Role of circulating platelets and granulocytes in PAF-induced pulmonary dysfunction in awake sheep

The effects of a single intravascular bolus injection of platelet-activating factor (PAF) on pulmonary hemodynamics, lung mechanics, and lung fluid and solute exchange were studied in 13 chronically instrumented unanesthetized sheep. Since PAF has profound effects on both platelets and granulocytes, we investigated the effects of platelet and granulocyte depletion on the sheep's response to exogenous PAF. Sheep received PAF when granulocyte and platelets counts were normal and after platelet depletion with rabbit antisheep platelet antibodies (n = 5) or granulocyte depletion with hydroxyurea (n = 5). Sheep served as their own controls, and the order of experimentation was varied. Bolus injections of PAF had reproducible effects on pulmonary hemodynamics (pulmonary arterial pressure increased acutely to 85 +/- 3.7 cmH2O) and lung mechanics (dynamic compliance of the lungs decreased to 24.5 +/- 3.8% of base line and resistance to airflow across the lungs increased greater than 10-fold) and caused marked increases in lung lymph concentrations of thromboxane B2 and 6-ketoprostaglandin F1 alpha. The single bolus injection of PAF also caused marked prolonged elevations in lung lymph flow and increases in the lymph-to-plasma protein concentration ratio for 3 h after PAF. PAF had profound effects despite platelet and granulocyte depletion. Platelet depletion slightly attenuated the pulmonary hypertension observed after PAF injection. Platelet depletion also attenuated the increases in thromboxane B2 concentrations in lung lymph, and lung mechanics normalized more rapidly in platelet-depleted sheep. There were no statistically significant effects of granulocyte depletion to less than 200 granulocytes/mm3 on any of the measured variables.(ABSTRACT TRUNCATED AT 250 WORDS)     

CVF-induced decomplementation: effect on lung transvascular protein flux after thrombin

We examined the effects of cobra venom factor (CVF) on the changes in pulmonary hemodynamics and transvascular fluid and protein exchange following thrombin-induced pulmonary microembolism. Studies were made in unanesthetized sheep prepared with lung lymph fistulas. The animals received tranexamic acid (100 mg) to suppress fibrinolysis and were then challenged with an intravenous infusion of alpha-thrombin (80 U/kg). Control-thrombin challenged sheep were compared with the CVF-treated sheep challenged with the same thrombin dosage. CVF treatment (187 U X kg-1 X day-1 for 4 days) decreased the total hemolytic complement activity by 45% of control. Thrombin infusion in control sheep increased the mean pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), and lymph protein clearance (pulmonary lymph flow X lymph-to-plasma protein concentration ratio, Clym). Thrombin infusion in CVF-treated sheep produced smaller increments in Ppa, PVR, and Clym. Pulmonary lymph obtained from control-thrombin and CVF-thrombin sheep induced migration of granulocytes obtained from normal unchallenged sheep. The granulocytes obtained from CVF-treated sheep responded relatively less to the migratory and O-2-generating stimuli (i.e., zymosan-treated serum, pulmonary lymph from sheep after thrombin challenge, and plasma from sheep after CVF treatment) compared with normal granulocytes. The attenuation of the thrombin-induced increases in Ppa, PVR, and lung transvascular fluid and protein exchange by CVF treatment may be the result of impaired function of granulocytes.     

Effect of inhibition of 5-lipoxygenase metabolism of arachidonic acid on response to endotoxemia in sheep

We studied the effects of a 5-lipoxygenase inhibitor, L-651,192, on the pulmonary dysfunction caused by endotoxemia in chronically instrumented unanesthetized sheep. The efficacy and selectivity of L-651,392 were tested by measuring in vivo production of leukotriene B4 (LTB4) and cyclooxygenase products of arachidonic acid after endotoxemia before and after pretreatment with L-651,392 and ex vivo from granulocytes and whole blood stimulated with calcium ionophore from sheep before and 24 h after pretreatment with L-651,392. A novel assay for LTB4 by high-performance liquid chromatography/gas chromatography/mass spectrometry techniques was developed as a measure of 5-lipoxygenase metabolism of arachidonic acid. L-651,392 proved to be an effective in vivo 5-lipoxygenase inhibitor in sheep. L-651,392 blocked the increase in LTB4 observed in lung lymph after endotoxemia in vivo in sheep as well as inhibited by 80% the ex vivo production of LTB4 by granulocytes removed from sheep treated 24 h earlier with L-651,392. Although L-651,392 blocked the increase in cyclooxygenase products of arachidonic acid observed in lung lymph after endotoxemia in vivo in sheep, the drug probably did not function directly as a cyclooxygenase inhibitor. L-651,392 did not attenuate the ex vivo production of thromboxane B2 by whole blood from sheep treated 24 h earlier with the drug. L-651,392 attenuated the alterations in pulmonary hemodynamics, lung mechanics, oxygenation, and lung fluid and solute exchange observed after endotoxemia in sheep. We speculate that 5-lipoxygenase products are a major stimulus for cyclooxygenase metabolism of arachidonic acid after endotoxemia in sheep.     

