Interleukin-16-producing NK cells and T-cells in the blood of tobacco smokers with and without COPD

Figures & data

Table 1 Clinical characteristics of nine smokers with COPD, eight asymptomatic smokers, and 13 never-smokers

	Smokers with COPD	Asymptomatic smokers	Never-smokers
n	9	8	13
Age (years)	64 (47–68)	57 (40–68)	66 (39–72)
Male/female	6/3	4/4	6/7
Pack-years	36 (21–75)	33 (17–46)	0
FEV1 (% pred)	58 (30–66)	114 (95–129)	108 (87–131)
FEV1/FVC	42 (29–56)	76 (71–85)	78 (65–85)
DLCO (% pred)	55 (24–71)	87 (78–99)	96 (80–121)
Leukocytes (10^6/mL)	7.7 (4.5–10)	7.3 (6.5–9)	5.9 (4–9.6)
High sensitivity CRP (mg/L)	1.7 (0.45–5.8)	0.8 (0.42–6.2)	0.94 (0.21–3)

Note: The results are presented as median with range.

Abbreviations: COPD, chronic obstructive pulmonary disease; FEV₁, forced expiratory volume in 1 second; FVC, forced vital capacity; CRP, C-Reactive protein.

Table 2 Cell characteristics of nine smokers with COPD, eight asymptomatic smokers, and 13 never-smokers

	Smokers with COPD	Asymptomatic smokers	Never-smokers
	NK cells median (range)	NK cells median (range)	NK cells median (range)
n	9	8	13
Relative number (% of total cells)	3.1 (0.7–7)	2.4 (1.2–6.6)	3.6 (0.5–10.3)
Total number (×10^6/ml)	0.2 (0.04–0.6)	0.2 (0.08–0.4)	0.2 (0.03–1)
	CD8^+ T-cells median (range)	CD8^+ T-cells median (range)	CD8^+ T-cells median (range)
n	9	8	13
Relative number (% of T-cells)	21.8 (10.6–57.4)	24 (14.5–44.7)	27.2 (16.3–45)
Total number (×10^6/ml)	0.6 (0.1–1.7)	0.5 (0.3–1.1)	0.6 (0.2–1.1)
	CD4^+ T-cells median (range)	CD4^+ T-cells median (range)	CD4^+ T-cells median (range)
n	9	8	13
Relative number (% of T-cells)	71.9 (33.4–85.8)	67.1 (50.1–73.6)	64.9 (50.6–78.3)
Total number (×10^6/ml)	1.2 (0.8–2.4)	1.2 (0.8–2.5)	1.1 (0.7–2.9)
	B-cells median (range)	B-cells median (range)	B-cells median (range)
n	9	8	13
Relative number (% of total cells)	4.8 (2.9–16.3)	7.8 (5.1–10.4)	4.8 (1.7–13.9)
Total number (×10^6/ml)	0.35 (0.24–1.24)a	0.62 (0.38–0.78)a	0.27 (0.13–0.8)a
	Monocytes median (range)	Monocytes median (range)	Monocytes median (range)
n	9	8	13
Relative number (% of total cells)	0.2 (0.1–5.2)	0.1 (0.02–5)	0.1 (0.03–4.7)
Total number (×10^3/ml)	14.6 (8.8–257.5)	9.1 (1.5–352.1)	8.8 (2.1–107)

Notes: The relative and total numbers of NK cells, CD8⁺ T-cells, CD4⁺ T-cells, B-cells, and monocytes are presented as median with range.

a In the pooled groups of smokers, there was a slight increase in total number of blood B-cells.

Abbreviation: COPD, chronic obstructive pulmonary disease.

Figure 1 IL-16 protein (A) and IL-1β protein (B) in plasma.

Notes: AS are indicated by [Image], NS are indicated by ◯, and smokers with COPD by ●. Data are presented as individual and median values (bold lines). The Mann–Whitney U-test was performed (P>0.05).
Abbreviations: AS, asymptomatic smokers; IL, interleukin; NS, never-smokers.

Table 3 Flow cytometry analysis of intracellular IL-16 protein plus the relative and total number of NK cells, CD8⁺ T-cells, CD4⁺ T-cells, B-cells, and monocytes as well as rMFI analyzed by flow cytometry

