1 Institute of Anatomy and Cell Biology, 2 Department of Internal Medicine, and 3 Department of Pathology, Justus-Liebig-University Giessen, 35385 Giessen, Germany
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ABSTRACT |
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Enhanced prostanoid generation has been
implicated in vascular abnormalities occurring during endotoxemia and
sepsis, and the lung is particularly prone to such events. Prostanoids
are generated from arachidonic acid (AA) via cyclooxygenase (COX)-1 or
-2, both isoenzymes recently demonstrated to be expressed in different
lung cell types. Upregulation of COX may underlie the phenomenon that
endotoxin [lipopolysaccharide (LPS)]-exposed lungs show
markedly enhanced vasoconstrictor responses to secondarily applied
stimuli (priming). Isolated rat lungs were perfused with a
physiological salt buffer solution in the absence and presence of 1.5%
rat plasma and exposed to different concentrations of LPS (1,000 or
10,000 ng/ml) during a 2-h priming period. No change in
physiological variables was noted during this period, although enhanced
baseline liberation of both thromboxane (Tx) A2 and
PGI2 as well as of tumor necrosis factor (TNF)- was
evident compared with that in control lungs in the absence of LPS. LPS
priming caused a significant elevation in AA-induced pulmonary arterial pressure, ventilation pressure, and lung weight gain. Concomitant increased levels of TxA2 were found in the buffer
perfusate. All changes were largely suppressed by three selective,
structurally unrelated COX-2 inhibitors (NS-398, DUP-697, and SC-236)
in both buffer- and buffer-plasma-perfused lungs. Anti-TNF-
neutralizing antibodies were ineffective under conditions of buffer
perfusion. In the presence of plasma components, manyfold augmented
TNF-
generation was noted, and anti-TNF-
antibodies significantly suppressed the increase in ventilation pressure but not in the vascular
pressor response and lung edema formation. We conclude that the
propensity of LPS-primed lungs to respond with enhanced vasoconstriction, edema formation, and bronchoconstriction to a
secondarily applied stimulus proceeds nearly exclusively via COX-2 and
increased Tx formation, with TNF-
generation being involved in the
change in bronchomotor reactivity in the presence of plasma
constituents. In context with recent immunohistological investigations,
LPS-induced upregulation of the COX-2-thromboxane synthase axis in
vascular and bronchial smooth muscle cells is suggested to underlie
these events.
lipopolysaccharide; isolated perfused rat lung; tumor necrosis factor; selective cyclooxygenase-2 inhibition
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INTRODUCTION |
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ENDOTOXEMIA-INDUCED pathophysiological changes are
thought to contribute largely to circulatory abnormalities and organ
failure under conditions of sepsis, with persistently high mortality
rates in critically ill patients (5, 41). Among the organs affected under these conditions, acute lung injury [acute respiratory
distress syndrome (ARDS)] is of major importance (5,
27). Endotoxin, a lipopolysaccharide (LPS) from gram-negative bacteria,
is known to provoke proinflammatory cytokine synthesis similar to that of tumor necrosis factor (TNF)- and enhanced generation of
eicosanoids. Among the latter, prostaglandins (PGs) and thromboxane
(Tx), which have been implicated in pulmonary vascular abnormalities
and changes in bronchomotor tone in acute lung injury (38, 40), are
generated by conversion of arachidonic acid (AA) to the unstable
intermediate PGG2/PGH2,
catalyzed by two cyclooxygenase (COX) isoenzymes. Initially, COX-1 was
noted to be constitutively expressed in various organ systems, being
involved in the regulation of physiological functions, whereas COX-2
was assumed to be quiescent under normal physiological conditions,
being upregulated only during inflammatory circumstances, then
providing the enzymatic basis for proinflammatory prostanoid effects.
Meanwhile, however, both COX isoenzymes have been shown to occur
constitutively in several organ systems (18, 21, 26, 33).
