Lung Neutrophil Accumulation Research Articles

Mepacrine decreases lung leak in rats given interleukin-1 intratracheally.

We hypothesized that phospholipase A2 (PLA2) metabolites contribute to the acute, neutrophil-dependent, edematous lung leak that develops after administration of interleukin-1 (IL-1) intratracheally to rats and tested this premise by using mepacrine to inhibit PLA2 activity in vivo. We found that lung PLA2 activity, lung lavage phospholipid content, lung leak index, lung weight gain, and lung lavage protein concentrations were increased in rats given IL-1 intratracheally compared with sham-treated control rats. By comparison, lungs of mecaprine and IL-1-treated rats had decreased PLA2 activity, lavage phospholipid content, leak, weight gain, and lavage protein increases compared with rats given IL-1 intratracheally. Mepacrine treatment also decreased lung neutrophil accumulation, but not lung lavage cytokine-induced neutrophil chemoattractant (CINC) levels, in rats given IL-1 intratracheally. In parallel experiments, mepacrine treatment reduced the adhesion of human neutrophils to IL-1-treated human umbilical vein endothelial cells in vitro. Our results indicate that PLA2 activity participates in the lung neutrophil retention and pulmonary vascular leak that develops in rats given IL-1 intratracheally.

Effects of inhaled nitric oxide on pulmonary edema and lung neutrophil accumulation in severe experimental hyaline membrane disease.

To determine the effects of inhaled NO (iNO) on pulmonary edema and lung inflammation in experimental hyaline membrane disease (HMD), we measured the effects of iNO on pulmonary hemodynamics, gas exchange, pulmonary edema, and lung myeloperoxidase (MPO) activity in extremely premature lambs (115 d of gestation, 0.78 term). In protocol 1, we measured the effects of iNO (20 ppm) on lung vascular endothelial permeability to 125I-labeled albumin (indexed to blood volume using 57Cr-tagged red blood cells) during 1 h (n = 10) and 3 h (n = 14) of conventional mechanical ventilation with FiO2 = 1.00. In comparison with controls, iNO improved pulmonary hemodynamics and gas exchange, but did not alter lung weight-to-dry weight ratio or vascular permeability to albumin after 1 or 3 h of mechanical ventilation. To determine whether low dose iNO (5 ppm) would decrease lung neutrophil accumulation in severe HMD, we measured lung MPO activity after 4 h of mechanical ventilation with or without iNO (protocol 2). Low dose iNO improved gas exchange during 4 h of mechanical ventilation (PaO2 at 4 h: 119 +/- 35 mm Hg iNO versus 41 +/- 7 mm Hg control, p < 0.05), and reduced MPO activity by 79% (p < 0.05). We conclude that low dose iNO increases pulmonary blood flow, without worsening pulmonary edema, and decreases lung neutrophil accumulation in severe experimental HMD. We speculate that in addition to its hemodynamic effects, low dose iNO decreases early neutrophil recruitment and may attenuate lung injury in severe HMD.

Neutrophils mediate pulmonary vasomotor dysfunction in endotoxin-induced acute lung injury.

The major hemodynamic feature of endotoxin (ETX)-induced acute lung injury is pulmonary hypertension secondary to increased pulmonary vascular resistance. Endotoxin causes dysfunction of pulmonary vasorelaxation, which is associated with increased lung neutrophil accumulation. We hypothesized that neutrophils mediate the dysfunction of cGMP-mediated pulmonary vasorelaxation in acute lung injury. In a rat model of ETX-induced lung injury, our purpose was to determine the effect of neutrophil depletion on the following mechanisms of pulmonary vasomotor control: endothelium-dependent cGMP-mediated relaxation (response to acetylcholine) and endothelium-independent relaxation (response to sodium nitroprusside). Rats were studied 6 hours after ETX (20 mg/kg). Neutropenia (< 75 neutrophils/microL) was induced with anti-neutrophil serum 24 hours before ETX. Saline injected rats were controls. Dose-response curves to acetylcholine and sodium nitroprusside were generated in isolated pulmonary artery rings preconstricted with phenylephrine (n = 10 rings/5 rats per group). Lungs were harvested (n = 4 rats/group) and lung neutrophil accumulation was assessed with a myeloperoxidase assay. Endothelium-dependent and -independent cGMP-mediated pulmonary vasorelaxation was dysfunctional in ETX-induced ALI. Neutrophil depletion prevented lung neutrophil accumulation and attenuated pulmonary vasomotor dysfunction after endotoxin. These data suggest that neutrophils contribute to pulmonary endothelium and smooth muscle dysfunction in acute lung injury induced by endotoxemia.

