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Following Segmental Antigen Challenge, Airway Eosinophils Exhibit Differential Cytokine Regulation of the STAT and ERK Signaling Networks When Compared to Blood Eosinophils
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Tetracycline Mediated-Suppression of IgE Secretion by Peripheral Blood Mononuclear Cells (PBMC) in Allergic Asthmatics with History of Chlamydia pneumoniae (Cpn) Infection M. S. Dzhindzhikhashvili, R. Joks, T. Smith-Norowitz, M. R. Hammerschlag, H. G. Durkin, S. Kohlhoff; Center for Allergy and Asthma Research at SUNY Downstate Medical Center, Brooklyn, NY. RATIONALE: We previously reported that tetracycline treatment of allergic asthmatic humans improves their symptoms and suppresses their IgE responses in vivo, and suppresses human and murine memory responses in vitro. The present studies investigated the ability of Cpn, an important cause of asthma exacerbations, and doxycycline to regulate cytokine and IgE reponses by PBMC in vitro. METHODS: PBMC from serum IgE1 allergic asthmatic humans with (n 5 4) and without (n 5 7) evidence of prior Cpn infection (microimmunofluorescence IgG titer >16) (median serum IgG anti Cpn titers: 256, 0, respectively) (mean IgE: 881, 296, IU/ml, respectively), and from serum IgE- donors (n 5 12), were infected or not with Cpn (multiplicity of infection 0.01-10), and then cultured for 2-12 days 1/2 doxycycline. The levels of cytokines (IL-4, IFN-g) (day 2) and IgE (day 10) in culture supernatants were determined by ELISA. RESULTS: PBMC from serum IgE1 allergic asthmatic patients with evidence of previous Cpn infection secreted low levels of IgE on all days of culture, which were further increased when infected with Cpn in vitro (days 2-12;peak levels d10); this further increase in IgE was correlated with Cpn induced IL-4 (CC 5 0.979;p < 0.001), but not with IFN-g production. Inclusion of doxycycline in culture strongly suppressed these IgE responses (>48%, p 5 0.043) and IL-4 (>45%, p 5 0.018), but not IFN-g (5%, NS) production even at subinhibitory concentrations for Cpn. No IgE was secreted by PBMC from any of the control groups. CONCLUSIONS: Cpn plays an important role in regulation of IgE responses; tetracyclines suppress Cpn mediated regulation of IgE responses. Following Segmental Antigen Challenge, Airway Eosinophils Exhibit Differential Cytokine Regulation of the STAT and ERK Signaling Networks When Compared to Blood Eosinophils C. J. Koziol-White, M. E. Bates, P. J. Bertics; University of WisconsinMadison, Madison, WI. RATIONALE: Airway eosinophils (BALEOS) isolated following segmental antigen challenge (SBP-Ag) exhibit multiple phenotypic changes relative to peripheral blood eosinophils (PBEOS), including altered effector functions and cytokine (e.g., IL-5, GM-CSF, IL-3) receptor expression profiles. Here, we tested the hypothesis that these changes in airway versus blood EOS occur in parallel to a shift in the activation of key signaling molecules (STAT5, ERK1/2) that are stimulated by these cytokines in vivo. METHODS: PBEOS and BALEOS (recovered from bronchoalveolar lavage (BAL) fluid 48 hr following SBP-Ag) were stimulated in vitro with 1 nM IL-5, GM-CSF, or IL-3. Cell lysates were prepared and STAT5 and ERK1/2 phosphorylation was assessed via immunoblotting, quantified by densitometry, and normalized to loading controls. RESULTS: With respect to STAT5 phosphorylation, BALEOS are significantly less responsive than PBEOS to IL-5 and GM-CSF (p 5 0.003 and p 5 0.021, respectively, n 5 5), with a similar trend following IL-3 stimulation (n 5 2). However, ERK1/2 phosphorylation is not significantly different in BALEOS versus PBEOS after cytokine stimulation (IL-5 p 5 0.39, GM-CSF p 5 0.75). CONCLUSIONS: These data suggest that a regulatory mechanism for STAT5 pathway activation (distinct from ERK1/2 activation) is present in BALEOS and suggests that STAT5-specific transcriptional regulation is altered in BALEOS, with profound consequences for downstream gene expression.
