Endotoxin priming of the cyclooxygenase-2-thromboxane axis in isolated rat lungs

M. Ermert1, M. Merkle2, R. Mootz2, F. Grimminger2, W. Seeger2, and L. Ermert3

1 Institute of Anatomy and Cell Biology, 2 Department of Internal Medicine, and 3 Department of Pathology, Justus-Liebig-University Giessen, 35385 Giessen, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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)-alpha 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-alpha neutralizing antibodies were ineffective under conditions of buffer perfusion. In the presence of plasma components, manyfold augmented TNF-alpha generation was noted, and anti-TNF-alpha 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-alpha 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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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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)-alpha 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-alpha (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-alpha 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-alpha blockage on these phenomena was noted.


    MATERIALS AND METHODS
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ABSTRACT
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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-PGF1alpha and TxB2 were obtained from Cayman Chemical (Ann Arbor, MI). The TNF-alpha ELISA kit was supplied by Biosource (Deerfield, IL). An anti-TNF-alpha 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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Fig. 1.   Time schedule of lipopolysaccharide (LPS) administration, arachidonic acid (AA) challenge, and inhibitor administration in buffer- and buffer-plasma-perfused lungs. TNF, tumor necrosis factor; Ab, antibody. For details, see text.

The following concentrations and combinations of challenge and inhibitor were used to inhibit selectively COX-2 activity (Fig. 1). Lungs were challenged with either 1,000 or 10,000 ng/ml of LPS; after 110 min of perfusion, NS-398 was administered at a final concentration of either 10 or 25 µmol/l (n = 5 experiments for each combination of LPS dosage and inhibitor dosage). Two additional COX-2-selective inhibitors were applied after preceding high-dose LPS stimulation (10,000 ng/ml). DUP-697 was used at a concentration of 25 µmol/l, and SC-236 was used at a concentration of 10 µmol/l (n = 5 experiments/group). The inhibitors were applied 10 min before AA administration to guarantee a sufficient preincubation period for structural binding and irreversible inhibition of COX-2 (30).

Further experiments were conducted with the application of an anti-TNF-alpha neutralizing antibody at two different concentrations (125 and 250 µg/l). One microgram of anti-TNF-alpha antibody is required to neutralize 25 µg of TNF-alpha in the buffer perfusate (neutralizing capacity). Rat lungs were challenged with 10,000 ng/ml of LPS, and the anti-TNF-alpha antibody was administered either 5 min before or 60 min after LPS administration (n = 5 experiments for each combination of anti-TNF-alpha antibody dosage and timing). The perfusate admixture of AA (5 µmol/l) was administered as in the preceding protocols.

In selected experiments, samples for perfusate analysis were taken at the onset of the 2-h perfusion period (0 min), after 1 h of perfusion (60 min), after 2 h of perfusion before application of AA (120 min), and 2 and 5 min after AA admixture application (122 and 125 min, respectively).

Perfusate analysis. TxA2 and PGI2 were assayed by ELISA from the recirculating buffer fluid as their stable hydrolysis products TxB2 and 6-keto-PGF1alpha , respectively. In addition, TNF-alpha 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.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Buffer-perfused lungs. In control lungs, no weight gain (Delta 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 approx 5 mmHg within 5 min (Table 1, Fig. 2). Concomitantly, a Delta W was noted, amounting to approx 1 g within 5 min. In contrast, VP remained unchanged in response to AA administration.

                              
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Table 1.   Delta PAP, Delta W, and Delta VP in response to AA challenge: impact of LPS priming



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Fig. 2.   Impact of LPS priming on AA-elicited responses in absence and presence of a selective cyclooxygenase (COX)-2 inhibitor. Lungs were buffer perfused in absence (control) and presence of different concentrations of LPS, and AA challenge (5 µmol/l) was undertaken after 2 h. In parallel groups, selective COX-2 inhibitor NS-398 was admixed to buffer fluid 10 min before AA administration. A: increase in pulmonary arterial pressure (Delta PAP). B: increase in ventilation pressure (Delta VP). C: weight gain (Delta W). Values are means ± SE of changes occurring within 5 min of AA challenge; n = 5 independent experiments each. Significant difference from control lungs (no LPS, no AA) or between groups: * P < 0.05; ** P < 0.01; *** P < 0.001.

