (MAP) kinases. In the present study, we find that fibronectin can synergize with very low levels of CD3 stimulation to activate the extracellular signal-regulated kinase (ERK)1 and ERK2 MAP kinases but that fibronectin alone induces no detectable MAP kinase activation in CTL. Surprisingly, antibodies to beta1 or beta 3 integrins were also unable to stimulate MAP kinase activation, suggesting that although beta 1 integrins are capable of stimulating MAP kinase activation in other cells, they cannot do so in CTL. In CTL, phosphorylation of proline-rich tyrosine kinase 2 downstream of integrin stimulation did not result in recruitment of the adaptor protein Grb2. Additionally, we examined the role of MAP kinases in regulating integrin-mediated adhesion. Anti-CD3-triggered adhesion to fibronectin was largely insensitive to the MAP kinase kinase inhibitor PD98059. Triggered cell-spreading on fibronectin was inhibited by PD98059 but not by U0126. In summary, ligation of beta 3 integrin by antibodies or fibronectin or of beta1 integrin by monoclonal antibodies fails to activate ERK MAP kinases, but integrin ligation synergizes with T cell receptor stimulation upstream of MAP kinases.     

ADP/P2Y1 aggravates inflammatory bowel disease through ERK5-mediated NLRP3 inflammasome activation

Inflammasomes are essential for inflammation and pathogen elimination in response to microbial infection and endogenous danger signals. However, the mechanism of inflammasome activation by endogenous danger signals mediated posttranslational modification and the connection between inflammasomes and inflammatory diseases remains elusive. In this study, we found that ADP was highly released from injured colonic tissue as a danger signal during inflammatory bowel disease. Consequently, extracellular ADP activated the NLRP3 inflammasome through P2Y₁ receptor-mediated calcium signaling, which led to the maturation and secretion of IL-1β and further aggravation of experimental colitis. Genetic ablation or pharmacological blockade of the P2Y₁ receptor significantly ameliorated DSS-induced colitis and endotoxic shock through reducing NLRP3 inflammasome activation. Moreover, ERK5-mediated tyrosine phosphorylation of ASC was essential for activation of the NLRP3 inflammasome. Thus, our study provides a novel theoretical basis for posttranslational modification of ASC in NLRP3 inflammasome activation and revealed that ADP/P2Y₁ is a potential drug target for inflammatory bowel disease.     

Beta1 integrin activation on human neutrophils promotes beta2 integrin-mediated adhesion to fibronectin

Although the importance of beta1 integrin-mediated binding to adhesion molecules and extracellular matrix (ECM) molecules is well established for most types of leukocytes, the expression patterns and functional importance of beta1 integrins on neutrophils have remained controversial. Using flow cytometry, we found that human neutrophils express the alpha4, alpha5, alpha9 and beta1 integrin subunits. To examine whether the integrins VLA-4 (alpha4/beta1) and VLA-5 (alpha5/beta1) have a functional role on neutrophils, we studied adhesion to their ligand fibronectin. Treatment of neutrophils with antibody 8A2, which specifically binds and activates beta1 integrins, resulted in increased binding to fibronectin. However, addition of blocking mAb revealed that 8A2-induced adhesion did not depend on beta1 integrins, but on the beta2 integrin CD11b/CD18. Similarly, activation of beta1 integrins by 8A2 resulted in CD11b-dependent binding of neutrophils to fibrinogen. 8A2 treatment increased expression of an activation epitope of CD11b/CD18, which depended on phosphoinositide 3-OH kinase activity and an adequate concentration of intracellular free Ca2+. These data suggest that engagement of beta1 integrins on neutrophils results in a cross-talk signal that leads to activation of the beta2 integrin CD11b/CD18, followed by CD11b-mediated adhesion. As transmigrated neutrophils are surrounded by both beta1 and beta2 ligands in the ECM, this integrin cross-talk could play a role in modifying migration and cellular activation in inflamed tissues.     

