Pattern of injury and the role of neutrophils in reperfusion injury of rat lung

Using a rat lung model, we sought to characterize the time course for ischemia-reperfusion injury and the role of neutrophils in the development of injury. Adult male Long-Evans rats underwent left thoracotomy with dissection and clamping of the left pulmonary artery, bronchus, and vein for 90 min, resulting in complete left lung ischemia. The lungs were then ventilated and reperfused for up to 4 hr. Time-matched sham animals underwent the identical thoracotomy and hilar dissection, but the lungs were not rendered ischemic. Using vascular permeability of 125I-labeled bovine serum albumin as a measure of reperfusion injury, a bimodal pattern of injury was observed. Compared to sham controls, animals undergoing ischemia-reperfusion demonstrated a significant early phase of lung injury at 30 min of reperfusion (P < 0.0001), followed by partial recovery. A second peak of lung injury was noted after 4 hr of reperfusion (P < 0.001). Myeloperoxidase activity in reperfused lung tissue, a measure of neutrophil sequestration, increased during the reperfusion time course. To determine the role of neutrophils in the development of lung reperfusion injury, additional animals undergoing the identical ischemia-reperfusion protocol received either rabbit anti-rat neutrophil serum or preimmune serum the day prior to operation. Profound neutropenia (< 75/mm3 blood) was confirmed by differential leukocyte counts. Neutropenia had no protective effect against microvascular permeability at 30 min of reperfusion, but there was a significant reduction in lung injury at 4 hr (P < 0.005). We conclude that, during lung ischemia-reperfusion, there is a bimodal pattern of injury, consisting of both neutrophil-independent and neutrophil-mediated events.     

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.     

A CD18 antibody prevents lung injury but not hypotension after intestinal ischemia-reperfusion

Antibodies to the neutrophil CD18 integrin have been shown to ameliorate the local effects of intestinal ischemia and reperfusion (I/R). In addition to local mucosal injury, intestinal I/R results in systemic hypotension and injury to the lungs with lung leukosequestration. This study tests the effect of a CD18 monoclonal antibody on the hypotension and lung injury after intestinal I/R. In anesthetized rabbits, the superior mesenteric artery was clamped for 60 min followed by 3 h of reperfusion. Animals were treated with saline, an anti-CD18 monoclonal antibody (R15.7 MAb), or nonspecific immunoglobulin G. Another non-ischemic group were sham controls. Neutrophil sequestration was assessed by measure of lung myeloperoxidase (MPO) and permeability by lung-to-blood concentration ratio of 125I-labeled bovine serum albumin and wet-to-dry weight ratio. Immediately after reperfusion, mean arterial pressure fell to 49 +/- 2.1 mmHg and remained at this level. The hypotension was unaffected by treatment with R15.7 MAb. Thirty minutes after reperfusion, the circulating white blood cell count fell to 2.91 +/- 0.53 x 10(3)/mm3 vs. sham 6.40 +/- 0.66 x 10(3)/mm3 (P < 0.05). Treatment with R15.7 MAb prevented this fall in white blood cell count (5.75 +/- 1.59 x 10(3)/mm3). At 3 h of reperfusion in saline-treated animals there was increased MPO, 74.8 +/- 4.9 U/g vs. 42.0 +/- 4.8 U/g in sham animals (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

L-arginine treatment in ischemia/reperfusion injury

We tested whether treatment with exogenous L-arginine, the precursor of nitric oxide (NO), could protect the skeletal muscle from ischemia/reperfusion (I/R) injury. A rabbit hindlimb I/R model (2.5 h ischemia/2 h reperfusion) was used. Morphological changes were elucidated by morphometry. Plasma concentrations of malondialdehyde (pMDA), as well as L-arginine and L-citrulline content in the plasma and skeletal muscle were measured. I/R injury in the skeletal muscle was manifested by development of prominent interstitial edema (fraction of interfiber area was 26.23% vs 15.09% in sham operated control, p < .005) and severe microvascular constriction (capillary area was 11.41 microns2 vs 16.92 in control, p <.005). These changes were accompanied by increased pMDA levels, indicating a process of lipid peroxidation in the cell membranes. L-arginine treatment (4 mg/kg/min intravenously, for 1 h, infusion initiated 30 min before reperfusion) caused an intracellular accumulation of this amino acid in the SM. Intracellular concentrations of L-citrulline increased (201.0 mumol/dm3 after reperfusion vs 176.0 before ischemia onset, p < .005), suggesting stimulated endogenous NO synthesis. L-arginine treatment protected capillary constriction (capillary area was 17.64 microns2 vs 11.41 in the untreated animals, p < .0005) and reduced interstitial edema after reperfusion (fraction of interfiber area was 17.80% vs 26.23 in untreated animals, p < 0.005). The protective effect of L-arginine treatment on I/R injury of SM may be related to its ability to prevent microvascular constriction and reduce permeability disorders by the stimulation of endogenous NO production.     

