Pulmonary Leukocyte Accumulation Research Articles

Alveolar hypoxia induces organ-specific inflammasome-related inflammation in male mouse lungs.

Inflammation through activation of caspase-1, seems to play a role in pulmonary hypertension induced by alveolar hypoxia. Whether alveolar hypoxia induces caspase-1-mediated inflammation and influx of leukocytes in other organs than the lungs, is not known. Our aim was to explore sites of caspase-1-related inflammation in alveolar hypoxia. Wild type (WT) mice were exposed to environmental hypoxia or room-air, and organs were analyzed. Right heart catheterization was performed after 14 days of alveolar hypoxia in WT mice and mice transplanted with WT or caspase-1-/- bone marrow. Hypoxia induced leukocyte accumulation and increased caspase-1 protein in the lungs, not in other organs. WT mice transplanted with WT or caspase-1-/- bone marrow showed no difference in pulmonary leukocyte accumulation or development of pulmonary hypertension after alveolar hypoxia. Caspase-1 and IL-18 were detected in bronchial epithelium in WT mice, and hypoxia induced IL-18 secretion from bronchial epithelial cells. IL-18 stimulation generated IL-6 mRNA in monocytes. Phosphorylated STAT3 was increased in hypoxic lungs, not in other organs. Alveolar hypoxia induces caspase-1 activation and leukocyte accumulation specific to the lungs, not in other organs. Caspase-1 activation and IL-18 secretion from bronchial epithelial cells might initiate hypoxia-induced inflammation, leading to pulmonary hypertension.

Role of tumor endothelial marker 1 (Endosialin/CD248) lectin-like domain in lipopolysaccharide-induced macrophage activation and sepsis in mice

Deleterious hyper-inflammation resulting from macrophage activation may aggravate sepsis and lead to lethality. Tumor endothelial marker 1 (TEM1), a type I transmembrane glycoprotein containing six functional domains, has been implicated in cancer and chronic sterile inflammatory disorders. However, the role of TEM1 in acute sepsis remains to be determined. Herein we explored the functional significance of the TEM1 lectin-like domain (TEM1D1) in monocyte/macrophage activation and sepsis using TEM1D1-deleted (TEM1LeD/LeD) transgenic mice and recombinant TEM1D1 (rTEM1D1) protein. Under stimulation with lipopolysaccharides (LPS) or several other toll-like receptor agonists, TEM1LeD/LeD macrophages produced lower levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 than wild-type TEM1wt/wt macrophages. Compared with TEM1wt/wt macrophages, LPS-macrophage binding and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation were suppressed in TEM1LeD/LeD macrophages. In vivo, TEM1D1 deletion improved survival in LPS-challenged mice with reduction of circulating TNF-α and IL-6 and alleviation of lung injury and pulmonary leukocyte accumulation. In contrast, rTEM1D1 could bind to LPS and markedly suppress LPS-macrophage binding, MAPK/NF-κB signaling in macrophages and proinflammatory cytokine production. Treatment with rTEM1D1 improved survival and attenuated circulating TNF-α and IL-6, lung injury and pulmonary accumulation of leukocytes in LPS-challenged mice. These findings demonstrated differential roles for the TEM1 lectin-like domain in macrophages and soluble TEM1 lectin-like domain in sepsis. TEM1 in macrophages mediates LPS-induced inflammation via its lectin-like domain, whereas rTEM1D1 interferes with LPS-induced macrophage activation and sepsis.

Protective Activity of Programmed Cell Death Protein 1 Blockade and Synergy With Caspofungin in a Murine Invasive Pulmonary Aspergillosis Model.

Pharmacological immune checkpoint blockade has revolutionized oncological therapies, and its remarkable success has sparked interest in expanding checkpoint inhibitor therapy in infectious diseases. Herein, we evaluated the efficacy of programmed cell death protein 1 (PD-1) blockade in a murine invasive pulmonary aspergillosis model. We found that, compared with isotype-treated infected control mice, anti-PD-1-treated mice had improved survival, reduced fungal burden, increased lung concentrations of proinflammatory cytokines and neutrophil-attracting chemokines, and enhanced pulmonary leukocyte accumulation. Furthermore, combined treatment with anti-PD-1 and caspofungin resulted in a significant survival benefit compared with caspofungin or anti-PD-1 therapy alone, indicating a synergistic effect between PD-1 inhibitors and immunomodulatory antifungal agents.

