Abstract
The role of T cells in the pathophysiology of chronic obstructive pulmonary disease (COPD) is not yet certain, although varying reports have shown increases in T helper 1 (Th1) and/or Th2 cytokines in peripheral blood and bronchoalveolar lavage (BAL). No studies have examined cytokine production by intraepithelial T cells obtained by bronchial brushing (BB). Intracellular cytokine analysis of T cell subsets from peripheral blood, BAL and BB from smoker and ex-smoker COPD patients, COPD patients receiving inhaled corticosteroids and smoker and non-smoker control subjects was studied using multi-parameter flow cytometry. CD4 : CD8 inversion was noted in the peripheral blood of smoker and ex-smoker COPD groups, in BAL and BB from smoker controls and BAL of COPD smokers. There was an increase in intracellular CD8− T cell Th1 proinflammatory cytokines in some COPD groups in the peripheral blood and in CD8− T cell tumour necrosis factor (TNF)-α in some COPD groups and smoker controls in BAL and BB. There was an increase in proinflammatory cytokines in COPD smokers compared with ex-smokers and a decrease in COPD smokers receiving inhaled corticosteroids in the airways. There was a negative correlation between forced expiratory volume in 1 s (FEV1) and the percentage of BAL and intraepithelial CD8− T cells producing TNF-α. COPD patients exhibit systemic inflammation as evidenced by increased intracellular Th1 proinflammatory cytokines in blood, BAL and intraepithelial CD8− T cells, whereas smoker controls showed localized Th1 response in the lung only. Systemic therapeutic targeting of TNF-α production by CD8− T cells may improve morbidity in COPD patients while targeting of TNF-α in the lung may prevent smokers progressing to COPD.
Keywords: COPD, intracellular Th1 proinflammatory cytokines, CD8− T cells, flow cytometry
Introduction
Chronic obstructive pulmonary disease (COPD) is a serious global epidemic that is predicted to become the third most common cause of death by 2020. It is caused mainly by cigarette smoking in developed countries but why only 10–20% of smokers develop progressive airflow limitation is unknown [1]. The role of T cells in the pathophysiology of COPD is not yet certain [2]. COPD is characterized by inflammation in the airways, an increase in the total number of T cells in lung parenchyma, peripheral and central airways, with the greater increase in CD8− than CD4− cells [3]. Several previous studies have attempted to characterize the pattern of lymphocyte cytokine production in COPD, but results are conflicting. One study showed that CD4− T cells produce increased interferon (IFN)-γ and decreased interleukin (IL)-4 in the peripheral blood of COPD patients, but no changes were noted for CD8− T cells [4]. Other studies have shown a predominance of T helper 2 (Th2) cytokines [5,6], while others have shown a Th1 pattern in CD4− T cells in bronchoalveolar lavage (BAL) but a Th2 pattern in CD8− T cells [7]. Differences are probably related to different tissue samples, purification of cells of interest or different stimuli and other experimental methodology. It has been shown previously that purification of cells results in selective cell loss [8] and apoptosis of specific cell subsets [9], which may account for some reported differences. No studies have compared intracellular cytokines from T cells in blood and BAL and intraepithelial T cells from bronchial brushings (BB) for individual patients. Recently, we have shown compartmentalization of T cell subsets and cytokine production in a group of stable lung transplant patients [10] and control volunteers and a multi-compartmental approach may also elucidate the systemic role of T cells in the pathophysiology of COPD. As COPD has been shown to be a systemic disease [2], we hypothesized that we would find changes in T cell cytokines in blood, BAL and BB compartments for these patients, whereas smokers that have not progressed to COPD would only show localized changes in the lung. We also hypothesized that further perturbations in T cell cytokines would be found in COPD smokers compared with the COPD ex-smokers, while COPD patients receiving inhaled corticosteroids would show a decrease in proinflammatory cytokines.
