- Email: [email protected]
Munc18 (SM) regulator, Munc18c
J ALLERGY CLIN IMMUNOL VOLUME 111, NUMBER 2
51.1%-+9.0%, respectively). C O N C L U S I O N S : LIRs are potent regulatory molecules on human basophils, and may be critical in the initiation, maintenance, and resolution of allergic inflammation.
Funding: Glaxo Smith Kline, N1H
570 Eosinophil SNAREs Are Critical Componentsfor Eosinophi, Peroxidase (EPO) Release and Are Associated with the Sec1/Munc18 (SM) Regulator,Munc18c M. R. Logan, S. O. Odemuyiwa, P. Lacy, R. Moqbel; Medicine, University of Alberta, Edmonton, AB, CANADA. RATIONALE: Exocytotic release of eosinophil granule-derived mediators is an important effector response contributing to allergic inflammation. Exocytosis of transport vesicles is generally dependent on the interaction of vesicle (v-) and target (plasma) membrane (t-) SNAREs (SNAP receptor), which is specifically blocked by botulinum (BoNT) and tetanus toxins. Eosinophils express the SNARE isoforms, VAMP-2, SNAP-23 and syntaxin-4. Sec 1/Munc 18 (mammalian homologue unc 18) proteins bind a "closed" conformation of syntaxin, unavailable for complex assembly, and are postulated to play a central role in SNARE assembly. Previous reports indicated that Muncl8c binds syntaxin-2 and 4. We sought to determine if SNAREs are required for granule-derived mediator release and examine the expression of Munc 18c in eosinophils. METHODS: Human eosinophils were preincubated with inhibitors in the presence of streptolysin-O, stimulated with Ca 2+ and GTPq,S and supernatants examined for EPO release. Expression of Munc 18c mRNA and protein was determined using RT-PCR and Western blot analysis of subcellular tractions, respectively. Syntaxin-4 immunoprecipitates from cell lysate and membrane fractions were generated and subjected to Western blotting. RESULTS: EPO was released in response to GTPTS and Ca 2§ in control permeabilized eosinophils (41 _+ 4%). Secretion was inhibited by 5-96% (_+ 2-8%) and 22-84% (_+ 2-5%) using anti-VAMP-2 and anti-syntaxin-4 antibodies (1-10 ~g/ml) respectively. BoNT-B (5-20 nM) inhibited release up to 98% ( - 1-7%). Muncl8c mRNA and protein was expressed in eosinophils. SNAP-23 and Muncl8c were coprecipitated in syntaxin-4 immunoprecipitates. C O N C L U S I O N S : Eosinophil mediator release is SNARE-dependent, and Muncl8c may be a critical molecule involved in the signaling cascade leading to vesicle docking.
Funding: C1HR, AHFMR
571 ExM~176 H u mand a n Role vIni Vitro v oof StudiesBI~176 Eosinophils in Angiogenesis: I. Puxeddu I, A. Alian 2, A. M. Piliponsky 1, A. Panet 2, F. Levi-Schafferi; Department of Pharmacology, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, ISRAEL, 2Department of Virology, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, ISRAEL. RATIONALE: Eosinophils (EOS) play a crucial role in late/chronic stages of allergic inflammation and are involved in reactions against parasites and in some autoimmune and neoplastic diseases as well as in fibrotic conditions. Angiogenesis, the growth and proliferation of new blood vessels from pre-existing microcirculati0n, has been observed during some of these conditions. EOS produce and release angiogenic factors such as Vas-
Abstracts
S211
cular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor-b, suggesting a link between EOS and angiogenesis. The aim of this study was to investigate the direct effect of EOS in angiogenesis. METHODS: Human peripheral blood EOS were purified (MACS), sonicated (EOSs) and added to rat aorta rings embedded in collagen and rat aortic endothelial cells (EC). Sprout formation from aorta rings, EC proliferation (3[H]-thymidine) and their VEGF production (semiquantitative RT-PCR) were evaluated. RESULTS: EOSs enhanced sprout tormation (6 • 105/400 ,ttl, 4 folds vs medium alone, p<0.001). Addition of increasing concentrations of EOSs to sub-confluent EC induced their proliferation in a concentration-dependent manner (10 s and 106 EOS/200 IM, 4.8 folds and 11.6 folds increase respectively, p<0.001). To assess the specific role of VEGFI2 I, ]65, EOSs were preincubated with anti-VEGF isoforms. This treatment significantly reduced EOSs-induced EC proliferation (500 ng/ml, 3.6 folds, p<0.001). EC incubated with l04 EOS were found to synthesize mRNA for VEGF (24 hrs). CONCLUSIONS: We have demonstrated that EOS induce angiogenesis in our models and that their mitogenic effect on EC is through preformed VEGF and EOS-induced EC VEGF production. Therefore, our data corroborate the importance of EOS in angiogenesis.