The role of endogenous nitric oxide in modulating ischemia-reperfusion injury in the isolated, blood-perfused rat lung

Ischemia-reperfusion (IR) lung injury occurs after various clinical procedures, including cardiopulmonary bypass. It is not clear whether endogenous nitric oxide (NO) is protective or injurious in lungs subjected to IR. Thus, in this study we examined the contribution of endogenous NO to IR injury in isolated, blood-perfused rat lungs. Lungs of male Wistar rats (300 g) were subjected to 30 min ischemia and 180 min reperfusion (I30R180). Lungs were sampled for inducible nitric oxide synthase (i-NOS) mRNA expression (each n = 3) and NOS enzyme activity (each n = 4) at different time points. NOS inhibitors NG-nitro-L-arginine-methyl ester (10[-4] M) and aminoguanidine (10[-4] M) were used to study the contribution of NO to IR injury in lungs subjected to I30R30 and I30R180. The contribution of i-NOS to IR lung injury was studied by inducing i-NOS enzyme with Salmonella lipopolysaccharide, followed by I30R30. We found that ischemia-reperfusion alone can upregulate i-NOS mRNA and i-NOS enzyme activity (p < 0.05, ANOVA), but downregulate constitutive NOS enzyme activity over 180 min reperfusion. Endogenously produced NO is protective against lung injury in I30R180 in normal rats and lung injury in I30R30 in septic rats. NO is also pivotal in maintaining pulmonary vascular homeostasis in septic rat lungs undergoing IR.     

Phosphotyrosine phosphatase and tyrosine kinase inhibition modulate airway pressure-induced lung injury

We determined whether drugs which modulate the state of protein tyrosine phosphorylation could alter the threshold for high airway pressure-induced microvascular injury in isolated perfused rat lungs. Lungs were ventilated for successive 30-min periods with peak inflation pressures (PIP) of 7, 20, 30, and 35 cmH2O followed by measurement of the capillary filtration coefficient (Kfc), a sensitive index of hydraulic conductance. In untreated control lungs, Kfc increased by 1.3- and 3.3-fold relative to baseline (7 cmH2O PIP) after ventilation with 30 and 35 cmH2O PIP. However, in lungs treated with 100 microM phenylarsine oxide (a phosphotyrosine phosphatase inhibitor), Kfc increased by 4.7- and 16.4-fold relative to baseline at these PIP values. In lungs treated with 50 microM genistein (a tyrosine kinase inhibitor), Kfc increased significantly only at 35 cmH2O PIP, and the three groups were significantly different from each other. Thus phosphotyrosine phosphatase inhibition increased the susceptibility of rat lungs to high-PIP injury, and tyrosine kinase inhibition attenuated the injury relative to the high-PIP control lungs.     

Platelet-activating factor mediates pulmonary macromolecular leak following intestinal ischemia-reperfusion

Platelet-activating factor (PAF) causes hypotension, cardiac dysfunction, increased vascular permeability, intestinal necrosis, and pulmonary microvascular injury when administered experimentally. Receptor antagonism attenuates or abolishes many of these effects in animal models of bacteremia, endotoxemia, and intestinal ischemia/reperfusion (I/R). The purpose of this study was to further examine the role of PAF in intestinal I/R-induced pulmonary injury using the PAF receptor antagonist WEB 2086. Sprague-Dawley rats were anesthetized and cannulated for measurement of mean arterial pressure, heart rate, and cardiac output. Laparotomy and thoracotomy were performed and the superior mesenteric artery was occluded for 45 min and reperfused for 120 min. Sham animals were treated similarly but without I/R. In the treatment groups, iv WEB 2086 (20 mg/kg/l cc NS) was administered as a bolus 15 min prior to reperfusion. Hemodynamic and videomicroscopic data were obtained before and during ischemia, and after reperfusion at 30-min intervals. Alveolar leak index was calculated offline via computer analysis of videomicroscopic images. Intestinal I/R caused pulmonary macromolecular leakage and hemodynamic instability. Treatment with WEB 2086 attenuated the pulmonary leak during the entire reperfusion period but improved cardiac output only during the first 30 min of reperfusion and had no effect on other hemodynamic variables. These data suggest that PAF is an important, but not the exclusive, mediator of pulmonary injury after intestinal I/R. PAF appears to play a minor role in the hemodynamic derangements observed after rat intestinal I/R.     

