The role of CD14 in neutrophil recruitment within the liver microcirculation during endotoxemia

During Gram-negative sepsis and endotoxemia, CD14 is essential for the recognition of LPS by the TLR4 complex and subsequent generation of systemic inflammation. However, CD14-independent responses to LPS have been reported in vitro and in vivo in selected tissues including the skin. As the liver is a key target organ for neutrophil sequestration and inflammatory pathology during sepsis and endotoxemia, we investigated the role of CD14 in the recruitment of neutrophils into the liver in a mouse model of endotoxemia. Using dynamic in vivo imaging of the liver, we observed that neutrophil recruitment within the sinusoids and post-sinusoidal venules occurred equivalently between LPS-treated wild-type and CD14-knockout mice. Neutrophil recruitment within the liver was completely independent of CD14 regardless of whether it was expressed on cells of hematopoietic or nonhematopoietic origin or in serum as soluble CD14. Whereas CD14 expression was essential for activation of circulating neutrophils and for the development of LPS-induced systemic inflammation (pulmonary neutrophil sequestration, leukopenia, and increased serum proinflammatory cytokine levels), deficiency of CD14 did not limit the adhesion strength of neutrophils in vitro. Furthermore, wild-type and CD14-knockout mice displayed identical deposition of serum-derived hyaluronan-associated protein within liver sinusoids in response to LPS, indicating that the sinusoid-specific CD44/hyaluronan/serum-derived hyaluronan-associated protein-dependent pathway of neutrophil adhesion is activated independently of CD14. Therefore, the liver microcirculation possesses a unique CD14-independent mechanism of LPS detection and activation of neutrophil recruitment.     

PECAM-1 dampens cytokine levels during LPS-induced endotoxemia by regulating leukocyte trafficking

AimsTo investigate the mechanism by which platelet endothelial cell adhesion molecule 1 (PECAM-1/CD31), an immunoglobulin (Ig)-superfamily cell adhesion and signaling receptor, regulates pro-inflammatory cytokine levels. The purpose of the present investigation was to test the hypothesis that PECAM-1 influences circulating cytokine levels by regulating the trafficking of activated, cytokine-producing leukocytes to sites of inflammation.Main methodsPECAM-1^+/+ and PECAM-1^?/? mice were subjected to lipopolysaccharide (LPS)-induced endotoxemia, and systemic cytokine levels were measured by Bioplex multiplex cytokine assays. Flow cytometry was employed to enumerate leukocytes at inflammatory sites and to measure cytokine synthesis in leukocyte sub-populations. Enzyme-linked immunosorbent assay (ELISA) was used to measure cytokine levels in tissue samples and in supernatants of in vitro-stimulated leukocytes.Key findingsWe confirmed earlier reports that mice deficient in PECAM-1 had greater systemic levels of pro-inflammatory cytokines following intraperitoneal (IP) LPS administration. Interestingly, expression of PECAM-1, in mice, had negligible effects on the level of cytokine synthesis by leukocytes stimulated in vitro with LPS and in peritoneal macrophages isolated from LPS-injected mice. There was, however, excessive accumulation of macrophages and neutrophils in the lungs of PECAM-1-deficient, compared with wild-type, mice — an event that correlated with a prolonged increase in lung pro-inflammatory cytokine levels.SignificanceOur results demonstrate that PECAM-1 normally functions to dampen systemic cytokine levels during LPS-induced endotoxemia by diminishing the accumulation of cytokine-producing leukocytes at sites of inflammation, rather than by modulating cytokine synthesis by leukocytes.     

