Chronic cigarette smoking enhances spontaneous release of tumour necrosis factor-α from alveolar macrophages of rats

Some biological effects of chronic cigarette smoking (two cigarettes for 2 h, daily for 4 months) in rats were evaluated. During the smoking period, body weight of smoker rats was always significantly lower than that of control rats. Immediately after the last smoking session the carboxyhaemoglobin concentration in the blood was about 8.5% and the polymorphonuclear cells in the bronchoalveolar fluid increased significantly. At the same time, enzymatic analyses on the supernatants of bronchoalveolar fluid revealed a significant increase of β-glucuronidase in the smoker group. Alveolar macrophages, collected 0, 8 and 24 h after the last smoking session, significantly increased the generation of superoxide anion and, after incubation for 24 h at 37° C in a humidified atmosphere, released significantly high amounts of TNF-α. When challenged with lipopolysaccharide, alveolar macrophages of smoker rats released much more TNF-α but, in such a case, TNF-α release was about one half of that observed in the control group. Peritoneal macrophages of both control and smoker rats were unable either to generate high levels of superoxide anion or to release significant amounts of TNF-α. The results clearly demonstrated the activated state of alveolar macrophages and the resting state of peritoneal macrophages.


Introduction
The lung represents a frequent site of inflammation because of its direct exposure to noxious agents, antigenic materials and invasion by microorganisms. Cigarette smoking, exposing the surface of the lower respiratory tract to more than 4000 chemical constituents such as polynuclear aromatic hydrocarbons and tobacco specific nitrosamines, has been associated with inflammatory processes. 2 The development of pulmonary inflammation is known to involve cellular events controlled by a variety of cytokines. These mediate interactions between macrophages (Mb), lymphocytes and granulocytes and represent an important event in the regulation of airway inflammation. 3 '4 In fact there is evidence that alveolar MqS, which are derived from blood monocytes, may mediate many of the harmful effects of smoking, exerting their activity at the inflammatory sites partly directly and partly through the action of tumour necrosis factor (TNF) and other immunological mediators. Moreover interactions between TNF and other cytokines are of pivotal importance in the mediation of the activation state of Mb. 6 TNF-o is a protein with a wide range of biological and inflammatory activities. The pleiotropic nature of its action is due to the fact that TNF receptors are present on virtually all cells examined and TNF action leads to the activation of a large array of cellular genes and of multiple signal transduction pathways. 7 Moreover TNF is chemotactic for monocytes and polymorphonuclear cells (PMN), stimulates phagocytosis, adherence to endothelium and superoxide anion production by these cells, induces procoagulant activity and activates Mb resulting in interleukin-1 and prostaglandin-E2 production. Thus, the locally regulated generation of TNF<z at the sites of injury represents an important autocrine-paracrine control mechanism operative in the inflammatory response.
Despite Control animals (air-sham exposed rats) were subjected to the same procedure except that the chamber was insufl:lated with air only.
Cell isolation and culture" The recovered BAL and PF were centrifuged at 300 x g, for 10 min at 4C; supernatants were collected and stored at -80C; cells were washed twice in cold RPMI 1640 medium (Gibco) and resuspended in a small volume of the same medium. After counting in a Biirker chamber, cells were diluted to the desired final concentration (106 viable cells/ml) with LPS-free RPMI 1640 containing 10% of heat-inactivated (56C for 60 min) foetal calf serum (FCS), 2 mM L-glutamine, 100 U/ml penicillin G and 100/ig/ml streptomycin.
The sedimented cells were proved to be macrophages (about 80% in the smokers) by nonspecific esterase staining. Separated Mb were tested for viability by blue exclusion method and more than 95% were found to be viable. Macrophages were cultured at a final volume of 0.2 ml per well in 96-well culture plates (Costar) and incubated for 24 h at 37C in a humidified atmosphere (95% air: 5% COg). This period of time was chosen after preliminary experiments had shown that the highest TNF-0 concentration was obtained after 24 h. 9 Plates were then centrifuged at 300 g" for 10 min at 4C, supernatants were collected and stored at -80C until TNF and IFN determinations. To verify cell responsiveness other lots of Mb suspension were challenged with 1/ig/ml bacterial lipopolysaccharide (LPS) from Escherichia coli (Sigma) and treated as above.