Effects of a metalloproteinase that truncates P-selectin glycoprotein ligand on neutrophil-induced cardiac dysfunction in ischemia/reperfusion

This study was designed to test the effects of polymorphonuclear leukocytes (PMNs) in the presence and absence of a P-selectin blocker, mocarhagin, in provoking cardiac dysfunction in isolated perfused rat hearts following ischemia and reperfusion. Control rat hearts not subjected to ischemia were perfused without blood cells for 80 min. Additional control rat hearts were perfused with 100 x 10(6) PMNs in the presence and absence of 0.2 microgram/ml mocarhagin over a 5-min perfusion followed by a 45-min observation period. No significant reduction in coronary flow (CF), left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dt max) was observed at the end of the observation period in any non-ischemic group. Similarly, global ischemia (I) for 20 min followed by 45 min of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of PMNs. I/R hearts perfused with PMNs exhibited decreases of 50-60% in all measurements of cardiac function (P < 0.001). These PMN perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity indicating a significant PMN infiltration, and enhanced P-selection expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were attenuated markedly by the metalloproteinase, mocarhagin, which inhibits P-selectin-mediated cell adhesion by cleaving its high-affinity receptor, PSGL-1, present on neutrophils. These results provide evidence that neutrophils provoke post-reperfusion cardiac dysfunction, and that this may be largely due to P-selectin-induced adherence of neutrophils to the endothelium.     

Optical measurement of isolated canine lung filtration coefficients after alloxan infusion

In this study, lung filtration coefficient (Kfc) was measured in eight isolated canine lung preparations by using three methods: standard gravimetric (Std), blood-corrected gravimetric (BC), and optical. The lungs were held in zone III conditions and were subjected to an average venous pressure increase of 8.79 +/- 0.93 (mean +/- SD) cmH2O. The permeability of the lungs was increased with an infusion of alloxan (75 mg/kg). The resulting Kfc values (in milliliters . min-1 . cmH2O-1 . 100 g dry lung weight-1) measured by using Std and BC gravimetric techniques before vs. after alloxan infusion were statistically different: Std, 0.527 +/- 0.290 vs. 1. 966 +/- 0.283; BC, 0.313 +/- 0.290 vs. 1.384 +/- 0.290. However, the optical technique did not show any statistical difference between pre- and postinjury with alloxan, 0.280 +/- 0.305 vs. 0.483 +/- 0. 297, respectively. The alloxan injury, quantified by using multiple-indicator techniques, showed an increase in permeability and a corresponding decrease in reflection coefficient for albumin (sigmaf). Because the optical method measures the product of Kfc and sigmaf, this study shows that albumin should not be used as an intravascular optical filtration marker when permeability is elevated. However, the optical technique, along with another means of measuring Kfc (such as BC), can be used to calculate the sigmaf of a tracer (in this study, sigmaf of 0.894 at baseline and 0.348 after injury). Another important finding of this study was that the ratio of baseline-to-injury Kfc values was not statistically different for Std and BC techniques, indicating that the percent contribution of slow blood-volume increases does not change because of injury.     

Contribution of tumor necrosis factor-alpha to pulmonary cytokine expression and lung injury after hemorrhage and resuscitation

OBJECTIVE: To examine the role of tumor necrosis factor-alpha (TNF-alpha) in producing acute inflammatory lung injury after hemorrhage and resuscitation. DESIGN: Prospective, controlled animal study. SETTING: Research laboratory. SUBJECTS: Male BALB/c mice. INTERVENTIONS: Treatment with rat antimouse monoclonal anti-TNF-alpha antibodies or control rat immunoglobulin G 1 hr after 30% blood volume hemorrhage and resuscitation. MEASUREMENTS AND MAIN RESULTS: Therapy with monoclonal anti-TNF-alpha antibodies prevented the posthemorrhage increases in pulmonary TNF-alpha and interferon-gamma protein levels that normally occur after blood loss. Administration of monoclonal anti-TNF-alpha antibodies also diminished the increases in interleukin-1 beta, interleukin-6, and interleukin-10 mRNA, but not the increases in TNF-alpha and interferon-gamma mRNA, which are found in the lungs following hemorrhage. In addition, therapy with monoclonal anti-TNF-alpha antibodies was associated with significant improvement in the histologic parameters of posthemorrhage lung injury, particularly intra-alveolar hemorrhage and pulmonary vascular congestion. CONCLUSIONS: These results indicate that TNF-alpha has an important role in the development of acute inflammatory lung injury after blood loss. Blockade of TNF-alpha with monoclonal antibodies significantly reduces hemorrhage-induced lung injury.     

