Hyperosmolarity abrogates neutrophil cytotoxicity provoked by post-shock mesenteric lymph

Hypertonic saline (HTS) resuscitation inhibits acute lung injury in animal models of shock, but some argue this may simply represent more efficient fluid resuscitation. Inflammatory mediators within mesenteric lymph have been identified as a link between splanchnic hypoperfusion and acute respiratory distress syndrome (ARDS). We hypothesize that HTS resuscitation abrogates post-shock lymph-mediated neutrophil (PMN) priming and PMN-mediated human endothelial cell cytotoxicity. Mesenteric lymph was collected from rats (n = 5) before (control), during non-lethal hemorrhagic shock, defined as a mean arterial pressure (MAP) of 40 mmHg for 30 min, and after resuscitation (shed blood + 2 x lactated Ringers (LR) versus 7.5% NaCl, 4 cc/kg, over 5 min). Isolated human PMNs were primed with physiologic concentrations (5% v:v) of lymph either from animals resuscitated with LR or HTS and activated with either PMA or fMLP. In a separate set of experiments, human PMNs were primed with LR lymph after incubation with HTS (180 mM NaCl). The maximal rate of superoxide production was measured by reduction of cytochrome C. In addition, the effect of HTS pretreatment on PMN adherence to human pulmonary microvascular endothelial cells (HMVEC) and PMN-mediated cytotoxicity was determined after lymph-mediated PMN priming. PHSML primed isolated PMNs above buffer controls and pre-shock lymph in a normotonic environment; HTS resuscitation abrogated this effect. HTS preincubation of isolated PMNs inhibited PHSML-induced PMN priming, adherence to HMVECs, and PMN-mediated HMVEC cytotoxicity. Hypertonic resuscitation (HTS) abrogates PHSML pniming of the PMN and PMN-mediated HMVEC cytotoxicity. Furthermore, incubation of PMNs in clinically relevant HTS (180 mM NaCl) prevents PHSML PMN priming and PMN:HMVEC interactions. These studies suggest inhibition of PMN signal transduction is a mechanism whereby HTS resuscitation abrogates acute lung injury.     

Stimulated neutrophils evoke signal transduction to increase vascular permeability in rat lungs

The mechanisms by which stimulated neutrophils (PMNs) damage pulmonary vascular endothelium were investigated using twenty-four perfused lung preparations isolated from rats. We tested the ability of unstimulated and mechanically stimulated PMNs to adhere to pulmonary endothelial cells and, thereby, alter pulmonary vascular permeability (measured as the pulmonary filtration coefficient) and hemodynamics. To stimulate PMNs, they were gently agitated in a glass vial for 10 seconds. Perfusing lungs with the stimulated PMNs (stimulated group) elicited a 3-fold increase in the filtration coefficient as compared to lungs perfused with unstimulated cells (unstimulated group). This increase in filtration was completely blocked by preincubation of stimulated PMNs with CD18 monoclonal antibody (MoAb group). This increase in filtration coefficient was also completely blocked by GF109203X, a protein kinase C inhibitor (GF group). Pulmonary vascular resistance increased when the stimulated PMNs were injected to the isolated lungs. Although, preincubation of stimulated PMNs with CD18 MoAb successfully blocked and GF109203X partly blocked this increase in pulmonary vascular resistance. The accumulation of stimulated PMNs within the lungs, as assessed by myeloperoxidase (MPO) levels, was blocked by preincubation of stimulated PMNs with CD18 MoAb. However, GF109203X did not decrease MPO levels. These findings suggest that stimulated PMN-induced increases in pulmonary vascular filtration, resulted from endothelial cell injury caused by adhesion to the endothelial cells, evoke intracellular signaling within the endothelial cells.     

Phorbol myristate acetate-induced lung injury: involvement of reactive oxygen species.

