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Cultivated human lung mast cells express multiple serotonin receptors
Abstracts S187
J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
Differentiation of a Commercial Hematopoetic Stem Cell Line (KG-1) Into Basophilic Leukocytes L. H. K. Lim, G. E. K. Fong; Department of Physiology, Faculty of Medicine, National University of Singapore, Singapore, SINGAPORE. RATIONALE: Allergy is a type-1 hypersensitivity inflammatory response involving basophils and mast cells in asthmatic and allergic tissues when exposed to allergens after first exposure. The mechanisms involved in the activation and movement of basophils from the bloodstream to the tissues are not clearly defined, particularly in the relationship between basophil and mast cell. However, due to the low numbers of basophils in peripheral blood, the difficulty in obtaining relatively high levels of basophils to perform these studies was high. Thus in this study, we used a different approach of acquiring basophils using a hematopoeitic stem cell line KG-1 (ATCC), and characterized their differentiation into basophils using various concentrations and incubations of the basophil-activating cytokine IL-3. METHODS: KG-1 cells were pulsed with increasing concentrations of IL-3 (10, 20, 50 & 100ng/ml) for 4h, and consequently grown in IL-3-free media for 28 days. In some cases, some cells were repulsed at the 14th day. RESULTS: KG-1 cells pulsed with 10 ng/ml and 100 ng/ml IL-3 showed a slight expression of FcR1 by flow cytometry and western blotting after 28 days, while repulsing with 10ng/ml at day 14and pulsing with 50 ng/ml of IL-3 resulted in an increased presence of granular structures via staining Wright-giemsa staining. Further characterization of these cells showed increased expression of FCRI, BSP-1 and 2D7 staining at day 28, indicating that KG-1 cells have developed similar characteristics to basophils. CONCLUSION: These results are the first demonstration that commercially available hematopoeitic stem cell lines can be used to differentiate stem cells into basophils. Funding: Academic Research Fund
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Primary Human Mast Cells Express Specific Isoforms of SNARE Proteins L. E. Sander1, S. Bolat1, J. Klempnauer2, M. P. Manns1, S. C. Bischoff1, A. Lorentz1; 1Department of Gastroenterology, Hepatology and
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Endocrinology, Medical School of Hannover, Hannover, GERMANY, of Visceral- and Transplantsurgery, Medical School of Hannover, Hannover, GERMANY. RATIONALE: The signals leading to mast cell degranulation, namely Fc RI crosslinking, have been subject to intense investigations. However, the last events facilitating the fusion of granule and plasma membrane are still elusive. SNARE (Soluble NSF Attachment Protein Receptors) proteins are known to mediate the synaptic transmission. We analyzed whether human mast cell express SNAREs and which of them might be crucial for mast cell degranulation. METHODS: Human mast cells were isolated and purified from surgical specimen of intestinal mucosa, using enzymatic digestion and MACStechnique. SNARE-protein expression was demonstrated employing RTPCR and Western blot. Interaction of SNAREs was analyzed by immunoprecipitation. RESULTS: On the mRNA as well as on the protein level human mast cells express the tSNAREs (=target SNAREs) Stx-1B (not Stx-1A), Stx2, Stx-3, Stx-4 and SNAP-23, but not SNAP-25. mRNA was found for the vSNAREs (=vesicular SNAREs) VAMP-1, VAMP-2, VAMP-3, VAMP-7, and VAMP-8. So far, VAMP-1, VAMP-3, and VAMP-8 were analyzed and found on the protein level. VAMP-2, which has been demonstrated to play a key role in eosinophil exocytosis, was only expressed at very low levels. SNAP-23 formed very stable complexes with other SNAREs e.g. VAMP-3 and VAMP-8. CONCLUSIONS: Human mast cells express a specific pattern of SNARE isoforms which are able to form stable complexes. SNAREs might play a major role in human MC exocytosis; inhibition of which could represent a novel therapeutical approach in treatment of allergic disorders. 2Department
