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Sputum Mast Cell Markers Reflect Invasive Measures and Represent a Noninvasive Biologic Marker of Asthma Control and Severity
Abstracts AB131
J ALLERGY CLIN IMMUNOL VOLUME 127, NUMBER 2
Sputum Mast Cell Markers Reflect Invasive Measures and Represent a Noninvasive Biologic Marker of Asthma Control and Severity M. L. Fajt1, S. Balzar1, J. F. Alcorn2, J. B. Trudeau1, S. E. Wenzel1; 1The Asthma Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, 2 The Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, PA. RATIONALE: Prior studies have shown that chymase positive mast cells (MCTC) predominate in the submucosa, epithelium [Balzar, AJRCCM, epub 2010] and bronchoalveolar (BAL) cells of severe asthmatics (SA) [Fajt, ATS, 2009] and predict a poorly controlled SA phenotype. We hypothesized that sputum MC markers would reflect those measured using bronchoscopy. METHODS: Sputum from 6 NC, 13 mild/moderate asthmatics (MMA) (10 on inhaled corticosteroids) and 15 SA was evaluated for the MC markers, tryptase and carboxypeptidase A3 (CPA3), by quantitative realtime-PCR. A subgroup (N517) had matching bronchoscopy MC mRNA. The average time between sputum induction and bronchoscopy was 32.1 days. Data were normalized to GAPDH, log transformed and compared between groups. RESULTS: Sputum CPA3 mRNA levels were highest in SA [39.25 (21.48- 71.74) arbitrary units (AU)] compared to MMA [3.94 (2.935.28) AU] and NC [6.62 (4.59-9.55) AU] [overall p50.004, SA vs. MMA p <0.05]. Tryptase mRNA was also increased in SA [overall p50.03]. Sputum CPA3 and tryptase mRNA correlated [r5 0.89, p<0.0001]. Importantly, BAL cell and sputum CPA3 mRNA also correlated [r50.68, p50.0026]. Similar to BAL, sputum CPA3 mRNA predicted asthma exacerbations and negatively correlated with FEV1% predicted. CONCLUSIONS: CPA3 mRNA is increased in SA sputum and reflects a loss of asthma control. The levels correlate strongly with BAL cell CPA3 mRNA suggesting sputum could be a useful noninvasive method to predict MC phenotype and asthma severity. As sputum and bronchoscopy were obtained >1 month apart, the MC signature appears stable. Sputum analysis may be useful in longitudinal clinical and research evaluation of severe asthma.
495
Mast Cell Restricted, Tetramer-forming Tryptases Hinder Thrombin-Induced Coagulation of Plasma by Proteolytically Destroying Fibrinogen A. Prieto-Garcıa1,2, M. Castells1, S. Wang1, R. L. Stevens1; 1Brigham and Women Hospital, Harvard Medical School, Boston, MA, 2Hospital Gregorio Mara~ n on, Madrid, SPAIN. RATIONALE: hTryptase-beta is stored in the cytoplasmic granules of human mast cells (MCs). Although the presence of this protease in the blood has been used diagnostically to identify patients undergoing systemic anaphylaxis, its preferred substrate in the circulation has not been identified. METHODS: Fibrinogen was incubated with recombinant hTryptase-beta at 37oC for 1-15 min, and SDS-PAGE was carried out on the treated samples. The resulting digestion products were excised from the gels and subjected to C-terminal amino acid sequence analysis via LC MS/MS. Sodium citrate-treated human plasma also was incubated at 37oC for 60 min in the absence or presence of hTryptase-beta (6.6 mg) or heparin (100 mg), and the times required for thrombin to clot the samples were determined. RESULTS: As assessed by SDS-PAGE electrophoresis, hTryptase-beta preferentially cleaved fibrinogen’s alpha chain at Lys556 or Lys562, thereby markedly delaying the ability of thrombin to clot hTryptase-beta treated plasma. CONCLUSION: Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Even though only a small amount of thrombin is needed to convert fibrinogen into fibrin, fibrin clots are not present in MC-induced edema sites. It has been known for some time that MC heparin hinders coagulation by promoting the inactivation of thrombin by antithrombin/serpin-C1. In the alternate anti-cogulation mechanism we uncovered, the MC’s tryptases proteolytically destroy fibrinogen before it has a chance to be converted to fibrin by thrombin. The finding that recombinant hTryptase-beta is a more efective anticoagulant than heparin on a weight basis has important clinical implications.