Using lucigenin-enhanced chemiluminescence, isolated rat lungs perfused with physiological salt-Ficoll solution were studied to test whether phorbol myristate acetate (PMA)-induced lung injury was mediated by reactive oxygen species (ROS). PMA (0.03 micrograms ml-1) caused small but significant increases in lung ROS levels and pulmonary arterial perfusion pressure (Ppa) but did not induce lung oedema. PMA (0.15 micrograms ml-1) induced lung oedema with large increases in ROS production and Ppa. Superoxide dismutase (SOD) inhibited the increases in ROS, Ppa, and lung oedema. Catalase and dimethylthiourea inhibited lung oedema but did not attenuate the increases in ROS and Ppa entirely. Indomethacin attenuated lung oedema partially but did not inhibit the increases in ROS and Ppa. These data indicate that PMA-induced lung injury is dependent on PMA concentration and ROS are responsible for such lung injury. Thromboxane plays a minor role for PMA-induced lung injury. The different effects of oxygen radical scavengers suggest that different radical species contribute to the increased pulmonary vascular response and lung injury.     

Phorbol myristate acetate-induced lung injury: Involvement of reactive oxygen species

Using lucigenin-enhanced chemiluminescence, isolated rat lungs perfused with physiological salt-Ficoll solution were studied to test whether phorbol myristate acetate (PMA)-induced lung injury was mediated by reactive oxygen species (ROS). PMA (0.03 pg ml-I) caused small but significant increases in lung ROS levels and pulmonary arterial perfusion pressure (Ppa) but did not induce lung oedema. PMA (0.15 pg ml-I) induced lung oedema with large increases in ROS production and Ppa. Superoxide dismutase (SOD) inhibited the increases in ROS, Ppa, and lung oedema. Catalase and dimethylthiourea inhibited lung oedema but did not attenuate the increases in ROS and Ppa entirely. Indomethacin attenuated lung oedema partially but did not inhibit the increases in ROS and Ppa. These data indicate that PMA-induced lung injury is dependent on PMA concentration and ROS are responsible for such lung injury. Thromboxane plays a minor role for PMA-induced lung injury. The different effects of oxygen radical scavengers suggest that different radical species contribute to the increased pulmonary vascular response and lung injury.     

The effect of tumor necrosis factor-alpha on microvascular permeability in an isolated,perfused lung

This study examines the hypotheses that TNF-alpha causes a dose-dependent increase in the microvascular permeability of ex vivo buffer perfused lungs that is quantitatively similar to that caused by lipopolysaccharide (LPS) or thromboxane A2 (TxA2). We also postulated that TNF-alpha potentiates the effect of interleukin-1beta (IL-1beta) or TxA2 receptor activation on pulmonary microvascular permeability. Lungs harvested from Wistar rats were perfused ex vivo with Krebs-Henseleit buffer containing 0, 10, 100, or 1000 ng/mL recombinant rat TNF-alpha. Twenty minutes later pulmonary microvascular permeability was determined by measuring the capillary filtration coefficient (Kf) using a gravimetric technique. The effect of TNF-alpha (100 ng/mL) on pulmonary Kf was compared with that of lungs exposed to LPS (400 microg/mL; E. coli 0111:B4) or a TxA2 receptor agonist (U-46619; 7 x 10(-8)). In other experiments, perfused lungs were exposed to TNF-alpha plus IL-1beta (1 ng/mL) or TNF-alpha plus U-46619 after which Kf was measured. Exposure of ex vivo buffer perfused lungs to 10-1000 ng/mL TNF-alpha had no effect on Kf whereas LPS and U-46619 was associated with a two- and six-fold increase in Kf, respectively (P < 0.05). The Kf of lungs exposed to TNF-alpha plus IL-1 was similar to that of lungs exposed to TNF-alpha alone. Lastly, the Kf of lungs exposed to TNF-alpha plus U-46619 was not different than that of lungs exposed to U-46619 alone. In conclusion, TNF-alpha at least when administered for a relatively brief period of time does not affect microvascular permeability in an isolated, buffer-perfused lung model.     

