Polymorphonuclear Neutrophil Sequestration Research Articles

Antibodies to major histocompatibility complex class II antigens directly prime neutrophils and cause acute lung injury in a two-event in vivo rat model.

Transfusion-related acute lung injury (TRALI) is a significant cause of mortality, especially after transfusions containing antibodies to major histocompatibility complex (MHC) class II antigens. We hypothesize that a first event induces both 1) polymorphonuclear neutrophils (PMNs) to express MHC class II antigens, and 2) activation of the pulmonary endothelium, leading to PMN sequestration, so that the infusion of specific MHC class II antibodies to these antigens causes PMN-mediated acute lung injury (ALI). Rats were treated with saline (NS), endotoxin (lipopolysaccharide [LPS]), or cytokines (interferon-γ [IFNγ], macrophage colony-stimulating factor [MCSF], tumor necrosis factor-α [TNFα]); the PMNs were isolated; and the surface expression of the MHC class II antigen OX6 and priming by OX6 antibodies were measured by flow cytometry or priming assays. A two-event model of ALI was completed with NS, LPS, or IFNγ/MCSF/TNFα (first events) and the infusion of OX6 (second event). Compared with NS incubation, rats treated with either LPS or IFNγ/MCSF/TNFα exhibited OX6 PMN surface expression, OX6 antibodies primed the formyl-methionyl-leucyl phenylalanine (fMLF)-activated respiratory burst, and PMN sequestration was increased. OX6 antibody infusion into LPS-incubated or IFNγ/MCSF/TNFα-incubated rats elicited ALI, the OX6 antibody was present on the PMNs, and PMN depletion abrogated ALI. Proinflammatory first events induce PMN MHC class II surface expression, activation of the pulmonary endothelium, and PMN sequestration such that the infusion of cognate antibodies precipitates TRALI.

Plasma ATP is Required for Neutrophil Activation in a Mouse Sepsis Model

Our previous work has shown that polymorphonuclear neutrophils (PMNs) require cellular adenosine triphosphate (ATP) release and autocrine purinergic signaling for their activation. Here we studied in a mouse model of cecal ligation and puncture (CLP) whether sepsis affects this purinergic signaling process and thereby alters PMN responses after sepsis. Using high-performance liquid chromatography, we found that plasma ATP, adenosine diphosphate (ADP), and adenosine monophosphate (AMP) concentrations increased up to 6-fold during the first 8 h after CLP, reaching top levels that were significantly higher than those in sham control animals without CLP. Although leukocyte and PMN counts in sham animals increased significantly after 4 h, these blood cell counts decreased in sepsis animals. CD11b expression on the cell surface of PMNs of septic animals was significantly higher compared with sham and untreated control animals. These findings suggest increased PMN activation and sequestration of PMN from the circulation after sepsis. Plasma ATP levels correlated with CD11b expression, suggesting that increased ATP concentrations in plasma contribute to PMN activation. We found that treatment of septic mice with the ATP receptor antagonist suramin diminished CD11b expression, indicating that plasma ATP contributes to PMN activation by stimulating P2 receptors of PMNs. Increased PMN activation can protect the host from invading microorganisms. However, increased PMN activation can also be detrimental by promoting secondary organ damage. We conclude that pharmacological targeting of P2 receptors may allow modulation of PMN responses in sepsis.

