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Human Mast Cells Express Neurokinin and Vasoactive Intestinal Peptide Receptors
Estradiol Enhances the Release of Allergic Mediator from Bone Marrow-derived Mast Cells (BMMCs) Via Estrogen Receptor (ER) S. Narita, R. M. Goldblum, C. K. Lambert, M. Zaitsu, M. D. Estes, E. G. Brooks, T. Midoro-Horiuti; Pediatrics, UTMB, Galveston, TX. RATIONALE: A role for female hormones in the increased prevalence of asthma in women has been suggested. We have reported that estradiol (E2) potentiates the release of allergic mediators from a rat mast cell line. To define the role of classic ERs in this effect, we examined the expression and role of these receptors in BMMCs from wild-type (WT) and ER knockout (KO) mice. METHODS: The expression of ER and were assessed by RT-PCR. The effects of E2 ± a nominal allergen (DNP-BSA) on degranulation was assessed by -hexosaminidase release, expressed as the mean+SE of the percentage of the total cellular content. RESULTS: BMMCs of WT mice expressed mRNA for ER, but not ER. E2 (100 pM) induced release of -hexosaminidase from WT BMMCs (7.5+1.5 %), but not from the ER KO. IgE-dependent release from WT BMMCs (3.5+1.0 %) increased (6.1+1.0 %) in the presence of 10 pM of E2 (p<0.05). CONCLUSIONS: These results indicate that BMMCs, like rat mast cell lines, express ER. Exposure to physiological doses of E2 induce the release of a substantial portion of their preformed mediators and augmented IgE-dependent release. The lack of E2-mediated release of hexosaminidase from ER KO BMMCs suggests that ER is required for this activity. This finding may help to identify new approaches for treating women with asthma. Funding: Center for Interdisciplinary Research in Women`s health UTMB
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Alpha 4 Integrins and VCAM-1, but not MAdCAM-1, are Essential for Recruitment of Mast Cell Progenitors to Inflamed Lung M. F. Gurish1, J. Hallgren1, T. Jones1, J. Boyce1, J. Abonia2; 1Rheumatology, Immunology, Allergy, Brigham and Women’s Hospital, Boston, MA, 2Pediatric Allergy and Immunology, Cincinnati Children’s Hospital, Cincinnati, OH. RATIONALE: There are increased numbers of mast cell progenitors (MCp) in the lungs of sensitized mice after exposure to aerosolized antigen. This increase in MCp is attenuated in mice lacking the 7 integrins or VCAM-1 addressin but not in mice lacking E integrins. As this represents an atypical integrin/addressin pairing associated with inflammatory recruitment to the lung, we chose to further define which of the 4 integrins participate in this recruitment and investigated which endothelial ligands they bind to, MAdCAM-1 and/or VCAM-1. METHODS: Mice were sensitized by ip injection with ovalbumin (OVA) adsorbed to alum. Following 3 daily challenges with aerosolized OVA, the number of MCp in the lungs were evaluated using a clonogenic assay. The involvement of different adhesive molecules was tested by injecting the animals with blocking antibody just prior to the aerosol challenge. RESULTS: In mice receiving antibody to 4 or 1 integrins, the increment in MCp was reduced by 69% and 63% respectively, while in mice receiving anti-7 integrin, only a 33% reduction was noted. Mice given anti-E integrin showed normal increments in lung MCp. Mice given anti-VCAM-1 also showed reduced numbers of MCp (76%) while mice given MAdCAM-1 had a slightly greater than normal increment. CONCLUSIONS: These observations provide the first insight into the adhesion pathways required for MCp recruitment to the inflamed lung, demonstrating the interaction of both the 41 and 47 integrins, with VCAM-1, and not MAdCAM-1; a process crucial to the development of the reactive intraepithelial mastocytosis. Funding: NIH
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Expression of Neuropilin-1 (NP-1) on Human Basophils Following In Vitro IL-3 Exposure: Implications for NP-1 as a Marker of Basophil Priming J. T. Schroeder, A. P. Bieneman, M. C. Liu; Department of Medicine, Johns Hopkins University, Baltimore, MD. RATIONALE: Since basophils functionally respond to IL-3 by secreting enhanced levels of mediators important in allergic inflammation, we investigated whether novel phenotypic markers are associated with this priming effect. METHODS: Blood basophils were purified (>97%) using density centrifugation and negative selection. Surface staining was assessed by flow cytometry, gene expression by RT-PCR, basophil cytokine and histamine release (HR) by ELISA and fluorimetry, respectively. RESULTS: Basophils cultured in IL-3 dose-dependently up-regulated the neuronal receptor NP-1, as defined