To investigate differences in cytokines between both COPD smokers and COPD ex-smokers, COPD patients receiving inhaled corticosteroids and smoking and non-smoking control volunteers, intracellular cytokine analysis of T cell subsets from peripheral blood, BAL and BB from these patient and control groups was studied using multi-parameter flow cytometry.
Materials and methods
Subject population
COPD patients and healthy control subjects were recruited for the study and fully informed consent obtained. There was no exacerbation of COPD for 6 weeks prior to involvement in the study. Ethics approval was obtained from the Royal Adelaide Hospital. The diagnosis of moderate COPD was established using the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria [11] of a relevant history and post-bronchodilator forced expiratory volume in 1 s (FEV1) 30–80% of predicted and and FEV1/forced vital capacity (FVC) < 70%. Blood, BAL and BB were collected from 25 patients with COPD (Table 1), of whom 13 of 25 were ex-smokers (> 1-year abstinence) and 12 of 25 were current smokers. Six COPD ex-smoker and five COPD smoker patients were receiving inhaled corticosteroids and 14 patients were receiving no treatment.
Table 1.
Demographic details of the chronic obstructive pulmonary disease (COPD) and control groups and bronchoalveolar lavage (BAL) leucocyte counts.
| Subjects | N | S | C | CI | CS | CSI |
|---|---|---|---|---|---|---|
| No. of subjects | 8 | 7 | 7 | 6 | 7 | 5 |
| Age (years) | 44 (± 8) | 49 (± 13) | 58 (± 16)* | 59 (± 17)* | 60 (± 18)* | 61 (± 17)* |
| FEV1, % pred | 110·4 (± 9) | 76·6 (± 8) | 68 (± 16) | 52 (± 24) | 76 (± 8) | 52 (± 10) |
| FEV1, % FVC | 96 (± 12) | 82 (± 16) | 68 (± 15)* | 51 (± 15)* | 66 (± 14)* | 42 (± 14)* |
| Male/female | 4/4 | 4/3 | 4/3 | 3/3 | 4/3 | 3/2 |
| BAL yield (ml) | 75 (± 12) | 58 (± 14)* | 54 (± 12)* | 50 (± 13)* | 47 (± 15)* | 45 (± 16)* |
| BAL leuc | 0·19 (± 0·01) | 0·41 (± 0·01)* | 0·20 (± 0·02) | 0·24 (± 0·02) | 0·26 (± 0·05) | 0·30 (± 0·05)* |
| BAL lymph | 0·03 (± 0·04) | 0·04 (± 0·05) | 0·03 (± 0·02) | 0·04 (± 0·02) | 0·04 (± 0·06) | 0·04 (± 0·07) |
N: non-smokers; S: smokers; C: COPD ex-smokers; CI: COPD ex-smokers receiving inhaled corticosteroids; CS: COPD smokers; CSI: COPD smokers receiving inhaled corticosteroids. Results are expressed as mean ± s.d. FEV1: forced expiratory volume in 1 s; FVC: forced vital capacity; leuc: leucocyte count (×109/l); lymph: lymphocyte count (×109/l)
P < 0·05 compared to non-smokers.
Specimens were also obtained from 15 healthy volunteer subjects (eight non-smoking and seven smoking) (Table 1) with no history of airways disease. Subjects underwent spirometry and chest X-ray as part of their routine clinical assessment.
BAL
Aspirated BAL samples (3 × 50 ml aliquots) were collected as described previously [12] and transferred to 50 ml polypropylene tubes. For each collection from an individual, BAL specimen 1 was processed for microbial testing and specimens 2 and 3 were pooled. All BAL cultures were negative except for Aspergillus spp., isolated from one ex-smoker COPD patient and one current smoker COPD patient.
Cell counts were determined as described previously [3] using a haemocytometer and standard techniques and cells were then pelleted by centrifugation at 500 g for 5 min. Supernatant was discarded and cells resuspended at 4 × 105 cells/ml in RPMI-1640 media supplemented with 125 U/ml penicillin and 125 U/ml streptomycin (Gibco, New York, USA).