Intermittent ischemia: energy metabolism, cellular volume regulation, adenosine and insights into preconditioning

Interruption of ischemia by brief reperfusions (I/R) is better tolerated by the heart than continuous ischemia. The present study aims to determine the metabolic profiles of isolated rat hearts during intermittent ischemia, the possible cardioprotective role of adenosine and the influence of I/R on intracellular volumes, using multinuclear NMR spectroscopy. After five I/R (5/5 min) episodes, hearts paced at 5 Hz developed pressures comparable to those of hearts continuously perfused for 50 min at 37 degrees C (CP). Following the first 5 min episode of no-flow ischemia, [ADP] dropped from 72 +/- 9 to 43 +/- 5 microM (P < 0.001) and remained stable at the end of the following reperfusions, despite a 2.5-4-fold increase during each episode of 5 min ischemia. Intracellular volumes were stable during CP at a value of 2.50 +/- 0.06 ml/g dry weight, and decreased by 4, 8, and 12% after 1, 3, and 5 I/R episodes. The phosphorylation potentials decreased from 54 +/- 8 to 4 mM-1 during each period of 5 min ischemia and were 40 +/- 6 and 28 +/- 6 mM-1 after CP and I/R5, respectively. Cardiac glycogen had decreased during 50 min of CP from 103 +/- 13 to 81 +/- 9 mumol/g dry weight and lactate production was 116 +/- 15 mumol/heart. Five I/R episodes decreased glycogen to 46 +/- 7 mumol/g dry weight (P < 0.005 v CP) and increased lactate efflux to 262 +/- 31 mumol/ heart (P < 0.005 v CP). These findings suggest that a brief ischemia/reperfusion episode increases anaerobic metabolism of exogenous glucose, reduces [ADP] and induces cellular shrinkage. Administration of the adenosine receptor blocker 8-phenyl theophylline (8PT) during intermittent perfusion depressed the developed pressure to 78 +/- 7%, accentuated the decrease in phosphorylation potential (14 +/- 4 mM-1), abolished cellular shrinkage, reduced lactate efflux and blunted the decrease in ADP following the first I/R episode. In variance, no detectable changes were observed during intermittent ischemia when the ATP-sensitive potassium channel blocker glibenclamide was administered. These data demonstrate: (a) a brief episode of ischemia/reperfusion stimulates anaerobic metabolism of exogenous glucose and lowers intracellular ADP concentration: (b) adenosine receptors are partially responsible for the glycolytic stimulation during intermittent ischemia; (c) cellular shrinkage is related to the rate of glycolysis during intermittent ischemia/reperfusion.     

A comparison of the new preservation solution Celsior to Euro-Collins and University of Wisconsin solutions in lung reperfusion injury

BACKGROUND: The lung is particularly susceptible to reperfusion injury, both experimentally and clinically after transplantation. The extracellular-type preservation solution Celsior, which has been predominantly studied in cardiac preservation, has components designed to prevent cell swelling, free radical injury, energy depletion, and calcium overload. Using an isolated blood-perfused rat lung model, we investigated whether Celsior would decrease preservation injury and improve lung function after cold ischemic storage and reperfusion compared to Euro-Collins (EC) and University of Wisconsin (UW) solutions. METHODS: Lewis rat lungs were isolated, flushed with the respective cold preservation solution, and then stored at 4 degrees C for 6 or 12 hr. After ischemic storage, the lung block was suspended from a force transducer, ventilated with 100% O2, and reperfused for 90 min with fresh blood via a cannula in the pulmonary artery. Lung compliance, alveolar-arterial oxygen difference, and outflow oxygen tension were all measured. The capillary filtration coefficient (Kf), a sensitive measure of changes in microvascular permeability, was determined. RESULTS: For 6 hr of cold storage, lungs stored in Celsior had lower Kf values than those stored in EC, indicating decreased microvascular permeability. No other significant differences were noted between Celsior and EC or UW. For 12 hr of cold storage, Celsior provided increased oxygenation, decreased alveolar-arterial O2 differences, increased compliance, and decreased Kf values as compared to both EC and UW. CONCLUSIONS: Celsior provides better lung preservation than EC or UW as demonstrated by increased oxygenation, decreased capillary permeability, and improved lung compliance, particularly at 12-hr storage times. These results are highly relevant, inasmuch as EC and UW are the most common clinically used lung preservation solutions. Further studies of Celsior in experimental and clinical lung transplantation, as well as in other solid organs, are indicated.     