P2Y12 antagonist attenuates eosinophilic inflammation and airway hyperresponsiveness in a mouse model of asthma

Leukotriene E4 (LTE4) that plays a key role in airway inflammation is expressed on platelets and eosinophils. We investigated whether blocking of the P2Y12 receptor can suppress eosinophilic inflammation in a mouse model of asthma because platelets and eosinophils share this receptor to be activated. BALB/c mice were sensitized by intraperitoneal injection of ovalbumin (OVA), followed by OVA nebulization. On each challenge day, clopidogrel, a P2Y12 antagonist was administered 30 min. before each challenge. Forty‐eight hours after the last OVA challenge, mice were assessed for airway hyperresponsiveness (AHR), cell composition and cytokine levels, including chemokine ligand 5 (CCL5), in bronchoalveolar lavage (BAL) fluid. EOL cells were treated with LTE4, with or without clopidogrel treatment, and intracellular and extracellular eosinophil cationic protein (ECP) expressions were measured to find the inhibiting function of P2Y12 antagonist on eosinophilic activation. The levels of P2Y12 expression were increased markedly in the lung homogenates of OVA‐sensitized and ‐challenged mice after platelet depletion. Administration of clopidogrel decreased AHR and the number of airway inflammatory cells, including eosinophils, in BAL fluid following OVA challenge. These results were associated with decreased levels of Th2 cytokines and CCL5. Histological examination showed that inflammatory cells as well as mucus‐containing goblet cells were reduced in clopidogrel‐administered mice compared to vehicle‐treated mice. Clopidogrel inhibited extracellular ECP secretion after LTE4 stimulation in EOL‐1 cells. Clopidogrel could prevent development of AHR and airway inflammation in a mouse model of asthma. P2Y12 can be a novel therapeutic target to the suppression of eosinophils in asthma.     

Extracellular Ca(2+) suppresses endotoxin-inducible tissue factor activation in monocytic THP-1 cells

BACKGROUND: Monocytic tissue factor (TF), an initiator of extrinsic blood coagulation, is often activated under various inflammatory conditions including endotoxemia. This activation could be a contributing factor to the manifestation of disseminated intravascular coagulation following septic shock. HYPOTHESIS: We herein determine if extracellular Ca(2+) ([Ca(2+)](ex)) regulates bacterial endotoxin (LPS)-inducible monocytic TF activation. METHODS: We have employed a model monocytic cell line (THP-1) to explore the mode of action of [Ca(2+)](ex) on the modulation of LPS-induced TF activation. TF activity was measured by a single stage clotting assay, while TF expression as well as LPS recognition and its receptor expression were studied in immunofluorescent approaches. RESULTS: LPS-induced TF activation was inversely correlated to [Ca(2+)](ex). Upon exposure of THP-1 cells to LPS (1.5 microg ml(-1)) for 6 h in the Hanks' medium without CaCl(2), TF was activated by nearly 10-fold. TF activation appreciably decreased with the increasing [Ca(2+)](ex). No more than 3.5-fold TF activation was detected at 5 mM [Ca(2+)](ex). Consistent with the significantly lower degree of TF activation, LPS-induced TF expression at 5 mM [Ca(2+)](ex) was 60 per cent less than that without [Ca(2+)](ex). FACScan analysis showed that LPS recognition was significantly blocked at 5 mM [Ca(2+)](ex) which however had no effect on the expression of CD14 and CD11b, the proposed major LPS receptors. Moreover, LPS binding in vitro was significantly inhibited by 5 mM CaCl(2). CONCLUSION: Our results demonstrate that [Ca(2+)](ex) blocked LPS recognition without affecting its receptor expression on THP-1 monocytes. This insensitivity to LPS thereby resulted in the depressed inducible monocytic TF expression and activation.     

Rac1 activation induces tumour necrosis factor‐α expression and cardiac dysfunction in endotoxemia

