- Email: [email protected]
Mast Cell Survival Following Gamma-Irradiation is Dependent on Bcl-2 and Bcl-xL
47
Mast Cell Survival Following Gamma-Irradiation is Dependent on Bcl-2 and Bcl-xL J. M. Brown, B. P. Soule, J. B. Mitchell, D. D. Metcalfe; NIH, Bethesda, MD. RATIONALE: We have reported that mast cells are resistant to the effects of gamma-radiation and retain their ability to respond to both FceRI and TLR-mediated signals. In contrast, we demonstrated that mast cells are susceptible to the cytotoxic effects of UV-irradiation. Therefore, the aim of this study was to determine what mechanisms allow survival of mast cells following gamma-irradiation, but not UV-irradiation. METHODS: Mouse bone marrow-derived mast cells (BMMC) were exposed to gamma-radiation (0-50 Gy) or exposed to UV irradiation (302 nm) for 4 minutes. Two hours post-irradiation, RNA was collected from BMMC and gene expression was examined using apoptosis pathway focused PCR arrays. Following identification of potential genes involved in mast cell resistance to gamma-irradiation, protein expression was confirmed by Western blot. RESULTS: Following UV-irradiation, we identified >90 genes related to apoptosis that were upregulated more than 10-fold as compared to untreated cells. However, following gamma-irradiation, only 2 antiapoptotic genes (Bcl-2 and Bcl-xL) were upregulated more than 10-fold, while the majority of pro-apoptotic genes were unchanged as compared to untreated cells. Protein expression of Bcl-2 and Bcl-xL was dose-dependently increased following gamma-irradiation at doses between 0 and 50 Gy. However, UV-irradiation resulted in a decrease in expression of both Bcl-2 and Bcl-xL. CONCLUSIONS: Mast cells are resistant to the cytotoxic effects of gamma-irradiation, but highly susceptible to UV-irradiation. We have identified the overexpression of pro-survival proteins Bcl-2 and Bcl-xL associated with the ability of mast cells to survive high doses of gammaradiation.
Esophageal Epithelial Cells as Antigen-Presenting Cells? Implications for Eosinophilic Esophagitis C. Justinich, A. Pooni, N. Mak, D. Mulder, S. Basta; Queen’s University, Kingston, ON, CANADA. RATIONALE: Through interactions with T-cells, antigen presenting cells (APC’s) play a crucial role in directing immune responses. While the function of professional APC’s are well defined, the contribution of nonprofessional APC’s including epithelial cells to GI inflammation is not known. The APC capability of esophageal epithelial cells may have importance in esophageal inflammation, especially eosinophilic esophagitis. We hypothesize that esophageal epithelial cells may play a role in esophagitis by acting as non-professional APC’s. METHODS: A human esophageal epithelial cell line (HET-1A) was used to study MHC Class II, co-stimulatory molecules CD80 and 86 by RT-PCR and flow cytometry. We also assessed HET-1A cells ability to capture and processes antigen using incorporation and processing of DQ-OVAÒ and a phagocytosis assay using PI treated, killed HEK cells using flow cytometry. Cells were stimulated with interferon-gamma and interleukin-4 (IL-4) [both 50 ug/ml], known to induce Class II antigen expression. RESULTS: Interferon and IL-4 induced the expression of MHC Class II mRNA and antigen by flow cytometry on HET-1A (51% and 15% above control respectively) and to a much lesser extent, CD80-86 by RT-PCR and flow cytometry. Unstimulated HET-1A cells were capable of phagocytosis of HEK cells (93% uptake at 24 hr) and process DQ-OVA antigen (45.9% at 4 hr); these effects were diminished by interferon and IL-4. CONCLUSION: Esophageal epithelial cells possess characteristics that allow them to function as non-professional APC’s. The increase in MHC Class II with interferon-gamma and IL-4 suggests a novel immunologic role for esophageal epithelium in conditions such as eosinophilic esophagitis by acting as APC’s. Funding: Physicians Services Inc
48
50
