Lung Dendritic Cells Drive Natural Killer Cytotoxicity in Chronic Obstructive Pulmonary Disease via IL-15Rα

Abstract

Rationale: Lung natural killer cells (NKs) kill a greater percentage of autologous lung parenchymal cells in chronic obstructive pulmonary disease (COPD) than in nonobstructed smokers. To become cytotoxic, NKs require priming, typically by dendritic cells (DCs), but whether priming occurs in the lungs in COPD is unknown.

Methods: We used lung tissue and in some cases peripheral blood from patients undergoing clinically indicated resections to determine in vitro killing of CD326+ lung epithelial cells by isolated lung CD56+ NKs. We also measured the cytotoxicity of unprimed blood NKs after preincubation with lung DCs. To investigate mechanisms of DC-mediated priming, we used murine models of COPD induced by cigarette smoke (CS) exposure or by polymeric immunoglobulin receptor (pIgR) deficiency, and blocked IL-15Rα (IL-15 receptor α subunit) trans-presentation by genetic and antibody approaches.

Results: Human lung NKs killed isolated autologous lung epithelial cells; cytotoxicity was increased (P = 0.0001) in COPD, relative to smokers without obstruction. Similarly, increased lung NK cytotoxicity compared with control subjects was observed in CS-exposed mice and pIgR-/- mice. Blood NKs both from smokers without obstruction and subjects with COPD showed minimal epithelial cell killing, but in COPD, preincubation with lung DCs increased cytotoxicity. NKs were primed by CS-exposed murine DCs in vitro and in vivo. Inhibiting IL-15Rα trans-presentation eliminated NK priming both by murine CS-exposed DCs and by lung DCs from subjects with COPD.

Conclusions: Heightened NK cytotoxicity against lung epithelial cells in COPD results primarily from lung DC-mediated priming via IL-15 trans-presentation on IL-15Rα. Future studies are required to test whether increased NK cytotoxicity contributes to COPD pathogenesis.

Keywords: chronic obstructive pulmonary disease; cigarette smoke; human; small-airway epithelial cells.

Figures

Figure 1.

Autologous lung epithelial cells are killed more avidly by lung natural killer cells (NKs) from patients with chronic obstructive pulmonary disease (COPD) than from smokers without obstruction. From dispersed human lung tissue, we sequentially isolated CD56+ NKs and then CD326+ (epithelial cell adhesion molecule) cells, using magnetic beads. Epithelial cells were cultured either alone or with CD56+ cells, and then after 4 hours all cells were collected; stained with CD45, annexin-V, and 7-aminoactinomycin D; and analyzed by flow cytometry. Epithelial cells were identified as CD45− with high side scatter. Representative histograms are shown of annexin-V staining from (A) a smoker without COPD and (B) a subject with COPD, showing epithelial cells cultured alone (dark gray histograms) and epithelial cells cultured with NKs (light gray histograms). (C) The percent cytotoxicity (calculated as described in Methods) is shown for individual subjects: n = 19 smokers without obstruction and n = 30 subjects with COPD. Lines represent means ± SEM, offset to facilitate visualization. Open symbols, former smokers; solid symbols, active smokers. The Mann-Whitney t test was used to determine significance.

Figure 2.

Lung natural killer cells (NKs), not lung epithelial cells, drive the increased cytotoxicity in chronic obstructive pulmonary disease (COPD). CD56+ NKs and CD326+ epithelial cells were isolated from human lung tissue. (A) Lung epithelial cells from smokers without obstruction (n = 9) or patients with COPD (n = 14) were cultured either alone or with NK-92 cells to determine the percent cytotoxicity; (B) K562 target cells were cultured either alone or with CD56+ cells from smokers without obstruction (n = 9) or from patients with COPD (n = 16) to determine the percent cytotoxicity. For both panels, lines represent means ± SEM, offset to facilitate visualization. Open symbols, former smokers; solid symbols, active smokers. The same subjects were used to generate the data in A and B, although we were unable to perform the experiment in A on two of the subjects with COPD. The Mann-Whitney t test was used to determine significance. N.S. = not significant.

Figure 3.

Lung natural killer cells (NKs) are more cytotoxic against epithelial cells in a murine model of cigarette smoke (CS) exposure. C57BL/6 mice were exposed to air or CS for 8 weeks, and then lung tissue was collected for the isolation of NKs (CD49b+) and CD326+ epithelial cells. Epithelial cells from air-exposed mice were cultured either alone or with NKs from air-exposed or CS-exposed mice. Likewise, epithelial cells from CS-exposed mice were cultured either alone or with NKs from air-exposed or CS-exposed mice. After 4 hours, cells were collected and stained for CD45, annexin-V, and 7-aminoactinomycin D for flow cytometry. Epithelial cells were identified as CD45− with high side scatter. The percent cytotoxicity is shown for four separate experiments (five mice per group per experiment). Box plots show the first and third quartiles plus the median, and the whiskers represent the minimum and maximum values. Repeated measures one-way ANOVA with Tukey’s multiple comparison test was used to determine significance. **P < 0.01.

Figure 4.

