Hemorrhagic Shock-induced Acute Lung Injury Research Articles

ESTROGEN ALLEVIATES POSTHEMORRHAGIC SHOCK MESENTERIC LYMPH-MEDIATED LUNG INJURY THROUGH AUTOPHAGY INHIBITION.

Background: Hemorrhagic shock-induced acute lung injury (ALI) is commonly associated with the posthemorrhagic shock mesenteric lymph (PHSML) return. Whether excessive autophagy is involved in PHSML-mediated ALI remains unclear. The relationship between estrogen treatment and PHSML or autophagy needs to verify. The current study will clarify the role of estrogen in reducing PHSML-mediated ALI through inhibition of autophagy. Methods: First, a hemorrhagic shock model in conscious rats was used to observe the effects of 17β-estradiol (E2) on intestinal blood flow, pulmonary function, intestinal and pulmonary morphology, and expression of autophagy marker proteins. Meanwhile, the effect of PHSML and autophagy agonist during E2 treatment was also investigated. Secondly, rat primary pulmonary microvascular endothelial cells were used to observe the effect of PHSML, PHSML plus E2, and E2-PHSML (PHSML obtained from rats treated by E2) on the cell viability. Results: Hemorrhagic shock induced intestinal and pulmonary tissue damage and increased wet/dry ratio, reduced intestinal blood flow, along with pulmonary dysfunction characterized by increased functional residual capacity and lung resistance and decreased inspiratory capacity and peak expiratory flow. Hemorrhagic shock also enhanced the autophagy levels in intestinal and pulmonary tissue, which was characterized by increased expressions of LC3 II/I and Beclin-1 and decreased expression of p62. E2 treatment significantly attenuated these adverse changes after hemorrhagic shock, which was reversed by PHSML or rapamycin administration. Importantly, PHSML incubation decreased the viability of pulmonary microvascular endothelial cells, while E2 coincubation or E2-treated lymph counteracted the adverse roles of PHSML. Conclusions: The role of estrogen reducing PHSML-mediated ALI is associated with the inhibition of autophagy.

VALPROIC ACID INHIBITS CLASSICAL MONOCYTE-DERIVED TISSUE FACTOR AND ALLEVIATES HEMORRHAGIC SHOCK-INDUCED ACUTE LUNG INJURY IN RATS.

Background: Monocytes and monocyte-derived tissue factor (TF) promote the development of sepsis-induced acute lung injury (ALI). Classical monocytes (C-Mcs) can be induced to express TF. Valproic acid (VPA) alleviates hemorrhagic shock (HS)-induced ALI (HS/ALI) and inhibits TF expression in monocytes. We hypothesized that C-Mcs and C-Mc-derived TF promoted HS/ALI and that VPA could inhibit C-Mc-derived TF expression and attenuate HS/ALI. Methods: Wistar rats and THP-1 cells were used to evaluate our hypothesis. Monocyte subtypes were analyzed by flow cytometry; mRNA expression was measured by fluorescence quantitative polymerase chain reaction; protein expression was measured by Western blotting, immunofluorescence, or immunohistology; inflammatory cytokines levels were measured by enzyme-linked immunosorbent assay; and ALI scores were used to determine the degree of ALI. Results: The blood %C-Mcs and C-Mcs/non-C-Mcs ratios, monocyte TF levels, serum and/or lung inflammatory cytokine levels, and ALI scores of HS rats were significantly increased ( P < 0.05). After monocyte depletion and thrombin inhibition, the inflammatory cytokine levels and ALI scores were significantly decreased ( P < 0.05). VPA reduced the %C-Mcs and C-Mc/non-C-Mc ratios, TF expression, inflammatory cytokine levels, and ALI scores during HS ( P < 0.05) and inhibited HS-induced monocyte Egr-1 and p-ERK1/2 expression ( P < 0.05). VPA inhibited hypoxia-induced TF expression in THP-1 cells by regulating the p-ERK1/2-Egr-1 axis. Conclusion: C-Mcs and C-Mc-derived TF accelerate the development of HS/ALI by increasing thrombin production. VPA inhibits HS-induced C-Mc production of TF by regulating the p-ERK1/2-Egr-1 axis and alleviates HS/ALI.