The NO-donor FK409 improves mechanical properties of activated neonatal PMN

Activated polymorphonuclear neutrophils (PMN) play an important role in the microcirculation. Nitric oxide (NO) reduces the sequestration of PMN in the narrow vessels of various organs and, therefore, may reduce organ injury during inflammation. Since PMN of term neonates show various functional differences compared to PMN in adults (decreased chemotaxis, decreased intracellular killing, decreased adhesion), we studied the influence of the semi-synthetical NO-donor FK-409 (4-Ethyl-2-hydroxyimino-5-nitro-3-hexenamide) on the deformability of IL-8 activated PMN in term neonates and adults. A cell transit analyzer (CTA) was used to study transit times of individual PMN through 8 μm filter pores, neutrophil elastase concentrations were determined by enzyme-immunoessay and activation of PMN was classified by mircroscopic evaluation. The transit times of PMN activated by IL-8 in adults were 9.3 ± 2.9 s, in term neonates 10.7 ± 3.3 s. FK-409 improved the transit time of activated PMN in adults (5.4 ± 1.6 s) and in term neonates (5.6 ± 1.1 s). Despite of the functional differences of PMN in term neonates and adults, the improvement of the transit times by FK-409 was not different between the two groups. The NO donor decreased the neutrophil elastase concentrations and the morphological signs of activation in neonates and adults. We conclude that the NO-donor FK-409 improves the microcirculation by increasing the deformability of IL-8 activated PMN. NO may reduce in neonates tissue damage by reduced PMN sequestration due to decreased PMN rigidity.

The mechanism of myocardium and pancreas injury in rabbits with acute renal failure might be related to myeloperoxidase and membrane pump activities

There is increasing evidence indicating that the distant organ injury is a major contributor of high mortality in patients subjected to acute renal failure (ARF). However, sources and mechanisms that ARF causes distant organ injury remain to be determined. The aim of this study is to explore the mechanism from polymorphonuclear neutrophil (PMN) sequestration and membrane pump suppression. To achieve this, we examined myeloperoxidase (MPO), a marker of PMN accumulation in tissues, and membrane pump activities of heart, pancreas, and kidney in two ARF rabbit models. Rabbits are randomly assigned to control, HgCl2-treated, and glycerin-treated groups. ARF animal models are established by hypodermic injection of 1% HgCl2 with 1.3 mL/kg bodyweight (bw) in HgCl2-treated group or intramuscular injection of 50% glycerin with 10 mL/kg bw in glycerin-treated group, respectively, and all animals in each group are further divided into 12 h, 24 h, and 48 h subgroups with each consisting of six rabbits. Six healthy rabbits serve as control group. Results have shown that MPO activities of kidney, myocardium, and pancreas in two model groups were significantly increased than control group at diverse time points. Membrane pump activities of kidney in two model groups are significantly lower than the control group at multiple time points. Moreover, Na+-K+-, Ca2+-, Mg2+-, and Ca2+-Mg2+-ATPase activities of myocardium and pancreas in two model groups are gradually declined with the development of ARF. These findings suggest that PMN sequestration and membrane pump suppression plays an important role in the pathogenesis of ARF and also a major mechanism of myocardium and pancreas injury during the process of ARF.

GLYCOCALYX PROTECTION REDUCES LEUKOCYTE ADHESION AFTER ISCHEMIA/REPERFUSION

Adhesion of polymorphonuclear neutrophils (PMN) to coronary endothelium is a key event for cardiac ischemia/reperfusion injury. Adhesion molecules are normally harbored within the glycocalyx, clothing every healthy vascular endothelium, but shed by ischemia/reperfusion. Our aim was to show whether protection of the glycocalyx with either hydrocortisone or antithrombin can reduce postischemic leukocyte adhesion. Isolated guinea pig hearts, perfused with Krebs-Henseleit buffer, were subjected to 20 min of warm (37 degrees C) no-flow ischemia and consecutive 10 min of reperfusion, either in the absence or presence of hydrocortisone (10 microg/mL) or antithrombin (1 U/mL). An intracoronary bolus of 3 x 10 PMN was applied at the end of reperfusion but without prior contact to the drugs. The sequestration of PMN was calculated from the difference between coronary input and output of cells. Expression of the integrin CD11b on PMN was measured before and after coronary passage. Ischemia/reperfusion induced severe degradation of the glycocalyx (coronary venous syndecan-1 release, 171 +/- 15 ng/g heart vs. basal, 19 +/- 2 ng/g; heparan sulfate, 5.27 +/- 0.28 microg/g vs. basal, 0.26 +/- 0.06 microg/g) and increased PMN adhesion (38.1% +/- 3.5% vs. basal, 11.7% +/- 3.1%). Hydrocortisone and antithrombin both not only reduced glycocalyx shedding (syndecan-1 release, 34 +/- 6 ng/g and 26 +/- 5 ng/g; heparan sulfate, 1.96 +/- 0.24 microg/g and 1.28 +/- 0.2 microg/g, respectively), but also PMN adhesion (17.3% +/- 2.2% and 25.4% +/- 3.3%, respectively) after ischemia/reperfusion. Electron microscopy revealed a mostly intact coronary glycocalyx after pretreatment with either drug. Activation of PMN upon coronary passage was not influenced. Preservation of the glycocalyx mitigates postischemic PMN adhesion. Preconditioning with either hydrocortisone or antithrombin should, thus, alleviate vascular leakage, tissue edema, and inflammation.

