- Email: [email protected]
Rottlerin induces apoptosis of human blood eosinophils: A possible role for PKC-δ in mediating eosinophil survival
Abstracts S193
J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
Eosinophils (EOS) Enhance CD4+ T-Lymphocyte Secretion of Th1 and Th2 Cytokines S. K. Mathur, L. Y. Liu, E. A. B. Kelly, N. N. Jarjour, W. W. Busse; Dept. of Medicine, University of Wisconsin, Madison, WI. RATIONALE: Regulation of EOS function by T-lymphocyte derived cytokines such as IL-5 is well appreciated. Although there is evidence in mouse models that EOS may regulate T-lymphocyte function, there is little evidence that such interactions occur with human cells and the conditions under which they may occur. METHODS: To evaluate the regulatory interactions between human EOS and T-lymphocytes, an in vitro co-culture system was used to model this process. Isolated purified human peripheral blood EOS and CD4+ Tlymphocytes were incubated with superantigen, Staphyloccal enterotoxin B (SEB). After 48 hours of co-culture, supernatants were collected and analyzed by ELISA for secretion of Th1 and Th2 cytokines. Proliferation was measured by 3H-thymidine incorporation. Statistical analysis was performed using paired t-test. RESULTS: Co-culture of EOS and CD4+ T-lymphocytes with SEB resulted in significantly increased secretion of both Th1 and Th2 cytokines compared to CD4+ T-lymphocytes alone with SEB: IFNgamma (1260pg/mL vs 9430pg/mL), IL-5 (19pg/mL vs 92pg/mL), and IL-13 (69pg/mL vs 292pg/mL), p<0.005 for all comparisons. Increased 3H-thymidine incorporation upon co-culture indicated increased proliferation of the CD4+ T-lymphocytes. Co-culture performed with separation of EOS and CD4+ T-lymphocytes by a semi-permeable membrane resulted in no detectable enhancement of cytokine secretion. CONCLUSIONS: EOS can interact with CD4+ T-lymphocytes to enhance cytokine secretion, which is, in part, due to increased proliferation of the CD4+ T-lymphocytes. The enhanced cytokine secretion is also dependent on cell contact between EOS and the CD4+ T-lymphocytes. Thus, EOS are able to regulate T-lymphocyte function and may enhance airway inflammation in human asthma. Funding: NIH
770
Airway Eosinophil (EOS) Expression of Urokinase-Type Plasminogen Activator Receptor (uPAR) Following Segmental Allergen Challenge Correlates With Airway Reactivity of Patients With Asthma A. M. Brooks, M. Bates, R. F. Vrtis, W. W. Busse, J. B. Sedgwick; Department of Medicine, University of Wisconsin, Madison, WI. RATIONALE: uPAR (CD87) is expressed on leukocytes and may contribute to integrin-dependent functions including cell migration. As the EOS is the major leukocyte recruited to the airways of asthmatic patients following allergen challenge, uPAR may participate in its migration. METHODS: Baseline airway reactivity was measured as methacholine PC20. Following segmental allergen challenge, cells were isolated from bronchoalveolar lavage (BAL) fluid and peripheral blood. Soluble (s)uPAR was measured by ELISA. Cellular uPAR expression was assessed by flow cytometry and Western blots; mRNA was determined by real time PCR. RESULTS: suPAR was significantly higher in post-challenge BAL fluid compared with pre-challenge fluid and correlated with BAL EOS counts. It did not correlate with cellular expression of this protein. Expression of uPAR was significantly increased on airway EOS obtained 48h-post-allergen challenge when compared with the corresponding peripheral blood EOS (51.3+/-5.2% vs 12.4+/-3.7% positive EOS, p<0.001, n=8). PCR demonstrated a 2-fold increase in uPAR mRNA in airway EOS compared to blood EOS. Although BAL EOS uPAR protein and mRNA expression was similar to peripheral blood neutrophils, the molecular weight of the protein varied considerably between the two granulocytes. Finally, uPAR expressed by BAL EOS 48h-post allergen challenge had a strong inverse correlation with airway hyperresponsiveness (methacholine PC20) of the asthmatic subjects (rs= -0.85, p<0.001). CONCLUSIONS: Enhanced airway eosinophil uPAR expression is a feature of asthma patients with increased airway responsiveness and, presumably, more severe disease. One could speculate that decreasing uPAR expression would result in decreased eosinophil presence in, and inflammation of, the asthmatic airway. Funding: NIH
