Introduction

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Increased levels of cytokines are a hallmark of inflammatory diseases. With respect to lung disorders, cytokines appear to play particularly prominent roles in the pathogenesis of adult respiratory distress syndrome and asthma. In the lungs, cytokines not only orchestrate and perpetuate the inflammatory response, but may also cause functional alterations such as bronchoconstriction and airway hyperresponsiveness. However, because most studies on cytokines have focused on their expression and release rather than on their effects on lung functions, we know a lot about the formation but relatively little about the functional consequences of cytokines.

Three of the most important proinflammatory cytokines are tumor necrosis factor (TNF)-, interleukin (IL)- 1, and interferon (IFN)-. There are, however, only few studies on their effects on lung functions. In sheep, infusion of human recombinant (hr) TNF caused bronchoconstriction within 30 min (1, 2). TNF- also appears to play a prominent role in airway hyperresponsiveness, as suggested by the finding that anti-TNF- antibodies blocked lipopolysaccharide (LPS)-induced airway hyperresponsiveness in rats (3). These findings may apply to humans, where inhaled rTNF- caused decreased forced expiratory volume at 1 s as well as airway hyperresponsiveness (4). IFN- exacerbated airway hyperresponsiveness in mice (5) and in asthmatic patients (6). In contrast to TNF and IFN-, IL-1 has been shown in vitro to relax rather than to constrict airway smooth muscle (7).

Most of these studies have been performed in vivo. However, because most cytokines have profound actions on leukocytes, in vivo it is very difficult to differentiate between the direct effects of cytokines and the indirect effects by activated leukocytes. Such mechanistic information, however, will be mandatory to fully understand the effects of cytokines on lung functions.

The major disadvantages of most in vitro systems are the disintegration of lung tissue and the impossibility of measuring lung functions. Recently a new in vitro model, the precision-cut lung slice, has been developed (8). This model allows investigators to take living lung slices into culture, expose them to cytokines, and study their functional responses by videomicroscopy. In this system it is possible not only to study the effects of cytokines in a tissue with an intact microanatomy at the level of mediators and gene expression, but at the same time also to examine functional responses such as bronchoconstriction. In the present study we have used precision-cut lung slices to examine the effects of TNF, IFN-, and IL-1 on airway tone in the absence of blood-derived leukocytes.

	Material and Methods

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Animals

Lungs were taken from 8-wk-old female Wistar rats (220 ± 20 g) obtained from Harlan Winkelman GmbH (Borchen, Germany) and kept under controlled conditions (22°C, 55% humidity, 12 h day/night rhythm) on standard laboratory chow.

Preparation of Precision-Cut Lung Slices

Rat lungs were prepared and perfused essentially as described recently (9, 10). After preparation, the lungs were suspended by the trachea and ventilated by negative pressure with 80 breaths/min at a tidal volume of 2 ml. Lungs were perfused at constant hydrostatic pressure (12 cm H₂O) through the pulmonary artery with Krebs-Henseleit buffer (37°C, 2% albumin, 0.1% glucose, and 0.3% N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid [Hepes]) for 5 to 10 min until they were free of blood. The lungs were removed from the perfusion apparatus and were filled with 10 ml agarose solution (0.75% in Eagle's minimum essential medium [MEM], 44 ml/kg) and a bolus of 1 ml air (8). For instillation and incubation, MEM with sodium pyruvate, amino acids, vitamins, and Hepes was used. After cooling of the agarose to 4°C, tissue cores were prepared by advancing a rotating sharpened metal tube (8 mm in diameter). From these cores, tissue slices (250 ± 20 µm) were prepared by using a Krumdieck tissue slicer (Alabama Research and Development, Munford, AL). Lung slices were floated on Teflon mesh and cultured in glass vials containing 1 ml of MEM. The vials were placed on a roller system housed in a humidified incubator. They were incubated at 37°C in a humid atmosphere and rotated at 10 rpm. The medium was changed hourly for 4 h; after 4 h of washing and preincubation the experiments were started. Notably, the incubation of the precision-cut lung slices was performed in MEM without Phenol Red, because Phenol Red acts as an inhibitor of the thromboxane receptor (11).