Induction of tumour necrosis factor‐α (TNF‐α) expression leads to myocardial depression during sepsis. However, the underlying molecular mechanisms are not fully understood. The aim of this study was to investigate the role of Rac1 in TNF‐α expression and cardiac dysfunction during endotoxemia and to determine the involvement of phosphoinositide‐3 kinase (PI3K) in lipopolysaccharide (LPS)‐induced Rac1 activation. Our results showed that LPS‐induced Rac1 activation and TNF‐α expression in cultured neonatal mouse cardiomyocytes. The response was inhibited in Rac1 deficient cardiomyocytes or by a dominant‐negative Rac1 (Rac1N17). To determine whether PI3K regulates Rac1 activation, cardiomyocytes were treated with LY294002, a PI3K selective inhibitor. Treatment with LY294002 decreased Rac1 activity as well as TNF‐α expression stimulated by LPS. Furthermore, inhibition of PI3K and Rac1 activity decreased LPS‐induced superoxide generation which was associated with a significant reduction in ERK1/2 phosphorylation. To investigate the role of Rac1 in myocardial depression during endotoxemia in vivo, wild‐type and cardiomyocyte‐specific Rac1 deficient mice were treated with LPS (2 mg/kg, i.p.). Deficiency in Rac1 significantly decreased myocardial TNF‐α expression and improved cardiac function during endotoxemia. We conclude that PI3K‐mediated Rac1 activation is required for induction of TNF‐α expression in cardiomyocytes and cardiac dysfunction during endotoxemia. The effect of Rac1 on TNF‐α expression seems to be mediated by increased NADPH oxidase activity and ERK1/2 phosphorylation.     

MiR‐643 inhibits lipopolysaccharide‐induced endometritis progression by targeting TRAF6

Endometritis is a prevalent disease with inflammation of uterus endangering women reproductive health. MicroRNAs (miRNAs) play important roles in inflammatory disorders, including endometritis. However, the role and mechanism of miR‐643 in endometritis development remain unclear. This study aimed to investigate the effect of miR‐643 on lipopolysaccharide (LPS)‐induced inflammatory response and clarify the potential mechanism. LPS‐treated human endometrial epithelial cells (HEECs) were cultured to investigate the role of miR‐643 in vitro. The expression levels of miR‐643 and tumor necrosis factor receptor‐associated factor 6 (TRAF6) were measured via quantitative real‐time polymerase chain reaction and western blot, respectively. LPS‐induced inflammatory response was assessed by inflammatory cytokines secretion via enzyme‐linked immunosorbent assay. The activation of nuclear factor‐κB (NF‐κB) pathway was investigated by western blot. The interaction between miR‐643 and TRAF6 was validated by bioinformatics analysis, luciferase reporter assay, and RNA immunoprecipitation. The expression of miR‐643 was decreased and TRAF6 protein level was enhanced in LPS‐treated HEECs. The overexpression of miR‐643 suppressed LPS‐induced secretion of inflammatory cytokines (tumor necrosis factor‐α, interleukin‐1β [IL‐1β], and IL‐6) and activation of NF‐κB pathway. The knockdown of TRAF6 inhibited LPS‐induced inflammatory response in HEECs. TRAF6 was validated as a target of miR‐643 and TRAF6 restoration reversed the effect of miR‐643 on inflammation response in LPS‐treated HEECs. Collectively, miR‐643 attenuated LPS‐induced inflammatory response by targeting TRAF6, indicating a novel avenue for the treatment of endometritis.     

Transient receptor potential ankyrin 1 (TRPA1) positively regulates imiquimod‐induced,psoriasiform dermal inflammation in mice

Transient receptor potential ankyrin 1 (TRPA1), a membrane protein ion channel, is known to mediate itch and pain in skin. The function of TRPA1, however, in psoriasiform dermatitis (PsD) is uncertain. Herein, we found that expression of TRPA1 is highly up‐regulated in human psoriatic lesional skin. To study the role of TRPA1 in PsD, we assessed Psoriasis Severity Index (PSI) scores, transepidermal water loss (TEWL), skin thickness and pathology, and examined dermal inflammatory infiltrates, Th17‐related genes and itch‐related genes in c57BL/6 as wild‐type (WT) and TRPA1 gene knockout (KO) mice following daily application of topical IMQ cream for 5 days. Compared with WT mice, clinical scores, skin thickness change and TEWL scores were similar on day 3, but were significantly decreased on day 5 in IMQ‐treated TRPA1 KO mice (vs WT mice), suggesting reduced inflammation and skin barrier defects. Additionally, the relative area of epidermal Munro's microabscesses and mRNA levels of neutrophil inducible chemokines (S100A8, S100A9 and CXCL1) were decreased in the treated skin of TRPA1 KO mice, suggesting that neutrophil recruitment was impaired in the KO mice. Furthermore, mast cells, CD31⁺ blood vascular cells, CD45⁺ leukocytes and CD3⁺ T cells were all reduced in the treated skin of TRPA1 KO mice. Lastly, mRNA expression levels of IL‐1β, IL‐6, IL‐23, IL‐17A, IL‐17F and IL‐22 were decreased in TRPA1 KO mice. In summary, these results suggest a key role for TRPA1 in psoriasiform inflammation and raising its potential as a target for therapeutic intervention.     