Effect of ventilation on vascular permeability and cyclic nucleotide concentrations in ischemic sheep lungs

Ventilation during ischemia attenuates ischemia-reperfusion lung injury, but the mechanism is unknown. Increasing tissue cyclic nucleotide levels has been shown to attenuate lung ischemia-reperfusion injury. We hypothesized that ventilation prevented increased pulmonary vascular permeability during ischemia by increasing lung cyclic nucleotide concentrations. To test this hypothesis, we measured vascular permeability and cGMP and cAMP concentrations in ischemic (75 min) sheep lungs that were ventilated (12 ml/kg tidal volume) or statically inflated with the same positive end-expiratory pressure (5 Torr). The reflection coefficient for albumin (sigmaalb) was 0.54 +/- 0.07 and 0.74 +/- 0. 02 (SE) in nonventilated and ventilated lungs, respectively (n = 5, P < 0.05). Filtration coefficients and capillary blood gas tensions were not different. The effect of ventilation was not mediated by cyclic compression of alveolar capillaries, because negative-pressure ventilation (n = 4) also was protective (sigmaalb = 0.78 +/- 0.09). The final cGMP concentration was less in nonventilated than in ventilated lungs (0.02 +/- 0.02 and 0.49 +/- 0. 18 nmol/g blood-free dry wt, respectively, n = 5, P < 0.05). cAMP concentrations were not different between groups or over time. Sodium nitroprusside increased cGMP (1.97 +/- 0.35 nmol/g blood-free dry wt) and sigmaalb (0.81 +/- 0.09) in nonventilated lungs (n = 5, P < 0.05). Isoproterenol increased cAMP in nonventilated lungs (n = 4, P < 0.05) but had no effect on sigmaalb. The nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester had no effect on lung cGMP (n = 9) or sigmaalb (n = 16) in ventilated lungs but did increase pulmonary vascular resistance threefold (P < 0.05) in perfused sheep lungs (n = 3). These results suggest that ventilation during ischemia prevented an increase in pulmonary vascular protein permeability, possibly through maintenance of lung cGMP by a nitric oxide-independent mechanism.     

In vivo cyclin E expression as a marker for early cervical neoplasia

BACKGROUND: Heat shock has been associated with the acquisition of tolerance to a wide variety of stressful conditions, including ischemia. This is partly mediated by the production of various heat shock proteins (HSP), including HSP70. One novel approach to the reduction of ischemia-reperfusion injury after lung transplantation is the induction of HSP70 by heat pretreatment of the donor. The purpose of this study was to investigate the feasibility of this approach in an animal model of lung transplantation. METHODS: Animals were divided into six main groups, with groups I to III representing transplanted animals: In groups I and II, donor animals were anesthetized and then underwent heat stress 6 and 12 hours before organ harvest, respectively. Control animals underwent general anesthesia but no heat stress. After harvest, left lungs from groups I to III were preserved for 18 hours at 40 degrees C and then implanted into isogeneic recipients, which were killed 24 hours after reperfusion to assess graft function. Group IV and V animals underwent heat stress followed by a recovery period of 6 and 12 hours, respectively. Lungs were collected both at the time of harvest (right lungs) and after 18 hours of cold preservation (left lungs). Group VI served as nontransplanted controls. Groups IV to VI did not undergo lung transplantation. RESULTS: At the time of harvest but before implantation, HSP70 was significantly increased in heat-shocked nontransplanted donor lungs (groups IV and V) compared with group VI controls. After 18 hours of cold preservation, HSP70 levels were higher in group IV compared with group V and group VI controls. At 24 hours after reperfusion, mean arterial oxygenation was significantly higher in group I compared with group II and group III controls (290.25+/-24.5 vs 154.5+/-23.9 and 119.6+/-11.3 mm Hg, respectively; P < .001). Myeloperoxidase activity was improved in group I compared with group III controls (0.048+/-0.018 vs 0.137+/-0.036 deltaOD/mg/min, respectively; P < .05). The wet/dry weight ratio was also improved in group I compared with group III controls (6.2+/-0.3 vs. 7.8+/-0.4, respectively; P < .05). CONCLUSIONS: Heat pretreatment of the donor 6 hours before harvest results in increased synthesis of HSP70, which offers a dramatic protective effect against subsequent ischemia-reperfusion injury in the lung isograft.     