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Mast Cells in the Innate Immune Response of the Middle Ear
J. Ebmeyer1,2, M. Furukawa1,3, K. K. Pak1, U. Ebmeyer1, H. H. Sudhoff4, D. H. Broide5, A. F. Ryan1, S. I. Wasserman5; 1Surgery/Otolaryngology, UCSD, La Jolla, CA, 2Otolaryngology, Head and Neck Surgery, University of Wuerzburg, Wuerzburg, GERMANY, 3Otolaryngology, Head and Neck Surgery, Tohoku University School of Medicine, Sendai, JAPAN, 4 Otorhinolaryngology, Ruhr-University of Bochum, Bochum, GERMANY, 5 Medicine/Allergology, UCSD, La Jolla, CA. RATIONALE: New pathophysiologic concepts are needed to explain the clinically observed connection between the allergic diathesis and otitis media (OM). Since mast cells are the only leucocytes present in substantial numbers in the normal middle ear mucosa and are known to participate in innate immunity, we examined their contributions to the pathogenesis of OM. METHODS: Wild type (WT) mice, mast cell deficient (W/Wv) mice and W/Wv mice whose mast cell populations were restored by transplantation of bone marrow derived mast cells were challenged by injection of the middle ear with non-typeable H. influenzae (NTHi) to generate an inflammatory response dependent upon the innate immune system. Allergic responses were elicited by injection of ovalbumin into the middle ears of sensitized mice. The magnitude of the resulting otitis media was compared by measurement of mucosal hyperplasia and influx of inflammatory cells. RESULTS: The strength of the early phase of the inflammatory reaction to NTHi was significantly greater in WT compared to W/Wv mice (p=0.036). Restoring the mast cell population returned the reaction to that of WT animals. Combining bacteria with allergen challenge of sensitized WT but not W/Wv mice prolonged the inflammatory reaction. CONCLUSIONS: Our results indicate that mast cells account for a majority of the innate immune response of the middle ear and may link responses to allergy and infection in the middle ear mucosa. Thus the mast cell may be a critical control and integration element in the pathogenesis of otitis media and may be pertinent to its persistence. Funding: NIH, DFG
MONDAY
Cultivated Human Lung Mast Cells Express Multiple Serotonin Receptors E. S. Schulman1, H. C. Davidson2, H. Nishi1, B. A. Jameson2; 1Medicine, Drexel University College of Medicine, Philadelphia, PA, 2Biochemistry, Drexel University College of Medicine, Philadelphia, PA. RATIONALE: Serotonin (5HT) is a pluripotent biogenic amine that exerts a wide range of cellular responses via interaction with one or more of its 14 different cognate receptors (5HTR). It is not known if human lung mast cells (HLMC) express 5-HTR. METHODS: HLMC purified by previously reported techniques were cultured in stem cell factor and interleukin-4. At 5-8 weeks of culture, homogeneous populations were challenged with buffer or with a FcRI cross-linking antibody (22E7). RNA was isolated, quantified, and RTPCR performed to study 5HTR expression. In select experiments, cHLMC were challenged in the presence of pharmacological agents active at 5HTR and histamine release examined. RESULTS: Unactivated cHLMC consistently express receptors for 5HT1B, -1D, -1E, -2A, -2B, -4, -5A, and -7. Expressions of 5-HT1A, and -6 were weak and/or variable. In no case was expression detected for 5HT2C, 3A or 3B (n=5). Following 22E7 challenge, there were no significant changes in the mRNA expression levels for the 5HTRs except for the upregulation of 1f and 2A. HLMCs constitutively express mRNAs for THP1, the rate limiting enzyme for conversion of tryptophan to serotonin and the Serotonin Transporter (SERT), the cell surface 5HT uptake site. Structurally unrelated 5HTR-1A antagonists enhance, and -1B antagonists inhibit allergic degranulation. CONCLUSION: We have shown for the first time that multiple 5HTRs are expressed on HLMCs. HLMCs also express THP1 and SERT, key enzymes in 5HT synthesis and transport. Agents active at 5HT1A and -1B are potent modulators of degranulation. Funding: State of Pennsylvania Tobacco Formula Fund and Margaret Wolf Memorial Fund
