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Inhibition of Eosinophil Differentiation in vitro by a PPARγ Agonist
AB120 Abstracts
455
SUNDAY
Membrane Cholesterol Removal from Human Eosinophils Disrupts Cholesterol Rich Membrane Microdomains Resulting in Down Regulated MAP Kinase Signaling but not JAK/STAT Signaling M. E. Burnham, M. E. Bates, P. J. Bertics; University of Wisconsin School of Medicine and Public Health, Madison, WI. RATIONALE: Eosinophils contribute to allergic asthma exacerbation and can undergo excessive recruitment to the lungs where their activation leads to tissue damage and fibrosis. Interleukin-5 (IL-5) family cytokine receptors, which are critical for eosinophil recruitment/activation, are proposed to exist in membrane microdomains. Because cholesterol-rich microdomains are linked to signal regulation in diverse receptor systems, and because hypercholesterolemia is an asthma risk-factor, we tested the hypothesis that cholesterol-rich plasma membrane microdomains are central to IL-5-family cytokine action in eosinophils. METHODS: Purified human blood eosinophils were incubated (1 hr) with the cholesterol-chelating agent methyl-b-cyclodextrin (MbCD) or soluble cholesterol (MbCD pre-loaded with cholesterol), followed by cholesterol/ membrane microdomain analyses via flow cytometry and confocal microscopy or stimulation with IL-5. Eosinophil activation was determined via immunoblotting for activated p38 MAPK, activated transcriptional regulator STAT5, cyclin D3 (MAPK-dependent), or Pim1 (STAT-dependent). RESULTS: MbCD decreases, and soluble cholesterol increases, membrane cholesterol content in a dose-dependent manner as assessed by flow cytometry. Likewise, confocal microscopy confirmed MbCD disrupts membrane microdomains. Furthermore, MbCD attenuates IL-5-induced p38 phosphorylation compared to control (p<0.001, N510), whereas soluble cholesterol restores IL-5-induced p38 phosphorylation and significantly elevates basal phosphorylation (p<0.05, N510). Cyclin D3 upregulation is blocked by MbCD treatment but unaffected by soluble cholesterol addition (N53). Neither MbCD nor soluble cholesterol appears to alter IL-5-induced STAT5 phosphorylation (N53) or Pim1 up-regulation, suggesting a selective action of these agents (N52). CONCLUSIONS: These studies reveal that disturbances in eosinophil membrane cholesterol content selectively affect eosinophil signaling, suggesting that in vivo cholesterol levels may direct eosinophilic function and inflammatory capacity. Inhibition of Eosinophil Differentiation in vitro by a PPARg Agonist S. G. Smith, M. Hill, A. J. Baatjes, K. Howie, R. M. Watson, R. Sehmi, G. M. Gauvreau; McMaster University, Hamilton, ON, CANADA. RATIONALE: Peroxisome proliferator-activated receptor (PPAR) agonists have been observed to have an inhibitory effect on differentiation of several cell types, including erythroid cells. However, no study has examined the effects of PPAR agonists on IL-5 induced eosinophil differentiation. METHODS: Peripheral blood was drawn from forty atopic donors. Nonadherent mononuclear cells (NAMNCs) or CD34+ cells were grown for 2 weeks in MethocultÒ cultures stimulated with 10ng/mL IL-5 plus/minus 25ng/mL IL-3, in the presence of 1-1000nM PPARa agonist (GW9578), PPARb/d agonist (GW501516), PPARg agonist (rosiglitazone) or diluent. The number of eosinophil/basophil colony forming units (Eo/Bo CFU) was quantified using an inverted microscope at 40x magnification. The data were analyzed using a repeated measures ANOVA. RESULTS: The number of Eo/Bo CFU grown from NAMNC and CD34+ cells increased significantly in response to IL-5 and IL-5+IL-3 stimulation (p50.0001 and p<0.0001 respectively). Incubation of NAMNC and CD34+ cells with 10-1000nM rosiglitazone significantly inhibited the number of IL-5-induced Eo/Bo CFU (p50.0011 and p50.0078 respectively) and the number of IL-5+IL-3-induced Eo/B CFU (p50.0144) with no effect of GW9578 (P50.3) or GW501516 (P50.7). CONCLUSION: We observed an inhibitory effect of rosiglitazone on eosinophil differentiation in vitro, suggesting a mechanism whereby PPARg agonist can regulate the development of eosinophilia.
