Evan Boetticher

Blockade of Inflammation and Airway Hyperresponsiveness in Immune-sensitized Mice by Dominant-Negative Phosphoinositide 3-Kinase-TAT

Phosphoinositide 3-kinase (PI3K) is thought to contribute to the pathogenesis of asthma by effecting the recruitment, activation, and apoptosis of inflammatory cells. We examined the role of class IA PI3K in antigen-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of Δp85 protein, a dominant negative form of the class IA PI3K regulatory subunit, p85α, which was fused to HIV-TAT (TAT-Δp85). Intraperitoneal administration of TAT-Δp85 caused time-dependent transduction into blood leukocytes, and inhibited activated phosphorylation of protein kinase B (PKB), a downstream target of PI3K, in lung tissues in mice receiving intranasal FMLP. Antigen challenge elicited pulmonary infiltration of lymphocytes, eosinophils and neutrophils, increase in mucus-containing epithelial cells, and airway hyperresponsiveness to methacholine. Except for modest airway neutrophilia, these effects all were blocked by treatment with 3–10 mg/kg of TAT-Δp85. There was also significant reduction in IL-5 and IL-4 secretion into the BAL. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by systemic pretreatment with TAT-Δp85. We conclude that PI3K has a regulatory role in Th2-cell cytokine secretion, airway inflammation, and airway hyperresponsiveness in mice.

Human Group V Phospholipase A2 Induces Group IVA Phospholipase A2-independent Cysteinyl Leukotriene Synthesis in Human Eosinophils

We previously reported that exogenously added human group V phospholipase A2 (hVPLA2) could elicit leukotriene B4 biosynthesis in human neutrophils through the activation of group IVA phospholipase A2 (cPLA2) (Kim, Y. J., Kim, K. P., Han, S. K., Munoz, N. M., Zhu, X., Sano, H., Leff, A. R., and Cho, W. (2002) J. Biol. Chem. 277, 36479-36488). In this study, we determined the functional significance and mechanism of the exogenous hVPLA2-induced arachidonic acid (AA) release and leukotriene C4 (LTC4) synthesis in isolated human peripheral blood eosinophils. As low a concentration as 10 nm exogenous hVPLA2 was able to elicit the significant release of AA and LTC4 from unstimulated eosinophils, which depended on its ability to act on phosphatidylcholine membranes. hVPLA2 also augmented the release of AA and LTC4 from eosinophils activated with formyl-Met-Leu-Phe + cytochalasin B. A cellular fluorescent PLA2 assay showed that hVPLA2 had a lipolytic action first on the outer plasma membrane and then on the perinuclear region. hVPLA2 also caused the translocation of 5-lipoxygenase from the cytosol to the nuclear membrane and a 2-fold increase in 5-lipoxygenase activity. However, hVPLA2 induced neither the increase in intracellular calcium concentration nor cPLA2 phosphorylation; consequently, cPLA2 activity was not affected by hVPLA2. Pharmacological inhibition of cPLA2 and the hVPLA2-induced activation of eosinophils derived from the cPLA2-deficient mouse corroborated that hVPLA2 mediates the release of AA and leukotriene in a cPLA2-independent manner. As such, this study represents a unique example in which a secretory phospholipase induces the eicosanoid formation in inflammatory cells, completely independent of cPLA2 activation.

Role of Mitogen-Activated Protein Kinase-Mediated Cytosolic Phospholipase A2 Activation in Arachidonic Acid Metabolism in Human Eosinophils