Dendritic cells (DCs) from cigarette smoke (CS)–exposed mice prime natural killer cells (NKs) to become cytotoxic, both in vitro and in vivo. C57BL/6 mice were exposed either to air or CS for 8 weeks. Lung tissue was collected and dispersed for isolation of NKs (CD49b+), CD326+ epithelial cells, and pan-DCs. (A) Lung NKs from air-exposed mice were preincubated for 18 hours alone or with DCs from air- or CS-exposed mice, and then pooled lung epithelial cells from CS-exposed mice were added for an additional 4 hours. Cytotoxicity was then assayed. Data are from three separate experiments (each with five mice per group); a repeated measures one-way ANOVA with Tukey’s multiple comparison test was used to determine significance. (B) DCs from either air- or CS-exposed mice (CD45.2 congenic strain) were adoptively transferred (200,000 cells intranasal) into naive recipient mice (CD45.1 congenic strain). Recipient mice were killed 48 hours later and lungs were collected to isolate NKs and epithelial cells. Pooled epithelial cells from all mice were cultured either alone or with NKs from individual mice. After 4 hours, cells were collected and cytotoxicity was assayed. Data represent means ± SEM of three mice per group in each of three separate experiments, indicated by symbols. The Mann-Whitney t test was used to determine significance. (C) Representative flow staining of lung tissue from recipient mouse that received adoptive transfer of DCs. Cells that are double-positive for CD45.2 and CD11c are indicated by the square gate. (D) Lung tissue and mediastinal lymph nodes (LNs) were collected from recipient mice that received adoptive transfer of either air-exposed DCs or CS-exposed DCs. Antibodies against CD11c, MHC class II, CD11b, and CD103 were used to identify all lung and LN DCs. CD45.2 staining was used to determine the percentage of DCs that were positive for CD45.2, indicating they had been adoptively transferred from donor mice. There were no significant differences between groups, as determined by the Mann-Whitney t test. *P < 0.05.

Figure 5.

Lung natural killer cells (NKs) from polymeric immunoglobulin receptor–deficient (pIgR−/−) mice have increased cytotoxicity against epithelial cells, which is driven by dendritic cell (DC)–mediated priming. (A) Paraffin-embedded lung tissue from 6-month-old pIgR+/+ and pIgR−/− mice was stained with anti-CD69 and anti-NCR1. Red arrowheads indicate NCR1 single-positive cells, and yellow arrowheads indicate NCR1 and CD69 double-positive cells. (B) The percentage of NKs coexpressing CD69 is shown. Data are pooled from six mice per group. (C) Lung tissue from 2- and 6-month-old pIgR+/+ and pIgR−/− mice was used to isolate NK (CD49b+) cells. CD326+ epithelial cells were isolated only from pIgR+/+ mice. Epithelial cells were cultured either alone or with pIgR+/+ NKs or pIgR−/− NKs. There was no difference in the percent cytotoxicity of the 2- and 6-month-old mice, and therefore these data have been combined (open symbols, 2-mo-old mice; solid symbols, 6-mo-old mice). (D) Lung NKs from pIgR+/+ mice were cocultured with lung DCs from either pIgR+/+ or pIgR−/− mice (2 and 6 mo old) for 18 hours. Epithelial cells from pIgR+/+ mice were added to the culture for an additional 4 hours, and then percent cytotoxicity was determined. For C and D, data are from two separate experiments, 5–10 total mice per group. The Mann-Whitney t test was used to determine significance.

Figure 6.

Blocking IL-15 trans-presentation prevents dendritic cell (DC)–mediated priming of natural killer cell (NK) cytotoxicity. (A) Lung NKs from air-exposed mice were cocultured for 18 hours with DCs from either air-exposed mice, cigarette smoke (CS)–exposed mice, or CS-exposed mice in the presence of anti-mouse IL-15R (IL-15 receptor)/IL-15. Epithelial cells from CS-exposed mice were added to the culture for an additional 4 hours, and then cytotoxicity was determined. (B) Lung NKs from air-exposed mice were cultured for 18 hours either by themselves, or with DCs from CS-exposed wild-type (WT) mice or CS-exposed IL-15Rα−/− mice. Lung epithelial cells pooled from CS-exposed WT mice were added for an additional 4 hours, and then cytotoxicity was determined. Data in A and B are from three separate experiments (five mice per group per experiment); repeated measures one-way ANOVA with Tukey’s multiple comparison test was used to determine significance. (C) DCs from either CS-exposed WT mice or CS-exposed IL-15Rα−/− mice were adoptively transferred via intranasal administration of 200,000 DCs into untreated recipient WT mice; 48 hours later, lungs were collected to isolate NKs and epithelial cells. Epithelial cells pooled from all mice were cultured alone or with NKs from individual mice for 4 hours, and then cytotoxicity was assayed. Lines represent means ± SEM of two independent experiments (indicated by symbols), each containing three recipient mice per group; significance was determined by the Mann-Whitney t test. *P < 0.05; **P < 0.01.

Figure 7.

Lung dendritic cells (DCs) from subjects with chronic obstructive pulmonary disease (COPD) increase natural killer cell (NK) cytotoxicity via IL-15Rα (IL-15 receptor α subunit) trans-presentation. Blood NKs from smokers without COPD (Sm) or subjects with COPD were cocultured either alone or with autologous lung DCs for 18 hours. Autologous lung epithelial cells were added for an additional 4 hours, and then cytotoxicity was measured via annexin and 7-aminoactinomycin D staining. (A) Representative annexin-V staining of gated CD326+ epithelial cells from a smoker without COPD (left) and subject with COPD (right). Dark gray histograms, coculture with blood NKs alone; light gray histograms, cocultured with NKs plus lung DCs. (B) Combined data of healthy smokers (n = 4) and subjects with COPD (n = 4), one-way ANOVA with Tukey’s multiple comparison test. *P < 0.05; no other comparisons are significant. (C) Blood NKs from subjects with COPD (n = 3) were cocultured for 18 hours alone, with autologous lung DCs, or with autologous lung DCs in the presence of IL-15Rα Fc chimera; autologous lung epithelial cells were added for an additional 4 hours, and then cytotoxicity was assayed. Repeated measures one-way ANOVA with Tukey’s multiple comparison test was used to determine significance.