American Journal of Respiratory and Critical Care Medicine

The role of tumor necrosis factor-α (TNF-α) as a mediator of cigarette smoke–induced disease is controversial. We exposed mice with knocked-out p55/p75 TNF-α receptors (TNF-α–RKO mice) to cigarette smoke and compared them with control mice. Two hours after smoke exposure, increases in gene expression of TNF-α, neutrophil chemoattractant, macrophage inflammatory protein-2, and macrophage chemoattractant, protein-1 were seen in control mice. By 6 hours, TNF-α, macrophage inflammatory protein-2, and macrophage chemoattractant protein-1 gene expression levels had returned to control values in control mice and stayed at control values through 24 hours. In TNF-α–RKO mice, no changes in gene expression of these mediators were seen at any time. At 24 hours, control mice demonstrated increases in lavage neutrophils, macrophages, desmosine (a measure of elastin breakdown), and hydroxyproline (a measure of collagen breakdown), whereas TNF-α–RKO mice did not. In separate experiments, pure strain 129 mice, which produce low levels of TNF-α, showed no inflammatory response to smoke at 24 hours or 7 days. We conclude that TNF-α is central to acute smoke-induced inflammation and resulting connective tissue breakdown, the precursor of emphysema. The findings support the idea that TNF-α promoter polymorphisms may be of importance in determining who develops smoke-induced chronic obstructive pulmonary disease.

Studies have suggested that polymorphisms in the human tumor necrosis factor-α (TNF-α) promoter at position −308 are associated with the presence of cigarette smoke–induced chronic obstructive pulmonary disease (COPD) and chronic bronchitis (1, 2) or the rate of progression of COPD (3). However, this finding is controversial and other groups have denied that TNF-α polymorphisms influence the appearance of COPD (46).

Attempts to examine the effects of smoke in causing TNF-α production are similarly inconsistent. Kuschner and coworkers (7) reported that human smokers had higher levels of lavage TNF-α than nonsmokers, but Keatings and coworkers (8), examining induced sputum, observed elevated TNF-α levels only in smokers with COPD and not in asymptomatic smokers. Takabatake and coworkers (9) found that patients with COPD who were chronically hypoxic had elevated TNF-α levels; they suggested that chronic hypoxemia results in increased TNF-α levels and weight loss, although it is also possible that the reverse is true and elevated TNF-α levels drive the inflammatory process and make COPD worse. The same group (10) found that smokers with COPD had higher serum TNF-α levels than healthy nonsmoking control subjects; however, the difference was only approximately 20%. Bresser and coworkers (11) noted that patients chronically infected with Haemophilus influenzae and who had chronic bronchitis and COPD had higher TNF-α levels than similar chronic bronchitis patients without COPD. But other reports claim that smoke either suppresses TNF-α production by lavaged or cultured alveolar macrophages and peripheral blood monocytes of humans and animals (1216), or has no effect at all (17).

The idea that cytokine mediators could be important in the development of COPD is supported by a study by Lucey and coworkers (18). They found that porcine pancreatic elastase-induced emphysema is considerably ameliorated in mice that have knocked-out TNF-α receptors (TNF-α–RKO mice) or interleukin (IL)-1β receptors. They suggested that elastase-induced emphysema might be driven in large measure through ongoing TNF-α and IL-1β–induced inflammation, and possibly also through TNF-α and IL-1β–mediated inhibition of elastin and collagen repair.

TNF-α is central to induction of inflammatory infiltrates in a variety of pulmonary diseases (see Discussion), and because cigarette smoke typically causes both acute and chronic inflammation (7, 19), the failure to show a consistent role for TNF-α, or even upregulation of TNF-α production, in cigarette smoke–induced disease is surprising. In this study we have further examined this process by using genetically altered mice that either lack TNF-α receptors or that produce only low levels of TNF-α (strain 129J mice) (20).

Sources of Materials

Mice with knocked-out p55 and p75 TNF-α receptors (TNF-α–RKO) were obtained from Immunex (Seattle, WA). The original mice were created in strain 129 stock and were backcrossed for five generations into C57BL/6 stock. C57BL/6 mice are known to react to cigarette smoke with a rapid inflammatory infiltrate (19). As a comparison group, strain 129J mice (obtained from Charles River Laboratories, Montreal, PQ, Canada) were backcrossed for five generations into C57BL/6 stock. These backcrossed animals are referred to as “control” in this article. 2R1 research cigarettes were obtained from the University of Kentucky (Lexington, KY).

