Division of Pulmonary Pharmacology, Research Center Borstel, D-23845 Borstel, Germany
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ABSTRACT |
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Tumor necrosis factor (TNF)-
and interleukin (IL)-1
are formed simultaneously under inflammatory
conditions such as asthma and acute respiratory distress syndrome. Here
we investigated the effects of TNF-
(10 ng/ml) and/or IL-1
(10 ng/ml) in isolated blood-free perfused rat lungs. In lungs
precontracted with methacholine, IL-1
alone and
IL-1
/TNF-
decreased airway resistance 10 min after
administration, whereas TNF-
alone had no effect. In untreated lungs, airway resistance was unaltered by either cytokine alone but
started to increase 40 min after treatment with both cytokines together, indicating bronchoconstriction. The bronchoconstriction was
accompanied by a steroid-sensitive increase in cyclooxygenase (COX)-2
mRNA expression and thromboxane formation. The cytokine-induced bronchoconstriction was blocked by the thromboxane receptor antagonist SQ-29548, indomethacin, the selective COX-2 inhibitor NS-398, and the
steroid dexamethasone. We conclude that IL-1
has an early bronchodilatory effect (after 10 min) that is unchanged by TNF-
. However, at later time points (after 40 min), IL-1
and TNF-
in
concert cause a COX-2- and thromboxane-dependent
bronchoconstriction. Our findings show that TNF-
and IL-1
exert complex and time-dependent effects on lung functions that cannot
be predicted by studying each cytokine alone.
tumor necrosis factor-; interleukin-1
; cyclooxygenase-2; thromboxane
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INTRODUCTION |
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INTERLEUKIN
(IL)-1 and tumor necrosis factor (TNF)-
are mediators of the
innate immune system that are released from inflamed sites to organize
the inflammatory response. In the lungs, conditions in which these two
cytokines are elevated include acute respiratory distress syndrome
(ARDS), asthma, and acute severe asthma (3, 15, 28).
Bronchoconstriction has been reported not only in asthmatic patients
but also in ARDS patients (37), but the mechanisms that
are responsible for airway contraction during acute severe asthma and
ARDS are largely unidentified. We therefore wondered whether cytokines
such as TNF-
and IL-1
that are released under these circumstances
might increase airway resistance and thereby contribute to the
increased airway tone in inflamed lungs.
Only a few studies have investigated whether TNF- and/or IL-1 can
cause bronchospasm. There is some evidence that TNF-
when given
directly may cause bronchoconstriction (35) and airway hyperresponsiveness (27). In contrast to TNF-
, IL-1
failed to increase airway resistance (17) and relaxed
rather than constricted airway smooth muscle (25). Other
studies on the effects of IL-1
treatment on airways reported a
decreased response to
-agonists (12) and
hyperreactivity to bradykinin (29). However, in these experiments, IL-1
and TNF-
have always been examined alone, dismissing the fact that under inflammatory conditions, TNF-
and
IL-1
usually occur in concert. The notion that IL-1
and TNF-
may have synergistic effects is supported by a number of previous
studies (reviewed in Ref. 5). However, most of these previous studies examined the synergistic effects on gene expression or
cytokine release but only rarely the functional physiological consequences thereof. Using the model of precision-cut lung slices, we
have recently shown a thromboxane-dependent airway contraction in the
simultaneous presence of TNF-
and IL-1
but not in the presence of
either cytokine alone (14).
However, the utilization of precision-cut lung slices to study dynamic
airway responses is rather new (13), and it is not known
whether mechanisms observed in this model also apply to the whole
intact organ, in particular, if events as intricate as those induced by
cytokines are under investigation. Therefore, and because of the
possible clinical implications of additive or synergistic actions of
TNF- and IL-1
in the lung, here we investigated the effects of
TNF-
and IL-1
in whole perfused lungs. To gain further insight
into the complex pulmonary reactions evoked by these cytokines, we also
studied their effects in lungs with precontracted airways. These
investigations will help to better understand the consequences of
pulmonary inflammation in the course of diseases such as ARDS and asthma.
