Regulatory effects of interleukin-10 on lung ischemia-reperfusion injury

OBJECTIVE: Interleukin-10, a cytokine with antiinflammatory activities, was studied to determine its effects on development of early lung reperfusion injury. METHODS: Adult male rats underwent 90 minutes of left lung ischemia followed by 4 hours of reperfusion. Time-matched sham-operated control rats underwent hilar dissection but not lung ischemia. Lung injury was measured by vascular permeability to bovine serum albumin tagged with iodine 125. To evaluate the effect of exogenous interleukin-10, additional animals received interleukin-10 intravenously before ischemia. To assess the role of endogenous interleukin-10, animals received rabbit antimouse interleukin-10 immunoglobin G (or preimmune rabbit immunoglobin G) before ischemia. RESULTS: Compared with sham control rats, ischemia-reperfusion control rats demonstrated significantly more lung injury. Animals receiving interleukin-10 had significantly less lung injury than did ischemia-reperfusion control rats. Animals receiving antiinterleukin-10 had significantly more lung injury than did animals receiving preimmune immunoglobin G. Alveolar macrophages from animals after 90 minutes of lung ischemia produced more tumor necrosis factor-alpha in culture than did unstimulated macrophages; this production was reduced significantly by the addition of interleukin-10 to the culture medium. CONCLUSION: Endogenous interleukin-10 has a protective effect against early lung reperfusion injury, and interleukin-10 administration can reduce lung reperfusion injury, perhaps in part through its ability to reduce production by alveolar macrophages of tumor necrosis factor-alpha, a known proinflammatory cytokine.     

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.     

Immunosuppressants decrease neutrophil chemoattractant and attenuate ischemia/reperfusion injury of the liver in rats

BACKGROUND: Neutrophils may play an important role in the development of liver ischemia/reperfusion injury. We investigated the effects of the immunosuppressants azathioprine (AZA), cyclosporine A (CsA), tacrolimus (FK506), and rapamycin (RPM) on the expression of cytokine-induced neutrophil chemoattractant (CINC) after ischemia/reperfusion of the liver. METHODS: Liver ischemia was induced in male Wistar rats by occluding the portal vein with a microvascular clip for 30 minutes. Rats received two intramuscular injections of AZA (4 mg/kg), CsA (5 mg/kg), FK506 (0.5 mg/kg), or RPM (0.5 mg/kg) 3 and 24 hours before ischemia/reperfusion of the liver. RESULTS: Serum CINC concentrations in untreated animals increased, peaked 6 hours after reperfusion, and thereafter decreased gradually. Pretreatment with AZA, CsA, FK506, and RPM, however, inhibited the increase in serum CINC concentrations after reperfusion. CINC mRNA in liver tissue increased and peaked 3 hours after reperfusion, but was significantly lower in animals treated with AZA, CsA, FK506, and RPM. In vitro CINC production by Kupffer cells harvested from animals treated with AZA, CsA, FK506, or RPM 3 hours after reperfusion was also significantly lower than that observed in untreated animals. Both myeloperoxidase activity and the number of neutrophils accumulating in the liver 24 hours after reperfusion in animals treated with AZA, CsA, FK506, and RPM were significantly lower than in untreated animals. This correlated with lower serum aspartate transaminase, alanine transaminase, and lactate dehydrogenase levels in animals treated with AZA, CsA, FK506, and RPM 24 hours after reperfusion. CONCLUSION: The immunosuppressants AZA, CsA, FK506, and RPM reduce neutrophil accumulation and attenuate ischemia/reperfusion injury of the liver.     