Smoke Exposure and Lavage Procedures

Experimental groups consisted of five mice. The mice were exposed to the whole smoke from four whole 2R1 cigarettes, using a standard smoking apparatus (described by us elsewhere [19]). Control mice were sham smoked. Twenty-four hours after smoke exposure, mice were killed by halothane overdose and the lungs were removed from the chest cavity. A 20-gauge catheter was inserted into the trachea and the lungs were lavaged six times with 1 ml of ice-cold saline for cell counts, or with distilled water for connective tissue degradation analysis. Water is used because the concentration of salts during sample preparation for the high-performance liquid chromatography (HPLC) procedure interferes with the analysis of desmosine (DES). Separate sets of animals were used for these experiments.

For inflammatory cell measurements, the saline lavage was centrifuged at 200 × g at 4°C for 10 minutes. The supernatants were decanted and the cell pellets were resuspended in 200 μl of saline. Total cell counts were performed in a hemacytometer and differential cell counts were performed on a 10-μl drop of the cell suspension heat fixed on a slide and stained with hematoxylin and eosin.

Hydroxyproline and DES Analyses

Hydroxyproline (HP) and DES analyses were performed as previously described, using the water lavage samples (21).

Expression of Inflammatory Mediators by Reverse Transcriptase-Polymerase Chain Reaction

In separate experiments groups of three mice were treated as described above and RNA was extracted from whole lung by the method of Chomczynski and Sacchi (22). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed as previously described (23), using the following primers: sense GAPDH (glyceraldehyde-3-phosphate dehydrogenase; 5′-CGG ATT TGG CCG TAT TGG GC) and antisense GAPDH (3′-TGA TGG CAT GCA CTG TGG TC) (Davis and coworkers [24]); sense TNF-α (5′-CCT CTC ATC AGT TCT ATG GC) and antisense TNF-α (3′-TCA CAG AGC AAA GAC TCC AA) (Davis and coworkers [24]); sense macrophage inflammatory protein-2 (MIP-2; 5′-GGC ACA TCA GGT ACG ATC CAG) and antisense MIP-2 (3′-ACC CTG CCA AGG GTT GAC TTC) (Zhao and coworkers [25]); and sense macrophage chemoattractant protein-1 (MCP-1; 5′-GCC CAG GAC CAG CAC CAG) and antisense MCP-1 (3′-GGC ATC ACA TGC CGA GTC ACA C) (Zhao and coworkers [25]).

Statistical Analysis

Groups were compared by analysis of variance. Values of p ⩽ 0.05 were considered significant.

Figure 1

shows gene expression of TNF-α, MIP-2, and MCP-1 in control and TNF-α–RKO mice 2 hours after initial cigarette smoke exposure. There was a small increase in TNF-α gene expression, a marked increase in MIP-2 expression, and an approximate doubling of MCP-1 expression in the control mice exposed to smoke compared with those sham smoked, whereas the TNF-α–RKO mice exposed to smoke showed no increases in any of these gene products and there was, in fact, a small decrease in TNF-α expression. By 6 hours TNF-α, MIP-2, and MCP-1 expression levels had returned to baseline values in the control mice and the 129 mice still showed no effects (Figure 2) . At 24 hours there were no elevations in gene expression in either strain (data not shown).

Figure 3

shows lavage inflammatory cells and connective tissue breakdown products at 24 hours in control and TNF-α–RKO mice. In the control mice, a marked increase was seen in lavage neutrophils and a smaller increase was seen in lavage macrophages. Similarly, there was a significant increase in lavage desmosine and hydroxyproline in these animals. TNF-α–RKO mice were protected from all these effects.

Figure 4

shows a separate experiment using control and pure strain 129 mice. The control mice again demonstrated elevations in lavage neutrophils and macrophages 24 hours after smoke, but no elevation was seen in 129 mice. Similarly, with daily exposure to smoke for 7 days there was again a persisting increase in lavage neutrophils and macrophages in the control animals and no response in the 129 mice.

TNF-α is a powerful proinflammatory cytokine that is a key mediator of inflammation, and also plays an important role in host defense against a variety of fungal, bacterial, and viral pathogens. TNF-α operates by binding to two different cell surface receptors, p55 and p75. The p55 receptor appears to be responsible for activating inflammatory responses and host defense. The role of p75 is less clear, but there is evidence that it functions to modulate TNF-α–mediated inflammation (26, 27).