Effects of thromboxane synthase inhibition on tumor necrosis factor-induced lung injury in sheep.

To examine the role of thromboxane (Tx) A2 in the pathogenesis of acute lung injury caused by tumor necrosis factor alpha (TNF), we tested the effects of OKY-046, a selective thromboxane synthase inhibitor, on pulmonary hemodynamics, lung lymph balance, circulating leukocytes, arterial blood gas analysis, and TxA2 (as TxB2) and prostacyclin (as 6-keto-prostaglandin F1 alpha) levels in plasma and lung lymph after TNF infusion in awake sheep. Infusion of human recombinant TNF (3.5 micrograms/kg) into a chronically instrumented awake sheep caused a transient increase in pulmonary arterial pressure (Ppa). The Ppa peaked within 15 min of the start of TNF infusion from 23.3 +/- 1.1 (SE) cmH2O of baseline to 42.3 +/- 2.3 cmH2O and then decreased toward baseline. The pulmonary hypertension was accompanied by transient hypoxemia, peripheral leukopenia, and the increases in TxB2 in plasma and lung lymph. These changes were followed by an increase in flow of protein-rich lung lymph, consistent with an increase in pulmonary microvascular permeability. OKY-046 significantly prevented the rises of Ppa and TxB2 concentrations in plasma and lung lymph during early phase after TNF infusion. OKY-046, however, did not attenuate the increase of lung lymph flow, transient hypoxemia, and leukopenia. From these data, and by comparison with our previous studies of OKY-046-pretreated sheep during endotoxemia, we conclude that TxA2 has an important role of the increase in the early pulmonary hypertension, but it is not related to the early hypoxemia, leukopenia, and lung lymph balances in TNF-induced lung injury.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lung vascular injury after Escherichia coli endotoxin and phorbol myristate acetate infusion in dogs

The effects of Escherichia coli endotoxin and phorbol myristate acetate (PMA), a potential stimulator of polymorphonuclear leukocyte (PMN), on circulating PMN counts, gas exchange, protein concentration of lavage fluid, pulmonary hemodynamics and pathology of the lung were studied in ten anesthetized dogs. Six dogs were infused with 1 microgram/kg endotoxin plus 10 micrograms/kg of PMA; four other dogs were infused with the same amount of endotoxin but 5 micrograms/kg of PMA. After administration of endotoxin plus 10 micrograms/kg PMA, the number of circulating PMN (per mm3) decreased dramatically from 4081 +/- 1041 to 303 +/- 119, arterial oxygen partial pressure (PaO2) dropped to 49.1 +/- 2.4 mmHg and the arterial alveolar oxygen partial pressure difference (A-a DO2) increased significantly above baseline. Lungs from this group appeared to be grossly damaged: edema with distinct petechial hemorrhage and areas of hemorrhagic consolidation; frothy edema fluid often emanated from the tracheas. The group infused with endotoxin plus 5 micrograms/kg PMA showed no significant decrease in the number of PMN; PaO2 and A-a DO2 maintained comparatively stable. Protein concentration of lavage fluid and lung wet/dry weight ratios in dogs of 10 micrograms/kg PMA group were significantly increased (P less than 0.05) as compared to those of 5 micrograms/kg PMA group. Our study showed that the magnitude of leukopenia after endotoxin and PMA was paralleled with the severity of lung vascular injury. These results support the potential role of PMN in the pathogenesis of acute edematous lung injury.     