Hepatic Estrogen Sulfotransferase Distantly Sensitizes Mice to Hemorrhagic Shock-Induced Acute Lung Injury.

Hemorrhagic shock (HS) is a potential life-threatening condition that may lead to injury to multiple organs, including the lung. The estrogen sulfotransferase (EST, or SULT1E1) is a conjugating enzyme that sulfonates and deactivates estrogens. In this report, we showed that the expression of Est was markedly induced in the liver but not in the lung of female mice subject to HS and resuscitation. Genetic ablation or pharmacological inhibition of Est effectively protected female mice from HS-induced acute lung injury (ALI), including interstitial edema, neutrophil mobilization and infiltration, and inflammation. The pulmonoprotective effect of Est ablation or inhibition was sex-specific, because the HS-induced ALI was not affected in male Est-/- mice. Mechanistically, the pulmonoprotective phenotype in female Est-/- mice was accompanied by increased lung and circulating levels of estrogens, attenuated pulmonary inflammation, and inhibition of neutrophil mobilization from the bone marrow and neutrophil infiltration to the lung, whereas the pulmonoprotective effect was abolished upon ovariectomy, suggesting that the protection was estrogen dependent. The pulmonoprotective effect of Est ablation was also tissue specific, as loss of Est had little effect on HS-induced liver injury. Moreover, transgenic reconstitution of human EST in the liver of global Est-/- mice abolished the pulmonoprotective effect, suggesting that it is the EST in the liver that sensitizes mice to HS-induced ALI. Taken together, our results revealed a sex- and tissue-specific role of EST in HS-induced ALI. Pharmacological inhibition of EST may represent an effective approach to manage HS-induced ALI.

Penehyclidine hydrochloride inhibits TLR4 signaling and inflammation, and attenuates blunt chest trauma and hemorrhagic shock-induced acute lung injury in rats.

Blunt chest trauma with hemorrhagic shock (THS) frequently induces pulmonary inflammation that leads to acute lung injury (ALI). Penehyclidine hydrochloride (PHC) possesses anti-inflammatory properties that may attenuate the systemic inflammatory response. The present study aimed to evaluate the molecular mechanism of PHC in modifying THS-induced ALI in rats. Rats underwent either THS or a sham procedure. At 6 h subsequent to blunt chest trauma, arterial blood was drawn for blood gas and pro-inflammatory factors analyses, and lung tissue samples were collected to examine pulmonary histopathological alterations, the wet/dry weight ratio, myeloperoxidase activity, and the protein expression levels of Toll-like receptor 4 (TLR4), phosphorylated (p-)p38 mitogen-activated protein kinase (MAPK), nuclear factor (NF)-κB and activator protein-1 (AP-1). THS caused significant reductions in heart rate and mean arterial blood pressure, and was associated with significant increases in tumor necrosis factor-α, interleukin (IL)-6, IL-1β, p-p38MAPK, NF-κB and AP-1 activation, in addition to TLR4 expression, in the lung. PHC effectively attenuated THS-induced ALI, and inhibited TLR4 expression, reduced the activation of p-p38MAPK, NF-κB and AP-1, and downregulated the expression of pro-inflammatory mediators. In conclusion, the results of the present study demonstrated that PHC may exert an anti-inflammatory effect and attenuate THS-induced ALI by inhibiting the TLR4 signaling pathway. These preclinical findings may offer a novel therapeutic strategy to restrict TLR4 overactivation in ALI.

Effect of BML-111 on the intestinal mucosal barrier in sepsis and its mechanism of action.