Chemokine expression during hepatic ischemia/reperfusion-induced lung injury in the rat. The role of epithelial neutrophil activating protein

The liver is highly susceptible to a number of pathological insults, including ischemia/reperfusion injury. One of the striking consequences of liver injury is the associated pulmonary dysfunction that may be related to the release of hepatic-derived cytokines. We have previously employed an animal model of hepatic ischemia/reperfusion injury, and demonstrated that this injury causes the production and release of hepatic-derived TNF, which mediates a neutrophil-dependent pulmonary microvascular injury. In this study, we have extended these previous observations to assess whether an interrelationship between TNF and the neutrophil chemoattractant/activating factor, epithelial neutrophil activating protein-78 (ENA-78), exists that may be accountable for the pathology of lung injury found in this model. In the context of hepatic ischemia/reperfusion injury, we demonstrated the following alterations in lung pathophysiology: (a) an increase in pulmonary microvascular permeability, lung neutrophil sequestration, and production of pulmonary-derived ENA-78; (b) passive immunization with neutralizing TNF antiserum resulted in a significant suppression of pulmonary-derived ENA-78; and (c) passive immunization with neutralizing ENA-78 antiserum resulted in a significant attenuation of pulmonary neutrophil sequestration and microvascular permeability similar to our previous studies with anti-TNF. These findings support the notion that pulmonary ENA-78 produced in response to hepatic-derived TNF is an important mediator of lung injury.     

Chlorine gas induced acute lung injury in isolated rabbit lung

This study was designed to investigate the pathogenesis of chlorine gas (Cl2) induced acute lung injury and oedema. Isolated blood-perfused rabbit lungs were ventilated either with air (n=7) or air plus 500 parts per million (ppm) of Cl2 (n=7) for 10 min. Capillary pressure, measured by analysing the pressure/time transients of pulmonary arterial, venous and double (both arterial and venous) occlusions, was unchanged in both groups. In Cl2-exposed lungs, the fluid filtration rate increased from -0.228+/-0.25 to 1.823+/-1.23 mL min(-1) x 100 g(-1) (p<0.001) and the filtration coefficient increased from 0.091+/-0.01 to 0.259+/-0.07 mL x min(-1) x cmH2O(-1) x 100 g(-1) (p<0.001). No changes were observed in the control lungs. The extravascular lung water/blood-free dry weight ratio was 8.6+/-1.6 in the Cl2 group and 4.0+/-0.5 in the control group (p<0.001), confirming that the increase in lung weight was related to accumulation of extravascular fluid. Although the alveolar flooding by oedema is explained, in part, by the Cl2-induced epithelial injury, our results suggest that Cl2 exposure induces acute lung injury and oedema due to an increased microvascular permeability.     

Interleukin-10 reduces the systemic inflammatory response in a murine model of intestinal ischemia/reperfusion

BACKGROUND: Intestinal ischemia/reperfusion (I/R) is known to increase systemic cytokine levels, as well as to activate neutrophils in distant organs. This study was designed to investigate the effect of interleukin-10 (IL-10) on cytokine release, pulmonary neutrophil accumulation, and histologic changes in a murine model of I/R. METHODS: Forty female Swiss-Webster mice were divided into four groups. Group 1 underwent 45 minutes of superior mesenteric artery occlusion followed by 3-hour reperfusion (I/R). Group 2 underwent laparotomy alone (Sham). Group 3 underwent I/R, but was treated with IL-10, 10,000 units IP every 2 hours, starting 1 hour before reperfusion (Pretreatment). Group 4 was treated with an equal dose of IL-10, starting 1 hour after reperfusion (Posttreatment). All animals were killed at 3 hours, standard assays were performed for serum cytokine levels, and lung myeloperoxidase activity and intestinal histology were scored. RESULTS: Serum cytokines (TNF-alpha and IL-6), lung myeloperoxidase levels, and histologic score were significantly reduced when IL-10 was administered either before or after reperfusion. CONCLUSIONS: IL-10 reduced the severity of local and systemic inflammation in a murine model of intestinal I/R when given before or after reperfusion injury. These observations suggest that IL-10 may exert its effect by blocking cytokine production and distant organ neutrophil accumulation.     