Compartmental Apoptosis and Neutrophil Accumulation in Severe Peritonitis

Migration and activation of polymorphonuclear neutrophils (PMN) and apoptosis are central to inflammatory tissue damage. This study examines the relation of these processes, and their expression in the abdominal, systemic, and bronchoalveolar compartments in patients with severe peritonitis. Thirty-one consecutive patients undergoing laparotomy for severe secondary peritonitis. Eight operated patients without peritonitis and 10 long-term mechanically ventilated noninfected patients served as controls. Peritoneal fluid, blood, and bronchoalveolar lavage fluid (BALF) was obtained on d 0 (day of initial laparotomy), 2, and 3. Levels of chemokines (interleukin (IL)-8 and monocyte chemotactic protein (MCP)-1), PMN-counts, PMN activation [myeloperoxidase (MPO), elastase] and apoptosis (nucleosomes) were determined. In peritonitis patients, levels of chemokines and markers of PMN sequestration were increased in all compartments. IL-8 levels were higher in BALF than in plasma, and did not originate from the circulation or from lysis of alveolar cells. Pulmonary nucleosome levels were higher in patients who died (P=0.020), and corresponded with PMN-count in BALF (P<0.001), levels of chemokines (IL-8, P=0.003; MCP-1, P=0.001), and PMN-activation (MPO, P<0.001; elastase P=0.007). Severe peritonitis produces an early pulmonary expression of chemoattractants creating a gradient for PMN sequestration and activation into the lung. These parameters are associated with expression of apoptosis in the lung, which is increased in nonsurviving peritonitis patients.

Developing Specific Therapeutic Strategies for Transfusion-Related Acute Lung Injury. An Overview of Potentially Useful Animal Models

Transfusion-related (TR)- acute lung injury (ALI) is the leading cause of transfusion-related morbidity and mortality. The pathogenesis of TRALI is thought to be a "two hit"-entity: the "first hit" is (any) proinflammatory pulmonary condition (e.g., pneumonia, sepsis or lung contusion) resulting in activation of lung endothelium with sequestration of polymorphonuclear neutrophils - the "second hit" is provided by transfusion of a blood product. Either antibodies against neutrophils are thought to be implicated in the activation of the sequestrated neutrophils, or bioactive lipids (which accumulate during storage of blood products) induce the "second hit", finally resulting in lung injury. Preventive measures do not prevent all TRALI cases. Also, TRALI is most probably underdiagnosed. In this review, we call for the development of therapeutic approaches for this potentially life-threatening disease. Several interventions which are beneficial in ALI and may also be beneficial in TRALI are discussed. The application of these interventions requires the development of clinically relevant TRALI animal models. We discuss the present TRALI animal models and their shortcomings and propose future animal models, in which clinically relevant "first hits" can be applied, thereby imitating the complex clinical situation.