Regulation of adhesion of AML14.3D10 cells by surface clustering of beta2-integrin caused by ERK-independent activation of cPLA2

We examined the role of cell surface clustering of beta2-integrin caused by protein kinase C (PKC)-activated-cPLA2 in adhesion of eosinophilic AML14.3D10 (AML) cells. Phorbol 12-myristate 13-acetate (PMA) caused time- and concentration-dependent adhesion of AML cells to plated bovine serum albumin (BSA), which was blocked by anti-CD11b or anti-CD18 monoclonal antibodies (mAb) directed against beta2-integrin. Inhibition of PKC with Ro-31-8220 or rottlerin blocked PMA-induced cell adhesion in a concentration-dependent fashion. Inhibition of cytosolic phospholipase A2 (cPLA2) with trifluoromethyl ketone or methyl arachidonyl fluorophosphonate also blocked PMA-induced cell adhesion. PMA caused time-dependent p42/44 mitogen-activated protein kinase (MAPK) (ERK) phosphorylation in these cells. U0126, a MAPK/extracellular signal-regulated protein kinase kinase (MEK) inhibitor, at the concentrations that blocked PMA-induced ERK phosphorylation, had no effect on PMA stimulated AML cell adhesion. Neither p38 MAPK nor c-Jun N-terminal kinase (JNK) was phosphorylated by PMA. PMA also caused increased cPLA2 activity, which was inhibited by Ro-31-8220, but not U0126. Confocal immunofluorescence microscopy showed that PMA caused clustering of CD11b on the cell surface, which was blocked by either PKC or cPLA2 inhibition. PMA stimulation also caused up-regulation of CD11b on the AML cell surface. However, this up-regulation was not affected by cPLA2- or PKC-inhibition. Using the mAb, CBRM1/5, we also demonstrated that PMA does not induce the active conformation of CD11b/CD18. Our data indicate that PMA causes AML cell adhesion through beta2-integrin by PKC activation of cPLA2. This pathway is independent of MEK/ERK and does not require change of CD11b/CD18 to its active conformation. We find that avidity caused by integrin surface clustering - rather than conformational change or up-regulation of CD11b/CD18 - causes PMA stimulated adhesion of AML cells.     

IL-5 enhances the in vitro adhesion of human eosinophils, but not neutrophils, in a leucocyte integrin (CD11/18)-dependent manner.

In an attempt to explain the preferential accumulation of eosinophils at sites of allergic tissue reactions, we have studied the effects of interleukin-5 (IL-5) on the adherence of human eosinophils and neutrophils to plasma-coated glass (PCG) or human microvascular endothelial cells (HMVEC). IL-5 was compared with IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF) and platelet-activating factor (PAF), since all these agents have biological properties associated with eosinophil activation and/or survival in vitro. IL-5, IL-3 and GM-CSF induced a time-dependent increase in adherence of normal density eosinophils to PCG optimal at 60 min, whereas the effect of PAF was greater at 15 min. Similar results were obtained with neutrophils, with the exception that IL-5 had minimal and non-significant effects on this cell type. Unstimulated eosinophils and neutrophils also adhered to PCG or HMVEC, but in low numbers. Preincubation of eosinophils with IL-5, GM-CSF or PAF resulted in dose-dependent increases in the numbers of adherent cells to PCG. IL-3 had a smaller but significant effect on enhanced eosinophil adhesion to PCG, while IL-2 and lyso-PAF were ineffective. Neutrophils gave similar levels of baseline and stimulated adhesion to PCG as eosinophils, IL-5 again had no significant stimulatory effect. IL-5 also increased eosinophil, but not neutrophil, adherence to HMVEC in a concentration-dependent manner. Preincubation with the protein synthesis inhibitor cycloheximide had no effect on IL-5-, GM-CSF- or PAF-stimulated eosinophil adhesion. The contribution of the CD11/18 leucocyte integrins to IL-5- and PAF-induced eosinophil hyperadherence was investigated by inhibition experiments utilizing monoclonal antibodies (mAb). Enhanced adhesion to PCG (by PAF) or HMVEC (by IL-5) was inhibited by (ranked in order of potency) anti-CR3 alpha = common beta-chain greater than LFA-1 alpha. Anti-p150,95 alpha had no measurable effect. Baseline adhesion by unstimulated eosinophils was not significantly influenced by prior incubation with these mAb. Using flow cytometry, IL-5 and IL-3 were found to up-regulate cosinophil but not neutrophil CR3 expression. These findings demonstrate that IL-5 enhances cosinophil, but not neutrophil, adherence reactions, by a mechanism dependent, at least in part, on the CD11/18 family of adhesion glycoproteins.     