496
Strain-dependent Influences On C-kit Function Alter The Mast Cell Phenotype Of Hyperresponsive A/J Mice H. J. Chong1, E. Cozzi2, A. Lundquist1, J. Boyce1, D. Beier2; 1Brigham and Women’s Hospital, Boston, MA, 2Harvard Medical School, Boston, MA. RATIONALE: Compared with C57BL/6 control mice, na€ıve A/J mice exhibit increased responsiveness to inhaled methacholine that is abrogated by the introduction of the W/v mutation into the A/J background) We sought to determine if A/J mice exhibited an aberrant mast cell phenotype. METHODS: Tracheal mast cells were enumerated by immunohistochemistry using an Ab against mouse MC protease (mMCP)-6. Bone marrowderived mast cells (BMMCs) were grown with IL-3 (10ng/ml) and SCF (10ng/ml) for 3 wk. Western blotting was done using an anti-mMCP4 and anti- mMCP6 Abs. BMMC were also stained with safranin to assess heparin content. Flow cytometry was performed using anti-FceRIalpha and anti CD117. Proliferation was assessed by thymidine incorporation. RESULTS: Na€ıve A/J mice had twice as many tracheal MCs as did C57BL/6 mice. BMMCs from A/J mice showed markedly increased safranin, as well as increased content of both mMCP-4 and mMCP-6 proteins compared to C57BL/6 BMMC. Although the levels of surface c-kit protein were identical on BMMCs from the two strains, BMMCs from A/J mice proliferated more rapidly than C57BL/6 BMMCS when stimulated with SCF, but not when stimulated with IL-3. CONCLUSION: Phenotypic differences between mast cells in A/J versus B6 mice may reflect aberrant regulation of c-kit signaling. Further studies will determine whether the c-kit-dependent na€ıve AHR is causally related to the accompanying mast cell phenotype.
497
Inhibitors of Dipeptidyl Peptidase I (DPPI) as Mast Cell Stabilising Agents: The Contribution of DPPI in Mast Cell Activation G. El-Feki1, X. Zhou1, L. C. Lau1, J. Pedersen2, A. F. Walls1; 1University of Southampton, Southampton, UNITED KINGDOM, 2Unizyme Laboratories A/S, Hørsholm, DENMARK. RATIONALE: Inhibitors of mast cell tryptase and chymase are potent inhibitors of mast cell activation. These abundant mast cell proteases may be converted into catalytically active forms by dipeptidyl peptidase 1 (DPPI), but the potential role of DPPI in degranulation is not known. METHODS: Cells of the LAD2 human mast cell line were sensitized with myeloma IgE and activated with anti-IgE antibody or calcium ionophore A23817. Concentrations of b-hexosaminidase were determined in supernatants and cell lysates, and also tryptase and DPPI using specific ELISA or chromogenic substrates. Cells were pre-incubated with the DPPI inhibitor Gly-Phe-CHN2 (GFCN) or with the small molecule inhibitors of DPPI, PZ610, PZ709 or PZ889 for 0, 5 or 30 min. Parallel studies were conducted with mast cells dispersed from human tonsil, lung or skin in which histamine release was also determined. RESULTS: IgE-dependent release of b-hexosaminidase from LAD2 cells was inhibited by pre-incubating cells with GFCN and other DPPI inhibitors. With 0.3mM GFCN inhibition of b-hexosaminidase release of at least 30% was observed with all incubation periods. In contrast, there was no significant inhibition of calcium ionophore-triggered mast cell activation. DPPI inhibitors stabilised mast cells dispersed from human tissues, with inhibition of mediator release of up to 80% in tonsil mast cells with antiIgE (but not calcium ionophore). Mast cell release of b-hexosaminidase, tryptase, histamine and also DPPI followed similar patterns. CONCLUSIONS: DPPI may play a role in mast cell degranulation, acting within the cell or following secretion. Inhibitors of DPPI may be of value as mast cell stabilizing compounds.