NEUTROPHIL NAD(P)H OXIDASE IS REQUIRED FOR HEMORRHAGIC SHOCK-ENHANCED TLR2 UP-REGULATION IN ALVEOLAR MACROPHAGES IN RESPONSE TO LPS

Alveolar macrophages (AMs) play an important role in the development of posttrauma lung inflammation through initiating polymorphonuclear neutrophil (PMN) migration by direct interactions with PMN, which is in turn mediated by the expression of chemokines and cytokines. We have recently reported that hemorrhagic shock-activated PMN sensitize AM to bacteria LPS for the up-regulation of Toll-like receptor (TLR)2; in turn, this TLR2 up-regulation results in the amplification of expression of cytokines and chemokines in the AM in response to the bacterial products LPS and peptidoglycan, associated with enhanced PMN sequestration in the lung. We sought to address the mechanism underlying the augmentation of TLR2 in AM by shock-activated PMN. We found that hemorrhagic shock/resuscitation (shock) followed by a low dose of i.t. LPS markedly increased TLR2 mRNA expression in AM in wild-type (WT) mice. In contrast, in mice lacking the gp91 subunit of nicotinamide adenine dinucleotide phosphate (reduced form) oxidase (gp91) or in neutropenic WT mice, the increase in TLR2 mRNA was attenuated. Coculture of AM with PMN derived from WT-shocked mice caused a significantly higher level of TLR2 expression in the AM in response to LPS. However, this increase in TLR2 expression was less evident when the AMs were cocultured with PMN derived from gp91 mice subjected to shock. The antioxidant polyethylene glycol catalase markedly decreased MyD88-dependent activation of IL-1 receptor associated kinase 4 and TLR2 expression in the AM in response to LPS. Thus, PMN nicotinamide adenine dinucleotide phosphate (reduced form) oxidase sensitizes hemorrhagic shock-primed AM to LPS, at least in part via enhancing IL-1 receptor associated kinase 4 activity.

Activation of Toll‐like Receptors is a Critical Determinant of Lung Neutrophil Sequestration and Injury Induced by High Tidal Volume Mechanical Ventilation

The infiltration of polymorphonuclear neutrophils (PMNs) into lungs is an important feature of ventilator-induced lung injury (VILI) associated with pneumonia, but the mechanisms that recruit PMNs are poorly understood. Using Toll-like receptor 4 knockout mice (TLR4−/−), we addressed the role of TLR4 signaling in PMN recruitment. Wild type (WT) or TLR4−/− mice were challenged by intratracheal instillation of LPS (3.0 mg/kg) for 2 hr and then subjected to high tidal volume mechanical ventilation (28 ml/kg) for 2 hr. Ventilated WT mice exhibited significant increases in PMN sequestration (5-fold), bronchoalveolar lavage PMN count (3.6-fold), airway fluid albumin concentration (2.4-fold), and edema formation (1.6-fold). Quantitative lung histopathology demonstrated PMN infiltration, alveolar hemorrhage, edema, and alveolar wall thickening in lungs of ventilated, LPS-challenged WT mice. However, TLR4−/− mice showed negligible PMN sequestration, microvascular barrier breakdown, and edema formation. Thus, high tidal volume ventilation during pneumonia induces PMN sequestration and lung injury via TLR4-dependent signaling pathways. The results suggest the important role of lung innate immunity regulated by TLRs in the mechanism of PMN sequestration and injury resulting from mechanical ventilation at high tidal volumes. Supported by NIH R01 HL 071626, P01 HL 60678, and P01 HL 077806.

Inhibition of Matrix Metalloproteinase on Hepatic Transforming Growth Factorβ1 and Caspase-3 Activation in Hemorrhage