Myeloid‐specific GPCR kinase‐2 negatively regulates NF‐κB1p105‐ERK pathway and limits endotoxemic shock in mice

G‐protein‐coupled receptor kinase 2 (GRK2) is a member of a kinase family originally discovered for its role in the phosphorylation and desensitization of G‐protein‐coupled receptors. It is expressed in high levels in myeloid cells and its levels are altered in many inflammatory disorders including sepsis. To address the physiological role of myeloid cell‐specific GRK2 in inflammation, we generated mice bearing GRK2 deletion in myeloid cells (GRK2^?mye). GRK2^?mye mice exhibited exaggerated inflammatory cytokine/chemokine production, and organ injury in response to lipopolysaccharide (LPS, a TLR4 ligand) when compared to wild‐type littermates (GRK2^fl/fl). Consistent with this, peritoneal macrophages from GRK2^?mye mice showed enhanced inflammatory cytokine levels when stimulated with LPS. Our results further identify TLR4‐induced NF‐κB1p105‐ERK pathway to be selectively regulated by GRK2. LPS‐induced activation of NF‐κB1p105‐MEK‐ERK pathway is significantly enhanced in the GRK2^?mye macrophages compared to GRK2^fl/fl cells and importantly, inhibition of the p105 and ERK pathways in the GRK2^?mye macrophages, limits the enhanced production of LPS‐induced cytokines/chemokines. Taken together, our studies reveal previously undescribed negative regulatory role for GRK2 in TLR4‐induced p105‐ERK pathway as well as in the consequent inflammatory cytokine/chemokine production and endotoxemia in mice. J. Cell. Physiol. 226: 627–637, 2011. © 2010 Wiley‐Liss, Inc.     

Gene expression profile of mouse white adipose tissue during inflammatory stress: age‐dependent upregulation of major procoagulant factors

Tolerance to physiological stress resulting from inflammatory disease decreases significantly with age. High mortality rates, increased cytokine production, and pronounced thrombosis are characteristic complications of aged mice with acute systemic inflammation induced by injection with lipopolysaccharide (LPS). As adipose tissue is now recognized as an important source of cytokines, we determined the effects of aging on visceral white adipose tissue gene expression during LPS‐induced inflammation in male C57BL/6 mice. Microarray analysis revealed that the expression of 6025 genes was significantly changed by LPS; of those, the expression of 667 showed an age‐associated difference. Age‐associated differences were found in many genes belonging to the inflammatory response and blood clotting pathways. Genes for several procoagulant factors were upregulated by LPS; among these, tissue factor, thrombospondin‐1, and plasminogen activator inhibitors‐1 and ‐2, exhibited age‐associated increases in expression which could potentially contribute to augmented thrombosis. Further analysis by qRT–PCR, histological examination, and cell fraction separation revealed that most inflammatory and coagulant‐related gene expression changes occur in resident stromal cells rather than adipocytes or infiltrating cells. In addition, basal expression levels of 303 genes were altered by aging, including increased expression of component of Sp100‐rs (Csprs). This study indicates that adipose tissue is a major organ expressing genes for multiple inflammatory and coagulant factors and that the expression of many of these is significantly altered by aging during acute inflammation. Data presented here provide a framework for future studies aimed at elucidating the impact of adipose tissue on age‐associated complications during sepsis and systemic inflammation.     