Oxidative organ damage in a rat model of thermal injury: the effect of cyclosporin A

Animal models of thermal trauma implicate oxygen radicals as a causative agent in local wound response, development of burn shock and distant organ injury. It has been proposed that the source of reactive oxygen metabolites could be neutrophils sequestered in systemic organs as a result of the systemic inflammatory reaction to a local burn insult. Recent studies have suggested that cyclosporin A (CsA), a potent immunosuppressive drug, may have effects on neutrophils by modulating the rate of their accumulation during acute inflammatory reactions. This study aimed to assess the role of neutrophils in the early and late phases of burn injury in rats with second-degree skin burn. We also aimed to determine whether CsA has protective effects on organs remote from the thermal injury. The results demonstrate that there is significant neutrophil accumulation in the gastric mucosa, liver and lung tissues during the early phase of a burn injury and that CsA failed to protect these organs. In conclusion, the data of this study suggest that neutrophil accumulation in liver, lung and gastric mucosa following burn injury may be involved in the pathogenesis of remote organ damage. The results also indicate that CsA failed to reduce the severity of damage in these organs, probably due to its own toxic effects.

Impairment of endothelial-dependent pulmonary vasorelaxation after mesenteric ischemia/reperfusion

A major hemodynamic feature of acute lung injury is pulmonary hypertension caused by pulmonary vasoconstriction. Impairment of the mechanisms of pulmonary vasorelaxation may contribute to this pulmonary vasoconstriction. This study examined the effect of mesenteric ischemia/reperfusion (I/R) on lung neutrophil accumulation and endothelial-dependent and -independent cyclic 3'-5' guanosine monophosphate-mediated pulmonary vasorelaxation in rats. Rats were studied after 1 hour of superior mesenteric artery occlusion and 2 hours of reperfusion. Lung neutrophil accumulation was determined by myeloperoxidase assay (MPO). The following mechanisms of pulmonary vasorelaxation were studied in isolated pulmonary artery rings by generating dose response curves (10(-9) to 10(-6)mol/L): (1) receptor-dependent, endothelial-dependent relaxation (response to acetylcholine), (2) receptor-independent, endothelial-dependent relaxation (response to the calcium ionophore, A23187), and (3) endothelial-independent relaxation (response to sodium nitroprusside [SNP]). Lung MPO activity was significantly increased from 2.4 +/- 0.2 units/gm lung weight in controls to 10.3 +/- 0.4 after mesenteric I/R (p < 0.05). The vasorelaxation response to SNP was not different after mesenteric I/R, but vasorelaxation by both acetylcholine and A23187 were significantly impaired. Endothelial-dependent pulmonary vasorelaxation is significantly impaired after mesenteric I/R. Such impairment of pulmonary vasorelaxation may help tip the net balance of pulmonary vasomotor tone toward vasoconstriction and contribute to the pulmonary hypertension seen in acute lung injury.

Neutrophil depletion attenuates endotoxin-induced dysfunction of cGMP-mediated pulmonary vasorelaxation