Wortmannin, a potent antineutrophil agent, exerts cardioprotective effects in myocardial ischemia/reperfusion

Ischemia followed by reperfusion in the presence of polymorphonuclear leukocytes (PMNs) results in a marked cardiac contractile dysfunction. Wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, suppresses superoxide production from PMNs. Therefore, we hypothesized that wortmannin could attenuate PMN-induced cardiac dysfunction by suppression of superoxide production from PMNs. We examined the effects of wortmannin in isolated ischemic (20 min) and reperfused (45 min) rat hearts perfused with PMNs. Wortmannin at 10, 20, or 40 nM given to hearts during the first 5 min of reperfusion, significantly improved left ventricular developed pressure (P < .01), and the maximal rate of development of left ventricular developed pressure (P < .01) compared with ischemic/reperfused hearts perfused with PMNs in the absence of wortmannin. In addition, wortmannin significantly reduced PMN infiltration into the myocardium by 50 to 75% (P < .001). Superoxide radical release also was significantly reduced in N-formylmethionyl-leucylphenylalanine-stimulated PMNs pretreated with 10 or 40 nM wortmannin by 70 and 95%, respectively (P < .001 versus untreated PMNs). Rat PMN adherence to rat superior mesenteric artery endothelium exposed to 2 U/ml thrombin was significantly attenuated by 10 to 40 nM wortmannin compared with untreated vessels (P < .001). These results provide evidence that wortmannin can significantly attenuate PMN-induced cardiac contractile dysfunction in the ischemic/reperfused rat heart via attenuation of PMN infiltration into the myocardium and suppression of superoxide release by PMNs.     

Plasma and lipids from stored packed red blood cells cause acute lung injury in an animal model.

Transfusion-related acute lung injury (TRALI) is a serious complication of hemotherapy. During blood storage, lipids are generated and released into the plasma. In this study, the role of these lipids in TRALI was investigated using an isolated, perfused rat lung model. Rats were pretreated with endotoxin (LPS) or saline in vivo and the lungs were isolated, ventilated, and perfused with saline, or (a) 5% (vol/ vol) fresh human plasma, (b) plasma from stored blood from the day of isolation (D.0) or from the day of outdate (D.42), (c) lipid extracts from D.42 plasma, or (d) purified lysophosphatidylcholines. Lungs from saline or LPS-pretreated rats perfused with fresh (D.0) plasma showed no pulmonary damage as compared with saline perfused controls. LPS pretreatment/D.42 plasma perfusion caused acute lung injury (ALI) manifested by dramatic changes in both pulmonary artery pressure and edema. Incubation of LPS pre-tx rats with mibefradil, a Ca2+ channel blocker, or WEB 2170, a platelet-activating factor (PAF) receptor antagonist, inhibited ALI caused by D.42 plasma. Lung histology showed neutrophil sequestration without ALI with LPS pretreatment/saline or D.0 plasma perfusion, but ALI with LPS pretreatment/D.42 plasma perfusion, and inhibition of D.42 plasma induced ALI with WEB 2170 or mibefradil. A significant increase in leukotriene E4 was present in LPS-pretreated/D.42 plasma-perfused lungs that was inhibited by WEB 2170. Lastly, significant pulmonary edema was produced when lipid extracts of D.42 plasma or lysophosphatidylcholines were perfused into LPS-pretreated lungs. Lipids caused ALI without vasoconstriction, except at the highest dose employed. In conclusion, both plasma and lipids from stored blood produced pulmonary damage in a model of acute lung injury. TRALI, like the adult respiratory distress syndrome, may be the result of two insults: one derived from stored blood and the other from the clinical condition of the patient.     

Coagulation-mediated hypoxia and neutrophil-dependent hepatic injury in rats given lipopolysaccharide and ranitidine

