Chronic cigarette smoke exposure generates pathogenic T cells capable of driving COPD-like disease in Rag2-/- mice

Abstract

Rationale: Pathogenic T cells drive, or sustain, a number of inflammatory diseases. Chronic obstructive pulmonary disease (COPD) is an inflammatory lung disease associated with the accumulation of activated T cells. We previously demonstrated that chronic cigarette smoke (CS) exposure causes oligoclonal expansion of lung CD4(+) T cells and CD8(+) T cells in a mouse model of COPD, thus implicating these cells in disease pathogenesis.

Objectives: To determine whether T cells are pathogenic in a CS-induced mouse model of COPD.

Methods: We transferred lung CD3(+) T cells from filtered air (FA)- and CS-exposed mice into Rag2(-/-) recipients. Endpoints associated with the COPD phenotype were then measured.

Measurements and main results: Here, we demonstrate that chronic CS exposure generates pathogenic T cells. Transfer of CD3(+) T cells from the lungs of CS-exposed mice into Rag2(-/-) recipients led to substantial pulmonary changes pathognomonic of COPD. These changes included monocyte/macrophage and neutrophil accumulation, increased expression of cytokines and chemokines, activation of proteases, apoptosis of alveolar epithelial cells, matrix degradation, and airspace enlargement reminiscent of emphysema.

Conclusions: These data formally demonstrate, for the first time, that chronic CS exposure leads to the generation of pathogenic T cells capable of inducing COPD-like disease in Rag2(-/-) mice. This report provides novel insights into COPD pathogenesis.

Figure 1.

Experimental design. BALB/c wild-type (WT) mice were exposed to either filtered air (FA) or cigarette smoke (CS) for 24 weeks as described in the Methods section. Leukocytes from the perfused lungs of these mice were pooled, and CD3⁺ T cells were purified by fluorescence-activated cell sorting (>99.7% pure). Purified T cells from FA- or CS-exposed WT mice were then injected into BALB/c Rag2^−/− mice. BALB/c Rag2^−/− FA and CS T cell–recipient mice were rested 3 weeks to allow for reconstitution of the lymphopenic environment. BALB/c WT, BALB/c Rag2^−/−, BALB/c Rag2^−/− FA T cell–recipient, and BALB/c Rag2^−/− CS T cell–recipient mice were then exposed to either FA or CS (eight experimental groups, n = 16 per group) for 13 weeks (1 wk of smoke acclimation followed by 12 wk of exposure). At the end of the study, tissues were harvested and analyzed as described in the Methods section. The CD3⁺ T-cell purification histograms depicted are actual sort data representative of all sorts. This experimental protocol was performed independently two times.

Figure 2.

Cigarette smoke (CS) T cells drive lung inflammation in the bronchoalveolar lavage (BAL). (A) Mice were lavaged and total BAL cells were counted as described in Methods. (B) Total monocyte/macrophage numbers in the BAL. (C) Total neutrophil numbers in the BAL. (D) Total lymphocyte numbers in the BAL. (E) Representative photomicrographs from BAL cytospins showing neutrophil accumulation and “foamy” macrophages. Original magnification 400×. Bar, 50 μM. (F) Percentage of leukocytes found in the BAL. All data representative of two experiments. n = 5–8 mice per group. Data are presented as means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.

Figure 3.

Histological examination of lungs. (A) Representative photomicrographs of hematoxylin and eosin (H&E)-stained lung sections from Rag2^−/− filtered air (FA) T cell recipients. Original magnification 200×. Bar, 100 μM. (B) Representative photomicrographs of H&E-stained lung sections of Rag2^−/− cigarette smoke (CS) T cell recipients showing alveolitis as well as perivascular and peribronchiolar inflammatory aggregates. Original magnification 200×. Bar, 100 μM. (C) Representative photomicrographs of H&E-stained lungs from Rag2^−/− FA T cell recipients. Original magnification 40×. Bar, 500 μM. (D) Representative photomicrographs of H&E-stained lungs of Rag2^−/− CS T cell recipients showing alveolitis. Original magnification 40×. Bar, 500 μM. (E) Representative photomicrograph of lungs of BALB/c Rag2^−/− CS T cell recipient mice showing inflammatory aggregates that contain significant numbers Mac3⁺ cells. Original magnification 100×. Bar, 200 μM. (F) Representative lung photomicrograph of CS T cell BALB/c Rag2^−/− recipient mice highlighting multinucleated giant cells of inflammation. Original magnification 400×. Bar, 50 μM.