Effects of methylprednisolone on lung oxygen toxicity in awake sheep

The purpose of this study was to measure the effects of high doses of corticosteroids on the response to breathing 100% O2 in sheep. Sheep were prepared for chronic measurement of vascular pressures, cardiac output, gas exchange, and for collection of lung lymph. Tracheostomies were made for accurate delivery of gas mixtures. Eight sheep received methylprednisolone 30 mg/kg body wt every 6 h for eight doses, four for the first 48 h, and four for the final 24-48 h of 100% O2 breathing. Eight control sheep breathed 100% O2 without methylprednisolone, four sheep breathed compressed air without methylprednisolone, and two breathed compressed air and received methylprednisolone. Sheep had daily measurements of hypoxic vasoconstriction (fractional concentration of O2 in inspired gas = 0.12), gas exchange, lymph flow, and lymph and plasma protein concentration. Polymorphonuclear leukocyte (granulocyte) function in experimental and control sheep was assessed ex vivo by tests of chemotaxis, aggregation, and superoxide production. The number of granulocytes in peripheral lung was measured in biopsy tissue taken at the time of original surgery and postmortem. Methylprednisolone did not affect the time course nor magnitude of gas exchange abnormality, lymph flow and composition, loss of hypoxic vasoconstriction, lung granulocyte accumulation, nor postmortem lung water caused by 100% O2 breathing. Sheep receiving methylprednisolone had a shorter survival by several h, independent of the timing of the drug. Granulocytes from methylprednisolone-treated sheep showed normal function ex vivo by all three assays. We conclude that high doses of methylprednisolone unfavorably affect the rate and progression of lung injury in sheep breathing 100% O2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pulmonary microcirculatory responses to leukotrienes B4, C4 and D4 in sheep

The pulmonary microvascular responses to leukotrienes B4, C4, and D4 (total dosage of 4 micrograms/kg i.v.) were examined in acutely-prepared halothane anesthetized and awake sheep prepared with lung lymph fistulas. In anesthetized as well as unanesthetized sheep, LTB4 caused a marked and transient decrease in the circulating leukocyte count. Pulmonary transvascular protein clearance (pulmonary lymph flow X lymph-to-plasma protein concentration ratio) increased transiently in awake sheep, suggesting a small increase in pulmonary vascular permeability. The mean pulmonary artery pressure (Ppa) also increased. In the acutely-prepared sheep, the LTB4-induced pulmonary hemodynamic and lymph flow responses were damped. Leukotriene C4 increased Ppa to a greater extent in awake sheep than in anesthetized sheep, but did not significantly affect the pulmonary lymph flow rate (Qlym) and lymph-to-plasma protein concentration (L/P) ratio in either group. LTD4 increased Ppa and Qlym in both acute and awake sheep; Qlym increased without a significant change in the L/P ratio. The LTD4-induced rise in Ppa occurred in association with an increase in plasma thromboxane B2 (TxB2) concentration. The relatively small increase in Qlym with LTD4 suggests that the increase in the transvascular fluid filtration rate is the result of a rise in the pulmonary capillary hydrostatic pressure. In conclusion, LTB4 induces a marked neutropenia, pulmonary hypertension, and may transiently increase lung vascular permeability. Both LTC4 and LTD4 cause a similar degree of pulmonary hypertension in awake sheep, but had different lymph flow responses which may be due to pulmonary vasoconstriction at different sites, i.e. greater precapillary constriction with LTC4 because Qlym did not change and greater postcapillary constriction with LTD4 because Qlym increased with the same rise in Ppa.     

Role of interstitium in clearance of alveolar fluid in normal and injured lungs

We investigated the contribution of the pulmonary interstitial space to the removal of alveolar fluid and solute. We prepared anesthetized sheep for the collection of lung lymph. A balloon-tipped catheter was advanced into a lower lung lobe, and 20 ml Ringer lactate solution (RL) were instilled in one group. Other groups received 20 ml RL with 4 mg/ml Evans blue dye (EB) or 10 micrograms/kg phorbol myristate acetate (PMA) or both. Instillation of 20 ml RL and EB resulted in an increase in lymph flow over RL alone, presumably by an osmotic mechanism. After 4 h, small perivascular fluid cuffs, which contained little EB, were present, and 1.9% of the instilled EB was removed by the lymphatics. An average of 9.2 ml of excess water remained in the lung. Instillation of RL, EB, and PMA resulted in an increase in lymph flow and large perivascular fluid cuffs, which contained large amounts of EB. Lymphatic removal of the instilled EB accounted for 1.2% of the total amount instilled. An average of 19.1 ml water was present in the lung after 4 h. We conclude that alveolar instillation of PMA results in epithelial and endothelial membrane injury and that when lung injury is present interstitial fluid reservoirs may be important sites of alveolar fluid accumulation and important routes of fluid removal from the air space.     