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METHODS |
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Animals
Lungs were taken from 8-wk-old female Wistar rats (220 ± 20 g) obtained from Harlan Winkelman (Borchen, Germany) and kept under controlled conditions (22°C, 55% humidity, 12:12-h day-night rhythm) on a standard laboratory chow.Chemicals
Rat recombinant (r) IL-1Isolated Perfused Rat Lung Preparation
The rat lungs were prepared and perfused as previously described (30). Briefly, the lungs were perfused with Krebs-Henseleit buffer (37°C; 2% albumin, 0.1% glucose, and 0.3% HEPES) through the pulmonary artery at a constant hydrostatic pressure (12 cmH2O), resulting in a flow rate of ~25 ml/min. The total amount of circulating buffer was 100 ml. The lungs were suspended by the trachea and were ventilated by negative pressure ventilation with 80 breaths/min and a tidal volume between 1.6 and 2 ml. Every 5 min, a sigh (Thromboxane Enzyme Immunoassay
Thromboxane release into the supernatant was assessed by measuring the stable metabolite thromboxane B2 with a commercially available enzyme immunoassay (Cayman Chemical, Ann Arbor, MI).RT-PCR
The lungs were treated with cytokines to induce gene expression. In some experiments, to block gene expression, dexamethasone (10 mM) was added 30 min before cytokine treatment. The lungs were removed from the thoracic chamber and transferred into Eppendorf cups, frozen immediately in liquid nitrogen, and stored atThe primers were specific for COX-1 (5' forward primer, CTA TCC ATG CCA
GAA CCA GG and 3' reverse primer, TCA CGT TGG AGA AGG ACT CC), COX-2
(5' forward primer, CAA GAC AGA TCA GAA GCG AGG and 3' reverse primer,
AAT GTT GAA GGT GTC CGG C), thromboxane synthase (TxS; 5' forward
primer, ACG TGA CAG AAG ATG GGA GG and 3' reverse primer, CCA GCA GGT
ATG TGA TGA AGG), and -actin (5' forward primer, ATG TAC GTA GCC ATC
CAG GC and 3' reverse primer, TGT GGT GGT GAA GCT GTA GC). These primer
pairs were found to yield PCR products of 323, 562, 692, and 216 bp for
COX-1, COX-2, TxS, and
-actin, respectively. Amplified cDNA bands
were detected by ethidium bromide staining.
The yield of the amplified product was always linear for the amount of
input cDNA and PCR cycle number (data not shown). For amplification, 25 cycles were used for COX-1, 20 cycles were used for COX-2 and
-actin, and 30 cycles were used for TxS.
Statistics
Data are expressed as means ± SD. Data were examined by analyzing the maximum values (see Figs. 1 and 2) or the area under the curve (see Fig. 4) with two-sided t-tests. Homoscedasticity was confirmed by F-test. The
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RESULTS |
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Action of Cytokines on Isolated Perfused Lungs
Two different designs were used to determine the effects of IL-1Airway resistance and thromboxane release.
Under control conditions, the airway resistance in isolated perfused
rat lungs remained stable throughout the whole experiment (Table 1, Fig.
1A). Perfusion
with rat rTNF- alone did not alter airway resistance. Administration
of rat rIL-1
alone resulted only in a weak nonsignificant increase
in airway resistance. However, perfusion with both cytokines together
caused a significant increase in airway resistance over time (Fig.
1A) as well as a decrease in pulmonary compliance and tidal
volume (Table 1). There was no effect of either cytokine alone or in
combination on vascular resistance or lung weight gain as a measure of
edema formation (Table 1).
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Pharmacological interventions.
The use of a thromboxane receptor antagonist (10 µM SQ-29548) 10 min
before cytokine treatment completely prevented the cytokine-induced bronchoconstriction (Fig. 2A).
Moreover, the nonspecific COX inhibitor indomethacin as well as the
selective COX-2 inhibitor NS-398 both prevented the increase in airway
resistance elicited by IL-1/TNF-
(Fig. 2A) as
well as the release of thromboxane into the perfusate (Fig.
2B). Similarly, the glucocorticoid dexamethasone inhibited both production of thromboxane (Fig. 2B) and
cytokine-induced bronchoconstriction (Fig. 2A).
