Critical role of tumor necrosis factor receptor 1 in the pathogenesis of pulmonary emphysema in mice

Figures & data

Figure 1 Generation of TNF-tg × TNFR1−/−, TNF-tg × TNFR2−/−.

Notes: Mice were identified by DNA typing using PCR methods. (A) Identification of TNF-tg. Arrow indicates the TNF-tg band. (B) Identification of TNFR1 deletion. Left arrow (→) indicates wt mice, and right arrow (←) indicates TNFR1 deletion. (C) Identification of TNFR2 deletion. Left arrow indicates wt mice. TNFR2 deletion is documented by the loss of the bands, as indicated by the arrow. In the case of identification of TNF-tg × TNFR1−/−, DNA typing of both (A) and (B) was used. In the case of TNF-tg × TNFR2−/−, (A) and (C) were used.
Abbreviations: PCR, polymerase chain reaction; tg, transgene; TNF-α, tumor necrosis factor-α; TNF-tg, TNF-α transgene; TNFR, TNF receptor; wt, wild type.

Table 1 Mice used in this experiment

	Wild-type mice	TNF-tg	P-value	TNFR1−/−	TNF-tg × TNFR1−/−	P-value	TNFR2−/−	TNF-tg × TNFR2−/−	P-value
Number	3	4		5	3		3	4
Body weight (g)	23.7±1.38	23.3±2.51	NS	23.9±0.60	26.0±0.08	NS	27.7±1.04	26.6±0.98	NS
Lung volume (mL)	0.32±0.08	0.87±0.05	*	0.29±0.03	0.32±0.05	NS	0.31±0.06	0.59±0.03	*
RV/LV + S	0.18±0.00	0.27±0.02	*	0.18±0.01	0.18±0.00	NS	0.17±0.00	0.21±0.01	NS

Notes:

* Significant differences between TNF-tg-positive mice and transgene-negative mice. Data presented as mean ± standard deviation.

Abbreviations: LV, left ventricular; NS, no significance; RV, right ventricular; S, septa; TNF-α, tumor necrosis factor-α; TNF-tg, TNF-α transgene; TNFR, TNF receptor.

Figure 2 Macroscopic lung findings.

Notes: Lungs containing the bronchus were excised from sacrificed mice en bloc. 1: wild-type mice (C57BL/6), 2: TNF-tg, 3: TNF-tg × TNFR1−/−, 4: TNF-tg × TNFR2−/−. Overexpression of TNF-α leads to the development of large emphysematous lungs. Deletion of TNFR2 attenuated the emphysematous changes. Deletion of TNFR1 completely diminished the emphysematous changes.
Abbreviations: TNF-α, tumor necrosis factor-α; TNF-tg, TNF-α transgene; TNFR, TNF receptor.

Figure 3 Histology of the lungs.

Notes: Lung sections from the mice were stained with hematoxylin–eosin (H–E). All panels are at the same magnification. Original magnification, 40×. The lungs from wild-type mice (C57BL/6), TNFR1−/−, TNFR2−/−, TNF-tg, TNF-tg × TNFR1−/−, and TNF-tg × TNFR2−/−. There is no histological difference between wild-type mice (C57BL/6), TNFR1−/−, and TNFR2−/− (top panel). TNF-tg showed alveolar enlargement and interstitial inflammation. The degree of involvement was consistent with the macroscopic findings. The histology of lungs from TNF-tg × TNFR1−/− mice was similar to that of wild-type mice (C57BL/6) and TNFR1−/−.

Abbreviations: TNF-α, tumor necrosis factor-α; TNF-tg, TNF-α transgene; TNFR, TNF receptor.

Figure 4 Investigation of emphysema by morphometry and physiologic study.

Notes: (A) Lm between TNF-tg- and tg-negative mice; *P<0.05. TNF-tg also have a significantly higher Lm compared with TNF-tg × TNFR2−/−. However, there is no difference in Lm between TNF-tg × TNFR1−/− and TNFR1−/−. Each group consisted of eight mice. (B) P–V curves from mice. The P–V curve documented hyperinflation of the lung, consistent with the macroscopic findings and histology. Emphysematous changes in the mice are as follows: TNF-tg > TNF-tg × TNFR2−/−. all other mice. The curve indicates the expiratory phase of the P–V curve. Each group consisted of three to four mice. *P<0.05.
Abbreviations: Lm, mean linear intercept; NS, no significance; P–V, pressure–volume; tg, transgene; TNF-α, tumor necrosis factor-α; TNF-tg, TNF-α transgene; TNFR, TNF receptor.

Figure 5 Analysis of BAL fluids.

Notes: (A) The cell counts were determined in the BAL fluids from mice. A substantial neutrophil accumulation was observed in the mice. This accumulation was significantly smaller in the TNF-tg and TNF-tg × TNFR2−/− than in the wild-type mice (C57BL/6) and TNF-tg × TNFR1−/−. Each group consisted of eight mice. *A significant difference compared with the wild-type mice (C57BL/6). (B) BAL fluids from mice were analyzed by gelatin zymography. Gelatinolytic activity (clear bands) was observed in BAL fluids from TNF-tg and TNF-tg × TNFR2−/− but not in wild-type mice (C57BL/6), TNFR1−/−, TNF-tg × TNFR1−/−, and TNFR2−/−. This figure represents the data from one of the three experiments.
Abbreviations: BAL, bronchoalveolar lavage; TNF-α, tumor necrosis factor-α; TNF-tg, TNF-α transgene; TNFR, TNF receptor.

Figure 6 Apoptosis in COPD pathogenesis.

Notes: (A) There were no signals in wild-type mice. (B) TUNEL staining revealed positive signals in the lung from TNF-tg mice as indicated by arrows. This figure represents data from one of two experiments. (C) Electron microscopy showed that apoptotic cells were phagocytosed by macrophages, as indicated the arrow. Magnification ×80.
Abbreviations: TNF-α, tumor necrosis factor-α; TNF-tg, TNF-α transgene; TUNEL, Tdt nick-end labeling.