BB
Fibreoptic bronchoscopy and BB were performed as described previously [3]. Bronchial epithelial cells were obtained from the third- or fourth-order bronchi by several passages of the brush into each airway so as to avoid bleeding. Cells were deposited by washing the brush in 10 ml RPMI-1640 in 10 ml conical polypropylene tubes (Johns Professional Products, Sydney, Australia), supernatant discarded and cells resuspended at 4 × 105 cells/ml in RPMI-1640 media supplemented with 125 U/ml penicillin and 125 U/ml streptomycin and kept on ice. Cells were processed within 1 h of collection.
CD4− and CD8− T cell counts
One hundred μl of peripheral blood, BAL and BB was stained with appropriately diluted fluorescently conjugated monoclonal antibodies to CD8 fluorescein isothiocyanate (FITC) (BD Biosciences, Sydney, Australia), CD4 phycoerythrin (PE) (BD) and CD3 PC5 (Beckman Coulter, Sydney, Australia) as described previously [3,12]. Samples were analysed by gating using forward scatter (FSC) versus side scatter (SSC) to exclude platelets and debris. Control staining of leucocytes with anti-mouse IgG1-FITC/IgG1a-PE/IgG1-PC5 was performed on each sample and background readings of < 2% were obtained. A minimum of 5000 CD3− cells from blood and BAL and 3000 CD3− T cells from BB was acquired in list mode format for analysis.
Leucocyte stimulation
The potential of T cells to produce cytokines was determined by stimulating cells with a protein kinase activator (phorbol myristate) and calcium ionophore (ionomycin) in the presence of a Golgi block (brefeldin A). The accumulation of intracellular cytokines can be determined by flow cytometry, as reported previously [10,13,14]. Two-ml aliquots of prepared BAL or BB or 1-ml aliquots of blood (diluted 1 : 2 with RPMI-1640 medium) were placed in a 10-ml sterile conical polyvinylchloride (PVC) tubes (Johns Professional Products, Sydney, Australia). Phorbol myristate (25 ng/ml) (Sigma, Sydney, Australia), ionomycin (1 µg/ml) (Sigma) and brefeldin A (10 µg/ml) were added (Sigma) and the tubes reincubated in a humidified 5% CO2/95% air atmosphere at 37°C for 16 h.
Cytokine determination
Cytokine determination was performed as reported previously [10,13,14]. Briefly, at 16 h 100 µl 20 mm ethylemediamine tetraacetic acid/phosphate-buffered saline (EDTA/PBS) was added to the culture tubes, which were vortexed vigorously for 20 s to remove adherent cells. To lyse red blood cells in the blood cultures, 2 ml of FACSlyse solution (BD) was added and tubes incubated for 10 min at room temperature in the dark. After centrifugation at 500 g for 5 min and decanting, 0·5 ml 1 : 10 diluted FACSperm (BD) was added to each blood, BAL and BB culture tube, mixed, and incubated for a further 10 min at room temperature in the dark. Two ml 0·5% bovine serum albumin (Sigma)/Isoton II (Beckman Coulter) was then added and the tubes centrifuged at 300 g for 5 min. After decanting supernatant, Fc receptors were blocked with 10 µl human immunoglobulin (Intragam, CSL, Parkville, Australia) for 10 min at room temperature. Five µl of appropriately diluted anti-CD8 FITC (BD), anti-CD3 PC5 (Coulter/Immunotech) and PE-conjugated anti-cytokine monoclonal antibodies to interleukin (IL)-2, IL-4, IFN-γ, tumour necrosis factor (TNF)-α (BD) and transforming growth factor (TGF)-β (IQ Products, Groningen, the Netherlands) or isotype control monoclonal antibody was added for 15 min in the dark at room temperature. Due to the limitation of sample size, TGF-β staining was not performed on BAL or BB samples. Two ml of 0·5% bovine serum albumin (Sigma)/Isoton II (Beckman Coulter) was then added and the tubes centrifuged at 300 g for 5 min. After decanting, cells were analysed within 1 h on a FACSCalibur flow cytometer using CellQuest software (BD). Samples were analysed by live gating using FL3 staining versus side scatter (SSC). A minimum of 5000 CD3+low SSC events from blood and BAL and 3000 CD3+low SSC events from BB were acquired in list-mode format for analysis. Control staining of cells with anti-mouse IgG1-PE/IgG-PC5 was performed on each sample and background readings of < 2% were obtained.