by staining with Blood Dendritic Cell Antigen (BDCA)-4 antibody. NP-1 expression was observed within 16h on 18±1% of the basophils cultured in 10 ng/ml IL-3. This frequency increased to 33±2% of the cells after 24h and was maintained at 39±4% after 72h incubation in IL-3. NP-1 was not evident on basophils activated with anti-IgE antibody alone, or after stimulation with other secretagogues. However, the 5±1% of basophils expressing NP-1 with a suboptimal concentration of IL-3, increased to 12±3% (n=8, p<0.05) when additionally activated with anti-IgE. NP-1 expression by basophils following IL-3 exposure was confirmed at the mRNA level by detection of a 409 bp amplicon. IgE-mediated HR and IL-4 secretion were not affected by NP-1 ligation, and IL-3-dependent IL-13 production was only marginally affected. Evidence for NP-1 expression with in vivo priming was supported by preliminary data that bronchoalveolar lavage basophils recovered after segmental allergen challenge co-purified with BDCA-4+ cells. CONCLUSIONS: Basophils expressing NP-1 could indicate a primed phenotype, and further implicate presently unknown cell-to-cell functional consequences between basophils and other immune cells during allergic responses. Funding: NIAID, NIH
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Human Mast Cells Express Neurokinin and Vasoactive Intestinal Peptide Receptors M. Kulka, C. Sheen, S. Toth, B. P. Tancowny, L. C. Grammer, R. P. Schleimer; Allgery-Immunology Division, Northwestern University Feinberg Sch. Med., Chicago, IL. RATIONALE: The neuropeptides substance P (SP) and vasoactive intestinal polypeptide (VIP) activate human mast cells in an Fc RI-independent manner. We determined human mast cell expression of neurokinin (NK) and VIP receptors and characterized some of the signaling pathways activated by these receptors. METHODS: SP and VIP affects on human mast cell degranulation were measured and expression of neurokinin and VIP receptors by human CD34+-derived human mast cells (HCMC) and cultured human mast cells (LAD2) was analyzed by real-time PCR and flow cytometry. RESULTS: Real-time PCR analysis showed that LAD2 cells expressed mRNA for both the C3a receptor (C3aR) and the C5a receptor (C5aR). LAD2 cells also expressed mRNA for the SP receptors, NK1 and NK2, and for the VIP receptor type 2 (VPAC2) but not VPAC1. Receptor protein expression was confirmed by flow cytometry which showed that LAD2 express surface C3aR, NK1R, NK2R, NK3R and VPAC2 but not VPAC1 or C5aR. Activation of human mast cells by IgE/anti-IgE downregulated expression of C3aR. VIP- and SP-activated human mast cell degranulation was blocked by the protein kinase A (PKA) inhibitor, H89, and the phosphoinositol-3 kinase (PI3K) inhibitor, wortmannin, but not by Ro-31-8220, a PKC inhibitor. VIP, but not substance P activated degranulation was also blocked by forskolin, a cAMP agonist. CONCLUSIONS: Human mast cells express receptors for SP, VIP and C3a. Activation of these receptors results in human mast cell degranulation, a process requiring the activation of PI3K and sensitive to changes in intracellular cAMP. Funding: NIH
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Abstracts S65
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2
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Alpha 4 Integrins and VCAM-1, but not MAdCAM-1, are Essential for Recruitment of Mast Cell Progenitors to Inflamed Lung M. F. Gurish1, J. Hallgren1, T. Jones1, J. Boyce1, J. Abonia2; 1Rheumatology, Immunology, Allergy, Brigham and Women’s Hospital, Boston, MA, 2Pediatric Allergy and Immunology, Cincinnati Children’s Hospital, Cincinnati, OH. RATIONALE: There are increased numbers of mast cell progenitors (MCp) in the lungs of sensitized mice after exposure to aerosolized antigen. This increase in MCp is attenuated in mice lacking the 7 integrins or VCAM-1 addressin but not in mice lacking E integrins. As this represents an atypical integrin/addressin pairing associated with inflammatory recruitment to the lung, we chose to further define which of the 4 integrins participate in this recruitment and investigated which endothelial ligands they bind to, MAdCAM-1 and/or VCAM-1. METHODS: Mice were sensitized by ip injection with ovalbumin (OVA) adsorbed to alum. Following 3 daily challenges with aerosolized OVA, the number of MCp in the lungs were evaluated using a clonogenic assay. The involvement of different adhesive molecules was tested by injecting the animals with blocking antibody just prior to the aerosol challenge. RESULTS: In mice receiving antibody to 4 or 1 integrins, the increment in MCp was reduced by 69% and 63% respectively, while in mice receiving anti-7 integrin, only a 33% reduction was noted. Mice given anti-E integrin showed normal increments in lung MCp. Mice given anti-VCAM-1 also showed reduced numbers of MCp (76%) while mice given MAdCAM-1 had a slightly greater than normal increment. CONCLUSIONS: These observations provide the first insight into the adhesion pathways required for MCp recruitment to the inflamed lung, demonstrating the interaction of both the 41 and 47 integrins, with VCAM-1, and not MAdCAM-1; a process crucial to the development of the reactive intraepithelial mastocytosis. Funding: NIH
