Effects of acute cigarette smoke exposure on macrophage kinetics and release of tumour necrosis factor α in rats

Some biological parameters before and after an acute episode of cigarette smoking in rats have been evaluated. The carboxyhaemoglobin levels depended either on the number of cigarettes, or on the time of exposure to cigarette smoke and returned to pre-smoking values in about 2 h. The evaluation of the kinetics of alveolar and peritoneal macrophages in rats after a smoking session of three cigarettes within an hour, indicated that alveolar macrophages in the bronchoalveolar lavage fluid significantly increased 8 h after the smoking, whereas the number of peritoneal macrophages remained practically constant. The incubation of these cells for various times at 37°C in a humidified atmosphere, resulted in a spontaneous release, 24 h thereafter, of variable amounts of tumour necrosis factor α (TNFα), which remained practically constant during the following days. Neither alveolar macrophages of control rats, nor peritoneal macrophages of both control and smoking rats were able to release TNFα. Moreover, after lipopolysaccharide induction of alveolar macrophages of both control and smoking rats, an increased release of TNFα was observed, indicating that these cells were in an active state.


Introduction
Cigarette smoking is a useful human model of chronic inflammation and it is well known that inflammation may contribute to the destruction and remodelling of normal lung architecture. Cigarette smoking, exposing the surface of the lower respiratory tract to more than 4000 chemical constituents, 2 has been associated with an inflammatory process. 3 Moreover, inflammation leads to an emigration, either of monocytes or polymorphonuclear cells, from the vasculature to the tissue. Alveolar macrophages, which are derived from blood monocytes, exert their inflammatory activities at the various sites either directly, or through the action of tumour necrosis factor (TNF) and other immunological mediators. 4 TNFz is chemotactic for monocytes and polymorphonucleate cells (PMN), stimulates phagocytosis, causes adherence to endothelium and production of oxygen-derived metabolites, induces procoagulant activities in cultured human endothelial cells, and activates macrophages resulting in interleukin-1 and prostaglandin E2 production. [4][5][6] Thus the locally regulated generation of TNFz at sites of injury represents an important autocrine-paracrine control mechanism which operates in acute inflammatory response. 4 Evaluation of the acute effect of cigarette smoking in human non-smokers is uncertain because they do not inhale cigarette smoke deeply.
(C) 1993 Rapid Communications of Oxford Ltd For this reason research has been carried out to study either the acute effect of cigarette smoking in human smokers, or in small animals, or the effect of cigarette smoke in blood cells in vitro. 9'1 Assuming that the activation of macrophages is an important event for the pleiotropic effects of these cells, the present study was undertaken to investigate some biological parameters after acute tobacco smoking and the ability of cigarette smoke to activate rat alveolar macrophages, thus causing the release of TNFo. We also examined whether the release of TNF by alveolar macrophages of rats exposed to an episode of acute, passive cigarette smoking, would be influenced by the presence of lipopolysaccharide (LPS).

Materials and Methods
Animals: Outbred Wistar male rats (Charles River) of about 280 g body weight were used throughout the experiments. Groups of six rats were each lodged in the smoke chamber for various periods of time as indicated in Table 1 Five washes of 10 ml each were carried out in rapid succession and the lavage fluids were pooled. Recovery was about 90%. Lavage fluid was then centrifuged at 300 g for 10min at 4C and the sedimented cells shown to be macrophages (more than 95%) by nonspecific esterase staining. Separated macrophages were tested for viability by the Trypan blue exclusion method and more than 95% of cells were found to be viable. The number of macrophages was evaluated by at least three countings in a Biirker chamber.
Control animals (air-sham exposed rats) were subjected to the same procedure except that the chamber was insulated with air only. Cell culture" For the experiments evaluating the eventual production of TNF0, cells from BAL and PF, collected immediately after (0 h) and 4, 8 and 24 h after the smoking session of three cigarettes for 1 h, were diluted to the desired final concentration (106 cells/ml) with RPMI 1640 containing 10% heat-inactivated foetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin G and 100 #g/ml streptomycin. One hundred microlitres of the cell suspensions (about 105 cells) were added to each well in 96-well culture plates (Costar) and were incubated for 24,48 and 72 h at 37C in a humidified atmosphere (95% air/5% CO2). A second batch of cells, collected 0, 8 and 24 h after the smoking session and layered as above, was challenged with 1/g/ml bacterial lipopolysaccharide (LPS) from Escherichia coli (Sigma)and incubated for 24 h at 37C. Then the plates were centrifuged and supernatants were stored at -80C until TNF was determined.
Sediments, after two washings with normal saline, were dissolved in 0.1 N NaOH (0.1 ml per well, 12 h at room temperature) and proteins were measured by the method of Lowry et al. 11

