Enhanced effector function of cytotoxic cells in the induced sputum of COPD patients

The Nottingham Local Research Ethics Committee approved the study protocol and written informed consent was obtained from the 26 subjects before entering the blinded study. Of these, the 11 participants diagnosed as having COPD (COPD subjects), according to the ATS guidelines, were current or ex-smokers who had accrued at least a 20 pack year smoking history and had an FEV₁ below 80% of predicted with an FEV₁/FVC ratio of <70% and reversibility to an inhaled beta-2 agonist of <10% or <200 mls absolute improvement. Ten healthy smokers (smokers), defined as smokers without airflow limitation, and five healthy non-smokers (HNS), with an FEV₁ above 80% of predicted, were recruited and matched for age and for the healthy smokers, smoking history, as closely as possible. Table 1 details the demographic and spirometric data of the subjects. Participants were excluded if they had a history of tuberculosis (TB), a clinical suspicion of current infection, history of physician diagnosed asthma or a positive skin prick test response to grass pollen, house dust mite, cat dander and dog hair (ALK-Abelló). COPD subjects were also excluded if they had had an exacerbation within the previous 6 weeks, were α1-anti-trypsin deficient or had other lung disease.

Sputum was induced by inhalation of hypertonic saline as described previously [13] with the difference that the subject was given salbutamol (400 mg) via a volumatic, rather than 1 mg terbutaline. The sample was placed on ice and processed within 1 hour.

The sputum was concentrated in a petri dish on ice, weight was calculated and 0.1% dithiothreitol (DTT) was added at a 4× weight/volume ratio. Sputum was dispersed, vortexed and then placed on a roller mixer. An equal volume of PBS was then added, vortexed and filtered through 48 μm nylon gauze. An aliquot for cell counting to assess viability and squamous cell contamination was taken. The remaining sample was centrifuged and resuspended.

CD56⁺ cells were isolated using α-CD56 microbeads (Miltenyi Biotech Ltd) according to manufacturers' instructions. Briefly, cells were incubated for 15 minutes at 4°C with α-CD56 microbeads and separated on a refrigerated MS column using cold PBS containing 1% FCS and 0.4% EDTA. The resulting positive fraction was washed and resuspended in RPMI 1640 medium. Following isolation, all fractions were washed, counted and purity confirmed at ≥90% by flow cytometric analysis.

Cells were washed with PBA, fixed in 3% formaldehyde in isotonic azide free solution and given a final wash with PBA - 0.04% saponin with 10% FCS. Labelled antibodies (Table 2) were added at the recommended concentration and the cells were incubated for two hours at 4°C in the dark. Excess antibody was removed by washing and cells were stored in 0.5% formaldehyde in isotonic azide free solution at 4°C. Flow cytometric analysis of these antibody labelled cells was performed using an EPICS Altra (Beckman Coulter). Fifty thousand live-gated events were collected for each sample and isotype matched antibodies were used to determine binding specificity. Data were analysed using WEASEL version 2.3 (WEHI). Necrotic cells were excluded from analysis according to their forward and side scatter characteristics.

A commercially available lactate dehydrogenase (LDH) kit (CytoTox 96 Non-Radioactive Cytotoxicity Assay, Promega) was used with erythroleukaemic K562 cells (ECACC) as the target cell line. Briefly, the effector and target cells were mixed at a ratio of 5:1, plated in quadruplicate on a 96-well U-bottomed plate and incubated for 4 h at 37°C in a humidified atmosphere containing 5% CO₂. After incubation, the plate was centrifuged, the supernatants collected and incubated for 30 min at room temperature with the Substrate Mix provided with the kit to detect LDH activity. A stop solution was added and the absorbance of the sample was measured at 490 nm on an Emax precision microplate reader (Molecular Devices) using SOFTmax software (Molecular Devices). The amount of cell-mediated cytotoxicity was calculated by subtracting the spontaneous LDH released from the target and effector cells from the LDH released by lysed target cells, using the following equation:

Specific lysis (%) = {(effector/target cell mix - spontaneous effector LDH release - spontaneous target LDH release)/(maximum target LDH release - spontaneous target LDH release)}x100

The biological lytic ability was then calculated by multiplying the lytic activity per cell by the total number of CD56⁺ cells per gram of induced sputum.

The viability of the cells (% total) in induced sputum did not differ between COPD subjects, smokers and HNS (median, range): 83 (75-94), 82 (72-93) and 86 (75-94), respectively. In all samples, more than 90% of the cells were non-squamous cells. The total cell count (TCC; g^-1) was significantly higher in COPD subjects than in smokers and HNS (Table 3). The TCC was also higher in smokers than in HNS (Table 3). The differences in cellular parameters amongst the three groups are presented in Table 3. The proportions of neutrophils and lymphocytes were higher in COPD subjects than in the other two groups, with the proportion of macrophages lower.