Protein phosphatase 1 is involved in IL-2-induced IL-5 and IL-13 expression in human T cells

IL-2 plays an important role in immunological and other biological functions. This cytokine directly induces the production of several cytokines, such as IL-5 and IL-13. The mechanisms of IL-2-mediated cytokine synthesis are mostly unclear; however, the involvement of IL-2 receptor (IL-2R)β has been suggested. In this study, the signaling molecule downstream of IL-2Rβ was investigated, employing a proteomic approach. Full-length IL-2Rβ and its mutant in which the intracellular component was truncated were introduced in an IL-2Rα- and IL-2Rγ-stably transfected T cell hybridoma, S1. The differential phosphorylation profiles of protein tyrosine residues in these cells upon IL-2 stimulation were examined by two-dimensional gel electrophoresis. The candidate phosphoproteins of interest were re-covered, in-gel digested and mass spectrometry fingerprinted. Among proteins specifically phosphorylated in full-length IL-2Rβ-expressing cells in response to IL-2 stimulation, protein phosphatase (PP)1β and FK506-binding protein 4 were identified. Particularly, PP1β augmented IL-5 and IL-13 expression stimulated by IL-2 but not by anti-CD3 antibody in human peripheral CD4+ T cells upon ectopic expression. IL-2-induced cytokine expression was suppressed by overexpression of PP1 regulatory subunit 2. A PP1 inhibitor, tautomycin, but not a PP2A inhibitor, okadaic acid, also inhibited the IL-2R-mediated responses. It was conclusively shown that PP1 is crucially involved in IL-2-mediated IL-5 and IL-13 synthesis in human T cells.     

IL-9 expression by human eosinophils: regulation by IL-1beta and TNF-alpha

BACKGROUND: IL-9 is a pleiotropic cytokine that exhibits biologic activity on cells of diverse hemopoietic lineage. IL-9 stimulates the proliferation of activated T cells, enhances the production of IgE from B cells, and promotes the proliferation and differentiation of mast cells and hematopoietic progenitors. OBJECTIVE: In this study we evaluated the expression of IL-9 messenger (m)RNA and protein by human peripheral blood eosinophils. We also investigated the role of IL-1beta and TNF-alpha in the release of IL-9 from human peripheral blood eosinophils. METHODS: RT-PCR, in situ hybridization, and immunocytochemistry were used to investigate the presence of IL-9 mRNA and protein in human peripheral blood eosinophils from asthmatic patients and normal control subjects. Furthermore, biologic assay was used to investigate the release of IL-9 protein from IL-1beta- or TNF-alpha-stimulated eosinophils in vitro. RESULTS: RT-PCR analysis showed the presence of IL-9 mRNA in human peripheral blood eosinophil RNA preparations from subjects with atopic asthma, as well as in the eosinophil-differentiated HL-60 cell line. By using in situ hybridization, a significant difference (P <.01) in IL-9 mRNA expression was detected in human peripheral blood eosinophils freshly isolated from asthmatic subjects compared with those isolated from normal control subjects. Furthermore, the percentage of IL-9 immunoreactive eosinophils from asthmatic patients was increased compared with that found in normal control subjects (P <.01). We also demonstrate that cultured human peripheral blood eosinophils from asthmatic subjects synthesize and release IL-9 protein, which is upregulated on stimulation with TNF-alpha and IL-1beta. CONCLUSION: Human eosinophils express biologically active IL-9, which suggests that these cells may influence the recruitment and activation of effector cells linked to the pathogenesis of allergic disease. These observations provide further evidence for the role of eosinophils in regulating airway immune responses.     