Image Acquisition

The incubation chamber was placed on the stage of an inverted microscope (Zeiss Axiovert 35; Zeiss, Oberkochen, Germany) and warmed to 37°C. The slices were screened for airways and transferred to the incubation chamber. The airways were focused, imaged with a video camera (Kappa CF8RCC; Kappa, Gleichen, Germany), and digitized using a frame grabber board (CFG-KIT-C2 AT; Imaging Technology, Inc., Bedford, MA). A saved image of 2.8 mm² was represented by 512 × 768 pixels.

After preincubation for 10 min with 1 ml of MEM, the first image was acquired. The airway area obtained from this first image served as the reference area (100%). The liquid was removed and 1 ml of medium containing the cytokines was transferred into the incubation cell. Initially, the airways were imaged every minute for an hour at the beginning to determine the time course of bronchoconstriction; later they were imaged after 4 h of incubation. The cytokine concentrations used were rat rIL-1 10 ng/ml, rat rTNF- 10 ng/ml, and rat rIFN- ng/ml. The cytokines were obtained from R&D Systems (Wiesbaden-Nordenstadt, Germany).

Image Analysis

The images were analyzed by an image analysis program (OPTIMAS 6.0; Optimas Corp., Bothell, WA). The lumen area was taken as the area enclosed by the epithelial luminal border and was quantified after setting the appropriate threshold. Control airway area was defined as 100%.

Reverse Transcription/Polymerase Chain Reaction

Lung slices were incubated in MEM (control), in MEM plus cytomix plus dexamethasone (Dex) (10 mM), or MEM plus a cytokine mix. Six slices were removed from the incubator, transferred into Eppendorf cups, frozen immediately in liquid nitrogen, and stored at 70°C. The frozen slices were ultrathoraxed for 30 s in an RNA-extracting solution (RNA-Clean; AGS, Heidelberg, Germany). After centrifugation at 15,000 × g, the supernatant was transferred into a new cup. After extraction with chloroform and addition of isopropanol the solution was stored on ice for 15 min. The solution was centrifuged and the pellet was washed with 1 ml of ethanol solution (70%) and dissolved in ultrapure water. The RNA content was determined spectrometrically. A total of 4 µg of RNA were used for an unspecific RT (oligo-dt-primer) with Superscript reverse transcriptase (GIBCO, Eggenstein, Germany).

After removal of excess dt primers, polymerase chain reaction (PCR) was performed using the complementary DNA (cDNA) template with the following nested primer pairs: for cyclooxygenase (Cox)-1: PCOX1MR2 (5'-ACCCGTCATCTCCAGGGTAA-3'), PCOX1F1 (5'-CAGCCCTTCAATGAGTACCG-3'); for Cox-2: PCOX2MR2 (5'-ATCTAGTCTGGAGTGGGAGG-3'), PCOX2F1 (5'-AATGAGTACCGCAAACGCTT-3'); and for thromboxane synthase (TXS): PTXSRR1 (5'-CTCTCCTTCATCACATACCTGCTG-3'), PTXSRF1 (5'-GACGCATTCGACATC-CAGAGGTGT-3').

The reactions were cycled 35 times (30 s at 94°C), 30 s at 56°C, and 30 s at 72°C after a 5-min denaturation step at 95°C (Gen-Amp 9600; Perkin Elmer, Weiterstadt, Germany). Products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide. No amplification products were found without specific primer or with the PCR reaction lacking template. Samples were examined in various dilutions to ensure the proportionality in the yield of PCR products. The identity of the fragments was evaluated by their molecular mass and restriction enzyme analysis.

Measurement of Thromboxane Release: Enzyme Immunoassay

In each roller incubator, three slices were incubated for up to 24 h. After a preincubation of 4 h, the slices were incubated in presence and absence of arachidonic acid (AA). The slices were divided into three groups: control (with medium alone), cyto (incubation with cytokines), and Dex (incubation with cytokines and Dex). The medium of the slices was changed every 4 h and new medium with or without AA, cytokines, or Dex was added. The collected medium samples were gassed with nitrogen and frozen in liquid nitrogen. The samples were then stored at 80°C. Thromboxane release into the supernatant was assessed by measuring the stable metabolite thromboxane B₂ with a commercially available enzyme immunoassay (Cayman, Ann Arbor, MI).

Measurement of Peptido-Leukotriene Release: Enzyme Immunoassay

The slices were distributed into a 24-well plate (six slices per well). The medium was collected after 8 and 16 h, frozen, and stored as described earlier. Total amount of leukotriene was measured in the medium with a commercially available enzyme immunoassay (Cayman).