5(S),6(R)-7-trihydroxymethyl heptanoate (BML-111) is an lipoxin A4 receptor agonist, which modulates the immune response and attenuates hemorrhagic shock-induced acute lung injury. However, the role of BML-111 in sepsis and in the intestinal mucosal barrier are not well understood. Therefore, the present study was designed to investigate the effect of BML-111 on the intestinal mucosal barrier in a rat model of sepsis. Furthermore, the molecular mechanism of action of BML-111 was evaluated. The cecal ligation and puncture-induced rat model of sepsis was constructed, and BML-111 was administered at three different doses. The results revealed that BML-111 suppressed the elevation of the pro-inflammatory cytokines tumor necrosis factor-α and interleukin-6, while enhancing the elevation of the anti-inflammatory cytokine transforming growth factor-β in the intestine. In addition, BML-111 significantly upregulated rat defensin-5 mRNA expression levels and downregulated the induction of cell apoptosis as well as caspase-3 activity in the intestine. All these results demonstrated that BML-111 exerted protective effects on the intestinal mucosal barrier in sepsis. Further, it was indicated that alterations in the expression of toll-like receptor (TLR)2 and TLR4 may be one of the molecular mechanisms underlying the protective effect of BML-111. The present study therefore suggested that BML-111 may be a novel therapeutic agent for sepsis.

Protective effects of peroxisome proliferation activated receptor-β on hemorrhagic shock-induced acute lung injury in rats

Objective To observe the protective effects of peroxisome proliferation activated receptorβ (PPAR-b) on acute lung injury in the wake of hemorrhagic shock in rats in order to illuminate the mechanism.Methods After ALI model of rat was established following hemorrhagic shock,96 SD rats were divided randomly (random number) into 4 groups:group A (sham operation group),group B (ALI group),group C (GSK0660,an antagonist to PPAR-b given prior to exsanguinations for pretreatment) and group D (GW0742,an agonist of PPAR-b given before exsanguinations for pretreatment).Each group was further divided into 1 h,2 h,4 h and 6 h subgroups.Six mice of each subgroup were sacrificed at each interval.Expression of PPAR-β receptor in lung tissues was detected at 1 h,2 h,4 h and 6 hours.Pathological examination and lung wet/dry weight ratio were detected,and lung tissue antioxidant enzyme SOD and 8-iso-PGF2α,the oxidation product of activated GSH-Px were evaluated.Results (①)Antioxidant enzymes SOD and activated GSH-Px were significantly higher in ALI group than those in the sham operation group (P＜0.01).(②Tne antioxidant enzyme SOD and activated GSH-Px in lung tissue in GW0742 group decreased at each interval especially at 2 h and4 h compared with ALI group (P ＜0.01).(③) GW0742 used alone can improvePaO2,W/D ratio of lung tissue of rats,lung pathology integral,and inhibit the activity of SOD and GSH-Px in lung tissue (P ＜ 0.01) and decrease the content of 8-iso-PGF-2α.The antagonist GSK0660 can lessen the above effects (P ＜ 0.01).Conclusions (①)ALI emerged from hemorrhagic shock in rats leading to significantly increased activity of SOD and GSH-Px in lung tissue,suggesting rats subjected to acute oxidative stress.(②)GW0742 can up-regulate the levels of SOD and GSH-Px in lung tissue of rats with ALI.It also decreased the content of 8-iso-PGF2 α,the oxidation products of GSH-Px.(③) It was suggested that GW0742 might inhibit the expression of TNF-α mRNA and level of TNF-a protein,and decreased SOD activity in lung tissue,protecting lung tissue to some degree from ALI and improving PaO2.The mechanism of it may be attributed to preventing NF-κ B activation. Key words: Acute lung injury; Peroxisome proliferator-activated receptor; Oxidative stress; Hemorrhagic shock ; Acute respiratory distress syndrome; Inflammatory response ; Apoptosis ; Mortality

Effects of penehyclidine hydrochloride on hemorrhagic shock-induced acute lung injury in rats