Gene Expression
Because the selective COX-2 inhibitor NS-398 prevented IL-1
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Action of Cytokines on Methacholine-Contracted Perfused Lungs
Because a previous study (25) had shown that pretreatment of bronchial segments with IL-1
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DISCUSSION |
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TNF- and IL-1
are two cytokines present at inflamed sites.
The major finding of this study is that in the simultaneous presence of
IL-1
and TNF-
, airways may either relax or contract, depending largely on the time scale studied. Thus, almost immediately after administration of TNF-
/IL-1
, a brief relaxation of
precontracted airways occurs that is solely due to IL-1
. At later
time points, however, starting ~30 min after administration, IL-1
and TNF-
in concert constrict airways. Because the airway relaxation
is very short-lived, we speculate that under clinical conditions such
as asthma or ARDS, where both cytokines usually occur together, the
bronchoconstriction may become more important. Our findings also
indicate that the role of cytokines in situ may be difficult to predict
from studies with single cytokines only. The present data further
suggest that the mechanism responsible for the effects of
TNF-
/IL-1
on bronchoconstriction is independent of
blood-derived leukocytes and involves induction of COX-2 followed by
production of thromboxane.
Synergistic actions between IL-1 and TNF-
are well known in the
literature, although the mechanism remained obscure (5). A
synergism between IL-1
and TNF-
was reported before with respect to mortality (33), pulmonary edema (18, 34),
and prostaglandin production (5). In contrast to IL-1
,
IL-1
together with TNF-
failed to show such synergism on
pulmonary edema (24), thromboxane production
(11), and pulmonary vascular resistance (11).
Despite the plethora of studies on TNF-
and IL-1
, there is very
little information about the effects of these cytokines on airway
functions. A direct effect of IL-1
on airway tone has not been
described before. Previously, IL-1
was studied in bronchial or
tracheal smooth muscle preparations where pretreatment decreased the
response to
-agonists (12), induced hyperreactivity to
bradykinin (29), and reduced the contractile responses to
acetylcholine, histamine, and KCl (25). Here we show that
IL-1
relaxes precontracted airways. The mechanism responsible for
this airway relaxation is unknown at present but may involve dilatory
prostaglandins or NO. The finding that IL-1
can relax precontracted
airways extends previous observations made in canine bronchial segments of attenuated airway contractions after preincubation with IL-1
for
150 min (25). It should be noted, however, that these
authors failed to observe a direct relaxation with IL-1
in
precontracted airway segments. Notable differences between that study
and the present one that might explain this discrepancy include the use of human IL-1
instead of homologous IL-1
and the use of bronchial segments in contrast to the whole organ.
In contrast to IL-1, bronchoconstriction by TNF-
alone in vivo
was demonstrated before (35). However, as with IL-1
,
most studies were performed in vitro where it was shown that
preincubation with TNF-
caused bronchial hyperresponsiveness
(1, 20). In general, cytokines are thought to orchestrate
the immune response, with indirect rather than direct effects on lung
physiology. Therefore, it is important to stress the fact the
synergistic bronchoconstriction by IL-1
and TNF-
in otherwise
untreated lungs occurred in blood-free perfused lungs, ruling out the
participation of blood-derived leukocytes (for confirmation of the
absence of blood cells in our model, see Ref. 31). Further
evidence for the fact the IL-1
/TNF-
-induced bronchoconstriction does not depend on blood-derived leukocytes is
given by the fact that a similar response also occurs in precision-cut lung slices from rats (14). The good agreement of the
present findings with those obtained in precision-cut lung slices
indicates that the slice model is a good representative of the whole
organ. This conclusion is particularly important for studies with
precision-cut lung slices from humans (Wohlsen A, Martin C, Vollmer E,
and Uhlig S, unpublished observations) in which comparisons
between different models cannot be easily made. Of course, as with all
such model systems that allow for control of parameters such as
cytokine concentration, the caveat should be made that under in vivo
conditions, factors such as blood and a functioning nerve supply might
modify the responses.