Statistical analysis
Statistical analysis was performed using the non-parametric Kruskal–Wallis test and when P < 0·05, post hoc analysis was performed using the Mann–Whitney test. For post hoc analyses, all groups were compared with non-smoker control subjects for all parameters. Control smokers were also compared with COPD ex-smokers and COPD smokers. To determine the effects of inhaled corticosteroids, COPD ex-smokers were compared with COPD ex-smokers receiving inhaled corticosteroids and COPD smokers were compared with COPD smokers receiving inhaled corticosteroids. Pearson's correlation tests were also performed with spss software and differences between groups of P < 0·05 considered significant.
Results
Blood CD4− and CD8− T cell counts
There was a significant increase in the absolute CD8 T cells in COPD ex-smokers and COPD smokers compared with non-smoker controls (0·43 ± 0·22, 0·42 ± 0·20 and 0·33 ± 0·16 (× 109/l) for COPD ex-smokers, COPD smokers and controls, respectively, P < 0·05). There were no other differences between CD3−, CD4− or CD8− absolute counts between any of the groups (P > 0·05). The percentage of CD8− T cells was increased significantly and CD4− T cells decreased significantly in COPD ex-smokers and COPD smokers compared to the smoking and non-smoking control group (Table 2, P < 0·05). There were no changes in CD4− or CD8− T cells between any other group. The percentage of CD4− CD8− and CD4− CD8− T cells was < 3% for all patient and control subjects.
Table 2.
The percentage of T cells producing intracellular cytokines in blood of chronic obstructive pulmonary disease (COPD) and control groups (median and range).
| CD3 | IFN-γ | IL-2 | IL-4 | TGF-β | TNF-α | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | |
| N | 65 | 35 | 16 | 22 | 34 | 7 | 0·5 | 0·4 | 3·5 | 1·8 | 40 | 22 |
| 47–74 | 18–53 | 10–31 | 8–41 | 5–62 | 2–19 | 0–2·2 | 0–1·3 | 0·6–13 | 0·5–9 | 27–56 | 11–46 | |
| S | 75 | 26 | 19 | 15 | 47 | 6 | 1 | 0·1 | 1·1* | 0·4 | 45 | 14 |
| 54–80 | 20–46 | 14–37 | 10–37 | 37–67 | 5–11 | 0·5–1·4 | 0·1-.4 | 0·7–1·4 | 0·1-.7 | 36–51 | 14–18 | |
| C | 52* | 48* | 19 | 42* | 31 | 11 | 0·6 | 0·9 | 2* | 0·6 | 44 | 44* |
| 37–70 | 30–63 | 15–34 | 23–56 | 10–59 | 6–15 | 0·2-.8 | 0·2–1 | 1·6–4·7 | 0·5-.6 | 27–57 | 24–60 | |
| CI | 67 | 33 | 17 | 22† | 51 | 9 | 0·6 | 0·4 | 2·2* | 0·9 | 51 | 29 |
| 53–75 | 25–47 | 13–18 | 21–28 | 33–57 | 6–16 | 0·2–1·3 | 0-.8 | 1–3·7 | 0·5–1·4 | 36–55 | 22–40 | |
| CS | 55* | 45* | 23 | 34* | 29 | 6 | 0·6 | 0·7 | 1·1* | 0·7 | 37 | 46* |
| 36–67 | 33–64 | 11–35 | 23–44 | 10–42 | 2–26 | 0·3–1·4 | 0·2–1·4 | 0·3–1·9 | 0·3–1·1 | 22–50 | 25–46 | |
| CSI | 57 | 43 | 18 | 32 | 36 | 8 | 0·7 | 0·7 | 2* | 1·3 | 40 | 33 |
| 56–70 | 30–44 | 13–29 | 11–40 | 30–47 | 3–24 | 0·3–1 | 0·1–1 | 1–4 | 1–4 | 20–43 | 22–43 | |
N: non-smokers; S: smokers; C: COPD ex-smokers; CI: COPD ex-smokers receiving inhaled corticosteroids; CS: COPD smokers; CSI: COPD smokers receiving inhaled corticosteroids). There was a significant decrease in the percentage of CD4− and increase in the percentage of CD8− T cells in C and CS compared with N (*P < 0·05). There was a significant increase in the percentage of CD8− T cells producing interferon (IFN)-γ and tumour necrosis factor (TNF)-α in C and CS compared with S and N (*P < 0·05). There was a significant decrease in the percentage of CD4− T cells producing transforming growth factor (TGF)-β in S, C, CI, CS, CSI compared with N (*P < 0·05). There was a significant decrease in the percentage of CD8− T cells producing IFN-γ in CI compared with C (†P < 0·5). IL: interleukin.