Proteins were measured directly in the wells using the method of Lowry eta/., 1 after two washings with saline and dissolving sedimented cells with 0.1 ml of 0.1 N NaOH for 12 h at room temperature. All media used were proved negative for endotoxin contamination by using the Limulus amoebocyte lysate gel test. Because the exposure of macrophages to endotoxin during the collection procedure cannot be absolutely excluded, supernatants of either BAL or PF were tested for endotoxin contamination and proved to be negative.
COHb levels: Blood COHb levels were estimated as an indicator for the biological effects of cigarette smoking. In order to check whether the smoke exposure was constant for all animals, the change of COHb in the blood of rats before and immediately after the smoking session was measured by a spectrophotometric method. 11 Cytokine determination: Supernatants were titrated for antiviral activity (AA) with a microplaque reduction assay 12  Because some subclones of L929 cells have been shown to be sensitive to either other cytokines or cytotoxic agents, in order to compare TNF-0 biological activity with the TNF-o antigen really present in our samples, we performed some determinations using both biological assay and a commercial enzyme-linked immunosorbent assay (TNF-ALPHA-EASIA, Medgenix Diagnostics, Belgium), which showed no cross-reaction with other cytokines such as IFN and interleukins.
Other chemical and enzymatic determinations: Superoxide anion generation by alveolar Mq was measured as the superoxide dismutase-inhibitable reduction of ferricytochrome c in presence and absence of zymosan. 14 Results were expressed as nM/105 cells.
Lactate dehydrogenase and fl-glucuronidase were measured in the supernatants of BAL and PF by colorimetric kits (Sigma) and the results were reported as U/ml. Statistical anasis" Evaluation of experimental data was performed using two-tail:ed Student's t-test with p < 0.05 as the minimal level of significance.

Results
Body weight measured monthly during the 4 months of experiments was each time significantly lower (p < 0.001) in the smoker group than in air-sham exposed rats (Fig. 1).
After the last smoking session the blood COHb concentration in smoker rats was 8.5 -t-1.2% (Table  1). The total recovery of alveolar Mq from BAL remained practically constant in both smoker and control rats. In contrast, PMN in the BAL of smoker rats significantly increased in respect to air-sham exposed rats and, at the same time, peritoneal Mq in the PF decreased (Table 1).
When alveolar M were collected immediately after the last smoking session (0 h) and 8 (Table 2).
Enzymatic analyses of BAL of smokers revealed a significant increase of /-glucuronidase and a decrease of lactate dehydrogenase, but both remained practically unvaried in the PF ( Table 3).
As the results were consistent with a state of metabolic activation of the alveolar M population recovered from chronic smoke-exposed rats, we investigated whether these cells release TNF-o. and incubated for 24 h at 37C in a humidified atmosphere, spontaneously released 77 -t-28, 80 -t- 23 and 84-t-34 units of TNF-0 per 106 cells respectively, these values being significantly higher than those obtained after incubation of alveolar collected from sham-exposed control rats. Moreover alveolar Mq obtained from BAL of both smoker and control rats challenged with 1/,g/ml of LPS and incubated Cas above, released much more TNF-, but in such cases, TNFproduction by alveolar M of the smoker group was significantly decreased to about one half of that of the control group (Fig. 2, top panel). Immunoenzymatic determinations on samples, the biological activity of which is reported in Fig. 2, gave practically the same TNF-0 units per 106 cells (data not shown).  *p < 0.5 (n 6), **p < 0.01 (n 6) and ***p < 0.001 (n 18), in respect to control values. Alveolar M collected from both smoker and control rats were unable to release any antiviral activity and, either TNF-, or IFN were also absent in the supernatants of BAL and PF and in plasma (data not shown). Instead peritoneal Mq of both smoker and control rats released, either spontaneously, or after LPS induction, little and extremely variable amounts of TNF-0 and IFN (Table 4). We attempted to correlate individual ratios of TNF-0 released by alveolar Mq compared with TNF-0 released by peritoneal Mq5 of both control and smoker rats. As shown in Table 5 ratios obtained from smokers were always higher than those obtained from controls although extremely variable.