Concerning the lung architecture, Ermert et al. (14) recently showed COX-2 to be constitutively expressed in various pulmonary cell types including bronchial epithelial cells, bronchial smooth muscle cells, macrophages, mast cell-like cells, and smooth muscle cells of partially muscular vessels, which are operative as pulmonary resistance vessels (31). Moreover, an immunostaining study (13) also demonstrated the presence of thromboxane synthase in the smooth muscle cells of the partially muscular vessels in noninflamed lungs. In contrast, no COX-2 or thromboxane synthase immunostaining was noted in endothelial cells under baseline conditions. In accordance with these morphological data, a recent pharmacological study (11) indeed demonstrated that vasoconstrictor responses to AA in noninflamed lungs, proceeding via Tx generation that supervenes the simultaneously enhanced prostacyclin formation, are operated largely via COX-2 and not via COX-1 activity.
Lung reactivity to vasoactive stimuli is known to be altered under
inflammatory conditions. This was impressively demonstrated for
endotoxin under experimental conditions: "priming" of perfused lungs with LPS for 2-3 h in doses that per se did not affect
physiological variables resulted in dramatically enhanced
vasoconstrictor responses in association with ventilation-perfusion
mismatch to secondarily applied stimuli such as bacterial exotoxins,
platelet-activating factor, and AA; and markedly increased Tx synthesis
was noted to underlie this altered reactivity (8, 36, 38, 44). This is
of interest in view of recent immunohistological studies (12, 13) that
demonstrated upregulation of both COX-2 and thromboxane synthase in
several lung cell types during the LPS-priming period. In these
histological studies, the upregulation of COX-2 was suggested to be
partially dependent on the presence of plasma constituents. This is of
interest against the background that many effects of LPS appear to be
mediated by the plasma protein LPS-binding protein (LBP) and the
membrane receptor CD14 (34, 39, 41, 45), but soluble CD14 might
substitute for membrane CD14 (15, 34, 39). In addition to the LBP-CD14
axis, however, CD14- and plasma component-independent signal
transduction pathways for endotoxin, partly but not yet fully
characterized, have been suggested (9, 20, 28, 45). Moreover, priming
phenomena in response to endotoxin might not be exerted via LPS itself
but rather could involve secondarily generated cytokines such as
TNF- (7, 10, 34). Pronounced, rapid generation of this cytokine was
previously noted to occur in LPS-challenged perfused lungs (29, 36,
44).
The present study, performed on LPS-primed rat lungs, employed
selective COX-2 inhibitors to question whether the enhanced Tx-mediated
vasoconstrictor response to AA, applied after preceding endotoxin
exposure, indeed proceeds via this COX isoenzyme. Moreover, LPS
exposure was performed in the absence and presence of rat plasma and
anti-TNF- neutralizing antibodies for functional analysis of a
putative contribution of plasma constituents. This prototype cytokine
to the priming of the COX-2-Tx axis was suggested to underlie the
enhanced vasoconstrictor reactivity. Interestingly, edema formation and
bronchoconstrictor events were noted to be prominent features
accompanying the vascular pressor response in the LPS-primed rat lungs,
and a differential impact of TNF-
blockage on these phenomena was noted.
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MATERIALS AND METHODS |
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Reagents. AA and acetylsalicylic acid were obtained from Paesel
and Lorei (Frankfurt, Germany). LPS (Salmonella abortus equi, S-form) was purchased from Cytogen (Bodenheim, Germany). All other biochemicals were obtained from Merck (Darmstadt, Germany). ELISA kits
for the determination of 6-keto-PGF1 and
TxB2 were obtained from Cayman Chemical (Ann Arbor, MI).
The TNF-
ELISA kit was supplied by Biosource (Deerfield, IL). An
anti-TNF-
neutralizing antibody (ICC-TNF-9A) was obtained from Ic
Chemikalien (Ismaning, Germany). The COX-2 inhibitor NS-398 was
purchased from BIOMOL (Hamburg, Germany). The COX-2 inhibitor DUP-697
was kindly provided by DuPont Pharmaceuticals (Wilmington, DE), and the
COX-2 inhibitor SC-236 was a gift from Searle (St. Louis, MO).
Animals. CD rats (Sprague-Dawley) were obtained from Charles River (Sulzfeld, Germany). All experimental procedures were performed in conformity with the guidelines of the National Institutes of Health Guide for the Care and Use of Laboratory Animals [DHEW Publication (NIH) No. 86-23, Revised 1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD 20892].