Neutrophil elastase inhibitor reduces neutrophil chemoattractant production after ischemia-reperfusion in rat liver

BACKGROUND & AIMS: Neutrophils are important in the development of tissue injury induced by ischemia-reperfusion. The ability of an inhibitor of neutrophil elastase (ONO-5046) to protect against ischemia-reperfusion injury in rat liver was investigated by measuring serum concentrations of cytokine-induced neutrophil chemoattractant. METHODS: Liver ischemia was induced in rats by occluding the portal vein for 30 minutes, and ONO-5046 or anticoagulants were injected intravenously 5 minutes before vascular clamping. RESULTS: Serum concentration of cytokine-induced neutrophil chemoattractant increased after reperfusion, reached a maximum at 6 hours, and then gradually decreased. However, pretreatment of animals with heparin (50 U/kg), antithrombin III (250 U/kg), or ONO-5046 (10 mg/kg) resulted in significantly smaller increases in the serum concentration of cytokine-induced neutrophil chemoattractant after reperfusion. Pretreatment with both ONO-5046 and heparin, or both ONO-5046 and antithrombin III, produced additive effects. Pretreatment of rats with both ONO-5046 and heparin or both ONO-5046 and antithrombin III also inhibited the increase in cytokine-induced neutrophil chemoattractant mRNA in liver. These combined treatments significantly reduced the increases in both the number of neutrophils accumulated in the liver and the hepatic activity of myeloperoxidase. CONCLUSIONS: Cytokine-induced neutrophil chemoattractant production after ischemia-reperfusion in the liver is mediated by neutrophil elastase and activation of coagulation within the hepatic microcirculation.     

Microcirculatory studies of frostbite injury

Frostbite represents a spectrum of injury ranging from irreversible cellular destruction to reversible changes seen after rewarming. These changes include increases in tissue edema, circulatory stasis, and progressive thrombosis leading to further tissue necrosis. For this reason, it is often difficult at the time of surgical debridement to determine the extent of frostbite injury. This delayed tissue injury is similar to that seen in muscle during ischemia/reperfusion injury. Muscle that initially appears viable on reperfusion may subsequently necrose due to collapse of the microcirculation. Adherent neutrophils have been specifically cited as important components in ischemia/reperfusion injury and have also been suggested to play a role in frostbite injury. We have used an intravital microscopic muscle preparation to study microcirculatory changes carefully in frostbite injury during rewarming. The right gracilis muscle of male Wistar rats is dissected free from its primary vascular pedicle and the rat is positioned on a specially constructed microsurgical stage. Temperature changes of the muscle are recorded. The prepared axial pattern flap is transilluminated with a microscope and projected on a video screen, allowing measurement of arteriolar diameters and changes in the numbers of stuck and rolling neutrophils before frostbite, during rewarming, and for several hours later. Cold silicone oil is used to freeze the muscle to -5+/-2 degrees C in 2 to 3 minutes and to hold this temperature for 5 minutes. The muscle is rewarmed with 42 degrees C normal saline placed directly on the muscle surface. Baseline vessel diameter and leukocyte counts in 100-mm segments of the microvasculature are recorded as well as at 5, 15, and 30 minutes, and at 1, 2, and 3 hours postrewarming of frozen muscle. Observations from our initial 11 animals show that reperfusion of the muscle following freezing is varied temporally and spatially, with circulation to most vascular segments restored 5 to 10 minutes after rewarming. In 9 of 11 animals we observed the shedding of "white clots" in small arterioles and venules occurring as soon as 5 minutes after thawing. In some instances shedding continued for as long as 1 hour after rewarming. Microvascular hemorrhage was widespread 1 hour following the thaw, but there was no significant increase in neutrophil adherence observed until 3 hours following rewarming. The exact nature of the vascular injury and the composition of the "white clots" are now being determined from ultrastructural studies. Blood flow in microcirculation stops during freezing, but small-vessel perfusion returns immediately on thawing. This suggests that the vascular architecture is maintained during the freezing and thawing. Unlike ischemia/reperfusion injury, neutrophil adhesion plays a smaller role in the early response to frostbite injury. The early microcirculatory observations seen after rewarming suggest progressive and severe perturbations in platelet function and fibrin formation that are significantly different from ischemia/reperfusion injury.     