Hemorrhage initiates an inflammatory response that induces the systemic release of cytokines and sequestration of polymorphonuclear neutrophils. Sequestered polymorphonuclear neutrophils release proteases, including matrix metalloproteinases (MMPs) that degrade elements of the extracellular matrix, contributing to the morbidity and mortality seen from hemorrhage. Activation of MMPs may be associated with changes in transforming growth factor beta1 (TGF-beta1) and caspase-3 signaling pathways. In this study, the authors examined hemorrhage-induced changes in the expression of rat hepatic MMP-9, tissue inhibitor of metalloproteinase-1 (TIMP-l), TGF-beta1, and caspase-3 activities in the presence and absence of the MMP inhibitor hydroxamate. Hemorrhagic shock was induced in fasted, anesthetized, and cannulated rats by rapid phlebotomy to a mean arterial pressure level of 40 mm Hg, maintained for 90 minutes by withdrawal and infusion of blood, followed by a resuscitation period of lactated Ringer's infusion. Rats received either hydroxamate (25 mg/kg) or vehicle by gavage before hemorrhage. Twenty-four hours after resuscitation, plasma and liver samples were collected. Liver MMP-9, TGF-beta1, and caspase-3 levels were quantified by Western immunoblotting. Plasma glutamic oxaloacetic transaminase (GOT) and plasma glutamic pyruvic transaminase (GPT) were determined enzymatically. Plasma GOT, plasma GPT, and liver MMP-9, TGF-beta1, and caspase-3 levels were all significantly elevated at 24 hours postresuscitation when compared with the control values. Hepatic TIMP-1, an in vivo inhibitor of MMP-9, was unaltered at 24 hours. Hydroxamate treatment reduced GOT, GPT, MMP-9, TGF-beta1, and caspase-3 levels at 24 hours. The mortality of hemorrhaged untreated rats was 29% after 24 hours, and pretreatment with hydroxamate reduced mortality to 0%. These results indicate the beneficial effects of MMP inhibitor in preventing an increase in GOT, GPT, MMP-9, TGF-beta1, and caspase-3 activity with the potential for improvement of hepatic injury due to hemorrhage.

Effect of exogenous carbon monoxide on sequestration of polymorphonuclear neutrophils in the lung following limb ischemia-reperfusion: an experimental study

To investigate the effect of exogenous carbon monoxide (CO) in inhibiting the sequestration of polymorphonuclear neutrophils (PMNs) in the lung following limb ischemia-reperfusion (IR) and the mechanism thereof. PMNs of peripheral blood were isolated from the venous blood of a healthy volunteer. Serum was collected from a patient undergoing bilateral knee joint replacement as IR serum. Human pulmonary microvascular endothelial cells (PMVECs) were cultured and divided into 4 groups: control group (cultured under the condition of room air containing 5% CO2 for 5 h and cultured in normal human serum instead of medium during the last 4 hours of experiment), IR group (cultured under the condition of air containing 5% CO2 for 5h and cultured in the serum of IR patient during the last 4 hours), IR + CO group (cultured under the condition of air containing 0.025% CO and 5% CO2 for 5 hours and cultured in IR serum during the last 4 hours), and control + CO group (cultured under the condition of air containing and 0.025% CO and 5% CO2 for 5 hours and cultured in normal human serum during the last 4 hours). Immunofluorescence flow cytometry was used to detect the expression of intercellular adhesion molecule (ICAM)-1 and integrin CD11b in the PMVECs. Human PMVECs were put into the wells of a 96-well plate and added with PMNs to calculate the PMVEC-PMN adhesion rate. Tourniquettes were bound at the bilateral hind thighs of 32 healthy male SD rats for 4 hours so as to establish a rate IR model. The rats were randomly divided into 4 equal groups: control group (undergoing the same operation without causing limb ischemia and exposed to room air), IR group (undergoing bilateral hind limb ischemia for 4 h and reperfusion for 4 h and exposed to room air), IR + CO group (exposed to the containing 0.025% CO one hour before reperfusion till 4 hours after reperfusion), and control + CO group (exposed to air containing 0.025% CO at the corresponding time point as that of the IR + CO group). Then the rats were killed and their middle pulmonary lobes were taken out for microscopy and calculation of the number of PMNs in alveolar septum. Western blotting was used to examine the ICAM-1 protein expression in the lung. The ICAM-1 expression and integrin CD11b expression of the IR group PMVECs were significantly stronger than those of the IR + CO group PMVECs (both P < 0.05) and there were no significant differences in the ICAM-1 expression and CD11b expression between the control + CO and control groups (both P > 0.05). The PMN-PMVEC adhesion rate of the IR group PMVECs was 30 +/- 2.9%, significantly higher than those of the IR + CO group and control group PMVECs (19.8 +/- 1.5% and 13.4 +/- 1.1% respectively, both P < 0.05) and there was no significant difference in the PMN-PMVEC adhesion rate between the CO + control group and control group (P > 0.05). The lung tissues of the IR group rats showed edema and hemorrhage. The number of PMNs in the alveolar septum was 60.6 +/- 1.7/10 high power fields, significantly higher than those of the IR + CO group and control group (36.4 +/- 1.6 and 22.5 +/- 1.6 respectively, both P < 0.05) and there was no significant difference between the latter 2 groups (P > 0.05). The ICAM-1 protein expression in the lung of the IR group was the strongest, followed by the IR + CO group, control + CO group, and control group. Exogenous CO inhibits the limb/IR-induced PMN sequestration in the lung, probably by the mechanism of down-regulation of the expression of adhesion molecules and suppression of the PMN\PMVEC adhesion following IR.