Decreased Leukocyte Accumulation and Delayed Bordetella pertussis Clearance in IL-6−/− Mice

IL-6, a pleiotropic cytokine primarily produced by the innate immune system, has been implicated in the development of acquired immune responses, though its roles are largely undefined and may vary in the context of different diseases. Using a murine model of infection, we established that IL-6 influences the adaptive immune responses against the endemic human respiratory pathogen Bordetella pertussis. IL-6 was induced in the lungs of C57BL/6 mice by B. pertussis. IL-6(-/-) mice showed a protracted infectious course and were less efficiently protected by B. pertussis vaccination than wild-type mice. Abs from IL-6(-/-) mice, though lower in titer, efficiently reduced B. pertussis numbers in IL-6-sufficient mice. Pulmonary leukocyte recruitment and splenic or pulmonary T cell cytokine responses to B. pertussis, including Th1 and Th17 cytokine production, were lower in IL-6(-/-) mice than in wild-type mice. Adoptive transfer of immune wild-type CD4(+) cells ameliorated the defect of IL-6(-/-) mice in the control of B. pertussis numbers. Together, these results reveal the dysregulation of multiple aspects of adaptive immune responses in B. pertussis-infected IL-6(-/-) mice and suggest that IL-6 is involved in regulating Ab generation, pulmonary leukocyte accumulation, and T cell cytokine production in response to B. pertussis as well as the generation of effective vaccine-induced immunity against this pathogen.

Immunopathology and dexamethasone therapy in a new model for malaria-associated acute respiratory distress syndrome

Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. To develop a new animal model for MA-ARDS, mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was characterized by increased lung weight, edema, leukocyte infiltration and hemorrhages (Figure ​(Figure1).1). The pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with P. chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8+ T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation. Figure 1 Histopathology of P. berghei NK65-induced MA-ARDS. Frozen sections of lungs of mice infected for 10 days with P. berghei NK65 or control mice were stained with H&E.The black bar corresponds with 100 μm. We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.

Immunopathology and Dexamethasone Therapy in a New Model for Malaria-associated Acute Respiratory Distress Syndrome

Malaria infection is often complicated by malaria-associated acute respiratory distress syndrome (MA-ARDS), characterized by pulmonary edema and hemorrhages. No efficient treatments are available for MA-ARDS and its pathogenesis remains poorly understood. Development of a new animal model for MA-ARDS to explore the pathogenesis and possible treatments. C57BL/6 mice were infected with Plasmodium berghei NK65, and the development of MA-ARDS was evaluated by the analysis of lung weight, histopathology, and bronchoalveolar lavages. Cytokine and chemokine expression in the lungs was analyzed by reverse transcription-polymerase chain reaction, and the accumulation of leukocyte subclasses was determined by flow cytometric analysis. In this model, the pulmonary expression of several cytokines and chemokines was increased to a higher level than in mice infected with Plasmodium chabaudi AS, which does not cause MA-ARDS. By depletion experiments, CD8(+) T lymphocytes were shown to be pathogenic. High doses of dexamethasone blocked MA-ARDS, even when administered after appearance of the complication, and reduced pulmonary leukocyte accumulation and the expression of a monocyte/macrophage-attracting chemokine. We developed a novel model of MA-ARDS with many similarities to human MA-ARDS and without cerebral complications. This contrasts with the more classical model with P. berghei ANKA, characterized by fulminant cerebral malaria. Hence, infection with P. berghei NK65 generates a broader time window to study the pathogenesis and to evaluate candidate treatments. The finding that high doses of dexamethasone cured MA-ARDS suggests that it might be more effective against MA-ARDS than it was in the clinical trials for cerebral malaria.