Objective To observe the effects of penehyclidine hydrochloride (PHC) on hemorrhagic shock/resuscitation-induced acute lung injury in rats.Methods Fifteen healthy adult Sprague-Dawley rats were randomly allocated equally into three groups:groups P,C and S.The rats in group S received sham operation,and those in groups P and C were subjected to bloodletting for shock,then shock state maintained for 60 min,and resuscitation was done with autoblood.The rats in group P received PHC treatment (0.03 mg/kg) before resuscitation of hemorrhagic shock,and those in group C and group S received 1.0 ml normal saline instead.Blood samples were collected via the carotid artery for interleukin-8 (IL-8),tumor necrosis factor-α (TNF-α) and nuclear factor-κB (NF-κB) monitoring at the time of subsequence carotid arteriopuncture (T0),before shock induction (T1),5 min after shock (T2),before resuscitation of shock (T3),5 min (T4),30 min (T5),1.5 h (T6) and 2.5 h (T7) after mean arterial pressure (MAP) reached expected resuscitation goal.The arterial blood gas analyses were detected at T0,T3 and T7.The morphological changes of lungs were observed under microscope.Results The ratio of PO2/FiO2 in groups P and C were significantly lower than in group S at T7 [(289.5 ± 67.1) mmHg (1 mmHg =0.133 kPa),(280.0 ±73.3) mmHg vs.(458.1 ±31.0) mmHg,P ＜0.01].There was marked diversity among group P,C and S in the morphological score (4.4 ± 1.7,7.4 ± 1.9 vs.0.8 ± 1.1,P ＜0.01) ; The peak concentrations of IL-8 [(7.35 ±0.14),(8.44 ±0.13) ng/L vs.(3.75 ±0.06) ng/L,P ＜0.01],TNF-α [(8.45 ±0.24,(9.69 ±0.52) ng/L vs.(4.19 ± 0.04) ng/L,P＜0.01] and NF-κB [(272.34 ± 12.60),(310.00 ± 16.47) ng/L vs.(133.98 ± 1.91) ng/L,P ＜ 0.01] respectively,with significant difference among three groups.Conclusion Treatment of PHC might have a protective effect against lung injury induced by hemorrhagic shock/resuscitation.Its action mechanisms are probably involved in the suppression of inflammatory factor production and inhibition of the NF-κB pathway. Key words: Hemorrhagic shock ; Acute lung injury; Penehyclidine hydrochloride ; Inflammatory cytokine

BML-111 attenuates hemorrhagic shock-induced acute lung injury through inhibiting activation of mitogen-activated protein kinase pathway in rats

BackgroundHemorrhagic shock activates cellular stress signals and can lead to systemic inflammatory response, organ injury, and death. Mitogen-activated protein kinase (MAPK) acts as a sensor of tissue injury in models of ischemia-reperfusion injury. Lipoxins are endogenous lipid mediators with potent anti-inflammatory and pro-resolving actions. We hypothesized that BML-111 (a lipoxin A4-receptor agonist) attenuates hemorrhagic shock-induced acute lung injury (ALI) through inhibiting activation of the MAPK pathway. MethodsWe randomized Sprague-Dawley rats into four groups: sham, hemorrhagic shock–resuscitation (HS), HS plus BML-111 (BML-111), and HS plus BML-111 and BOC-2 (BOC-2). Two hours after resuscitation, we collected samples of lung. We obtained bronchoalveolar lavage fluid for neutrophil count. We performed optical microscopy to examine pathologic changes in lungs. Wet/dry ratios, myeloperoxidase expression, interleukin (IL)-1β and IL-6 levels in lung were measured. We evaluated MAPK activation and the DNA binding activity of activator protein-1 in lung. ResultsTreatment with BML-111 reduced the lung damage and wet/dry ratio, neutrophil count in bronchoalveolar lavage fluid, expression of myeloperoxidase, and production of IL-1β and IL-6 in lung. Phosphorylation of MAPK was also decreased by BML-111 in lung. Furthermore, the DNA binding activity of activator protein-1 was blocked by BML-111. An antagonist of the lipoxin A4-receptor, BOC-2, reversed the protective effect of BML-111 on ALI induced by hemorrhagic shock. ConclusionsThis study indicates that BML-111 attenuated hemorrhagic shock-induced ALI via the MAPK/activator protein-1 signaling pathway. Therefore, BML-111 may have therapeutic potential for hemorrhagic shock-induced ALI.