Statistics

Data are expressed as means ± standard deviation (SD). Data were analyzed by analysis of variance (ANOVA) followed by Student's t test. The  level was adjusted according to Shaffer's modified sequentially rejective multiple test procedure (12). In case of heteroscedasticity (F-test, P < 0.05), Welch's ANOVA (JMP; SAS Institute, Cary, NC) and Welch's t test were used. Percentage data were transformed by the arcsin transformation (13) before hypothesis testing. P < 0.05 was considered significant.

	Results

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Because in inflamed tissue in vivo usually a cytokine mixture rather than a single cytokine is found, we started by investigating such a mixture. Precision-cut lung slices were observed in an incubation chamber under the microscope during incubation with a mixture consisting of 10 ng/ml of each of the three rat recombinant cytokines, IL-1, TNF-, and IFN-. Incubation of the slices with this cytokine mixture resulted in a time-dependent contraction of single airways that started after 2 h and was maximal after 4 h (Figure 1).

View larger version (71K): [in this window] [in a new window]   Figure 1.   Cytokine-induced bronchoconstriction in rat lung slices. (A) The bronchus shown was treated with a mixture of cytokines (10 ng/ml IFN-, 10 ng/ml IL-1, and 10 ng/ml TNF-) for 4 h. Images were taken after 0, 1, 3, and 4 h of cytokine exposure. (B) The airway area shown in A is expressed as percentage of the initial airway area (time point 0 h).

Because previous studies have shown that TNF-mediated bronchoconstriction is at least partly mediated by thromboxane (2), we tested this hypothesis in our model. Initial airway area was determined after a preincubation period of 4 h. Slices were incubated for another 4 h in the presence or absence of the cytokine mixture. At this time, airway area in cytokine-treated samples was reduced to 67% of the initial area (P < 0.01 versus control). The thromboxane receptor antagonist SQ29548 inhibited bronchoconstriction completely (Figure 2). The glucocorticoid Dex, the unspecific Cox inhibitor indomethacin, and the specific Cox-2 inhibitor NS398 partially prevented the cytokine- induced bronchoconstriction (Figure 2). The 5-lipoxygenase inhibitor AA861 had no effect on cytokine induced bronchoconstriction. None of these inhibitors showed any effect on airway area in controls.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Cytokine-induced bronchoconstriction. Lung slices were treated with AA861 (10 µM), Dex (10 µM), indomethacin (Indo, 10 µM), NS398 (1 µM), or SQ29548 (10 µM) 10 min before exposure to the cytokine mixture (10 ng/ml IFN-, 10 ng/ml IL-1, and 10 ng/ml TNF-) for 4 h. Data are means ± SD; the number of independent experiments is shown inside the bars. The luminal area of cytokine-treated airways (second bar) was smaller than that of all other groups (P < 0.05). *P < 0.05 versus control; #P < 0.05 versus SQ29548/cytokine mixture.

Recently, it was shown that the bronchoconstriction by LPS, which is also thromboxane-mediated, depends on induction of Cox-2 (14). Therefore, and because NS398 was protective in our model, the expression of messenger RNA (mRNA) for Cox-1, Cox-2, and TXS in the lung slices was determined by RT-PCR. Cox-1 and TXS mRNA amounts were not altered by incubation with the cytokines or with Dex (Figure 3). However, Cox-2 mRNA was more strongly expressed in slices incubated with the cytokine mixture, a response which was blocked in the presence of Dex (Figure 3). Interestingly, we found that after 20 h of cytokine exposure the airways were still contracted, but this contraction was not affected by either Cox-inhibitors, lipoxygenase-inhibitors, or platelet-activating factor antagonists (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 3.   RT-PCR analysis of precision-cut lung slices after 4 h of exposure to a cytokine mixture. The cytokine mixture consisted of IFN-, IL-1, and TNF- at 10 ng/ml each. Slices were treated with medium (lane 1), a combination of cytokine mixture and Dex (lane 2), or the cytokine mixture alone (lane 3). The cDNA dilutions were as follows: Cox-2, 1:5; Cox-1, 1:1; TXS, 1:2; and -actin, 1:40.