The neurotoxin MPTP increases calbindin-D28k levels in mouse midbrain dopaminergic neurons

Interleukin (IL)-2-induced microvascular lung injury is an experimental paradigm commonly used to investigate the pathogenesis of the adult respiratory distress syndrome. Since tumor necrosis factor-alpha (TNF-alpha) is known to induce such an injury in vivo and since TNF-alpha is involved in other models of lung injury, we postulated that it might also mediate pulmonary toxicity after IL-2 administration. The present study tested this hypothesis by evaluating the effect of TNF-alpha inhibition on IL-2-induced lung injury in the rat. Recombinant human IL-2 (10(6) U IV per rat, n = 6) elevated lung water, myeloperoxidase activity, and protein accumulation in bronchoalveolar lavage fluid and induced tissue hypoxia. Also, IL-2 enhanced lung tissue TNF-alpha mRNA and peptide (1543 +/- 496 pg/g lung wet weight) localized to alveolar macrophages by in situ hybridization. In marked contrast, IL-2 failed to affect serum TNF-alpha, which remained at undetectable levels. Pretreatment with anti-TNF-alpha monoclonal antibody (25 mg/kg IV, n = 7) or the TNF-alpha synthesis inhibitor rolipram (200 micrograms/kg IV, n = 7) attenuated lung injury and reverted tissue hypoxia. Furthermore, TNF-alpha inhibition prevented the upregulation of lung tissue IL-1 beta, IL-6, cytokine-induced neutrophil chemoattractant, and E-selectin (ELAM-1) but not intercellular adhesion molecule-1 mRNAs in response to IL-2. These data imply that locally produced TNF-alpha mediates IL-2-induced lung inflammation and tissue injury and point to the potential utilization of TNF-alpha inhibitors in treating the pulmonary toxicity of IL-2 immunotherapy.     

Synergism between platelets and neutrophils in provoking cardiac dysfunction after ischemia and reperfusion: role of selectins

BACKGROUND: Neutrophils (PMNs) are known to contribute to both cardiac dysfunction and myocardial necrosis after reperfusion of an ischemic heart. Moreover, platelets are also important blood cells that can aggravate myocardial ischemic injury. This study was designed to test the effects of PMNs and platelets separately and together in provoking cardiac dysfunction in isolated perfused rat hearts after ischemia and reperfusion. METHODS AND RESULTS: Control rat hearts not subjected to ischemia were perfused without blood cells for 80 minutes. Additional control rat hearts were perfused with 75x106 PMNs, with 100x106 platelets, or with 75x106 PMNs+100x106 platelets over a 5-minute perfusion followed by a 75-minute observation period. No significant reduction in coronary flow, left ventricular developed pressure (LVDP), or the first derivative of LVDP (dP/dtmax) was observed at the end of the observation period in any nonischemic group. Similarly, global ischemia (I) for 20 minutes followed by 45 minutes of reperfusion (R) produced no sustained effects on the final recovery of any of these parameters in any group of hearts perfused in the absence of blood cells. However, I/R hearts perfused with either PMNs or platelets alone exhibited decreases in these variables of 10% to 12% (P<0.05 from control). Furthermore, I/R hearts perfused with both PMNs and platelets exhibited decreases of 50% to 60% in all measurements of cardiac function (P<0.001). These dual-cell-perfused I/R hearts also exhibited marked increases in cardiac myeloperoxidase (MPO) activity, indicating a significant PMN infiltration, and enhanced P-selectin expression on the coronary microvascular endothelium. All cardiodynamic effects as well as MPO accumulation and PMN infiltration were markedly attenuated by a sialyl LewisX-oligosaccharide or a recombinant soluble P-selectin ligand, which inhibits selectin-mediated cell adhesion. CONCLUSIONS: These results provide evidence that platelets and neutrophils act synergistically in provoking postreperfusion cardiac dysfunction and that this may be largely due to cell-to-cell interactions mediated by P-selectin. These findings may help explain the reperfusion injury phenomenon.