Enhanced susceptibility of staggerer (RORalphasg/sg) mice to lipopolysaccharide-induced lung inflammation

The retinoid-related orphan receptor alpha (RORalpha), a member of the ROR subfamily of nuclear receptors, has been implicated in the control of a number of physiological processes, including the regulation of several immune functions. To study the potential role of RORalpha in the regulation of innate immune responses in vivo, we analyzed the induction of airway inflammation in response to lipopolysaccharide (LPS) challenge in wild-type and staggerer (RORalpha(sg/sg)) mice, a natural mutant strain lacking RORalpha expression. Examination of hematoxylin and eosin-stained lung sections showed that RORalpha(sg/sg) mice displayed a higher degree of LPS-induced inflammation than wild-type mice. Bronchoalveolar lavage (BAL) was performed at 3, 16, and 24 h after LPS exposure to monitor the increase in inflammatory cells and the level of several cytokines/chemokines. The increased susceptibility of RORalpha(sg/sg) mice to LPS-induced airway inflammation correlated with a higher number of total cells and neutrophils in BAL fluids from LPS-treated RORalpha(sg/sg) mice compared with those from LPS-treated wild-type mice. In addition, IL-1beta, IL-6, and macrophage inflammatory protein-2 were appreciably more elevated in BAL fluids from LPS-treated RORalpha(sg/sg) mice compared with those from LPS-treated wild-type mice. The enhanced susceptibility of RORalpha(sg/sg) mice appeared not to be due to a repression of IkappaBalpha expression. Our observations indicate that RORalpha(sg/sg) mice are more susceptible to LPS-induced airway inflammation and are in agreement with the hypothesis that RORalpha functions as a negative regulator of LPS-induced inflammatory responses.     

Inhaled nitric oxide attenuates acute lung injury via inhibition of nuclear factor-kappa B and inflammation.

The effect of inhaled nitric oxide (NO) on inflammatory process in acute lung injury (ALI) is unclear. The aims of this study were to 1) examine whether inhaled NO affects the biochemical lung injury parameters and cellular inflammatory responses and 2) determine the effect of inhaled NO on the activation of nuclear factor-kappa B (NF-kappa B) in lipopolysaccharide (LPS)-induced ALI. Compared with saline controls, rabbits treated intravenously with LPS showed increases in total protein and lactate dehydrogenase in the bronchoalveolar lavage (BAL) fluid, indicating ALI. LPS-treated animals with NO inhalation (LPS-NO) showed significant decreases in these parameters. Neutrophil numbers in the BAL fluid, the activity of reactive oxygen species in BAL cells, and the levels of interleukin (IL)-1 beta and IL-8 in alveolar macrophages were increased in LPS-treated animals. In contrast, neutrophil numbers and these cellular activities were substantially decreased in LPS-NO animals, compared with LPS-treated animals. NF-kappa B activation in alveolar macrophages from LPS-treated animals was also markedly increased, whereas this activity was effectively blocked in LPS-NO animals. These results suggest that inhaled NO attenuates LPS-induced ALI and pulmonary inflammation. This attenuation may be associated with the inhibition of NF-kappa B activation.     

LPS-induced neutrophilic inflammation and Bcl-2 expression in metaplastic mucous cells

Our previous studies show that Bcl-2, a regulator of apoptosis, may be involved in the reduction of mucous cell metaplasia (MCM) during recovery from inflammatory responses. The present study was to determine whether neutrophilic inflammation mediates Bcl-2 expression in mucous cells. Rats were intratracheally instilled with 50-1000 microg of LPS. The number of neutrophils recovered by bronchoalveolar lavage (BAL) increased with the dose of LPS, and the percentage of Bcl-2-expressing cells increased with the numbers of neutrophils in the BAL. Depletion of neutrophils did not reduce MCM, but the percentage of Bcl-2-positive cells increased 1.8-fold in neutrophil-depleted compared with controls. Injection of rats with bezafibrate, an inducer of cytochrome P-450, doubled the number of neutrophils in the BAL, decreased MCM twofold compared with vehicle-injected controls, and reduced Bcl-2 expression. Bcl-2 mRNA levels decreased in a tracheal epithelial cell line treated with bezafibrate. These data demonstrate that Bcl-2 expression is independent of the number of neutrophils in the BAL and that bezafibrate may directly reduce Bcl-2 expression in epithelial cells.     