The thromboxane B₂ concentrations in the supernatants of control slices changed little during the first 16 h of incubation, whereas in cytokine-treated slices they were already about 2-fold increased after 4 h (Figure 4A). At later time points, the thromboxane production in both control as well as cytokine-treated lung slices fell to about 50% of the initial values. To investigate whether this decrease in thromboxane might be explained by exhaustion of AA, the same experiments were repeated in the presence of 1 µM AA (Figure 4B). In these experiments the absolute thromboxane levels were higher than before, but the decrease in thromboxane formation occurred nevertheless. Lung slices that were treated with Dex and the cytokine mixture behaved similarly to controls (Figure 4). Dex, indomethacin, and NS398 all reduced the cytokine-induced thromboxane formation completely, i.e., to levels that were similar to those in the absence of the cytokine mixture (Figure 5). Interestingly, both Cox-inhibitors, but not Dex, reduced the basal thromboxane formation (Figure 5).

View larger version (23K): [in this window] [in a new window]   Figure 4.   Time course of thromboxane B2 release into the supernatant of lung slices exposed to cytokine mixture in the absence (A) and presence (B) of AA (1 µM). The cytokine mixture consisted of IFN-, IL-1, and TNF- at 10 ng/ml each. Slices were treated with medium (squares), the cytokine mixture (diamonds), or a combination of cytokine mixture and Dex (circles). Data are means ± SD; n = 3. The initial thromboxane B2 concentration at 0 h was 122 ± 30 pg/slice.

View larger version (14K): [in this window] [in a new window]   Figure 5.   Pharmacologic interventions against cytokine-induced thromboxane release. Slices were incubated with a mixture of cytokines, i.e., IFN-, IL-1, and TNF- at 10 ng/ml each. AA861 (10 µM), Dex (10 µM), indomethacin (Indo, 10 µM), or NS398 (1 µM) were added 10 min before the cytokine mixture. Data are means ± SD; the number of independent experiments is shown inside the bars. The thromboxane release in cytokine-treated airways (second bar) was larger than in all other groups (P < 0.05); the thromboxane release in all slices treated with a Cox inhibitor (Indo or NS398) was smaller than in all other groups (P < 0.05); *P < 0.05 versus control. The initial thromboxane B2 concentration at 0 h was 122 ± 30 pg/slice.

The amount of leukotrienes in the supernatant was very low under basal conditions (12.5 ± 2.6 pg per slice) and was not different if slices were treated with the cytokine mix (Figure 6). The leukotriene concentrations in slices obtained from lungs that were not perfused free of blood was the same. To be able to detect leukotrienes in the supernatant we needed at least six slices, compared with three slices in the case of thromboxane. It is notable that after an incubation for 4 h no leukotrienes were detectable. Spiking the supernatant of the slices with 1 ng of leukotrienes showed a recovery of nearly 100%.

View larger version (27K): [in this window] [in a new window]   Figure 6.   Production of peptido-leukotrienes in cytokine-treated and untreated lung slices. Lung slices were obtained from either perfused or unperfused rat lungs and were subsequently incubated for 8 h in the absence or presence of a cytokine mixture (IFN-, IL-1, and TNF-, 10 ng/ml each). Leukotrienes were determined in the supernatants. As a positive control, 1 ng of peptido-leukotriene standard was added to the supernatant of untreated lung slices.

To investigate the contribution of each single cytokine, i.e., TNF-, IL-1, and IFN-, to the bronchoconstriction induced by this mixture, the cytokines were incubated alone and in all possible combinations, and the airway responses were followed (Figure 7). As before, bronchoconstriction was measured after 4 h of incubation and compared with control slices incubated in MEM only. Treatment with any cytokine alone in a concentration of 10 ng/ml showed a weak, and in the case of IL-1, significant, increase in airway area to about 110%. The combinations IL-1/IFN- or TNF-/IFN- had no effect on airway area. However, contraction of airways was observed in the simultaneous presence of IL-1 and TNF, which resulted in 75% of initial airway area. The response obtained with IL-1/ TNF was similar to the bronchoconstriction seen in the presence of all three cytokines.

View larger version (42K): [in this window] [in a new window]   Figure 7.   Airway areas of lung slices treated with cytokines alone and in combination for 4 h. The cytokines were used at the following concentrations: 10 ng/ml IFN-, 10 ng/ml IL-1, and 10 ng/ml TNF-. Data are means ± SD; the number of independent experiments is shown inside the bars. *P < 0.05 versus control (no cytokines); #P < 0.05 versus the mixture consisting of all three cytokines.