456
J ALLERGY CLIN IMMUNOL FEBRUARY 2012
457
Mast Cell Phenotypes and Distribution in Nasal Polyps with Allergic Airway E. E. Fouda1, S. Abdelgawad2, S. Said-Ahmed3, A. Ali4, E. Mohamed3; 1 Al-azhar university allergy &Immunology center, Cairo, EGYPT, 2Faculty of medicine(girls),Al-azhar university, Cairo, EGYPT, 3Faculty of medicine(Girls),Al-azhar university, Cairo, EGYPT, 4Faculty of medicine,Al-azhar university, Cairo, EGYPT. RATIONALE: Nasal polyps (NPs) is a multi-factorial disease, however, Four histological types were described; edematous, fibro-inflammatory, glandular and that with atypical stroma. We studied mast cell (MC) phenotypes and distribution in NPs of allergic patients. METHODS: Based on history, clinical examination, Para nasal C-T, high serum tIgE and +ve sIgE Ala top screening test, 31 allergic adults with NPs who underwent polypectomy were included. Paraffin sections of specimens from NPs and nasal turbinate mucosa were subjected to: staining with H&E,PAS and Masson trichrome to classify polyps and Tryptase and chymase immunohistochemical staining for MCs phenotypes. RESULTS: NPs were reclassified into: Edematous, fibrotic, MC tryptase (MCT) dominated the glandular type (11.33 6 0.95) while the tryptase Chymase (MCTC) dominated the fibrotic (12.96 6 11.06) Phenotype distribution showed dominant MCT at the stroma and at sub epithelium of glandular (11.33 6 0.95) and mixed polyps (4.38 6 3.28) respectively. While, MCTC dominated the stroma and sub epithelium of fibrotic(12.96 6 11.06) and edematous polyps (4.17 6 3.53) respectively. No MCs subset were found in epithelium of NPs except the fibrotic type((MCTC) (1.0660.55). MCT concentration expressed similarity in both; the polyps and nasal mucosa. While MCTC are preferentially increased in the edematous (2.54 6 1.31) and fibrotic polyps (12.96 6 11.06) than mucosa. CONCLUSIONS: Phenotypes and distribution of MCs suggests that allergy is one determining factor in NPs formation. MCT is responsible for glandular hyperplasia and MCTC for polyp fibrosis.
458
A Role for Mast Cell Chymase in Regulating Levels of Immunoglobulin E A. Fawzy1,2, T. Iwanaga1, A. R. McEuen1, B. L. Nicholas1, A. Mochizuki1, J. W. Holloway1, A. F. Walls1; 1University of Southampton, Southampton, UNITED KINGDOM, 2National Research Center, Cairo, EGYPT. RATIONALE: The His45Arg single nucleotide polymorphism (SNP) in the gene for mast cell chymase (CMA1) is associated with atopy and serum IgE levels in asthmatics. We have hypothesized that chymase is involved in IgE production, and that this SNP encodes an inactive form of the enzyme. METHODS: The cDNA for pre-pro-chymase was ligated into the baculovirus transfer vector BacPAK8 and transfected into SF9 cells. Prochymase was purified from supernatants by sequential application of heparin agarose and S-200 Sephacryl chromatography and subjected to Nterminal sequencing, SDS-PAGE and immunoblotting with chymasespecific antibodies. The recombinant pro-enzyme was activated with dipeptidyl peptidase I. The ability to cleave of suc-AAF-pNA and RETF-4NA was measured spectrophotometrically, and hydrolysis of angiotensin I was examined by size exclusion chromatography on HPLC. Cells of the U266B1 B cell myeloma line were incubated with chymases, and secretion of immunoglobulin E was measured by indirect ELISA. RESULTS: Recombinant wild-type chymase efficiently cleaved the chromogenic substrates and angiotensin I, but the variant was without any activity. Incubation of B cells with wild-type chymase (0.25-10ng/ml) stimulated an increase in supernatant IgE concentrations of more than fivefold after 5 days. However, at high concentrations of chymase (1001000ng/ml) there was a reduction in IgE levels as a consequence of IgE cleavage. The variant form of chymase or the heat-inactivated wild-type failed to provoke increased IgE secretion or cleavage of this immunoglobulin. CONCLUSIONS: Chymase may play key roles in the regulation of IgE levels. This may explain why expression of the inactive variant can offer protection from atopy in asthmatics.