Effects of Ethanol on Pulmonary Inflammation in Postburn Intratracheal Infection

Infectious complications are a major cause of mortality in trauma patients. Burn patients with prior ethanol exposure have a worse prognosis than those who sustain injury but had not been drinking. We examined pulmonary infection and lung pathology in mice given ethanol (1.2 g/kg) 30 minutes before being subjected to 13 to 15% total body surface area scald burn followed by intratracheal inoculation with Pseudomonas aeruginosa (1-2 x 10(3) colony-forming units [CFUs]). Survival was monitored for up to 48 hours. Sham control groups had 100% survival after intratracheal infection regardless of ethanol exposure. Infected burned animals had 55% survival; however, survival of infected mice exposed to ethanol and burn injury was significantly lower (27%, P < .0001). When pulmonary infection was evaluated, the lungs of sham groups were negative for bacterial colonies. In addition, at 24 hours there were no significant differences in lung CFUs from infected burned animals regardless of ethanol exposure (3.0 x 10(4)). However, pulmonary bacterial content significantly decreased (1.2 x 10, P < .02) at 48 hours in mice given burn injury alone, where CFUs from the lungs of mice exposed to ethanol prior to burn did not decline (5.4 x 10(5)). At the same time point, lungs from animals given ethanol and burn injury had about a 2-fold (P < .02) increase in leukocyte infiltration and vascular congestion, as well as decreased pulmonary oxygen saturation (82.8%, P < .02), when compared with other treatment groups. In summary, ethanol exposure in postburn intratracheal infection results in the inability to clear pulmonary infection marked by a prolonged pulmonary leukocyte accumulation and a decrease in pulmonary function.

Inhibition of leukotriene biosynthesis abrogates the host control of Mycobacterium tuberculosis

Leukotrienes produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. Recently, it has been demonstrated that leukotrienes also play an important role in host defense against microorganisms. In vitro studies have shown that leukotrienes augmented the anti-mycobacterial activity of neutrophils. In this study, we examined the role of leukotrienes in regulating host response and cytokine generation in a murine model of tuberculosis. Administration of the 5-LO pathway inhibitor MK 886, which reduced lung levels of both the leukotriene B 4 and the anti-inflammatory substance lipoxin A 4 by ∼50%, increased 60-day mortality from 14% to ∼57% in Mycobacterium tuberculosis-infected mice, and increased lung bacterial burden by ∼15-fold. Although MK 886-treated animals exhibited no reduction in pulmonary leukocyte accumulation, they did manifest reduced levels of nitric oxide generation and of the protective type 1 cytokines interleukin-12 and gamma interferon. Together our results demonstrate that 5-LO pathway product(s) – presumably leukotrienes – positively regulate protective Th1 responses against mycobacterial infection in vivo. Moreover, the immunosuppressive phenotype in infected mice observed with MK 886 is most consistent with inhibition of an activator (LTB 4) rather than a suppressor (LXA 4) of antimicrobial defense, suggesting the major effect of leukotrienes.

Antiinflammatory properties of inducible nitric oxide synthase in acute hyperoxic lung injury.

The objective of this study was to determine whether endogenous nitric oxide (NO), specifically the inducible NO synthase isoform (iNOS: NOS II), reduces or amplifies lung injury in mice breathing at a high oxygen tension. Previous studies have shown that exogenous (inhaled) NO protects against hyperoxia-induced lung injury, and that endogenous NO derived from iNOS inhibits leukocyte recruitment and protects against lung injury induced by lipopolysaccharide. In the present study, hyperoxia (> 98% O(2) for 72 h) induced acute lung injury in both wild-type and iNOS-deficient mice as determined by elevated albumin and lactate dehydrogenase levels in bronchoalveolar lavage fluid (BALF) and by increased extravascular lung water. Lung injury was greater in iNOS-deficient mice than in wild-type mice and was associated with an increased number of polymorphonuclear leukocytes in BALF. iNOS messenger RNA expression levels increased in the lungs of wild-type hyperoxic mice. Nitrotyrosine, a marker of reactive NO species, was expressed in both wild-type and iNOS-deficient mice in hyperoxia, indicating an iNOS-independent pathway for protein nitration. We conclude that iNOS is capable of reducing pulmonary leukocyte accumulation and lung injury. The data indicate that iNOS induction serves as a protective mechanism to minimize the effects of acute exposure to hyperoxia.

