Chemotactic Mediators of Th1 T-cell Trafficking in Smokers and COPD Patients

Figures & data

Table 1 Patient demographics of the population studied (data expressed as median ± data range), * p < 0.05 versus never smoker controls

	Never smoker	Current-smoker	Ex-smoker
	controls	COPD subjects	COPD subjects	Smokers
A. Peripheral blood
Numbers per group (n)	25	20	20	10
Smoking (pack-years)	0	50 [20–100]*	45 [5–175]*	30 [10–60]*
Age	51 [20–76]	57 [39–72]*	66 [54–74]*	52 [25–67]
FEV1 (% Predicted)	101 [91–150]	59 [24–105]*	66 [21–91]*	92 [77–112]
FEV1/FVC (%)	90 [76–98]	57 [28–70]*	54 [19–70]*	75 [70–92]
Gender (M/F)	12/13	11/9	13/7	5/5
B. BAL
Numbers per group (n)	22	19	19	11
Smoking (pack-years)	0	50 [26–100]*	50 [20–170]*	37 [10–120]*
BAL volume (ml)	75 [48–90]	48 [15–75]*	49 [16–84]*	60 [27–80]*
Age	51 [20–71]	58 [42–71]	66 [53–75]*	55 [25–75]
FEV1 (% Predicted)	104 [92–125]	63 [34–90]*	64 [31–91]*	96 [73–112]
FEV1/FVC (%)	84 [71–98]	54 [27–68]*	59 [31–70]*	75 [70–100]
Gender (M/F)	9/13	11/8	13/6	5/6

Table 2 Mabs used for investigation of chemokine receptor expression

Mab	Fluorochrome	Supplier	Details	Dilution
CCR3	PE	R&D	FAB155P	Undiluted
CCR4	Unconjugated	R&D	551121	1:50
CCR5	FITC	BD Biosciences	FAB1802F	Undiluted
CXCR3	PE	BD Biosciences	557185	Undiluted
CXCR4	PE	BD Biosciences	555974	Undiluted
CD3	PC5	Beckman Coulter	IM2635	Undiluted
CD8	FITC	BD Biosciences	347313	Undiluted
CD8	PE	BD Biosciences	555367	Undiluted
Biotinylated anti-mouse		Vector Laboratories	BA2000	1:5
Streptavidin	PE	Sigma	S3402	Undiluted
62L	FITC	BD Biosciences		Undiluted
CD45RO	PE	BD Biosciences		Undiluted
CD11a	FITC	BD Biosciences		Undiluted
CD28	PE	BD Biosciences		Undiluted

Figure 1 Representative flow cytometry dot plots of chemokine receptor expression on T-cells from blood (A-E) and BAL (F-G) from a never smoker control and a COPD subject. A. and F. CCR5; B and G. CXCR3; C. CCR4; D. CXCR4 and E. intracellular CCR3. T cells were selected using SSC vs. CD3 (data not shown). Quadrant markers were set using a cell population known to lack expression of the marker of interest. The chemokine [PE stained] of interest is on the y-axis and CD8 [FITC] on the x-axis (with the exception of CCR5 [FITC] on the x-axis with CD8 [PE] on the y-axis). The percentage of cells expressing chemokine receptors are shown in the relevant quadrants.

Table 3 Differential cell counts

	Never smoker	Current-smoker	Ex-smoker
	controls	COPD subjects	COPD subjects	Smokers
A. Peripheral blood
Total WCC (×10^6/ml)	5.65 [3.3–9.0]	7.5 [4.7–11.1]*	6.8 [5.1–10.0]*	7.6 [5.2–10.7]
Lymphocytes (×10^6/ml)	2.1 [1.2–3.3]	2.5 [1.4–5.4]	2.2 [0.8–3.8]	2.2 [1.6–3.6]
CD8+ T Cells (% CD3)	29.8 [20.0–44.0]	37.2 [16.8–59.7]*	44.7 [20.3–64.5]*	28.7 [15.0–45.9]
CD8+ T-cells (×10^6/ml)	0.09 [0.04–0.31]	0.17 [0.03–0.36]*	0.18 [0.02–0.53]*	0.14 [0.03–0.21]
B. BAL
Total WCC (×10^3/ml)	100 [40–700]	285 [20–1500]*	200 [40–2000]*	280 [40–1300]*
Lymphocytes (×10^3/ml)	14.1 [0–90]	11.1 [0–236]	17.0 [0–200]	13.0 [1–85]
CD8+ T Cells (% CD3)	24.5 [12–49]	51.5 [14–73]*	36.8 [13–85]*	48.5 [14–78]*
CD8+ T-cells (×10^3/ml)	3.7 [0–21]	5.1 [1–126]	6.5 [0.4–89]*	8.0 [0.9–170]