Myocardial infarction and apoptosis after myocardial ischemia and reperfusion: role of the terminal complement components and inhibition by anti-C5 therapy

BACKGROUND: Myocardial ischemia and reperfusion (MI/R)-induced tissue injury involves necrosis and apoptosis. However, the precise contribution of apoptosis to cell death, as well as the mechanism of apoptosis induction, has not been delineated. In this study, we sought to define the contribution of the activated terminal complement components to apoptosis and necrosis in a rat model of MI/R injury. METHODS AND RESULTS: Monoclonal antibodies (mAbs; 18A and 16C) raised against the rat C5 complement component bound to purified rat C5 (ELISA). 18A effectively blocked C5b-9-mediated cell lysis and C5a-induced chemotaxis of rat polymorphonuclear leukocytes (PMNs), whereas 16C had no complement inhibitor activity. A single dose (20 mg/kg i.v.) of 18A blocked >80% of serum hemolytic activity for >4 hours. Administration of 18A before myocardial ischemia (30 minutes) and reperfusion (4 hours) significantly reduced (91%) left ventricular free wall PMN infiltration compared with 16C treatment. Treatment with 18A 1 hour before ischemia or 5 minutes before reperfusion significantly reduced infarct size compared with 16C treatment. A significant reduction in infarct size (42%) was also observed in 18A-treated rats after 30 minutes of ischemia and 7 days of reperfusion. DNA ladders and DNA labeling (eg, TUNEL assay) demonstrated a dramatic reduction in MI/R-induced apoptosis in 18A-treated compared with 16C-treated rats. CONCLUSIONS: Anti-C5 therapy in the setting of MI/R significantly inhibits cell apoptosis, necrosis, and PMN infiltration in the rat despite C3 deposition. We conclude that the terminal complement components C5a and C5b-9 are key mediators of tissue injury in MI/R.     

The role of xanthine dehydrogenase (xanthine oxidase) in ischemia-reperfusion injury in rat kidney

Xanthine dehydrogenase (XDH) and xanthine oxidase (XO) are enzymes involved in the metabolism of purines in various organisms. XO produces superoxide radicals, suggesting that is responsible for tissue ischemia-reperfusion injury. To test this notion further studies were performed on rat kidneys and the time course of changes in purine nucleotides, oxypurines and XDH and XO activity was determined. At 24 hours after reperfusion subsequent to 30-minute ischemia, serum creatinine increased to 0.83 +/- 0.74 mg/dl from 0.28 +/- 0.06 mg/dl (the level prior to ischemia, the control). Renal ATP and ADP contents were reduced after ischemia lasting for 30 minutes and restored 10 minutes after reperfusion following 30 minutes of ischemia. The renal AMP content increased after 30 minutes of ischemia and recovered within 10 minutes after reperfusion. The total adenine nucleotide (TAN) content was reduced gradually during ischemia-reperfusion in the rat kidney. Although the energy charge was reduced following 30 minutes of ischemia, it was restored to the control level 10 minutes following reperfusion. Hypoxanthine (HX) and xanthine (X), which had accumulated at 30 minutes after ischemia, were reduced to the control levels 10 minutes after reperfusion. There were no significant changes in the pre-ischemia values of total XDH and XO activities or XDH/XO ratio during the period nor at various time intervals (up to 24 hours) during reperfusion. It was shown that HX and X accumulate without significant conversion of XDH to XO during ischemia. Therefore the putative role of XO in ischemia-reperfusion injury seems to more complex than initially predicted.     

Supraceliac, but not infrarenal, aortic cross-clamping upregulates neutrophil integrin CD11b

OBJECTIVE: To evaluate the effects of supraceliac and infrarenal aortic cross-clamping on the expression of neutrophil integrin in CD11b (a marker of systemic cytokine release). DESIGN: Two groups, determined by anatomic placement of aortic cross-clamp. Laboratory personnel were blinded as to group assignment. SETTING: University teaching and community hospitals. Laboratory facilities used were university and Veteran's Affairs medical centers. PARTICIPANTS: Patients scheduled for aortic surgery. INTERVENTIONS: Blood sampling was performed at baseline, after 30 minutes of aortic cross-clamp duration, 30 and 90 minutes after reperfusion (for tumor necrosis factor-alpha plasma levels in infrarenal cross-clamp group), and at baseline and 90 minutes reperfusion (for neutrophil CD11b expression quantification) in both groups. MEASUREMENTS AND MAIN RESULTS: Tumor necrosis factor-alpha measured by ELISA technique did not change at any time period in the infrarenal clamping group. Neutrophil CD11b expression, measured by double antibody staining and FACScan analysis, did not change significantly at 90 minutes of reperfusion in the infrarenal group, but increased significantly (p < 0.05) in the supraceliac aortic cross-clamp group. CONCLUSION: Neutrophil integrin CD11b has been demonstrated to be the primary adhesive glycoprotein responsible for neutrophil organ entrapment and subsequent neutrophil-mediated reperfusion injury. These results suggest that upregulation of neutrophil integrin CD11b after supraceliac aortic clamping may in part be responsible for the higher incidence of acute lung injury after thoracic aortic aneurysm repair requiring supraceliac clamping when compared with infrarenal aneurysm surgery.     