Detergent inhibits 70-90% of responses to intravenous endotoxin in awake sheep.

Sheep have reactive pulmonary intravascular macrophages, which are essential for the marked pulmonary vascular response to infusions of small quantities of endotoxin. In another species with reactive pulmonary intravascular macrophages, horses, our laboratory found that an intravenous biosafe detergent, tyloxapol, inhibited some systemic and pulmonary responses to endotoxin (Longworth KE, Smith BL, Staub NC, Steffey EP, and Serikov V. Am J Vet Res 57: 1063-1066, 1996). We determined whether the same detergent would inhibit endotoxin responses in awake sheep. In 10 awake, instrumented sheep with chronic lung lymph fistulas, we did a control experiment by intravenously infusing 1 microg/kg Escherichia coli endotoxin. One week later, we gave 40 micromol/kg tyloxapol intravenously 1-4 h before infusing the same dose of endotoxin. In these paired studies, we compared pulmonary hemodynamics, lung lymph dynamics, body temperature, circulating leukocyte concentrations, and circulating tumor necrosis factor for 6 h. In all 10 sheep, tyloxapol blocked 80-90% of the pulmonary responses and 70-90% of the systemic responses. Tyloxapol is safe, inexpensive, easy to use, and effective immediately. It may be a clinically useful approach to contravening many of the effects of endotoxemia, in humans as well as animals.     

Effects of ONO-6240, a platelet-activating factor antagonist, on endotoxin shock in unanesthetized sheep

To determine the role of platelet-activating factor (PAF) in endotoxin shock, we studied the effects of ONO-6240, a PAF antagonist, on endotoxin shock in unanesthetized sheep. Changes in hemodynamics, lung lymph balance, leukocyte and platelet counts, and arterial blood gas tensions were measured in four groups; endotoxin alone; endotoxin plus ONO-6240; ONO-6240 alone; vehicle control. Pretreatment with ONO-6240 in sheep given endotoxin significantly prevented the decreases in systemic arterial pressure, left atrial pressure and cardiac output observed in sheep given endotoxin alone. A partial effect on diminishing the magnitude of peripheral leukopenia was also noted. However, pretreatment with ONO-6240 had little effect on pulmonary hypertension and lung lymph balance. We conclude that endotoxin causes two different effects: vascular collapse and direct lung injury; and that PAF is involved only in the circulatory manifestations.     

Oleic acid lung injury in sheep

Intravenous infusion of oleic acid into experimental animals causes acute lung injury resulting in pulmonary edema. We investigated the mechanism of oleic acid lung injury in sheep. In experiments with anesthetized and unanesthetized sheep with lung lymph fistulas, we measured pulmonary arterial and left atrial pressures, cardiac output, lung lymph flow, and lymph and plasma protein concentrations. We injured the lungs with intravenous infusions of oleic acid at doses ranging from 0.015 to 0.120 ml/kg. We found that oleic acid caused reproducible dose-related increases in pulmonary arterial pressure and pulmonary vascular resistance, arterial hypoxemia, and increased protein-rich lung lymph flow and extravascular lung water. The lung fluid balance changes were characteristic of increased permeability pulmonary edema. Infusion of the esterified fat triolein had no hemodynamic or lung fluid balance effects. Depletion of leukocytes with a nitrogen mustard or platelets with an antiplatelet serum had no effect on oleic acid lung injury. Treatment of sheep before injury with methylprednisolone 30 mg/kg or ibuprofen 12.5-15.0 mg/kg also had no effects. Unlike other well-characterized sheep lung injuries, injury caused by oleic acid does not require participation of leukocytes.     

Thromboxane increases pulmonary vascular resistance and transvascular fluid and protein exchange after pulmonary microembolism