BAL CD4− and CD8− T cell counts
The BAL yield was decreased significantly from smoker controls and all COPD groups compared with non-smoker controls (Table 1).
There was an increase in absolute leucocyte counts for smoker controls and COPD smokers receiving inhaled corticosteroids compared with non-smoker controls (Table 1), but no change in absolute lymphocyte counts.
The percentage of CD8− T cells was increased significantly and CD4− T cells decreased significantly in COPD smokers and smoker controls compared with non-smoker controls (P < 0·05) (Table 3).
Table 3.
The percentage of T cells producing intracellular cytokines by bronchoalveolar lavage (BAL) CD4− and CD8− T cells (median and range).
| CD3 | IFN-γ | IL-2 | IL-4 | TNF-α | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | |
| N | 72 | 28 | 2 | 1 | 1 | 1 | 7 | 3 | 2 | 1 |
| 38–92 | 8–62 | 1–4 | 0–3 | 0–4 | 0–3 | 0–20 | 0–9 | 0–11 | 0–9 | |
| S | 47* | 53* | 11* | 12* | 5* | 3 | 12 | 7 | 14* | 16* |
| 40–76 | 24–60 | 5–12 | 8–14 | 3–6 | 2–5 | 6–17 | 3–18 | 8–30 | 5–21 | |
| C | 70 | 29 | 5 | 4 | 5* | 2 | 9 | 4 | 18* | 7* |
| 10–77 | 3–90 | 0·3–9 | 1–14 | 1–8 | 1–5 | 0–43 | 1–10 | 4–20 | 3–22 | |
| CI | 59 | 41 | 15* | 12* | 1 | 1 | 8 | 3 | 22* | 12* |
| 52–75 | 25–48 | 2–26 | 1–15 | 0·1–5 | 0·5–2 | 2–11 | 1–7 | 2–30 | 1–44 | |
| CS | 50* | 50* | 10* | 18*† | 1 | 3 | 5 | 4 | 21* | 30*† |
| 42–55 | 45–59 | 5–20 | 13–21 | 0·3–4 | 1–5 | 2–9 | 2–8 | 10–28 | 12–41 | |
| CSI | 75 | 26 | 12* | 7* | 7* | 2 | 2 | 3 | 15* | 7* |
| 58–77 | 21–39 | 5–15 | 2–11 | 3–10 | 1–3 | 1–4 | 2–5 | 10–22 | 3–12 | |
N: non-smokers; S: smokers; C: chronic obstructive pulmonary disease (COPD) ex-smokers; CI: COPD ex-smokers receiving inhaled corticosteroids; CS: COPD smokers; CSI: COPD smokers receiving inhaled corticosteroids). There was a significant decrease in the percentage of CD4− and increase in the percentage of CD8− T cells in S and CS compared to N (*P < 0·05). The percentage of CD4− and CD8− BAL T cells producing interferon (IFN)-γ was increased in S, CI, CS and CSI compared with N (*P < 0·05). The percentage of CD8− BAL T cells producing IFN-γ and tumour necrosis factor (TNF)-α was increased in CS compared to C (†P < 0·05). The percentage of CD4− BAL T cells producing interleukin (IL)-2 was increased S, C and CSI compared to N (*P < 0·05). The percentage of CD4− and CD8− BAL T cells producing TNF-α was increased in S, CI, CS and CSI compared with N (*P < 0·05).