Lipoxin receptor agonist, may be a potential treatment for hemorrhagic shock-induced acute lung injury

The main pathogenesis of acute lung injury induced by hemorrhagic shock is increasingly recognized as an inflammatory process. BML-111, a lipoxin receptor agonist, has been demonstrated to promote acute inflammatory resolution by reduction of pro-inflammatory cytokines, attenuation of neutrophilic infiltration, and increasing macrophage phagocytosis of apoptotic neutrophils. Meanwhile, lipoxins and lipoxin analogues have been reported to play pro-resolving and anti-inflammatory effects in many disease models including cerebral ischemia, dorsal air pouch, peritonitis, and so on. Therefore, we hypothesize that BML-111 may be implicated in pathogenesis of hemorrhagic shock-induced acute lung injury.

Effects of remifentanil on lipid peroxidation during hemorrhagic shock-induced acute lung injury in rabbits

Objective To investigate the effects of remifentanil on lipid peroxidation druing hemorrhagic shock-induced acute lung injury （ALI） in rabbits. Methods Thirty-two healthy adult rabbits weighing 2.0-2.5 kg were randomly divided into 4 groups （n = 8 each）： sham operation group （group S）; ALI group; low-dose remifentanil group （group LR）; high-dose remifentanil group （group HR）. The left femoral artery was cannulated for blood-letting and blood sampling. The right femoral artery was cannulated for remifentanil administration. The model of hemorrhagic shock was established by modified Wigger＇ s methods. In group S, only cannulation was performed. In group LR and HR, remifentanil was infused intraperitoneally at 0.66 and 1.32 μg·kg-1 ·min-1 15 min before blood-letting respectively, while group ALI received equal volume of normal saline instead. Arterial blood samples were taken at 0, 20,70 and 100 min after blood-letting （T1-4） for blood gas analysis. The animals were then sacrificed and the lungs were immediately removed for histological examination with light microscope and determination of W/D lung weight ratio, MDA content and SOD activity. Results W/D ratio and MDA content were significantly increased, while SOD activity was significantly decreased in group ALI compared with group S （P 〈0.05）. The pH value at T2 and PaO2 at T2-4 in group LR and the pH value and PaCO2 at T2-4 in group HR were significantly higher than those in group ALI （P 〈 0.05）. W/D ratio and MDA content were significantly lower,while SOD activity was significantly higher in group LR and HR than in group ALI, and in group HR than in group LR （P 〈 0.05）. Remifentanil infusion significantly attenuated the pathologic changes in a dose-dependent manner. Conclusion Remifentanil pretreatment can attenuate hemorrhagic shock-induced ALI through inhibiting lipid peroxidation in rabbits. Key words: Piperidines; Respiratory distress syndrome,adult; Shock,hemorrhagic; Lipid peroxidation

