Role of LPS in the hepatic microvascular dysfunction elicited by cecal ligation and puncture in mice

BACKGROUND/AIMS: Sepsis remains a leading cause of death in critically ill patients. Because endotoxemia is viewed as a key mediator of sepsis-induced inflammation, administration of bacterial endotoxin (LPS) is often used to simulate sepsis in experimental animals. This study tests the hypothesis that LPS is a critical determinant of the hepatic microvascular dysfunction in mice made septic by cecal ligation and puncture (CLP). METHODS: Intravital videomicroscopy was used to quantify sinusoidal perfusion, and platelet and leukocyte adhesion in terminal hepatic venules (THV) and sinusoids in LPS-sensitive and LPS-insensitive mice subjected to CLP or LPS (i.p.). mRNA expression of TLR-2, TLR-4, MyD-88, and Ly-96 was also assessed. RESULTS: While LPS-sensitive mice responded to both CLP and LPS challenges with elevated leukocyte and platelet adhesion in THV and sinusoids, and a reduced sinusoidal perfusion density, LPS-insensitive mice exhibited comparable blood cell adhesion and sinusoidal malperfusion following CLP, but not LPS. Hepatic mRNA of MyD-88 and TLR-2 was elevated in the CLP and LPS groups. Endotoxin was not detectable in the blood of LPS-sensitive mice after CLP, but was elevated after LPS administration. CONCLUSIONS: These findings do not support a major role for LPS in the hepatic microvascular disturbances associated with polymicrobial sepsis.     

Independent roles of Macrophage Migration Inhibitory Factor and endogenous,but not exogenous glucocorticoids in regulating leukocyte trafficking

Objectives : Macrophage migration inhibitory factor (MIF) promotes leukocyte recruitment and antagonizes the anti‐inflammatory effects of glucocorticoids (GC). The aim of this study was to examine whether interaction between MIF and GC underlies the ability of MIF to promote leukocyte– endothelial cell (EC) interactions. Methods : Intravital microscopy was used to assess leukocyte‐EC interactions in wild‐type and MIF^?/? mice following treatment with lipopolysaccharide (LPS), the GC dexamethasone, and inhibition of endogenous GC, using the GC‐receptor antagonist, RU486. Results : Dexamethasone reduced LPS‐induced leukocyte interactions in wild‐type mice to levels similar to those observed in MIF ^?/? mice not treated with dexamethasone, whereas in MIF ^?/? mice, leukocyte interactions were not further inhibited by dexamethasone. RU486 increased LPS‐induced leukocyte adhesion and emigration to a similar extent in both wild‐type and MIF ^?/? mice, indicating that endogenous GC exert a similar inhibitory effect on leukocyte trafficking in wild‐type and MIF ^?/? mice. Both MIF deficiency and RU486 treatment reduced VCAM‐1 expression, while neither treatment modulated expression of ICAM‐1 or chemokines CCL2, KC, and MIP‐2. Conclusions : These results suggest that endogenous MIF and GC regulate leukocyte‐EC interactions in vivo reciprocally but through predominantly independent mechanisms, and that the anti‐inflammatory effect of MIF deficiency is comparable to that of exogenous GC.     

Endogenous estrogen regulation of inflammatory arthritis and cytokine expression in male mice,predominantly via estrogen receptor α

Objective

A number of experimental observations have associated elevated estrogen levels with amelioration of inflammation. The involvement of estrogen and estrogen receptor (ER) isotypes in the regulation of inflammation in males is not well understood. In this study, we used specific ERα and ERβ agonists in male mice deficient in estrogen because of a deletion of aromatase (aromatase‐knockout [ArKO] mice) to investigate ER isotype utilization in estrogen regulation of inflammation.

Methods

Lipopolysaccharide (LPS)‐induced cytokine expression and antigen‐induced arthritis (AIA) were investigated in male ArKO and WT littermate mice, as well as in response to selective agonists of ERα (16α‐LE2) and ERβ (8β‐VE2). The therapeutic effect of selective ER agonists was also examined in mice with collagen‐induced arthritis (CIA).