Serum amyloid A promotes LPS clearance and suppresses LPS‐induced inflammation and tissue injury

Lipopolysaccharide (LPS) is a major microbial mediator for tissue injury and sepsis resulting from Gram‐negative bacterial infection. LPS is an external factor that induces robust expression of serum amyloid A (SAA), a major constituent of the acute‐phase proteins, but the relationship between SAA expression and LPS‐induced tissue injury remains unclear. Here, we report that mice with inducible transgenic expression of human SAA1 are partially protected against inflammatory response and lung injury caused by LPS and cecal ligation and puncture (CLP). In comparison, transgenic SAA1 does not attenuate TNFα‐induced lung inflammation and injury. The SAA1 expression level correlates inversely with the endotoxin concentrations in serum and lung tissues since SAA1 binds directly to LPS to form a complex that promotes LPS uptake by macrophages. Disruption of the SAA1‐LPS interaction with a SAA1‐derived peptide partially reduces the protective effect and exacerbates inflammation. These findings demonstrate that acute‐phase SAA provides innate feedback protection against LPS‐induced inflammation and tissue injury.     

MCTR1 enhances the resolution of lipopolysaccharide‐induced lung injury through STAT6‐mediated resident M2 alveolar macrophage polarization in mice

Acute respiratory distress syndrome (ARDS) is a fatal disease characterized by excessive infiltration of inflammatory cells. MCTR1 is an endogenously pro‐resolution lipid mediator. We tested the hypothesis that MCTR1 accelerates inflammation resolution through resident M2 alveolar macrophage polarization. The mice received MCTR1 via intraperitoneal administration 3 days after LPS stimulation, and then, the bronchoalveolar lavage (BAL) fluid was collected 24 hours later to measure the neutrophil numbers. Flow cytometry was used to sort the resident and recruited macrophages. Post‐treatment with MCTR1 offered dramatic benefits in the resolution phase of LPS‐induced lung injury, including decreased neutrophil numbers, reduced BAL fluid protein and albumin concentrations and reduced histological injury. In addition, the expression of the M2 markers Arg1, FIZZ1, Remlα, CD206 and Dectin‐1 was increased on resident macrophages in the LPS + MCTR1 group. Resident macrophage depletion abrogated the therapeutic effects of MCTR1, and reinjection of the sorted resident macrophages into the lung decreased neutrophil numbers. Finally, treatment with MCTR1 increased STAT6 phosphorylation. The STAT6 inhibitor AS1517499 abolished the beneficial effects of MCTR1. In conclusion, MCTR1 promotes resident M2 alveolar macrophage polarization via the STAT6 pathway to accelerate resolution of LPS‐induced lung injury.     

Regulation of leukocyte adhesion to heart by the tripeptides feG and feG(NH2)

The role of the D-isomeric form of the salivary gland tripeptide FEG (feG) and its carboxyl-amidated derivative, feG(NH2), in regulating leukocyte adherence to nonfixed atrial slices from Sprague-Dawley rats was examined under static conditions. Optimal binding of the leukocytes was seen if the leukocytes were treated with platelet activating factor (PAF; 10(-9)M). The increased adherence of PAF-treated peripheral blood leukocytes was totally inhibited by both feG and feG(NH2) (10-9M), as well as by antibodies against CD18 and CD49d. In contrast, the binding of peritoneal leukocytes was blocked only by CD49d antibody. Circulating leukocytes obtained from lipopolysaccharide (LPS) treated (2 mg/kg ip) rats did not bind to atrial slices obtained from normal hearts, but readily bound to atrial slices obtained from LPS-treated rats. This leukocyte binding was inhibited by in vivo feG treatment (100 microg/kg ip, 24 h before harvest) or by treating the isolated cells with feG (10(-9)M). The amidated peptide feG(NH2) reduced neutrophil accumulation in the atrium elicited by ip injection of LPS, whereas feG was ineffective. The reduction in neutrophil infiltration into the myocardium by feG(NH2) and the prevention of leukocyte interaction with myocytes seen with both feG and feG(NH2) probably results in hindered leukocyte migration in the inflamed heart, resulting in less tissue damage. The inhibition by these tripeptides on neutrophil adhesion to myocytes suggests that salivary glands hormones regulate the severity of cardiac inflammation.     