Effect of heparin on antigen-induced airway responses and pulmonary leukocyte accumulation in neonatally immunized rabbits.

The effect of single administrations of aerosolized heparin, low molecular weight heparin (LMWH) and the linear polyanionic molecule, polyglutamic acid (PGA) were examined on antigen-induced airway hyperresponsiveness and leukocyte accumulation in neonatally immunized rabbits. Adult litter-matched NZW rabbits immunized within 24 h of birth with Alternaria tenuis antigen were treated with heparin, LMWH or PGA prior to or following antigen challenge (Alternaria tenuis). For each drug-treated group, a parallel group of rabbits were treated with the appropriate vehicle. In all groups, airway responsiveness to inhaled histamine and bronchoalveolar lavage (BAL) was performed 24 h prior to and following antigen challenge. Basal lung function in terms of resistance (R(L)) and dynamic compliance (C(dyn)) and acute bronchoconstriction was unaltered by pre-treatment with heparin, LMWH or PGA compared to their respective vehicles 24 h prior to or following antigen challenge. In vehicle-treated animals, airway hyperresponsiveness to inhaled histamine was indicated by an increase in the maximal responses of the cumulative concentration-effect curves to histamine and reductions in R(L)PC(50) and C(dyn)PC(35) values 24 h following antigen challenge. Heparin and LMWH given prior to antigen challenge significantly inhibited the development of airway hyperresponsiveness, whereas PGA did not. When given following antigen challenge, all three drugs failed to inhibit the development of airway hyperresponsiveness. Eosinophil and neutrophil cell numbers in BAL fluid increased significantly 24 h following antigen challenge. Heparin, LMWH and PGA failed to inhibit the increase in cell numbers following antigen challenge whether given prior to or following antigen challenge.

Lipoxin A 4 and aspirin-triggered 15-epi-LXA are protective in acute inflammation and reperfusion injury in leukotriene B 4 receptor transgenic mice

Leukotrienes produced from arachidonic acid by the action of 5-lipoxygenase (5-LO) are classical mediators of inflammatory responses. Recently, it has been demonstrated that leukotrienes also play an important role in host defense against microorganisms. In vitro studies have shown that leukotrienes augmented the anti-mycobacterial activity of neutrophils. In this study, we examined the role of leukotrienes in regulating host response and cytokine generation in a murine model of tuberculosis. Administration of the 5-LO pathway inhibitor MK 886, which reduced lung levels of both the leukotriene B4 and the anti-inflammatory substance lipoxin A4 by ∼50%, increased 60-day mortality from 14% to ∼57% in Mycobacterium tuberculosis-infected mice, and increased lung bacterial burden by ∼15-fold. Although MK 886-treated animals exhibited no reduction in pulmonary leukocyte accumulation, they did manifest reduced levels of nitric oxide generation and of the protective type 1 cytokines interleukin-12 and gamma interferon. Together our results demonstrate that 5-LO pathway product(s) – presumably leukotrienes – positively regulate protective Th1 responses against mycobacterial infection in vivo. Moreover, the immunosuppressive phenotype in infected mice observed with MK 886 is most consistent with inhibition of an activator (LTB4) rather than a suppressor (LXA4) of antimicrobial defense, suggesting the major effect of leukotrienes.

Involvement of bradykinin B 1 and B 2 receptors in pulmonary leukocyte accumulation induced by Sephadex beads in guinea pigs