253
Expression of Neuropilin-1 (NP-1) on Human Basophils Following In Vitro IL-3 Exposure: Implications for NP-1 as a Marker of Basophil Priming J. T. Schroeder, A. P. Bieneman, M. C. Liu; Department of Medicine, Johns Hopkins University, Baltimore, MD. RATIONALE: Since basophils functionally respond to IL-3 by secreting enhanced levels of mediators important in allergic inflammation, we investigated whether novel phenotypic markers are associated with this priming effect. METHODS: Blood basophils were purified (>97%) using density centrifugation and negative selection. Surface staining was assessed by flow cytometry, gene expression by RT-PCR, basophil cytokine and histamine release (HR) by ELISA and fluorimetry, respectively. RESULTS: Basophils cultured in IL-3 dose-dependently up-regulated the neuronal receptor NP-1, as defined by staining with Blood Dendritic Cell Antigen (BDCA)-4 antibody. NP-1 expression was observed within 16h on 18±1% of the basophils cultured in 10 ng/ml IL-3. This frequency increased to 33±2% of the cells after 24h and was maintained at 39±4% after 72h incubation in IL-3. NP-1 was not evident on basophils activated with anti-IgE antibody alone, or after stimulation with other secretagogues. However, the 5±1% of basophils expressing NP-1 with a suboptimal concentration of IL-3, increased to 12±3% (n=8, p<0.05) when additionally activated with anti-IgE. NP-1 expression by basophils following IL-3 exposure was confirmed at the mRNA level by detection of a 409 bp amplicon. IgE-mediated HR and IL-4 secretion were not affected by NP-1 ligation, and IL-3-dependent IL-13 production was only marginally affected. Evidence for NP-1 expression with in vivo priming was supported by preliminary data that bronchoalveolar lavage basophils recovered after segmental allergen challenge co-purified with BDCA-4+ cells. CONCLUSIONS: Basophils expressing NP-1 could indicate a primed phenotype, and further implicate presently unknown cell-to-cell functional consequences between basophils and other immune cells during allergic responses. Funding: NIAID, NIH
254
Human Mast Cells Express Neurokinin and Vasoactive Intestinal Peptide Receptors M. Kulka, C. Sheen, S. Toth, B. P. Tancowny, L. C. Grammer, R. P. Schleimer; Allgery-Immunology Division, Northwestern University Feinberg Sch. Med., Chicago, IL. RATIONALE: The neuropeptides substance P (SP) and vasoactive intestinal polypeptide (VIP) activate human mast cells in an Fc RI-independent manner. We determined human mast cell expression of neurokinin (NK) and VIP receptors and characterized some of the signaling pathways activated by these receptors. METHODS: SP and VIP affects on human mast cell degranulation were measured and expression of neurokinin and VIP receptors by human CD34+-derived human mast cells (HCMC) and cultured human mast cells (LAD2) was analyzed by real-time PCR and flow cytometry. RESULTS: Real-time PCR analysis showed that LAD2 cells expressed mRNA for both the C3a receptor (C3aR) and the C5a receptor (C5aR). LAD2 cells also expressed mRNA for the SP receptors, NK1 and NK2, and for the VIP receptor type 2 (VPAC2) but not VPAC1. Receptor protein expression was confirmed by flow cytometry which showed that LAD2 express surface C3aR, NK1R, NK2R, NK3R and VPAC2 but not VPAC1 or C5aR. Activation of human mast cells by IgE/anti-IgE downregulated expression of C3aR. VIP- and SP-activated human mast cell degranulation was blocked by the protein kinase A (PKA) inhibitor, H89, and the phosphoinositol-3 kinase (PI3K) inhibitor, wortmannin, but not by Ro-31-8220, a PKC inhibitor. VIP, but not substance P activated degranulation was also blocked by forskolin, a cAMP agonist. CONCLUSIONS: Human mast cells express receptors for SP, VIP and C3a. Activation of these receptors results in human mast cell degranulation, a process requiring the activation of PI3K and sensitive to changes in intracellular cAMP. Funding: NIH
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Abstracts S65
J ALLERGY CLIN IMMUNOL VOLUME 117, NUMBER 2