Funding: University Monies
572
w,,. ,ntegrin and Regulates Adhesion and Activation of Human Eosinophils
A. Terada, K. lijima, H. Kita; Medicine, Mayo Clinic, Rochester, MN. RATIONALE: A prominent feature of eosinophils, compared to neutrophils, is that eosinophils' effector functions, such as degranulation and superoxide production, are highly dependent on cellular adhesion. However, the molecular mechanism involved in this process is poorly understood. Because a tetraspanin, CD9, is uniquely expressed on eosinophils but not on neutrophils, we hypothesized that CD9 pivotally regulates eosinophil adhesion and the subsequent activation process. METHODS: Physical association between CD9 and a beta2 integrin, C D l l b / C D I 8 , was examined by immunoprecipitation and confocal microscopy. We examined the role of CD9 in eosinophil function by crosslinking CD9 with specific antibodies. RESULTS: lmmunoprecipitation of the eosinophils' surface CD9 with antiCD9 antibody co-precipitated CDI l b and CDI8 molecules; similarly, immunoprecipitation of CD 1 l b or CDI8 co-precipitated CD9. When examined by double immunofluorescence staining and confocal laser microscopy, CD9, but not CD32, was co-k)calized with C D I I b in pseudopods of eosinophils adherent to immobilized human serum albumin (HSA), a CDI lb/CDI8 ligand. Furthermore, crosslinking of surface CD9 by an antiCD9 antibody (clone PI/33/2) induced eosinophil adhesion to HSA; this cellular adhesion produced superoxide and released eosinophil-derived neurotoxin from the cells. Finally, antibodies to CDI lb or CDI8 inhibited both adhesion and activation of eosinophils induced by CD9-crosslinking. CONCLUSIONS: In human eosinophils, CD9 is physically associated with a beta2 integrin, CDI Ib/CDI8, and it positively regulates the function of this integrin and effector functions of eosinophils. CD9 is likely a critical accessory molecule in the inflammatory responses of human eosinophils.
Funding: NIH
51.1%-+9.0%, respectively). C O N C L U S I O N S : LIRs are potent regulatory molecules on human basophils, and may be critical in the initiation, maintenance, and resolution of allergic inflammation.
Funding: Glaxo Smith Kline, N1H
570 Eosinophil SNAREs Are Critical Componentsfor Eosinophi, Peroxidase (EPO) Release and Are Associated with the Sec1/Munc18 (SM) Regulator,Munc18c M. R. Logan, S. O. Odemuyiwa, P. Lacy, R. Moqbel; Medicine, University of Alberta, Edmonton, AB, CANADA. RATIONALE: Exocytotic release of eosinophil granule-derived mediators is an important effector response contributing to allergic inflammation. Exocytosis of transport vesicles is generally dependent on the interaction of vesicle (v-) and target (plasma) membrane (t-) SNAREs (SNAP receptor), which is specifically blocked by botulinum (BoNT) and tetanus toxins. Eosinophils express the SNARE isoforms, VAMP-2, SNAP-23 and syntaxin-4. Sec 1/Munc 18 (mammalian homologue unc 18) proteins bind a "closed" conformation of syntaxin, unavailable for complex assembly, and are postulated to play a central role in SNARE assembly. Previous reports indicated that Muncl8c binds syntaxin-2 and 4. We sought to determine if SNAREs are required for granule-derived mediator release and examine the expression of Munc 18c in eosinophils. METHODS: Human eosinophils were preincubated with inhibitors in the presence of streptolysin-O, stimulated with Ca 2+ and GTPq,S and supernatants examined for EPO release. Expression of Munc 18c mRNA and protein was determined using RT-PCR and Western blot analysis of subcellular tractions, respectively. Syntaxin-4 immunoprecipitates from cell lysate and membrane fractions were generated and subjected to Western blotting. RESULTS: EPO was released in response to GTPTS and Ca 2§ in control permeabilized eosinophils (41 _+ 4%). Secretion was inhibited by 5-96% (_+ 2-8%) and 22-84% (_+ 2-5%) using anti-VAMP-2 and anti-syntaxin-4 antibodies (1-10 ~g/ml) respectively. BoNT-B (5-20 nM) inhibited release up to 98% ( - 1-7%). Muncl8c mRNA and protein was expressed in eosinophils. SNAP-23 and Muncl8c were coprecipitated in syntaxin-4 immunoprecipitates. C O N C L U S I O N S : Eosinophil mediator release is SNARE-dependent, and Muncl8c may be a critical molecule involved in the signaling cascade leading to vesicle docking.
