Neutrophil inhibitory factor prevents neutrophil-dependent lung injury.

Neutrophil inhibitory factor (NIF) is a recently cloned 41-kDa protein from the canine hookworm that binds CD11b/CD18 and inhibits CD11b/CD18-dependent neutrophil adhesion. We evaluated NIF's effects on neutrophil-dependent lung injury in guinea pigs. Pulmonary vascular endothelial CD54 (ICAM-1) was induced in buffer-perfused lungs by 90-min exposure to 1000 U/ml TNF-alpha. Human neutrophils (2 x 10(7)) were added to the perfusate and activated by 5 x 10(-9) PMA; in some lungs, the neutrophils were pretreated with NIF (100 nM) before their addition to the perfusate. Lung injury was assessed by wet:dry weight ratio, and neutrophil uptake by lung myeloperoxidase (MPO) activity. HUVEC exposed to TNF-alpha for 90 min were assayed for neutrophil adhesion, and we compared PMA-stimulated neutrophil adhesion to endothelial cells and fibrinogen-coated plates. PMA-induced pulmonary edema (lung wet:dry ratio increased from 8.8 +/- 0.7 to 18.8 +/- 4.4) was inhibited by NIF (10.0 +/- 1.0). Lung MPO activity concomitantly decreased from 17.1 +/- 6.1 to 8.7 +/- 1.8 U/mg dry lung tissue in the NIF-treated group, similar to controls (6.9 +/- 2.0). Endothelial monolayer experiments confirmed that NIF reduced neutrophil adherence (basal adhesion of 11 +/- 3% increased to 30 +/- 5% with TNF-alpha pretreatment of endothelial cells, an increase that was reduced to 10 +/- 4% with NIF). Moreover, NIF prevented PMA-induced neutrophil adhesion to fibrinogen, a CD11b/CD18-dependent event, but produced a smaller decrease in adherence to endothelial cells, which also involves CD11a/CD18 integrins. These studies indicate that NIF prevents neutrophil-dependent lung vascular injury by inhibiting neutrophil adhesion to the TNF-alpha-activated endothelium.

Muscarinic agonists and antagonists cause vasodilation in isolated rat lung.

The present study investigated the ability of atropine and different muscarinic receptor subtypes to affect acetylcholine (ACh)-induced bronchoconstriction and vasodilation in the isolated rat lung model. ACh (10(-7) M) given after U-46619 decreased total (RT), precapillary, and postcapillary vascular resistances and increased peak airway pressure. Atropine (20 microM) decreased RT and precapillary and postcapillary vascular resistances and blocked ACh-induced increases in peak airway pressure. The M1-selective agonist McN-A-343 (1.3 x 10(-5) M) decreased RT from 40.27 +/- 2.98 to 29.20 +/- 2.81 cmH2O.l-1.min-100 g lung wt (P = 0.01), and ACh caused no further dilation. The M1-selective antagonist pirenzepine (1.6 x 10(-6) M) blocked ACh-induced vasodilation. The M2-selective antagonist gallamine (7.5 x 10(-7) M) decreased RT from 45.50 +/- 3.19 to 34.86 +/- 1.25 cmH2O.l-1.min.100 g lung wt (P < 0.05), and after gallamine, ACh further decreased RT to 28.59 +/- 1.75 cmH2O.l-1.min.100 g lung wt (P < 0.01). Neither the selective muscarinic agonists nor antagonists affected peak airway pressures. We conclude that ACh-induced vasodilation in isolated rat lungs preconstricted with U-46619 is mediated by M1 receptors. Atropine-induced vasodilation in this model is mediated through the inhibition of the M2 receptor. We postulate that this represents either a blockade of postganglionic receptors, permitting release of vasodilator substances from local nerve terminals, or a direct vasodilatory effect on the vascular smooth muscle.

Vascular permeability and epithelial transport effects on lung edema formation in ischemia and reperfusion.