Results
Because of the lack of a readily and simply measurable marker of environmental tobacco smoke, COHb levels were assessed as a reliable indicator of the biological eflects of cigarette smoking. Normally in non-smokers, the average of basal COHb levels was --1 +__ 1.1 and, as shown in Table 1, COHb levels critically depended on both the number of cigarettes and the time of exposure to cigarette smoke. The decay of COHb in the blood of the rats was also evaluated after they had smoked three cigarettes for 1 h and after they were returned to the normal environment. As shown in Fig. 1 the COHb reached the maximum at the end of the smoking session but in about 2 h returned to pre-smoking values. The evaluation of the kinetics of alveolar and peritoneal macrophages was carried out in rats that, after undergoing smoking (three cigarettes within a hour), showed a COHb increase up to 16.4%. Figure 2 (panel A) indicates that the total number of macrophages Cigarette smoke exposure in rats present in the bronchoalveolar lavage significantly increased 8 h after smoking (U _< 0.05) in respect to control rats (Fig. 2, panel B). On the other hand the number of peritoneal macrophages remained practically constant in both smokers and controls ( Fig. 2, panels A and B). An increase of polymorphonuclear cells (PMN) in the bronchoalveolar lavage was also observed at 8 and 24 h after an acute smoking episode but it was not statistically significant (data not shown).  The appraisal of the kinetics of alveolar macrophages after acute smoking compelled us to evaluate the eventual activity of these cells by measuring the release of TNF0. As shown in Table  2, alveolar macrophages, collected at various times after acute smoking and incubated for 24, 48 and 72h at 37C in a humidified atmosphere, spontaneously released variable amounts of TNF0, which were higher 8 h after the smoking session. Moreover TNF0 production increased up to 24 h and varied little in the following days of incubation ( Table 2). Neither alveolar macrophages of air-sham exposed rats, nor peritoneal macrophages of both control and smoking rats were able to release TNF0, which was also absent either in the supernatants of BAL and PL or in plasma. When alveolar macrophages of control and smoking rats, collected 0, 8 and 24 h after the smoking session and incubated for 24 h at 37C, were challenged with LPS, they released much more TNF0 (Fig. 3) but, in such a case, the TNF0 release was significantly lower for the smoking group with respect to the control rats.

Discussion
These studies provide data on the short-term effects of acute cigarette smoking in rats. It is known that cigarette smoking in rabbits and in man causes a  macrophages, collected after an acute smoking episode, were in an active state and spontaneously released TNF. The absence of this cytokine in the BAL could be due to both the dilution and the short time for the cell collection. Additionally, the absence of TNFo in plasma could be explained by both the dilution in body fluids and the rapid clearance of this substance. 2'21 At a later date the eventual local catabolism of TNFo will be reported. Peritoneal macrophages, representing a resident population remote from the primary site of smoke and perhaps not having direct contact with smoke pollutants and products, were unable to spontaneously release TNFo. This clearly indicates that these cells were in a resting state. However, differences in regulation of TNF production by either human, 22 or murine 23 alveolar macrophages stimulated with LPS are well known. Finally we wanted to ascertain whether a classical inducer such as LPS antagonizes or synergizes with the eect of smoke on alveolar macrophages. It was found that alveolar macrophages collected from control rats released much more TNFo than those collected from smokers, probably because of a negative feed-back and enhanced breakdown due to proteinases released during the incubation period. 24