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Neuropeptides Activate Human Mast Cell Degranulation and Chemokine Production
S68 Abstracts
SATURDAY
Vespid Venom-Induced IL-4- and IL-13-Production of Basophils is Inhibited By IL-10 C. Bauer, J. Walter, F. Ruëff, B. Przybilla; Ludwig-Maximilians-Universität München, Klinik und Poliklinik für Dermatologie und Allergologie, München, GERMANY. RATIONALE: Basophils are target cells of venom immunotherapy (VIT) especially in the early period of treatment. They show a decreased allergen-induced histamine and leukotriene release after reaching the maintenance dose. A decreased release of these mediators is also achieved by incubating basophils with IL-10. As basophils also synthesize the Th2cytokines IL-4 and IL-13, we were interested whether the vespid venom(VV-) induced IL-4- and IL-13-production of basophils from VV-allergic patients might be inhibited by IL-10 as well. METHODS: 20 individuals with anaphylaxis to a yellow jacket sting and sensitization to VV were investigated. After preincubation with IL-3 patients’ basophils were challenged with VV (1000 ng/ml) in the presence of different concentrations of IL-10 (0, 1, 10 or 100 ng/ml). The IL-4- and IL-13-concentration in the supernatants was measured by ELISA. RESULTS: There was a significant (p<0.05) reduction of the VVinduced IL-4-production from 1.182 to 0.796 pg/ml in the presence of 100 ng/ml of IL-10. Lower concentrations of IL-10 exerted no significant effect on the IL-4-production. The IL-13-production was inhibited by IL10 significantly (p<0.01) in a dose-dependent manner: The median decreased from 4.96 pg/ml (without IL-10) to 2.86 pg/ml (1 ng/ml), 1.28 pg/ml (10 ng/ml), or 0.80 pg/ml (100 ng/ml), respectively. CONCLUSIONS: IL-10, which is produced by T regulatory cells, is able to inhibit the IL-4- and IL-13-production of basophils. Changes of mediator release from these cells might not only play an important role in the early treatment period of VIT, but also in the long-term downregulation of the Th2 immune response.
264
CysLT1 and CysLT2 Receptors Form Functional Heterodimers on Human Mast Cells and Neutrophils Y. Jiang, J. Boyce; Medicine, Brigham and Women’s Hospital, Boston, MA. RATIONALE: CysLT1 and CysLT2 receptors are G protein-coupled receptors (GPCRs) that are specific in their binding of cysteinyl leukotrienes (cys-LTs). Several GPCRs are reported to function as homoor heterodimeric complexes. Our group has previously demonstrated that human mast cells (hMCs) derived in vitro from cord blood express both of these receptor subtypes, but that certain cys-LT-dependent signaling functions (particularly calcium flux and ERK MAP kinase phosphorylation) were completely dependent on CysLT1 receptors. METHODS: We used specific polyclonal antibodies and immunoprecipitation from hMCs and from neutrophils to determine whether CysLT1 and CysLT2 receptor proteins form heteromeric complexes. RESULTS: CysLT1 and CysLT2 receptors co-precipitate with one another from extracts of both hMCs and peripheral blood neutrophils. This association was specific and did not occur between either cys-LT receptor and other related GPCRs. Neutrophils responded to a partial CysLT2 receptor agonist, BAY-u9773, with a calcium flux that was nearly completely blocked by pre-treatment of the cells with a CysLT1 receptorselective antagonist, MK571. CONCLUSIONS: Collectively, these observations indicate that CysLT1 and CysLT2 receptors function as a heterodimeric complex to mediate cys-LT-dependent proinflammatory functions in hematopoietic effector cells. Funding: NIH/NIAID
265
Neuropeptides Activate Human Mast Cell Degranulation and Chemokine Production B. P. Tancowny, C. Sheen, L. C. Grammer, R. P. Schleimer, M. Kulka; Allergy-Immunology, Northwestern University Feinberg Sch. Med., Chicago, IL.