NAC Attenuates LPS-Induced Toxicity in Aspirin-Sensitized Mouse Macrophages via Suppression of Oxidative Stress and Mitochondrial Dysfunction

Bacterial endotoxin lipopolysaccharide (LPS) induces the production of inflammatory cytokines and reactive oxygen species (ROS) under in vivo and in vitro conditions. Acetylsalicylic acid (ASA, aspirin) is a commonly used anti-inflammatory drug. Our aim was to study the effects of N-acetyl cysteine (NAC), an antioxidant precursor of GSH synthesis, on aspirin-sensitized macrophages treated with LPS. We investigated the effects of LPS alone and in conjunction with a sub-toxic concentration of ASA, on metabolic and oxidative stress, apoptosis, and mitochondrial function using J774.2 mouse macrophage cell line. Protection from LPS-induced toxicity by NAC was also studied. LPS alone markedly induced ROS production and oxidative stress in macrophage cells. When ASA was added to LPS-treated macrophages, the increase in oxidative stress was significantly higher than that with LPS alone. Similarly, alteration in glutathione-dependent redox metabolism was also observed in macrophages after treatment with LPS and ASA. The combination of LPS and ASA selectively altered the CYP 3A4, CYP 2E1 and CYP 1A1 catalytic activities. Mitochondrial respiratory complexes and ATP production were also inhibited by LPS-ASA treatment. Furthermore a higher apoptotic cell death was also observed in LPS-ASA treated macrophages. NAC pre-treatment showed protection against oxidative stress induced apoptosis and mitochondrial dysfunction. These effects are presumed, at least in part, to be associated with alterations in NF-κB/Nrf-2 mediated cell signaling. These results suggest that macrophages are more sensitive to LPS when challenged with ASA and that NAC pre-treatment protects the macrophages from these deleterious effects.     

Identification with MRI of the pleura as a major site of the acute inflammatory effects induced by ovalbumin and endotoxin challenge in the airways of the rat

Inflammatory effects in the rat lung have been investigated, non-invasively by MRI, at early time points (3 and 6 h) after ovalbumin (OA) or endotoxin (LPS) challenges. Six hours after challenge with OA, a strong, even inflammatory signal was present around the periphery of the lung in a region corresponding to the pleura. Histological analysis confirmed the presence of marked edema associated with the pleural cavity of OA-treated animals. Lower levels of pleural edema were observed in MRI and histological evaluation of LPS-treated animals and no abnormality was observed in actively sensitized and na?ve, saline-treated groups. Diffuse edematous signals were detected in the lung 3 and 6 h after challenge with OA or LPS; the signal volumes were larger at both time points following OA instillation. Bronchoalveolar lavage (BAL) fluid analysis performed 6 h after challenge revealed increased levels of protein and greater cellular activation in OA- than in LPS-treated animals. Furthermore, increased levels of peribronchial edema were found by histology 6 h after OA. BAL fluid and histological assessments demonstrated that the inflammatory signals were due to edema and not mucus as no significant changes in BAL mucin concentrations or differences in goblet cells were identified between OA or LPS challenge and their respective vehicle groups. Our data show that MRI is able to detect, non-invasively, inflammatory signals in both the lung and the pleura in spontaneously breathing animals, highlighting its potential to study the consequences of pulmonary insults on both sites.     