PAP, VP, and Delta 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 Delta W of approx 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 Delta 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, Delta 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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Table 2.   Impact of COX-2 inhibition in AA-elicited response in LPS-primed lungs

In buffer-perfused lungs undergoing priming with 10,000 ng/ml of LPS for 2 h, the administration of anti-TNF-alpha neutralizing antibodies either 5 min before or 60 min after LPS administration did not affect the AA-induced responses of PAP, Delta W, or VP (Table 3, Fig. 3).

                              
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Table 3.   Delta PAP, Delta W, and Delta VP in response to AA challenge: effect of anti-TNF-alpha neutralizing antibody



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Fig. 3.   Impact of anti-TNF-alpha antibody (2 doses) on AA-induced Delta VP in LPS-primed lungs perfused in absence (A) and presence (B) of plasma. Lungs were buffer or buffer-plasma perfused in presence of 10,000 ng/ml of LPS, and AA challenge (5 µmol/l) was undertaken after 2 h. Antibody was admixed to perfusate either 5 min before (-5 min) or 60 min after LPS administration. Values are means ± SE of changes occurring within 5 min of AA challenge; n = 5 independent experiments each. Significant difference from lungs perfused in absence of antibody: * P < 0.05; ** P < 0.01.

In control lungs, only minor amounts of TxB2 and 6-keto-PGF1alpha 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-PGF1alpha ). 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 approx 250 pg/ml in control lungs and approx 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-PGF1alpha 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 PGF1alpha levels to approx 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 PGF1alpha were elevated in the presence of NS-398 in the LPS-primed lungs.


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Fig. 4.   AA-induced prostanoid release and impact of COX-2 inhibition. TxB2, thromboxane B2. Lungs were buffer perfused in absence (control) and presence of LPS, and AA challenge (5 µmol/l) was undertaken after 2 h. Selective COX-2 inhibitor was admixed to buffer fluid 10 min before AA administration. Values are means ± SE of TxB2 and 6-keto-PGF1alpha detected in perfusate 5 min after AA challenge; n = 5 independent experiments each. Significant difference from control lungs or between groups: * P < 0.05; ** P < 0.01; *** P < 0.001.

Although in control lungs only negligible levels of TNF-alpha were detected in the perfusion medium, addition of LPS caused a dose-dependent increase of TNF-alpha 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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Fig. 5.   Time course of TNF-alpha release into perfusate of LPS-primed lungs undergoing AA challenge. Lungs were buffer (-plasma) or buffer-plasma (+plasma) perfused in absence (control) and presence of LPS. AA was admixed to perfusate at a final concentration of 5 µmol/l after a 2-h recirculation period. Values are means ± SE; n = 5 independent experiments each. Note interruption of time scale after 2 h. Significant difference from respective control lungs: ** P < 0.01; *** P < 0.001.

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-alpha 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-alpha 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 Delta W in response to AA application were not altered by prior administration of TNF-alpha neutralizing antibodies in the buffer-plasma-perfused lungs (Table 3).

The amounts of TxB2 and PGF1alpha measured after 2 h of LPS priming within the buffer-plasma perfusate before and after administration of AA were comparable to those in buffer-perfused lungs.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-alpha 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-alpha 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-alpha liberation was, however, markedly increased, and anti-TNF-alpha 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-alpha 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-alpha 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 (approx 1-g Delta W within 5 min), and this, too, was markedly enhanced in the LPS-primed lungs (approx 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-alpha 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-alpha per se did not apparently suffice to provoke lung edema formation because no Delta W was noted after the 2-h LPS recirculation period and the TNF-alpha levels were maximally increased at this time point, with no further elevation on subsequent AA administration. Moreover, no reduction in Delta W was observed in the experiments employing anti-TNF-alpha 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 Delta 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-alpha 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-alpha 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-alpha , 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-alpha neutralizing antibodies applied at concentrations sufficient to bind the total amount of perfusate TNF-alpha 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-alpha in the bronchial compartment (the priming of the bronchoconstrictor response under conditions of plasma admixture), and thus high levels of TNF-alpha generation were markedly suppressed by anti-TNF-alpha antibodies. This finding thus supports the concept that proinflammatory cytokines like TNF-alpha 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-alpha 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-alpha generation. In addition, COX-2-dependent bronchoconstriction is noted in the LPS-primed lungs, with high levels of TNF-alpha 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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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