266
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
RATIONALE: During stress-induced inflammatory disease, mast cells may respond to stimuli such as neuropeptides in an Fc RI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to other stimuli such as IgE/antiIgE and 48/80. METHODS: Primary cultured mast cells, generated from CD34+ progenitors in the presence of SCF and IL-6, and human cultured mast cells (LAD2) were stimulated with SP, NGF, CGRP, VIP, C3a, C5a and 48/80 and their degranulation and chemokine production was assessed. RESULTS: VIP, SP, C3a and C5a stimulated primary human mast cells and LAD cells to degranulate; CGRP and NGF did not activate degranulation. While anti-IgE stimulation induced relatively small amounts of MCP-1, stimulation with VIP, SP or 48/80 potently induced production of MCP-1. Similarly, anti-IgE stimulation did not induce production of IP10, MIG, RANTES or IL-8. However, VIP, SP and 48/80 induced production of IP-10, RANTES and IL-8. C5a and C3a induced production of IP-10 and MCP-1. VIP, C5a, SP and 48/80 activated TNF, and GM-CSF release, but not IL-4, IFN- or eotaxin. CONCLUSIONS: SP, VIP, C3a and C5a activate human mast cell degranulation and chemokine production. Furthermore, the chemokine profile activated by SP and VIP is distinct from that of other G-protein coupled receptor activators such as C3a and C5a. Therefore, SP and VIP activate a unique signaling pathway in human mast cells, which may be relevant to stress-induced inflammatory diseases. Funding: NIH
267
Opiates Activate Human Mast Cell Chemokine Production
C. Sheen, L. C. Grammer, R. P. Schleimer, M. Kulka; Allgery-Immunology Division, Northwestern University Feinberg Sch. Med., Chicago, IL. RATIONALE: Activation of mast cells and the systemic release of histamine is a common side effect of opioids. In some individuals, codeine not only elicits a sizable early response due to degranulation, but can also lead to late cutaneous allergic inflammation characterized by leukocyte recruitment. Therefore, we determined whether activation of human mast cells leads to the generation of chemokines. METHODS: Cultured human mast cells (LAD2) were stimulated with codeine and meperidine and degranulation and chemokine production was measured. Mast cells were also pretreated with inhibitors to examine signaling pathways involved in opiate activation. RESULTS: Codeine and meperidine activated human mast cell degranulation within 30 min in a concentration-dependent manner. Degranulation was blocked by the protein kinase A (PKA) inhibitor, H89 and the phosphoinositol-3 kinase (PI3K) inhibitor, wortmannin, but not by Ro-318220, a PKC inhibitor or forskolin, a cAMP agonist. Codeine also activated human mast cells to release monocyte chemoattractant protein-1 (MCP-1, 2.23 + 0.07 ng/106 cells), RANTES (61.3 + 6.15 ng/106 cells) and interleukin (IL)-8 (180.7 + 3.3 ng/106 cells) but not inducible protein10 (IP-10) or monokine induced by IFN-gamma (MIG) suggesting that opiates activate the nuclear factor (NF)-kappaB pathway but not the JAK/STAT signaling pathways. CONCLUSIONS: Codeine and meperidine activate human mast cell degranulation and chemokine production by activating PKA and PI3 kinase possibly leading to NF-kappaB activation. Activation by codeine led to release of the chemokines MCP-1, RANTES and IL-8. Therefore, opiates may regulate allergic inflammation by activating chemokine production by human mast cells. Funding: Northwestern University
SATURDAY
Vespid Venom-Induced IL-4- and IL-13-Production of Basophils is Inhibited By IL-10 C. Bauer, J. Walter, F. Ruëff, B. Przybilla; Ludwig-Maximilians-Universität München, Klinik und Poliklinik für Dermatologie und Allergologie, München, GERMANY. RATIONALE: Basophils are target cells of venom immunotherapy (VIT) especially in the early period of treatment. They show a decreased allergen-induced histamine and leukotriene release after reaching the maintenance dose. A decreased release of these mediators is also achieved by incubating basophils with IL-10. As basophils also synthesize the Th2cytokines IL-4 and IL-13, we were interested whether the vespid venom(VV-) induced IL-4- and IL-13-production of basophils from VV-allergic patients might be inhibited by IL-10 as well. METHODS: 20 individuals with anaphylaxis to a yellow jacket sting and sensitization to VV were investigated. After preincubation with IL-3 patients’ basophils were challenged with VV (1000 ng/ml) in the presence of different concentrations of IL-10 (0, 1, 10 or 100 ng/ml). The IL-4- and IL-13-concentration in the supernatants was measured by ELISA. RESULTS: There was a significant (p<0.05) reduction of the VVinduced IL-4-production from 1.182 to 0.796 pg/ml in the presence of 100 ng/ml of IL-10. Lower concentrations of IL-10 exerted no significant effect on the IL-4-production. The IL-13-production was inhibited by IL10 significantly (p<0.01) in a dose-dependent manner: The median decreased from 4.96 pg/ml (without IL-10) to 2.86 pg/ml (1 ng/ml), 1.28 pg/ml (10 ng/ml), or 0.80 pg/ml (100 ng/ml), respectively. CONCLUSIONS: IL-10, which is produced by T regulatory cells, is able to inhibit the IL-4- and IL-13-production of basophils. Changes of mediator release from these cells might not only play an important role in the early treatment period of VIT, but also in the long-term downregulation of the Th2 immune response.
