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Eosinophil Adhesion to Junctional Adhesion Molecules In Vitro
CONCLUSIONS: Tissue staining for cellular and extracellular EDN is a useful tool to evaluate eosinophilic inflammation in EE. Reliance solely on eosinophil counts may overlook patients with marked eosinophil degranulation, but with few intact eosinophils. Funding: NIH Grant AI 34486
CONCLUSIONS: Eosinophil adhesion to junctional adhesion molecules is a novel finding and may provide a therapeutic target in ocular and other types of allergic inflammation. Funding: NIH/NEI
175
Phospho-Protein Analysis of Intracellular Signaling Molecules in Eosinophils using Luminex System K. Yamamura, T. Adachi, T. Masuda, H. Nagase, K. Ohta; Teikyo University School of Medicine, Tokyo, JAPAN. RATIONALE: Eosinophils play a pivotal role in the pathogenesis of asthma, especially in epithelial remodeling of airways. Thus it is of paramount importance to investigate the mechanism of eosinophil activation. Although a number of factors including cytokines/chemokines activate eosinophils, the potency of each stimulus to phosphorylate intracellular molecules and activate eosinophils remains to be elucidated. In the present study, we performed inclusive analyses of protein phosphorylation and the functional relevance in cytokine production of eosinophils. METHODS: Blood eosinophils were purified using Percoll and antiCD16 antibody-coated magnetic beads. Purified eosinophils were stimulated with the various stimuli. The lysates of eosinophils were subjected to phospho-protein analysis using Luminex System. In some experiments with the application of Luminex, we studied the effect of some signaling inhibitors on cytokine production from eosinophils. RESULTS: We found that several factors such as IL-5, eotaxin, PAF, and PGD2 phosphorylate Akt, ATF-2, CREB, ERK1/2, GSK-3, and p38 in eosinophils. Because eotaxin most potently induced production of various cytokines, we performed the inhibition study in eosinophils stimulated with eotaxin. The eotaxin-induced production of G-CSF, IL-1b, IL-6, IL-8, MIP-1b, and TNF-a was significantly reduced by an inhibitor against MEK (PD98059), p38 (SB203580), or PI3K (LY294002). In contrast, the GSK-3 inhibitor SB216763 blocked only IL-1b and TNF-a production from eosinophils. CONCLUSIONS: In terms of phosphorylation of intracellular signaling molecules, we could quantify the potency of various stimuli that activate eosinophils. The strategy using Luminex System is extremely useful to examine the mechanism of eosinophil activation. Funding: Ministry of Health, Labor and Welfare of Japan
CLC-3 Channels and Migration of Eosinophils in Asthma: Effect of TGF-b1 B. Moore1, G. Cheng1, Z. Shao1, A. K. Bewtra2, D. K. Agrawal1; 1Department of Biomedical Sciences, Creighton University, Omaha, NE, 2Department of Internal Medicine, Creighton University, Omaha, NE. RATIONALE: Asthma is a chronic airway disease characterized by inflammation and remodeling of the airway tissue. Eosinophils are major effector cells in inflammation and remodeling in asthma, and TGF-b is important in the asthmatic response, particularly in airway remodeling. Therefore, we examined the effect of TGF-b on the function of human eosinophils, as well as differences between allergic asthmatic and nonasthmatic eosinophils. METHODS: Eosinophils were isolated from the peripheral blood of healthy and allergic asthmatic human donors. They were stimulated with TGF-b1, or rottlerin, NPPB, or DIDS prior to TGF-b1 stimulation. Protein was extracted for Western blotting, and RNA was extracted for RT-PCR. Patch-clamping was used to measure chloride channel activity, and chemotaxis was assessed with a Boyden microchamber. Transmigration was measured using a transwell, and shape change was determined by flow cytometry. RESULTS: In normal eosinophils, TGF-b1 increased CLC-3 mRNA and protein expression. CLC-3 mRNA and protein