Transient neutrophil infiltration after allergen challenge is dependent on specific antibodies and Fc gamma III receptors

Following allergen challenge of sensitized mice, neutrophils are the first inflammatory cells found in bronchoalveolar lavage (BAL) fluid. To determine the underlying mechanism for their accumulation, mice were sensitized to OVA on days 0 and 14, and received, on day 28, a single intranasal challenge (s.i.n.) with either OVA or ragweed. Eight hours after the s.i.n., BAL fluid was obtained. BALB/c mice sensitized and challenged with OVA showed significantly higher total cell counts and numbers of neutrophils in BAL fluid compared to the OVA-sensitized and ragweed-challenged or nonsensitized mice. Levels of neutrophil chemokines in BAL fluid supernatants were markedly elevated in the sensitized and OVA-challenged mice; Fc epsilon RI-deficient mice showed comparable numbers of neutrophils and neutrophil chemokines in BAL fluid after s.i.n. But in sensitized mice lacking the Fc common gamma-chain and B cell-deficient mice, the number of neutrophils and levels of neutrophil chemokines in BAL fluid were significantly lower. Further, mice lacking the FcgammaRIII did not develop this early neutrophil influx. Neutrophil infiltration could be induced in naive mice following intranasal instillation of allergen combined with allergen-specific IgG1. In addition, macrophages from sensitized mice were stimulated with allergen and activated to produce neutrophil chemokines. These results demonstrate that neutrophil influx after allergen challenge requires prior sensitization, is allergen-specific, is mediated through FcgammaRIII, and is dependent on the presence of Ab.     

Effects of certain inducers of leukocytes migration into the bovine mammary gland on neutrophil apoptosis manifestation in a subsequent in vitro cultivation

The aim of the study was to elucidate the effects of induced leukocyte migration into the bovine mammary gland on the manifestations of early and late apoptotic features of neutrophils cultivated in vitro. The Latin square design was used in two experiments, each involving four experimental repetitions in 4 clinically healthy virgin heifers. The neutrophil early apoptotic features were detected by flow cytometric detection (FCM) of phosphatidyl-serine translocation. Late neutrophil apoptotic features were detected by ELISA quantitation of histone-complexed DNA fragments. Leukocyte influx induction was accomplished by using four inducers: i) sterile buffered saline solution (PBS); ii) 5 % glucose solution (GLU); iii) synthetic muramyl dipeptide analogue (MDP); and iv) lipopolysaccharide (LPS), administered into the mammary gland lumen. Leukocytes from mammary glands were obtained by mammary gland lumen lavages after influx induction. The total cell counts in lavages increased after treatment by all inducers in comparison to the counts before influx induction (P<0.001). Cell counts were higher and differed significantly by MDP and LPS (P<0.01) in contrast to PBS. The highest proportion of neutrophils was induced by LPS (P<0.01). After three-hour incubation, light microscopy examination revealed the highest manifestation of neutrophil apoptosis after induction by GLU (P<0.05). The lowest apoptosis manifestation, though statistically non-significant, was detected after induction by MDP and LPS. Determination of early manifestation of neutrophil apoptosis revealed the lowest manifestation of neutrophil apoptosis after induction by LPS (P<0.01). The results of late manifestation of neutrophil apoptosis revealed the highest proportion of apoptotic neutrophils after induction by GLU (P<0.05). The manifestation of secondary necrosis of apoptotic neutrophils or neutrophil lysis after 3 h of incubation was low and not significant. In conclusion, certain inducers of neutrophil migration into the lumen of bovine mammary glands (GLU and LPS in the present experiments) significantly influence the manifestation of neutrophil apoptosis during their subsequent in vitro incubation.     