Lung isolation and perfusion. The rats (male, body weight 350-400 g) were deeply anesthetized with pentobarbital sodium (100 mg/kg body wt ip). After local anesthesia with 2% xylocaine and a median incision, the trachea was dissected and a tracheal cannula (Hugo Sachs Elektronik, March-Hugstetten, Germany) was immediately inserted. A median laparotomy was performed, and the rats were anticoagulated with 1,000 U of heparin. Subsequently, mechanical ventilation was started with 4% CO2, 17% O2, and 79% N2 (tidal volume 4 ml, frequency 65 breaths/min, end-expiratory pressure 3 cmH2O) with a small-animal respirator KTR-4 (Hugo Sachs Elektronik). After a midsternal thoracotomy, the right ventricle was incised, a cannula (2.0-mm diameter; Hugo Sachs Elektronik) was fixed in the pulmonary artery, and the apex of the heart was cut off to allow pulmonary venous outflow. Simultaneously, pulsatile perfusion with the buffer solution was started. The buffer contained 2.4 mM CaCl2, 1.3 mM MgCl2, 4.3 mM KCl, 1.1 mM KH2PO4, 125.0 mM NaCl, and 25 mM NaHCO3 as well as 13.32 mM glucose (pH ranged between 7.35 and 7.40).
The lungs were carefully excised, with any damage being avoided, while being perfused with the buffer solution and placed in an upright position. Next, a cannula (Hugo Sachs Elektronik) was fixed in the left atrium through the left ventricle to obtain a closed perfusion circuit. After extensive rinsing of the vascular bed, the lungs were recirculatingly perfused with a pulsatile flow of 13 ml/min. The alternate use of two separate perfusion circuits, each containing 100 ml, allowed the repetitive exchange of perfusion fluid. Perfusion pressure, ventilation pressure (VP), and weight of the isolated organ were registered continuously (Combitrans Monitoring Set Med. II for arterial blood pressure measurement, Braun Melsungen, Germany; transducer, Marquette Hellige, Neudrossenfeld, Germany). The left atrial pressure was set at 2 mmHg under baseline conditions (0 referenced at the hilum) to guarantee zone III conditions at end expiration throughout the lung. Lungs selected for the study were those that 1) had a homogeneous white appearance without signs of hemostasis or edema formation, 2) had pulmonary arterial pressure (PAP) and VP in the normal range (PAP 5-7 mmHg, VP 4-5 mmHg), and 3) were isogravimetric during a steady-state period of 30 min.Experimental protocol. All experiments were undertaken both in buffer-perfused lungs and in lungs perfused with an admixture of 1.5% rat plasma in the buffer fluid. In total, 140 isolated lung experiments were performed. Control lungs were perfused with recirculating fluid under standard conditions for 125 min without any drug application (n = 5 experiments for each buffer and buffer-plasma perfusion). In experiments without LPS application (n = 5 each), free AA was admixed to the perfusate at a concentration of 5 µmol/l after 2 h of perfusion.
As shown in Fig. 1, two different dosages of LPS were admixed to the perfusate at the onset of the 2-h perfusion period, resulting in concentrations of 1,000 or 10,000 ng/ml in the recirculating buffer fluid (n = 5 experiments each). At the end of the 2-h perfusion period, 5 µmol/l of AA were administered as in the lungs perfused in the absence of LPS.
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Perfusate analysis.
TxA2 and PGI2 were assayed by ELISA from the
recirculating buffer fluid as their stable hydrolysis products
TxB2 and 6-keto-PGF1, respectively. In
addition, TNF-
in the buffer perfusate was measured by the ELISA technique.
Statistical analysis. Analysis of variance followed by the Newman-Keuls post hoc test and Student's t-test for unpaired data was used to evaluate differences among different groups. A value of P < 0.05 was considered significant. All data are means ± SE.
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RESULTS |
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Buffer-perfused lungs. In control lungs, no weight
gain (W) or change in PAP or VP was registered over the entire
observation period of 2 h. In the absence of preceding LPS
administration, 5 µmol/l of free AA provoked a rapid increase in PAP
by
5 mmHg within 5 min (Table 1, Fig.