The effect of neutrophil depletion on endotoxin-induced dysfunction of guanosine 3',5'-cyclic monophosphate (cGMP)-mediated pulmonary vasorelaxation was studied in rats. Two mechanisms of neutrophil depletion were used: vinblastine (0.75 mg/kg iv) and rabbit anti-rat neutrophil antiserum (0.15 ml iv). Concentration-response curves were generated (10(-9) to 10(-6) M) for acetylcholine (ACh), A-23187, and sodium nitroprusside (SNP) in isolated pulmonary arterial rings preconstricted with phenylephrine 6 h after endotoxin (20 mg/kg ip). Absolute neutrophil count was significantly lowered from 1,050 +/- 206 (neutrophils/ml; mean +/- SE) in controls to 100 +/- 41 by vinblastine and to 50 +/- 29 by antiserum. Endotoxin produced histological evidence of pulmonary vascular endothelial damage and significantly increased lung neutrophil accumulation (myeloperoxidase assay, 5.1 +/- 0 vs. 1.2 +/- 0.1 in controls; 0.1 +/- 0.1 and 0.8 +/- 0.0 U/g lung wt after endotoxin in neutrophil-depleted rats by vinblastine and antiserum, respectively). Endotoxin produced significant impairment of endothelium-dependent cGMP-mediated pulmonary vasorelaxation by receptor-dependent (ACh) and -independent (A-23187) pathways as well as endothelium-independent relaxation (SNP). Neutrophil depletion significantly attenuated the endotoxin-induced impairment of all three of these mechanisms. We conclude that neutrophils contribute to endotoxin-induced impairment of GMP-mediated pulmonary vasorelaxation.

Protective effect of B464, a lipid A analog, on endotoxin-induced cellular responses and acute lung injury.

B464 is a novel synthetic analog of lipid A, a toxic component of endotoxin (LPS; lipopolysaccharide). We investigated the effects of B464 on both LPS-induced cellular responses in vitro and acute lung injury in vivo. In the in vitro study, B464 inhibited tumor necrosis factor-alpha (TNF-alpha) production from human monocytes, priming and stiffening of neutrophils, and expression of adhesion molecules on endothelial cells induced by LPS. We then studied the effects of B464 pretreatment on acute lung injury elicited by intravenous LPS administration in vivo. Guinea pigs were divided into saline control, B464 alone, LPS alone, and LPS + B464 groups. Animals were observed for 4 h after LPS administration, and lung injury was evaluated by extravascular lung water, 125I-albumin leakage in lung tissue, and lung neutrophil accumulation. In the LPS alone group, rapid and sustained peripheral neutropenia (p < 0.001 versus saline at 15 min and at 1, 2, and 4 h), an increased plasma TNF-alpha concentration (p < 0.005 at 1 h), and increases in lung injury parameters (p < 0.05) were observed. In the LPS + B464 group, no changes were observed in either plasma TNF-alpha or lung injury parameters. Transient peripheral neutropenia and subsequent rapid recovery (p > 0.05, p < 0.001, p < 0.01, and p > 0.05 at 15 min and 1, 2, and 4 h, respectively) were observed in the LPS + B464 group. These in vivo data, together with in vitro evidence of suppressed cellular responses, suggest that B464 (1) inhibits neutrophil accumulation in lung tissue, and (2) attenuates the development of acute lung injury by blocking the activation of neutrophils and mononuclear cells as well as the interaction between neutrophils and endothelial cells.

Tumor necrosis factor induced acute lung leak in rats: less than with interleukin-1.

Although local tumor necrosis factor-alpha (TNF) release by alveolar macrophages has been postulated to contribute to the development of acute respiratory distress syndrome (ARDS), the effects of instilling TNF intratracheally on the development of acute lung leak are not known. Our goal was to determine the effect of intratracheally administered TNF on the development of acute lung leak in rats. We found that rats given TNF (500 ng) 5 hours previously intratracheally had increased (p < 0.05) lung lavage cytokine induced neutrophil chemoattractant (CINC) concentrations, lung lavage neutrophils, lung myeloperoxidase (MPO) activity, and lung leak compared to saline-treated control rats. However, all of the responses following TNF instillation were much lower than the responses to interleukin-1 alpha (IL-1) instillation. For example, instilling 50 ng of IL-1 caused 6.4 times the increases in lung lavage CINC concentrations, 15.5 times the increase in lung lavage neutrophils, 3.6 times the increase in lung MPO activity and 3.8 times the increase in lung leak caused by giving 500 ng of TNF intratracheally. Co-treatment with TNF-binding protein decreased both lung MPO and lung leak increases in rats given TNF intratracheally. These observations suggest that locally elevated levels of TNF may induce lung neutrophil recruitment and acute lung leak but that IL-1 is a much more potent agent than TNF in causing lung neutrophil accumulation and lung leak.

Inhaled nitric oxide prevents pulmonary endothelial dysfunction after mesenteric ischemia-reperfusion.