Cellular and molecular mechanisms underlying LPS-associated myocyte impairment

Recently we reported that Toll-like receptor 4 (TLR4)-positive immune cells of unknown identity were responsible for the LPS-induced depression of cardiac myocyte shortening. The aim of this study is to identify the TLR4-positive cell type that is responsible for the LPS-induced cardiac dysfunction. Neither neutrophil depletion alone nor mast cell deficiency had any impact on the impairment of myocyte shortening during LPS treatment. In contrast, LPS-treated, macrophage-deficient mice demonstrated a partial reduction in shortening compared with saline-treated, macrophage-deficient mice. Because the removal of macrophages could only partially restore myocyte shortening, we also investigated the effects of removing both neutrophils and macrophages on myocyte shortening. Interestingly, endotoxemic, neutrophil-depleted, and macrophage-deficient mice had completely restored myocyte shortening. Because both macrophages and neutrophils can produce nitric oxide (NO) and TNF-alpha, we examined LPS-treated inducible NO synthase knockout (iNOSKO) mice and TNF receptor (TNFR)-deficient mice. Eliminating both TNFR1 and TNFR2 was required to restore myocyte shortening during LPS treatment, whereas iNOS deficiency had no effect. These data suggest that macrophages and to a lesser degree neutrophils cause cardiac impairment, presumably via TNF-alpha.     

Eotaxin represents the principal eosinophil chemoattractant in a novel murine asthma model induced by house dust containing cockroach allergens

Asthma represents a serious health problem particularly for inner city children, and recent studies have identified that cockroach allergens trigger many of these asthmatic attacks. This study tested the concept that asthma-like pulmonary inflammation may be induced by house dust containing cockroach allergens. An aqueous extract was prepared from a house dust sample containing endotoxin and high levels of cockroach allergens. BALB/c mice were immunized with the house dust extract (HDE) and received two additional pulmonary challenges. Bronchoalveolar lavage (BAL) eosinophil counts and eotaxin levels were significantly increased in immunized mice exposed to the HDE, whereas neutrophils were the predominant BAL inflammatory cell in the unimmunized mice. Kinetics studies in immunized mice demonstrated a peak pulmonary inflammatory response 48 h after the last challenge. The allergic response in this model was further confirmed by histological and physiological studies demonstrating a significant influx of eosinophils and lymphocytes in the peribronchial area, and severe airway hyperreactivity through whole-body plethysmography. The specificity of the response was established by immunizing with HDE and challenging with purified cockroach allergen, which induced pulmonary eosinophilia and airway hyperreactivity. Ab inhibition of eotaxin significantly inhibited the number of BAL eosinophils. These data describe a novel murine model of asthma-like pulmonary inflammation induced by house dust containing endotoxin and cockroach allergens and further demonstrate that eotaxin represents the principal chemoattractant for the recruitment of the pulmonary eosinophils.     

Neutrophils recruited to the site of Mycobacterium bovis BCG infection undergo apoptosis and modulate lipid body biogenesis and prostaglandin E production by macrophages

Neutrophil influx to sites of mycobacterial infections is one of the first events of tuberculosis pathogenesis. However, the role of early neutrophil recruitment in mycobacterial infection is not completely understood. We investigated the rate of neutrophil apoptosis and the role of macrophage uptake of apoptotic neutrophils in a pleural tuberculosis model induced by BCG. Recruited neutrophils were shown to phagocyte BCG and a large number of neutrophils undergo apoptosis within 24 h. Notably, the great majority of apoptotic neutrophils were infected by BCG. Increased lipid body (lipid droplets) formation, accompanied by prostaglandin E(2) (PGE(2)) and TGF-beta1 synthesis, occurred in parallel to macrophage uptake of apoptotic cells. Lipid body and PGE(2) formation was observed after macrophage exposure to apoptotic, but not necrotic or live neutrophils. Blockage of BCG-induced lipid body formation significantly inhibited PGE(2) synthesis. Pre-treatment with the pan-caspase inhibitor zVAD inhibited BCG-induced neutrophil apoptosis and lipid body formation, indicating a role for apoptotic neutrophils in macrophage lipid body biogenesis in infected mice. In conclusion, BCG infection induced activation and apoptosis of infected neutrophils at the inflammatory site. The uptake of apoptotic neutrophils by macrophages leads to TGF-beta1 generation and PGE(2)-derived lipid body formation, and may have modulator roles in mycobacterial pathogenesis.     