2). Concomitantly, a
W was noted, amounting to
1 g within 5 min. In contrast, VP remained unchanged in
response to AA administration.
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PAP, VP, and W were within normal ranges over 2 h in lungs perfused
with 1,000 or 10,000 ng/ml of LPS. However, a markedly enhanced
responsiveness to the subsequent AA challenge was noted. The PAP
increase registered 5 min after AA administration was more than
doubled, a rapid
W of
6-8 g occurred, and the VP increased to approximately twofold baseline values (Table 1, Fig. 2). The response in the presence of 10,000 ng/ml pf LPS slightly surpassed that
in the presence of 1,000 ng/ml of LPS.
Preapplication of the selective COX-2 inhibitors NS-398 (10 µmol/l),
SC-236 (10 µmol/l), or DUP-697 (25 µmol/l) 10 min before the
administration of AA suppressed the AA-induced PAP response to near
baseline values both in control lungs and in lungs undergoing a
preceding 2-h LPS incubation period (Table
2, Fig. 2). In parallel, the
AA-induced W and VP, which were prominent only in the LPS-primed lungs, was markedly reduced by all three COX-2 inhibitors (Table 2,
Fig. 2). Application of a higher dose of the COX-2 inhibitor NS-398 (25 µmol/l) nearly fully extinguished the PAP increase,
W, and
increase in VP in response to AA administration in lungs primed with
1,000 or 10,000 ng/ml of LPS (Table 2, Fig. 2).
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In buffer-perfused lungs undergoing priming with 10,000 ng/ml of LPS
for 2 h, the administration of anti-TNF- neutralizing antibodies
either 5 min before or 60 min after LPS administration did not affect
the AA-induced responses of PAP,
W, or VP (Table 3, Fig.
3).
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In control lungs, only minor amounts of TxB2 and
6-keto-PGF1 were liberated into the recirculating buffer
fluid over the entire observation period (all data <50 pg/ml for
TxB2 and <750 pg/ml for 6-keto-PGF1
). LPS
priming with 1,000 and 10,000 ng/ml of LPS caused a progressive but
moderate accumulation of TxB2, amounting to 115 ± 20 and
176 ± 23 pg/ml, respectively, within 2 h. Subsequent administration
of AA increased the perfusate TxB2 levels to
250 pg/ml
in control lungs and
500-750 pg/ml in LPS-primed lungs within 5 min (Fig. 4). This AA-induced additional TxB2 liberation was largely suppressed by NS-398 (10 µmol/l) being administered to the perfusate 10 min before AA
application (Fig. 4). In addition, a more pronounced accumulation of
6-keto-PGF1
was noted in LPS-primed lungs, amounting to
1,360 ± 314 (1,000 ng/ml of LPS) and 1,986 ± 226 (10,000 ng/ml of
LPS) pg/ml within 2 h. AA administration resulted in a further increase
in perfusate PGF1
levels to
2,500-3,500 ng/ml
within 5 min (Fig. 4). This increase was not suppressed by 10 µmol/l
of NS-398, but rather the levels of 6-keto PGF1
were
elevated in the presence of NS-398 in the LPS-primed lungs.
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Although in control lungs only negligible levels of TNF- were
detected in the perfusion medium, addition of LPS caused a dose-dependent increase of TNF-
within 60-120 min (Fig.
5). As anticipated, this increase was not
further enhanced by the admixture of AA to the perfusate.
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Buffer-plasma-perfused lungs.