Results

Estrogen deficiency in ArKO mice was associated with significant increases in LPS‐induced serum interleukin‐6 (IL‐6), tumor necrosis factor, monocyte chemotactic protein 1, and interferon‐γ levels, which were significantly abrogated by administration of 16α‐LE2, but not 8β‐VE2. In contrast, both 16α‐LE2 and 8β‐VE2 significantly increased LPS‐induced IL‐10 levels. Estrogen deficiency was also associated with significant exacerbation of AIA and antigen‐specific T cell proliferation, which was reversed by administration of either 16α‐LE2 or 8β‐VE2. ArKO mice showed increased antigen‐specific T cell proliferation in response to immunization with type II collagen (CII). Administration of 16α‐LE2, but not 8β‐VE2, significantly reduced the severity of CIA, which was associated with inhibition of anti‐CII–specific IgG.

Conclusion

These data indicate that endogenous estrogen plays an essential inhibitory role in inflammation in male mice and that ERα is the dominant receptor that mediates these effects.

     

Blocking very late antigen-4 integrin decreases leukocyte entry and fatty streak formation in mice fed an atherogenic diet

Atherosclerotic lesion development is characterized by the recruitment of leukocytes, principally monocytes, to the vessel wall. Considerable interest has been focused on the adhesion molecule(s) involved in leukocyte/endothelial interactions. The goal of the present study was to determine the role of the very late antigen-4 (VLA-4) integrin/ligand interaction in fatty streak development using murine models. Because alpha4 null mice are not viable, a peptidomimetic was used to block VLA-4-mediated leukocyte binding. The ability of a synthetic peptidomimetic of connecting segment-1 (CS-1 peptide) to block the recruitment of leukocytes and the accumulation of lipid in the aortic sinus of either wild-type mice (strain C57BL/6J) or mice with a low-density lipoprotein null mutation (LDLR-/-) maintained on an atherogenic diet was assessed. The active (Ac) CS-1 peptide or scrambled (Sc) CS-1 peptide was delivered subcutaneously into mice using a mini osmotic pump. Mice were exposed to the peptide for 24 to 36 hours before the onset of the atherogenic diet. In C57BL/6J mice, leukocyte entry into the aortic sinus, as assessed by en face preparations, was inhibited by the active peptide (Ac=28+/-4, Sc=54+/-6 monocytes/valve; P=0.004). Additionally, frozen sections stained with Oil Red O were analyzed to assess lipid accumulation in the aortic sinus. C57BL/6J mice that received the (Ac) compound demonstrated significantly reduced lesion areas as compared with mice that received the (Sc) peptide (Ac=4887+/-4438 microm2, Sc=15 009 +/-5619 microm2; P<0.0001). In a separate study, LDLR-/- mice were implanted with pumps containing either the (Ac) or (Sc) peptide before initiation of the atherogenic diet. Because LDLR-/- mice fed a chow diet displayed small lesions at 14 weeks, the effects of the peptide seen in these animals represented a change in early lipid accumulation rather than initiation. By using whole-mount preparations, the (Ac) but not the (Sc) peptide significantly reduced the area of lipid accumulation in the aortic sinus, resulting in an approximate 66% decrease. Plasma analysis from all studies revealed concentrations of peptide to be present at levels previously determined by in vitro analysis to block adhesion. (Ac) CS-1 peptide, which blocks VLA-4 on the leukocyte surface, is effective in reducing leukocyte recruitment and lipid accumulation in the aortic sinus. The present study provides in vivo evidence that the VLA-4 integrin plays an important role in the initiation of the atherosclerotic lesion and lipid accumulation, and it suggests a potential therapeutic strategy for this disease.     