	Smokers with COPD	Asymptomatic smokers	Never-smokers
	IL-16^+ NK cells median (range)	IL-16^+ NK cells median (range)	IL-16^+ NK cells median (range)
n	9	8	13
Relative number (% of NK cells)	24 (4.3–94.7)*	13 (2.9–67.2)*	66 (2.2–96.4)*
Total number (×10^6/mL)	0.04 (0.004–0.6)	0.01 (0.01–0.3)	0.08 (0.003–1)
rMFI	2.6 (1.6–8.1)	1.9 (1.7–2.6)	2.2 (1.8–13.8)
	IL-16^+ CD8^+ T-cells median (range)	IL-16^+ CD8^+ T-cells median (range)	IL-16^+ CD8^+ T-cells median (range)
n	9	8	13
Relative number (% of CD8^+ T-cells)	36.2 (9.1–88)	9.3 (0.2–81.4)	40.6 (1.6–89.4)
Total number (×10^6/mL)	0.1 (0.05–1.5)	0.05 (0.0006–0.6)	0.2 (0.007–0.9)
rMFI	2.4 (1.6–7.5)	2.2 (1.7–4.4)	2 (1.9–9.1)
	IL-16^+ CD4^+ T-cells median (range)	IL-16^+ CD4^+ T-cells median (range)	IL-16^+ CD4^+ T-cells median (range)
n	9	8	13
Relative number (% of CD4^+ T-cells)	26 (7.1–96.8)	19 (1.6–91)	55 (1.9–94.2)
Total number (×10^6/mL)	0.5 (0.08–2.4)	0.2 (0.02–1.7)	0.5 (0.02–2.7)
rMFI	2.6 (2–13.5)	2.2 (2–5.8)	2.5 (2.1–11.1)
	IL-16^+ B-cells median (range)	IL-16^+ B-cells median (range)	IL-16^+ B-cells median (range)
n	9	8	13
Relative number (% of B-cells)	32.1 (3.3–94.8)	9.7 (1.5–58.9)	32.1 (2.5–94.4)
Total number (×10^6/mL)	0.2 (0.01–0.4)	0.04 (0.006–0.4)	0.07 (0.003–0.6)
rMFI	2.2 (1.8–6.7)	2.2 (2–3.1)	2.6 (2–8.6)
	IL-16^+ monocytes median (range)	IL-16^+ monocytes median (range)	IL-16^+ monocytes median (range)
n	7	5	6
Relative number (% of monocytes)	7.7 (0.3–32.7)	1.9 (0.05–20.2)	8.1 (0.2–23.9)
Total number (×10^3/mL)	1 (0.3–4.5)	0.2 (0.03–1.4)	0.4 (0.1–2)
rMFI	2.3 (1.7–4.8)	2.4 (1.7–4.1)	2.4 (1.4–3.4)

Note: Data are presented as median with range.

* The Mann–Whitney U-test was performed between the pooled group of long-term smokers (COPD + AS) and never-smokers. P<0.05.

Abbreviations: AS, asymptomatic smokers; IL, interleukin; rMFI, relative mean fluorescence index.

Figure 2 The relative number of IL-16⁺ NK cells in blood (A). AS are indicated by [Image], NS are indicated by ◯, and smokers with COPD by ●. Data is presented as individual and median values (bold lines). The Mann–Whitney U-test was performed (P<0.05.) A representative flow cytometry analysis was conducted for IL-16⁺ CD3⁻ CD16⁺ CD56⁺ cells (B).

Notes: The cells were sequentially gated on intact (a), mononuclear (b), CD3⁻ (c), and CD16⁺ CD56⁺ double positive cells (d). The gate for IL-16 intracellular expression was set by staining with the matching IL-16 IC, and based on that, the NK cells expressing IL-16 were evaluated (e and f). Numbers represent the percentage of NK cells stained with the isotype control and the IL-16 antibody, respectively. *P<0.05.
Abbreviations: AS, asymptomatic smokers; COPD, chronic obstructive pulmonary disease; IL, interleukin; IL-16 IC, IL-16 isotype control; NS, never-smokers.

Figure 3 Correlations between, on one hand, the relative (A) and the total (B) numbers of NK cells and CD4⁺ T-cells (C) in blood from the pooled group of smokers (COPD + AS) and, on the other, tobacco load in pack-years (Spearman rank correlation: P<0.05, ρ: −0.56 [A and B] and −0.62 [C]).

Figure 4 Total number of activated (CD69⁺) CD8⁺ T-cells in blood for never-smokers, asymptomatic smokers and smokers with COPD.

Notes: The results are presented as individual and median values (bold lines). (Kruskal–Wallis test followed by Mann–Whitney U-test, P<0.05). *P<0.05.
Abbreviation: COPD, chronic obstructive pulmonary disease.

Figure 5 Concentrations of extracellular IL-16 protein measured by ELISA in conditioned medium from human blood NK cell cultured for 20 hours in vitro, exposed to either water-soluble tobacco smoke components (CSE) or vehicle solution (VEH) and treated with glutathione (10⁻² M).

Notes: The results are presented as individual as well as median values (bold lines). (Kruskal–Wallis test followed by Mann–Whitney U-test, P<0.05). *P<0.05.
Abbreviations: CSE, cigarette smoke extract; IL, interleukin; VEH, vehicle.

Figure 6 Flow cytometry analysis of human NK cells cultured for 20 hours in vitro exposed to either water-soluble tobacco smoke components (CSE) or vehicle solution (VEH) and treated with glutathione (10⁻² M).

Notes: Shown are 7AAD⁻ cells (A), IL-16⁺ cells (B) and IL-16 rMFI (C). The results are presented as individual as well as median values (bold lines). (Kruskal–Wallis test followed by Mann–Whitney U-test, P<0.05). *P<0.05.
Abbreviations: CSE, cigarette smoke extract; IL, interleukin; rMFI, relative mean fluorescence index; VEH, vehicle.

Figure 7 Result of flow cytometry analysis of human NK cells cultured for 20 hours in vitro, exposed to either water-soluble tobacco smoke components (CSE) or vehicle solution (VEH) and treated with glutathione (10⁻² M).

Notes: Representative flow cytometry plots showing IFN-γ⁺ and IL-16⁺ cells under different conditions (A). Shown are IFN-γ⁺ cells (B), and IFN-γ rMFI (C). The results are presented as individual as well as median values (bold lines). (Kruskal–Wallis test followed by Mann–Whitney U-test, P<0.05). *P<0.05.
Abbreviations: CSE, cigarette smoke extract; rMFI, relative mean fluorescence index; VEH, vehicle.