The presence of 1.5% rat plasma admixed to the buffer fluid throughout
the experiments did not affect the baseline values of PAP, lung weight,
and VP in the control lungs. Moreover, no significant change in these
variables was noted when 1,000 or 10,000 ng/ml of LPS was recirculated
for 2 h in the presence of rat plasma. In contrast, a markedly more
pronounced liberation of TNF- was noted on LPS exposure in the
presence of plasma constituents (Fig. 5). Concerning the responses to
AA challenge, all changes in physiological variables (PAP, weight, and
VP) were more moderate compared with those in the buffer-perfused
lungs, but again, a marked amplification of these changes after
preceding LPS incubation was noted (Table 1). In accord with the
buffer-perfused lungs, all AA-elicited changes were virtually fully
suppressed in the presence of the selective COX-2 inhibitors NS-398,
SC-236, and DUP-697 (Table 2). In contrast to lungs perfused in the
absence of plasma, the impact of the anti-TNF-
neutralizing
antibodies on the AA-elicited elevation in VP was noted in
buffer-plasma-perfused organs (Table 3, Fig. 3); this was significantly
reduced by preapplication of the antibody either 5 min before or 60 min
after LPS administration. In contrast, PAP increase and
W in
response to AA application were not altered by prior administration of
TNF-
neutralizing antibodies in the buffer-plasma-perfused lungs
(Table 3).
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DISCUSSION |
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In the present study, a 2-h period of LPS priming did not provoke any
change in PAP, VP, and lung weight in buffer-perfused rat lungs,
although enhanced baseline liberation of prostanoids and TNF- was
noted. Subsequent administration of AA did, however, provoke markedly
increased PAP responses in association with enhanced Tx release in the
LPS-primed lungs. Moreover, rapid lung edema formation and an increase
in VP were elicited by AA under these conditions. All changes were
virtually fully suppressed by three structurally unrelated COX-2
inhibitors, whereas anti-TNF-
antibodies were ineffective.
Performing the studies in the presence of rat plasma forwarded less
impressive changes in the physiological variables, but corresponding
efficacy of the COX-2 inhibitors was noted. Under these circumstances,
the LPS-elicited TNF-
liberation was, however, markedly increased,
and anti-TNF-
antibodies significantly reduced the AA-induced
increase in VP.
It is well in line with previous studies (8, 36, 38, 44) in
buffer-perfused rabbit and rat lungs that recirculation of substantial
quantities of LPS did not provoke overt changes in functional variables
such as pulmonary vascular pressure, lung weight, and VP assessed in
the present investigation. The enhanced baseline liberation of
TxA2 noted during the LPS-priming period was obviously
counterbalanced by increased generation of PGI2 and
possibly other vasodilators occurring under these conditions. Moreover,
the accumulation of TNF- in response to the LPS challenge apparently
did not suffice to alter the physiological variables in the absence of
a secondary stimulus, which is in accordance with the observation that
exogenously administered TNF-
does not per se provoke
vascular abnormalities in buffer-perfused lungs (6).
In contrast to the unchanged baseline variables, administration of AA after a 2-h LPS-priming period provoked a markedly increased PAP elevation, and this was clearly attributable to a "hyperreactive" COX-2-Tx axis. First, enhanced Tx liberation in response to AA administration was observed to occur in parallel with the elevated pressor response, and Tx is a well-known potent vasoconstrictor agent in the pulmonary circulation (11, 32, 35). Second, the Tx release reaction was not accompanied by a corresponding increase in PGI2 liberation. Third, and most importantly, the pressor response was largely suppressed by three structurally unrelated selective COX-2 inhibitors (NS-398, DUP-697, and SC-236). In the concentration range currently applied, these agents were shown to be highly selective for COX-2, with no effect on COX-1 activity (30). This is even true for the higher NS-398 dose (25 µmol/l) presently employed, which virtually fully blocked any AA-elicited pressure elevation as similarly noted for 25 µmol/l of DUP-697. In addition to the recently noted fact that Tx-mediated vasoconstrictor responses proceed via COX-2 in noninflamed lungs (11), the present data thus demonstrate that this is also true for the markedly enhanced Tx and vasoconstrictor responses in lungs undergoing LPS exposure. Moreover, these findings are fully compatible with recent immunohistological studies in endotoxin-challenged rat lungs (12, 13), in which markedly enhanced expression of both COX-2 and thromboxane synthase was noted in the smooth muscle cells of large arteries and, in particular, of small partially muscularized arteries known to be intimately involved in the regulation of lung vascular tone (31). In contrast, COX-1 expression, predominant, e.g., in endothelial cells, was found to be fully unchanged in these investigations. Correspondingly, the COX-2 but not the COX-1 message was noted to be increased in the homogenate of lungs exposed to LPS for 2 h (8, 13). The finding that the present use of selective COX-2 inhibitors did not suppress PGI2 formation in the LPS-primed rat lungs indeed suggests that the generation of this vasodilatory agent proceeds largely via COX-1 also under conditions of LPS exposure.