Glycine modulates cytokine secretion,inhibits hepatic damage and improves survival in a model of endotoxemia in mice

Background and aim: There is substantial experimental evidence that the amino acid glycine may have a role in protecting tissues against insults such as ischemia, hypoxia and reperfusion. Our aim was to investigate the ability of the amino acid glycine to prevent hepatic damage induced by injection of lipopolysaccharide and d ‐galactosamine (d ‐Gal), to modulate pro‐ and anti‐inflammatory cytokine levels, and to improve survival. Methods: Mice were challenged with an intraperitoneal injection of d ‐Gal (16 mg/mouse) and lipopolysaccharide (LPS, 1 μg/mouse). The intervention group also received an intraperitoneal injection of glycine (150 mg/kg) in two doses: 24 h before and just after LPS challenge. Serum cytokine levels were measured 2 h after challenge, and liver enzymes and histology were determined 16 h after LPS. Separate groups of mice received the same treatment and the survival rate was determined 24 h and ten days after endotoxin administration. In in vitro experiments, cultured mononuclear cells were stimulated by LPS, and TNF‐α and IL‐10 secretion were measured, in the presence or absence of glycine. Results: In the glycine‐treated mice, the serum levels of liver enzymes and TNF‐α, the histologic necroinflammation score and the mortality rate were significantly reduced compared to control mice (P<0.001). Serum IL‐10 levels in the glycine‐treated mice were increased (P<0.01). In vitro studies in cultured lymphocytes isolated from either normal or glycine pretreated mice, demonstrated a significant and dose‐dependent inhibition of LPS‐induced TNF‐α secretion and increase in IL‐10 response after treatment with glycine (P<0.01). In conclusion, glycine reduces hepatic damage and improves survival rate in this mouse model of endotoxemia. The protective effect of glycine is associated with modulation of TNF‐α and IL‐10 secretion.     

Effects of N-acetylcysteine and tirilazad mesylate on intestinal functional capillary density, leukocyte adherence, mesenteric plasma extravasation and cytokine levels in experimental endotoxemia in rats

INTRODUCTION: The study's objective was to determine the effects of the administration of N-acetylcysteine (NAC) and of tirilazad mesylate (TM) on intestinal functional capillary density, mesenteric plasma extravasation, leukocyte adherence and on cytokine release during experimental endotoxemia in rats. METHODS: In a prospective, randomized, controlled animal study, 80 male Wistar rats were examined in 2 test series. Both series were divided into 4 groups. Group 1 served as control group (CON group). Group 2 (LPS group), group 3 (NAC group) and group 4 (TM group) received endotoxin infusions (10 mg/kg over 2 h). In NAC group 150 mg/kg body weight NAC was administered after the first 30 minutes of endotoxemia intravenously. In TM group, 10 mg/kg body weight TM was administered after the first 30 minutes of endotoxemia intravenously. Animals of the series 1 underwent studies of leukocyte adherence on submucosal venular endothelium of the small bowel wall and intestinal functional capillary density (FCD) in the intestinal mucosa and the circular as well as the longitudinal muscle layer by intravital fluorescence microscopy (IVM). Plasma levels of interleukin 1beta (IL-1beta), interferone gamma (IFN-gamma) and soluble intercellular adhesion molecule1 (s-ICAM 1) as well as white blood cell count (WBC) were estimated. In the animals of the series 2 mesenteric plasma extravasation was determined by IVM and plasma levels of tumor necrosis factor alpha (TNF-alpha), IL-4, IL-6, IL-10 and malondialdehyde (MDA) were estimated. RESULTS: After LPS administration, FCD in the villi intestinales was unchanged and in the longitudinal muscularis layer it was increased. There was no effect of NAC or TM administration on FCD.Although the plasma extravasation was not significantly influenced by LPS administration, TM administration resulted in a lower plasma extravasation in the TM group compared to the other groups. After endotoxin challenge, the firmly adherence of leukocytes to vascular endothelium as a parameter of leukocyte activation in endotoxemia was increased but NAC or TM administration had no influence on leukocyte adherence. The plasma levels of IL-1beta, IL-6, IL-10, TNF-alpha, IFN-gamma and sICAM-1 were increased in the endotoxemic groups (LPS group, NAC group and TM group) and the WBC was decreased compared to controls. IL-4 levels were unchanged during observation period. Plasma MDA levels were not influenced by LPS administration compared to controls. The administration of NAC resulted in lower sICAM-1 and MDA levels compared to the LPS group. The IL-1beta, IL-6, IL-10, TNF-alpha and IFN-gamma plasma levels were not influenced by NAC or TM administration. CONCLUSIONS: In this posttreatment sepsis model in rats, NAC administration resulted in lower sICAM-1 and MDA levels compared to the LPS treated animals. TM administration reduced the plasma extravasation in this model.     

