Light cigarette smoke-induced emphysema and NFkappaB activation in mouse lung

Abstract

Light cigarette (LC) exposure is supposed to be less hazardous with a decreased incidence of cancer and tobacco-associated diseases. C57BL/6 mouse groups were subjected to smoke from 3, 6 or 12 LC for 60 days and compared with mice exposed to ambient air (EAA) in order to study lung injury by morphometrical and biochemical methods. Bronchoalveolar lavage (BAL) analysis and histology and stereology were performed. Tissue from the right lung was used for measuring thiobarbituric acid reactive substances (TBARS) and Western blot analysis. One way anova was performed followed by the Student-Newman Keuls post-test (P < 0.05). The cellular content of BAL was 95% alveolar macrophages in all groups except in mice exposed to 3 LC, where 23% neutrophils were observed. Emphysema was not observed in three and 6 LC, but it was found in 12 LC parallel to increased volume density (Vv) of airspaces from 61.0 +/- 0.6 (EAA) to 80.9 +/- 1.0 (12 LC) and decreased Vv of elastic fibres from 17.8 +/- 0.9 (EAA) to 11.8 +/- 0.6 (12 LC). All exposed groups to LC showed low TBARS levels compared with mice EAA. Lung tissue from animals exposed to 12 LC showed decreased tissue inhibitor of metalloprotease-2 and increased matrix metalloprotease-12 detection, which suggests an imbalance in extracellular matrix (ECM). Increased tumour necrosis factor-alpha and nuclear factor-kappaB detection were observed in exposed groups to LC when compared with mice EAA. The data suggest that LC is so dangerous to lungs as full-flavour cigarettes inducing ECM imbalance and emphysema.

Figure 1

Lung morphology of control mice and light cigarette (LC) groups: (a) normal histology in exposed to ambient air (EAA) where airspaces and alveolar septa are preserved; (b) and (c) 3 LC and 6 LC with normal histology and alveolar macrophages (AM) in airspaces; (d) 12 LC with large AM influx and airspace enlargement. H&E, scale bar 100μm.

Figure 2

Lung elastic fibres of control mice and light cigarette (LC) groups: (a) exposed to ambient air (EAA) with normal delicate branching fibres; (b) and (c) 3 LC and 6 LC with no apparent changes in elastic fibres; (d) 12 LC deposits of elastin with fragmented and irregular aspect. Orcein, scale bar 100μm.

Figure 3

Morphometry on alveolar macrophages (AM) in lung sections of controls, 3 LC, 6 LC and 12 LC. There is a clear increase of macrophage numbers correlating light cigarette (LC) dose in mice compared with control. Tissue macrophages were counted in Giemsa stained sections (mean ± SEM).

Figure 4

Cellular influx into the bronchoalveolar lavage (BAL) of control mice, 3 LC, 6 LC and 12 LC. Neutrophils were present only in 3 LC, whereas alveolar macrophages (AM) increased according to cigarette dose compared with control. At least 200 cells/BAL sample were counted using standard morphological criteria (mean ± SEM).

Figure 5

Tissue inhibitor of metalloprotease-2 (TIMP-2) expression in response to 3 LC, 6 LC, 12 LC and control. After 60 days of exposure to light cigarette (LC) or exposed to ambient air (EAA), lungs were removed en bloc, perfused with saline until free of blood, and then frozen and total lung tissue extracts were prepared. Equal amounts of protein were subjected to western blot analysis using an antibody specific for TIMP-2. Figure under lane represents densitometry (%). LC exposure decreased TIMP-2 in 3 and 12 LC, but increase it expression in 6 LC group compared with control (n = 2).

Figure 6

Matrix metalloprotease-12 (MMP-12) expression in response to 3 LC, 6 LC, 12 LC and control. Procedures were performed as for Figure 5. LC exposure induced little MMP-12 expression in 3 LC and 12 LC, but the lane is strong in 12 LC. The control mice showed little MMP-12 expression (n = 2).

Figure 7

Tumour necrosis factor (TNF)-α expression in response to 3 LC, 6 LC, 12 LC and control. Procedures were performed as for Figure 5. TNF-α is strongly expressed in all LC exposed groups but there is little expression in control (n = 2).

Figure 8

NFκB expression in response to 3 LC, 6 LC, 12 LC and control. After 60 days of exposure to light cigarette (LC) or exposed to ambient air (EAA), lungs were removed en bloc, perfused with saline until free of blood, and then frozen and total lung tissue nuclear extracts were prepared. Equal amounts of protein were subjected to western blot analysis using an antibody specific for NFκB. LC exposure induced strong NFκB expression in 3 LC, 6 LC and 12 LC, but the lane is weak for the control mice (n = 2).