A small volume technique to examine and compare alveolar macrophage phagocytosis of apoptotic cells and non typeable Haemophilus influenzae (NTHI)

Miranda Ween, Jessica Ahern, Alexander Carroll, Greg Hodge, Susan Pizzutto, Hubertus Jersmann, Paul Reynolds, Sandra Hodge

    Research output: Contribution to journalArticleResearchpeer-review

    Abstract

    A defective ability of alveolar macrophages to phagocytose both apoptotic airway epithelial cells and bacteria in chronic lung diseases potentially associated with inflammation and bacterial colonisation of the lower airways, often with non-typeable Haemophilus influenzae (NTHi), has been shown. We routinely assess phagocytosis in the airway of children: however, the small volume of BAL obtained usually precludes the investigation of phagocytosis of both (potentially equally relevant) apoptotic cells and NTHi.
    Methods: We established a 'one-tube, dual target' flow-cytometric assay for investigating alveolar macrophage phagocytosis of both apoptotic cells and NTHi. The effect of bacterial presence on phagocytosis of apoptotic cells was assessed by comparing results using this technique to standard 'two tube, single target' methods. The comparative ability of alveolar macrophages to phagocytose NTHi or apoptotic cells was assessed in 10/group of healthy adult controls and patients with COPD, 12 children with bronchiectasis, and 10 children controls. We then assessed the influence of increasing concentrations of NTHi targets on the ability of THP-1 macrophages to simultaneously phagocytose apoptotic cells.
    Results: Alveolar macrophages phagocytosed NTHi more avidly than apoptotic cells (mean ± SEM: apoptotic cells 15.4% ± 0.5 vs. NTHi 17.2% ± 0.7, p < 0.05). The presence of NTHi targets (ratio of 1:100 macrophage: NTHi; 2 x107 CFU routinely applied in our assay) had no effect on the ability of macrophages to simultaneously phagocytose apoptotic cells. However, when bacterial numbers were increased (up to 4-fold) there was a small but significant suppressive effect on the ability of macrophages to phagocytose apoptotic cells.
    Conclusions: We describe a small volume 'one tube, dual target' technique to measure phagocytosis of apoptotic cells and NTHi. We show that alveolar macrophages phagocytose NTHi more avidly than apoptotic cells, and that an increased presence of NTHi in the airway may reduce the ability of alveolar macrophages to phagocytose apoptotic cells. 
    Original languageEnglish
    Pages (from-to)7-14
    Number of pages8
    JournalJournal of Immunological Methods
    Volume429
    DOIs
    Publication statusPublished - 2016

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    Cytophagocytosis
    Haemophilus influenzae
    Alveolar Macrophages
    Phagocytosis
    Macrophages
    Dimercaprol
    Bronchiectasis
    Chronic Obstructive Pulmonary Disease
    Lung Diseases
    Chronic Disease

    Cite this

    Ween, Miranda ; Ahern, Jessica ; Carroll, Alexander ; Hodge, Greg ; Pizzutto, Susan ; Jersmann, Hubertus ; Reynolds, Paul ; Hodge, Sandra. / A small volume technique to examine and compare alveolar macrophage phagocytosis of apoptotic cells and non typeable Haemophilus influenzae (NTHI). In: Journal of Immunological Methods. 2016 ; Vol. 429. pp. 7-14.
    @article{083ea7279bce484f98285e305d64bf82,
    title = "A small volume technique to examine and compare alveolar macrophage phagocytosis of apoptotic cells and non typeable Haemophilus influenzae (NTHI)",
    abstract = "A defective ability of alveolar macrophages to phagocytose both apoptotic airway epithelial cells and bacteria in chronic lung diseases potentially associated with inflammation and bacterial colonisation of the lower airways, often with non-typeable Haemophilus influenzae (NTHi), has been shown. We routinely assess phagocytosis in the airway of children: however, the small volume of BAL obtained usually precludes the investigation of phagocytosis of both (potentially equally relevant) apoptotic cells and NTHi.Methods: We established a 'one-tube, dual target' flow-cytometric assay for investigating alveolar macrophage phagocytosis of both apoptotic cells and NTHi. The effect of bacterial presence on phagocytosis of apoptotic cells was assessed by comparing results using this technique to standard 'two tube, single target' methods. The comparative ability of alveolar macrophages to phagocytose NTHi or apoptotic cells was assessed in 10/group of healthy adult controls and patients with COPD, 12 children with bronchiectasis, and 10 children controls. We then assessed the influence of increasing concentrations of NTHi targets on the ability of THP-1 macrophages to simultaneously phagocytose apoptotic cells.Results: Alveolar macrophages phagocytosed NTHi more avidly than apoptotic cells (mean ± SEM: apoptotic cells 15.4{\%} ± 0.5 vs. NTHi 17.2{\%} ± 0.7, p < 0.05). The presence of NTHi targets (ratio of 1:100 macrophage: NTHi; 2 x107 CFU routinely applied in our assay) had no effect on the ability of macrophages to simultaneously phagocytose apoptotic cells. However, when bacterial numbers were increased (up to 4-fold) there was a small but significant suppressive effect on the ability of macrophages to phagocytose apoptotic cells.Conclusions: We describe a small volume 'one tube, dual target' technique to measure phagocytosis of apoptotic cells and NTHi. We show that alveolar macrophages phagocytose NTHi more avidly than apoptotic cells, and that an increased presence of NTHi in the airway may reduce the ability of alveolar macrophages to phagocytose apoptotic cells. ",
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    author = "Miranda Ween and Jessica Ahern and Alexander Carroll and Greg Hodge and Susan Pizzutto and Hubertus Jersmann and Paul Reynolds and Sandra Hodge",
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    A small volume technique to examine and compare alveolar macrophage phagocytosis of apoptotic cells and non typeable Haemophilus influenzae (NTHI). / Ween, Miranda; Ahern, Jessica; Carroll, Alexander; Hodge, Greg; Pizzutto, Susan; Jersmann, Hubertus; Reynolds, Paul; Hodge, Sandra.