肠源性内毒素血症促使水肿型胰腺炎向坏死型胰腺炎转化

目的 探讨肠源性内毒素血症对急性坏死型胰腺炎（ANP）的促成作用及其可能机制。方法Ｃ５７ＢＬ／６小鼠随机分成4组：Ⅰ组,急性水肿型胰腺炎（AEP）组(n=20); Ⅱ组,AEP 脂多糖（LPS）处理组（n=20);Ⅲ组,LPS组（n=20);Ⅳ组,正常对照组（n=5).腹腔内间隔1h共7次,注射50μg/kg体重雨蛙素诱导小鼠AEP。第6小时胃管内灌入5mg/kg体重LPS溶液。监测12、24、48h和5d血清淀粉酶、乳酸脱氢酶（LDH）活性;胰组织髓过氧化物酶（MPO）活性和组织变化;免疫组化观察胰组织Mac-1(CD11b/CD18),E选择素、P选择素和细胞间黏附分子（ICAM－1）表达;RT－PCR和Southern印迹检测胰组织TNFα,IL-1β,IL-6和IFNγmRNA变化。结果 雨蛙素诱导小鼠AEP。LPS单独并不引起胰组织明显病理变化和血清淀粉酶、LDH活性改变。然而,同剂量LPS则使小鼠AEP胰损伤加重,血清淀粉酶和LDH活性显著增加;胰组织Mac-1表达增强;MPO活性增加;E选择素、P选择素和ICAM－1表达增强;TNFα,IL-1β,IL-6和IFNγmRNA表达上调。结论 肠源性内毒素血症可促使AEP转化为ANP,黏附分子介导的中性粒细胞过度聚集和大量细胞因子释放,造成胰腺损伤可能为其重要机制。     

Bikunin suppresses lipopolysaccharide-induced lethality through down-regulation of tumor necrosis factor- alpha and interleukin-1 beta in macrophages

BACKGROUND: Lipopolysaccharide (LPS) is the primary mediator of gram-negative sepsis; it induces the production of macrophage-derived cytokines. It has been shown that bikunin, a Kunitz-type protease inhibitor, inhibits LPS-induced cytokine expression. METHODS: To explore the role of bikunin, bikunin knockout (Bik(-/-)) mice were used for in vitro cytokine experiments and in vivo animal models. RESULTS: We show that a higher level of LPS-mediated death was induced in Bik(-/-), compared with wild-type (wt), mice; the administration of bikunin caused a significant reduction in LPS-induced lethality; LPS significantly increased tumor necrosis factor (TNF)- alpha and interleukin-1 beta levels in Bik(-/-), relative to wt, mice after LPS challenge; concomitant administration of bikunin inhibited the LPS-induced plasma levels of these cytokines; bikunin suppressed the LPS-induced up-regulation of cytokine expression through the suppression of the phosphorylation of ERK1/2, JNK, and p38 in macrophages; and LPS-induced up-regulation of TNF- alpha expression was not enhanced in Bik(-/-) macrophages without endogenous bikunin. CONCLUSIONS: These data allow us to speculate that the increased sensitivity of Bik(-/-) mice to LPS-induced death in vivo is due to a lack of circulating bikunin in plasma. Bikunin may play a role as a potent anti-inflammatory agent.     

Endotoxemia in transgenic mice overexpressing human glutathione peroxidases

In response to endotoxemia induced by administration of lipopolysaccharide, a complex series of reactions occurs in mammalian tissues. During this inflammation response, cells produce different mediators, such as reactive oxygen species, a number of arachidonic acid metabolites, and cytokines. The reactive oxygen species thus generated have been suggested to produce tissue injury as a result of macromolecular damage or by interfering with regulatory processes. They may also act as important signaling molecules to induce redox-sensitive genes. We report here that transgenic mice overexpressing 2 major forms of human glutathione peroxidases (GPs), intra- and extracellular GP, are able to modulate host response during endotoxemic conditions. We show that these animals have a decreased hypotension and increased survival rate after administration of a high dosage of lipopolysaccharide. Overexpression of GPs alters vascular permeability and production of cytokines (interleukin-1 beta and tumor necrosis factor-alpha) and NO, affects arachidonic acid metabolism, and inhibits leukocyte migration. These results suggest an important role for peroxides in pathogenesis during endotoxemia, and GPs, by regulating their level, may prove to be good candidates for antioxidant therapy to protect against such injury.     