This study examined the effect of inhaled nitric oxide (NO) on lung neutrophil accumulation and endothelial-dependent and -independent guanosine 3',5'-cyclic monophosphate (cGMP)-mediated mechanisms of pulmonary vasorelaxation after mesenteric ischemia-reperfusion (I/R) in mechanically ventilated rats. Inhaled NO (20 ppm) was administered in two protocols: 1) throughout mesenteric I/R and 2) during mesenteric reperfusion alone. Concentration-response curves were generated (10(-9) to 10(-8) M) for acetylcho-line (ACh), A23187, and sodium nitroprusside (SNP) in isolated pulmonary arterial rings preconstricted with phenylephrine. Lung neutrophil accumulation [myeloperoxidase assay (MPO)] was significantly increased from 2.4 +/- 0.2 units/g lung wt in controls to 10.3 +/- 0.4 after 1 h of superior mesenteric artery occlusion and 2 h of reperfusion. Lung MPO activity was not different from controls in rats receiving inhaled NO either 1) during mesenteric I/R or during mesenteric reperfusion alone. The concentration-response curves demonstrated significant impairment of pulmonary vasorelaxation by endothelial-dependent mechanisms (response to ACh and A23187) but not endothelial-independent pulmonary vasorelaxation (response to SNP) after mesenteric I/R. This pulmonary vasomotor dysfunction was prevented by administration of inhaled NO during either mesenteric I/R or during mesenteric reperfusion alone. We conclude that inhaled NO prevents lung neutrophil accumulation and pulmonary vascular endothelial dysfunction after mesenteric I/R.

NO Prevents Neutrophil-Mediated Pulmonary Vasomotor Dysfunction in Acute Lung Injury

The purpose of this study was to examine the effect of administration of inhaled nitric oxide (NO) on lung neutrophil accumulation and pulmonary vascular endothelial cell function in endotoxin-induced acute lung injury. Mechanically ventilated rats were studied 4 hr after endotoxin (0.5 mg/kg IP). Inhaled NO (20 ppm) was administered for either the entire 4 hr after endotoxin (continuous group) or for only the first 2 of 4 hr after endotoxin (abbreviated group). Endothelial-dependent (acetylcholine, ACh) and -independent cGMP-mediated relaxation (nitroprusside, SNP) pulmonary vasorelaxation were studied in isolated pulmonary arterial rings. Lung neutrophil accumulation was determined by myeloperoxidase assay (MPO). Inhaled NO prevented endotoxin-induced lung neutrophil accumulation as well as pulmonary endothelial cell dysfunction. However, this protection required continuous administration of inhaled NO. We conclude that inhaled NO prevents neutrophil-mediated pulmonary vascular endothelial cell dysfunction in acute lung injury.

MICE DEFICIENT IN ICAM-1 EXPRESSION AND WITH REDUCED CD18 EXPRESSION HAVE INCREASES IN HYPEROXIC LUNG INFLAMMATION AND INJURY. • 2337

Treatment with supplemental oxygen is frequently used in premature infants because of abnormal lung function. While frequently essential to sustain life, the treatment may cause lung injury. Recently, there has been renewed interest in lung inflammation in hyperoxic lung injury. Investigators have determined that inhibition of the adhesion molecules, intercellular adhesion molecule-1(ICAM-1), and neutrophil (PMN) β2 integrins with monoclonal antibodies (mab) inhibits lung inflammation and injury in hyperoxic mice. However, mab administration may not be entirely specific so that mabs may have effects other than inhibiting adhesion molecules. We therefore placed mice with absent ICAM-1 expression, and mice with reduced β2-integrin expression in >95% oxygen and measured lung inflammation and injury as a function of hyperoxia-exposure time. In all studies wild-type mice on the same genetic background were used as controls. In the two mutant strains of mice there were increases in lung PMN counts when compared to the wild-type controls. In addition, lung weights were significantly greater in ICAM-1 deficient mice than in C57/BL6 at 72 h of hyperoxia-exposure. When mice were exposed to hyperoxia for 84 hours there were 12/12, 3/6 and 0/6 survivors in C57/BL6, ICAM-1 and β2 integrin mice respectively (P<0.05). In conclusion, mice with defects in ICAM-1 and β2-integrin expression are more susceptible to hyperoxic lung injury and have increased lung inflammation than their wild type counterparts. The apparent contradiction in our results with the mab studies suggests that the role of lung neutrophil accumulation in hyperoxic lung injury is more complex than previously appreciated, and that PMN accumulation occurring late in the course of hyperoxia-exposure is ICAM-1/β2 integrin independent.Table

Role for macrophage inflammatory protein-2 in lipopolysaccharide-induced lung injury in rats.