An Experimental Model for Self-sustained Activation (Bistability) of the ERK1/2 Signaling Module-a Precursor for Memory-driven Chronic Inflammation? W. Liu, K. Tundwal, Q. Liang, R. Alam; National Jewish Medical and Research Center, Denver, CO. RATIONALE: We hypothesize that chronic airway inflammation in asthma is partially driven by a self-sustained mechanism that is intermittently fueled by environmental factors. The objective was to establish an experimental model of a self-sustained signaling process that could drive inflammation. METHODS: Airway epithelial cells and genetically-modified fibroblasts were stimulated with IL-13, IL-6, eotaxin, TNF, or EGF and their effect on signaling and cell activation was studied by western blotting, kinase assay, immunostaining, microarray, real-time PCR and ELISA. RESULTS: A single stimulation of cells with cytokines causes rapid ERK1/2 activation, which returns to baseline in 12-24 hr. Repeated stimulation leads to sustained activation of ERK1/2 but not STAT6, p38 or AKT. The ERK1/2 activation lasts for 3-7 days. This sustained activation depends upon a positive feedback mechanism involving the induction of sprouty-2. Overexpression of sprouty-2 induces and its homozygous genetic deletion abrogates ERK1/2 bistability. Sprouty-2 directly activates Fyn kinase, which then induces ERK1/2 activation. Unlike the acutely activated pERK1/2, the memory-driven pERK1/2 does not induce a high level gene transcription as shown by microarray analysis (33,300 probe sets). This is in part due to its compartmentalization in the Rab4 and Rab51 endosomes and reduced translocation to the nucleus. Instead, the memory-driven pERK1/2 functions as a latent signaling process and primes cells for heightened cytokine (SCF & IL-13) and chemokine (eotaxin-1 & -3) production. CONCLUSIONS: We have developed a model for self-sustained ERK1/2 activation. We previously reported increased ERK1/2 activation in asthmatic airways. Our model may provide a mechanistic understanding of sustained ERK1/2 activation and chronic inflammation in asthma.
Role of Lyn Kinase in Leukotriene C4 (LTC4) Secretion from Mast Cells upon High Affinity IgE Receptor (FceRI) Stimulation H. Sun, M. McLane, B. M. Vonakis; The Johns Hopkins University, Baltimore, MD. RATIONALE: Factors that fine tailor the Yin-Yang effect of Lyn on mast cell functions remain elusive, and few specific functions have been assigned to LynA and LynB isoforms. Here we examined how membrane raft-exclusion of LynA and LynB unique domains can antagonize FceRI signaling resulting in inhibition of LTC4 secretion. METHODS: We stably transfected the RBL-2H3 mast cell line with membrane raft-excluded (Tac) chimeras of the unique domain of LynA, LynB or the cytoplasmic domain of Tac (Tac-WT, control). The cells were sensitized with IgE and stimulated with antigen (2 - 10 minutes). The phosphorylation of MAP kinases (MEK, ERK2) and cytosolic phospholipase A2 (cPLA2) was examined using Western blotting and normalized to total ERK protein. RESULTS: No significant inhibition of MEK phosphorylation was observed (n 5 4). ERK2 phosphorylation was significantly inhibited (35%-87%) in Tac-Lyn A and B unique domain transfectants at all time points compared to Tac-WT (n 5 8-12; P < 0.05). Unexpectedly, the inhibition gradually reversed in Tac-LynA clones with time, but was sustained in Tac-LynB clones. Inhibition of cPLA2 phosphorylation ranged from 57%-67% in Tac-Lyn A clones (n 5 12) and 10%-54% in Tac-LynB clones (n 5 7). CONCLUSIONS: LynA and LynB kinases localized outside of membrane rafts positively regulate the ERK2 activation required for LTC4 secretion. This ERK2 activation may not be mediated via the canonical ras-raf-MEK pathway. LynA may positively regulate a MAP kinase phosphatase, given the transient inhibition of ERK2 phosphorylation observed in TacLynAunique domain transfectants. In contrast, the higher specific activity of Lyn B may permit the sustained inhibition detected in Tac-LynB unique domain transfectants.
