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Human CD56+ cytotoxic lung lymphocytes kill autologous lung cells in chronic obstructive pulmonary disease.

Freeman CM, Stolberg VR, Crudgington S, Martinez FJ, Han MK, Chensue SW, Arenberg DA, Meldrum CA, McCloskey L, Curtis JL - PLoS ONE (2014)

Bottom Line: Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining.Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV1 % predicted.Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation.

View Article: PubMed Central - PubMed

Affiliation: Research Service, VA Ann Arbor Healthcare System, Ann Arbor, Michigan, United States of America; Pulmonary & Critical Care Medicine Division, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, United States of America.

ABSTRACT

Unlabelled: CD56+ natural killer (NK) and CD56+ T cells, from sputum or bronchoalveolar lavage of subjects with chronic obstructive pulmonary disease (COPD) are more cytotoxic to highly susceptible NK targets than those from control subjects. Whether the same is true in lung parenchyma, and if NK activity actually contributes to emphysema progression are unknown. To address these questions, we performed two types of experiments on lung tissue from clinically-indicated resections (n = 60). First, we used flow cytometry on fresh single-cell suspension to measure expression of cell-surface molecules (CD56, CD16, CD8, NKG2D and NKp44) on lung lymphocytes and of the 6D4 epitope common to MICA and MICB on lung epithelial (CD326+) cells. Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining. Lung NK cells (CD56+ CD3-) and CD56+ T cells (CD56+ CD3+) were present in a range of frequencies that did not differ significantly between smokers without COPD and subjects with COPD. Lung NK cells had a predominantly "cytotoxic" CD56+ CD16+ phenotype; their co-expression of CD8 was common, but the percentage expressing CD8 fell as FEV1 % predicted decreased. Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV1 % predicted. Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation. Such natural cytotoxicity was increased in subjects with severe COPD and was unexplained in multiple regression analysis by age or cancer as indication for surgery. These data show that as spirometry worsens in COPD, CD56+ lung lymphocytes exhibit spontaneous cytotoxicity of autologous structural lung cells, supporting their potential role in emphysema progression.

Trial registration: ClinicalTrials.gov NCT00281229.

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Related in: MedlinePlus

Human lung CD56+ cells spontaneously kill autologous lung CD45− cells in vitro.CD56+ cells were isolated from dispersed human lung tissue using magnetic beads. CD8+ cells were isolated from the CD56 depleted fraction and CD4+ cells were isolated from the CD56− and CD8− depleted fraction. The remaining cells were used as autologous target cells. Target cells were cultured either alone or with CD56+ cells, CD8+ cells, or CD4+ cells at a ratio of 1 target to 10 effectors. After 4 hours, all cells were collected and stained with CD45, Annexin-V, and 7-AAD for flow cytometry. Target cells were identified as CD45− with a high side scatter. (A) Representative staining of Annexin-V on target cells that were cultured with CD56+ cells (left panel), CD8+ cells (middle panel), and CD4+ cells (right panel). (B) % Cytotoxicity (y-axis) for target cells cultured with CD56+ cells (blue circles), CD8+ cells (orange circles), or CD4+ cells (green circles); n = 28. Lines represent the mean ± SEM. The Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test was used to determine significant differences between groups.
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pone-0103840-g004: Human lung CD56+ cells spontaneously kill autologous lung CD45− cells in vitro.CD56+ cells were isolated from dispersed human lung tissue using magnetic beads. CD8+ cells were isolated from the CD56 depleted fraction and CD4+ cells were isolated from the CD56− and CD8− depleted fraction. The remaining cells were used as autologous target cells. Target cells were cultured either alone or with CD56+ cells, CD8+ cells, or CD4+ cells at a ratio of 1 target to 10 effectors. After 4 hours, all cells were collected and stained with CD45, Annexin-V, and 7-AAD for flow cytometry. Target cells were identified as CD45− with a high side scatter. (A) Representative staining of Annexin-V on target cells that were cultured with CD56+ cells (left panel), CD8+ cells (middle panel), and CD4+ cells (right panel). (B) % Cytotoxicity (y-axis) for target cells cultured with CD56+ cells (blue circles), CD8+ cells (orange circles), or CD4+ cells (green circles); n = 28. Lines represent the mean ± SEM. The Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test was used to determine significant differences between groups.