expression was also increased in allergic asthmatic subjects as compared to non-asthmatics. Increases in chloride channel activity, chemotaxis, transmigration, and shape change were induced by TGF-b1 in normal eosinophils. The chloride channel inhibitors, DIDS and NPPB, reduced the TGF-b1-stimulated chemotaxis, transmigration, and shape change. The PKC-d inhibitor, rottlerin, blocked TGF-b1-induced chloride channel activity and chemotaxis. CONCLUSIONS: These results demonstrate an important role for TGFb1 in the migration of eosinophils involving CLC-3 channels in asthmatic patients. Thus, the CLC-3 channels could be a target in the treatment of chronic asthma. Funding: NIH
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Eosinophil Adhesion to Junctional Adhesion Molecules In Vitro E. B. Cook, J. L. Stahl, K. E. Fox, J. B. Sedgwick, N. P. Barney, F. M. Graziano; University of Wisconsin-Madison, Madison, WI. RATIONALE: Maintenance of eosinophils on the conjunctival epithelium is an important feature of ocular allergic inflammation, yet the specific receptors involved have not been identified. The purpose of this study was to examine eosinophil adhesion to junctional adhesion molecules (JAM) A, B and C; and the potential role of JAM in eosinophil adhesion to conjunctival epithelial cells. METHODS: Peripheral blood eosinophil adhesion to recombinant JAM-A, JAM-B or JAM-C coated plates and a human conjunctival epithelial cell line (IOBA-NHC, Valladolid, Spain) were evaluated using an eosinophil peroxidase assay of adherent eosinophils. Eosinophil adhesion was stimulated with fMLP or PAF. IOBA-NHC cells were pre-treated with proinflammatory cytokines and/or blocking antibody to JAM-A. IOBA-NHC cells were also examined for surface expression of JAM-A and JAM-C using flow cytometry. RESULTS: Unstimulated eosinophil adhesion to recombinant JAM-A, B, or C was increased compared to uncoated wells (p < 0.05). Moreover, eosinophils stimulated with fMLP or PAF were more adherent to JAM-A, B, and C than to uncoated wells (p < 0.05). IOBA-NHC cells expressed JAM-A and JAM-C. Pro-inflammatory cytokine stimulation of IOBA-NHC cells enhanced eosinophil adhesion, while a blocking JAM-A antibody inhibited eosinophil adhesion to levels below that of unstimulated IOBA-NHC cells.
Neutrophil Proteases Activate Eosinophil Function In Vitro Y. Noma, K. Hosoki, M. Nagao, R. Tokuda, T. Fujisawa; Institute for Clinical Research, Mie National Hospital, Tsu, Mie, JAPAN. RATIONALE: Recent evidence suggests that both neutrophilic and eosinophilic inflammation persists in the airways of patients with severe asthma. Neutrophils secrete a variety of serine proteases such as neutrophil elastase, cathepsin G and myeloblastin (proteinase 3: PR3), which have been shown to increase in BALF of severe asthmatic patients. To investigate possible interaction between eosinophils and neutrohils in severe asthma, we examined the effect of neutrophil proteases on eosinophil functions. METHODS: Peripheral blood eosinophils were stimulated with elastase, cathepsin G and PR3 and superoxide generation was measured with cytochrome c reduction method. Eosinophils were also cultured with the proteases and a panel of cytokines and chemokines in the supernatants were measured with a multiplex beads array system. RESULTS: Superoxide production was induced by the proteases and the order of relative potency was elastase > cathepsin G >> PR3. A serine protease inhibitor, PMSF, suppressed the reaction, suggesting that the effect of proteases were mediated through proteinase-activated receptor 2 on eosinophils. The proteases also induced production of IL-6 and TNFa, proinfalmmatory cytokines, and IL-8 and GROa, neutrophilotactic chemokines, with the order of potency being cathepsin G, PR3 > elastase. CONCLUSIONS: These results suggest that massive neutrophil infiltration in the airways seen in severe asthma may further aggravate inflammation through activation and cytokine/chemokine production from eosinophils. Funding: Ministry of Health, Welfare, and Labor of Japan