Like in precision-cut lung slices, in perfused lungs, the
IL-1/TNF-
-induced bronchoconstriction is most likely
caused by COX-2-dependent thromboxane formation. The evidence for this
mechanism is as follows. 1) After 60 min of perfusion with
IL-1
/TNF-
, we found significant induction of COX-2 mRNA
compared with weak or hardly detectable induction in the presence of
IL-1
or TNF-
alone. Previous cell culture studies provide ample
evidence that COX-2 expression is inducible in parenchymal lung cells,
i.e., lipopolysaccharide (LPS) or IL-1
and TNF-
elicited
expression of COX-2 in pulmonary epithelial cells (16),
airway macrophages (19), and airway smooth muscle cells
(2). In addition, pulmonary COX-2 expression was recently
confirmed by immunohistochemical staining with anti-COX-2 antibodies
(7). 2) Thromboxane was released into the
perfusate with a time course matching that of the bronchoconstriction.
As for COX-2 synthase, recent histochemical data (6) have
provided evidence that TxS is also present in parenchymal lung cells.
3) Inhibition of COX-2 induction by dexamethasone or COX-2
enzyme by NS-398 prevented thromboxane formation as well as
bronchoconstriction. 4) Blockade of the thromboxane receptor SQ-29548 completely abrogated the IL-1
/TNF-
-induced bronchoconstriction.
A number of different animal models support the notion that expression of COX-2 and/or thromboxane in the lungs is frequently associated with different forms of lung failure. Perfusion of rat lungs with LPS causes a very similar type of bronchoconstriction, as described here, that is also COX-2 and thromboxane dependent but neutrophil independent (32). In another model, in vivo pretreatment with granulocyte-macrophage colony-stimulating factor or granulocyte colony-stimulating factor resulted in increased bronchial responsiveness to LPS that is neutrophil dependent and, again, causally related to COX-2 expression and thromboxane formation (36). In murine lungs, LPS causes COX-2- and thromboxane-dependent vascular hyperresponsiveness (4, 10). In dogs, priming with endotoxin for oleic acid-induced lung injury was also partly attributed to COX-2 induction (9). Therefore, enhanced expression of COX-2 followed by increased thromboxane formation appears to play an important role in the pathophysiology of many animal models.
The present findings may have bearings on our understanding of the
pathophysiology of ARDS and asthma. Several studies have shown
increased airway resistance in ARDS patients (e.g., Ref. 37). Because both TNF- and IL-1
clearly occur in the
airways of ARDS patients (15), TNF-
and IL-1
in
concert might contribute to the bronchoconstriction in these patients.
And also in the case of asthma, there is some evidence for a
contribution of IL-1
and/or TNF-
. 1) In the
bronchoalveolar lavage fluid and in biopsies from asthmatic patients,
IL-1
and TNF-
are present, with the highest concentrations in
acute severe asthma (3, 28). 2) In sensitized
guinea pigs, an IL-1
receptor antagonist attenuated the infiltration
of neutrophils, airway hyperresponsiveness toward substance P, and the
late asthmatic bronchoconstriction (17, 23). 3)
Th1 cell-derived TNF-
is required for recruitment of inflammatory
cells to the airways in a murine asthma model (22). 4) Subsequent to antigen challenge, increased TNF-
production by mast cells during the early-phase response
(3) and by macrophages during the late-phase response has
been demonstrated (8, 21). Because, in our model, the
effect of IL-1
/TNF-
is mediated by thromboxane, it is of
interest to note that a recent study (26) has reported
improvements in lung functions in a subpopulation of asthma patients
treated with a thromboxane receptor antagonist.
We conclude that IL-1 and TNF-
have time-dependent effects on
airway tone that are not mediated by blood-derived leukocytes. An early
bronchodilating effect that is mainly caused by IL-1
is followed by
a COX-2- and thromboxane-dependent bronchoconstriction. Our findings
suggest that IL-1
and TNF-
contribute to the altered lung
functions in inflamed lungs where these two cytokines usually occur in concert.
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ACKNOWLEDGEMENTS |
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We acknowledge the perfect technical assistance of Cornelia Rodde and Darja Buchholz.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft Grants DFG Uh 88/2-2 and DFG Uh 88/3-1.
Address for reprint requests and other correspondence: S. Uhlig, Division of Pulmonary Pharmacology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany (E-mail: suhlig{at}fz-borstel.de).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 4 October 2000; accepted in final form 15 November 2000.
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