Macrophage inflammatory protein-2 (MIP-2) is a C-X-C chemokine that possesses chemotactic activity for neutrophils. Rat MIP-2 was cloned and expressed as a 7.9-kDa peptide that exhibited dose-dependent neutrophil chemotactic activity at concentrations from 10 to 250 nM. Rabbit polyclonal Ab to the 7.9-kDa peptide showed reactivity by western blot analysis and suppressed its in vitro chemotactic activity. Cross-desensitization chemotaxis experiments suggested that the chemotactic responses elicited by MIP-2 and the related chemokine, cytokine-induced neutrophil chemoattractant, may be mediated through a common receptor. Also, chemotactic responses to human GRO-alpha were blocked by exposure of human neutrophils to either GRO-alpha or rat MIP-2, suggesting conservation of this receptor-mediated response. After LPS instillation into rat lung, mRNA for MIP-2 was up-regulated in a time-dependent manner, peaking at 6 h. MIP-2 protein was detected in bronchoalveolar lavage fluids of these animals and a significant amount of chemotactic activity present in these fluids was attributed to MIP-2. On the basis of intrapulmonary instillation of Ab to MIP-2, neutrophil accumulation in lungs after airway instillation of LPS was found to be MIP-2-dependent. These data indicate that MIP-2 plays a significant role in LPS-induced inflammatory response in rat lungs and is required for the full recruitment of neutrophils.

P-selectin is upregulated early in the course of hyperoxic lung injury in mice

While treatment with supplemental oxygen is often essential in patients with lung disease, prolonged therapy may cause lung injury by itself. Although the mechanisms responsible for initiating hyperoxic lung damage almost certainly involve primary oxidative transformations, the possible contributions of inflammation to the tissue injury have been attracting increasing research activity. Increases in intercellular adhesion molecule-1 (ICAM-1) coincide with the inflammation, but in other models of inflammation transient adhesion mediated by members of the Selectin gene family was found to be essential before ICAM-1/β 2 interactions could occur. We, therefore, wondered whether a similar sequence of initial transient adhesion followed by subsequent responses would be observed in hyperoxic lung inflammation. We, therefore, determined the effects of hyperoxia exposure on lung mRNA for P-and E-Selectin in mouse lungs. We found that there was no detectable mRNA for E-Selectin through 72 h of hyperoxia exposure by Northern blotting, but that mRNA for P-Selectin was detectable as early as 48 h after initiation of hyperoxia. To determine the location of P-Selectin upregulation we examined hyperoxia-exposed mouse lungs by in situ hybridization and found that the upregulation of P-Selectin at 48 h was localized to large muscularized vessels, at 72 h expression was detected in some medium size muscularized vessels, and at 96 h abundant expression was observed also on nonmuscularized small vessels. In conclusion, increases in mRNA for P-Selectin early in the course of hyperoxia exposure suggest that P-Selectin expression in hyperoxic lungs increases in parallel with upregulation of ICAM-1, leading to the accumulation of neutrophils in hyperoxic lungs, and that interventions targeting these two adhesion molecules may lead to a diminution in hyperoxic lung inflammation and lung injury.

Detection of O 2− generation and neutrophil accumulation in rat lungs after acute necrotizing pancreatitis

Polymorphonuclear neutrophils (PMNs) and PMN-derived superoxide anions (O2-) have been found to mediate acute respiratory distress syndrome (ARDS). But the evidence has been obtained only indirectly, (for example, by treatment with free radical scavengers or by measuring lipid peroxides). We used chemiluminescence technique to measure O2- generation directly from the surface of rat lungs with necrotizing pancreatitis. Myeloperoxidase activity in the lung tissue was measured by a new chemiluminescence method to quantitate pulmonary neutrophil sequestration. Transient bursts of chemiluminescence that reflected O2- generation were observed at the lung surface 3 to 5 hours after necrotizing pancreatitis was induced. No O2- dependent chemiluminescence was detected at the lung surfaces of the neutropenic rat made by pretreatment with polyclonal antineutrophil antibody. Myeloperoxidase activity was significantly high (mean, 1690 cell numbers/mg wet tissue) in the necrotizing pancreatitis rat lung (p < 0.005). These data confirmed that activated neutrophils and neutrophil-derived superoxide anions were implicated in lung injury after acute severe pancreatitis.