In addition to the vasoconstrictor response, AA administration provoked
a moderate pulmonary edema formation in the control lungs (1-g
W
within 5 min), and this, too, was markedly enhanced in the LPS-primed
lungs (
6-8 g within 5 min). Both enhanced vascular permeability
and increased capillary filtration pressure might underlie this edema
formation. Because partitioning of the pulmonary vascular resistance
and direct assessment of lung capillary permeability were not performed
in the present study, this question may not be definitely settled.
However, Tx-mediated postcapillary vasoconstriction with subsequently
elevated hydrostatic pressure in the capillary bed offers a most
plausible explanation for this finding: 1) the edema formation
occurred very rapidly after the administration of AA, whereas
elevations in the capillary filtration coefficient in response to this
fatty acid, mostly attributed to lipoxygenase pathways (17, 43), were
commonly noted to represent a more protracted event; 2)
selective COX-2 inhibition fully blocked edema formation, strongly
supporting its pathogenesis via prostanoid and, in particular, Tx
generation, and none of the prostanoids were hitherto found to increase
directly the capillary permeability in rat lungs; and 3) Tx is
known to provoke postcapillary in addition to precapillary
vasoconstriction in the rat lung vasculature and may thus well be
operative to increase the microvascular filtration pressure (19, 37).
In addition to such a role for Tx in the edematous response of the
LPS-primed lungs, the endotoxin-related enhanced TNF-
generation
might be attributed to the readiness of the isolated organs for
pressure-induced fluid accumulation because this agent was noted in
vivo to increase pulmonary vascular permeability (3, 16, 25); however,
such an effect was lacking in other investigations (4, 6). In the
present study, TNF-
per se did not apparently suffice to
provoke lung edema formation because no
W was noted after the 2-h
LPS recirculation period and the TNF-
levels were maximally
increased at this time point, with no further elevation on subsequent
AA administration. Moreover, no reduction in
W was observed in the
experiments employing anti-TNF-
neutralizing antibodies, thus
largely excluding a role for this cytokine in the edematous response of
the LPS-primed lungs.
In addition to the vascular abnormalities, a doubling in VP was provoked by AA in the LPS-primed lungs, which was fully lacking in the organs perfused in the absence of endotoxin. Under the presently employed conditions of volume-controlled ventilation at a preset frequency, such an increase in VP might reflect both alterations in lung compliance and changes in bronchial resistance. Random performance of static pressure-volume curves (data not shown) clearly demonstrated that increased bronchial resistance and not loss of tissue compliance is the predominant phenomenon underlying the VP elevations in the LPS-primed lungs. This is well in line with the observation that the VP increase occurred very rapidly in response to the AA admixture, even preceding edema formation (which might reduce static compliance), and the previous findings that LPS-related Tx generation provokes marked bronchoconstriction in rat lungs (40) in contrast to that in rabbit lungs (36, 38, 44). Indeed, COX-2 inhibition was presently found to inhibit significantly the VP increase in response to AA in the LPS-primed lungs. This is of interest against the background that a previous study (3) demonstrated coexpression of COX-1 and COX-2 in cultured bronchial smooth muscle cells, with COX-2 upregulation in response to LPS. In accordance with these in vitro studies, immunostaining experiments in intact rat lungs showed the presence of both COX-1 and COX-2 in bronchial epithelial cells and bronchial smooth muscle cells under baseline conditions (14), and upregulation of both COX-2 and thromboxane synthase was recently noted to occur in response to LPS in both cell types (12, 13). Thus compartmentalized Tx generation in the bronchial tissue in response to AA, occurring largely via the COX-2-Tx axis being upregulated during the period of LPS exposure and forwarding a marked bronchoconstrictor response, is the most plausible explanation for the rapid increase in VP in the endotoxin-primed lungs.