Infection of tobacco or Arabidopsis plants by CMV counteracts systemic post-transcriptional silencing of nonviral (trans)genes

BACKGROUND: Inflammatory cytokine production contributes to lung injury after lung ischemia reperfusion and during lung transplant rejection. Although nitric oxide has been demonstrated to reduce lung injury associated with the adult respiratory distress syndrome, it remains unknown whether the mechanism of nitric oxide's beneficial effects involves reducing lung macrophage inflammatory cytokine production. The purpose of this study was to determine whether nitric oxide downregulates lung macrophage inflammatory cytokine production. METHODS: Lung macrophages were harvested by bronchoalveolar lavage (10(6) macrophage per milliliter from normal Sprague-Dawley rats, 6 animals per group) and treated under ex vivo tissue culture conditions with the nitric oxide releasing compound S-nitoso-N-acetyl-D, L-penicillamine (0, 10(-5) 10(-4), 10(-3), 10(-2) mol/L) before induction of inflammatory cytokines with endotoxin, (50 ng/mL for 24 hours). Supernatants were assayed for inflammatory cytokine production (tumor necrosis factor alpha, interleukin-1beta) by enzyme-linked immunosorbent assay. RESULTS: Continuous nitric oxide release by S-nitoso-N-acetyl-D, L-penicillamine decreased lung macrophage tumor necrosis factor-alpha and interleukin-1beta production in a dose-dependent fashion (6 rats per group; data were analyzed for significance [p < 0.05] using two-way analysis of variance with Tukey's post-hoc correction). CONCLUSIONS: Nitric oxide decreases inflammatory cytokine production by lung macrophage. The mechanism of nitric oxide's beneficial effects may be partially attributable to decreased production of inflammatory cytokines. Nitric oxide may serve an expanded role for reducing inflammatory cytokine production during acute lung injury, ischemia-reperfusion-induced inflammation, or lung transplant rejection.     

Controlled reperfusion after lung ischemia: implications for improved function after lung transplantation

OBJECTIVES: Despite improvements in organ preservation, reperfusion injury remains a major source of morbidity and mortality after lung transplantation. This pilot study was designed to investigate the effects of controlled reperfusion after lung ischemia. METHODS: Twenty adult pigs underwent 2 hours of warm lung ischemia by crossclamping the left bronchus and pulmonary artery. In five (group 1), the clamp was simply removed at the end of ischemia (uncontrolled reperfusion). The 15 other pigs underwent modified reperfusion using blood from the femoral artery to perfuse the lung through the pulmonary artery (pressure 40 to 50 mm Hg) for 10 minutes before removing the pulmonary artery clamp. In five (group 2), the blood was mixed with crystalloid, resulting in a substrate-enriched, hypocalcemic, hyperosmolar, alkaline solution. In five (group 3), the blood was circulated through a leukocyte-depleting filter, and the last five (group 4) underwent reperfusion with both a modified solution and white blood cell filter. Lung function was assessed 60 minutes after reperfusion, and biopsy specimens were taken. RESULTS: Controlled reperfusion with both a white blood cell filter and modified solution (group 4) completely eliminated the reperfusion injury that occurred with uncontrolled reperfusion (group 1), resulting in complete preservation of compliance (98% +/- 1% vs 77% +/- 1%; p < 0.001, and arterial/alveolar ratio (97% +/- 2% vs 27% +/- 2%; p < 0.001); no increase in pulmonary vascular resistance (106% +/- 1% vs 198% +/- 1%; p < 0.001); lowered tissue edema (82.1% +/- 0.4% vs 84.3% +/- 0.2%; p < 0.001), and myeloperoxidase activity (0.18 +/- 0.02 vs 0.35 +/- 0.02 deltaOD/min/mg protein; p < 0.001). In contrast, using either a white blood cell filter or modified solution separately improved but did not avoid the reperfusion injury, resulting in pulmonary function and tissue edema levels that were intermediate between group 1 (uncontrolled reperfusion) and group 4 (white blood cell filter and modified solution). CONCLUSION: After 2 hours of warm pulmonary ischemia, (1) a severe lung injury occurs after uncontrolled reperfusion, (2) controlled reperfusion with either a modified reperfusion solution or white blood cell filter limits, but does not avoid, a lung reperfusion injury, (3) reperfusion using both a modified reperfusate and white blood cell filter results in complete preservation of pulmonary function. We therefore believe surgeons should control the reperfusate after lung transplantation to improve postoperative pulmonary function.     