SATURDAY
Abstracts S45
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
CONCLUSIONS: Eosinophil adhesion to junctional adhesion molecules is a novel finding and may provide a therapeutic target in ocular and other types of allergic inflammation. Funding: NIH/NEI
175
Phospho-Protein Analysis of Intracellular Signaling Molecules in Eosinophils using Luminex System K. Yamamura, T. Adachi, T. Masuda, H. Nagase, K. Ohta; Teikyo University School of Medicine, Tokyo, JAPAN. RATIONALE: Eosinophils play a pivotal role in the pathogenesis of asthma, especially in epithelial remodeling of airways. Thus it is of paramount importance to investigate the mechanism of eosinophil activation. Although a number of factors including cytokines/chemokines activate eosinophils, the potency of each stimulus to phosphorylate intracellular molecules and activate eosinophils remains to be elucidated. In the present study, we performed inclusive analyses of protein phosphorylation and the functional relevance in cytokine production of eosinophils. METHODS: Blood eosinophils were purified using Percoll and antiCD16 antibody-coated magnetic beads. Purified eosinophils were stimulated with the various stimuli. The lysates of eosinophils were subjected to phospho-protein analysis using Luminex System. In some experiments with the application of Luminex, we studied the effect of some signaling inhibitors on cytokine production from eosinophils. RESULTS: We found that several factors such as IL-5, eotaxin, PAF, and PGD2 phosphorylate Akt, ATF-2, CREB, ERK1/2, GSK-3, and p38 in eosinophils. Because eotaxin most potently induced production of various cytokines, we performed the inhibition study in eosinophils stimulated with eotaxin. The eotaxin-induced production of G-CSF, IL-1b, IL-6, IL-8, MIP-1b, and TNF-a was significantly reduced by an inhibitor against MEK (PD98059), p38 (SB203580), or PI3K (LY294002). In contrast, the GSK-3 inhibitor SB216763 blocked only IL-1b and TNF-a production from eosinophils. CONCLUSIONS: In terms of phosphorylation of intracellular signaling molecules, we could quantify the potency of various stimuli that activate eosinophils. The strategy using Luminex System is extremely useful to examine the mechanism of eosinophil activation. Funding: Ministry of Health, Labor and Welfare of Japan
CLC-3 Channels and Migration of Eosinophils in Asthma: Effect of TGF-b1 B. Moore1, G. Cheng1, Z. Shao1, A. K. Bewtra2, D. K. Agrawal1; 1Department of Biomedical Sciences, Creighton University, Omaha, NE, 2Department of Internal Medicine, Creighton University, Omaha, NE. RATIONALE: Asthma is a chronic airway disease characterized by inflammation and remodeling of the airway tissue. Eosinophils are major effector cells in inflammation and remodeling in asthma, and TGF-b is important in the asthmatic response, particularly in airway remodeling. Therefore, we examined the effect of TGF-b on the function of human eosinophils, as well as differences between allergic asthmatic and nonasthmatic eosinophils. METHODS: Eosinophils were isolated from the peripheral blood of healthy and allergic asthmatic human donors. They were stimulated with TGF-b1, or rottlerin, NPPB, or DIDS prior to TGF-b1 stimulation. Protein was extracted for Western blotting, and RNA was extracted for RT-PCR. Patch-clamping was used to measure chloride channel activity, and chemotaxis was assessed with a Boyden microchamber. Transmigration was measured using a transwell, and shape change was determined by flow cytometry. RESULTS: In normal eosinophils, TGF-b1 increased CLC-3 mRNA and protein expression. CLC-3 mRNA and protein expression was also increased in allergic asthmatic subjects as compared to non-asthmatics. Increases in chloride channel activity, chemotaxis, transmigration, and shape change were induced by TGF-b1 in normal eosinophils. The chloride channel inhibitors, DIDS and NPPB, reduced the TGF-b1-stimulated chemotaxis, transmigration, and shape change. The PKC-d inhibitor, rottlerin, blocked TGF-b1-induced chloride channel activity and chemotaxis. CONCLUSIONS: These results demonstrate an important role for TGFb1 in the migration of eosinophils involving CLC-3 channels in asthmatic patients. Thus, the CLC-3 channels could be a target in the treatment of chronic asthma. Funding: NIH