The effects of selected bradykinin receptor antagonists on leukocyte infiltration into the lungs were studied in a model of guinea pig lung inflammation induced by the intravenous injection of Sephadex beads. The bradykinin B 1 receptor antagonist, [Leu 8]desArg 9-BK (40 mg kg −1 24 h −1) and the bradykinin B 2 receptor antagonist, dArg[Hyp 3,Thi 5, dTic 7,Oic 8]BK (code name HOE 140; 4 mg kg −1 24 h −1), administered intravenously by osmotic pumps, significantly reduced eosinophil counts by 33% and 42% in bronchoalveolar fluid, respectively. HOE 140 decreased neutrophil counts by 35%. LysLys[Hyp 3,Igl 5, d-Igl 7,Oic 8]desArg 9BK (code name B 9858), a newly described bradykinin B 1 receptor antagonist, administered intraperitoneally (1 mg kg −1), decreased eosinophil and neutrophil counts by 45% in bronchoalveolar fluid. d-Arg[Hyp 3,Igl 5, d-Igl 7,Oic 8]BK (code name B 9430), a non-selective bradykinin B 1/B 2 receptor antagonist, also administered intraperitoneally (1 mg kg −1), decreased eosinophil and macrophage counts by 62% and 80% in bronchoalveolar fluid. These results suggest that bradykinin B 1 and B 2 receptors are involved in leukocyte recruitment in our model of lung inflammation.

Effects of Various Doses of Antithrombin III on Endotoxin-Induced Endothelial Cell Injury and Coagulation Abnormalities in Rats

We previously demonstrated that antithrombin III reduced the injury to endothelial cells caused by activated leukocytes in rats administered endotoxin. This occurred via the increase of the endothelial release of prostaglandin I 2, which is a potent inhibitor of leukocyte activation. We evaluated the dose of antithrombin III required to prevent such endothelial cell injury in rats administered endotoxin, by comparing the effects of various antithrombin III doses on the pulmonary vascular injury. The intravenous administration of endotoxin, 5 mg/kg, produced a transient accumulation of leukocytes in the lung, followed by pulmonary vascular injury, as indicated by an increase in the pulmonary vascular permeability, and coagulation abnormalities. The dose of 250 U/kg significantly inhibited all such effects of endotoxin. While lower doses of antithrombin III (50 and 100 U/kg) significantly inhibited such coagulation abnormalities, they failed to prevent either the pulmonary accumulation of leukocytes or the subsequent pulmonary vascular injury. Rats administered endotoxin exhibited an accumulation of neutrophils and edematous changes in the pulmonary interstitial space. Although such changes were reduced after 250 U/kg of antithrombin III, they were unaffected by lower doses of 50 and 100 U/kg. Plasma levels of 6-keto-PGF 1α were markedly increased in rats 90 min after the administration of endotoxin, and were significantly decreased in the endotoxin-treated rats administered the lower doses of antithrombin III (50 and 100 U/kg), but not altered in those endotoxin-treated rats receiving 250 U/kg of antithrombin III. These findings suggest that a higher antithrombin III dose is necessary to prevent endothelial cell injury than is required to inhibit coagulation abnormalities in an animal model of sepsis. These observations support the notion that antithrombin III may prevent endotoxin-induced endothelial cell injury by promoting endothelial release of prostaglandin I 2 and thus inhibiting leukocyte activation.

Antithrombin III (AT III) prevents LPS-induced pulmonary vascular injury: novel biological activity of AT III.

Acute respiratory distress syndrome (ARDS) adversely affects the outcome of patients with disseminated intravascular coagulation (DIC) associated with sepsis. To determine whether antithrombin III (AT III) is useful for the treatment of ARDS in sepsis, we evaluated the effect of AT III on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. Although the intravenous administration of AT III (250 U/kg) prevented LPS-induced pulmonary accumulation of leukocytes, increases in pulmonary vascular permeability, and coagulation abnormalities, inactivated factor Xa, a selective inhibitor of thrombin generation, did not prevent such events other than the coagulation abnormalities. AT III promotes the endothelial release of prostacyclin by interacting with cell surface glycosaminoglycans in vivo. Trp49-modified AT III, which lacks affinity for heparin, did not prevent LPS-induced pulmonary vascular injury. Plasma levels of 6-keto-prostaglandin F1alpha were markedly increased in rats after the administration of LPS and significantly decreased in the LPS-treated rats administered Trp49-modified AT III, but not altered in those LPS-treated rats receiving AT III. Preventive effects of AT III were not observed in rats pretreated with indomethacin, which inhibits prostacyclin biosynthesis. Prostacyclin prevents LPS-induced pulmonary vascular injury by inhibiting leukocyte accumulation in the lungs. These observations strongly suggest that AT III prevents pulmonary vascular injury induced by LPS by promoting the endothelial release of prostacyclin, a potent inhibitor of leukocyte activation.