To determine the role of various Na+ transport systems in the edema fluid accumulation after ischemia and reperfusion in the lung, we evaluated the effect of amiloride (a Na+ channel blocker), ouabain (a Na(+)-K(+)-adenosinetriphosphatase blocker), and phloridzin (a Na(+)-glucose cotransport blocker) in isolated rat lungs. Ischemia and reperfusion (I/R) significantly increased the edema accumulation, with the wet-to-dry weight ratios increasing to 10.14 +/- 0.58 from 6.03 +/- 0.05 in control lungs (P < 0.04). Amiloride significantly augmented the amount of edema fluid (wet-to-dry weight ratio 12.26 +/- 0.77), and ouabain further increased the amount of edema (wet-to-dry weight ratio 18.58 +/- 1.00). Phloridzin did not significantly affect edema formation associated with I/R. Isoproterenol decreased the amount of edema formation in the presence and absence of amiloride. This occurred because the endothelial permeability as assessed by filtration coefficient was restored to normal values and less edema formed. The present study indicates that Na+ channels and Na(+)-K(+)-adenosinetriphosphatase, components of the active Na+ absorption transport system, are very important in opposing edema fluid accumulation in rat lungs subjected to I/R injury and operate as an edema safety factor. However, if the endothelial damage associated with I/R is allowed to persist, then the transport processes, even if operative, are insufficient to prevent continuous edema accumulation.

Reversal of pulmonary capillary ischemia-reperfusion injury by rolipram, a cAMP phosphodiesterase inhibitor.

Isoproterenol (ISO) and forskolin, agents that increase adenosine 3',5'-cyclic monophosphate (cAMP) via adenylyl cyclase activation, reverse lung injury associated with increased microvascular permeability. We studied the role of rolipram, a relatively isozyme-selective cAMP phosphodiesterase (PDE) inhibitor, in reversing increased capillary permeability due to ischemia-reperfusion (I/R), a form of oxidant injury in the lung, by using the isolated perfused rat lung model. Rolipram (2 microM) administered after 45 min of ischemia and 45 min of reperfusion reduced I/R-increased permeability as measured by the capillary filtration coefficient to control lung values. Computer image analysis of air space edema and perivascular cuffing, as well as wet-to-dry weight ratios, confirms the permeability reversal by rolipram administration. Rolipram inhibition of cAMP PDE in the lung was assessed by using [3H]adenine prelabeling adapted for the whole lung and perfusate [3H]cAMP accumulation. Rolipram failed to increase perfusate cAMP alone but dramatically increased perfusate cAMP above ISO alone. Dose-response relationships of ISO or rolipram show a close correlation of the half-maximal effective dose (ED50) for injury reversal and perfusate cAMP production. The combination of rolipram and ISO produced synergistic reversal of I/R injury. We conclude that reversal of I/R-induced increased microvascular permeability can be achieved with rolipram and that the mechanism of action of rolipram is probably through PDE isozyme-selective inhibition. The similarity of the ED50 values for cAMP efflux and reversal of permeability increases also supports a close coupling between cAMP accumulation and endothelial cell permeability.

Effect of nitric oxide and cyclooxygenase products on vascular resistance in dog and rat lungs.

Pulmonary vascular resistance decreases with increased cardiac output. Because nitric oxide (NO) and prostacyclin are potent vasodilators that are released with increased shear stress, their roles in the control of pulmonary vascular pressure were evaluated using isolated blood-perfused rat and dog lungs. Lungs were perfused with an initial arteriovenous pressure gradient (Ppa-Ppv) of 15 cmH2O; Ppa and Ppv were increased by the same amount, and the flow was measured. In rat lung (n = 6), the NO synthesis inhibitor NG-nitro-L-arginine methyl ester (L-NAME) decreased pulmonary blood flow by approximately 50% at the same pressure (P < 0.05), whereas the cyclooxygenase inhibitor indomethacin (n = 6) had no effect. In dog lungs (n = 6), indomethacin decreased pulmonary blood flow by approximately 50% at the same pressure gradient (P < 0.05), whereas L-NAME (n = 6) had no effect. Furthermore, the flow increase that occurs as venous and arterial pressures are elevated together (so that Ppa-Ppv is constant) was inhibited by L-NAME in rat lungs and by indomethacin in dog lungs (P < 0.05 for each). Plasma guanosine 3',5'-cyclic monophosphate (cGMP) rose with increased absolute pressure in rat lung [from 71 +/- 17 to 274 +/- 104 pM (P < 0.05)], and this increase was blocked by L-NAME. Plasma cGMP was unchanged in dog lung, but the ratio of prostacyclin to thromboxane tended to be higher.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine prevents PMA-induced lung injury via an A2 receptor mechanism.