Flow cytometric analysis of the lymphocytes showed that the proportions of CD8⁺ T lymphocytes, NK (CD56⁺CD3^-) cells and NKT-like (CD56⁺CD3⁺) cells were significantly higher in COPD subjects compared to the other two groups (Figure 1). However, the CD4⁺/CD8⁺ ratio was significantly lower in COPD subjects (0.70) compared to smokers (1.4; p < 0.001) and HNS (1.0; p < 0.001). There was also a significant increase in the proportion of B lymphocytes in COPD patients compared to smokers and HNS (Figure 1).

Further analysis of the CD8⁺ T lymphocytes by flow cytometry revealed that COPD subjects had an increased proportion of memory cells (CD45RO⁺RA^-) and a reduction in the proportion of naïve cells (CD45RO^-RA⁺) compared to the other two groups (Figure 2A). The proportion of T_EMRA cells (CD45RO⁺RA⁺) was also higher in COPD subjects.

The subpopulation split of NK cells showed a higher proportion of immunoregulatory CD56^brightCD16^- NK cells in COPD subjects, compared to the other two groups (Figure 2B ). There was no measurable difference in the proportion of NK cells expressing CD8 in any of the three groups (data not shown).

NKT-like (CD56⁺CD3⁺) cells in COPD subjects showed an increased proportion expressing CD8 and a decreased proportion expressing CD4 (Figure 2C), compared to the other two groups. There was no difference between groups for the double negative fraction.

The expression of perforin and granzyme B were studied in CD8⁺ T lymphocytes, CD56^dimCD16⁺ NK cells, CD56^brightCD16^- NK cells and NKT-like (CD56⁺CD3⁺) cells (Figure 3).

The proportion of CD8⁺ T lymphocytes expressing both perforin and granzyme B was significantly higher in COPD subjects (43.9) compared to HNS (18.6; p < 0.01) (Figure 3B). The proportions of both CD56^dimCD16⁺ and CD56^brightCD16^- NK cells expressing both perforin and granzyme B were also significantly higher in COPD subjects (74.6 and 44.8, respectively) compared to both HNS (46.2; p < 0.001 and 18.5; p < 0.01) and smokers (55.9; p < 0.01 and 24.9; p < 0.05)(Figure 3B). The proportion NKT-like (CD56⁺CD3⁺) cells expressing both perforin and granzyme B were also significantly higher in COPD subjects (63.8) compared to both HNS (14.5; p < 0.001) and smokers (43.4; p < 0.01)(Figure 3B).

The proportion of CD8⁺ T lymphocytes that expressed only perforin and no granzyme B was significantly higher in both smokers (8.0; p < 0.01) and COPD subjects (4.6; p < 0.05) than in HNS (1.4) (data not shown). The proportion of NKT-like (CD56⁺CD3⁺) cells that expressed only perforin and no granzyme B was significantly higher in both smokers (14.0; p < 0.001) and HNS (13.8; p < 0.01) than in COPD subjects (5.4) (data not shown). There was no difference in the proportions of CD56^dimCD16⁺ NK cells and CD56^brightCD16^- NK cells expressing only perforin and no granzyme B. Examining the proportion of cells expressing only granzyme B and no perforin revealed no differences between groups and cell types (data not shown).

To establish if the increased proportions of CD56⁺ cells that expressed both perforin and granzyme B were cytotoxic in the LDH release assay, CD56⁺ cells were immunomagnetically purified from induced sputum. All samples were ≥ 90% pure with respect to B lymphocytes, helper T lymphocytes, CD8⁺ T lymphocytes, neutrophils and monocytes (Table 4).

Using the same number of effector cells (effector to target ratio of 5:1) the CD56⁺ cells from COPD subjects were significantly more cytotoxic (36.8% specific lysis) than those from smokers (22.4%; p < 0.01) and HNS (16.1%; p < 0.001) (Figure 4A). When taking into account for the differences in cell numbers on overall cytotoxicity, by examining the product of CD56 cell numbers and lytic activity of the CD56⁺ cells from COPD subjects (which we have termed biological lytic activity) the significant differences remain (Figure 4B) and this inversely correlated with lung function, as assessed by FEV₁ measurement (r = -0.75; p = 0.0098) (Figure 4C).

To examine a possible mechanism of selected cell recruitment to the lung the adhesion molecules CXCR3 and VLA-4 were measured. The proportion of CD8+ T lymphocytes, CD56^dimCD16⁺ NK cells, CD56^brightCD16^- NK cells and NKT-like cells expressing CXCR3 was significantly higher in COPD subjects than in smokers (Figure 5A). The proportions of these cells expressing VLA-4 were also significantly higher in COPD subjects than in smokers (Figure 5B).