The objective of this investigation was to determine the role of secretory and cytosolic isoforms of phospholipase A2 (PLA2) in the induction of arachidonic acid (AA) and leukotriene synthesis in human eosinophils and the mechanism of PLA2 activation by mitogen-activated protein kinase (MAPK) isoforms in this process. Pharmacological activation of eosinophils with fMLP caused increased AA release in a concentration (EC50 = 8.5 nM)- and time-dependent (t1/2 = 3.5 min) manner. Both fMLP-induced AA release and leukotriene C4 (LTC4) secretion were inhibited concentration dependently by arachidonic trifluoromethyl ketone, a cytosolic PLA2 (cPLA2) inhibitor; however, inhibition of neither the 14-kDa secretory phospholipase A2 by 3-(3-acetamide-1-benzyl-2-ethylindolyl-5-oxy)propanephosphonic acid nor cytosolic Ca2+-independent phospholipase A2 inhibition by bromoenol lactone blocked hydrolysis of AA or subsequent leukotriene synthesis. Pretreatment of eosinophils with a mitogen-activated protein/extracellular signal-regulated protein kinase (ERK) kinase inhibitor, U0126, or a p38 MAPK inhibitor, SB203580, suppressed both AA production and LTC4 release. fMLP induced phosphorylation of MAPK isoforms, ERK1/2 and p38, which were evident after 30 s, maximal at 1–5 min, and declined thereafter. fMLP stimulation also increased cPLA2 activity in eosinophils, which was inhibited completely by 30 μM arachidonic trifluoromethyl ketone. Preincubation of eosinophils with U0126 or SB203580 blocked fMLP-enhanced cPLA2 activity. Furthermore, inhibition of Ras, an upstream GTP-binding protein of ERK, also suppressed fMLP-stimulated AA release. These findings demonstrate that cPLA2 activation causes AA hydrolysis and LTC4 secretion. We also find that cPLA2 activation caused by fMLP occurs subsequent to and is dependent upon ERK1/2 and p38 MAPK activation. Other PLA2 isoforms native to human eosinophils possess no significant activity in the stimulated production of AA or LTC4.

Blockade of Focal Clustering and Active Conformation in β2-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 β2-integrin adhesion, neither TAT-dnRas nor PD98059 blocked the eosinophil adhesion to VCAM-1. Thus, a substantially different signaling mechanism was identified for β1-integrin adhesion. We conclude that H-Ras-mediated activation of ERK is critical for β2-integrin adhesion and that Ras-protein functions as the common regulator for cytokine-, chemokine-, and G-protein-coupled receptors in human eosinophils.

Forkhead Box F1 Is Essential for Migration of Mesenchymal Cells and Directly Induces Integrin-Beta3 Expression

ABSTRACT The Forkhead box f1 (Foxf1) transcription factor is expressed in mesenchymal cells of the lung, liver, and gallbladder. Although Foxf1 deficiency causes severe abnormalities in the development of these organs, the molecular mechanisms underlying Foxf1 function remain uncharacterized. In this study we inactivated Foxf1 function in lung mesenchymal cells and mouse embryonic fibroblasts (MEFs) by use of either short interfering RNA duplexes or a membrane-transducing Foxf1 dominant negative (DN) mutant protein (Foxf1 DN), the latter of which is fused to the human immunodeficiency virus TAT protein transduction domain. Although Foxf1 did not influence DNA replication or cell survival, Foxf1 depletion severely diminished mesenchyme migration. Foxf1 deficiency in mesenchymal cells was associated with reduced expression of the integrin-beta3 (Itgβ3) subunit. Furthermore, we generated transgenic mice containing a tetracycline-inducible Foxf1 DN transgene. Adenovirus-mediated activation of Foxf1 DN in transgenic MEFs caused diminished cell migration and reduced Itgβ3 expression. A chromatin immunoprecipitation assay demonstrated that Foxf1 protein binds to the bp −871 to −815 region of the mouse Itgβ3 promoter. Deletion of the −871 to −815 Itgβ3 promoter region completely abolished the ability of Foxf1 to activate transcription of the Itgβ3 promoter in cotransfection experiments, indicating that the mouse Itgβ3 is a direct transcriptional target of Foxf1 protein. Foxf1 plays an essential role in mesenchyme migration by transcriptionally regulating Itgβ3.

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 A2 (cPLA2) activation in human eosinophil adhesion to plate-coated fibronectin (FN). In the control state, eosinophil adhesion was maximal, with 10 μg/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 cPLA2 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 cPLA2 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 cPLA2 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 cPLA2 phosphorylation caused by adhesion to FN. These results demonstrate that ERK1/2 mediating cPLA2 activation is essential for eosinophil adhesion to FN.