Previous studies indicate that adenosine attenuates phorbol myristate acetate-(PMA) induced canine lung injury, but the mechanism has not been explained. To evaluate adenosine's protective mechanism, isolated and blood-perfused dog lungs were challenged by PMA (50 micrograms) under control conditions and after both pre- and post-treatment with adenosine and pretreatment with 2-chloro-N6-cyclopentyladenosine (CCPA), 2-[p-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido adenosine (CGS 21680C), 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; PD-116948), or isoproterenol. Injury was assessed by measurement of the capillary filtration coefficient (Kf,c), and pulmonary vascular resistance was measured. PMA increased the Kf,c (0.170 +/- 0.015 to 1.030 +/- 0.167 ml.min-1.cmH2O-1.100 g lung wet wt-1) and the total pulmonary vascular resistance (18.2 +/- 3.8 to 110.2 +/- 60.8 cmH2O.l-1.min.100 g lung wet wt). Pretreatment with adenosine, A2 agonist, A1 antagonist, and isoproterenol blocked the increase in Kf,c induced by PMA. These agents also slightly attenuated the resistance increase induced by PMA, with the exception of the A1 antagonist, which completely prevented the resistance increase (24.3 +/- 7.8 to 23.4 +/- 8.1 cmH2O.l-1.min.100 g lung wet wt). The A1 agonist also slightly attenuated the increase in Kf,c (0.174 +/- 0.022 to 0.486 +/- 0.128 ml.min-1.cmH2O-1.100 g lung wet wt-1) and did not affect the resistance increase. Posttreatment with adenosine did not significantly affect the changes induced by PMA. These data show that PMA-induced increases in capillary permeability in the isolated blood-perfused dog lung can be blocked by pretreatment with adenosine, which binds the adenosine A2 receptors.

Evaluation of prostaglandin F2 alpha and prostacyclin interactions in the isolated perfused rat lung.

To characterize the interactions between prostaglandin F2 alpha and prostacyclin in controlling tone in the pulmonary circulation, isolated rat lungs were ventilated, perfused with blood, and subjected to challenge by prostaglandin F2 alpha in increasing doses. The pulmonary resistance was evaluated using occlusion techniques that separate the resistance into segments of large and small arteries and veins. The total vascular compliance was evaluated using outflow occlusion. Resistance increased after prostaglandin F2 alpha, and this resistance change was primarily in the small artery segment. The maximum resistance increase by prostaglandin F2 alpha (Rmax,PGF2 alpha), calculated from the Michaelis-Menton equation, was 16.6 +/- 3.6 cmH2O.l-1.min.100 g-1 for total vascular resistance with a concentration required to produce 50% Rmax (K0.5) of 5.26 +/- 3.57 nM. The Rmax,PGF2 alpha for small artery resistance was 13.5 +/- 2.4 cmH2O.l-1.min.100 g-1 with a K0.5 of 2.35 +/- 1.57 nM. The vascular compliance decreased during vasoconstriction by prostaglandin F2 alpha, and the maximum decrease in compliance (Cmin,PGF2 alpha) was -0.43 +/- 0.12 ml/cmH2O with a K0.5 of 2.84 +/- 2.99 nM. At each dose of prostaglandin F2 alpha, prostacyclin was administered in increasing doses to reverse the vasoconstriction caused by prostaglandin F2 alpha. For each concentration of prostaglandin F2 alpha, prostacyclin almost completely reversed the resistance increases and approximately one-half the compliance decrease. The maximum change in vascular resistance or compliance produced by prostacyclin was dependent on the dose of prostaglandin F2 alpha; yet the K0.5 for prostacyclin was within the picomolar range for all doses of prostaglandin F2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of thromboxane and prostacyclin effects on pulmonary vascular resistance.

Although thromboxane and prostacyclin (PGI2) have long been described as major controllers of pulmonary vascular resistance, little has been reported on the characteristics of the interactions between the two arachidonic acid products. The current study uses segmental vascular resistance and compliance measurements to evaluate the actions of thromboxane and PGI2 in isolated blood-perfused rat lung. The thromboxane analogue U-46619 increases pulmonary vascular resistance by increasing only small artery resistance and decreases pulmonary vascular compliance in the middle compartment. Among the vascular effects of U-46619 are a maximum increase in resistance (RmaxU-46619) of 60.3 +/- 15.6 cmH2O.l-1.min.100 g-1 and a concentration required for 50% of maximum increase (K0.5,U-46619) of 1.60 +/- 0.85 nM for small artery resistance, a minimum vascular compliance (CminU-46619) of -0.93 +/- 0.58 cmH2O, and a K0.5,U-46619 of 1.10 +/- 1.60 nM for middle compartment compliance. Similar results were obtained for total resistance and total compliance. The effects of PGI2 on thromboxane-induced resistance and compliance changes were evaluated using K0.5,PGI2, RmaxPGI2, and CmaxPGI2 at each dose of thromboxane. PGI2 was more effective in reversing the thromboxane constriction at higher concentrations of thromboxane. These data show that the absolute concentration of PGI2 and thromboxane and not a simple ratio of thromboxane to PGI2 determines vascular tone.