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J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
Differentiation of a Commercial Hematopoetic Stem Cell Line (KG-1) Into Basophilic Leukocytes L. H. K. Lim, G. E. K. Fong; Department of Physiology, Faculty of Medicine, National University of Singapore, Singapore, SINGAPORE. RATIONALE: Allergy is a type-1 hypersensitivity inflammatory response involving basophils and mast cells in asthmatic and allergic tissues when exposed to allergens after first exposure. The mechanisms involved in the activation and movement of basophils from the bloodstream to the tissues are not clearly defined, particularly in the relationship between basophil and mast cell. However, due to the low numbers of basophils in peripheral blood, the difficulty in obtaining relatively high levels of basophils to perform these studies was high. Thus in this study, we used a different approach of acquiring basophils using a hematopoeitic stem cell line KG-1 (ATCC), and characterized their differentiation into basophils using various concentrations and incubations of the basophil-activating cytokine IL-3. METHODS: KG-1 cells were pulsed with increasing concentrations of IL-3 (10, 20, 50 & 100ng/ml) for 4h, and consequently grown in IL-3-free media for 28 days. In some cases, some cells were repulsed at the 14th day. RESULTS: KG-1 cells pulsed with 10 ng/ml and 100 ng/ml IL-3 showed a slight expression of FcR1 by flow cytometry and western blotting after 28 days, while repulsing with 10ng/ml at day 14and pulsing with 50 ng/ml of IL-3 resulted in an increased presence of granular structures via staining Wright-giemsa staining. Further characterization of these cells showed increased expression of FCRI, BSP-1 and 2D7 staining at day 28, indicating that KG-1 cells have developed similar characteristics to basophils. CONCLUSION: These results are the first demonstration that commercially available hematopoeitic stem cell lines can be used to differentiate stem cells into basophils. Funding: Academic Research Fund
746
Primary Human Mast Cells Express Specific Isoforms of SNARE Proteins L. E. Sander1, S. Bolat1, J. Klempnauer2, M. P. Manns1, S. C. Bischoff1, A. Lorentz1; 1Department of Gastroenterology, Hepatology and
747
Endocrinology, Medical School of Hannover, Hannover, GERMANY, of Visceral- and Transplantsurgery, Medical School of Hannover, Hannover, GERMANY. RATIONALE: The signals leading to mast cell degranulation, namely Fc RI crosslinking, have been subject to intense investigations. However, the last events facilitating the fusion of granule and plasma membrane are still elusive. SNARE (Soluble NSF Attachment Protein Receptors) proteins are known to mediate the synaptic transmission. We analyzed whether human mast cell express SNAREs and which of them might be crucial for mast cell degranulation. METHODS: Human mast cells were isolated and purified from surgical specimen of intestinal mucosa, using enzymatic digestion and MACStechnique. SNARE-protein expression was demonstrated employing RTPCR and Western blot. Interaction of SNAREs was analyzed by immunoprecipitation. RESULTS: On the mRNA as well as on the protein level human mast cells express the tSNAREs (=target SNAREs) Stx-1B (not Stx-1A), Stx2, Stx-3, Stx-4 and SNAP-23, but not SNAP-25. mRNA was found for the vSNAREs (=vesicular SNAREs) VAMP-1, VAMP-2, VAMP-3, VAMP-7, and VAMP-8. So far, VAMP-1, VAMP-3, and VAMP-8 were analyzed and found on the protein level. VAMP-2, which has been demonstrated to play a key role in eosinophil exocytosis, was only expressed at very low levels. SNAP-23 formed very stable complexes with other SNAREs e.g. VAMP-3 and VAMP-8. CONCLUSIONS: Human mast cells express a specific pattern of SNARE isoforms which are able to form stable complexes. SNAREs might play a major role in human MC exocytosis; inhibition of which could represent a novel therapeutical approach in treatment of allergic disorders. 2Department
748