Table 4 Summary of results

			Current-smoker COPD		Ex-smoker COPD		Smoker
	Technique		BAL	Blood	BAL	Blood	BAL	Blood
Cytokine
IFN-γ	Flow cytometry	T-cell intracellular	↑*	↑*	↑*	↑*	↑*	ns
Chemokines
MCP-1	ELISA	Soluble	↑*	↑ (trend)	↑*	↑ (trend)	↑*	↑ (trend)
MCP-3	ELISA	Soluble	ns	ns	ns	ns	ns	ns
MIG	CBA	Soluble	↑*	ns	↑*	↑*	↑*	ns
IP-10	CBA	Soluble	ns	ns	ns	ns	ns	ns
ITAC	ELISA	Soluble	↑*	↑*	↑*	↑*	↑*	ns
RANTES	CBA	Soluble	↑*	ns	↑*	ns	↑*	ns
IL-8	CBA	Soluble	↑*	ns	↑*	ns	↑ *	ns
Chemokine receptors
CCR5 (Th1)	Flow cytometry	T-cell surface	↑*	↑*	↑*	↑*	↑*	ns
CXCR3 (Th1)	Flow cytometry	T-cell surface	↑*	↑*	↑*	↑*	↑*	↑*
CCR4	Flow cytometry	T-cell surface	nt	↑*	nt	↑*	nt	↑*
CXCR4	Flow cytometry	T-cell surface	ns	ns	ns	ns	ns	ns
CCR3	Flow cytometry	T-cell intracellular	nt	↑*	nt	↑*	nt	↑ (trend)
Th1 and cytotoxic T-cell markers
CD28^bright	Flow cytometry	CD8+ T-cell surface	↑*	ns	↑*	nt	ns	nt
CD11A^bright	Flow cytometry	CD8+ T-cell surface	nt	↑*	nt	↑*	nt	ns
62L^−CD45RO^+	Flow cytometry	Surface CD3+ T-cell	↑ (trend)	↑*	↑*	↑*	↑ (trend)	↑ *

Figure 2 A. Cell membrane expression of CCR5 and CXCR3 (as a percentage of CD3+ T-cells) in peripheral blood (mean ± SEM). Similar results were noted for CD8+ T-cells (not presented). B. BAL. Cell membrane expression of CCR5 and CXCR3 (as a percentage of CD3+ T-cells). * p < 0.05 versus never-smoker controls.

Figure 3 Intracellular expression of CCR3 and cell membrane expression of CCR4 (as a percentage of CD3+ T-cells) in peripheral blood (mean ± SEM). * p < 0.05 versus never smoker controls. Note increased expression of CCR4 and intracellular CCR3 in all groups compared to never smoker controls. Similar results were noted for CD3+ T-cells (not presented). Data is presented as box plots showing median ± 25th and 75th percentiles (solid box) and the 10th and 90th percentiles shown by whiskers outside the box.

Figure 4 Cytotoxic and Th1 markers in A. peripheral blood and B. BAL (mean ± SEM). Th1 type T-cells were considered to be CD3⁺CD45RO⁺CD62L⁻ T-cells. Cytotoxic T-cells were considered to be CD8⁺CD11a^bright or CD8⁺CD28^bright. * p < 0.05 versus never smokers. Note the significantly increased percentage of Th1 type T-cells in both COPD groups and healthy smokers but an increase in cytotoxic T-cells only in the COPD groups.

Figure 5 Intracellular expression of IFN-γ (as a percentage of CD8+ T-cells) in A. blood and B. BAL from COPD subjects, asymptomatic smokers and healthy never-smokers measured current and ex-smoker by flow cytometry (mean ± SEM). * p < 0.05 versus never-smokers. There were significantly increased levels of IFN-γ in blood CD8+ T-cells from both current and ex-smoker COPD subjects but no change in healthy smokers compared to never-smoker controls.

Figure 6 Soluble MCP-1, MIG and ITAC levels in plasma and BAL from current and ex-smoker COPD subjects, healthy smokers and never-smoker controls (mean ± SEM). MCP-1 and ITAC were measured by ELISA and MIG by cytometric bead array A. plasma B. BAL. * p < 0.05 versus never-smokers. All chemokines were increased in BAL from all groups compared to controls. Note increased levels of MIG and ITAC in plasma from both current and ex-smoker COPD subjects but no change in healthy smokers.

Figure 7 Soluble cytokine levels in BAL from current and ex-smoker COPD subjects, healthy smokers and never-smoker controls measured by cytometric bead array (mean ± SEM). A. RANTES B. IL-8. * p < 0.05 versus never-smokers. ** p < 0.05 versus current smokers. ns: no significant differences between groups. There were no significant differences in levels of these soluble chemokines in plasma (not shown).

Figure 8 Correlation between A. % CCR5 expression and B. % CXCR3 expression by peripheral blood CD8+ T-cells and COPD disease severity (FEV₁).