455
SUNDAY
Membrane Cholesterol Removal from Human Eosinophils Disrupts Cholesterol Rich Membrane Microdomains Resulting in Down Regulated MAP Kinase Signaling but not JAK/STAT Signaling M. E. Burnham, M. E. Bates, P. J. Bertics; University of Wisconsin School of Medicine and Public Health, Madison, WI. RATIONALE: Eosinophils contribute to allergic asthma exacerbation and can undergo excessive recruitment to the lungs where their activation leads to tissue damage and fibrosis. Interleukin-5 (IL-5) family cytokine receptors, which are critical for eosinophil recruitment/activation, are proposed to exist in membrane microdomains. Because cholesterol-rich microdomains are linked to signal regulation in diverse receptor systems, and because hypercholesterolemia is an asthma risk-factor, we tested the hypothesis that cholesterol-rich plasma membrane microdomains are central to IL-5-family cytokine action in eosinophils. METHODS: Purified human blood eosinophils were incubated (1 hr) with the cholesterol-chelating agent methyl-b-cyclodextrin (MbCD) or soluble cholesterol (MbCD pre-loaded with cholesterol), followed by cholesterol/ membrane microdomain analyses via flow cytometry and confocal microscopy or stimulation with IL-5. Eosinophil activation was determined via immunoblotting for activated p38 MAPK, activated transcriptional regulator STAT5, cyclin D3 (MAPK-dependent), or Pim1 (STAT-dependent). RESULTS: MbCD decreases, and soluble cholesterol increases, membrane cholesterol content in a dose-dependent manner as assessed by flow cytometry. Likewise, confocal microscopy confirmed MbCD disrupts membrane microdomains. Furthermore, MbCD attenuates IL-5-induced p38 phosphorylation compared to control (p<0.001, N510), whereas soluble cholesterol restores IL-5-induced p38 phosphorylation and significantly elevates basal phosphorylation (p<0.05, N510). Cyclin D3 upregulation is blocked by MbCD treatment but unaffected by soluble cholesterol addition (N53). Neither MbCD nor soluble cholesterol appears to alter IL-5-induced STAT5 phosphorylation (N53) or Pim1 up-regulation, suggesting a selective action of these agents (N52). CONCLUSIONS: These studies reveal that disturbances in eosinophil membrane cholesterol content selectively affect eosinophil signaling, suggesting that in vivo cholesterol levels may direct eosinophilic function and inflammatory capacity. Inhibition of Eosinophil Differentiation in vitro by a PPARg Agonist S. G. Smith, M. Hill, A. J. Baatjes, K. Howie, R. M. Watson, R. Sehmi, G. M. Gauvreau; McMaster University, Hamilton, ON, CANADA. RATIONALE: Peroxisome proliferator-activated receptor (PPAR) agonists have been observed to have an inhibitory effect on differentiation of several cell types, including erythroid cells. However, no study has examined the effects of PPAR agonists on IL-5 induced eosinophil differentiation. METHODS: Peripheral blood was drawn from forty atopic donors. Nonadherent mononuclear cells (NAMNCs) or CD34+ cells were grown for 2 weeks in MethocultÒ cultures stimulated with 10ng/mL IL-5 plus/minus 25ng/mL IL-3, in the presence of 1-1000nM PPARa agonist (GW9578), PPARb/d agonist (GW501516), PPARg agonist (rosiglitazone) or diluent. The number of eosinophil/basophil colony forming units (Eo/Bo CFU) was quantified using an inverted microscope at 40x magnification. The data were analyzed using a repeated measures ANOVA. RESULTS: The number of Eo/Bo CFU grown from NAMNC and CD34+ cells increased significantly in response to IL-5 and IL-5+IL-3 stimulation (p50.0001 and p<0.0001 respectively). Incubation of NAMNC and CD34+ cells with 10-1000nM rosiglitazone significantly inhibited the number of IL-5-induced Eo/Bo CFU (p50.0011 and p50.0078 respectively) and the number of IL-5+IL-3-induced Eo/B CFU (p50.0144) with no effect of GW9578 (P50.3) or GW501516 (P50.7). CONCLUSION: We observed an inhibitory effect of rosiglitazone on eosinophil differentiation in vitro, suggesting a mechanism whereby PPARg agonist can regulate the development of eosinophilia.
