Increased Airway Granzyme b and Perforin in Current and Ex-Smoking COPD Subjects

Figures & data

Table 1 Demographic characteristics of the population studied and BAL leucocyte counts

	Healthy Never-smokers	Current smokers COPD	Ex smokers COPD	Asymptomatic smokers
N	30	30	30	20
Smoking (pack-yrs)	0	50 (20–150)	40 (10–175)	37 (10–75)
Age	53 (30–75)	60 (43–75)	65 (49–75)	52 (25–75)
FEV1 (% pred)	100 (86–145)	59 (24–100)	59 (21–92)	91 (75–112)
FEV1/FVC (%)	104 (70–155)	54 (26–101)	56 (19–84)	82 (70–109)
Mild COPD (n)	0	9	7	0
Moderate-severe COPD (n)	0	21	23	0
Gender (m/f)	15/15	18/12	17/13	10/8
BAL leucocyte count (×10^9/L)	0.13 (0.04–4.4)	0.31 (0.13–1.2)	0.21 (0.04–4.4)	0.52 (0.2601.6)
BAL lymphocyte count (×10^9/L)	0.02 (0.002–0.1)	0.02 (0.01–0.24)	0.03 (0.01–0.33)	0.04 (0.01–0.19
BAL CD8+ T-cell numbers (×10^9/L)	0.003 (0.001–0.01)	0.005 (0.001–0.12)	0.008 (0.002–0.09)	0.01 (0.002–0.07)

Results are expressed as median (data range). FEV₁: forced expiratory volume in 1 second; FVC: forced vital capacity; BAL: bronchoalveolar lavage.

COPD was categorized using GOLD criteria with clinical correlation. Mild COPD: FEV₁/FVC < 70% but FEV₁ ≥ 80% predicted; Moderate COPD FEV₁ 30% ≤ 80% predicted, Severe COPD FEV₁ < 30% predicted. *p < 0.05 compared to control group.

Figure 1 Representative flow cytometric dot plots of granzyme-b and perforin expression by NK cells and T-cells. (A,B) Peripheral blood-derived NK cells were identified in R1 as staining brightly with CD56PC5 (not shown). Subsequent analysis was carried out on cells from R1. NK cells expressing (A) granzyme-b or (B) perforin were identified in quadrant 1 (Q1) by negative staining with CD3 (ie. excluding NKT cells) and bright staining with Mabs to granzyme-b or perforin. Data was expressed as a percentage of total CD3 negative NK cells. (C,D) Peripheral blood-derived T-cells were identified in R1 as staining brightly with CD3PC5 (not shown). Subsequent analysis was carried out on cells from R1. T-cells expressing granzyme-b or perforin were identified by bright staining with Mabs to (C) granzyme-b or (D) perforin (Q1 +Q2). Data was expressed as a percentage of total CD3 T-cells and as a percentage of CD8+ T-cells (Q2/Q2+Q3). (E,F) BAL-derived T-cells. Analysis of BAL was performed as described for blood.

Figure 2 Intracellular expression of granzyme-b and perforin in peripheral blood derived T-cells. Expression of granzyme-b and perforin was quantified on A. CD3+ T-cells and B. CD8+ T-cells by flow cytometry. Note significantly increased expression of granzyme-b and perforin in current- (COPD current smokers, n = 30) and ex- (COPD ex-smokers, n = 30) smokers with COPD but no changes in asymptomatic smokers (Smokers, n = 18) compared to never-smokers (n = 30). *significant increase (p ≤ 0.05) in granzyme-b compared to control; **(p ≤ 0.05) significant increase in perforin compared to never-smokers. Box plots shows median, quartiles, and ranges.

Figure 3 Intracellular expression of granzyme-b and perforin in BAL derived T-cells. Expression of granzyme-b and perforin was quantified on A. CD3+ T-cells and B. CD8+ T-cells by flow cytometry. Note significantly increased expression of granzyme-b and perforin in current- (COPD current smokers, n = 15) and ex- (COPD ex-smokers, n = 15) smokers with COPD and asymptomatic smokers (Smokers, n = 10) compared to never-smokers (n = 12). *significant increase (p ≤ 0.05) in granzyme-b compared to never-smokers; **(p ≤ 0.05) significant increase in perforin compared to never-smokers. Box plots shows median, quartiles, and ranges.

Figure 4 Soluble granzyme-b levels in BAL. Granzyme-b levels were measured by ELISA in BAL. Note significantly increased expression of granzyme-b in current- (COPD current smokers, n = 10), ex- (COPD ex-smokers, n = 13) smokers with COPD but no changes in asymptomatic smokers (Smokers, n = 11) compared to never-smokers (n = 24). *significant increase (p ≤ 0.05) in granzyme-b compared to never-smokers. Box plot shows median, quartiles, and ranges.

Figure 5 Correlation between T-cell granzyme-b levels in BAL and bronchial epithelial apoptosis. Apoptosis was measured in bronchial brushing derived epithelial cells by 7AAD staining. Note good correlation between intracellular granzyme-b in BAL-derived T-cells and apoptosis of bronchial epithelial cells.