SUNDAY
494
J ALLERGY CLIN IMMUNOL VOLUME 127, NUMBER 2
Sputum Mast Cell Markers Reflect Invasive Measures and Represent a Noninvasive Biologic Marker of Asthma Control and Severity M. L. Fajt1, S. Balzar1, J. F. Alcorn2, J. B. Trudeau1, S. E. Wenzel1; 1The Asthma Institute, University of Pittsburgh Medical Center, Pittsburgh, PA, 2 The Children’s Hospital of Pittsburgh of the University of Pittsburgh Medical Center, Pittsburgh, PA. RATIONALE: Prior studies have shown that chymase positive mast cells (MCTC) predominate in the submucosa, epithelium [Balzar, AJRCCM, epub 2010] and bronchoalveolar (BAL) cells of severe asthmatics (SA) [Fajt, ATS, 2009] and predict a poorly controlled SA phenotype. We hypothesized that sputum MC markers would reflect those measured using bronchoscopy. METHODS: Sputum from 6 NC, 13 mild/moderate asthmatics (MMA) (10 on inhaled corticosteroids) and 15 SA was evaluated for the MC markers, tryptase and carboxypeptidase A3 (CPA3), by quantitative realtime-PCR. A subgroup (N517) had matching bronchoscopy MC mRNA. The average time between sputum induction and bronchoscopy was 32.1 days. Data were normalized to GAPDH, log transformed and compared between groups. RESULTS: Sputum CPA3 mRNA levels were highest in SA [39.25 (21.48- 71.74) arbitrary units (AU)] compared to MMA [3.94 (2.935.28) AU] and NC [6.62 (4.59-9.55) AU] [overall p50.004, SA vs. MMA p <0.05]. Tryptase mRNA was also increased in SA [overall p50.03]. Sputum CPA3 and tryptase mRNA correlated [r5 0.89, p<0.0001]. Importantly, BAL cell and sputum CPA3 mRNA also correlated [r50.68, p50.0026]. Similar to BAL, sputum CPA3 mRNA predicted asthma exacerbations and negatively correlated with FEV1% predicted. CONCLUSIONS: CPA3 mRNA is increased in SA sputum and reflects a loss of asthma control. The levels correlate strongly with BAL cell CPA3 mRNA suggesting sputum could be a useful noninvasive method to predict MC phenotype and asthma severity. As sputum and bronchoscopy were obtained >1 month apart, the MC signature appears stable. Sputum analysis may be useful in longitudinal clinical and research evaluation of severe asthma.
495
Mast Cell Restricted, Tetramer-forming Tryptases Hinder Thrombin-Induced Coagulation of Plasma by Proteolytically Destroying Fibrinogen A. Prieto-Garcıa1,2, M. Castells1, S. Wang1, R. L. Stevens1; 1Brigham and Women Hospital, Harvard Medical School, Boston, MA, 2Hospital Gregorio Mara~ n on, Madrid, SPAIN. RATIONALE: hTryptase-beta is stored in the cytoplasmic granules of human mast cells (MCs). Although the presence of this protease in the blood has been used diagnostically to identify patients undergoing systemic anaphylaxis, its preferred substrate in the circulation has not been identified. METHODS: Fibrinogen was incubated with recombinant hTryptase-beta at 37oC for 1-15 min, and SDS-PAGE was carried out on the treated samples. The resulting digestion products were excised from the gels and subjected to C-terminal amino acid sequence analysis via LC MS/MS. Sodium citrate-treated human plasma also was incubated at 37oC for 60 min in the absence or presence of hTryptase-beta (6.6 mg) or heparin (100 mg), and the times required for thrombin to clot the samples were determined. RESULTS: As assessed by SDS-PAGE electrophoresis, hTryptase-beta preferentially cleaved fibrinogen’s alpha chain at Lys556 or Lys562, thereby markedly delaying the ability of thrombin to clot hTryptase-beta treated plasma. CONCLUSION: Fibrinogen is a major constituent of the edema fluid that accumulates in tissues when MCs degranulate. Even though only a small amount of thrombin is needed to convert fibrinogen into fibrin, fibrin clots are not present in MC-induced edema sites. It has been known for some time that MC heparin hinders coagulation by promoting the inactivation of thrombin by antithrombin/serpin-C1. In the alternate anti-cogulation mechanism we uncovered, the MC’s tryptases proteolytically destroy fibrinogen before it has a chance to be converted to fibrin by thrombin. The finding that recombinant hTryptase-beta is a more efective anticoagulant than heparin on a weight basis has important clinical implications.