The mechanisms of serum-treated zymosan (STZ)-induced oxidative metabolism by human eosinophils and the effects of IL-5 priming

BACKGROUND: The aim of this work was to study the mechanisms of action of IL-5 on the subsequent stimulation of the oxidative metabolism of blood eosinophils by serum-treated zymosan (STZ), in terms of signal transduction characteristics, and by comparing the response of cells from healthy and allergic subjects during environmental exposure to birch pollen. METHODS: Eosinophils from healthy controls and allergic patients were purified to over 95% by Percoll gradients and the MACS system. Oxidative metabolism was measured by a lucigenin-enhanced chemiluminescence (CL) assay. Eosinophils were primed with IL-5 and subsequently stimulated with STZ. The signal transduction mechanisms of IL-5 priming were studied with the MEK inhibitor PD 98059,the PkC inhibitors staurosporine and Ro 318220, and the PI3 kinase inhibitor wortmannin. RESULTS: IL-5 increased the maximum radical production (P=0.0079) and reduced the t(1/2) rise (0.000018) of the CL reactions. The t(1/2) rise was PkC dependent and MEK independent, while the maximum radical production was PkC, MEK, and PI3 kinase dependent. During the pollen season, IL-5 reduced the total STZ-induced CL response in the patients' cells (P=0.016), but not in the control cells, whereas it primed the response to STZ of both cell populations in terms of the t(1/2) rise (P=0.012 and 0.00066, respectively). CONCLUSION: STZ-induced oxidative metabolism consists of different stages. The initial stage (t(1/2) rises of the curves) is PkC dependent and MEK independent, while the end stage (maximum radical production) is PkC, MEK, and PI3 kinase dependent. IL-5 shortened the initial stage, and increased the end stage. During allergen exposure, however, the end stage was reduced by IL-5. This could be due to increased amounts of hypodense eosinophils and/or some abnormality in cell responses.     

IL-10 suppresses CD2-mediated T cell activation via SHP-1

Interleukin (IL)-10 is an essential suppressive cytokine and plays a key role in peripheral T cell tolerance to allergens, autoantigens, transplantation antigens and tumor antigens. However, the molecular mechanisms of direct T cell suppression by IL-10 are not fully understood. Here, we demonstrate that IL-10 directly inhibits CD2 signaling in T cells. T cell stimulation via CD2 alone induces activation and proliferation, when endogenous IL-10 sources are eliminated from cultures. IL-10 utilizes the src-homology-2 domain containing tyrosine phosphatase (SHP-1) to directly suppress T cell activation. The role of SHP-1 in IL-10-mediated suppression of CD2 co-stimulation on T cells is demonstrated by using dominant-negative SHP-1 over-expressing T cells and silencing endogenous SHP-1 by small inhibitory RNA. Findings are confirmed using both SHP-1-deficient mice and IL-10-deficient mice. CD2-induced proliferation is suppressed by exogenous IL-10 in IL-10-deficient, but not SHP-1-deficient murine T cells. In conclusion, SHP-1-mediated inhibition of CD2 signaling represents a novel mechanism for direct T cell suppression by IL-10.     

Syk-dependent ERK activation regulates IL-2 and IL-10 production by DC stimulated with zymosan

Zymosan is a particulate yeast preparation that elicits high levels of IL-2 and IL-10 from dendritic cells (DC) and engages multiple innate receptors, including the Syk-coupled receptor dectin-1 and the MyD88-coupled receptor TLR2. Here, we show that induction of IL-2 and IL-10 by zymosan requires activation of ERK MAP kinase in murine DC. Surprisingly, ERK activation in response to zymosan is completely blocked in Syk-deficient DC and unaffected by MyD88 deficiency. Conversely, ERK activation in response to the TLR2 agonist Pam3Cys is completely MyD88 dependent and unaffected by Syk deficiency. The inability of TLR2 ligands in zymosan to couple to ERK may explain the Syk dependence of the IL-2 and IL-10 response in DC and emphasises the importance of Syk-coupled pattern recognition receptors such as dectin-1 in the detection of yeasts. Furthermore, the lack of receptor compensation observed here suggests that responses induced by complex innate stimuli cannot always be predicted by the signalling pathways downstream of individual receptors.     