Idiosyncrasy-like liver injury occurs in rats cotreated with nonhepatotoxic doses of ranitidine (RAN) and bacterial lipopolysaccharide (LPS). Hepatocellular oncotic necrosis is accompanied by neutrophil (PMN) accumulation and fibrin deposition in LPS/RAN-treated rats, but the contribution of PMNs to injury has not been shown. We tested the hypothesis that PMNs are critical mediators of LPS/RAN-induced liver injury and explored the potential for interaction between PMNs and hemostasis-induced hypoxia. Rats were given either LPS (44.4 x 10(6) endotoxin units/kg) or its vehicle and then RAN (30 mg/kg) or its vehicle 2 h later. They were killed 3 or 6 h after RAN treatment, and hepatocellular injury was estimated from serum alanine aminotransferase activity and liver histopathology. Plasma PMN chemokine concentration and the number of PMNs in liver increased after LPS treatment at 3 h and were not markedly altered by RAN cotreatment. Depletion of circulating PMNs attenuated hepatic PMN accumulation and liver injury and had no effect on coagulation system activation. Anticoagulation with heparin attenuated liver fibrin deposition and injury in LPS/RAN-treated rats; however, heparin had little effect on liver PMN accumulation or plasma chemokine concentration. Liver hypoxia occurred in LPS/RAN-cotreated rats and was significantly reduced by heparin. In vitro, hypoxia enhanced the killing of rat hepatocytes by PMN elastase and shortened its onset, indicating a synergistic interaction between PMNs and hypoxia. The results suggest that PMNs are involved in the hepatocellular injury caused by LPS/RAN-cotreatment and that hemostasis increases sensitivity to PMN-induced hepatocellular injury by causing liver hypoxia.     

Quantitative and functional alterations of peripheral blood neutrophils after 10% and 30% thermal injury

Polymorphonuclear leukocytes (PMNs) comprise the majority of early nonspecific inflammatory responses to infection or trauma and, as such, must be of sufficient number and qualitative function to properly limit and combat inflammation. Peripheral PMNs isolated from rats that received 10% or 30% body surface area full-thickness thermal injuries were quantitated and examined for functional alterations in membrane potential and cytosolic hydrogen peroxide production for 35 days after thermal injury. With 10% thermal injury, leukocytes increased quantitatively to experimental maximums that were 70% above normal on day 7 before a return to normal by day 28. Platelet levels showed a nonsignificant decrease for 2 days after thermal injury before increasing to levels 20% to 40% above normal through day 28. Phorbol myristate acetate-induced PMN membrane depolarization was inhibited as much as 30% for 21 days after 10% thermal injury. No changes in oxidative activity were apparent except for day 14, when hydrogen peroxide production was 40% above normal. With 30% thermal injury, leukocyte quantities were three to five times normal, with increased relative numbers of PMNs and decreased lymphocytes through day 28. Platelet levels decreased for 4 days before increasing to levels 30% to 47% above normal through day 21. Compared with 10% thermal injury, 30% thermal injury further reduced the ability of PMN membranes to depolarize through day 35. In addition, PMN hydrogen peroxide production was 30% lower on day 1 and increased thereafter to levels that were 40% above normal on day 21.     

Effects of prostaglandin E1 on platelet attenuation of oxidant-induced edema in isolated rabbit lungs

Numerous studies suggest that platelets may contribute to preservation of normal endothelial cell permeability in models of lung injury. We have previously shown that washed human platelets prevent xanthine oxidase-induced edema in the isolated perfused lung and that protective mechanisms depend on the platelet glutathione redox cycle. It is uncertain, however, whether platelets preserve endothelial function by reducing toxic oxygen metabolites or by aggregating and releasing endothelial cell supportive factors-an activity that may require the glutathione redox cycle. In this study, we present data demonstrating that platelet prevention of oxidant lung injury occurs independent of platelet aggregation and release. Isolated rabbit lungs perfused with a cell-free medium were instilled with purine (2 mmol/L) and xanthine oxidase (0.003 U/ml) to generate oxidant lung edema. The infusion of washed human platelets (1 x 10(10) cells) prevented lung edema formation as measured by lung weight gain, wet-to-dry lung weight ratios, and lung histology. Incubation of platelets with prostaglandin E1 (PGE1), a potent inhibitor of platelet aggregation and release, did not inhibit platelet attenuation of lung edema. Additionally, with the instillation of PGE1 into the perfusate to further inhibit platelet aggregation, no prevention of lung protection by PGE1-treated platelets was seen when these results were compared with those from studies in which lungs were infused with xanthine oxidase and PGE1. Aggregometry studies documented that the inhibitory effect of PGE1 on platelet aggregation persisted for up to 60 minutes, which was the duration of the isolated lung protocol. We conclude that platelet aggregation and release of platelet factors is not required for platelet attenuation of oxidant lung edema.     

