Cigarette smoke-induced pulmonary emphysema in scid-mice. Is the acquired immune system required?

Abstract

Background: Chronic obstructive pulmonary disease is associated with a chronic inflammatory response of the host to chronic exposure to inhaled toxic gases and particles. Although inflammatory cells of both the innate and adaptive immune system infiltrate the lungs in pulmonary emphysema and form lymphoid follicles around the small airways, the exact role of the acquired immune system in the pathogenesis of emphysema is not known.

Methods: In this study, wild type Balb/c mice and immunodeficient scid mice--which lack functional B- and T-cells--were exposed to mainstream cigarette smoke (CS) for 5 weeks or 6 months.

Results: Subacute CS-exposure for 5 weeks significantly increased innate inflammatory cells (neutrophils, macrophages and dendritic cells) in the bronchoalveolar lavage (BAL) fluid of wild type mice and scid mice, which correlated with the CS-induced upregulation of the chemokines Monocyte Chemotactic Protein-1, Macrophage Inflammatory Protein-3alpha and KC (= mouse Interleukin-8). Chronic CS-exposure for 6 months significantly increased the number of neutrophils, macrophages, dendritic cells, CD4+ and CD8+ T-lymphocytes in BAL fluid and lungs of wild type mice compared to air-exposed littermates, and augmented the size and number of peribronchial lymphoid follicles. In contrast, neither B-lymphocytes, nor T-lymphocytes, nor lymphoid follicles could be discerned in the lungs of air- or CS-exposed scid mice. Importantly, chronic CS-exposure induced pulmonary emphysema in both wild type animals and scid mice, as evidenced by a significant increase in the mean linear intercept and the destructive index of CS-exposed versus air-exposed animals. The CS-induced emphysema was associated with increased mRNA expression of matrix metalloproteinase-12 in the lungs and increased protein levels of Tumor Necrosis Factor-alpha in the BAL fluid of CS-exposed Balb/c and scid mice compared to air-exposed littermates.

Conclusion: This study suggests that the adaptive immune system is not required per se to develop pulmonary emphysema in response to chronic CS-exposure, since emphysema can be induced in scid mice, which lack lymphoid follicles as well as functional B- and T-cells.

Figure 1

Cell differentiation in bronchoalveolar lavage fluid upon air or cigarette smoke exposure. Effect of cigarette smoke (CS) exposure for 1 month or 6 months on cell differentiation in bronchoalveolar lavage fluid (absolute numbers in BAL fluid) in wild type mice and scid mice (air-exposed mice: open bar, CS-exposed mice: closed bar): (A) total BAL cells, (B) neutrophils, (C) macrophages, (D) dendritic cells, (E) CD4⁺ T-cells and (F) CD8⁺ T-cells. Animals (n = 8) were exposed to 5 cigarettes/group/exposure; 4 exposures/day; 5 days/week. Results are expressed as means ± SEM (** p < 0.001 versus wild type mice at corresponding timepoint). Numbers of total BAL cells, neutrophils, macrophages, dendritic cells, CD4⁺ and CD8⁺ T-cells are significantly increased in CS-exposed versus air-exposed wild type mice and scid mice. No CD4⁺ and CD8⁺ T-cells were detected in BAL of scid mice.

Figure 2

T- and B-cell populations in the lungs upon air or cigarette smoke exposure. Effect of cigarette smoke (CS) exposure for 6 months on T-cell populations in the lungs (absolute numbers in the lungs; determined by flow cytometry) in wild type mice and scid mice (air-exposed mice: open squares, CS-exposed mice: closed squares): (A) lung CD4⁺ T-lymphocytes, (B) lung CD8⁺ T-lymphocytes, (C) activated lung CD4⁺ T-lymphocytes, (D) activated lung CD8⁺ T-lymphocytes and (E) lung B-lymphocytes. Animals (n = 8) were exposed to 5 cigarettes/group/exposure; 4 exposures/day; 5 days/week (* p < 0.05 and ** p < 0.001 versus air-exposed mice).

Figure 5

Lymphoid follicles in the lungs upon air or cigarette smoke exposure. Photomicrographs of lymphoid follicles in lungs of air- and cigarette smoke (CS)-exposed wild type mice and scid mice at 6 months (magnification × 100). (A)-(E) B220 staining (brown = B220 positive cells): (A) air-exposed wild type mice, (B) CS-exposed wild type mice, (C) air-exposed scid mice, (D) CS-exposed scid mice and (E) CS-exposed wild type mice (magnification × 200). (F) CD3/B220 staining (brown = CD3 positive cells; blue = B220 positive cells): CS-exposed wild type mice.

Figure 3

Quantification of pulmonary emphysema in wild type mice and scid mice. Effect of cigarette smoke (CS) exposure for 6 months on emphysema in wild type mice and scid mice (air-exposed mice: open squares, CS-exposed mice: closed squares). (A) mean linear intercept (μm) and (B) destructive index (* p < 0.05 and ** p < 0.01 versus air-exposed mice).

Figure 4

Pulmonary emphysema in wild type versus scid mice after 6 months air or CS-exposure. Photomicrographs of hematoxylin and eosin stained lung tissue of air- and cigarette smoke (CS)-exposed wild type mice and scid mice at 6 months (magnification × 200). (A) air-exposed wild type mice, (B) CS-exposed wild type mice, (C) air-exposed scid mice and (D) CS-exposed scid mice.

Figure 6

Expression of MMP-12 and MIP-3α RNA in lungs upon air or cigarette smoke exposure. (A) MMP-12 and (B) MIP-3α RNA expression in lung tissue of wild type mice and scid mice to air or cigarette smoke (CS) exposure for 1 month (air-exposed mice: open bar, CS-exposed mice: closed bar). RT-PCR results are expressed as a ratio target mRNA with hprt (hypoxanthine guanine phosphoribosyl transferase) mRNA (mean ± SEM, n = 5 animals per group, * p < 0.05 and ** p < 0.01). MMP-12: matrix metalloproteinase-12; MIP-3α: Macrophage Inflammatory Protein-3α.

Figure 7

Protein levels of cytokines and chemokines in bronchoalveolar lavage fluid upon air or cigarette smoke exposure. Effect of cigarette smoke (CS) exposure for 6 months on the release of cytokines and chemokines in bronchoalveolar lavage fluid of wild type mice and scid mice (air-exposed mice: open squares, CS-exposed mice: closed squares). (A) TNF-α, (B) MCP-1, (C) MIP-3α and (D) KC (* p < 0.05, ** p < 0.001). TNF-α (Tumor Necrosis Factor-α) and MCP-1 (Monocyte Chemotactic Protein-1) were measured by Cytometric Bead Array; MIP-3α (Macrophage Inflammatory Protein-3α) and KC (mouse Interleukin-8) were determined by ELISA.