    In: Journal of Immunological Methods, Vol. 429, 2016, p. 7-14.

    Research output: Contribution to journalArticleResearchpeer-review

    TY - JOUR

    T1 - A small volume technique to examine and compare alveolar macrophage phagocytosis of apoptotic cells and non typeable Haemophilus influenzae (NTHI)

    AU - Ween, Miranda

    AU - Ahern, Jessica

    AU - Carroll, Alexander

    AU - Hodge, Greg

    AU - Pizzutto, Susan

    AU - Jersmann, Hubertus

    AU - Reynolds, Paul

    AU - Hodge, Sandra

    PY - 2016

    Y1 - 2016

    N2 - A defective ability of alveolar macrophages to phagocytose both apoptotic airway epithelial cells and bacteria in chronic lung diseases potentially associated with inflammation and bacterial colonisation of the lower airways, often with non-typeable Haemophilus influenzae (NTHi), has been shown. We routinely assess phagocytosis in the airway of children: however, the small volume of BAL obtained usually precludes the investigation of phagocytosis of both (potentially equally relevant) apoptotic cells and NTHi.Methods: We established a 'one-tube, dual target' flow-cytometric assay for investigating alveolar macrophage phagocytosis of both apoptotic cells and NTHi. The effect of bacterial presence on phagocytosis of apoptotic cells was assessed by comparing results using this technique to standard 'two tube, single target' methods. The comparative ability of alveolar macrophages to phagocytose NTHi or apoptotic cells was assessed in 10/group of healthy adult controls and patients with COPD, 12 children with bronchiectasis, and 10 children controls. We then assessed the influence of increasing concentrations of NTHi targets on the ability of THP-1 macrophages to simultaneously phagocytose apoptotic cells.Results: Alveolar macrophages phagocytosed NTHi more avidly than apoptotic cells (mean ± SEM: apoptotic cells 15.4% ± 0.5 vs. NTHi 17.2% ± 0.7, p < 0.05). The presence of NTHi targets (ratio of 1:100 macrophage: NTHi; 2 x107 CFU routinely applied in our assay) had no effect on the ability of macrophages to simultaneously phagocytose apoptotic cells. However, when bacterial numbers were increased (up to 4-fold) there was a small but significant suppressive effect on the ability of macrophages to phagocytose apoptotic cells.Conclusions: We describe a small volume 'one tube, dual target' technique to measure phagocytosis of apoptotic cells and NTHi. We show that alveolar macrophages phagocytose NTHi more avidly than apoptotic cells, and that an increased presence of NTHi in the airway may reduce the ability of alveolar macrophages to phagocytose apoptotic cells. 

    AB - A defective ability of alveolar macrophages to phagocytose both apoptotic airway epithelial cells and bacteria in chronic lung diseases potentially associated with inflammation and bacterial colonisation of the lower airways, often with non-typeable Haemophilus influenzae (NTHi), has been shown. We routinely assess phagocytosis in the airway of children: however, the small volume of BAL obtained usually precludes the investigation of phagocytosis of both (potentially equally relevant) apoptotic cells and NTHi.Methods: We established a 'one-tube, dual target' flow-cytometric assay for investigating alveolar macrophage phagocytosis of both apoptotic cells and NTHi. The effect of bacterial presence on phagocytosis of apoptotic cells was assessed by comparing results using this technique to standard 'two tube, single target' methods. The comparative ability of alveolar macrophages to phagocytose NTHi or apoptotic cells was assessed in 10/group of healthy adult controls and patients with COPD, 12 children with bronchiectasis, and 10 children controls. We then assessed the influence of increasing concentrations of NTHi targets on the ability of THP-1 macrophages to simultaneously phagocytose apoptotic cells.Results: Alveolar macrophages phagocytosed NTHi more avidly than apoptotic cells (mean ± SEM: apoptotic cells 15.4% ± 0.5 vs. NTHi 17.2% ± 0.7, p < 0.05). The presence of NTHi targets (ratio of 1:100 macrophage: NTHi; 2 x107 CFU routinely applied in our assay) had no effect on the ability of macrophages to simultaneously phagocytose apoptotic cells. However, when bacterial numbers were increased (up to 4-fold) there was a small but significant suppressive effect on the ability of macrophages to phagocytose apoptotic cells.Conclusions: We describe a small volume 'one tube, dual target' technique to measure phagocytosis of apoptotic cells and NTHi. We show that alveolar macrophages phagocytose NTHi more avidly than apoptotic cells, and that an increased presence of NTHi in the airway may reduce the ability of alveolar macrophages to phagocytose apoptotic cells. 

    KW - adult

    KW - airway

    KW - apoptosis

    KW - Article

    KW - bronchiectasis

    KW - child

    KW - chronic obstructive lung disease

    KW - clinical article

    KW - cohort analysis

    KW - controlled study

    KW - correlational study

    KW - female

    KW - flow cytometry

    KW - human

    KW - human cell

    KW - infant

    KW - lung alveolus macrophage

    KW - male

    KW - nontypeable Haemophilus influenzae

    KW - phagocytosis

    KW - priority journal

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    U2 - 10.1111/resp.12495

    DO - 10.1111/resp.12495

    M3 - Article

    VL - 429

    SP - 7

    EP - 14

    JO - Journal of Immunological Methods

    JF - Journal of Immunological Methods

    SN - 0022-1759

    ER -