Peroxynitrite inhibits leukocyte-endothelial cell interactions and protects against ischemia-reperfusion injury in rats.

Peroxynitrite (ONOO-) anion, formed by the interaction of superoxide with nitric oxide (NO), has previously been implicated as a cytotoxic agent. However, the effects of this free radical species on neutrophil (PMN)-endothelial cell interactions is largely unknown. We investigated the direct actions of ONOO- on PMN adhesion to endothelial cells in vitro and in vivo, as well as the effects of ONOO- on PMN-mediated myocardial ischemia-reperfusion injury. In vitro, peroxynitrite (100-1,000 nM) inhibited the adhesion of rat PMNs to the endothelium of isolated thrombin- or H2O2-stimulated rat mesenteric artery (P < 0.01 vs. thrombin or H2O2 alone). In vivo, in the rat mesentery, thrombin (0.5 U/ml) or N(G)-nitro-L-arginine-methyl ester (50 microM) significantly increased venular leukocyte rolling and adherence, which were also significantly (P < 0.01) attenuated by ONOO (800 nM) accompanied by reduced P-selectin expression on the endothelial cell surface. Isolated perfused rat hearts were subjected to global ischemia and reperfusion with rat PMNs (10(8) cells), which resulted in profound cardiac depression (i.e., a marked reduction in left ventricular developed pressure and maximal rate of development of left ventricular pressure). Infusion of ONOO- reversed the myocardial contractile dysfunction of ischemic-reperfused rat hearts to near baseline levels, and markedly attenuated the accumulation of PMNs in the postischemic heart. The present study provides strong evidence that nanomolar concentrations of ONOO- both inhibit leukocyte-endothelial cell interactions and exert cytoprotective effects in myocardial ischemia-reperfusion injury. Furthermore, our results suggest that the inhibition of P-selectin expression by peroxynitrite is a key mechanism of the modulatory actions of ONOO- on leukocyte-endothelial cell interactions.     

Cyclin-dependent kinase inhibition reduces lung damage in a mouse model of ventilator-induced lung injury

Mechanical ventilation (MV) has the potential to induce lung damage in healthy lungs or aggravate existing lung injury. Polymorphonuclear neutrophil (PMN) recruitment plays an important role in driving the inflammatory response in ventilator-induced lung injury (VILI). The cyclin-dependent kinase inhibitor r-roscovitine has been shown to induce apoptosis in PMNs. In this study, we investigated the potential of r-roscovitine treatment in reducing lung damage in a mouse model of VILI. Mice were tracheotomized and subjected to lung-protective MV with lower (～7.5 mL/kg) or lung-injurious MV with higher (～15 mL/kg) tidal volume (VT). R-roscovitine treatment enhanced apoptosis in PMNs in vitro. Ventilator-induced lung injury was associated with pulmonary PMN influx in low and high VT MV. During lung-injurious MV, r-roscovitine treatment reduced the number of PMNs and lowered levels of the lung damage markers RAGE (receptor for advanced glycation end products) and total immunoglobulin M in bronchoalveolar lavage fluid. R-roscovitine did not affect cytokine or chemokine levels in the bronchoalveolar space, neither during lung-protective nor lung-injurious MV. Thus, r-roscovitine treatment reduces lung damage in VILI, possibly dependent on increased apoptosis of PMNs.     

Tumor necrosis factor mediates experimental pulmonary edema by ICAM-1 and CD18-dependent mechanisms.