Role of high mobility group box1 in hemorrhagic shock-induced acute lung injury

Objective To investigate the role of high mobility group box1 (HMGB1) in hemorrhagic shock-induced acute lung injury (ALI) in mouse model. Method Forty-two healthy male BALB/c mice were randomly divided into three groups: control group, hemorrhagic shock (HS) group and anti-HMGB1 treatmeut group. Car-diac puncture, was performed to induce ALI in mice. IL-β and TNF-α in lung tissues were detected by ELISA. Ex-pression of HMGB1 was determined by Westemblot. Myeloperoxidase (MPO) and Evens blue dye (EBD) in lung tissues were measured by colorimetry. Pathology of lung tissue was observed. ANOVA and LSD-t test were used in statistical analysis. Results Pulmonary blood vessel permeability increased 2 h after HS, pathology results re-vealed a diffused inflammatory cell infiltration in mouse lung tissue. Levels of IL-1β and TNF-α in lung tissues sig-nificantly increased at the early stage of ALI (4 h) compared to controls [IL-1β(333.83±31.18) vs. (284.83± 30.49), P =0.014; TNF-α(805.00±114.67) vs. (584.17±181.17), P =0.023]. Significant increase of HMGB1 in lung tissues occurred at the late stage of ALI (16～48 h) [HMGB1/β-actin (0.99±0.16) vs. (0.50 ±0.18), P =0.003]. The levels of MPO and EBD in ALl group significantly increased compared to controls, and reached the peak at4 h and 24 h respectively [MPO (38.33±3.88) vs. (8.00±0.89), P <0.01;EBD (20.05±2.79) vs. (4.63±0.43), P <0.01]. The peak level of MPO and EBD significantly decreased in anti-HMGB1 groups compared to that of ALI group [MPO (25.67±1.63) vs. (38.33±3.88), P < 0.01 ; EBD (5.68±0.53) vs. (20.05±2.79), P <0.001]. The infiltration of neutrophils and the destruction of the pul-monary cell walls ameliorated in anti-HMGB1 groups, especially in mice treated with anti-HMGB1 antihedy for 24 h. Conclusions HMGBI acts as a late pminfiammatory mediator in HS-induced ALI. Blockade of HMGB1 de-creases HS-induced pathophysiologic change of ALI in mice. Key words: High mobility group boxl ; Inflammatory mediator; Acute lung injury; Hemorrhagic shock; Patho-physiologic

Protective effects of N-acetylcysteine and erdosteine on hemorrhagic shock-induced acute lung injury

The drugs N-acetylcysteine and erdosteine were used to evaluate their protective effects in hemorrhagic shock-induced acute lung injury in an animal model. Forty rats were used and randomly allocated into four groups (n=10). Animals in group III were fed with water containing 1 mg/dl erdosteine, and those in group IV were given 0.5 mg/dl N-acetylcysteine 3 days before the experiment. Group I was taken as the control and group II was taken as the hemorrhagic shock group. Hemorrhagic shock was initiated by blood withdrawal and reduction of the mean arterial pressure to 40 mmHg within 10 min via the right carotid artery. After a hypotensive period of 2 h, animals were resuscitated by transfusion of the shed blood and Ringer lactate in a volume equal to the shed blood. After a period of 1 h, blood samples were taken via the carotid artery. Bronchoalveolar lavage was performed to recover cells from the alveolar space with 40 ml of bronchoalveolar lavage fluid. Lung tissues were also resected to measure tissue malondialdehyde and L-gamma-glutamyl-L-cysteinyl-glycine levels with high performance liquid chromatography. The numbers of neutrophils and alveolar macrophages in bronchoalveolar lavage fluid were counted. Serum malondialdehyde levels were significantly higher in the shock groups (P<0.05), but there was no significant difference (P>0.05). Lung malondialdehyde levels were also significantly increased in the shock groups (P<0.05). In the erdosteine-applied group, tissue malondialdehyde levels were lower than in group II and the NAC-applied group (P<0.05). In the analyses of serum and lung tissue L-gamma-glutamyl-L-cysteinyl-glycine, the values of groups I, II and IV were found to be below the calibration graphics. The alveolar macrophage count was found to be the highest and the neutrophil count the lowest in group III when compared with the other groups in bronchoalveolar lavage fluid analyses (P<0.05). We may say that in the model of hemorrhagic shock-induced acute lung injury, it was found that erdosteine has a protective effect on lung tissue.