Rhs-TM prevents ET-induced increase in pulmonary vascular permeability through protein C activation.

We have previously demonstrated that recombinant human soluble (rhs) thrombomodulin (TM) inhibits the endotoxin (ET)-induced increase in pulmonary vascular permeability by inhibiting leukocyte activation. In the present study, we examined whether rhs-TM could inhibit the ET-induced increase in pulmonary vascular permeability in rats by activating protein C. rhs-TM did not inhibit ET-induced increases in pulmonary vascular permeability when its protein C activation ability was selectively inhibited by a monoclonal antibody (MAb) against rhs-TM (MAb R5G12). Histological examination revealed that neutrophil infiltration in lung tissues after ET administration was significantly reduced by rhs-TM, but infiltration was not reduced by MAb R5G12-pretreated rhs-TM. ET-induced intravascular coagulation was prevented by rhs-TM and by MAb R5G12-pretreated rhs-TM. However, ET-induced coagulation was not prevented by rhs-TM that had been treated with MAb F2H5, which cannot bind thrombin or activate protein C. These observations strongly suggest that rhs-TM prevents ET-induced pulmonary vascular injury by inhibiting pulmonary accumulation of leukocytes through thrombin binding and the subsequent protein C activation and may prevent ET-induced intravascular coagulation through thrombin binding.

Intercellular adhesion molecule-1 deficiency prolongs survival and protects against the development of pulmonary inflammation during murine lupus.

One of the characteristic features of the lupus syndrome in humans and mice is the organ-specific accumulation of leukocytes within a variety of different tissues; however, the etiology of this phenomenon remains unclear. The work presented here determined the role of intercellular adhesion molecule (ICAM)-1 in the development of pulmonary leukocyte accumulation by generating MRL/MpJ-Faslpr mice that are genetically deficient in this critical adhesion molecule. Interestingly, these MRL/MpJ-Faslpr ICAM-1 knockout mice exhibit prolonged survival times compared to littermates expressing ICAM-1. We have determined that lack of ICAM-1 completely abrogates the development of pulmonary inflammation but does not prevent the development of autoantibodies, lymphadenopathy, and glomerulonephritis. Furthermore, the lack of pulmonary inflammation was found to be due to decreased migration of leukocytes to the lung rather than decreased in situ proliferation of cells.

Effect of nafamostat mesilate on pulmonary vascular injury induced by lipopolysaccharide in rats.

Nafamostat mesilate (NM) is a synthetic protease inhibitor that is capable of inhibiting the various coagulation factors such as factor VIIa and thrombin. To determine whether NM may also be useful in treating adult respiratory distress syndrome (ARDS) related in sepsis, we investigated the effect of NM on lipopolysaccharide (LPS)-induced pulmonary vascular injury in rats. The intraperitoneal administration of NM prevented the pulmonary vascular injury and coagulation abnormalities induced by LPS. DEGR-factor VIIa, a selective inhibitor of factor VIIa, prevented the coagulation abnormalities, but not the pulmonary vascular injury, induced by LPS. NM did not reduce LPS-induced increase in pulmonary accumulation of leukocytes. NM did not inhibit the increase in the plasma concentration of tumor necrosis factor-alpha (TNF-alpha) observed after administration of LPS. NM did not inhibit the function of activated neutrophils in vitro. Plasma values of total serum hemolytic complement (CH50) were markedly decreased after the administration of LPS. NM inhibited the LPS-induced decrease in plasma CH50 values. Findings suggest that NM may reduce the pulmonary vascular injury as well as the coagulation abnormalities induced by LPS. The former effect may be independent of the anticoagulant effect but dependent on the inhibitory effect of the activation of the complement system in rats administered LPS.