Prevention of rapid reperfusion-induced lung injury with prostaglandin E1 during the initial period of reperfusion

We have found that the instantaneous restoration of blood flow causes acute dysfunction and massive edema in rat lungs after 4 hours of room temperature ischemia. This is associated with an early increase in pulmonary artery pressure (Ppa) and can be prevented by a stepwise increase in flow rate during the first 10 minutes of reperfusion. The objectives of this study were to determine whether rapid reperfusion causes lung injury after hypothermic preservation, and whether this injury can be attenuated by a short-course of prostaglandin E1 (PGE1). Rat lungs were flushed preserved with low-potassium dextran solution for 12 hours at 4 degrees C and randomly divided into three groups: (1) control (no PGE1); (2) PGE1 only in the flush solution; and (3) PGE1 in both flush solution and blood perfusate during the first 10 minutes of reperfusion. Postpreservation pulmonary function was assessed in an isolated rat lung reperfusion model developed previously. We found that rapid initiation of reperfusion led to significant pulmonary dysfunction, which was attenuated by a short-course of PGE1 in the blood perfusate. The addition of PGE1 to the flush solution alone did not have such an effect. Administration of PGE1 to the blood perfusate during the first 10 minutes resulted in significant lower Ppa and airway pressure and better gas exchange. There was a positive correlation between the peak Ppa during the first 10 minutes of reperfusion and the final shunt fraction. The physical forces generated by the rapid initiation of blood reperfusion appear to induce severe injury. The first 10 minutes of reperfusion seem to be a transition phase in which mechanical factors play an important role relating to ultimate post reperfusion lung function. A short course of PGE1 may be a useful maneuver to prevent rapid reperfusion-induced lung injury.     

CD18-independent mechanism of neutrophil emigration in the rabbit lung after ischemia-reperfusion

BACKGROUND: Reperfusion of ischemic lung causes an inflammatory pulmonary vascular injury characterized by increased vascular permeability and migration of inflammatory cells into the alveoli. Migration of neutrophils into the alveolus during reperfusion after 24 hours of unilateral pulmonary artery occlusion has been shown to be in part dependent on the CD18 adhesion molecule on the cell surface. The current study investigated whether reperfusion lung injury after a 1-hour period of complete lung ischemia was CD18 dependent. METHODS: Eighteen rabbits were assigned to one of three groups. Groups 1 and 2 were subjected to one hour of in situ right hilar occlusion followed by 2 hours of reperfusion. Group 3 was subjected to identical surgical dissection but the right hilum was never occluded. Group 1 rabbits received saline solution (1 mL/kg) before hilar occlusion and group 2 rabbits, monoclonal antibody 60.3, a blocking antibody for the CD18 adhesion molecule on the neutrophil surface (2 mg/kg). In 3 of the antibody-treated rabbits, flow cytometry was performed on blood neutrophils before and after administration of the antibody and 120 minutes after reperfusion. RESULTS: The rabbits in groups 1 and 2 had significantly increased alveolar neutrophil infiltrate and increased pulmonary vascular resistance compared with the rabbits in group 3. However, there was no significant difference between group 1 (saline solution treated) and group 2 (antibody treated). Antibody treatment did not block migration of neutrophils into the alveoli. Flow cytometry of circulating neutrophils demonstrated that CD18 was upregulated after reperfusion and that CD18 was fully blocked after antibody treatment for the duration of the study. CONCLUSIONS: We conclude that a 1-hour period of warm ischemia followed by reperfusion results in upregulation of CD18 but that emigration of the neutrophils into the alveoli is not CD18 dependent in this injury.     