771
Rottlerin Induces Apoptosis of Human Blood Eosinophils: A Possible Role for PKC- in Mediating Eosinophil Survival A. I. Berro, A. Bharadwaj, D. K. Agrawal; School of Medicine, Creighton University, Omaha, NE. RATIONALE: Many aspects of eosinophil apoptosis mechanisms have yet to be identified, with one of them being the role different PKC isoforms in modulating eosinophils survival and apoptosis. METHODS: Human eosinophils were purified from allergic and atopic asthmatic subjects. Eosinophil apoptosis was induced by incubating in RPMI supplemented with only1% fetal bovine serum (FBS) (serumdeprived group), and eosinophil survival was conferred by incubating cells with 10% FBS and 15ng/ml IL-5 in RPMI (serum-sufficient). Eosinophils apoptosis was determined by Annexin V/PI labeling and caspases-3, -8, and -9 activities. Protein expression was measured by western blotting using PKC isoforms-specific antibodies. RESULTS: We observed decreased protein expression of many PKC isoforms in serum-deprived apoptotic eosinophils as compared to freshly isolated eosinophils and serum-sufficient eosinophils incubated for up to 48 hours. However, PKC- expression displayed less significant change, coupled with the appearance of an approximately 40 KDa fragment in the apoptotic serum-deficient cells. Incubating eosinophils with 10M rottlerin for different time periods resulted in significant increase in eosinophil apoptosis starting at 4-hour post-incubation as compared to the control groups. After 12-hour incubation under serum deprivation conditions, eosinophil apoptosis increased by >100% in the presence of rottlerin as compared to the no-rottlerin group. However, rottlerin’s effect on eosinophil apoptosis was considerably less evident in eosinophils incubated in the presence of 10% FBS and 15ng/ml IL-5. CONCLUSIONS: Our observations revealed that rottlerin, which at the concentration of 10M acts as a selective inhibitor of PKC- , induced eosinophil apoptosis. This suggests a pivotal role for PKC- in modulating eosinophil survival. Funding: NIH RO1 HL070885 to DKA
772
MONDAY
Dual Expression of Histamine Receptors H2 and H4 Regulates Eosinophil Chemotaxis and Superoxide Generation by Histamine T. Fujisawa1, Y. Kato1, T. Nakayama2, K. Hirai3, O. Yoshie2; 1Institute for Clinical Research, Mie National Hospital, Tsu, JAPAN, 2Microbiology, Kinki University School of Medicine, Osaka, JAPAN, 3Bioregulatory Function, Tokyo University Graduate School of Medicine, Tokyo, JAPAN. RATIONALE: Histamine receptor H4 is expressed by various immune cells including eosinophils and responsible for chemotactic responses of eosinophils to histamine. However, expression profile and function of histamine receptor subtypes in eosinophils have not been fully clarified. We quantified gene expression of histamine receptors in eosinophils and examined histamine-induced chemotaxis and superoxide generation in vitro. METHODS: Gene expression of H1, H2, H3, H4 receptors in highly purified peripheral blood eosinophils from normal donors was quantified with real-time PCR. Migratory responses to histamine were examined using Chemotaxicell™ chambers and superoxide generation was measured by the reduction of cytochrome c in the presence or absence of histamine receptor antagonists. RESULTS: Eosinophils highly expressed H2 and H4 at comparable levels, while expression of H1 was significantly lower than those of H2 and H4. H3 was barely detectable. Since H2 and H4 are coupled with Gs and Gi, respectively, they are likely to counter-regulate each other in responses to histamine. Consistently, although histamine itself did not cause superoxide generation, it induced significant superoxide release in the presence of H2 antagonist, famotidine. H3/H4 antagonist, thioperamide, inhibited the reaction. Histamine-induced chemotaxis of eosinophil was also enhanced by pretreatment with famotidine. IL-5 and GM-CSF, cytokines known to activate eosinophils, strongly unregulated the expression of H4, but not H1, H2 or H3, and enhanced migratory responses of eosinophils to histamine. CONCLUSIONS: Histamine participates in the pathogenesis of eosinophilic inflammation in allergy through H2 and H4 and their mutual counter regulation may modulate eosinophil responses to histamine. Funding: Ministry of Health, Labor and Welfare, Japan