Figure 4.

CD3⁺ T cells in the lungs of Rag2^−/− recipients. (A) Fluorescence image showing CD3⁺ T cells in Rag2^−/− filtered air (FA) T cell recipients. Original magnification 200×. Bar, 100 μM. Positive cells are indicated by white arrows. (B) Differential interference contrast (DIC) image of A. (C) Fluorescence image showing CD3⁺ T cells in inflammatory aggregates of Rag2^−/− cigarette smoke (CS) T cell recipients. Original magnification 200×. Bar, 100 μM. (D) DIC image of C showing inflammatory aggregate. (E) Fluorescence image showing CD3⁺ T cells in parenchymal inflammation of Rag2^−/− CS T cell recipients. Original magnification 200×. Bar, 100 μM. (F) DIC image of E showing inflammation. All data representative of two experiments. n = 5–8 mice per group.

Figure 5.

Cigarette smoke (CS) T cells induce expression of inflammatory cytokines. Bronchoalveolar lavage from mice was assayed for cytokines as described in the Methods section. Raw data were analyzed using the Mayday software (http://www-ps.informatik.uni-tuebingen.de/mayday/wp/). The means of the raw data for each group were z-score transformed and are presented as a heatmap. The transformation was performed for each analyte individually. nd = not detectable. Data representative of two experiments. n = 4–6 mice per group. FA = filtered air; G-CSF = granulocyte colony-stimulating factor; GM-CSF = granulocyte-macrophage colony-stimulating factor; IP = inducible protein; KC = keratinocyte chemoattractant; LIX = lipopolysaccharide-induced CXC chemokine; M-CSF = macrophage colony-stimulating factor; MCP = monocyte chemoattractant protein; MIP = macrophage inflammatory protein; RANTES = regulated upon activation, normal T-cell expressed and secreted; TNF = tumor necrosis factor; WT = wild type.

Figure 6.

Cigarette smoke (CS) T-cell transfer results in protease activation. (A) Matrix metalloproteinase (MMP)-12 mRNA expression in whole lungs. (B) CATHEPSIN-S mRNA expression in whole lungs. (C) Broad-spectrum MMP activity in bronchoalveolar lavage (BAL). All data representative of two experiments. n = 5–8 mice per group. Data are presented as means ± SEM. * P < 0.05; ** P < 0.01. RFU = relative fluorescent units.

Figure 7.

Cigarette smoke (CS) T cell transfer causes lung damage and emphysematous changes. (A) Representative photomicrographs of hematoxylin and eosin–stained lungs highlighting emphysematous changes in lungs of CS T cell BALB/c Rag2^−/−–recipient mice. Original magnification 100×. Bar, 200 μM. (B) Quantification of emphysema. (C) Hyaluronan ELISA of bronchoalveolar lavage. All data representative of two experiments. n = 5–8 mice per group. Data are presented as means ± SEM. * P < 0.05; ** P < 0.01.

Figure 8.

Cigarette smoke (CS) T-cell transfer causes apoptosis. (A) Representative fluorescence images highlighting increase in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)⁺ cells in lungs of CS T cell BALB/c Rag2^−/−–recipient mice. Original magnification 200×. Bar, 100 μM. (B) Quantification of TUNEL⁺ cells. (C) TUNEL labeling and pro–SP-C double staining shows apoptosis of type II alveolar epithelial cells. Representative fluorescence images presented are of the lungs of CS T cell BALB/c Rag2^−/− recipient mice. Original magnification 200×. Bar, 5 μM. All data representative of two experiments. n = 5–8 mice per group. Data are presented as means ± SEM. * P < 0.05; ** P < 0.01.