Effect of abdominal trauma on hemorrhagic shock-induced acute lung injury in rats

To evaluate the effects of abdominal trauma on hemorrhagic shock-induced acute lung injury in rats. Five groups were allocated (n = 8) in the study. Group I was taken as the control group, group II as the hemorrhagic shock group, group III as hemorrhagic shock + laparotomy, group IV as hemorrhagic shock + splenectomy and group V as splenectomy + omentectomy + hemorrhagic shock group. Hemorrhagic shock was induced by drawing blood and reducing mean arterial pressure (MAP) to 40 mmHg within 10 min. After a hypotensive period of 1 h, animals were resuscitated. Bronchoalveolar lavage (BAL) was performed to recover cells from the alveolar space with 40 mL of BAL fluid after resuscitation malondialdehyde (MDA) and L-gamma-glutamyl-L-cysteinyl-glycine (GSH) levels were measured in serum, erythrocytes and lung tissue. Serum, erythrocyte, lung tissue MDA and GSH levels were significantly increased in hemorrhagic shock groups II-V (P < 0.05). Lymphocyte, neutrophil and alveolar macrophage counts in BAL fluid indicated a significant difference between control and shock groups (P < 0.05). The degree of trauma increases hemorrhagic shock-induced acute lung injury.

Post-hemorrhagic shock mesenteric lymph activates human pulmonary microvascular endothelium for in vitro neutrophil-mediated injury: the role of intercellular adhesion molecule-1.

Splanchnic hypoperfusion is believed to be central in the pathogenesis of hemorrhagic shock-induced acute respiratory distress syndrome and multiple organ failure. Our previous work focused on the portal circulation as the conduit for gut-derived mediators of acute respiratory distress syndrome. Our current focus is the proinflammatory effects of postshock mesenteric lymph. We hypothesize that postshock lymph induces neutrophil (PMN)-mediated endothelial cell damage in an intercellular adhesion molecule-1 (ICAM-1)-dependent fashion, and devised a two-insult model to test this hypothesis. Rats (n > or = 5) underwent hemorrhagic shock (mean arterial pressure, 40 mm Hg for 30 minutes) and resuscitation (shed blood plus two times crystalloid) with lymph collection. Human pulmonary microvascular endothelial cells (HMVECs) were divided into three groups and grown to near confluence. Group 1 was incubated for 6 hours in 1% preshock or postshock lymph and ICAM-1 was measured by flow cytometry. Group 2 consisted of coculture of HMVECs and PMNs after endothelial cell activation to determine whether postshock lymph would stimulate PMN adherence. Group 3 was incubated under identical conditions, but PMNs were added for 30 minutes, and then activated with 4.5 micromol/L lysophosphatidylcholine (lyso-PC) for 1 hour to ascertain cytotoxicity. HMVEC density was measured using microscopy and recorded as HMVECs per millimeter squared. ICAM-1-blocking antibody and isotype control were used to assess the effects of ICAM-1 on PMN cytotoxicity. A buffer control was used for comparison using analysis of variance with Tukey's correction. Postshock lymph activated HMVECs for increased surface expression of ICAM-1 and stimulated PMNs to adhere to endothelial cell monolayers. Activation of PMNs with lyso-PC in the presence of postshock lymph resulted in marked HMVEC death. The addition of an ICAM-1-blocking antibody abrogated this effect. Neither postshock lymph alone (758 +/- 35 HMVECs/mm(2)), nor postshock lymph in the presence of quiescent PMNs alone (734 +/- 28 HMVECs/mm(2)), nor lymph plus lyso-PC (834 +/- 21 HMVECs/mm(2)) provoked endothelial cell damage. Postshock mesenteric lymph activates endothelial cells for increased ICAM-1 expression and PMN adherence. Furthermore, postshock lymph acts as an inciting event in a two-event in vitro model of PMN-mediated endothelial cell injury. These findings further substantiate the key mechanistic role of mesenteric lymph in hemorrhagic shock-induced acute lung injury and suggest that ICAM-1 expression is pivotal in the two-event model of multiple organ failure.