二草清肝汤对内毒素性肝损害防护作用的实验研究

目的：探讨中药二草清肝汤对内毒素/脂多糖（LPS）性肝损害的防护机制。方法：采用LPS腹腔注射（10mg/kg）制备小鼠内毒素血症肝损害模型,BALB/c小鼠200只随机分为正常对照组、LPS组、二草清肝汤小剂量组和二草清肝汤大剂量组;光镜观察肝组织病理学改变,全自动生化分析仪检测血浆丙氨酸氨基转移酶（ALT）水平,酶联免疫吸附法检测血浆白细胞介素-12（IL-12）、肿瘤坏死因子（TNF）-α浓度,免疫组织化学法检测Toll样受体4（TLR4）表达水平,逆转录聚合酶链反应（RT-PCR）检测肝组织TLR4 mRNA表达水平。结果：二草清肝汤能明显提高小鼠存活率,肝组织病理损害程度减轻;二草清肝汤小剂量组及大剂量组小鼠的IL-12、TNF-α水平显著低于LPS组,差异有显著性意义（均P（0.05）;二草清肝汤小剂量组和大剂量组小鼠肝组织TLR4 mRNA水平也明显低于LPS组,差异均有显著性意义（均P（0.05）,但二草清肝汤大、小剂量组间差异无显著性意义（P〉0.05）。结论：二草清肝汤能明显减轻LPS所致的肝损害,其机制可能与其下调肝脏各种细胞的TLR4表达,同时下调IL-12、TNF-α的水平有关。     

Reduced leukocyte-endothelial cell interactions in the inflamed microcirculation of macrophage migration inhibitory factor-deficient mice

OBJECTIVE: Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with established roles in a range of inflammatory conditions. However, it is not known whether MIF influences inflammation via the direct promotion of leukocyte-endothelial cell interactions. Therefore, the aim of these experiments was to investigate the ability of MIF to regulate leukocyte-endothelial cell interactions in the inflamed microvasculature. METHODS: Intravital microscopy was used to examine postcapillary venules in the cremaster muscle and synovium of wild-type and MIF(-/-) mice. Leukocyte-endothelial cell interactions (rolling, adhesion, emigration) were compared under a range of inflammatory conditions. RESULTS: In cremasteric postcapillary venules of MIF(-/-) mice, lipopolysaccharide (LPS)-induced leukocyte rolling, adhesion, and emigration were significantly reduced relative to that in wild-type mice. Similar responses were observed in response to tumor necrosis factor alpha and histamine. Examination of the synovial microvasculature following exposure to carrageenan revealed that leukocyte rolling and adhesion in synovial postcapillary venules and leukocyte entry into the joint space were also reduced significantly in MIF(-/-) mice. In each of these models, the level of P-selectin-dependent rolling was reduced in MIF(-/-) mice. Despite this, no difference in P-selectin expression was observed following LPS treatment. However, microvascular shear forces were elevated in MIF(-/-) mice, raising a possible mechanism to explain the reduced interactions in these animals. CONCLUSION: MIF(-/-) mice consistently displayed a reduction in P-selectin-dependent rolling, suggesting that MIF exerts proinflammatory effects, in part, via the promotion of P-selectin-mediated rolling. Together, these data indicate that MIF promotes interactions between leukocytes and endothelial cells, thereby enhancing the entry of leukocytes into sites of inflammation.     

