Differential activation of killer cells in the circulation and the lung: a study of current smoking status and chronic obstructive pulmonary disease (COPD)

Abstract

Background: CD8(+) T-lymphocytes, natural killer T-like cells (NKT-like cells, CD56(+)CD3(+)) and natural killer cells (NK cells, CD56(+)CD3(-)) are the three main classes of human killer cells and they are implicated in the pathogenesis of chronic obstructive pulmonary disease (COPD). Activation of these cells can initiate immune responses by virtue of their production of inflammatory cytokines and chemokines that cause lung tissue damage, mucus hypersecretion and emphysema. The objective of the current study was to investigate the activation levels of human killer cells in healthy non-smokers, healthy smokers, ex-smokers with COPD and current smokers with COPD, in both peripheral blood and induced sputum.

Methods/principal findings: After informed consent, 124 participants were recruited into the study and peripheral blood or induced sputum was taken. The activation states and receptor expression of killer cells were measured by flow cytometry. In peripheral blood, current smokers, regardless of disease state, have the highest proportion of activated CD8(+) T-lymphocytes, NKT-like cells and NK cells compared with ex-smokers with COPD and healthy non-smokers. Furthermore, CD8(+) T-lymphocyte and NK cell activation is positively correlated with the number of cigarettes currently smoked. Conversely, in induced sputum, the proportion of activated killer cells was related to disease state rather than current smoking status, with current and ex-smokers with COPD having significantly higher rates of activation than healthy smokers and healthy non-smokers.

Conclusions: A differential effect in systemic and lung activation of killer cells in COPD is evident. Systemic activation appears to be related to current smoking whereas lung activation is related to the presence or absence of COPD, irrespective of current smoking status. These findings suggest that modulating killer cell activation may be a new target for the treatment of COPD.

Figure 1. Representative dot plots gating for live/dead cells, identification of cell populations and expression of activation markers by CD8⁺ T lymphocytes from four groups.

(i)(A) Forward Scatter (FS) vs Side Scatter (SS) plot for identification of live and dead cells; (B) Identification of CD8⁺ T lymphocytes (CD3+CD8+); and (C) identification of NK cells (CD3-CD56+) and NKT-like cells (CD3+CD56+). (ii) Representative dot plots for activation of CD8+ T lymphocytes expressing either CD69 alone (lower right quadrant), CD69 and CD25 (upper right quadrant) or CD25 alone (upper left quadrant) in four groups: (A) Healthy non-smoking participant; (B) Healthy smoker; (C) Current smoker with COPD; and (D) ex-smoker with COPD.

Figure 2. Activation of killer cells from peripheral blood ex vivo of four groups.

Activation of CD8⁺ T-lymphocytes, NKT-like cells and NK cells was analysed. There was a significant increase in level of activation ex vivo of CD8⁺ T lymphocytes (panel A), NKT-like (CD56⁺CD3⁺) cells (panel B) and NK (CD56⁺CD3⁻) cells (panel C) from both healthy smokers (HS, open circles) and cuS-COPD (solid circles). Activation of NK (CD56⁺CD3⁻) cells from exS-COPD (grey solid circles) was also significantly increased compared with healthy non-smokers (HNS). *: p<0.05, **: p<0.01, ***: p<0.001. HNS (n = 21), HS (n = 21), cuS-COPD participants (n = 14) and exS-COPD (n = 10).

Figure 3. Correlation of the proportion of cells activated and number of cigarettes currently smoked per day.

Analysis was performed on peripheral blood CD8⁺ T lymphocytes (Panel A), NKT-like (CD56⁺CD3⁺) cells (Panel B) and NK (CD56⁺CD3⁻) cells (Panel C). A significant correlation was observed in CD8⁺ T lymphocytes and NK (CD56⁺CD3⁻) cells but not NKT-like (CD56⁺CD3⁺) cells.

Figure 4. Expression of KIR by peripheral blood cells in four groups.

(i) Representative dot plots of KIR expression: (Panel A) healthy non-smokers; (Panel B) healthy smokers; (Panel C) current smokers with COPD and (Panel D) ex-smokers with COPD. (ii) CD8⁺ T lymphocytes (Panel A), NKT-like (CD56⁺CD3⁺) cells (Panel B) and NK (CD56⁺CD3⁻) cells (Panel C). A significantly lower proportion of CD8⁺ T lymphocytes, NKT-like (CD56⁺CD3⁺) cells and NK (CD56⁺CD3⁻) cells from healthy smokers (HS, open circles) and cuS-COPD (solid circles) expressed KIR on the cell surface compared to healthy non-smokers (HNS). ***: p<0.001. HNS (n = 8); HS (n = 8); cuS-COPD participants (n = 9) and exS-COPD (n = 7).

Figure 5. Activation of killer cells in induced sputum of four groups.

CD8⁺ T-lymphocytes (Panel A), NKT-like (CD56⁺CD3⁺) cells (Panel B) and NK (CD56⁺CD3⁻) cells (Panel C). A significant increase in level of activation ex vivo of CD8⁺ T lymphocytes, NKT-like (CD56⁺CD3⁺) cells and NK (CD56⁺CD3⁻) cells from COPD patients with or without smoking history compared with healthy non-smokers (HNS). *: p<0.05, **: p<0.01, ***: p<0.001. HNS (n = 5), HS (n = 10), cuS-COPD participants (n = 5) and exS-COPD (n = 6).