We compared the effects of inhibition of thromboxane synthetase with antagonism of thromboxane A2 (TxA2)/prostaglandin H2 receptors on the changes in pulmonary hemodynamics and pulmonary transvascular fluid and protein exchange following thrombin-induced pulmonary microembolism. Studies were made in chronically instrumented unanesthetized sheep prepared with lung lymph fistulas. Control thrombin challenged sheep (n = 5) were compared to animals pretreated with Dazoxiben (the Dazoxiben-thrombin group, n = 8) or animals pretreated with L-640,035 (the L-640,035-thrombin group, n = 5). In the control-thrombin sheep, plasma TxA2 concentration rose after thrombin and the response was inhibited in the Dazoxiben-thrombin group. The rise in the plasma TxA2 concentration was greater in the L-640,035-thrombin group than in the control-thrombin group. In the control-thrombin group, thrombin produced a sustained increase in the pulmonary transvascular protein clearance (pulmonary lymph flow x lymph/plasma protein concentration ratio) and pulmonary vascular resistance (PVR). In the Dazoxiben-thrombin group, increases in both pulmonary transvascular protein clearance and PVR after thrombin were less than in the control-thrombin group. In the L-640,035-thrombin group, thrombin initially increased pulmonary transvascular protein clearance and PVR to the same levels as the control group; however, both protein clearance and PVR declined with time, in contrast to the sustained responses in the control-thrombin group. These differences may be related to the initially greater increase in plasma TxA2 concentrations after thrombin in the L-640,035-treated animals. The results indicate that TxA2 plays a role in mediating the increases in PVR and contributes to increases in pulmonary transvascular fluid and protein exchange after thrombin-induced pulmonary microembolism.     

Effects of ONO-6240, a platelet-activating factor antagonist, on endotoxin shock in unanesthetized sheep

To determine the role of platelet-activating factor (PAF) in endotoxin shock, we studied the effects of ONO-6240, a PAF antogonist, on endotoxin shock in unanesthetized sheep. Changes in hemodynamics, lung lymph balance, leukocyte and platelet counts, and arterial blood gas tensions were measured in four groups; (1) endotoxin alone; (2) endotoxin plus ONO-6240; (3) ONO-6240 alone; (4) vehicle control. Pretreatment with ONO-6240 in sheep given endotoxin significantly prevented the decreases in systemic arterial pressure, left atrial pressure and cardiac output observed in sheep given endotoxin alone. A partial effect on diminishing the magnitude of peripheral leukopenia was also noted. However, pretreatment with ONO-6240 had little effect on pulmonary hypertension and lung lymph balance. We conclude that endotoxin causes two different effects: vascular collapse and direct lung injury; and that PAF is involved only in the circulatory manifestations.     

Administration of a synthetic antiprotease reduces smoke-induced lung injury.

Our previous studies suggest that a neutrophil-mediated inflammatory injury causes a major fraction of the pulmonary edema that occurs after smoke inhalation. Because activated neutrophils extrude cytotoxic proteases, the current study was conducted to evaluate the role of proteases in the pulmonary microvascular injury. Twelve sheep, instrumented for collection of lung lymph, were insufflated with cotton smoke. The sheep were treated 30 min after smoke inhalation with either gabexate mesilate (an inhibitor of serine proteases) or vehicle. Smoke inhalation resulted in an increased protease activity in the lung interstitium, as evidenced by decreases in both antiprotease activity and immunoreactive alpha 2-macroglobulin. Intravenous infusion of gabexate mesilate prevented the decrease in antiprotease activity. The protease inhibitor significantly attenuated the smoke-induced increase in transvascular fluid and protein flux, with untreated animals exhibiting 460% increases in flux compared with 180% in the inhibitor treated sheep. The protease inhibitor also eliminated the functional degradation in gas exchange that was observed in the untreated sheep. These studies strongly suggest that an increase in pulmonary proteolytic enzyme activity is responsible for a significant fraction of the degradation in microvascular integrity and gas exchange that is associated with smoke inhalation injury.     

Prostaglandin E2 attenuation of sheep lung responses to endotoxin

Prostaglandin (PG) E2 can inhibit inflammatory responses of neutrophils and lymphocytes, including eicosanoid release. Diffuse lung injury after endotoxemia in sheep is accompanied by sequestration of neutrophils and lymphocytes in the lungs, and eicosanoids mediate some of the pathophysiology of the response. To determine whether exogenous PGE2 could prevent the endotoxin response, we measured pulmonary hemodynamics, gas exchange, and lung lymph responses to infusion of Escherichia coli endotoxin (0.5 micrograms/kg iv over 30 min) in unanesthetized sheep in the presence and absence of PGE2 (0.5 micrograms.kg-1.min-1) infused intravenously for 4 h beginning 0.5 h before endotoxin infusion. We also measured lung lymph concentrations of thromboxane B2 (TxB2) and prostacyclin metabolite, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), by radioimmunoassay and leukotriene B4 (LTB4) by gas chromatography-mass spectrometry. PGE2 decreased endotoxin-induced pulmonary hypertension and hypoxemia and markedly attenuated the lymph flow and lymph protein clearance responses. PGE2 also attenuated endotoxin-induced increases in lung lymph TxB2 and 6-keto-PGF1 alpha and decreased lymph LTB4 flow after endotoxin without decreasing lymph LTB4 concentrations. We conclude that PGE2 infusion attenuates lung dysfunction caused by endotoxemia, possibly by preventing endogenous release of other eicosanoids.     