NK cells and polymorphonuclear neutrophils are both critical for IL-2-induced pulmonary vascular leak syndrome.

The mechanism of IL-2-induced vascular leak syndrome (VLS) is still poorly understood. Cells of both innate and adaptive immune systems have been implicated, but no definitive conclusions have been reached concerning their respective roles. In this study we report a new mouse model of IL-2-induced pulmonary VLS used to obtain a detailed analysis of the early events (sequestration of polymorphonuclear neutrophils and bronchoconstriction) and late events (modifications in the cell and protein content of bronchoalveolar lavages, followed by edema) that characterize this lung injury. This model and knockout animals are used to reconsider the importance of the different leukocyte lineages in early and late events. Recombinase-activating gene 2(-/-) mice are used to demonstrate that adaptive lymphocytes, including NK T cells, are not required for pulmonary VLS induction. By contrast, results obtained with newly described recombinase-activating gene 2(-/-)/IL-15(-/-) mice indicate that NK cells play a key role in both early and late events. In parallel, polymorphonuclear neutrophil depletion is used to evaluate the contributions made by these cells to the late alterations occurring in the lung. Furthermore, when used in combination with inhibition of NO synthase, granulocyte depletion was completely effective in protecting mice from the late events of IL-2-induced pulmonary VLS. Together our results indicate that both NK and PMN cells play a central role in the late events of IL-2-induced VLS.

Inhibition of matrix metalloproteinases by chemically modified tetracyclines in sepsis.

Sepsis precipitates a systemic inflammatory stimulus that causes systemic release of cytokines and sequestration of polymorphonuclear neutrophils, resulting in degranulation of matrix metalloproteinases (MMPs), which causes extracellular matrix basement membrane degradation. One of the important anti-inflammatory properties of tetracyclines is their ability to inhibit MMPs. In this study, we focused on the regulation of MMPs in sepsis and their reduction by treatment with nonantimicrobial chemically modified tetracyclines (CMTs), which retain their anti-inflammatory activity. Sepsis was induced by cecal ligation and puncture (CLP) method. At 24 h and 1 h before CLP, some rats received CMT-3 (25 mg/kg), another group of rats received hydroxamate (H; an inhibitor of MMP; 25 mg/kg), and untreated rats received saline by gavage. At 0 h, 0.5 h, 1.5 h, and 24 h after CLP, blood and liver samples were collected. Plasma and liver MMP-9 by zymography and Western immunoblotting, plasma nitric oxide by measuring nitrate level, plasma glutamic oxaloacetic transaminase (GOT) and glutamic pyruvic transaminase (GPT) by enzymatic method, and liver gelatinase by radiolabeled gelatin lysis assay and 24 h mortality were determined. Plasma MMP-9 (92 kDa), nitrate, and GOT and GPT levels were elevated compared with the time 0 level and reached peak at 1.5 h CLP and remained high for 24 h. Both CMT-3 and H treatment reduced GOT,GPT, 92-kDa gelatinase, and nitrate levels throughout the 24 h. CMT-3 and H are equally effective in sepsis treatment. The 24-h mortality for CLP rats was 30%, whereas pretreatment with CMT-3 and H resulted in 0% mortality. Hepatic MMP-9 and gelatinase activity increased significantly after CLP, and pretreatment with CMT-3 and H inhibited these expressions. These results indicate the beneficial effect of CMT-3 in preventing the increase in GOT, GPT, NO, MMP-9, gelatinase activity, and the ensuing septic shock.