Internalization of Tissue Factor‐Rich Microvesicles by Platelets Occurs Independently of GPIIb‐IIIa,and Involves CD36 Receptor,Serotonin Transporter and Cytoskeletal Assembly

Platelets are important in hemostasis, but also detect particles and pathogens in the circulation. Phagocytic and endocytic activities of platelets are widely recognized; however, receptors and mechanisms involved remain poorly understood. We previously demonstrated that platelets internalize and store phospholipid microvesicles enriched in human tissue factor (TF+MVs) and that platelet‐associated TF enhances thrombus formation at sites of vascular damage. Here, we investigate the mechanisms implied in the interactions of TF+MVs with platelets and the effects of specific inhibitory strategies. Aggregometry and electron microscopy were used to assess platelet activation and TF+MVs uptake. Cytoskeletal assembly and activation of phosphoinositide 3‐kinase (PI3K) and RhoA were analyzed by western blot and ELISA. Exposure of platelets to TF+MVs caused reversible platelet aggregation, actin polymerization and association of contractile proteins to the cytoskeleton being maximal at 1 min. The same kinetics were observed for activation of PI3K and translocation of RhoA to the cytoskeleton. Inhibitory strategies to block glycoprotein IIb‐IIIa (GPIIb‐IIIa), scavenger receptor CD36, serotonin transporter (SERT) and PI3K, fully prevented platelet aggregation by TF+MVs. Ultrastructural techniques revealed that uptake of TF+MVs was efficiently prevented by anti‐CD36 and SERT inhibitor, but only moderately interfered by GPIIb‐IIIa blockade. We conclude that internalization of TF+MVs by platelets occurs independently of receptors related to their main hemostatic function (GPIIb‐IIIa), involves the scavenger receptor CD36, SERT and engages PI3‐Kinase activation and cytoskeletal assembly. CD36 and SERT appear as potential therapeutic targets to interfere with the association of TF+MVs with platelets and possibly downregulate their prothrombotic phenotype. J. Cell. Biochem. 117: 448–457, 2016. © 2015 Wiley Periodicals, Inc.     

Valsartan independent of AT1 receptor inhibits tissue factor,TLR‐2 and ‐4 expression by regulation of Egr‐1 through activation of AMPK in diabetic conditions

Patients suffering from diabetes mellitus (DM) are at a severe risk of atherothrombosis. Early growth response (Egr)‐1 is well characterized as a central mediator in vascular pathophysiology. We tested whether valsartan independent of Ang II type 1 receptor (AT₁R) can reduce tissue factor (TF) and toll‐like receptor (TLR)‐2 and ‐4 by regulating Egr‐1 in THP‐1 cells and aorta in streptozotocin‐induced diabetic mice. High glucose (HG, 15 mM) increased expressions of Egr‐1, TF, TLR‐2 and ‐4 which were significantly reduced by valsartan. HG increased Egr‐1 expression by activation of PKC and ERK1/2 in THP‐1 cells. Valsartan increased AMPK phosphorylation in a concentration and time‐dependent manner via activation of LKB1. Valsartan inhibited Egr‐1 without activation of PKC or ERK1/2. The reduced expression of Egr‐1 by valsartan was reversed by either silencing Egr‐1, or compound C, or DN‐AMPK‐transfected cells. Valsartan inhibited binding of NF‐κB and Egr‐1 to TF promoter in HG condition. Furthermore, valsartan reduced inflammatory cytokine (TNF‐α, IL‐6 and IL‐1β) production and NF‐κB activity in HG‐activated THP‐1 cells. Interestingly, these effects of valsartan were not affected by either silencing AT₁R in THP‐1 cells or CHO cells, which were devoid of AT₁R. Importantly, administration of valsartan (20 mg/kg, i.p) for 8 weeks significantly reduced plasma TF activity, expression of Egr‐1, TLR‐2, ‐4 and TF in thoracic aorta and improved glucose tolerance of streptozotocin‐induced diabetic mice. Taken together, we concluded that valsartan may reduce atherothrombosis in diabetic conditions through AMPK/Egr‐1 regulation.     

