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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

Identification and characterization of human lung NK cells and CD56+ T cells.Lung tissue was dispersed, stained with monoclonal antibodies against CD45, CD3, CD56, and CD16 and analyzed by flow cytometry to select a viable population comprised predominately of lung lymphocytes (CD45+, low side-scatter cells). (A, C, E) Representative staining: isotype controls on left, specific staining on right; (B, D, F) Frequency of various lung lymphocyte populations in individual subjects as a percentage of the total viable lung lymphocyte population; note difference in scale of panel B. (A) Ungated staining for CD3 and CD56 identifies four distinct populations: NK cells (CD56+ CD3−); CD56+ T cells (CD56+ CD3+); conventional T cells (CD56− CD3+); and double-negative cells (predominately B cells). (B) NK cells (blue bars) versus CD56+ T cells (orange bars). (C, D) After gating on CD3− cells, staining for CD56 and CD16 identifies two lung NK populations: CD56+ CD16+ (light blue circle & columns) and CD56+ CD16− (dark blue circle & columns). (E, F) After gating on CD3+ cells, staining for CD56 and CD16 identifies two lung CD56+ T cell populations: CD56+ CD16+ (dark orange circle & columns) and CD56+ CD16− (light orange circle & columns). By Kruskal-Wallis one-way ANOVA, there are no significant differences between subject groups for any of these three lung cell populations (B, D, F).
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pone-0103840-g001: Identification and characterization of human lung NK cells and CD56+ T cells.Lung tissue was dispersed, stained with monoclonal antibodies against CD45, CD3, CD56, and CD16 and analyzed by flow cytometry to select a viable population comprised predominately of lung lymphocytes (CD45+, low side-scatter cells). (A, C, E) Representative staining: isotype controls on left, specific staining on right; (B, D, F) Frequency of various lung lymphocyte populations in individual subjects as a percentage of the total viable lung lymphocyte population; note difference in scale of panel B. (A) Ungated staining for CD3 and CD56 identifies four distinct populations: NK cells (CD56+ CD3−); CD56+ T cells (CD56+ CD3+); conventional T cells (CD56− CD3+); and double-negative cells (predominately B cells). (B) NK cells (blue bars) versus CD56+ T cells (orange bars). (C, D) After gating on CD3− cells, staining for CD56 and CD16 identifies two lung NK populations: CD56+ CD16+ (light blue circle & columns) and CD56+ CD16− (dark blue circle & columns). (E, F) After gating on CD3+ cells, staining for CD56 and CD16 identifies two lung CD56+ T cell populations: CD56+ CD16+ (dark orange circle & columns) and CD56+ CD16− (light orange circle & columns). By Kruskal-Wallis one-way ANOVA, there are no significant differences between subject groups for any of these three lung cell populations (B, D, F).

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)

Identification and characterization of human lung NK cells and CD56+ T cells.Lung tissue was dispersed, stained with monoclonal antibodies against CD45, CD3, CD56, and CD16 and analyzed by flow cytometry to select a viable population comprised predominately of lung lymphocytes (CD45+, low side-scatter cells). (A, C, E) Representative staining: isotype controls on left, specific staining on right; (B, D, F) Frequency of various lung lymphocyte populations in individual subjects as a percentage of the total viable lung lymphocyte population; note difference in scale of panel B. (A) Ungated staining for CD3 and CD56 identifies four distinct populations: NK cells (CD56+ CD3−); CD56+ T cells (CD56+ CD3+); conventional T cells (CD56− CD3+); and double-negative cells (predominately B cells). (B) NK cells (blue bars) versus CD56+ T cells (orange bars). (C, D) After gating on CD3− cells, staining for CD56 and CD16 identifies two lung NK populations: CD56+ CD16+ (light blue circle & columns) and CD56+ CD16− (dark blue circle & columns). (E, F) After gating on CD3+ cells, staining for CD56 and CD16 identifies two lung CD56+ T cell populations: CD56+ CD16+ (dark orange circle & columns) and CD56+ CD16− (light orange circle & columns). By Kruskal-Wallis one-way ANOVA, there are no significant differences between subject groups for any of these three lung cell populations (B, D, F).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4117545&req=5

pone-0103840-g001: Identification and characterization of human lung NK cells and CD56+ T cells.Lung tissue was dispersed, stained with monoclonal antibodies against CD45, CD3, CD56, and CD16 and analyzed by flow cytometry to select a viable population comprised predominately of lung lymphocytes (CD45+, low side-scatter cells). (A, C, E) Representative staining: isotype controls on left, specific staining on right; (B, D, F) Frequency of various lung lymphocyte populations in individual subjects as a percentage of the total viable lung lymphocyte population; note difference in scale of panel B. (A) Ungated staining for CD3 and CD56 identifies four distinct populations: NK cells (CD56+ CD3−); CD56+ T cells (CD56+ CD3+); conventional T cells (CD56− CD3+); and double-negative cells (predominately B cells). (B) NK cells (blue bars) versus CD56+ T cells (orange bars). (C, D) After gating on CD3− cells, staining for CD56 and CD16 identifies two lung NK populations: CD56+ CD16+ (light blue circle & columns) and CD56+ CD16− (dark blue circle & columns). (E, F) After gating on CD3+ cells, staining for CD56 and CD16 identifies two lung CD56+ T cell populations: CD56+ CD16+ (dark orange circle & columns) and CD56+ CD16− (light orange circle & columns). By Kruskal-Wallis one-way ANOVA, there are no significant differences between subject groups for any of these three lung cell populations (B, D, F).
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