IL-10 enhances IL-2-induced proliferation and cytotoxicity by human intestinal lymphocytes

IL-10 modulation of human intestinal T lymphocyte functions was studied for the first time. Lymphocyte proliferation was determined by 3H-thymidine incorporation; cytokine production, by ELISA; expression of surface markers, by immunofluorescence and flow cytometric analysis; and cytotoxicity, by lysis of 51Cr-labelled target cells. IL-10 blocked phytohaemagglutinin (PHA)-induced activation and proliferation of CD8+ T cells from the epithelium and lamina propria. It was a greater inhibitor of IL-2, interferon-gamma, and tumour necrosis factor-alpha production than were IL-4 or transforming growth factor-beta. In contrast, IL-10 enhanced IL-2-stimulated proliferation of both CD4+ and CD8+ T cells by increasing cell division after activation. It also augmented IL-2- but not IL-15-induced cytotoxicity of intestinal lymphocytes against colon cancer by a mechanism independent of natural killer cells. In conclusion, IL-10 blocking of proinflammatory cytokine secretion probably reduces intestinal inflammation. IL-10 augmentation of IL-2-induced cytotoxicity may help to maintain host defence.     

Effect of IL-5, glucocorticoid, and Fas ligation on Bcl-2 homologue expression and caspase activation in circulating human eosinophils

IL-5 is a potent eosinophil viability-enhancing factor that has been strongly implicated in the pathogenesis of IgE-mediated inflammation in vivo. Recently published data have suggested that IL-5 (and related cytokines) may act by altering the expression of the anti-apoptotic regulator Bcl-2 or its homologues, but this is controversial. The behaviour of the recently described pro-apoptotic cysteine proteases (caspases) in eosinophils after IL-5 treatment has not been explored. We examined the effect of IL-5 on the expression of four major Bcl-2 homologues, as well as on the expression/activation of key members of the caspase cell death cascade in cultured circulating human eosinophils. The effect of relevant inducers of eosinophil apoptosis (glucocorticoid and Fas ligation) on these regulatory proteins was also examined. We observed baseline expression of the anti-apoptotic Mcl-1 and pro-apoptotic Bax proteins in immunoblots of eosinophil lysates, but not Bcl-x, Bcl-2. IL-5 treatment had the effect of maintaining this basal level of expression over time without altering the balance of Bcl-2 homologues. The (upstream) caspase 8 and (downstream) caspase 3 proenzymes were detected in eosinophils at baseline, and were processed during spontaneous and stimulated eosinophil death. IL-5 completely blocked caspase processing in spontaneous and dexamethasone-induced cell death, and significantly slowed processing during Fas ligation. Our data do not support the theory that IL-5 acts by altering the balance of anti-apoptotic and pro-apoptotic Bcl-2 homologues, but suggest that it may act by regulating activation of the caspase cell death cascade.     

Transforming growth factor-β stimulates IL-1β-induced monocyte chemoattractant protein-1 expression in human synovial cells via the ERK/AP-1 pathway

Objective and Design: Transforming growth factor- β (TGF-β) has not only a fibrogenic role, but also monocyte/ macrophage chemotactic properties in a synovial joint. However, little is known about the effects of TGF-β on monocyte chemoattractant protein-1 (MCP-1) expression in human synovial cells under inflammatory status. The aim of this study was to determine whether TGF-modulates MCP-1 production under the chronic inflammation, and to elucidate the cell signaling mechanism involved. Materials and methods: Human synovial cells were exposed to IL-1β, which mimics the environment of chronic inflammation. Production of MCP-1 protein and expression of MCP-1 mRNA were determined by ELISA and real-time PCR. Results: TGF-β upregulated the expression of MCP-1 mRNA and protein with or without IL-1β. TGF-β and IL-1β synergistically enhanced MCP-1 gene expression, and an AP-1 binding site was involved in the signal transduction. In addition, MEK inhibitor completely suppressed TGF-β-induced MCP-1 production. Conclusions: TGF-β and IL-1β synergistically enhance MCP-1 gene expression through the activation of the MEK/ERK1/2 pathways, which leads to AP-1 activation. The impairment of MCP-1 regulation by TGF-β in resident synovial cells might represent an important mechanism of chronic inflammation and tissue fibrosis in a synovial joint. MCP-1 should be considered a valid target for therapeutic intervention. Received 22 September 2005; returned for revision 3 February 2006; accepted by J. Skotnicki 29 May 2006