Toll-like receptor 4 modulates myocardial ischaemia-reperfusion injury: Role of matrix metalloproteinases

Toll-like receptor 4 (TLR4) mediates innate immune responses following endotoxemia and myocardial ischaemia-reperfusion (I/R) injury. Pre-treatment with the major TLR4 ligand lipopolysaccharide (LPS) reduces infarct size. Matrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play a crucial role in endotoxemia possibly also determining I/R injury. AIMS: We investigated the influence of TLR4 on infarct size and assessed the influence of MMP and TIMP regulation on I/R injury. METHODS: Left anterior descending artery (LAD) occlusion was performed on wild-type (C3H/HeN) and TLR4-deficient (C3H/HeJ) mice. Animals were stimulated with LPS (1 mg/kg) or PBS 16 h ahead of 60 min LAD ligation. After 24 h of reperfusion, triphenyltetrazolium chloride staining was performed and infarct size was measured by planimetry. MMP- and TIMP-mRNA expression were determined by RPA after 3 h of reperfusion. MMP zymographic activity was monitored 6 h after occlusion. RESULTS: TLR4-deficient mice and LPS-treated wild-type mice showed significantly reduced infarct areas. LPS-stimulation significantly increased the overall MMP/TIMP mRNA expression ratio due to elevated MMP-3, -8, -9, and TIMP-1 in wild-type mice. I/R overall reduced the MMP/TIMP ratio due to increased MMP-1, TIMP-1, and -3 mRNA expression. CONCLUSIONS: LPS pre-treatment and TLR4-deficiency led to a decreased infarct size. However, infarct area and MMP/TIMP ratio were not correlated. This means that in TLR4-deficient mice MMP/TIMP ratios are not determining the infarct size.     

Reduced leukocyte–endothelial cell interactions in the inflamed microcirculation of macrophage migration inhibitory factor–deficient mice

Objective

Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine with established roles in a range of inflammatory conditions. However, it is not known whether MIF influences inflammation via the direct promotion of leukocyte–endothelial cell interactions. Therefore, the aim of these experiments was to investigate the ability of MIF to regulate leukocyte–endothelial cell interactions in the inflamed microvasculature.

Methods

Intravital microscopy was used to examine postcapillary venules in the cremaster muscle and synovium of wild‐type and MIF^−/− mice. Leukocyte–endothelial cell interactions (rolling, adhesion, emigration) were compared under a range of inflammatory conditions.

Results

In cremasteric postcapillary venules of MIF^−/− mice, lipopolysaccharide (LPS)–induced leukocyte rolling, adhesion, and emigration were significantly reduced relative to that in wild‐type mice. Similar responses were observed in response to tumor necrosis factor α and histamine. Examination of the synovial microvasculature following exposure to carrageenan revealed that leukocyte rolling and adhesion in synovial postcapillary venules and leukocyte entry into the joint space were also reduced significantly in MIF^−/− mice. In each of these models, the level of P‐selectin–dependent rolling was reduced in MIF^−/− mice. Despite this, no difference in P‐selectin expression was observed following LPS treatment. However, microvascular shear forces were elevated in MIF^−/− mice, raising a possible mechanism to explain the reduced interactions in these animals.

Conclusion

MIF^−/− mice consistently displayed a reduction in P‐selectin–dependent rolling, suggesting that MIF exerts proinflammatory effects, in part, via the promotion of P‐selectin–mediated rolling. Together, these data indicate that MIF promotes interactions between leukocytes and endothelial cells, thereby enhancing the entry of leukocytes into sites of inflammation.

     

Rac1 mediates sex difference in cardiac tumor necrosis factor-α expression via NADPH oxidase-ERK1/2/p38 MAPK pathway in endotoxemia