Funding: C1HR, AHFMR
571 ExM~176 H u mand a n Role vIni Vitro v oof StudiesBI~176 Eosinophils in Angiogenesis: I. Puxeddu I, A. Alian 2, A. M. Piliponsky 1, A. Panet 2, F. Levi-Schafferi; Department of Pharmacology, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, ISRAEL, 2Department of Virology, Faculty of Medicine, The Hebrew University of Jerusalem, Jerusalem, ISRAEL. RATIONALE: Eosinophils (EOS) play a crucial role in late/chronic stages of allergic inflammation and are involved in reactions against parasites and in some autoimmune and neoplastic diseases as well as in fibrotic conditions. Angiogenesis, the growth and proliferation of new blood vessels from pre-existing microcirculati0n, has been observed during some of these conditions. EOS produce and release angiogenic factors such as Vas-
Abstracts
S211
cular Endothelial Growth Factor (VEGF) and Fibroblast Growth Factor-b, suggesting a link between EOS and angiogenesis. The aim of this study was to investigate the direct effect of EOS in angiogenesis. METHODS: Human peripheral blood EOS were purified (MACS), sonicated (EOSs) and added to rat aorta rings embedded in collagen and rat aortic endothelial cells (EC). Sprout formation from aorta rings, EC proliferation (3[H]-thymidine) and their VEGF production (semiquantitative RT-PCR) were evaluated. RESULTS: EOSs enhanced sprout tormation (6 • 105/400 ,ttl, 4 folds vs medium alone, p<0.001). Addition of increasing concentrations of EOSs to sub-confluent EC induced their proliferation in a concentration-dependent manner (10 s and 106 EOS/200 IM, 4.8 folds and 11.6 folds increase respectively, p<0.001). To assess the specific role of VEGFI2 I, ]65, EOSs were preincubated with anti-VEGF isoforms. This treatment significantly reduced EOSs-induced EC proliferation (500 ng/ml, 3.6 folds, p<0.001). EC incubated with l04 EOS were found to synthesize mRNA for VEGF (24 hrs). CONCLUSIONS: We have demonstrated that EOS induce angiogenesis in our models and that their mitogenic effect on EC is through preformed VEGF and EOS-induced EC VEGF production. Therefore, our data corroborate the importance of EOS in angiogenesis.
Funding: University Monies
572
w,,. ,ntegrin and Regulates Adhesion and Activation of Human Eosinophils
A. Terada, K. lijima, H. Kita; Medicine, Mayo Clinic, Rochester, MN. RATIONALE: A prominent feature of eosinophils, compared to neutrophils, is that eosinophils' effector functions, such as degranulation and superoxide production, are highly dependent on cellular adhesion. However, the molecular mechanism involved in this process is poorly understood. Because a tetraspanin, CD9, is uniquely expressed on eosinophils but not on neutrophils, we hypothesized that CD9 pivotally regulates eosinophil adhesion and the subsequent activation process. METHODS: Physical association between CD9 and a beta2 integrin, C D l l b / C D I 8 , was examined by immunoprecipitation and confocal microscopy. We examined the role of CD9 in eosinophil function by crosslinking CD9 with specific antibodies. RESULTS: lmmunoprecipitation of the eosinophils' surface CD9 with antiCD9 antibody co-precipitated CDI l b and CDI8 molecules; similarly, immunoprecipitation of CD 1 l b or CDI8 co-precipitated CD9. When examined by double immunofluorescence staining and confocal laser microscopy, CD9, but not CD32, was co-k)calized with C D I I b in pseudopods of eosinophils adherent to immobilized human serum albumin (HSA), a CDI lb/CDI8 ligand. Furthermore, crosslinking of surface CD9 by an antiCD9 antibody (clone PI/33/2) induced eosinophil adhesion to HSA; this cellular adhesion produced superoxide and released eosinophil-derived neurotoxin from the cells. Finally, antibodies to CDI lb or CDI8 inhibited both adhesion and activation of eosinophils induced by CD9-crosslinking. CONCLUSIONS: In human eosinophils, CD9 is physically associated with a beta2 integrin, CDI Ib/CDI8, and it positively regulates the function of this integrin and effector functions of eosinophils. CD9 is likely a critical accessory molecule in the inflammatory responses of human eosinophils.
Funding: NIH