Clopidogrel, a P2Y12 receptor antagonist, potentiates the inflammatory response in a rat model of peptidoglycan polysaccharide-induced arthritis

The P2Y12 receptor plays a crucial role in the regulation of platelet activation by several agonists, which is irreversibly antagonized by the active metabolite of clopidogrel, a widely used anti-thrombotic drug. In this study, we investigated whether reduction of platelet reactivity leads to reduced inflammatory responses using a rat model of erosive arthritis. We evaluated the effect of clopidogrel on inflammation in Lewis rats in a peptidoglycan polysaccharide (PG-PS)-induced arthritis model with four groups of rats: 1) untreated, 2) clopidogrel-treated, 3) PG-PS-induced, and 4) PG-PS-induced and clopidogrel-treated. There were significant differences between the PG-PS+clopidogrel group when compared to the PG-PS group including: increased joint diameter and clinical manifestations of inflammation, elevated plasma levels of pro-inflammatory cytokines (IL-1 beta, interferon (IFN) gamma, and IL-6), an elevated neutrophil blood count and an increased circulating platelet count. Plasma levels of IL-10 were significantly lower in the PG-PS+clopidogrel group compared to the PG-PS group. Plasma levels of platelet factor 4 (PF4) were elevated in both the PG-PS and the PG-PS+clopidogrel groups, however PF4 levels showed no difference upon clopidogrel treatment, suggesting that the pro- inflammatory effect of clopidogrel may be due to its action on cells other than platelets. Histology indicated an increase in leukocyte infiltration at the inflammatory area of the joint, increased pannus formation, blood vessel proliferation, subsynovial fibrosis and cartilage erosion upon treatment with clopidogrel in PG-PS-induced arthritis animals. In summary, animals treated with clopidogrel showed a pro-inflammatory effect in the PG-PS-induced arthritis animal model, which might not be mediated by platelets. Elucidation of the mechanism of clopidogrel-induced cell responses is important to understand the role of the P2Y12 receptor in inflammation.     

In silico and in vivo approach to elucidate the inflammatory complexity of CD14-deficient mice

The inflammatory phenotype of genetically modified mice is complex, and the role of Gram-negative lipopolysaccharide (LPS) in acute inflammation induced by surgical cannulation trauma, alone or in combination with hemorrhage and resuscitation ("hemorrhagic shock"), is both complex and controversial. We sought to determine if a mathematical model of acute inflammation could elucidate both the phenotype of CD14-deficient (CD14(-/-)) mice--following LPS, cannulation, or hemorrhagic shock--and the role of LPS in trauma/hemorrhage-induced inflammation. A mathematical model of inflammation initially calibrated in wild-type (C57Bl/6) mice subjected to LPS, cannulation, and hemorrhagic shock was recalibrated in CD14(-/-) mice subjected to the same insults, yielding an ensemble of models that suggested specific differences at the cellular and molecular levels (for example, 43-fold lower activation of leukocytes by LPS). The CD14(-/-)-specific model ensemble could account for complex changes in inflammatory analytes in these mice following LPS treatment. Model prediction of similar organ damage in CD14(-/-) and wild-type mice subjected to cannulation alone or with hemorrhagic shock was verified in vivo (similar ALT levels). These studies suggest that LPS-CD14 responses do not cause inflammation in surgical trauma/hemorrhagic shock and demonstrate a novel use of combined in silico and in vivo methods to elucidate the complex inflammatory phenotype of genetically modified animals.     

I. Suppression by compound 48/80 of bacterial endotoxin-inducible monocytic tissue factor activity: direct blockade of factor VII binding to THP-1 monocytes