The purpose of this study was to investigate the role of Rac1 and estrogen in sex difference of cardiac tumor necrosis factor-alpha (TNF-α) expression during endotoxemia. Endotoxemia was induced in male and female mice by peritoneal injection of lipopolysaccharide (LPS, 4 mg/kg). Compared with female mice, male mice produced more TNF-α in the heart 4 h after LPS treatment, which were correlated with higher Rac1 and NADPH oxidase activity, more phosphorylation of ERK1/2 and p38 MAPK, and up-regulation of toll-like receptor-4 (TLR-4) expression in male mice. Cardiac specific Rac1 knockout or administration of 17β-estradiol down-regulated Rac1 expression, attenuated gp91^phox-NADPH oxidase expression and activity, decreased phosphorylation of ERK1/2/p38 MAPK and inhibited cardiac TNF-α expression induced by LPS, suggesting an important role of Rac1 and estrogen in LPS-stimulated TNF-α expression in the heart. More importantly, the sex difference in TNF-α expression was abrogated by Rac1 knockout or gp91^phox knockout and by administration of apocynin or N-acetylcysteine in LPS-stimulated mice. To investigate the functional significance of sex difference in endotoxemia, heart function was measured in isolated hearts with a Langendorff system. Male mice exhibited worse myocardial dysfunction compared with female in endotoxemia. Treatment of male mice with 17β-estradiol attenuated myocardial dysfunction during endotoxemia. In conclusion, LPS induces Rac1 activation, which contributes to NADPH oxidase activity and phosphorylation of ERK1/2/p38 MAPK, leading to TNF-α expression in the heart. The sex difference in TNF-α expression is estrogen-dependent and mediated via Rac1 dependent NADPH oxidase/ERK1/2 and p38 MAPK pathway in LPS-stimulated hearts.     

Biomonitoring for assessment of organic dust-induced lung inflammation.

Inhalation exposure to particulate matter containing endotoxin (or lipopolysaccharide (LPS)) occurs in a variety of occupations. Nasal lavage and induced sputum have been used to evaluate lung inflammation resulting from such exposures. Whole blood assay (WBA) measures cytokine production of leukocytes after ex vivo stimulation with LPS. The present study examined the effectiveness of WBA for evaluating inflammatory responses and susceptibility. C3HeB/FEJ mice were tolerised by LPS injection or sham tolerised with saline. Animals then inhaled either swine barn dust extract containing endotoxin or saline. Bronchoalveolar lavage (BAL) fluid was assayed for leukocyte counts and pro-inflammatory cytokines (interleukin-6, tumour necrosis factor-alpha). Whole blood was stimulated with 10 or 100 ng.mL(-1) of LPS, incubated for 5 or 18 h and assayed for cytokines. Barn dust-exposed groups revealed significantly higher total cells, neutrophils and cytokines in BAL compared with saline-exposed groups. Animals tolerised to LPS and exposed to barn dust demonstrated lower cellular and cytokine BAL responses. Similarly, WBA yielded significantly elevated cytokines with barn dust exposure and reduced responses with tolerisation. This study demonstrates the efficacy of whole blood assay as a biomarker of inhalation exposure to inflammatory agents and its use for assessing susceptibility to organic dust-induced lung inflammation.     

Annexin 1 mediates the rapid anti-inflammatory effects of neutrophil-derived microparticles

Polymorphonuclear leukocyte (PMN)-derived microparticles display inhibitory properties on target cells as assessed in vitro; since PMNs contain abundant amounts of the endogenous anti-inflammatory protein annexin 1 (AnxA1), we tested here whether biologically active AnxA1 could be present in PMN-derived microparticles. PMN adhesion to human umbilical vein endothelial cell (HUVEC) monolayers led to the generation of microparticles that contained AnxA1, as detected by Western blotting, flow cytometry, and mass spectrometry analyses. Addition of these microparticles to recipient PMNs prior to flow over HUVEC monolayers significantly inhibited cell adhesion, an effect abrogated by a neutralizing anti-AnxA1 antibody, or an antibody raised against the AnxA1 receptor, that is termed lipoxin A(4) receptor or ALX. Intravenous delivery of human PMN-derived microparticles markedly inhibited PMN recruitment to an air pouch inflamed with IL-1beta. This anti-inflammatory effect was also dependent on endogenous AnxA1, since injection of microparticles produced from wild-type PMNs (bone marrow derived), but not from AnxA1-null PMNs, inhibited IL-1beta-induced leukocyte trafficking. In conclusion, PMN-derived microparticles contain functionally active AnxA1 that confers them anti-inflammatory properties; generation of these microparticles in the microcirculation could promote inflammatory resolution by time-dependent dampening of cell recruitment.