(TNF alpha)-induced sequestration of neutrophils (PMN) in lungs and of the resultant PMN-dependent pulmonary edema. Guinea pig lungs perfused with Ringers-albumin were challenged with TNF alpha (1,000 U/ml) for 90 min, followed by addition of fresh perfusate containing 2 x 10(7) human PMN. TNF alpha challenge caused sequestration of PMN in the pulmonary vascular bed as indicated by a threefold increase in lung tissue myeloperoxidase activity (MPO). The activation of the sequestered PMN with phorbol 12-myristate 13-acetate (PMA; 5 x 10(-9) M) produced threefold increases in pulmonary artery (Ppa) and pulmonary capillary hydrostatic (Pcap) pressures, and twofold increases in lung wet-to-dry weight (W/D) ratio and capillary filtration coefficient (Kf,c) over baseline. TNF alpha prestimulation was required for these responses since activation of PMN with PMA in control lungs produced smaller increases in Ppa and Pcap (P less than 0.01) and did not change the W/D and Kf,c. TNF alpha prestimulation also induced the expression of intercellular adhesion molecule (ICAM-1) on pulmonary vascular endothelial cells. Monoclonal antibodies (mAbs) to the neutrophil CD18 integrin (beta-chain of CD11/CD18 complex) (mAb IB4) and to its endothelial cell ligand ICAM-1 (mAb RR1/1) were used to examine the role of PMN adhesion in the TNF alpha-induced responses. Pretreatment of PMN with mAb IB4 prevented PMN uptake and increases in Ppa, Pcap, Kf,c, and W/D ratio. Addition of mAb RR1/1 to the perfusate reduced PMN uptake by 58%, and prevented the increases in Ppa, Pcap, Kf,c, and W/D ratio, as with mAb IB4. The findings indicate that TNF alpha prestimulation of lungs mediates PMN uptake and that this requires the expression of ICAM-1 and its interaction with CD18 integrin on PMN. The activation of PMN sequestered by ICAM-1-dependent mechanism contributes to the development of pulmonary vascular injury and edema.     

Critical role of endothelial CXCR2 in LPS-induced neutrophil migration into the lung

In models of acute lung injury, CXC chemokine receptor 2 (CXCR2) mediates migration of polymorphonuclear leukocytes (PMNs) into the lung. Since CXCR2 ligands, including CXCL1 and CXCL2/3, are chemotactic for PMNs, CXCR2 is thought to recruit PMNs by inducing chemotactic migration. In a model of PMN recruitment to the lung, aerosolized bacterial LPS inhalation induced PMN recruitment to the lung in wild-type mice, but not in littermate CXCR2-/- mice. Surprisingly, lethally irradiated wild-type mice reconstituted with CXCR2-/- BM still showed about 50% PMN recruitment into bronchoalveolar lavage fluid and into lung interstitium, but CXCR2-/- mice reconstituted with CXCR2-/- BM showed no PMN recruitment. Conversely, CXCR2-/- mice reconstituted with wild-type BM showed a surprisingly large defect in PMN recruitment, inconsistent with a role of CXCR2 on PMNs alone. Cell culture, immunohistochemistry, flow cytometry, and real-time RT-PCR were used to show expression of CXCR2 on pulmonary endothelial and bronchial epithelial cells. The LPS-induced increase in lung microvascular permeability as measured by Evans blue extravasation required CXCR2 on nonhematopoietic cells. Our data revealed what we believe to be a previously unrecognized role of endothelial and epithelial CXCR2 in LPS-induced PMN recruitment and lung injury.     

Pulmonary leukostasis and the inhibition of airway neutrophil recruitment are early events in the endotoxemic rat

Neutrophil (polymorphonuclear leukocyte [PMN]) migration into pulmonary airspaces is a prerequisite for clearance of bacteria commonly found in nosocomial pneumonia. Patients at risk for nosocomial pneumonia often experience endotoxemia, and neutrophil dysfunction is associated with endotoxemia in both humans and animals. Using a rodent model of endotoxemia-associated pneumonia, we characterized the altered kinetics of pulmonary PMN trafficking and addressed the roles of platelets, tumor necrosis factor (TNF), and products of complement activation as potential mediators in the modulation of PMN migratory function. In male Sprague-Dawley rats made endotoxemic with intravenously (i.v.) administered endotoxin (lipopolysaccharide [LPS]), recruitment of PMNs into the lung airspaces in response to intratracheally (i.t.) instilled LPS was inhibited. In animals given IT LPS alone (0.5 mg/rat), numbers of airway PMNs were significantly elevated by 2 h, and immunohistochemical evaluation revealed PMNs in alveolar airspaces, alveolar walls, and in interstitium surrounding large airways. LPS (2 mg/kg i.v.) caused neutropenia and pulmonary PMN sequestration within 15 min of administration. Inhibition of airway PMN accumulation occurred by 30 min and lasted for at least 6 h after i.v. LPS. Factors present or activated after 30 min of endotoxemia were hypothesized to mediate the inhibitory effect of i.v. LPS. We found that pretreatment of rats with cobra venom factor to deplete complement (and C5a production) or immunodepletion of platelets or TNF did not affect the ability of i.v. LPS to inhibit pulmonary PMN recruitment or to cause pulmonary leukostasis. In summary, our results show that the inhibitory effects of i.v. LPS on PMN trafficking are rapid and persist for several hours and suggest that neither TNF, C5a, nor platelets are sufficient to mediate the inhibitory response.     