Lung-restricted macrophage activation in the pearl mouse model of Hermansky-Pudlak syndrome

Pulmonary inflammation, abnormalities in alveolar type II cell and macrophage morphology, and pulmonary fibrosis are features of Hermansky-Pudlak Syndrome (HPS). We used the naturally occurring "pearl" HPS2 mouse model to investigate the mechanisms of lung inflammation observed in HPS. Although baseline bronchoalveolar lavage (BAL) cell counts and differentials were similar in pearl and strain-matched wild-type (WT) mice, elevated levels of proinflammatory (MIP1gamma) and counterregulatory (IL-12p40, soluble TNFr1/2) factors, but not TNF-alpha, were detected in BAL from pearl mice. After intranasal LPS challenge, BAL levels of TNF-alpha, MIP1alpha, KC, and MCP-1 were 2- to 3-fold greater in pearl than WT mice. At baseline, cultured pearl alveolar macrophages (AMs) had markedly increased production of inflammatory cytokines. Furthermore, pearl AMs had exaggerated TNF-alpha responses to TLR4, TLR2, and TLR3 ligands, as well as increased IFN-gamma/LPS-induced NO production. After 24 h in culture, pearl AM LPS responses reverted to WT levels, and pearl AMs were appropriately refractory to continuous LPS exposure. In contrast, cultured pearl peritoneal macrophages and peripheral blood monocytes did not produce TNF-alpha at baseline and had LPS responses which were no different from WT controls. Exposure of WT AMs to heat- and protease-labile components of pearl BAL, but not WT BAL, resulted in robust TNF-alpha secretion. Similar abnormalities were identified in AMs and BAL from another HPS model, pale ear HPS1 mice. We conclude that the lungs of HPS mice exhibit hyperresponsiveness to LPS and constitutive and organ-specific macrophage activation.     

Vanadium-induced apoptosis and pulmonary inflammation in mice: Role of reactive oxygen species

Pulmonary exposure to metals and metal-containing compounds is associated with pulmonary inflammation, cell death, and tissue injury. The present study uses a mouse model to investigate vanadium-induced apoptosis and lung inflammation, and the role of reactive oxygen species (ROS) in this process. Aspiration of the pentavalent form of vanadium, V (V), caused a rapid influx of polymorphonuclear leukocytes into the pulmonary airspace with a peak inflammatory response at 6 h post-exposure and resolution by 72 h. During this period, the number of apoptotic lung cells which were predominantly neutrophils increased considerably with a peak response at 24 h accompanied by no or minimum necrosis. After 24 h when the V (V)-induced inflammation was in the resolution phase, an increased influx of macrophages and engulfment of apoptotic bodies by these phagocytes was observed, supporting the role of macrophages in apoptotic cell clearance and resolution of V (V)-induced lung inflammation. Electron spin resonance (ESR) studies using lavaged alveolar macrophages showed the formation of ROS, including O(2)(*-), H(2)O(2), and (*)OH radicals which were confirmed by inhibition with free radical scavengers. The mechanism of ROS generation induced by V (V) involved the activation of an NADPH oxidase complex and the mitochondrial electron transport chain. The ROS scavenger, catalase (H(2)O(2) scavenger), effectively inhibited both lung cell apoptosis and the inflammatory response, whereas superoxide dismutase (SOD) (O(2)(*-) scavenger) and the metal chelator, deferoxamine (inhibitor of (*)OH generation by Fenton-like reactions) had lesser effects. These results indicate that multiple oxidative species are involved in V (V)-induced lung inflammation and apoptosis, and that H(2)O(2) plays a major role in this process.     