Discussion
Some biological effects of chronic smoke in rats have been evaluated. During the 4 months of the smoking period the body-weight increments were always lower than those of control rats. Although the cigarette smoking effects in lowering the body-weight are well known, 15'16 little attention has been devoted to studying the effect of chronic smoke on the metabolic rate in rats where sympathoadrenal activation by nicotine appears to be primarily responsible for the metabolic eects of smoking. 17 Smoking rats were lodged in the smoking chamber daily for 2 h, during which time they 426 Mediators of Inflammation Vol 2.1993 breathed the smoke of two cigarettes. Evaluation of COHb blood levels, immediately after the last smoking session, gave values of about 8.5% which compared well with those observed in guinea pig and in human cigarette smokers. 18J9 The total number of alveolar M4 collected from BAL of chronic smoker rats was unchanged compared with that of control rats or acute smoker rats, but PMN increased significantly in the BAL of smoker rats. Previous work by Costabel and Guzman 2 and others 2'2'22 clearly shows a constant increase of PMN in the BAL after smoking, probably due to the action of chemotactic factors 23'24 produced by both alveolar and pulmonary cells.
The major aim of the investigation reported here was to determine whether resident alveolar Mq of chronic smoker rats produced TNF-0. The present results clearly demonstrated that alveolar Mq5 collected immediately after (0 h) and 8 and 24 h following the last smoking session and incubated for 24 h at 37C spontaneously released significant and constant amounts of TNF-0. Immunoenzymatic analyses on these samples gave about the same values, thus assuring the identity of the cytokine. We are unable to assess which substance stimulated the release of TNF-0 and at least IFN-25 can be excluded, because we were unable to detect any antiviral activity in our samples. TNF-z and IFN were also absent in the supernatants of BAle and PL, but this is probably due to the dilution and short time of contact of these fluids with the cells. Moreover the absence of TNF-0 in the plasma could be due to either cytokine inactivation during the transalveolar passage or the dilution in body fluids and the rapid clearance from plasma. 26'2v Alveolar Mq collected from both control and smoker rats were extremely sensitive to low LPS concentrations but, in the last case, TNF-0 release was about one-half of that observed in the control group, in agreement with results of others obtained in the mouse. 28 Peritoneal Mq of the control and smoker group, which represent a resident population without any direct contact with air pollutants and smoke products, released small amounts of TNF-o and IFN either spontaneously or after challenge with LPS. This is also clarified by the analysis of individual ratios of alveolar TNF-cz versus peritoneal TNF-o released various time after the last smoking session evaluated in both control and smoker rats. The high variability of the ratios is probably due to the scarce sensitivity of our bioassay especially when the units of TNF-o were below 5. Taken together, the results suggest that peritoneal Mq were in a resting state and alveolar Mq were primed, based on the fact that only primed Mq are able to release massive amounts of TNF-o after stimulation with LPS. 29'3 Moreover it is known 29 that at least two pathways govern the release of TNF-, one is LPS specific and the other phagocytic trigger specific, suggesting that the priming effect for the TNF-o release is variable and probably related to a particular triggering stimulus.
The activated state of alveolar Mq5 of chronic smoker rats, due to either smoke products and pollutants, 31'2 or a self-priming effect of TNF->-s is also suggested by the significant increase of superoxide anion generation and of fl-glucuronidase. At the same time lactate dehydrogenase in the BAL of smoker rats, which represents a cytoplasmic marker employed as a measure of the cell lysis, remained unchanged or only slightly decreased compared with the control group. This suggests that the increase of the fl-glucuronidase in the BAL was not due to a lytic effect on the cells, but to a secretagogue effect on alveolar Mb and PMN probably exerted by smoke products and/or TNFitself. 35