Transcellular Secretion of Group V Phospholipase A2 from Epithelium Induces β2-Integrin-Mediated Adhesion and Synthesis of Leukotriene C4 in Eosinophils

We examined the mechanism by which secretory group V phospholipase A2 (gVPLA2) secreted from stimulated epithelial cells activates eosinophil adhesion to ICAM-1 surrogate protein and secretion of leukotriene (LT)C4. Exogenous human group V PLA2 (hVPLA2) caused an increase in surface CD11b expression and focal clustering of this integrin, which corresponded to increased β2 integrin-mediated adhesion. Human IIaPLA2, a close homolog of hVPLA2, or W31A, an inactive mutant of hVPLA2, did not affect these responses. Exogenous lysophosphatidylcholine but not arachidonic acid mimicked the β2 integrin-mediated adhesion caused by hVPLA2 activation. Inhibition of hVPLA2 with MCL-3G1, a mAb against gVPLA2, or with LY311727, a global secretory phospholipase A2 (PLA2) inhibitor, attenuated the activity of hVPLA2; trifluoromethylketone, an inhibitor of cytosolic group IVA PLA2 (gIVA-PLA2), had no inhibitory effect on hVPLA2-mediated adhesion. Activation of β2 integrin-dependent adhesion by hVPLA2 did not cause ERK1/2 activation and was independent of gIVA-PLA2 phosphorylation. In other studies, eosinophils cocultured with epithelial cells were stimulated with FMLP/cytochalasin B (FMLP/B) and/or endothelin-1 (ET-1) before LTC4 assay. FMLP/B alone caused release of LTC4 from eosinophils, which was augmented by coculture with epithelial cells activated with ET-1. Addition of MCL-3G1 to cocultured cells caused ∼50% inhibition of LTC4 secretion elicited by ET-1, which was blocked further by trifluoromethylketone. Our data indicate that hVPLA2 causes focal clustering of CD11b and β2 integrin adhesion by a novel mechanism that is independent of arachidonic acid synthesis and gIVA-PLA2 activation. We also demonstrate that gVPLA2, endogenously secreted from activated epithelial cells, promotes secretion of LTC4 in cocultured eosinophils.

Blockade of Airway Inflammation and Hyperresponsiveness by HIV-TAT-Dominant Negative Ras

We have reported previously that HIV-TAT-dominant negative (dn) Ras inhibits eosinophil adhesion to ICAM-1 after activation by IL-5 and eotaxin. In this study, we evaluated the role of Ras in Ag-induced airway inflammation and hyperresponsiveness by i.p. administration into mice of dnRas, which was fused to an HIV-TAT protein transduction domain (TAT-dnRas). Uptake of TAT-dnRas (t1/2 = 12 h) was demonstrated in leukocytes after i.p. administration. OVA-sensitization significantly increased eosinophil and lymphocyte numbers in bronchoalveolar lavage fluid 24 h after final challenge. Treatment of animals with 3–10 mg/kg TAT-dnRas blocked the migration of eosinophils from 464 ± 91 × 103/ml to 288 ± 79 × 103/ml with 3 mg/kg of TAT-dnRas (p < 0.05), and further decreased to 116 ± 63 × 103/ml after 10 mg/kg TAT-dnRas (p < 0.01). Histological examination demonstrated that inflammatory cell infiltration (largely eosinophils and mononuclear cells) and mucin production around the airways caused by OVA were blocked by TAT-dnRas. OVA challenge also caused airway hyperresponsiveness to methacholine, which was dose dependently blocked by treatment with TAT-dnRas. TAT-dnRas also blocked Ag-induced IL-4 and IL-5, but not IFN-γ, production in lung tissue. Intranasal administration of IL-5 caused eosinophil migration into the airway lumen, which was attenuated by pretreatment with TAT-dnRas. By contrast, TAT-green fluorescent protein or dnRas lacking the TAT protein transduction domain did not block airway inflammation, cytokine production, or airway hyperresponsiveness. We conclude that Ras mediates Th2 cytokine production, airway inflammation, and airway hyperresponsiveness in immune-sensitized mice.