Effects of granulocyte depletion on pulmonary responsiveness to aerosol histamine

The effects of granulocyte depletion with hydroxyurea on pulmonary responsiveness to aerosol histamine were studied in 10 chronically instrumented unanesthetized sheep. Sheep were studied when granulocyte counts were normal (B), after 3 days of hydroxyurea but before granulocyte counts had dropped below 700 cells/mm3 (H), and after granulocyte counts had fallen below 200 cells/mm3 (D). Hydroxyurea itself had no effect on aerosol histamine responsiveness and the results were unaffected by the order of experimentation. All 10 sheep were less responsive (P less than 0.05) to aerosol histamine when granulocyte depleted effective dose of histamine that caused a reduction to 65% of control dynamic compliance (ED65Cdyn = 23.98 +/- 4.70 mg/ml) compared with base line (ED65Cdyn = 7.06 +/- 1.86 mg/ml). Those sheep initially most responsive to aerosol histamine had the greatest attenuation in their airway responsiveness to aerosol histamine (P less than 0.05). There was a significant negative correlation between absolute granulocyte counts in peripheral blood and pulmonary responsiveness to aerosol histamine during base-line (B) condition (r = -0.74, P less than 0.05) and for the data as a whole [r = -0.69, P less than 0.05 (B + H + D)]. Circulating granulocytes and/or pulmonary inflammation may contribute to pulmonary responsiveness to bronchial challenge.     

Lung ischemia-reperfusion injury in awake sheep: protection with verapamil.

We caused unilateral lung ischemia-reperfusion injury in awake sheep by simultaneously occluding the left pulmonary artery and left main stem bronchus for 12 h. The occluded left lung was inflated with nitrogen. Reperfusion resulted in an elevation of lung lymph flow from 1.3 to 5.0 ml/15 min and an increase in lymph-to-plsma protein concentration ratios. Reperfusion, but not ischemia alone, caused an increase in wet-to-dry weight ratios in both the reperfused left lung and the contralateral right lung. Granulocytes increased in both lungs during the ischemic period and after reperfusion, and hypoxemia developed after reperfusion. The calcium channel antagonist, verapamil, given just before reperfusion, caused a marked attenuation in the reperfusion-induced changes in the lung lymph variables and wet-to-dry weight ratio. However, verapamil did not affect the hypoxemia or granulocyte sequestration seen after reperfusion. We conclude that reperfusion of ischemic sheep lung results in increased microvascular permeability that can be partially prevented by verapamil.     

Human recombinant tumor necrosis factor alpha infusion mimics endotoxemia in awake sheep

The macrophage-derived cytokine tumor necrosis factor alpha (TNF alpha) has been proposed as the major mediator of endotoxin-induced injury. To examine whether a single infusion of human recombinant TNF alpha (rTNF alpha) reproduces the pulmonary effects of endotoxemia, we infused rTNF alpha (0.01 mg/kg) over 30 min into six chronically instrumented awake sheep and assessed the ensuing changes in hemodynamics, lung lymph flow and protein concentration, and number of peripheral blood and lung lymph leukocytes. In addition, levels of thromboxane B2, 6-ketoprostaglandin F1 alpha, prostaglandin E2, and leukotriene B4 were measured in lung lymph. Pulmonary arterial pressure (Ppa) peaked within 15 min of the start of rTNF alpha infusion [base-line Ppa = 22.0 +/- 1.5 (SE) cmH2O; after 15 min of rTNF alpha infusion, Ppa = 54.2 +/- 5.4] and then fell toward base line. The pulmonary hypertension was accompanied by hypoxemia and peripheral blood and lung lymph leukopenia, both of which persisted throughout the 4 h of study. These changes were followed by an increase in protein-rich lung lymph flow (base-line lymph protein clearance = 1.8 +/- 0.4 cmH2O; 3 h after rTNF alpha infusion, clearance = 5.6 +/- 1.2), consistent with an increase in pulmonary microvascular permeability. Cardiac output and left atrial pressure did not change significantly throughout the experiment. Light-microscopic examination of lung tissue at autopsy revealed congestion, neutrophil sequestration, and patchy interstitial edema. We conclude that rTNF alpha induces a response in awake sheep remarkable similar to that of endotoxemia. Because endotoxin is a known stimulant of TNF alpha production, TNF alpha may mediate endotoxin-induced lung injury.     