A potent inhibitor of cytosolic phospholipase A2, arachidonyl trifluoromethyl ketone, attenuates LPS-induced lung injury in mice.

Acute respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and sepsis syndrome is one of the most frequent causes of ARDS. Metabolites of arachidonic acid, including thromboxanes and leukotrienes, are proinflammatory mediators and potentially involved in the development of ARDS. A key enzyme for the production of these inflammatory mediators is cytosolic phospholipase A(2) (cPLA(2)). Recently, it has been reported that arachidonyl trifluoromethyl ketone (ATK) is a potent inhibitor of cPLA(2). In the present study, we hypothesized that pharmacological intervention of cPLA(2) could affect acute lung injury. To test this hypothesis, we examined the effects of ATK in a murine model of acute lung injury induced by septic syndrome. The treatment with ATK significantly attenuated lung injury, polymorphonuclear neutrophil sequestration, and deterioration of gas exchange caused by lipopolysaccharide and zymosan administration. The current observations suggest that pharmacological intervention of cPLA(2) could be a novel therapeutic approach to acute lung injury caused by sepsis syndrome.

Phosphated chitin (P-chitin) exerts protective effects by restoring the deformability of polymorphonuclear neutrophil (PMN) cells

In continuation of our on-going investigation on P-chitin, we screened 11 more formulations, out of which only two of them were found effective in vivo. It was not known at that moment, why all the formulations were not effective in vivo. Based on the results of the in vivo screening experiment, in vitro tests were carried out to determine the influence of two effective and non-effective samples on migrating ability of polymorphonuclear neutrophil (PMN) cells. PMN cells were made to loose migrating ability to mimic the in vivo condition of PMN sequestration as it occurs during the acute lung injury, by pre-incubating with chitosan activated serum (CAS). To our surprise, such CAS treated PMN cells with reduced ability to migrate, when subjected to P-chitin treatment were found to have restored the migrating ability. Interestingly, only those samples which were effective in vivo enhanced the deformability or the migrating ability of PMN cells, whereas non-effective samples hindered it. How effective P-chitin restores PMN cells deformability is under investigation.

Leukocyte and endothelial cell adhesion molecules in a chronic murine model of myocardial reperfusion injury.

Expression of endothelial and leukocyte cell adhesion molecules is a principal determinant of polymorphonuclear neutrophil (PMN) recruitment during inflammation. It has been demonstrated that pharmacological inhibition of these molecules can attenuate PMN influx and subsequent tissue injury. We determined the temporal expression of alpha-granule membrane protein-40 (P-selectin), endothelial leukocyte adhesion molecule 1 (E-selectin), and intercellular cell adhesion molecule 1 (ICAM-1) after coronary artery occlusion and up to 3 days of reperfusion. The expression of all of these cell adhesion molecules peaked around 24 h of reperfusion. We determined the extent to which these molecules contribute to PMN infiltration by utilizing mice deficient (-/-) in P-selectin, E-selectin, ICAM-1, and CD18. Each group underwent 30 min of in vivo, regional, left anterior descending (LAD) coronary artery ischemia and 24 h of reperfusion. PMN accumulation in the ischemic-reperfused (I/R) zone was assessed using histological techniques. Deficiencies of P-selectin, E-selectin, ICAM-1, or CD18 resulted in significant (P < 0.05) attenuation of PMN infiltration into the I/R myocardium (MI/R). In addition, P-selectin, E-selectin, ICAM-1, and CD18 -/- mice exhibited significantly (P < 0.05) smaller areas of necrosis after MI/R compared with wild-type mice. These data demonstrate that MI/R induces coronary vascular expression of P-selectin, E-selectin, and ICAM-1 in mice. Furthermore, genetic deficiency of P-selectin, E-selectin, ICAM-1, or CD18 attenuates PMN sequestration and myocardial injury after in vivo MI/R. We conclude that P-selectin, E-selectin, ICAM-1, and CD18 are involved in the pathogenesis of MI/R injury in mice.