Percentage of BB intraepithelial CD4− and CD8− T cells
The percentage of CD8− T cells was significantly increased and CD4− T cells decreased significantly in smoker controls and COPD ex-smokers receiving inhaled corticosteroids compared with non-smoker controls (Table 3). There were no changes in CD4− or CD8− T cells between any other group. The percentage of CD4− CD8− and CD4− CD8− T cells was < 3% for all patient and control subjects.
Percentage of blood T cells producing intracellular cytokines
There was a significant increase in the percentage of CD8− blood T cells producing IFN-γ and TNF-α in COPD ex-smokers and COPD smokers compared with smoker and non-smoker controls (Table 2), but no difference between smoker and non-smoker controls (Table 2). There was a significant decrease in the percentage of CD8− blood T cells producing IFN-γ in COPD ex-smokers receiving inhaled corticosteroids compared with COPD ex-smokers. There was a significant decrease in the percentage of CD4− T cells producing TGF-β in all COPD groups and smoker controls compared with non-smoker controls. There was no difference in the percentage of CD4− or CD8− T cells producing any other cytokine between any of the groups.
Percentage of BAL T cells producing intracellular cytokines
The percentage of CD4− and CD8− BAL T cells producing IFN-γ was increased significantly in all COPD groups (except COPD ex-smokers) and smoker controls compared with non-smoker controls (Table 3). The percentage of CD8− BAL T cells producing IFN-γ and TNF-α was increased significantly in COPD smokers compared with COPD ex-smokers. The percentage of CD4− BAL T cells producing IL-2 was increased significantly in COPD ex-smokers, COPD smokers receiving inhaled corticosteroids and smoker controls compared with non-smoker controls. The percentage of CD4− and CD8− BAL T cells producing TNF-α was increased significantly in all COPD groups and smoker controls compared with non-smoker controls.
Percentage of BB intraepithelial T cells producing intracellular cytokines
There was a significant increase in the percentage of CD8− BB T cells producing TNF-α in all COPD groups and smoker controls compared with non-smoker controls (Table 4). There was a significant decrease in the percentage of CD8− BB T cells producing IFN-γ and TNF-α in COPD smokers receiving inhaled corticosteroids compared with COPD smokers. Representative dot plots showing TNF-α production by blood, BAL and BB CD8− and CD8− (CD4−) T cells from a COPD smoker and non-smoker control subject are shown in Fig. 1.
Table 4.
The percentage of intraepithelial T cells producing intracellular cytokines in bronchial brushings (BB) of chronic obstructive pulmonary disease (COPD) and control groups (median and range).