Mast Cells in the Innate Immune Response of the Middle Ear
J. Ebmeyer1,2, M. Furukawa1,3, K. K. Pak1, U. Ebmeyer1, H. H. Sudhoff4, D. H. Broide5, A. F. Ryan1, S. I. Wasserman5; 1Surgery/Otolaryngology, UCSD, La Jolla, CA, 2Otolaryngology, Head and Neck Surgery, University of Wuerzburg, Wuerzburg, GERMANY, 3Otolaryngology, Head and Neck Surgery, Tohoku University School of Medicine, Sendai, JAPAN, 4 Otorhinolaryngology, Ruhr-University of Bochum, Bochum, GERMANY, 5 Medicine/Allergology, UCSD, La Jolla, CA. RATIONALE: New pathophysiologic concepts are needed to explain the clinically observed connection between the allergic diathesis and otitis media (OM). Since mast cells are the only leucocytes present in substantial numbers in the normal middle ear mucosa and are known to participate in innate immunity, we examined their contributions to the pathogenesis of OM. METHODS: Wild type (WT) mice, mast cell deficient (W/Wv) mice and W/Wv mice whose mast cell populations were restored by transplantation of bone marrow derived mast cells were challenged by injection of the middle ear with non-typeable H. influenzae (NTHi) to generate an inflammatory response dependent upon the innate immune system. Allergic responses were elicited by injection of ovalbumin into the middle ears of sensitized mice. The magnitude of the resulting otitis media was compared by measurement of mucosal hyperplasia and influx of inflammatory cells. RESULTS: The strength of the early phase of the inflammatory reaction to NTHi was significantly greater in WT compared to W/Wv mice (p=0.036). Restoring the mast cell population returned the reaction to that of WT animals. Combining bacteria with allergen challenge of sensitized WT but not W/Wv mice prolonged the inflammatory reaction. CONCLUSIONS: Our results indicate that mast cells account for a majority of the innate immune response of the middle ear and may link responses to allergy and infection in the middle ear mucosa. Thus the mast cell may be a critical control and integration element in the pathogenesis of otitis media and may be pertinent to its persistence. Funding: NIH, DFG
MONDAY
Cultivated Human Lung Mast Cells Express Multiple Serotonin Receptors E. S. Schulman1, H. C. Davidson2, H. Nishi1, B. A. Jameson2; 1Medicine, Drexel University College of Medicine, Philadelphia, PA, 2Biochemistry, Drexel University College of Medicine, Philadelphia, PA. RATIONALE: Serotonin (5HT) is a pluripotent biogenic amine that exerts a wide range of cellular responses via interaction with one or more of its 14 different cognate receptors (5HTR). It is not known if human lung mast cells (HLMC) express 5-HTR. METHODS: HLMC purified by previously reported techniques were cultured in stem cell factor and interleukin-4. At 5-8 weeks of culture, homogeneous populations were challenged with buffer or with a FcRI cross-linking antibody (22E7). RNA was isolated, quantified, and RTPCR performed to study 5HTR expression. In select experiments, cHLMC were challenged in the presence of pharmacological agents active at 5HTR and histamine release examined. RESULTS: Unactivated cHLMC consistently express receptors for 5HT1B, -1D, -1E, -2A, -2B, -4, -5A, and -7. Expressions of 5-HT1A, and -6 were weak and/or variable. In no case was expression detected for 5HT2C, 3A or 3B (n=5). Following 22E7 challenge, there were no significant changes in the mRNA expression levels for the 5HTRs except for the upregulation of 1f and 2A. HLMCs constitutively express mRNAs for THP1, the rate limiting enzyme for conversion of tryptophan to serotonin and the Serotonin Transporter (SERT), the cell surface 5HT uptake site. Structurally unrelated 5HTR-1A antagonists enhance, and -1B antagonists inhibit allergic degranulation. CONCLUSION: We have shown for the first time that multiple 5HTRs are expressed on HLMCs. HLMCs also express THP1 and SERT, key enzymes in 5HT synthesis and transport. Agents active at 5HT1A and -1B are potent modulators of degranulation. Funding: State of Pennsylvania Tobacco Formula Fund and Margaret Wolf Memorial Fund
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