Acute lung injury by sepsis and acid aspiration: a key role for cytosolic phospholipase A2.

Adult respiratory distress syndrome (ARDS) is characterized by acute lung injury with a high mortality rate and yet its mechanism is poorly understood. Sepsis syndrome and acid aspiration are the most frequent causes of ARDS, leading to increased lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration and respiratory failure. Using a murine model of acute lung injury induced by septic syndrome or acid aspiration, we investigated the role of cytosolic phospholipase A2 (cPLA2) in ARDS. We found that disruption of the gene encoding cPLA2 significantly reduced pulmonary edema, PMN sequestration and deterioration of gas exchange caused by lipopolysaccharide and zymosan administration. Acute lung injury induced by acid aspiration was similarly reduced in mice with a disrupted cpla2 gene. Our observations suggest that cPLA2 is a mediator of acute lung injury induced by sepsis syndrome or acid aspiration. Thus, the inhibition of cPLA2-initiated pathways may provide a therapeutic approach to acute lung injury, for which no pharmaceutical agents are currently effective.

Beneficial effects of novel nitric oxide donor (FK409) on pulmonary ischemia-reperfusion injury in rats

Background: Nitric oxide (NO) seems to play an important role in tissue injury during reperfusion of the lung. FK409 is the first spontaneous NO donor that increases plasma guanosine 3′:5′-cyclic monophosphate. It is reported that FK409 prevented myocardial infarction following occlusion and reperfusion in rat coronary arteries. In this study, we evaluated the effects of FK409 on pulmonary ischemia-reperfusion injury in an in situ warm ischemia model of rats. Methods Animals were divided into 2 groups: the FK409 study group that was administered FK409 (0.4 mg/kg) before reperfusion and the control group, administered a saline vehicle only. Following a thoracotomy, the bronchus, pulmonary artery and vein were separately clamped for 1 hour. Arterial oxygen tension (PaO 2), arterial oxygen saturation (SaO 2), and endothelin-I (ET-I) were measured after 2 hours of reperfusion. Histologic and immunohistochemical studies were performed; polymorphonuclear neutrophils (PMNs) were counted after 2 hours of reperfusion. Results PaO 2, SaO 2, ET-I after 2 hours of reperfusion and the 7-day survival rate were significantly ( p < 0.05) better in the FK409 group than the control group. Histologic damage was reduced in the FK409 group compared with the control group. PMN infiltration was also significantly ( p < 0.05) lower in the FK409 group than in the control group. Conclusion FK409 seems to protect against ischemia-reperfusion injury of the lung. This effect may be related to a homeostatic effect on pulmonary vascular beds and prevention of PMN sequestration.

Platelet-activating factor mediates acid-induced lung injury in genetically engineered mice.

Adult respiratory distress syndrome (ARDS) is an acute lung injury of high mortality rate, and the molecular mechanisms underlying it are poorly understood. Acid aspiration-induced lung injury is one of the most common causes of ARDS, characterized by an increase in lung permeability, enhanced polymorphonuclear neutrophil (PMN) sequestration, and respiratory failure. Here, we investigated the role of platelet-activating factor (PAF) and the PAF receptor (PAFR) gene in a murine model of acid aspiration-induced lung injury. Overexpression of the PAFR gene in transgenic mice enhanced lung injury, pulmonary edema, and deterioration of gas exchange caused by HCl aspiration. Conversely, mice carrying a targeted disruption of the PAFR gene experienced significantly less acid-induced injury, edema, and respiratory failure. Nevertheless, the efficiency of PMN sequestration in response to acid aspiration was unaffected by differences in PAFR expression level. The current observations suggest that PAF is involved in the pathogenesis of acute lung injury caused by acid aspiration. Thus, inhibition of this pathway might provide a novel therapeutic approach to acute lung injury, for which no specific pharmaceutical agents are currently available.

Hepatic Hemodynamics and Cell Functions in Human and Experimental Sepsis

Hepatic Hemodynamics and Cell Functions in Human and Experimental Sepsis