In the presence of plasma constituents, both the vasoconstrictor
response and the W provoked by the AA challenge were reduced, but
potent inhibition of these events by all three COX-2 inhibitors was
nevertheless apparent. Most probably, the lower responsiveness to AA is
attributable to some protein binding of this fatty acid and/or the
arising prostanoids, thus eliciting a reduced vasoconstrictor response
as previously described for buffer-perfused lungs (22). This
nonspecific effect of plasma proteins was apparently not "overcompensated" for by an enhanced priming potency of LPS in the presence of specialized plasma constituents, among which LBP and
soluble CD14 may be of major interest (15, 34, 39, 41, 45). It is in
line with such reasoning that a recent immunohistochemical study (13)
showed LPS-induced COX-2 upregulation in smooth muscle cells of
partially muscularized vessels to occur independently of plasma
components. In contrast, markedly enhanced TNF-
generation was noted
in the presence of plasma components, well compatible with a large body
of in vitro studies (23, 24, 29, 46) demonstrating LBP dependency of
the cytokine release reaction in different types of leukocytes and a
previous investigation (44) in perfused rabbit lungs in which TNF-
generation in both the intravascular and bronchoalveolar compartments
was noted to be manyfold increased in the presence of small quantities
of plasma proteins. Cytokines, and in particular TNF-
, are known to
elicit regulatory events in a large number of cell types, and they have also been implicated in the upregulation of COX-2 (1, 2). The current
employment of anti-TNF-
neutralizing antibodies applied at
concentrations sufficient to bind the total amount of perfusate TNF-
did not, however, support the view that the LPS-elicited upregulation
of COX-2 in the vascular smooth muscle cells, assumed to underlie the
enhanced Tx-mediated vasoconstrictor response as detailed above, might
be mediated in an indirect fashion via liberation of this cytokine in
both the absence and presence of plasma constituents. In contrast,
these experiments forwarded evidence for a role of TNF-
in the
bronchial compartment (the priming of the bronchoconstrictor response
under conditions of plasma admixture), and thus high levels of TNF-
generation were markedly suppressed by anti-TNF-
antibodies. This
finding thus supports the concept that proinflammatory cytokines like
TNF-
may be involved in the pathogenesis of airway hyperreactivity as suggested from models of asthma pathogenesis (42, 47). It may not be
fully settled by the current data whether this effect of TNF-
proceeded largely via upregulation of COX-2 because selective COX-2
inhibitors were invariably effective in suppressing the increase in VP
under these conditions or whether elicitation of additional
bronchoconstrictor agents might be involved as, e.g., suggested for
endothelin (42, 47).
In conclusion, LPS exposure of rat lungs profoundly altered the
readiness of these organs to respond to a subsequent AA challenge. Enhanced Tx generation occurring via upregulation of COX-2, in particular in vascular smooth muscle cells, is suggested to underlie an
increased vasoconstrictor response and rapid lung edema formation, and
these events occur independent of plasma constituents and LPS-induced
TNF- generation. In addition, COX-2-dependent bronchoconstriction is
noted in the LPS-primed lungs, with high levels of TNF-
arising in
the presence of endotoxin plus plasma components contributing to this
phenomenon. In agreement with a recent immunohistological study in
LPS-primed lungs (14), the present pharmacological data support the
view of a concordant induction of COX-2 and terminal thromboxane
synthase in both the vascular and bronchial smooth muscle cells as the
predominant event underlying the enhanced pulmonary readiness to
respond to a secondary stimulus after preceding endotoxin exposure.
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ACKNOWLEDGEMENTS |
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We thank G. Müller for excellent technical assistance. We are grateful to Dr. R. L. Snipes (Department of Anatomy, Justus-Liebig-University Giessen, Giessen, Germany) for linguistically reviewing the manuscript.
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FOOTNOTES |
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This work was supported by the Deutsche Forschungsgemeinschaft SFB 547 (Kardiopulmonales Gefässsystem).
This publication includes parts of the thesis of M. Merkle in partial fulfillment for the MD degree.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. Ermert, Institut fuer Anatomie und Zellbiologie, Aulweg 123, 35385 Giessen, Germany (E-mail: leander.ermert{at}anatomie.med.uni-giessen.de).
Received 27 September 1999; accepted in final form 19 January 2000.
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