456
J ALLERGY CLIN IMMUNOL FEBRUARY 2012
457
Mast Cell Phenotypes and Distribution in Nasal Polyps with Allergic Airway E. E. Fouda1, S. Abdelgawad2, S. Said-Ahmed3, A. Ali4, E. Mohamed3; 1 Al-azhar university allergy &Immunology center, Cairo, EGYPT, 2Faculty of medicine(girls),Al-azhar university, Cairo, EGYPT, 3Faculty of medicine(Girls),Al-azhar university, Cairo, EGYPT, 4Faculty of medicine,Al-azhar university, Cairo, EGYPT. RATIONALE: Nasal polyps (NPs) is a multi-factorial disease, however, Four histological types were described; edematous, fibro-inflammatory, glandular and that with atypical stroma. We studied mast cell (MC) phenotypes and distribution in NPs of allergic patients. METHODS: Based on history, clinical examination, Para nasal C-T, high serum tIgE and +ve sIgE Ala top screening test, 31 allergic adults with NPs who underwent polypectomy were included. Paraffin sections of specimens from NPs and nasal turbinate mucosa were subjected to: staining with H&E,PAS and Masson trichrome to classify polyps and Tryptase and chymase immunohistochemical staining for MCs phenotypes. RESULTS: NPs were reclassified into: Edematous, fibrotic, MC tryptase (MCT) dominated the glandular type (11.33 6 0.95) while the tryptase Chymase (MCTC) dominated the fibrotic (12.96 6 11.06) Phenotype distribution showed dominant MCT at the stroma and at sub epithelium of glandular (11.33 6 0.95) and mixed polyps (4.38 6 3.28) respectively. While, MCTC dominated the stroma and sub epithelium of fibrotic(12.96 6 11.06) and edematous polyps (4.17 6 3.53) respectively. No MCs subset were found in epithelium of NPs except the fibrotic type((MCTC) (1.0660.55). MCT concentration expressed similarity in both; the polyps and nasal mucosa. While MCTC are preferentially increased in the edematous (2.54 6 1.31) and fibrotic polyps (12.96 6 11.06) than mucosa. CONCLUSIONS: Phenotypes and distribution of MCs suggests that allergy is one determining factor in NPs formation. MCT is responsible for glandular hyperplasia and MCTC for polyp fibrosis.
458
A Role for Mast Cell Chymase in Regulating Levels of Immunoglobulin E A. Fawzy1,2, T. Iwanaga1, A. R. McEuen1, B. L. Nicholas1, A. Mochizuki1, J. W. Holloway1, A. F. Walls1; 1University of Southampton, Southampton, UNITED KINGDOM, 2National Research Center, Cairo, EGYPT. RATIONALE: The His45Arg single nucleotide polymorphism (SNP) in the gene for mast cell chymase (CMA1) is associated with atopy and serum IgE levels in asthmatics. We have hypothesized that chymase is involved in IgE production, and that this SNP encodes an inactive form of the enzyme. METHODS: The cDNA for pre-pro-chymase was ligated into the baculovirus transfer vector BacPAK8 and transfected into SF9 cells. Prochymase was purified from supernatants by sequential application of heparin agarose and S-200 Sephacryl chromatography and subjected to Nterminal sequencing, SDS-PAGE and immunoblotting with chymasespecific antibodies. The recombinant pro-enzyme was activated with dipeptidyl peptidase I. The ability to cleave of suc-AAF-pNA and RETF-4NA was measured spectrophotometrically, and hydrolysis of angiotensin I was examined by size exclusion chromatography on HPLC. Cells of the U266B1 B cell myeloma line were incubated with chymases, and secretion of immunoglobulin E was measured by indirect ELISA. RESULTS: Recombinant wild-type chymase efficiently cleaved the chromogenic substrates and angiotensin I, but the variant was without any activity. Incubation of B cells with wild-type chymase (0.25-10ng/ml) stimulated an increase in supernatant IgE concentrations of more than fivefold after 5 days. However, at high concentrations of chymase (1001000ng/ml) there was a reduction in IgE levels as a consequence of IgE cleavage. The variant form of chymase or the heat-inactivated wild-type failed to provoke increased IgE secretion or cleavage of this immunoglobulin. CONCLUSIONS: Chymase may play key roles in the regulation of IgE levels. This may explain why expression of the inactive variant can offer protection from atopy in asthmatics.