Four year''s experience with quality assurance in anesthesiology in Hamburg

50 eyes of 30 Sprague-Dawley rats were subjected to 60 minutes of pressure-induced ischemia, then fixed for light and electron microscopy with no reperfusion, or reperfusion after 30 minutes, 1, 2 or 4 hours, and 1 or 3 days from the time ocular ischemia was relaxed. The TdT-mediated dUTP-biotin nick end labeling (TUNEL) method revealed apoptotic signs at the inner retina as early as 1 hour after reperfusion. However, the incidence of apoptotic signs with the TUNEL method did not accord with the results of electron microscopic examination. During the time after the reperfusion started, especially after more than 4 hours, apoptotic signs became obvious and extended from the inner to the outer retina. These apoptotic findings could be seen with both the TUNEL method and electron microscopy. By 3 days after the reperfusion, necrotic cells in the ganglion cell layer, and the inner and outer nuclear layer became more prominent than apoptotic cells. These results may provide a baseline for therapeutic strategy and the prognosis of ischemia-reperfusion injury in the retina.     

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.     

Carvedilol, a new beta adrenoreceptor blocker and free radical scavenger, attenuates myocardial ischemia-reperfusion injury in hypercholesterolemic rabbits

Oxygen-derived free radicals play a critical role in atherogenesis and reperfusion injury. The present experiment evaluated the effects of carvedilol, a new beta adrenoreceptor blocker with potent free radical-scavenging activity, on myocardial ischemia and reperfusion injury in a hypercholesterolemic rabbit model. New Zealand rabbits were fed a normal diet, a high-cholesterol diet, or a high-cholesterol diet supplemented with 1200 ppm carvedilol or propranolol. Eight weeks later, the rabbits were subjected to 60 min of myocardial ischemia followed by 60 min of reperfusion. The nontreated cholesterol-fed animals experienced greater cardiac damage after ischemia and reperfusion than rabbits fed a normal diet (necrosis 51% +/- 4% vs. 28% +/- 3% in the normal-diet group, P < .01). In addition, nontreated cholesterol-fed rabbits showed a significantly decreased vasorelaxant response to ACh in U-46619-precontracted aortic rings (56% +/- 5% vs 90% +/- 3% in the control group, P < .001). Treatment with propranolol neither preserved endothelial function after cholesterol feeding nor reduced neutrophil accumulation in ischemic-reperfused myocardial tissue. Propranolol treatment did significantly decrease HR, pressure-rate index and infarct size (necrosis 33% +/- 4%). Despite their having essentially identical effects on HR and pressure-rate index, carvedilol exerted more profound cardiac protective effects than propranolol (necrosis 19% +/- 3%). Moreover, carvedilol treatment significantly preserved aortic endothelial function and markedly reduced neutrophil accumulation in ischemic-reperfused myocardial tissue. These results indicate that in addition to its beta blocking activity, the antioxidant and endothelial protective activities of carvedilol contributed significantly to its cardiac protective effects after ischemia and reperfusion.     

MaMi, a human endometrial stromal sarcoma cell line that constitutively produces interleukin-6, interleukin-8, and monocyte chemoattractant protein-1