SUNDAY
Abstracts S119
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2
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Tetracycline Mediated-Suppression of IgE Secretion by Peripheral Blood Mononuclear Cells (PBMC) in Allergic Asthmatics with History of Chlamydia pneumoniae (Cpn) Infection M. S. Dzhindzhikhashvili, R. Joks, T. Smith-Norowitz, M. R. Hammerschlag, H. G. Durkin, S. Kohlhoff; Center for Allergy and Asthma Research at SUNY Downstate Medical Center, Brooklyn, NY. RATIONALE: We previously reported that tetracycline treatment of allergic asthmatic humans improves their symptoms and suppresses their IgE responses in vivo, and suppresses human and murine memory responses in vitro. The present studies investigated the ability of Cpn, an important cause of asthma exacerbations, and doxycycline to regulate cytokine and IgE reponses by PBMC in vitro. METHODS: PBMC from serum IgE1 allergic asthmatic humans with (n 5 4) and without (n 5 7) evidence of prior Cpn infection (microimmunofluorescence IgG titer >16) (median serum IgG anti Cpn titers: 256, 0, respectively) (mean IgE: 881, 296, IU/ml, respectively), and from serum IgE- donors (n 5 12), were infected or not with Cpn (multiplicity of infection 0.01-10), and then cultured for 2-12 days 1/2 doxycycline. The levels of cytokines (IL-4, IFN-g) (day 2) and IgE (day 10) in culture supernatants were determined by ELISA. RESULTS: PBMC from serum IgE1 allergic asthmatic patients with evidence of previous Cpn infection secreted low levels of IgE on all days of culture, which were further increased when infected with Cpn in vitro (days 2-12;peak levels d10); this further increase in IgE was correlated with Cpn induced IL-4 (CC 5 0.979;p < 0.001), but not with IFN-g production. Inclusion of doxycycline in culture strongly suppressed these IgE responses (>48%, p 5 0.043) and IL-4 (>45%, p 5 0.018), but not IFN-g (5%, NS) production even at subinhibitory concentrations for Cpn. No IgE was secreted by PBMC from any of the control groups. CONCLUSIONS: Cpn plays an important role in regulation of IgE responses; tetracyclines suppress Cpn mediated regulation of IgE responses. Following Segmental Antigen Challenge, Airway Eosinophils Exhibit Differential Cytokine Regulation of the STAT and ERK Signaling Networks When Compared to Blood Eosinophils C. J. Koziol-White, M. E. Bates, P. J. Bertics; University of WisconsinMadison, Madison, WI. RATIONALE: Airway eosinophils (BALEOS) isolated following segmental antigen challenge (SBP-Ag) exhibit multiple phenotypic changes relative to peripheral blood eosinophils (PBEOS), including altered effector functions and cytokine (e.g., IL-5, GM-CSF, IL-3) receptor expression profiles. Here, we tested the hypothesis that these changes in airway versus blood EOS occur in parallel to a shift in the activation of key signaling molecules (STAT5, ERK1/2) that are stimulated by these cytokines in vivo. METHODS: PBEOS and BALEOS (recovered from bronchoalveolar lavage (BAL) fluid 48 hr following SBP-Ag) were stimulated in vitro with 1 nM IL-5, GM-CSF, or IL-3. Cell lysates were prepared and STAT5 and ERK1/2 phosphorylation was assessed via immunoblotting, quantified by densitometry, and normalized to loading controls. RESULTS: With respect to STAT5 phosphorylation, BALEOS are significantly less responsive than PBEOS to IL-5 and GM-CSF (p 5 0.003 and p 5 0.021, respectively, n 5 5), with a similar trend following IL-3 stimulation (n 5 2). However, ERK1/2 phosphorylation is not significantly different in BALEOS versus PBEOS after cytokine stimulation (IL-5 p 5 0.39, GM-CSF p 5 0.75). CONCLUSIONS: These data suggest that a regulatory mechanism for STAT5 pathway activation (distinct from ERK1/2 activation) is present in BALEOS and suggests that STAT5-specific transcriptional regulation is altered in BALEOS, with profound consequences for downstream gene expression.