769
J ALLERGY CLIN IMMUNOL VOLUME 115, NUMBER 2
Eosinophils (EOS) Enhance CD4+ T-Lymphocyte Secretion of Th1 and Th2 Cytokines S. K. Mathur, L. Y. Liu, E. A. B. Kelly, N. N. Jarjour, W. W. Busse; Dept. of Medicine, University of Wisconsin, Madison, WI. RATIONALE: Regulation of EOS function by T-lymphocyte derived cytokines such as IL-5 is well appreciated. Although there is evidence in mouse models that EOS may regulate T-lymphocyte function, there is little evidence that such interactions occur with human cells and the conditions under which they may occur. METHODS: To evaluate the regulatory interactions between human EOS and T-lymphocytes, an in vitro co-culture system was used to model this process. Isolated purified human peripheral blood EOS and CD4+ Tlymphocytes were incubated with superantigen, Staphyloccal enterotoxin B (SEB). After 48 hours of co-culture, supernatants were collected and analyzed by ELISA for secretion of Th1 and Th2 cytokines. Proliferation was measured by 3H-thymidine incorporation. Statistical analysis was performed using paired t-test. RESULTS: Co-culture of EOS and CD4+ T-lymphocytes with SEB resulted in significantly increased secretion of both Th1 and Th2 cytokines compared to CD4+ T-lymphocytes alone with SEB: IFNgamma (1260pg/mL vs 9430pg/mL), IL-5 (19pg/mL vs 92pg/mL), and IL-13 (69pg/mL vs 292pg/mL), p<0.005 for all comparisons. Increased 3H-thymidine incorporation upon co-culture indicated increased proliferation of the CD4+ T-lymphocytes. Co-culture performed with separation of EOS and CD4+ T-lymphocytes by a semi-permeable membrane resulted in no detectable enhancement of cytokine secretion. CONCLUSIONS: EOS can interact with CD4+ T-lymphocytes to enhance cytokine secretion, which is, in part, due to increased proliferation of the CD4+ T-lymphocytes. The enhanced cytokine secretion is also dependent on cell contact between EOS and the CD4+ T-lymphocytes. Thus, EOS are able to regulate T-lymphocyte function and may enhance airway inflammation in human asthma. Funding: NIH
770
Airway Eosinophil (EOS) Expression of Urokinase-Type Plasminogen Activator Receptor (uPAR) Following Segmental Allergen Challenge Correlates With Airway Reactivity of Patients With Asthma A. M. Brooks, M. Bates, R. F. Vrtis, W. W. Busse, J. B. Sedgwick; Department of Medicine, University of Wisconsin, Madison, WI. RATIONALE: uPAR (CD87) is expressed on leukocytes and may contribute to integrin-dependent functions including cell migration. As the EOS is the major leukocyte recruited to the airways of asthmatic patients following allergen challenge, uPAR may participate in its migration. METHODS: Baseline airway reactivity was measured as methacholine PC20. Following segmental allergen challenge, cells were isolated from bronchoalveolar lavage (BAL) fluid and peripheral blood. Soluble (s)uPAR was measured by ELISA. Cellular uPAR expression was assessed by flow cytometry and Western blots; mRNA was determined by real time PCR. RESULTS: suPAR was significantly higher in post-challenge BAL fluid compared with pre-challenge fluid and correlated with BAL EOS counts. It did not correlate with cellular expression of this protein. Expression of uPAR was significantly increased on airway EOS obtained 48h-post-allergen challenge when compared with the corresponding peripheral blood EOS (51.3+/-5.2% vs 12.4+/-3.7% positive EOS, p<0.001, n=8). PCR demonstrated a 2-fold increase in uPAR mRNA in airway EOS compared to blood EOS. Although BAL EOS uPAR protein and mRNA expression was similar to peripheral blood neutrophils, the molecular weight of the protein varied considerably between the two granulocytes. Finally, uPAR expressed by BAL EOS 48h-post allergen challenge had a strong inverse correlation with airway hyperresponsiveness (methacholine PC20) of the asthmatic subjects (rs= -0.85, p<0.001). CONCLUSIONS: Enhanced airway eosinophil uPAR expression is a feature of asthma patients with increased airway responsiveness and, presumably, more severe disease. One could speculate that decreasing uPAR expression would result in decreased eosinophil presence in, and inflammation of, the asthmatic airway. Funding: NIH
771