264
CysLT1 and CysLT2 Receptors Form Functional Heterodimers on Human Mast Cells and Neutrophils Y. Jiang, J. Boyce; Medicine, Brigham and Women’s Hospital, Boston, MA. RATIONALE: CysLT1 and CysLT2 receptors are G protein-coupled receptors (GPCRs) that are specific in their binding of cysteinyl leukotrienes (cys-LTs). Several GPCRs are reported to function as homoor heterodimeric complexes. Our group has previously demonstrated that human mast cells (hMCs) derived in vitro from cord blood express both of these receptor subtypes, but that certain cys-LT-dependent signaling functions (particularly calcium flux and ERK MAP kinase phosphorylation) were completely dependent on CysLT1 receptors. METHODS: We used specific polyclonal antibodies and immunoprecipitation from hMCs and from neutrophils to determine whether CysLT1 and CysLT2 receptor proteins form heteromeric complexes. RESULTS: CysLT1 and CysLT2 receptors co-precipitate with one another from extracts of both hMCs and peripheral blood neutrophils. This association was specific and did not occur between either cys-LT receptor and other related GPCRs. Neutrophils responded to a partial CysLT2 receptor agonist, BAY-u9773, with a calcium flux that was nearly completely blocked by pre-treatment of the cells with a CysLT1 receptorselective antagonist, MK571. CONCLUSIONS: Collectively, these observations indicate that CysLT1 and CysLT2 receptors function as a heterodimeric complex to mediate cys-LT-dependent proinflammatory functions in hematopoietic effector cells. Funding: NIH/NIAID
265
Neuropeptides Activate Human Mast Cell Degranulation and Chemokine Production B. P. Tancowny, C. Sheen, L. C. Grammer, R. P. Schleimer, M. Kulka; Allergy-Immunology, Northwestern University Feinberg Sch. Med., Chicago, IL.
266
J ALLERGY CLIN IMMUNOL FEBRUARY 2006
RATIONALE: During stress-induced inflammatory disease, mast cells may respond to stimuli such as neuropeptides in an Fc RI-independent manner. In this study, we characterized human mast cell responses to substance P (SP), nerve growth factor (NGF), calcitonin gene-related peptide (CGRP) and vasoactive intestinal polypeptide (VIP) and compared these responses to human mast cell responses to other stimuli such as IgE/antiIgE and 48/80. METHODS: Primary cultured mast cells, generated from CD34+ progenitors in the presence of SCF and IL-6, and human cultured mast cells (LAD2) were stimulated with SP, NGF, CGRP, VIP, C3a, C5a and 48/80 and their degranulation and chemokine production was assessed. RESULTS: VIP, SP, C3a and C5a stimulated primary human mast cells and LAD cells to degranulate; CGRP and NGF did not activate degranulation. While anti-IgE stimulation induced relatively small amounts of MCP-1, stimulation with VIP, SP or 48/80 potently induced production of MCP-1. Similarly, anti-IgE stimulation did not induce production of IP10, MIG, RANTES or IL-8. However, VIP, SP and 48/80 induced production of IP-10, RANTES and IL-8. C5a and C3a induced production of IP-10 and MCP-1. VIP, C5a, SP and 48/80 activated TNF, and GM-CSF release, but not IL-4, IFN- or eotaxin. CONCLUSIONS: SP, VIP, C3a and C5a activate human mast cell degranulation and chemokine production. Furthermore, the chemokine profile activated by SP and VIP is distinct from that of other G-protein coupled receptor activators such as C3a and C5a. Therefore, SP and VIP activate a unique signaling pathway in human mast cells, which may be relevant to stress-induced inflammatory diseases. Funding: NIH
267
Opiates Activate Human Mast Cell Chemokine Production
C. Sheen, L. C. Grammer, R. P. Schleimer, M. Kulka; Allgery-Immunology Division, Northwestern University Feinberg Sch. Med., Chicago, IL. RATIONALE: Activation of mast cells and the systemic release of histamine is a common side effect of opioids. In some individuals, codeine not only elicits a sizable early response due to degranulation, but can also lead to late cutaneous allergic inflammation characterized by leukocyte recruitment. Therefore, we determined whether activation of human mast cells leads to the generation of chemokines. METHODS: Cultured human mast cells (LAD2) were stimulated with codeine and meperidine and degranulation and chemokine production was measured. Mast cells were also pretreated with inhibitors to examine signaling pathways involved in opiate activation. RESULTS: Codeine and meperidine activated human mast cell degranulation within 30 min in a concentration-dependent manner. Degranulation was blocked by the protein kinase A (PKA) inhibitor, H89 and the phosphoinositol-3 kinase (PI3K) inhibitor, wortmannin, but not by Ro-318220, a PKC inhibitor or forskolin, a cAMP agonist. Codeine also activated human mast cells to release monocyte chemoattractant protein-1 (MCP-1, 2.23 + 0.07 ng/106 cells), RANTES (61.3 + 6.15 ng/106 cells) and interleukin (IL)-8 (180.7 + 3.3 ng/106 cells) but not inducible protein10 (IP-10) or monokine induced by IFN-gamma (MIG) suggesting that opiates activate the nuclear factor (NF)-kappaB pathway but not the JAK/STAT signaling pathways. CONCLUSIONS: Codeine and meperidine activate human mast cell degranulation and chemokine production by activating PKA and PI3 kinase possibly leading to NF-kappaB activation. Activation by codeine led to release of the chemokines MCP-1, RANTES and IL-8. Therefore, opiates may regulate allergic inflammation by activating chemokine production by human mast cells. Funding: Northwestern University