Mentions: We recruited and consented subjects undergoing clinically-indicated resections for pulmonary nodules, lung volume reduction surgery, or lung transplantation. Only non-neoplastic lung tissue remote from the nodules and lacking post-obstructive changes as judged by a Pathologist was collected. Because not all experiments can be performed on every lung sample, two cohorts of subjects were used to complete these experiments (Table 1). Cohort A (n = 35) was used for both flow cytometry and cell isolations. Cohort B (n = 25) was used exclusively for flow cytometric analyses. All subjects (n = 60) underwent preoperative spirometry, prospectively collected medication history and clinical evaluation by a pulmonologist. We categorized subjects using the 2008 classification of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) [26]. Subjects (n = 16) with a smoking history of 10 pack years or greater, a ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC) >0.70, normal spirometry, and no clinical diagnosis of COPD represent smoking controls (Smoker). Subjects (n = 19) with a smoking history, FEV1/FVC <0.7 and FEV1 % predicted ≥50% were considered to have mild COPD (Mild COPD). Subjects (n = 25) with a smoking history, FEV1/FVC <0.7 and FEV1 % predicted <50% were considered to have severe COPD (Severe COPD). Table 1 shows the male-to-female ratio, age range, smoking history, FEV1 % predicted, diffusing capacity of the lung for carbon monoxide (DLCO) % predicted, inhaled corticosteroid (ICS) usage, and the indication for lung resection for smokers, mild COPD, and severe COPD subjects for both Cohort A and Cohort B.


Human CD56+ cytotoxic lung lymphocytes kill autologous lung cells in chronic obstructive pulmonary disease.

Freeman CM, Stolberg VR, Crudgington S, Martinez FJ, Han MK, Chensue SW, Arenberg DA, Meldrum CA, McCloskey L, Curtis JL - PLoS ONE (2014)

Human lung CD56+ cells spontaneously kill autologous lung CD45− cells in vitro.CD56+ cells were isolated from dispersed human lung tissue using magnetic beads. CD8+ cells were isolated from the CD56 depleted fraction and CD4+ cells were isolated from the CD56− and CD8− depleted fraction. The remaining cells were used as autologous target cells. Target cells were cultured either alone or with CD56+ cells, CD8+ cells, or CD4+ cells at a ratio of 1 target to 10 effectors. After 4 hours, all cells were collected and stained with CD45, Annexin-V, and 7-AAD for flow cytometry. Target cells were identified as CD45− with a high side scatter. (A) Representative staining of Annexin-V on target cells that were cultured with CD56+ cells (left panel), CD8+ cells (middle panel), and CD4+ cells (right panel). (B) % Cytotoxicity (y-axis) for target cells cultured with CD56+ cells (blue circles), CD8+ cells (orange circles), or CD4+ cells (green circles); n = 28. Lines represent the mean ± SEM. The Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test was used to determine significant differences between groups.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4117545&req=5