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Eosinophil Adhesion to Junctional Adhesion Molecules In Vitro E. B. Cook, J. L. Stahl, K. E. Fox, J. B. Sedgwick, N. P. Barney, F. M. Graziano; University of Wisconsin-Madison, Madison, WI. RATIONALE: Maintenance of eosinophils on the conjunctival epithelium is an important feature of ocular allergic inflammation, yet the specific receptors involved have not been identified. The purpose of this study was to examine eosinophil adhesion to junctional adhesion molecules (JAM) A, B and C; and the potential role of JAM in eosinophil adhesion to conjunctival epithelial cells. METHODS: Peripheral blood eosinophil adhesion to recombinant JAM-A, JAM-B or JAM-C coated plates and a human conjunctival epithelial cell line (IOBA-NHC, Valladolid, Spain) were evaluated using an eosinophil peroxidase assay of adherent eosinophils. Eosinophil adhesion was stimulated with fMLP or PAF. IOBA-NHC cells were pre-treated with proinflammatory cytokines and/or blocking antibody to JAM-A. IOBA-NHC cells were also examined for surface expression of JAM-A and JAM-C using flow cytometry. RESULTS: Unstimulated eosinophil adhesion to recombinant JAM-A, B, or C was increased compared to uncoated wells (p < 0.05). Moreover, eosinophils stimulated with fMLP or PAF were more adherent to JAM-A, B, and C than to uncoated wells (p < 0.05). IOBA-NHC cells expressed JAM-A and JAM-C. Pro-inflammatory cytokine stimulation of IOBA-NHC cells enhanced eosinophil adhesion, while a blocking JAM-A antibody inhibited eosinophil adhesion to levels below that of unstimulated IOBA-NHC cells.
Neutrophil Proteases Activate Eosinophil Function In Vitro Y. Noma, K. Hosoki, M. Nagao, R. Tokuda, T. Fujisawa; Institute for Clinical Research, Mie National Hospital, Tsu, Mie, JAPAN. RATIONALE: Recent evidence suggests that both neutrophilic and eosinophilic inflammation persists in the airways of patients with severe asthma. Neutrophils secrete a variety of serine proteases such as neutrophil elastase, cathepsin G and myeloblastin (proteinase 3: PR3), which have been shown to increase in BALF of severe asthmatic patients. To investigate possible interaction between eosinophils and neutrohils in severe asthma, we examined the effect of neutrophil proteases on eosinophil functions. METHODS: Peripheral blood eosinophils were stimulated with elastase, cathepsin G and PR3 and superoxide generation was measured with cytochrome c reduction method. Eosinophils were also cultured with the proteases and a panel of cytokines and chemokines in the supernatants were measured with a multiplex beads array system. RESULTS: Superoxide production was induced by the proteases and the order of relative potency was elastase > cathepsin G >> PR3. A serine protease inhibitor, PMSF, suppressed the reaction, suggesting that the effect of proteases were mediated through proteinase-activated receptor 2 on eosinophils. The proteases also induced production of IL-6 and TNFa, proinfalmmatory cytokines, and IL-8 and GROa, neutrophilotactic chemokines, with the order of potency being cathepsin G, PR3 > elastase. CONCLUSIONS: These results suggest that massive neutrophil infiltration in the airways seen in severe asthma may further aggravate inflammation through activation and cytokine/chemokine production from eosinophils. Funding: Ministry of Health, Welfare, and Labor of Japan
SATURDAY
Abstracts S45
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2