BACKGROUND: Uterine endometrial stromal sarcoma is a rare neoplasm with a morphology that closely resembles that of the proliferative endometrial stroma. To understand its pathologic characteristics, we established a novel cell line, MaMi, from a primary culture of an endometrial stromal sarcoma obtained from a 65-year-old Japanese woman. METHODS: We observed the morphology of MaMi cells and performed immunohistochemical analysis on the primary tumor and transplants in nude mice. Prolactin, insulin-like growth factor-binding protein-1, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1, and fibronectin production in the culture medium of MaMi cells were also examined. RESULTS: MaMi cells were shown to exhibit a fibroblast-like morphology in vitro, and they adopted a more elongated appearance in response to 12-O-tetradecanoyl phorbol-13-acetate (TPA). On injection into nude mice, the cells gave rise to subcutaneous tumors. Immunohistologically, both the primary tumor and MaMi cell-induced tumors stained positively with antibodies to neuron-specific enolase or vimentin. MaMi cells constitutively produced IL-6, IL-8, and monocyte chemoattractant protein-1 in vitro. Interleukin-1beta, (100 pmol/L), tumor necrosis factor-alpha (1 nmol/L), and lipopolysaccharide (1 microg/mL) each increased the release of IL-6, IL-8, and monocyte chemoattractant protein-1 by MaMi cells. TPA (10 nmol/L) also stimulated the production of IL-6 and IL-8 by these cells, but inhibited that of monocyte chemoattractant protein-1. CONCLUSIONS: We demonstrated that MaMi cells closely resemble proliferative endometrial stromal cells not only morphologically, but also functionally. This cell line may prove valuable in understanding the role of cytokines produced by tumor cells in the pathogenesis of endometrial stromal sarcoma and may also be useful as an in vitro model of functioning endometrial stromal cells.     

Early release of proinflammatory cytokines after lung transplantation

BACKGROUND: Systemic hypotension may complicate the early postoperative period after lung transplantation. A release of proinflammatory cytokines secondary to lung ischemia/reperfusion injury could be involved in the pathogenesis of this early hemodynamic failure (EHF). Study objective: To assess prospectively whether the occurrence of EHF is associated with a release of cytokines in the systemic circulation. DESIGN: Blood samples were taken daily during the first postoperative week in 26 patients who underwent a double or a single-lung transplantation. These patients were divided into three groups: 7 patients who experienced EHF and subsequently died (EHF group); 15 patients without EHF (control group); and 4 patients without EHF but with an identified sepsis (sepsis group). The serum levels of interleukin (IL)-1beta, tumor necrosis factor-alpha (TNF-alpha), IL-6, and IL-8 were compared among the three groups. RESULTS: In the EHF group, the levels of each cytokine peaked at day 1 postoperatively. Cytokine levels at day 1 were significantly higher in the EHF group than in the control group (p<0.0006) or in the sepsis group (p<0.003 except for TNF-alpha). CONCLUSION: We conclude that EHF is associated with a massive release of proinflammatory cytokines that could play a determinant role in the pathogenesis of this complication.     

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.     

Effect of hypotension preceding death on the function of lungs from donors with nonbeating hearts

BACKGROUND: A shortage of suitable brain-dead donors continues to severely limit lung transplantation. Use of donors with nonbeating hearts has been suggested as a solution. Lungs are unique, in that aerobic metabolism can continue in the absence of blood circulation because oxygen is present in airways and alveoli. Animal studies have shown reasonable cadaveric graft function up to several hours after sudden death by drug administration. However, hemodynamic instability before death may worsen lung function through activation and pulmonary sequestration of neutrophils and release of inflammatory mediators. Because many potential cadaveric donors experience hypotension before death, this study was undertaken to assess the effect of hypotensive shock on cadaveric lung viability. METHODS: A rat isolated lung reperfusion model was used to assess pulmonary function over 3 hours of reperfusion or until gross pulmonary edema developed. Twenty-five rats were randomly allocated to the following study groups, which were based on status before lung harvest: (1) control: no interventions; (2) hypotensive: 1 hour of hypotension by exsanguination to a mean blood pressure of 30 to 40 mm Hg; (3) cadaver: death by cervical dislocation followed by 3 hours of in situ lung ischemia; (4) hypotensive + 3 hours cadaver: 1 hour of hemorrhagic shock, followed by death and 3 hours of in situ ischemia; (5) hypotensive + 2 hours cadaver: similar to group 4, except the in situ ischemia was abbreviated to 2 hours. RESULTS: No significant differences were found among group 1, 2, or 3 lungs with regard to wet to dry weight ratios, gas exchange, and pulmonary arterial or airway pressures. However, all group 4 lungs became grossly hemorrhagic and developed severe pulmonary edema within 10 minutes of reperfusion. Group 5 lungs fared only marginally better, with two of five lungs tolerating 3 hours of reperfusion. CONCLUSIONS: A period of hypotension before death severely impairs cadaveric lung viability.