Additive blockade of β2‐integrin adhesion of eosinophils by salmeterol and fluticasone propionate

Migration of human eosinophils is regulated by integrin expression, conformational change, and activation of cytosolic phospholipase A2 (cPLA2). Corticosteroids have been shown to inhibit cPLA2 hydrolysis in human eosinophils. The objective of this study was to determine the mechanisms of fluticasone propionate (FP) alone or in combination with salmeterol (SM) in blocking adhesion mediated by β2‐integrin in human eosinophils. Human eosinophils were isolated by negative magnetic selection. β2‐integrin-mediated eosinophil adhesion was measured by residual eosinophil peroxidase activity. Eosinophils were pretreated for 12 h to 24 h with FP and with or without SM for 30 min. Both SM alone and FP alone inhibited eosinophil adhesion in concentration- and time-dependent manner. SM alone modestly (∼30%) inhibited interleukin (IL)‐5‐induced eosinophil adhesion. Blockade of IL‐5‐induced eosinophil adhesion caused by 10−7 M FP at 24 h was augmented by 10−7 M SM from 41.5% to 72.5%. Similar blockade was also observed for eotaxin-induced eosinophil adhesion. Neither SM, FP, nor FP+SM blocked either: 1) upregulation of CD11b surface expression; or 2) phosphorylation of cPLA2. Blockade of β2‐integrin-mediated eosinophil adhesion by fluticasone propionate is augmented by salmeterol. Decreased adhesion results from augmented blockade of nuclear translocation of cytosolic phospholipase A2 caused by addition of salmeterol to fluticasone.Migration of human eosinophils is regulated by integrin expression, conformational change, and activation of cytosolic phospholipase A2 (cPLA2). Corticosteroids have been shown to inhibit cPLA2 hydrolysis in human eosinophils. The objective of this study was to determine the mechanisms of fluticasone propionate (FP) alone or in combination with salmeterol (SM) in blocking adhesion mediated by beta 2-integrin in human eosinophils. Human eosinophils were isolated by negative magnetic selection. beta 2-integrin-mediated eosinophil adhesion was measured by residual eosinophil peroxidase activity. Eosinophils were pretreated for 12 h to 24 h with FP and with or without SM for 30 min. Both SM alone and FP alone inhibited eosinophil adhesion in concentration- and time-dependent manner. SM alone modestly (approximately 30%) inhibited interleukin (IL)-5-induced eosinophil adhesion. Blockade of IL-5-induced eosinophil adhesion caused by 10(-7) M FP at 24 h was augmented by 10(-7) M SM from 41.5% to 72.5%. Similar blockade was also observed for eotaxin-induced eosinophil adhesion. Neither SM, FP, nor FP + SM blocked either: 1) upregulation of CD11b surface expression; or 2) phosphorylation of cPLA2. Blockade of beta 2-integrin-mediated eosinophil adhesion by fluticasone propionate is augmented by salmeterol. Decreased adhesion results from augmented blockade of nuclear translocation of cytosolic phospholipase A2 caused by addition of salmeterol to fluticasone.

Characterization of monoclonal antibodies specific for 14-kDa human group V secretory phospholipase A2 (hVPLA2).

Secretory phospholipase A2 (PLA2) consists of several 14-kDa isoforms with extensive homology, which makes it difficult to identify a specific isoform. In this study, we have developed and characterized monoclonal antibodies (MAbs) directed specifically against human group V sPLA2 (hVPLA2) derived from cultured hybridomas. These hybridomas were produced from the fusion of BALB/c-derived myeloma s/p20-Ag14 and splenocytes from mice immunized with purified recombinant hVPLA2. Three hybridomas secreting MAbs, MCL-3G1, MCL-2A5, and MCL-1B7, were selected and subcloned on the basis of their specificity to recognize hVPLA2 using solid-phase enzyme-linked immunoadsorbent assay (ELISA). The purified MAbs demonstrated a common pattern of immunoreactivity to hVPLA2, but not to human group IIa isoform (hIIaPLA2). Isotype analysis indicates that these hybridomas are of the IgG1 type. Under reducing conditions, MCL-3G1 sensitively detected hVPLA2 and demonstrated no cross-reactivity to either hIIaPLA2 or group IV cytosolic PLA2. Although specific for hVPLA2, a relatively modest signal was recognized with MCL-1B7 and MCL-2A5. These newly developed MAbs allow for determination of tissue distribution and cell-specific functions of hVPLA2.