Failure of therapy with 2,3-dihydroxybenzoic acid to modify the course of sepsis-induced lung injury

Oxidant-induced injury of the pulmonary microvasculature reportedly contributes to an increase in microvascular permeability and pulmonary hypertension, both of which are principal features of acute lung injury (ALI). We tested the hypothesis that antioxidant therapy with 2,3-dihydroxybenzoic acid (DHB), initiated in awake sheep after the development of sepsis-induced ALI, would ameliorate the progression of these lesions. DHB has many actions that suggested to us the potential for demonstrating benefit in ALI complicating sepsis; it is a nontoxic hydroxyl-radical scavenger that also inhibits the cyclooxygenase pathway and acts as a weak iron chelator. In preliminary experiments, we demonstrated that pretreatment with DHB prevented an increase in mean pulmonary arterial pressure, plasma thromboxane A2, measured as its metabolite thromboxane B2, and lymph total protein clearance that otherwise followed an infusion of zymosan-activated plasma (ZAP) in sheep. In subsequent experiments, 12 additional sheep were rendered septic by cecal ligation and perforation. Twenty-four to 36 h after cecal ligation and perforation, an increase in lung microvascular permeability was confirmed, because pulmonary lymph flow had increased by 82% while the mean lymph-to-plasma total protein ratio was unchanged from baseline. At this point, six sheep were then treated with parenteral DHB and six with DHB vehicle for the subsequent 24 h. In contrast to the demonstrated benefit of DHB pretreatment in preventing ALI secondary to an infusion of ZAP, the progressive increase in lymph total protein clearance that complicated septic lung injury in the DHB vehicle group throughout this 24-h study period was not ameliorated in the DHB treatment group. However, DHB did prevent a modest increase in mean pulmonary arterial pressures that was demonstrated in the DHB vehicle group throughout this 24-h treatment period. Although pretreatment prevented ALI after a ZAP infusion, we conclude that DHB only incompletely modified disease progression when administered after the onset of sepsis-induced ALI because it ameliorated the pulmonary hypertensive response without concurrently modifying an increase in lung microvascular fluid flux.     

Effect of prostacyclin on pulmonary vascular response to thrombin in awake sheep

We determined the effects of infusion of prostacyclin (PGI2) and 6-alpha-carba-PGI2 (6-cPGI2), a stable PGI2 analogue, on pulmonary transvascular fluid and protein fluxes after intravascular coagulation induced by thrombin. Studies were made in control awake sheep prepared with lung lymph fistulas (n = 6) and in similarly prepared awake sheep pretreated with either 6-cPGI2 (n = 5) or PGI2 (n = 5). Both prostacyclin compounds (500 ng X kg-1 X min-1) were infused intravenously. All groups were challenged with 80 U/kg thrombin. Pulmonary arterial pressure (Ppa), pulmonary vascular resistance (PVR), pulmonary lymph flow (Qlym), lymph protein clearance (Qlym X lymph/plasma protein concentration ratio), and neutrophil and platelet counts were determined. In vitro tests assessed sheep neutrophil chemotaxis and chemiluminescence and platelet aggregation. In both 6-cPGI2 and PGI2 groups, the increases in Qlym after thrombin were less than those in the control group. The increase in lymph protein clearance in the 6-cPGI2 group was the same as that in control, whereas the increase in clearance in the PGI2 group was reduced. PVR and Ppa increased to a greater extent in the 6-cPGI2 group than in the control group, whereas the increases in PVR and Ppa were inhibited in the PGI2 group. Neutrophil and platelet counts decreased after thrombin in PGI2 and 6-cPGI2 groups, as they did in the control group. Neither 6-cPGI2 altered neutrophil chemotaxis induced by thrombin and chemiluminescence induced by opsonized zymosan. Both prostacyclin compounds inhibited platelet aggregation induced by ADP or thrombin.(ABSTRACT TRUNCATED AT 250 WORDS)