Cytokine-induced neutrophil chemoattractant is necessary for interleukin-1-induced lung leak in rats.

Because interleukin-1 (IL-1) is not a potent chemoattractant intrinsically, our goal was to determine whether cytokine-induced neutrophil chemoattractant (CINC) mediates neutrophil accumulation and leak that occur in lungs of rats given IL-1 intratracheally. We found that rats given IL-1 intratracheally had increased lung lavage CINC concentrations, lung myeloperoxidase (MPO) activity, lung lavage neutrophil numbers, and lung leak compared with saline-treated control rats. In parallel, rats given increasing doses of anti-CINC antibody along with IL-1 intratracheally had progressively decreased lung lavage CINC concentrations, lung lavage neutrophil numbers, and lung leak, but the same lung MPO activity, as did rats given only IL-1 intratracheally. In addition, lavage CINC concentrations correlated with lavage neutrophil numbers in rats. Because CINC is a chemoattractant in vitro and because anti-CINC antibody decreased lung leak induced by IL-1, CINC was then given alone intratracheally. Rats given only CINC intratracheally had the same lung MPO activity, lung lavage neutrophil numbers, and lung leak as did saline-treated control rats. These observations suggest that instilling IL-1 intratracheally induces lung leak via a CINC-dependent pathway but that administering CINC alone in concentrations exceeding the levels measured in lung lavages from IL-1-treated rats is not sufficient to cause lung neutrophil accumulation and lung leak.

Up-regulation of lung vascular ICAM-1 in rats is complement dependent.

Intrapulmonary deposition of IgG immune complexes in rats causes acute inflammatory lung injury that is neutrophil, complement, cytokines (IL-1, TNF-alpha), and intercellular adhesion molecule (ICAM-1) dependent. In the current studies involving the same model of lung injury, complement depletion or complement blockade resulted in substantial reductions in up-regulation of pulmonary vascular ICAM-1, accompanied by reduced lung injury and neutrophil accumulation. Complement depletion neither reduced the amount of immune complex deposited in lung nor the TNF-alpha content in bronchoalveolar lavage fluids. In the same model of inflammatory lung injury, neutrophil depletion (which is highly protective) did not affect up-regulation of lung vascular ICAM-1. Up-regulation of lung vascular ICAM-1 by intratracheally administered TNF-alpha was also prevented by complement depletion. These studies indicate an unexpected in vivo relationship between complement and up-regulation of lung vascular ICAM-1.

A dual-binding antibody to E- and L-selectin attenuates sepsis-induced lung injury.

Many studies indicate a pivotal role for neutrophil adhesion in sepsis-associated lung injury. Neutrophil adhesion to endothelium depends on activation and expression of selectin and integrin adhesion receptors. We studied the effects of pretreatment with a dual-binding porcine anti-E- and anti-L-selectin monoclonal antibody (EL-246) on a porcine model of sepsis-induced lung injury. Four groups were studied for 5 h. Group 1 (control animals) received intravenous saline only. Group 2 (septic) received a 1-h infusion of Pseudomonas aeruginosa. Group 3 (EL-246 pretreatment) received EL-246 (1 mg/kg) prior to Pseudomonas infusion. Group 4 (EL-246 controls) received EL-246 infusion only. Group 2 animals showed rapid, significant decline in arterial pH and oxygen tension whereas, in Group 3, physiologic deterioration was significantly attenuated. Bronchoalveolar lavage at 5 h showed a significant increase in neutrophil count and protein content in Group 2. Group 3, however, showed no significant differences in these parameters compared with control animals. Despite severe neutropenia, lung myeloperoxidase content at 5 h was significantly reduced in Group 3 compared with Group 2. There was no significant difference in pulmonary and systemic hemodynamics between Groups 2 and 3. Group 4 animals exhibited a transient neutropenia, but otherwise no other differences in measured parameters were found compared with Group 1 control animals. In conclusion, EL-246 significantly reduced neutrophil accumulation in lung and attenuated sepsis-induced lung injury, but failed to attenuate deranged pulmonary and systemic hemodynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

Dysfunction of cGMP-mediated pulmonary vasorelaxation in endotoxin-induced acute lung injury.