TNF-α functions in several different ways regarding inflammation. Endothelial cells exposed to TNF-α upregulate a variety of surface adhesion molecules such as intercellular adhesion molecule-1 and selectins, which cause neutrophil and monocyte adhesion and eventual extravascular migration. TNF-α upregulates production of IL-6, a cytokine that causes hepatic production of acute phase proteins. In addition, TNF-α activates macrophages and epithelial and mesenchymal cells to produce various inflammatory cell chemoattractants such as IL-8 (murine MIP-2), MCP-1, and leukotriene B4 (26, 27).

Less is known about the role of TNF-α in inducing specific diseases. TNF-α is clearly important in fibrogenesis. Administration of anti–TNF-α antibodies protects mice against silica-induced or bleomycin-induced fibrosis (28, 29). Mice with knocked-out TNF-α receptors are similarly protected against the fibrogenic effects of bleomycin (30) and also against the fibrogenic effects of asbestos (31). Brass and coworkers (20) found that strain 129 mice also failed to develop asbestos-related disease, and noted that these mice produced little TNF-α. Further studies of strain 129 and TNF-α–RKO mice suggest they are protected against fibrogenic effects because one role of TNF-α in this setting is upregulation of production of the fibrogenic cytokine transforming growth factor-β1 in epithelial and, probably, mesenchymal cells (3234). It is interesting in this regard that TNF-α polymorphisms that cause higher levels of TNF-α production are associated with increased incidence/severity of both berylliosis and silicosis (35, 36), supporting the importance of TNF-α in fibrotic diseases in humans.

The controversies surrounding the potential role of TNF-α in cigarette smoke–induced disease have been described above. We have examined this question in two different ways: by using TNF-α–RKO mice lacking functional TNF-α receptors, and strain 129 mice, which, as low-level TNF-α producers, are, in effect, a model of humans with a low TNF-α–producing gene polymorphism. Our conclusion from both approaches is that TNF-α is crucial to at least the acute inflammatory responses evoked by smoke.

In the present experiments, not only were neutrophil numbers and gene expression of the neutrophil chemoattractant MIP-2 elevated, but macrophage numbers and gene expression of the macrophage chemoattractant MCP-1 were elevated in control animals exposed to smoke as well. We have found that, in our smoke model, elevations in macrophage numbers are much more variable than elevations in neutrophil numbers, and no elevations in macrophage numbers are usually observed if a smaller number of cigarettes is used than was employed in this study (19). The current experiments suggest that TNF-α is also important in the development of smoke-induced macrophage influxes.

Our data show a potential mechanism whereby TNF-α plays a role in smoke-induced disease. The protease–antiprotease hypothesis states that emphysema develops in cigarette smokers as a result of smoke-induced inflammatory cell influx into the lung and the release of inflammatory cell-derived proteolytic enzymes, which lead to connective tissue breakdown and eventual emphysema (19, 37). Although the exact cells and proteases responsible for this process are a matter of controversy, we have previously shown that there is a good correlation between neutrophil numbers and measures of connective tissue breakdown, both in animals exposed to cigarette smoke (19) and animals exposed to silica, a powerful neutrophil inducer (38). The present study supports this idea; as shown in Figure 4, connective tissue breakdown is seen in control mice, the strain that shows a neutrophil influx after smoke exposure, but is not found in TNF-α–RKO mice.

These results need to be interpreted with caution, because the experiments performed here are short-term, and the exact correlations of acute connective tissue breakdown and long-term appearance of emphysema are not known. But the data of Lucey and coworkers (18) do suggest that long-term elevations in TNF-α can be associated with emphysema under the correct conditions. As well, Fujita and coworkers (39) reported that transgenic mice overexpressing TNF-α under the control of the SpC promoter developed chronic inflammation and alveolar airspace enlargement, along with physiologic evidence of increased lung volumes and loss of elastic recoil. These findings also support the idea that persistent production of TNF-α by chronic exposure to cigarette smoke may be related to the development of emphysema.

Our findings thus emphasize the importance of TNF-α in cigarette smoke disease and support the idea that TNF-α polymorphisms may be important in determining which smokers actually develop disease.

The authors thank Immunex Corporation and Dr. J. Peschon for supplying the TNF-α receptor knockout mice.

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Correspondence and requests for reprints should be addressed to Andrew Churg, M.D., Department of Pathology, University of British Columbia, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 2B5. E-mail:


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