Inhibitory Effect of Substance P via NK-1R Tachykinin Receptor on NK Cell Function L. M. Shawver, J. Guo, J. P. Lai, H. Li, L. Kilpatrick, S. D. Douglas, J. S. Orange; The Childrens Hopsital of Philadelphia, Philadelphia, PA. RATIONALE: Substance P (SP) neurokinin is present at elevated levels during HIV disease and can serve as a potent immunoregulator. Thus, we hypothesized that SP may contribute to impaired NK cell function, and that an SP antagonist may reduce this effect. METHODS: The YTS NK cell line or ex vivo NK cells were pretreated with SP (10^-6M) and/or antagonist prior to a 4 hour chromium release assay. YTS cells were also activated with anti CD28 after treatment with SP and/or antagonist to determine the effects of SP upon activation signaling for NF-kB, and ERK phosphorylation, both which are essential for cytotoxicity. Presence of the SP receptor NK-1R was determined using PCR, and its stability and occupancy using fluorescently labeled SP. RESULTS: NK-1R is present on NK cells and SP binding is not affected by activation. Preincubating with SP produced a 12 6 3% inhibition of cytotoxicity at 30 minutes (p 5 0.005), and 17 6 7% after 72 hours (p 5 0.02). The SP Antagonist CP 96,345 reduced this effect by 20% (p 5 0.03) and 21% (p 5 0.03) respectively. Mechanistic insight into this effect was obtained through consideration of the effect of SP upon ERK and NF-kB activation as well as Ca21 flux after NK cell stimulation. CONCLUSIONS: We have identified a reproducible inhibitory effect of SP upon NK cells and have delineated a potential mechanism. This may provide insight into clinically relevant depressions of NK cell function associated with increased exposure to SP and present opportunities to therapeutically augment this activity. Work supported by NIH-P01MH76388 Funding: The Childrens Hospital of Philadelphia Pa NIH-P01MH76388
49
Expression of Glucocorticoid Receptor Translational Isoforms in Airway Epithelial and Monocytic Cells A. Konstantinidis, R. Schleimer, N. Lu; Division of Allergy-Immunology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL. RATIONALE: Since glucocorticoid receptor (GR) translational isoforms may mediate different glucocorticoid responses in allergic diseases, we determined the expression pattern of these newly described GR isoforms in airway epithelial and monocytic cell lines. METHODS: The airway epithelial cell lines BEAS-2B and A549 as well as the monocytic cell lines U937 and THP-1 were cultured in vitro. Western blot and densitometric analyses were performed to determine the level of GR translational isoforms. RESULTS: All GR translational isoforms including the GR-A, -B, -C, and -D isoforms were detected although the composition of the GR isoforms in each cell line differed significantly (n 5 3, ANOVA, p < 0.05). For example, the relative expression pattern in U937 cells was GR-B>-D>A>-C isoform whereas that in THP-1 cells was GR-A5-B>-C5-D isoform. In contrast, BEAS-2B and A549 cells both had high level of the GR-B isoform (60% of total GR protein) and negligible amount of the GR-D isoform. CONCLUSIONS: GR translational isoforms had different expression patterns in airway epithelial and monocytic cell lines. The cell typespecific expression pattern of GR isoforms may provide a basis for cellspecific responses to glucocorticoids.
SATURDAY
Abstracts S13
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2
Mast Cell Survival Following Gamma-Irradiation is Dependent on Bcl-2 and Bcl-xL J. M. Brown, B. P. Soule, J. B. Mitchell, D. D. Metcalfe; NIH, Bethesda, MD. RATIONALE: We have reported that mast cells are resistant to the effects of gamma-radiation and retain their ability to respond to both FceRI and TLR-mediated signals. In contrast, we demonstrated that mast cells are susceptible to the cytotoxic effects of UV-irradiation. Therefore, the aim of this study was to determine what mechanisms allow survival of mast cells following gamma-irradiation, but not UV-irradiation. METHODS: Mouse bone marrow-derived mast cells (BMMC) were exposed to gamma-radiation (0-50 Gy) or exposed to UV irradiation (302 nm) for 4 minutes. Two hours post-irradiation, RNA was collected from BMMC and gene expression was examined using apoptosis pathway focused PCR arrays. Following identification of potential genes involved in mast cell resistance to gamma-irradiation, protein expression was confirmed by Western blot. RESULTS: Following UV-irradiation, we identified >90 genes related to apoptosis that were upregulated more than 10-fold as compared to untreated cells. However, following gamma-irradiation, only 2 antiapoptotic genes (Bcl-2 and Bcl-xL) were upregulated more than 10-fold, while the majority of pro-apoptotic genes were unchanged as compared to untreated cells. Protein expression of Bcl-2 and Bcl-xL was dose-dependently increased following gamma-irradiation at doses between 0 and 50 Gy. However, UV-irradiation resulted in a decrease in expression of both Bcl-2 and Bcl-xL. CONCLUSIONS: Mast cells are resistant to the cytotoxic effects of gamma-irradiation, but highly susceptible to UV-irradiation. We have identified the overexpression of pro-survival proteins Bcl-2 and Bcl-xL associated with the ability of mast cells to survive high doses of gammaradiation.