Endothelial-dependent and -independent cGMP-mediated mechanisms of pulmonary vasorelaxation were studied in endotoxin-induced acute lung injury in the rat. Concentration-response curves were generated (10(-9) to 10(-6) M) for acetylcholine (ACh), A23187, and sodium nitroprusside (SNP) and for 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP) (10(-9) to 10(-4) M) in isolated pulmonary arterial rings preconstricted with phenylephrine 6 h after endotoxin treatment (20 mg/kg ip). Endotoxin treatment produced significantly increased lung neutrophil accumulation (myeloperoxidase assay, 28 +/- 6 units/g lung tissue vs. 1.8 +/- 1 in controls) and lung leakage (lung/blood 125I-labeled albumin ratio, 0.06 +/- 0.01 vs. 0.028 +/- 0.01 in controls) as well as histological evidence of pulmonary vascular endothelial damage. The concentration-response curves demonstrated that pulmonary vasorelaxation by mechanisms that require generation of cGMP by either endothelial-dependent (both receptor-dependent, ACh, and receptor-independent, A23187) or endothelial-independent (SNP) pathways were significantly impaired after endotoxin treatment. Relaxation by stimulation with the cGMP analogue 8-BrcGMP was not different from control. Pulmonary vascular smooth muscle is able to relax in response to cGMP after endotoxin treatment, but relaxation by endothelial-dependent and -independent pathways that require generation of cGMP is significantly impaired.

Endotoxin-induced pulmonary vascular injury is mainly mediated by activated neutrophils in rats

Acute respiratory failure is a common complication in patients with disseminated intravascular coagulation associated with sepsis. To elucidate the role of coagulation abnormalities in acute lung injury in sepsis, we investigated the effect of anticoagulants on the pulmonary vascular injury in rat induced by lipopolysaccharide (LPS). When administered intravenously, LPS (5 mg/kg body weight) significantly increased the accumulation of 111indium-labeled neutrophils in lung 30 min after administration. Subsequently, the pulmonary vascular permeability and the serum level of fibrin and fibrinogen degradation products (E) [FDP (E)] increased and remained elevated for several hours. Neither heparin alone, heparin plus antithrombin III, or dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa, a selective inhibitor of thrombin generation, prevented LPS-induced vascular injury 6 hours after LPS administration, whereas these substances significantly inhibited the increase in serum FDP (E) at that time. LPS-induced pulmonary vascular injury was significantly attenuated in rats with methotrexate-induced leukocytopenia or treated with ONO-5046, a potent granulocyte elastase inhibitor, although ONO-5046 did not inhibit the LPS-induced increase in serum FDP (E). Thus, activated leukocytes play a more important role than coagulation abnormalities in the pathogenesis of LPS-induced pulmonary vascular injury in an experimental rat model of endotoxemia.

Phosphatidic acid signaling mediates lung cytokine expression and lung inflammatory injury after hemorrhage in mice.

Because phosphatidic acid (PA) pathway signaling may mediate many basic reactions involving cytokine-dependent responses, we investigated the effects of CT1501R, a functional inhibitor of the enzyme lysophosphatidic acid acyltransferase (LPAAT) which converts lysophosphatidic acid (Lyso-PA) to PA. We found that CT1501R treatment not only prevented hypoxia-induced PA increases and lyso-PA consumption in human neutrophils, but also prevented neutrophil chemotaxis and adherence in vitro, and lung injury and lung neutrophil accumulation in mice subjected to hemorrhage and resuscitation. In addition, CT1501R treatment prevented increases in mRNA levels and protein production of a variety of proinflammatory cytokines in multiple lung cell populations after blood loss and resuscitation. Our results indicate the fundamental role of PA metabolism in the development of acute inflammatory lung injury after blood loss.