Anissa T. Lambertino

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.

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.

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.

Phosphodiesterase 4 inhibition of β2-integrin adhesion caused by leukotriene B4 and TNF-α in human neutrophils

Phosphodiesterase (PDE)4 inhibition attenuates neutrophilic inflammation in chronic obstructive pulmonary disease. The objective of the present study was to examine the efficacy and mechanism by which PDE4 inhibition blocks adhesion of β2-integrin to an endothelial counterligand. Neutrophils (polymorphonuclear leukocytes (PMNs)) were isolated from humans receiving no medication. Adhesion was analysed by myeloperoxidase activity. The effects of cilomilast±salmeterol on the following were determined: 1) surface CD11b expression; 2) adhesion; 3) intracellular cyclic adenosine monophosphate (cAMP) concentration; and 4) extracellular signal-regulated kinase (ERK)-1/2-mediated group IVA-phospholipase A2 (gIVA-PLA2) phosphorylation caused by leukotriene (LT)B4 or tumour necrosis factor (TNF)-α activation. Either cilomilast or rolipram±salmeterol caused concentration-related blockade of LTB4-induced adhesion to counterligand, but had no effect on TNF-α-activated PMNs. A comparable increase in intracellular cAMP concentration for PMNs activated with LTB4 and TNF-α was caused by 1 μM cilomilast and 0.1 μM salmeterol. Upregulation of surface CD11b expression and ERK-1/2 phosphorylation were blocked by cilomilast or rolipram±salmeterol for PMNs activated by LTB4, but not for cells stimulated by TNF-α. Cilomilast±salmeterol also blocked gIVA-PLA2 phosphorylation caused by LTB4 but not TNF-α. In conclusion, the current study demonstrates that both leukotriene B4 and tumour necrosis factor-α upregulate cyclic adenosine monophosphate. However, cyclic adenosine monophosphate does not block β2-integrin adhesion caused by tumour necrosis factor-α. It was concluded that tumour necrosis factor-α prevents inhibition of extracellular signal-regulated kinase-1/2-mediated group IVA-phospholipase A2 activation, which is essential for β2-integrin adhesion in polymorphonuclear leukocytes.

Quantitation of secretory group V phospholipase A2 in human tissues by sandwich enzyme-linked immunosorbent assay

We have developed a sensitive sandwich ELISA (sELISA) for quantitative determination of group V phospholipase A(2) (gVPLA(2)). This assay utilizes three monoclonal antibodies (mAbs) directed against human gVPLA(2) (MCL-1B7, MCL-2A5, and MCL-3G1), which recognize specifically different epitopes of gVPLA(2). A mixture of MCL-1B7 and MCL-2A5 was used as the capture mAb, and MCL-3G1 as the detector mAb; purified human gVPLA(2) was used as the standard protein. The limit of detection of the sELISA is 2 ng/ml; the intra- and inter-coefficients of variation were 4.97+/-0.81% and 8.42+/-3.4%. The validity of the sELISA was assured by the recovery of exogenous recombinant gVPLA(2), which was 99.7% to 102%, and demonstration of noninterference of the gVPLA(2) assay by a high concentrations of other protein from murine lung and heart. To assess the usefulness of this sELISA for tissue measurements, the amount of gVPLA(2) in cultured human epithelial cells and isolated human eosinophils was determined. Total gVPLA(2) mass in epithelial cells was 2.83+/-0.33 ng/10(7) cells; gVPLA(2) was not detected in eosinophils. The presence of high concentration of gVPLA(2) in epithelial cells was confirmed by immunoprecipitation/Western blot analysis and by flow cytometry. This assay allows for convenient differentiation between the highly homologous 14-kDa secretory PLA(2)s, gVPLA(2), gIIaPLA(2), gIbPLA(2) and gXPLA(2), and accurate quantitation of gVPLA(2) in biological samples.

Regulation of adhesion of AML14.3D10 cells by surface clustering of β2-integrin caused by ERK-independent activation of cPLA2

We examined the role of cell surface clustering of β2‐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 β2‐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 β2‐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.