Effect of human urinary thrombomodulin on endotoxin-induced intravascular coagulation and pulmonary vascular injury in rats.

Adult respiratory distress syndrome (ARDS) and disseminated intravascular coagulation (DIC) are serious complications of sepsis. Thrombomodulin, an important endothelial anticoagulant, binds thrombin to generate activated protein C (APC). To determine whether thrombomodulin purified from human urine (urinary thrombomodulin, UTM) is useful for the treatment of DIC and ARDS in sepsis, we examined the effect of UTM on endotoxin (ET)-induced coagulation abnormalities and pulmonary vascular injury in rats. Intravenous administration of UTM prevented the ET-induced pulmonary accumulation of leukocytes and the increase in pulmonary vascular permeability, as well as ET-induced histological changes such as leukocyte infiltration and pulmonary interstitial edema. On the other hand, dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, did not prevent these effects of ET. UTM did not prevent ET-induced pulmonary accumulation of leukocytes and pulmonary vascular injury in rats pretreated with DEGR-Xa. Our findings suggest that UTM attenuates ET-induced coagulation abnormalities and pulmonary vascular injury. Furthermore, the latter effect may be dependent on the capacity of UTM to activate protein C.

Recombinant thrombomodulin prevents endotoxin-induced lung injury in rats by inhibiting leukocyte activation

Acute respiratory distress syndrome (ARDS) is a serious complication of sepsis. Thrombomodulin, an important endothelial anticoagulant, binds thrombin to generate activated protein C (APC). We have previously demonstrated that APC prevents endotoxin (ET)-induced pulmonary vascular injury by inhibiting activated leukocytes. We therefore examined whether recombinant human soluble thrombomodulin (rhs-TM) prevents activated leukocyte-induced pulmonary vascular injury in rats receiving ET. Intravenous administration of rhs-TM prevented ET-induced pulmonary accumulation of leukocytes and increase in pulmonary vascular permeability, as well as ET-induced histological changes, such as leukocyte infiltration and pulmonary interstitial edema. Dansyl-Glu-Gly-Arg-chloromethyl ketone-treated factor Xa (DEGR-Xa), a selective inhibitor of thrombin generation, did not prevent these effects of ET. rhs-TM did not prevent ET-induced pulmonary accumulation of leukocytes and pulmonary vascular injury in rats pretreated with DEGR-Xa. These results suggest that rhs-TM prevents ET-induced pulmonary vascular injury by inhibiting pulmonary accumulation of leukocytes and that this effect may be mediated primarily by APC generation.

Attenuation of endotoxin-induced pulmonary vascular injury by antithrombin III

We evaluated the effects of antithrombin III (AT III) on the pulmonary vascular injury induced by injecting rats with lipopolysaccharide (LPS) to investigate the possible usefulness of AT III as a treatment for acute respiratory distress syndrome. The intravenous administration of AT III prevented the pulmonary accumulation of leukocytes (as evaluated by myeloperoxidase activity) and the increase in pulmonary vascular permeability to 125I-bovine serum albumin induced by LPS. The increase in pulmonary vascular permeability induced by LPS administration was unaffected by various anticoagulants but was inhibited by the leukocytopenia induced by nitrogen mustard or by the administration of a granulocyte elastase inhibitor, ONO-5046. AT III given alone, but not heparin plus AT III or Trp49-modified AT III, which lacks affinity for heparin, significantly increased the plasma concentration of 6-keto-prostaglandin F1alpha, suggesting that the interaction of AT III with heparin-like substances at the endothelial cell surface promotes the release of prostacyclin from endothelial cells in vivo. Trp49-modified AT III failed to prevent the LPS-induced accumulation of leukocytes and vascular injury. The pulmonary accumulation of leukocytes and vascular injury induced by LPS were not prevented by administering AT III to rats that were pretreated with indomethacin. The continuous intravenous infusion of prostacyclin prevented the LPS-induced pulmonary accumulation of leukocytes and vascular injury. Findings suggest that AT III depends on its ability to promote the release of prostacyclin, a potent inhibitor of leukocyte activation, from endothelial cells to prevent pulmonary vascular injury induced by LPS.