Rottlerin Induces Apoptosis of Human Blood Eosinophils: A Possible Role for PKC- in Mediating Eosinophil Survival A. I. Berro, A. Bharadwaj, D. K. Agrawal; School of Medicine, Creighton University, Omaha, NE. RATIONALE: Many aspects of eosinophil apoptosis mechanisms have yet to be identified, with one of them being the role different PKC isoforms in modulating eosinophils survival and apoptosis. METHODS: Human eosinophils were purified from allergic and atopic asthmatic subjects. Eosinophil apoptosis was induced by incubating in RPMI supplemented with only1% fetal bovine serum (FBS) (serumdeprived group), and eosinophil survival was conferred by incubating cells with 10% FBS and 15ng/ml IL-5 in RPMI (serum-sufficient). Eosinophils apoptosis was determined by Annexin V/PI labeling and caspases-3, -8, and -9 activities. Protein expression was measured by western blotting using PKC isoforms-specific antibodies. RESULTS: We observed decreased protein expression of many PKC isoforms in serum-deprived apoptotic eosinophils as compared to freshly isolated eosinophils and serum-sufficient eosinophils incubated for up to 48 hours. However, PKC- expression displayed less significant change, coupled with the appearance of an approximately 40 KDa fragment in the apoptotic serum-deficient cells. Incubating eosinophils with 10M rottlerin for different time periods resulted in significant increase in eosinophil apoptosis starting at 4-hour post-incubation as compared to the control groups. After 12-hour incubation under serum deprivation conditions, eosinophil apoptosis increased by >100% in the presence of rottlerin as compared to the no-rottlerin group. However, rottlerin’s effect on eosinophil apoptosis was considerably less evident in eosinophils incubated in the presence of 10% FBS and 15ng/ml IL-5. CONCLUSIONS: Our observations revealed that rottlerin, which at the concentration of 10M acts as a selective inhibitor of PKC- , induced eosinophil apoptosis. This suggests a pivotal role for PKC- in modulating eosinophil survival. Funding: NIH RO1 HL070885 to DKA
772
MONDAY
Dual Expression of Histamine Receptors H2 and H4 Regulates Eosinophil Chemotaxis and Superoxide Generation by Histamine T. Fujisawa1, Y. Kato1, T. Nakayama2, K. Hirai3, O. Yoshie2; 1Institute for Clinical Research, Mie National Hospital, Tsu, JAPAN, 2Microbiology, Kinki University School of Medicine, Osaka, JAPAN, 3Bioregulatory Function, Tokyo University Graduate School of Medicine, Tokyo, JAPAN. RATIONALE: Histamine receptor H4 is expressed by various immune cells including eosinophils and responsible for chemotactic responses of eosinophils to histamine. However, expression profile and function of histamine receptor subtypes in eosinophils have not been fully clarified. We quantified gene expression of histamine receptors in eosinophils and examined histamine-induced chemotaxis and superoxide generation in vitro. METHODS: Gene expression of H1, H2, H3, H4 receptors in highly purified peripheral blood eosinophils from normal donors was quantified with real-time PCR. Migratory responses to histamine were examined using Chemotaxicell™ chambers and superoxide generation was measured by the reduction of cytochrome c in the presence or absence of histamine receptor antagonists. RESULTS: Eosinophils highly expressed H2 and H4 at comparable levels, while expression of H1 was significantly lower than those of H2 and H4. H3 was barely detectable. Since H2 and H4 are coupled with Gs and Gi, respectively, they are likely to counter-regulate each other in responses to histamine. Consistently, although histamine itself did not cause superoxide generation, it induced significant superoxide release in the presence of H2 antagonist, famotidine. H3/H4 antagonist, thioperamide, inhibited the reaction. Histamine-induced chemotaxis of eosinophil was also enhanced by pretreatment with famotidine. IL-5 and GM-CSF, cytokines known to activate eosinophils, strongly unregulated the expression of H4, but not H1, H2 or H3, and enhanced migratory responses of eosinophils to histamine. CONCLUSIONS: Histamine participates in the pathogenesis of eosinophilic inflammation in allergy through H2 and H4 and their mutual counter regulation may modulate eosinophil responses to histamine. Funding: Ministry of Health, Labor and Welfare, Japan
769