Entry of gut lymph into the circulation primes rat neutrophil respiratory burst in hemorrhagic shock.

OBJECTIVE: Endothelial cell injury by polymorphonuclear neutrophil (neutrophil [PMN]) respiratory burst after trauma and hemorrhagic shock (T/HS) predisposes subjects to acute respiratory distress syndrome and multiple organ failure. T/HS mesenteric lymph injures endothelial cell and lymph duct ligation (LDL) before T/HS prevents pulmonary injury. We investigated the role of mesenteric lymph in PMN priming by T/HS. DESIGN: Prospective experiment in rats. SETTING: University hospital laboratory. SUBJECTS: Adult male rats. INTERVENTIONS: Mesenteric lymph was obtained from rats undergoing T/HS (30 mm Hg, 90 mins) or sham shock (T/SS). Plasma was harvested from uninstrumented control (UC), T/HS, T/SS, and T/HS+LDL rats. PMNs were isolated from UC, T/HS, and T/HS+LDL rats. MEASUREMENTS AND MAIN RESULTS: PMNs from UC rats were incubated in buffer, 1% T/HS lymph, and 1% T/SS lymph. PMNs from UC rats were incubated in UC, T/HS, T/SS, and T/HS+LDL plasma. PMN respiratory burst was initiated by using macrophage inflammatory protein (MIP)-2/platelet-aggregating factor (PAF) or phorbol myristate acetate. Cytosolic calcium ([Ca2+]i) responses to MIP-2/PAF were assayed in PMN from UC, T/HS, and T/HS+LDL rats. PMN preincubated in T/HS lymph showed significant elevations in MIP/PAF-elicited respiratory burst compared with T/HS lymph or buffer only (p <.05; analysis of variance/Tukey's test). T/HS lymph incubation also increased (p <.05) phorbol myristate acetate elicited respiratory burst compared with buffer or T/SS. Preincubation in T/HS plasma increased MIP-2/PAF-elicited respiratory burst (p <.05) compared with UC or T/SS plasma. LDL blocked T/HS priming of respiratory burst. Control PMN [Ca2+]i responses to MIP-2 and PAF were low. T/SS PMN were significantly more responsive, but the T/HS PMN showed still higher responses (p <.01). LDL reversed the priming of [Ca2+]i responses by T/HS (p <.01). CONCLUSIONS: PMNs are primed by T/HS lymph but not T/SS lymph and by T/HS plasma but not T/SS plasma. LDL before shock prevents T/HS plasma from priming PMN. The magnitude of respiratory burst found here paralleled the [Ca2+]i responses seen to receptor dependent initiating agonists. Mesenteric lymph is both necessary and sufficient to prime PMN after T/HS in the rat, and it primes PMN in part by enhancing [Ca2+]i responses to G-protein coupled chemoattractants. Mesenteric lymph mediates postshock PMN dysfunction.     