pone-0103840-g004: Human lung CD56+ cells spontaneously kill autologous lung CD45− cells in vitro.CD56+ cells were isolated from dispersed human lung tissue using magnetic beads. CD8+ cells were isolated from the CD56 depleted fraction and CD4+ cells were isolated from the CD56− and CD8− depleted fraction. The remaining cells were used as autologous target cells. Target cells were cultured either alone or with CD56+ cells, CD8+ cells, or CD4+ cells at a ratio of 1 target to 10 effectors. After 4 hours, all cells were collected and stained with CD45, Annexin-V, and 7-AAD for flow cytometry. Target cells were identified as CD45− with a high side scatter. (A) Representative staining of Annexin-V on target cells that were cultured with CD56+ cells (left panel), CD8+ cells (middle panel), and CD4+ cells (right panel). (B) % Cytotoxicity (y-axis) for target cells cultured with CD56+ cells (blue circles), CD8+ cells (orange circles), or CD4+ cells (green circles); n = 28. Lines represent the mean ± SEM. The Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test was used to determine significant differences between groups.
Mentions: We recruited and consented subjects undergoing clinically-indicated resections for pulmonary nodules, lung volume reduction surgery, or lung transplantation. Only non-neoplastic lung tissue remote from the nodules and lacking post-obstructive changes as judged by a Pathologist was collected. Because not all experiments can be performed on every lung sample, two cohorts of subjects were used to complete these experiments (Table 1). Cohort A (n = 35) was used for both flow cytometry and cell isolations. Cohort B (n = 25) was used exclusively for flow cytometric analyses. All subjects (n = 60) underwent preoperative spirometry, prospectively collected medication history and clinical evaluation by a pulmonologist. We categorized subjects using the 2008 classification of the Global Initiative for Chronic Obstructive Lung Disease (GOLD) [26]. Subjects (n = 16) with a smoking history of 10 pack years or greater, a ratio of forced expiratory volume in 1 second to forced vital capacity (FEV1/FVC) >0.70, normal spirometry, and no clinical diagnosis of COPD represent smoking controls (Smoker). Subjects (n = 19) with a smoking history, FEV1/FVC <0.7 and FEV1 % predicted ≥50% were considered to have mild COPD (Mild COPD). Subjects (n = 25) with a smoking history, FEV1/FVC <0.7 and FEV1 % predicted <50% were considered to have severe COPD (Severe COPD). Table 1 shows the male-to-female ratio, age range, smoking history, FEV1 % predicted, diffusing capacity of the lung for carbon monoxide (DLCO) % predicted, inhaled corticosteroid (ICS) usage, and the indication for lung resection for smokers, mild COPD, and severe COPD subjects for both Cohort A and Cohort B.

Bottom Line: Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining.Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV1 % predicted.Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation.

View Article: PubMed Central - PubMed

Affiliation: Research Service, VA Ann Arbor Healthcare System, Ann Arbor, Michigan, United States of America; Pulmonary & Critical Care Medicine Division, Department of Internal Medicine, University of Michigan Health System, Ann Arbor, Michigan, United States of America.

ABSTRACT

Unlabelled: CD56+ natural killer (NK) and CD56+ T cells, from sputum or bronchoalveolar lavage of subjects with chronic obstructive pulmonary disease (COPD) are more cytotoxic to highly susceptible NK targets than those from control subjects. Whether the same is true in lung parenchyma, and if NK activity actually contributes to emphysema progression are unknown. To address these questions, we performed two types of experiments on lung tissue from clinically-indicated resections (n = 60). First, we used flow cytometry on fresh single-cell suspension to measure expression of cell-surface molecules (CD56, CD16, CD8, NKG2D and NKp44) on lung lymphocytes and of the 6D4 epitope common to MICA and MICB on lung epithelial (CD326+) cells. Second, we sequentially isolated CD56+, CD8+ and CD4+ lung lymphocytes, co-cultured each with autologous lung target cells, then determined apoptosis of individual target cells using Annexin-V and 7-AAD staining. Lung NK cells (CD56+ CD3-) and CD56+ T cells (CD56+ CD3+) were present in a range of frequencies that did not differ significantly between smokers without COPD and subjects with COPD. Lung NK cells had a predominantly "cytotoxic" CD56+ CD16+ phenotype; their co-expression of CD8 was common, but the percentage expressing CD8 fell as FEV1 % predicted decreased. Greater expression by autologous lung epithelial cells of the NKG2D ligands, MICA/MICB, but not expression by lung CD56+ cells of the activating receptor NKG2D, correlated inversely with FEV1 % predicted. Lung CD56+ lymphocytes, but not CD4+ or CD8+ conventional lung T cells, rapidly killed autologous lung cells without additional stimulation. Such natural cytotoxicity was increased in subjects with severe COPD and was unexplained in multiple regression analysis by age or cancer as indication for surgery. These data show that as spirometry worsens in COPD, CD56+ lung lymphocytes exhibit spontaneous cytotoxicity of autologous structural lung cells, supporting their potential role in emphysema progression.

Trial registration: ClinicalTrials.gov NCT00281229.

Show MeSH
Related in: MedlinePlus