Hypercoagulation with upregulated monocytic tissue factor (TF) activity often occurs under a variety of inflammatory conditions including endotoxemia. The antagonism to bacterial endotoxin (LPS) signaling often results in the depression in TF upregulation. We herein report that compound 48/80 (48/80) significantly depressed LPS-induced TF activity in human and cebus monkey peripheral blood monocytes. Employing a model monocyte-like cell line (THP-1), we explored the regulatory mechanism to identify the inhibitory site(s) of 48/80. We determine whether the inhibition results from the blockade of LPS signaling. 48/80 dose-dependently inhibited LPS-induced TF activity. Chase of LPS-challenged cells with 48/80 also significantly offset TF upregulation. In immunofluorescent approaches, FACScan analysis revealed that 48/80 had no effect on either LPS recognition or the expression of its receptors (CD14 and CD11b). Moreover, LPS-induced TF expression as well as synthesis remained unaffected in the presence of 48/80. Consistent with the independence of LPS action, 48/80 was also able to inhibit TF activity induced by A23187, ionomycin, or Quin-2 AM. Interestingly, 48/80 significantly decreased the FVII binding to either resting or LPS-challenged cells. In conclusion, our results elucidate that the inhibitory action of 48/80 was independent of LPS signaling including recognition, receptor expression, and the induced TF expression/ synthesis. However, 48/80 was able to directly block FVII binding to monocytic TF, thereby resulting in such antagonism to LPS-induced TF-initiated extrinsic coagulation.     

CD44 is a negative regulator of acute pulmonary inflammation and lipopolysaccharide-TLR signaling in mouse macrophages

CD44 is a transmembrane adhesion molecule and hemopoietic CD44 has an essential role in hyaluronan clearance and resolution of noninfectious lung injury. In this study, we examined the role of CD44 in acute pulmonary inflammation and in the regulation of LPS-TLR signaling. Following intratracheally LPS treatment, CD44(-/-) mice demonstrated an exaggerated inflammatory response characterized by increased inflammatory cell recruitment, elevated chemokine expression in bronchoalveolar lavage fluid, and a marked increase in NF-kappaB DNA-binding activity in lung tissue in vivo and in macrophages in vitro. Furthermore, CD44(-/-) mice were more susceptible to LPS-induced shock. Reconstitution of hemopoietic CD44 reversed the inflammatory phenotype. We further found that the induction of the negative regulators of TLR signaling IL-1R-associated kinase-M, Toll-interacting protein, and A20 by intratracheal LPS in vivo and in macrophages in vitro was significantly reduced in CD44(-/-) mice. Collectively, these data suggest CD44 plays a previously unrecognized role in preventing exaggerated inflammatory responses to LPS by promoting the expression of negative regulators of TLR-4 signaling.     

Cooperative role of endogenous leucotrienes and platelet‐activating factor in ischaemia–reperfusion‐mediated tissue injury

Insufficient oxygen delivery to organs leads to tissue dysfunction and cell death. Reperfusion, although vital to organ survival, initiates an inflammatory response that may both aggravate local tissue injury and elicit remote organ damage. Polymorphonuclear neutrophil (PMN) trafficking to remote organs following ischaemia/reperfusion (I/R) is associated with the release of lipid mediators, including leucotriene (LT) B₄, cysteinyl‐LTs (CysLTs) and platelet‐activating factor (PAF). Yet, their potentially cooperative role in regulating I/R‐mediated inflammation has not been thoroughly assessed. The present study aimed to determine the cooperative role of lipid mediators in regulating PMN migration, tissue oedema and injury using selective receptor antagonists in selected models of I/R and dermal inflammation. Our results show that rabbits, pre‐treated orally with BIIL 284 and/or WEB 2086 and MK‐0571, were protected from remote tissue injury following I/R or dermal inflammation in an additive or synergistic manner when the animals were pre‐treated with two drugs concomitantly. The functional selectivity of the antagonists towards their respective agonists was assessed in vitro, showing that neither BIIL 284 nor WEB 2086 prevented the inflammatory response to IL‐8, C5a and zymosan‐activated plasma stimulation. However, these agonists elicited LTB₄ biosynthesis in isolated rabbit PMNs. Similarly, a cardioprotective effect of PAF and LTB₄ receptor antagonists was shown following myocardial I/R in mice. Taken together, these results underscore the intricate involvement of LTB₄ and PAF in each other's responses and provide further evidence that targeting both LTs and PAF receptors provides a much stronger anti‐inflammatory effect, regulating PMN migration and oedema formation.