Prevention of granulocyte-mediated oxidant lung injury in rats by a hydroxyl radical scavenger, dimethylthiourea

Toxic, partially reduced metabolites of oxygen (toxic oxygen radicals) are increasingly implicated in acute leukocyte-mediated tissue injury. To further probe the roles of oxygen radicals in acute lung edema, I studied the effects of a recently described and very potent oxygen radical scavenger, dimethylthiourea (DMTU) (Fox, R. B., R. N. Harada, R. M. Tate, and J. E. Repine, 1983, J. Appl. Physiol., 55:1456-1459) on polymorphonuclear leukocyte (PMN) oxidant function and on two types of lung injury mediated by oxygen radicals and PMN. DMTU (10 mM) blocked 79% of hydroxyl radical (OH) production by PMN in vitro without interfering with other PMN functions, such as O-2 production, myeloperoxidase activity, chemotaxis, degranulation, or aggregation. When isolated rat lung preparations were perfused with PMN activated to produce OH, lung weights were increased from 2.3 +/- 0.2 to 11.2 +/- 0.8 g. DMTU (10 mM) prevented 70% of these increases (lung weights, 5.0 +/- 1.1 g, P less than 0.005). Finally, when intact rats were exposed to 100% O2 for 66 h, lung weight:body weight ratios were increased from 5.78 +/- 0.33 to 8.87 +/- 0.16 g. DMTU (500 mg/kg) prevented 83% of this hyperoxia-induced lung edema in vivo (lung:body weight ratios, 6.05 +/- 0.21, P less than 0.001). Pharmacokinetic studies showed that DMTU diffused effectively into lung interstitial fluids and had a relatively long half-life (25-35 h) in the circulation. Because a variety of oxygen radicals, such as superoxide (O-2), hydrogen peroxide (H2O2), or OH are produced by PMN, there is usually some uncertainty about which one is responsible for injury. However, in these studies, DMTU did not scavenge O-2 and scavenged H2O2 only very slowly while scavenging OH very effectively. Therefore, DMTU may be useful in the investigation of the roles of oxygen radicals, especially OH, in acute granulocyte-mediated tissue injury.     

Tezosentan reduces the microvascular filtration coefficient in isolated lungs from rats subjected to cecum ligation and puncture

Introduction  

We recently demonstrated that the non-selective endothelin-1 (ET-1) receptor blocker tezosentan antagonizes ovine acute lung injury (ALI) following infusion of endotoxin or ET-1 by reducing the enhanced lung microvascular pressure, although we could not exclude the possibility of a simultaneous decline in microvascular permeability. In the present study, our aim was to find out if tezosentan reverses the rise in microvascular filtration coefficient (Kfc) in rat lungs that have been isolated and perfused 12 h after cecum ligation and puncture (CLP) or infusion of ET-1.     

Neutrophil interaction with the hemostatic system contributes to liver injury in rats cotreated with lipopolysaccharide and ranitidine

Cotreatment of rats with nontoxic doses of ranitidine (RAN) and lipopolysaccharide (LPS) causes liver injury, and this drug-inflammation interaction might be a model for idiosyncratic adverse drug responses in humans. Both polymorphonuclear neutrophils (PMNs) and the hemostatic system have been shown to be important in the injury. We tested the hypothesis that PMNs cause liver injury by interacting with the hemostatic system and producing subsequent hypoxia. In rats cotreated with LPS/RAN, PMN depletion by anti-PMN serum reduced fibrin deposition and hypoxia in the liver. PMN depletion also reduced the plasma concentration of active plasminogen activator inhibitor-1 (PAI-1), a major down-regulator of the fibrinolytic system. This suggests that PMNs promote fibrin deposition by increasing PAI-1 concentration. PMNs were activated in the livers of LPS/RAN-cotreated rats as evidenced by increased staining for hypochlorous acid-modified proteins generated by the myeloperoxidase-hydrogen peroxide-chloride system of activated phagocytes. Antiserum against the PMN adhesion molecule CD18 protected against LPS/RAN-induced liver injury. Because CD18 is important for PMN transmigration and activation, these results suggest that PMN activation is required for the liver injury. Furthermore, anti-CD18 serum reduced biomarkers of hemostasis and hypoxia, suggesting the necessity for PMN activation in the interaction between PMNs and the hemostatic system/hypoxia. Liver injury, liver fibrin, and plasma PAI-1 concentration were also reduced by eglin C, an inhibitor of proteases released by activated PMNs. In summary, PMNs are activated in LPS/RAN-cotreated rats and participate in the liver injury in part by contributing to hemostasis and hypoxia.