Lung protection against paraquat is calcium dependent.

Isolated, perfused, and ventilated rat lungs were challenged by paraquat (0.01 M) in the presence of 2.5 mM Ca2+, 2.5 mM Ca2+ with trifluoperazine (100 microM), 0.025 mM Ca2+, or 0.025 mM Ca2+ with sodium metavanadate (10 microM) to establish the effect of varying calcium concentration or calcium-dependent enzyme activities on injury induced by paraquat. Segmental vascular resistances, microvascular permeability (as assessed by the capillary filtration coefficient), lung tissue oxidized glutathione, and lung paraquat accumulation were measured. Exposure to paraquat for 2.5 h did not increase microvascular permeability or pulmonary vascular resistance in the presence of either normal extracellular calcium or low extracellular calcium and sodium metavanadate. Lungs exposed to paraquat were injured (as assessed by increased filtration coefficient) only in the presence of low extracellular calcium or after trifluoperazine was added. This injury was associated with decreased levels of oxidized glutathione and increased paraquat accumulation, suggesting that calcium's protective effect was both by inhibition of paraquat accumulation and maintenance of NADPH. Pulmonary vascular resistance was not increased with paraquat challenge.

Compounds that increase cAMP prevent ischemia-reperfusion pulmonary capillary injury.

This study evaluated the physiological effects of compounds that increase adenosine 3',5'-cyclic monophosphate (cAMP) on changes in pulmonary capillary permeability and vascular resistance induced by ischemia-reperfusion (I-R) in isolated blood-perfused rabbit lungs. cAMP was elevated by 1) beta-adrenergic stimulation with isoproterenol (ISO, 10(-5) M), 2) post-beta-receptor stimulation of adenylate cyclase with forskolin (FSK, 10(-5) M), 3) and dibutyryl cAMP (DBcAMP, 1 mM), a cAMP analogue. Vascular permeability was assessed by determining the capillary filtration coefficient (Kf,c), and capillary pressure was measured using the double occlusion technique. The total, arterial, and venous vascular resistances were calculated from measured pulmonary arterial, venous, and capillary pressures and blood flow. Reperfusion after 2 h of ischemia significantly (P less than 0.05) increased Kf,c (from 0.115 +/- 0.028 to 0.224 +/- 0.040 ml.min-1.cmH2O-1.100 g-1). These I-R-induced changes in capillary permeability were prevented when ISO, FSK, or DBcAMP was added to the perfusate at reperfusion (0.110 +/- 0.022 and 0.103 +/- 0.021, 0.123 +/- 0.029 and 0.164 +/- 0.024, and 0.153 +/- 0.030 and 0.170 +/- 0.027 ml.min-1.cmH2O-1.100 g-1, respectively). I-R significantly increased total, arterial, and venous vascular resistances. These increases in vascular resistance were also blocked by ISO, FSK, and DBcAMP. These data suggest that beta-adrenergic stimulation, post-beta-receptor activation of adenylate cyclase, and DBcAMP prevent the changes in pulmonary vascular permeability and vascular resistances caused by I-R in isolated rabbit lungs through a mechanism involving an increase in intracellular levels of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

Sustained effects of endothelin-1 on rabbit, dog, and rat pulmonary circulations.

Effects of endothelin-1 (10(-8) M) on the pulmonary vascular resistance-compliance profile were examined in isolated blood-perfused rabbit, dog, and rat lungs using occlusion techniques. Capillary permeability was assessed by filtration coefficient (Kfc). Cyclooxygenase products were assessed by radioimmunoassay. In rabbit lungs, endothelin-1 increased all resistances except large vein; ibuprofen reversed the constriction. Endothelin-1 decreased total vascular compliance (CT), which was reversed by ibuprofen. Cyclooxygenase products were unchanged by endothelin-1 or endothelin-1 plus ibuprofen. In dog lungs, large vein resistance increased after endothelin-1; ibuprofen increased large arterial resistance. Endothelin-1 decreased CT and middle compartment compliance, and endothelin-1 plus ibuprofen decreased large vessel compliance (CLV). Ibuprofen reversed the endothelin-1 increase in plasma 6-oxo-prostaglandin F1 alpha. In rat lungs, ibuprofen reversed the endothelin-1 increase in small arterial resistance. Endothelin-1 decreased CLV, and endothelin-1 plus ibuprofen returned the compliance to baseline values. Ibuprofen potentiated the endothelin-1 increase in plasma prostanoids. Endothelin-1 plus indomethacin increased vascular resistance and blocked prostaglandin production. Kfc was increased only in rat lung after endothelin-1 plus ibuprofen. In summary, endothelin-1 increased pulmonary vascular resistance, which was attenuated by prostacyclin in dogs and rats. In rabbits, the resistance increase was reversed by ibuprofen.