	Discussion

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The present study demonstrates that IL-1 and TNF together cause bronchoconstriction in the absence of blood-derived leukocytes. The proposed mechanism is induction of Cox-2, formation of prostanoids such as thromboxane, and finally activation of thromboxane receptors on airway smooth-muscle cells. This result is particularly interesting in view of the data showing that each cytokine alonein line with a previous in vitro study (7)relaxed airway smooth muscle. However, because in vivo the two cytokines usually occur together, the action of TNF and IL-1 in vivo is probably contraction rather than relaxation of airways. Synergistic actions of IL-1 and TNF in vivo have been documented before, and interestingly, in that case also the response was blocked by Cox inhibitors (15). These findings suggest that cytokines may interact in a fashion that is impossible to predict by studying each cytokine alone.

Only few previous studies have analyzed the effects of cytokines on lung functions in vitro (7, 16). These studies examined whether cytokines elicit airway hypo- or hyperresponsiveness, but none of them observed direct effects of cytokines on airway function. This failure to see direct effects of cytokines may be explained in part by the fact that only single rather than synergistic actions of cytokines were examined, and in part by the properties of the different in vitro models used. Thus, compared with tracheal preparations, precision-cut lung slices still possess a large part of intact lung parenchyma that may be required for the full response. And compared with cell cultures, the responses in precision-cut lung slices are much quicker: in isolated pulmonary smooth muscle and epithelial cells the induction of Cox-2 peaks after 18 to 24 h (20, 21), whereas in precision-cut lung slices it takes only about 2 h before Cox-2 induction and bronchoconstriction commence. This time course is more reminiscent of in vivo experiments with TNF (1, 2) and perfused lung experiments with endotoxin (14) in which bronchoconstriction and thromboxane release occur between 15 and 90 min. To a certain extent it appears as if the more simplified the models become, the longer it takes for the responses to develop.

Leukotrienes are potent proinflammatory mediators that contract airway smooth muscle, increase microvascular permeability, stimulate mucus secretion, decrease mucociliary clearance, and appear capable of recruiting eosinophils into the airways (22). Incubation of human smooth-muscle cells with IL-1 and TNF- led to a weak but significant increase of peptido-leukotrienes (23). However, in precision-cut lung slices leukotrienes were present only in small amounts and cytokines had no effect on leukotriene formation. The small basal leukotriene production was not altered if lung slices were prepared from lungs that were not perfused free of blood, suggesting that lung cells such as alveolar macrophages (24) rather than neutrophils are the source of leukotrienes under these conditions. In line with the lack of stimulated leukotriene production, a 5-lipoxygenase inhibitor had no effect on cytokine-induced bronchoconstriction. These results are in agreement with previous findings in another Cox-2 and thromboxane-dependent model, where lipoxygenase inhibitors and leukotriene receptor antagonists had no effect on the endotoxin-induced bronchoconstriction (14).

Synergistic effects of cytokines on eicosanoid metabolism have been described before, without, however, showing the functional consequences thereof. In such experiments, the combination of IL-1, IFN-, and TNF- caused a synergistic increase in Cox-2 mRNA or protein in airway smooth-muscle cells (20) and pulmonary epithelial cells (21), although IFN- may not be necessary (25). In pulmonary epithelial cells, lung fibroblasts, and endothelial cells, IL-1 alone may be sufficient for the induction of Cox-2 (26), although this is somewhat controversial (30). In precision-cut lung slices we found that IL-1 and TNF together but not alone caused bronchoconstriction, which is in agreement with the results on Cox-2 induction and thromboxane formation cited earlier (20, 21). Taking all these data together, it seems likely that in the lung slices the cell type in which Cox-2 was induced were airway smooth-muscle and/or pulmonary epithelial cells. This conclusion is also in line with a recent immunohistochemical study showing expression of Cox-2 and TXS in these two cells types in rats (31, 32).