| CD3 | IFN-γ | IL-2 | IL-4 | TNF-α | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | CD4 | CD8 | |
| N | 38 | 62 | 17 | 36 | 5 | 2 | 1 | 1 | 17 | 27 |
| 19–60 | 40–81 | 5–24 | 28–41 | 2–10 | 0–3 | 0–1 | 0–2 | 7–25 | 22–36 | |
| S | 23* | 77* | 12 | 21 | 5 | 6 | 0·5 | 0·9 | 17 | 53* |
| 20–28 | 72–80 | 5–14 | 16–62 | 3–9 | 3–9 | 0–1 | 0–1 | 11–19 | 38–64 | |
| C | 27 | 74 | 7 | 26 | 4 | 2 | 0·4 | 0·8 | 14 | 43* |
| 20–26 | 64–80 | 6–17 | 18–53 | 2–11 | 1–3 | 0·1-.5 | 0·4–1·1 | 10–23 | 37–51 | |
| CI | 23* | 77* | 9 | 54 | 6 | 6 | 0·1 | 0·3 | 17 | 49* |
| 18–26 | 74–82 | 6–11 | 42–63 | 4–8 | 4–9 | 0–·4 | 0–·5 | 14–19 | 44–54 | |
| CS | 29 | 71 | 17 | 42 | 6 | 3 | 1·3 | 2·2 | 21 | 43* |
| 17–45 | 55–83 | 6–35 | 37–61 | 0·2–13 | 1–8 | 0·5–2 | 0·3–5 | 9–32 | 39–53 | |
| CSI | 40 | 60 | 10 | 22† | 5 | 2 | 1·1 | 2 | 22 | 25† |
| 33–44 | 56–67 | 3–15 | 14–27 | 1–7 | 1–4 | 0·4–2 | 0·7–4 | 14–27 | 18–28 | |
N: non-smokers; S: smokers; C: COPD ex-smokers; CI: COPD ex-smokers receiving inhaled corticosteroids; CS: COPD smokers; CSI: COPD smokers receiving inhaled corticosteroids). There was a significant decrease in the percentage of CD4− and increase in the percentage of CD8− T cells in S and CI compared with N (*P < 0·05). There was a significant increase in the percentage of CD8− T cells producing tumour necrosis factor (TNF)-α in S, C, CI and CS compared with N (P < 0·05). There was a significant decrease in the percentage of CD8− T cells producing interferon (IFN)-γ and TNF-α in CSI compared with CS (†P < 0·05). IL: interleukin.
Fig. 1.
Representative dot plots showing tumour necrosis factor (TNF)-α production by blood, bronchoalveolar lavage (BAL) and bronchial brushings (BB) CD8− and CD8− (CD4−) T cells from a chronic obstructive pulmonary disease (COPD) smoker patient and non-smoker control (N). T cells were identified by CD3 PC5 versus side scatter characteristics. COPD smokers showed an increase in TNF-α in CD8− T cell subsets in blood and BB and both CD8− and CD8− (CD4−) T cell subsets in BAL. Note the reduced percentage of CD4− T cells and increased CD8− T cells in blood and BAL of COPD smokers compared to non-smoker controls.
Correlation between FEV1 and cytokine production
There was a negative correlation between FEV1 and TNF-α production by CD8− T cells in BB (R = − 0·712, P = 0·004) and by CD8− T cells in BAL (R = − 0·407, P = 0·045). There was no other correlation between FEV1 and any other cytokine or T cell subset between any of the groups.
Discussion
This is the first comprehensive report of intracellular pro- and anti-inflammatory T cell cytokines in the separate compartments of blood, BAL and BB intraepithelial T cells from COPD subjects and smokers. Our findings are consistent with our hypotheses that changes would be noted for T cell cytokines in all compartments in COPD patients compared with smokers who have not progressed to COPD; that COPD smokers would exhibit further pertubations in T cell cytokines compared with COPD ex-smokers; and that COPD smokers receiving inhaled corticosteroids would show reduced proinflammatory changes consistent with therapeutic strategy. Changes in intracellular Th1 proinflammatory cytokines were found in all compartments in COPD groups and, importantly, an increase in Th1 proinflammatory cytokines, IFN-γ and TNF-α in BAL CD8− T cells of COPD smokers compared with COPD ex-smoker patients. These findings suggest that smoking may cause an increase in inflammation in the airways of COPD patients or, alternatively, a decrease in Th1 cytokines in these patients following smoking cessation. In addition, smoking causes a significant Th1 proinflammatory response in the airways and in intraepithelial CD8− T cells in smoking individuals who have not progressed to COPD. One could hypothesize that if this Th1 response could be inhibited, particularly in CD8− T cells, these individuals may not progress to COPD disease.