Esophageal Epithelial Cells as Antigen-Presenting Cells? Implications for Eosinophilic Esophagitis C. Justinich, A. Pooni, N. Mak, D. Mulder, S. Basta; Queen’s University, Kingston, ON, CANADA. RATIONALE: Through interactions with T-cells, antigen presenting cells (APC’s) play a crucial role in directing immune responses. While the function of professional APC’s are well defined, the contribution of nonprofessional APC’s including epithelial cells to GI inflammation is not known. The APC capability of esophageal epithelial cells may have importance in esophageal inflammation, especially eosinophilic esophagitis. We hypothesize that esophageal epithelial cells may play a role in esophagitis by acting as non-professional APC’s. METHODS: A human esophageal epithelial cell line (HET-1A) was used to study MHC Class II, co-stimulatory molecules CD80 and 86 by RT-PCR and flow cytometry. We also assessed HET-1A cells ability to capture and processes antigen using incorporation and processing of DQ-OVAÒ and a phagocytosis assay using PI treated, killed HEK cells using flow cytometry. Cells were stimulated with interferon-gamma and interleukin-4 (IL-4) [both 50 ug/ml], known to induce Class II antigen expression. RESULTS: Interferon and IL-4 induced the expression of MHC Class II mRNA and antigen by flow cytometry on HET-1A (51% and 15% above control respectively) and to a much lesser extent, CD80-86 by RT-PCR and flow cytometry. Unstimulated HET-1A cells were capable of phagocytosis of HEK cells (93% uptake at 24 hr) and process DQ-OVA antigen (45.9% at 4 hr); these effects were diminished by interferon and IL-4. CONCLUSION: Esophageal epithelial cells possess characteristics that allow them to function as non-professional APC’s. The increase in MHC Class II with interferon-gamma and IL-4 suggests a novel immunologic role for esophageal epithelium in conditions such as eosinophilic esophagitis by acting as APC’s. Funding: Physicians Services Inc
48
50
Inhibitory Effect of Substance P via NK-1R Tachykinin Receptor on NK Cell Function L. M. Shawver, J. Guo, J. P. Lai, H. Li, L. Kilpatrick, S. D. Douglas, J. S. Orange; The Childrens Hopsital of Philadelphia, Philadelphia, PA. RATIONALE: Substance P (SP) neurokinin is present at elevated levels during HIV disease and can serve as a potent immunoregulator. Thus, we hypothesized that SP may contribute to impaired NK cell function, and that an SP antagonist may reduce this effect. METHODS: The YTS NK cell line or ex vivo NK cells were pretreated with SP (10^-6M) and/or antagonist prior to a 4 hour chromium release assay. YTS cells were also activated with anti CD28 after treatment with SP and/or antagonist to determine the effects of SP upon activation signaling for NF-kB, and ERK phosphorylation, both which are essential for cytotoxicity. Presence of the SP receptor NK-1R was determined using PCR, and its stability and occupancy using fluorescently labeled SP. RESULTS: NK-1R is present on NK cells and SP binding is not affected by activation. Preincubating with SP produced a 12 6 3% inhibition of cytotoxicity at 30 minutes (p 5 0.005), and 17 6 7% after 72 hours (p 5 0.02). The SP Antagonist CP 96,345 reduced this effect by 20% (p 5 0.03) and 21% (p 5 0.03) respectively. Mechanistic insight into this effect was obtained through consideration of the effect of SP upon ERK and NF-kB activation as well as Ca21 flux after NK cell stimulation. CONCLUSIONS: We have identified a reproducible inhibitory effect of SP upon NK cells and have delineated a potential mechanism. This may provide insight into clinically relevant depressions of NK cell function associated with increased exposure to SP and present opportunities to therapeutically augment this activity. Work supported by NIH-P01MH76388 Funding: The Childrens Hospital of Philadelphia Pa NIH-P01MH76388
49
Expression of Glucocorticoid Receptor Translational Isoforms in Airway Epithelial and Monocytic Cells A. Konstantinidis, R. Schleimer, N. Lu; Division of Allergy-Immunology, Department of Medicine, Northwestern University Feinberg School of Medicine, Chicago, IL. RATIONALE: Since glucocorticoid receptor (GR) translational isoforms may mediate different glucocorticoid responses in allergic diseases, we determined the expression pattern of these newly described GR isoforms in airway epithelial and monocytic cell lines. METHODS: The airway epithelial cell lines BEAS-2B and A549 as well as the monocytic cell lines U937 and THP-1 were cultured in vitro. Western blot and densitometric analyses were performed to determine the level of GR translational isoforms. RESULTS: All GR translational isoforms including the GR-A, -B, -C, and -D isoforms were detected although the composition of the GR isoforms in each cell line differed significantly (n 5 3, ANOVA, p < 0.05). For example, the relative expression pattern in U937 cells was GR-B>-D>A>-C isoform whereas that in THP-1 cells was GR-A5-B>-C5-D isoform. In contrast, BEAS-2B and A549 cells both had high level of the GR-B isoform (60% of total GR protein) and negligible amount of the GR-D isoform. CONCLUSIONS: GR translational isoforms had different expression patterns in airway epithelial and monocytic cell lines. The cell typespecific expression pattern of GR isoforms may provide a basis for cellspecific responses to glucocorticoids.
SATURDAY
Abstracts S13
J ALLERGY CLIN IMMUNOL VOLUME 121, NUMBER 2