An Experimental Model for Self-sustained Activation (Bistability) of the ERK1/2 Signaling Module-a Precursor for Memory-driven Chronic Inflammation? W. Liu, K. Tundwal, Q. Liang, R. Alam; National Jewish Medical and Research Center, Denver, CO. RATIONALE: We hypothesize that chronic airway inflammation in asthma is partially driven by a self-sustained mechanism that is intermittently fueled by environmental factors. The objective was to establish an experimental model of a self-sustained signaling process that could drive inflammation. METHODS: Airway epithelial cells and genetically-modified fibroblasts were stimulated with IL-13, IL-6, eotaxin, TNF, or EGF and their effect on signaling and cell activation was studied by western blotting, kinase assay, immunostaining, microarray, real-time PCR and ELISA. RESULTS: A single stimulation of cells with cytokines causes rapid ERK1/2 activation, which returns to baseline in 12-24 hr. Repeated stimulation leads to sustained activation of ERK1/2 but not STAT6, p38 or AKT. The ERK1/2 activation lasts for 3-7 days. This sustained activation depends upon a positive feedback mechanism involving the induction of sprouty-2. Overexpression of sprouty-2 induces and its homozygous genetic deletion abrogates ERK1/2 bistability. Sprouty-2 directly activates Fyn kinase, which then induces ERK1/2 activation. Unlike the acutely activated pERK1/2, the memory-driven pERK1/2 does not induce a high level gene transcription as shown by microarray analysis (33,300 probe sets). This is in part due to its compartmentalization in the Rab4 and Rab51 endosomes and reduced translocation to the nucleus. Instead, the memory-driven pERK1/2 functions as a latent signaling process and primes cells for heightened cytokine (SCF & IL-13) and chemokine (eotaxin-1 & -3) production. CONCLUSIONS: We have developed a model for self-sustained ERK1/2 activation. We previously reported increased ERK1/2 activation in asthmatic airways. Our model may provide a mechanistic understanding of sustained ERK1/2 activation and chronic inflammation in asthma.
Role of Lyn Kinase in Leukotriene C4 (LTC4) Secretion from Mast Cells upon High Affinity IgE Receptor (FceRI) Stimulation H. Sun, M. McLane, B. M. Vonakis; The Johns Hopkins University, Baltimore, MD. RATIONALE: Factors that fine tailor the Yin-Yang effect of Lyn on mast cell functions remain elusive, and few specific functions have been assigned to LynA and LynB isoforms. Here we examined how membrane raft-exclusion of LynA and LynB unique domains can antagonize FceRI signaling resulting in inhibition of LTC4 secretion. METHODS: We stably transfected the RBL-2H3 mast cell line with membrane raft-excluded (Tac) chimeras of the unique domain of LynA, LynB or the cytoplasmic domain of Tac (Tac-WT, control). The cells were sensitized with IgE and stimulated with antigen (2 - 10 minutes). The phosphorylation of MAP kinases (MEK, ERK2) and cytosolic phospholipase A2 (cPLA2) was examined using Western blotting and normalized to total ERK protein. RESULTS: No significant inhibition of MEK phosphorylation was observed (n 5 4). ERK2 phosphorylation was significantly inhibited (35%-87%) in Tac-Lyn A and B unique domain transfectants at all time points compared to Tac-WT (n 5 8-12; P < 0.05). Unexpectedly, the inhibition gradually reversed in Tac-LynA clones with time, but was sustained in Tac-LynB clones. Inhibition of cPLA2 phosphorylation ranged from 57%-67% in Tac-Lyn A clones (n 5 12) and 10%-54% in Tac-LynB clones (n 5 7). CONCLUSIONS: LynA and LynB kinases localized outside of membrane rafts positively regulate the ERK2 activation required for LTC4 secretion. This ERK2 activation may not be mediated via the canonical ras-raf-MEK pathway. LynA may positively regulate a MAP kinase phosphatase, given the transient inhibition of ERK2 phosphorylation observed in TacLynAunique domain transfectants. In contrast, the higher specific activity of Lyn B may permit the sustained inhibition detected in Tac-LynB unique domain transfectants.
SUNDAY
Abstracts S119
J ALLERGY CLIN IMMUNOL VOLUME 123, NUMBER 2