Our findings contrast with those of a recent report that showed an increase in TNF-α in non-smokers compared with smokers and COPD groups but no changes for IFN-γ [15]. This study also showed an increase in Th2 cytokines in BAL T cell subsets. Our findings of increased IFN-γ in CD8− blood T cells from COPD patients are consistent with a previous report [16]; however, others have found an increase in IFN-γ in CD4− blood T cells and not CD8− T cells [4,6]. These conflicting reports may be due partly to the diversity of methods used and possibly argue for a standardized technique in measuring intracellular T cell cytokines in these compartments. We have previously shown excellent reproducibility using non-purification techniques for several investigations of intracellular cytokines in intraepithelial T cells and T cells from blood and BAL from lung transplant patients [10,13,14].
Our findings of increased CD8− T cells in blood and BAL and BB of COPD patients are consistent with our previous findings [3] and similar to those of several other reports [17–19]. In this regard, smokers with normal lung function were also found to have increased numbers of BAL and BB CD8− T cells, suggesting that smoking per se may induce influx of CD8− T cells into the lung epithelium and airways and/or possibly increase the proliferation of resident cells [1,2].
We have previously shown up-regulation of TNF-α/TNF-αR1 in peripheral blood T cells from patients with COPD [20], and our current study extends these findings to airway and intraepithelial compartments. TNF-α production by CD8− T cells was increased in peripheral blood, BAL and BB of all COPD patient groups (except groups receiving inhaled corticosteroids in blood CD8− T cells) and also in BAL and BB of the smoking control group. Importantly, induction of TNF-α in the lung has been shown to result in emphysema in the mouse model [21]. Overall, these findings suggest a possible early causal link between smoking, CD8− TNF-α production and development of COPD. Interestingly, TNF-α has recently been described as the driving force behind COPD [22]. TNF-α has been shown to induce IL-2Rs and IFN-γ production by T cells and activate neutrophils, macrophages, endothelial cells and fibroblasts [23]: cells that play important roles in the pathogenesis of COPD [2]. Recently it has been shown that fractalkine, a potent chemoattractant for monocytes and T cells produced by airway smooth muscle cells, was induced in the presence of both IFN-γ and TNF-α [24]. Our current findings indicate that lung-infiltrating CD8− T cells are an important source of IFN-γ and TNF-α to induce this potent chemoattractant and as such may play an important role in the pathogenesis of COPD. Our findings of increased BAL T cell IL-2 production has not been reported previously in COPD patients, although cigarette smoking has been shown to be associated with increased plasma IL-2 in patients with stable coronary artery disease [25] and healthy smokers compared with non-smokers [26]. IL-2 has been shown to promote proliferation, cytotoxicity and chemotaxis and stimulate production of cytokines such as IFN-γ by T cells and as such may also play an important role in the pathogenesis of COPD. Although T cell regulatory cytokine IL-4 was unaltered in COPD patients in the present study, TGF-β, an important regulatory Th3 cytokine, shown previously to inhibit Th1 cytokines [27] was reduced in CD4− T cell subsets in the peripheral blood of COPD groups and smoker controls. Previously, we have found increased production of TGF-β by monocytes in the peripheral blood from COPD subjects [19] suggesting a cell-specific influence on levels of TGF-β in COPD. Recently, a single nucleotide polymorphism in the TGF-β gene, associated with increased TGF-β production, was shown to occur more commonly in control subjects compared to COPD patients [28]. Unfortunately, BAL and BB samples were insufficient to determine intracellular T cell TGF-β levels but this would be an important adjunct to this present study. One limitation of this study is the chance of a type 1 error due to the large number of ad hoc analyses between multiple groups where the number of patients is relatively small. Our ongoing study with a much larger cohort of patients and controls will be required to confirm these findings.
The correlation between severity of COPD as determined by FEV1 and intracellular TNF-α in BAL and BB intraepithelial CD8− T cells suggest that monitoring this intracellular T cell cytokine may be an appropriate indicator of patient morbidity.
Systemic therapeutic targeting of TNF-α production by CD8− T cells may improve morbidity in COPD patients, while targeting of TNF-α in the lung may prevent smokers progressing to COPD.
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