496
Strain-dependent Influences On C-kit Function Alter The Mast Cell Phenotype Of Hyperresponsive A/J Mice H. J. Chong1, E. Cozzi2, A. Lundquist1, J. Boyce1, D. Beier2; 1Brigham and Women’s Hospital, Boston, MA, 2Harvard Medical School, Boston, MA. RATIONALE: Compared with C57BL/6 control mice, na€ıve A/J mice exhibit increased responsiveness to inhaled methacholine that is abrogated by the introduction of the W/v mutation into the A/J background) We sought to determine if A/J mice exhibited an aberrant mast cell phenotype. METHODS: Tracheal mast cells were enumerated by immunohistochemistry using an Ab against mouse MC protease (mMCP)-6. Bone marrowderived mast cells (BMMCs) were grown with IL-3 (10ng/ml) and SCF (10ng/ml) for 3 wk. Western blotting was done using an anti-mMCP4 and anti- mMCP6 Abs. BMMC were also stained with safranin to assess heparin content. Flow cytometry was performed using anti-FceRIalpha and anti CD117. Proliferation was assessed by thymidine incorporation. RESULTS: Na€ıve A/J mice had twice as many tracheal MCs as did C57BL/6 mice. BMMCs from A/J mice showed markedly increased safranin, as well as increased content of both mMCP-4 and mMCP-6 proteins compared to C57BL/6 BMMC. Although the levels of surface c-kit protein were identical on BMMCs from the two strains, BMMCs from A/J mice proliferated more rapidly than C57BL/6 BMMCS when stimulated with SCF, but not when stimulated with IL-3. CONCLUSION: Phenotypic differences between mast cells in A/J versus B6 mice may reflect aberrant regulation of c-kit signaling. Further studies will determine whether the c-kit-dependent na€ıve AHR is causally related to the accompanying mast cell phenotype.
497
Inhibitors of Dipeptidyl Peptidase I (DPPI) as Mast Cell Stabilising Agents: The Contribution of DPPI in Mast Cell Activation G. El-Feki1, X. Zhou1, L. C. Lau1, J. Pedersen2, A. F. Walls1; 1University of Southampton, Southampton, UNITED KINGDOM, 2Unizyme Laboratories A/S, Hørsholm, DENMARK. RATIONALE: Inhibitors of mast cell tryptase and chymase are potent inhibitors of mast cell activation. These abundant mast cell proteases may be converted into catalytically active forms by dipeptidyl peptidase 1 (DPPI), but the potential role of DPPI in degranulation is not known. METHODS: Cells of the LAD2 human mast cell line were sensitized with myeloma IgE and activated with anti-IgE antibody or calcium ionophore A23817. Concentrations of b-hexosaminidase were determined in supernatants and cell lysates, and also tryptase and DPPI using specific ELISA or chromogenic substrates. Cells were pre-incubated with the DPPI inhibitor Gly-Phe-CHN2 (GFCN) or with the small molecule inhibitors of DPPI, PZ610, PZ709 or PZ889 for 0, 5 or 30 min. Parallel studies were conducted with mast cells dispersed from human tonsil, lung or skin in which histamine release was also determined. RESULTS: IgE-dependent release of b-hexosaminidase from LAD2 cells was inhibited by pre-incubating cells with GFCN and other DPPI inhibitors. With 0.3mM GFCN inhibition of b-hexosaminidase release of at least 30% was observed with all incubation periods. In contrast, there was no significant inhibition of calcium ionophore-triggered mast cell activation. DPPI inhibitors stabilised mast cells dispersed from human tissues, with inhibition of mediator release of up to 80% in tonsil mast cells with antiIgE (but not calcium ionophore). Mast cell release of b-hexosaminidase, tryptase, histamine and also DPPI followed similar patterns. CONCLUSIONS: DPPI may play a role in mast cell degranulation, acting within the cell or following secretion. Inhibitors of DPPI may be of value as mast cell stabilizing compounds.
SUNDAY
494