Effects of phorbol myristate acetate-stimulated human leukocytes on rat lung.

Human blood was separated into polymorphonuclear (PMN) and mononuclear (MN) leukocyte fractions, and 3 x 10(7) cells (PMN or MN) were added to isolated rat lungs perfused with 5% human albumin in buffer and stimulated with phorbol myristate acetate (PMA). Lungs perfused with either albumin alone, PMN, or MN but not stimulated with PMA showed no change in vascular resistance or endothelial permeability measured as the capillary filtration coefficient (Kf,c). Lungs that were stimulated with PMA with no cells showed no change in Kf,c (0.34 +/- 0.07 vs. 0.37 +/- 0.7), but vascular resistance increased in all segments of the circulation. Capillary pressure, the major force responsible for edema formation, nearly doubled in the absence of cells 40 min after PMA. Lungs perfused with either PMN or MN and stimulated with PMA were injured. Kf,c increased from 0.41 +/- 0.03 to 0.87 +/- 0.10 (PMN) and from 0.36 +/- 0.07 to 0.81 +/- 0.23 (MN) 90 min after PMA. In addition to the increased endothelial permeability, vascular resistances and pressures also increased in the cell-perfused PMA-stimulated lungs. These results demonstrate that cells other than granulocytes are capable of producing severe acute lung injury and cannot be ignored when the effects of PMA on neutrophil-depleted lungs are studied.

The role of activation of neutrophils and microvascular pressure in acute pulmonary edema.

Activated polymorphonuclear neutrophils (PMN) can mediate vascular injury in the lung. This study compared activated aggregate PMN (emboli) to activated PMN that were previously adhered to the microvasculature (non-embolic) in the isolated perfused rat lung. Permeability and microvascular pressure (Pmv), components of PMN-induced edema, were examined by continuous measurement of wet weight, pulmonary arterial and left atrial pressures, and by intermittent determination of double occlusion pressure. PMN that were activated with phorbol myristate acetate and then perfused into the lung formed aggregates that lodged primarily in the precapillary bed, increasing arterial resistance. Although these PMN had minimal direct contact with the capillary endothelium, edema rapidly developed and Pmv was progressively elevated. If PMN were allowed to adhere in the capillary bed, a minimal and nonprogressive increase in Pmv and lung weight occurred. When these adherent PMN were then activated, there was a progressive rise in both Pmv and lung weight. The free radical scavenger catalase prevented this edema formation but not the rise in pressure. In control lungs with matched elevation of Pmv, edema did not develop. In another group of lungs with activation of pre-adherent PMN in which Pmv was maintained at control levels, edema formation was greatly delayed. These data show that: (1) the activated PMN free radical products alone caused permeability injury in the lung because neither contact of the PMN with the capillary endothelium nor embolization was necessary, and (2) increased Pmv does not cause edema but greatly increases the rate of edema formation when the endothelium is injured.

Role of microvascular pressure in reactive oxygen-induced lung edema.

O2 radicals are important in the pathogenesis of acute lung injury. The purpose of this investigation was to determine the role that microvascular pressure plays in edema induced by reactive O2 species generated by xanthine oxidase. In isolated rat lungs perfused with Krebs buffer plus 4% albumin, 5 mM glucose, and 2 mM xanthine at constant flow (13 ml/min), addition of xanthine oxidase (0.02 U/ml) caused a progressive increase in both pulmonary arterial and microvascular pressure (double occlusion method), which preceded the onset of edema. Both the pressure rise and edema formation were blocked by catalase, suggesting that vascular injury was related to H2O2 production. Lungs not exposed to free radicals that had microvascular pressure elevated to match that of the xanthine oxidase-perfused lungs showed only a small, reversible (nonedematous) weight gain. Lungs exposed to xanthine oxidase but perfused at constant microvascular pressure (5 Torr, similar to control lungs) showed a significant delay in protein-rich edema formation. These data indicate that reactive O2 metabolites induced lung injury, which is accompanied by increased microvascular pressure. Although the rise in microvascular pressure was shown not to be essential for edema formation, it does play a role in acceleration of the rate of transvascular fluid loss.