In addition to Cox-2 induction by the cytokine mix, we also observed that selective inhibition of Cox-2 with NS398 reduced both thromboxane formation and bronchoconstriction. NS398 produces half-maximal inhibition for Cox-2 at 5 µM and shows no effect on Cox-1 even at a concentration of 100 µM (33). In addition, blocking Cox-2 induction with the steroid Dex similarly prevented the cytokine-induced bronchoconstriction. Taken together with the protective effect of the thromboxane receptor antagonist, these data suggest that induction of Cox-2 followed by activation of the thromboxane receptor by prostanoids is necessary for the airway constriction induced by the TNF and IL-1 in combination. Although in the present work we have largely focused on thromboxane, we cannot exclude the possibility that other prostaglandins (PGs) such as PGH₂ or PGF₂ that can also activate the thromboxane prostanoid receptor (TP-receptor) (34) also contribute to bronchoconstriction. Of interest in this regard is also PGD₂, which appears to play an important role in allergic asthma (35). However, the fact that PGD₂ was effective only in sensitized animals (35) and thus showed no direct effect on airway tone in otherwise untreated lung slices (data not shown), where known TP-receptor agonists such as U46619 provoke airway contraction (36), suggests that PGD₂ is not very active at the TP receptor, at least in rat airways.

Thromboxane is an active proinflammatory mediator that in addition to bronchoconstriction also causes vasoconstriction and platelet aggregation (37). Thus, treatment of precision-cut lung slices with a thromboxane receptor agonist leads to contraction of airways and vessels (36). Thromboxane is well known as a mediator of bronchoconstriction caused by a number of other proinflammatory agents such endotoxin (14), TNF (2), or IL-2 (38). However, a direct link between Cox-2 induction and alterations in lung functions had previously been shown only in the case of endotoxin-induced bronchoconstriction (14).

The source of thromboxane in the lung under these conditions remains unclear and has been discussed before (14). However, because the perfused lungs we used for the slice preparation were almost completely devoid of blood-derived leukocytes (39), the contribution of platelets or neutrophil seems rather unlikely. The participation of platelets is further made unlikely by our injecting heparin before lung perfusion. Therefore, pulmonary cells must be responsible for the thromboxane production and there is recent immunohistochemical evidence that parenchymal lung cells express TXS with an anatomical distribution similar to that of Cox-2. Accordingly, TXS is present in smooth-muscle cells, epithelial cells, and alveolar macrophages (32). Interestingly, incubation of alveolar macrophages or mixed pneumocytes with LPS (40) or of pulmonary epithelial or smooth-muscle cells with TNF/IL-1 (20, 21) does not cause a significant increase in thromboxane formation above basal levels, suggesting that an intact lung structure is required for this response.

Besides thromboxane and maybe PGH₂, another mediator must contribute to the cytokine-induced bronchoconstriction. This suggestion is made from the observation that the thromboxane receptor antagonist SQ29548 prevented the cytokine-induced bronchoconstriction completely, whereas Cox inhibitors or steroids were only partially effective. These observations suggest the existence of another mediator that is not formed by Cox but that still acts on the thromboxane receptor. Recently a series of F2-isoprostanes was discovered that is synthesized independently from the Cox pathway via free-radical peroxidation of AA. These isomers are potent vasoconstrictors and one of them, 8-epi-prostaglandin F2, leads to bronchoconstriction via the thromboxane receptor (41). Recently it was suggested that isoprostanes may contribute to the endotoxin-induced airway hyperresponsiveness (42).

Another interesting observation was the basal thromboxane release of the lung slices, which was reduced by both Cox inhibitors but not by steroids, even when exogenous AA was supplied. The latter facts demonstrate that this basal production was not due to Cox-2 induction but to constitutive Cox activity, suggesting that the precision-cut lung slices were not in a preactivated state. The fact that the selective Cox-2 inhibitor NS-398 was effective suggests that this basal thromboxane formation is caused by Cox-2, which is in line with reports documenting a constitutive expression of Cox-2 in the airways (31). This conclusion, of course, does not contradict our mRNA data, because these data show only ratios but not absolute amounts of the mRNAs. Although not explicitly stated so far, thromboxane production appears to be a basal property of at least rat lungs. This conclusion is derived not only from the present study but also from other experiments showing that even lung tissue of untreated rat lungs contains about 500 ng thromboxane/g lung tissue (D. Bundschuh, A. Wendel, and S. Uhlig, unpublished data).

The present study has shown that the model of the precision-cut lung slices allows us to recapitulate complex processes and to study lung functions with all the advantages of cell-culture methods. Our data show that the two cytokines, TNF- and IL-1, together exhibit actions markedly different from those elicited by either of them alone. In the lung, the functional consequence of the induction of Cox-2 by TNF- and IL-1 appears to be thromboxane receptor-dependent bronchoconstriction. Our studies raise the possibility that TNF and IL-1 may contribute to bronchospasm during inflammatory lung diseases.