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

Abstract

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.

Conflict of interest statement

Competing Interests: The authors have read the journal’s policy and the authors of this manuscript have the following competing interests: MKH participated in advisory boards for Boehringer Ingelheim, Pfizer, GlaxoSmithKline, Genentech, Novartis, Forest and Medimmune; participated on speaker’s bureaus for Boehringer Ingelheim, Pfizer, GlaxoSmithKline, Grifols Therapeutics, Forest and the National Association for Continuing Education, and WebMD; has consulted for Novartis and United Biosource Corporation; and has received royalties from UpToDate and ePocrates. She has served as an investigator for research sponsored by GlaxoSmithKline. FJM has been a member of advisory boards and/or consultant for Able Associates, Actelion, Almirall, Bayer, GSK, Ikaria, Janssen, MedImmune, Merck, Pearl, Pfizer and Vertex, American Institute for Research, AstraZeneca, Carden Jennings, Cardiomems, Grey Healthcare, HealthCare Research and Consulting, Merion, Nycomed/Takeda, and Sudler and Hennessey. He has been a member of steering committees for studies sponsored by Actelion, Centocor, Forest, GlaxoSmithKline, Gilead, Mpex, Nycomed/Takeda. He has participated in Food and Drug Administration mock panels for Boehringer Ingelheim, Forest and GSK. He has served on speaker’s bureaus or in continuing medical education activities sponsored by American College of Chest Physicians, American Lung Association, Astra Zeneca, Bayer, William Beaumont Hospital, Boehringer Ingelheim, Center for Health Care Education, CME Incite, Forest, France Foundation, GlaxoSmithKline, Lovelace, MedEd, MedScape/WebMD, National Association for Continuing Education, Network for Continuing Medical Education, Nycomed/Takeda, Projects in Knowledge, St Luke’s Hospital, the University of Illinois Chicago, University of Texas Southwestern, University of Virginia, UpToDate. He has served on DSMBs for Biogen and Novartis. He has received royalties from Castle Connolly and Informa. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

Figures

Figure 1. 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).

Figure 2. The frequency of lung NK cells, but not lung CD56+ T cells, co-expressing CD8 correlates with FEV1 % predicted.

(A–C) NK cells; (D–F) CD56+ T cells. (A) Representative staining for CD8 on CD56+ CD3− NK cells from a smoker without COPD (left panel) and a COPD subject (right panel); blue line, CD8+ staining; grey line, isotype control. (B) Subjects were categorized by pulmonary function (x-axis) versus the percentage of NK cells that co-express CD8 (y-axis); x, smokers without COPD (n = 5); □, mild COPD (n = 9); Δ, severe COPD (n = 9); Kruskal-Wallis one-way ANOVA testing was used to determine significance. (C) The percentage of NK cells that co-express CD8 (y-axis) versus FEV1 % predicted (x-axis). Spearman correlation was used to determine the p-value. (D) Representative staining for CD8 and CD4 on CD56+ CD3+ T cells from a smoker without COPD (left panel) and a COPD subject (right panel). The numbers in the quadrants are the % of each subset among all lung CD56+ T cells. (E) CD4 single-positive (brown bars), CD8 single-positive (orange bars), and CD8/CD4 double-negative (grey bars) are shown as the percentage of lung CD56+ T cells for individual subjects. No difference was seen between groups. (F) The percentage of lung CD56+ T cells (y-axis) that express CD4 (brown symbols) or CD8 (orange symbols) versus FEV1 % predicted (x-axis); x, smokers without COPD (n = 5); □, mild COPD (n = 9); Δ, severe COPD (n = 9).

Figure 3. The percentage of epithelial cells expressing MICA/MICB is increased with COPD severity.

Human lung tissue was dispersed and stained with monoclonal antibodies against CD45, CD56, NKG2D, CD326, and MICA/MICB. (A) Representative staining showing the expression of NKG2D on CD45+ CD56+ cells from a smoker without COPD (left panel) and a subject with COPD (right panel). Blue line, NKG2D+ staining; grey line, isotype control. (B) The percentage of CD56+ cells that express NKG2D (y-axis) versus FEV1 % predicted (x-axis). x, smokers without COPD (n = 10); □, mild COPD (n = 5); Δ, severe COPD (n = 10). N.S., not significant. (C) Representative staining showing the expression of MICA/MICB on CD45−, CD326 (EpCAM)+ epithelial cells from a smoker without COPD (left panel) and a subject with COPD (right panel). Blue line, MICA/MICB+ staining; grey line, isotype control. (D) The percentage of CD326+ epithelial cells that express MICA/MICB (y-axis) versus FEV1 % predicted (x-axis). x, smokers without COPD (n = 10); □, mild COPD (n = 5); Δ, severe COPD (n = 10). Spearman correlation was used to determine the p value.

Figure 4. 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.

Figure 5. Increased cytotoxicity by lung CD56+ cells correlates with decreased pulmonary function.

Human lung CD56+ cells were co-cultured with autologous lung target cells and % cytotoxicity was determined as described in the Methods. (A) Subjects were categorized by pulmonary function (x-axis) versus % cytotoxicity (y-axis). The Kruskal-Wallis one-way ANOVA with Dunn’s multiple comparison test was used to determine significant differences between groups. (B) FEV1 % predicted (x-axis) versus % cytotoxicity (y-axis). Spearman non-parametric correlation was used to determine the p-value. (C, D) The same subjects were separated into two groups based on their CD56+ cell function: non-cytotoxic (<8.5% cytotoxicity) or cytotoxic (≥8.5% cytotoxicity) and then FEV1 % predicted (C) and DLCO % predicted (D) were analyzed. The parametric unpaired Student t-test was used to determine significant differences between the two groups. In all figures, x, smokers without COPD (n = 6); □, mild COPD (n = 12); Δ, severe COPD (n = 10). Lines represent the mean ± SEM.