The presence of LPS in OVA inhalations affects airway inflammation and AHR but not remodeling in a rodent model of asthma

Ovalbumin (OVA) is the most frequently used allergen in animal models of asthma. Lipopolysaccharide (LPS) contaminating commercial OVA may modulate the evoked airway inflammatory response to OVA. However, the effect of LPS in OVA on airway remodeling, especially airway smooth muscle (ASM) has not been evaluated. We hypothesized that LPS in commercial OVA may enhance allergen-induced airway inflammation and remodeling. Brown Norway rats were sensitized with OVA on day 0. PBS, OVA, or endotoxin-free OVA (Ef-OVA) was instilled intratracheally on days 14, 19, 24. Bronchoalveolar lavage (BAL) fluid, lung, and intrathoracic lymph node tissues were collected 48 h after the last challenge. Immunohistochemistry for α-smooth muscle actin, Periodic-Acid-Schiff staining, and real-time qPCR were performed. Airway hyperresponsiveness (AHR) was also measured. BAL fluid macrophages, eosinophils, neutrophils, and lymphocytes were increased in OVA-challenged animals, and macrophages and neutrophils were significantly lower in Ef-OVA-challenged animals. The ASM area in larger airways was significantly increased in both OVA and Ef-OVA compared with PBS-challenged animals. The mRNA expression of IFN-γ and IL-13 in lung tissues and IL-4 in lymph nodes was significantly increased by both OVA and Ef-OVA compared with PBS and were not significantly different between OVA and Ef-OVA. Monocyte chemoattractant protein (MCP)-1 in BAL fluid and AHR were significantly increased in OVA but not in Ef-OVA. LPS contamination in OVA contributes to the influx of macrophages and MCP-1 increase in the airways and to AHR after OVA challenges but does not affect OVA-induced Th1 and Th2 cytokine expression, goblet cell hyperplasia, and ASM remodeling.     

Peptidylprolyl cis/trans isomerase,NIMA-interacting 1 (PIN1) regulates pulmonary effects of endotoxin and tumor necrosis factor-α in mice

Peptidylprolyl cis/trans isomerase, NIMA-interacting 1 (PIN1) modulates phospho-signaling by catalyzing rotation of the bond between a phosphorylated serine or threonine before proline in proteins. As depletion of PIN1 increased inflammatory protein expression in cultured endothelial cells treated with bacterial endotoxin (lipopolysaccharide, LPS) and interferon-γ, we hypothesized that PIN1 knockout would increase sensitivity to LPS-induced lung inflammation in mice. Mortality due to a high dose of LPS (30 mg/kg) was greater in knockout than wildtype mice. Lung myeloperoxidase activity, reflecting neutrophils, was increased to a 35% higher level in PIN1 knockout mouse lung, as compared with wildtype, after treatment with a sublethal dose of 3 mg LPS/kg, ip. Unexpectedly, plasma tumor necrosis factor-α (TNF) was approximately 50% less than in wildtype mice. Knockout mice, however, were more sensitive than wildtype to TNF-induced neutrophil accumulation. The neutrophil adhesion molecule, E-selectin, was also elevated in lungs of knockout mice treated with TNF, suggesting that PIN1 depletion increases endothelial sensitivity to TNF. Indeed, TNF induced more reactive oxygen species in cultured endothelial cells depleted of PIN1 with short hairpin RNA than in control cells. Collectively, the results indicate that while PIN1 normally facilitates TNF production in LPS-treated mice, it suppresses pulmonary and endothelial reactions to the cytokine. Tissue or cell-specific effects of PIN1 may affect the overall inflammatory response to LPS and other stimuli.     

Chemokine receptor 5 blockade modulates macrophage trafficking in renal ischaemic-reperfusion injury

Chemokine receptor 5 (CCR5) is a pivotal regulator of macrophage trafficking in the kidneys in response to an inflammatory cascade. We investigated the role of CCR5 in experimental ischaemic-reperfusion injury (IRI) pathogenesis. To establish IRI, we clamped the bilateral renal artery pedicle for 30 min and then reperfused the kidney. We performed adoptive transfer of lipopolysaccharide (LPS)-treated RAW 264.7 macrophages following macrophage depletion in mice. B6.CCR5^−/− mice showed less severe IRI based on tubular epithelial cell apoptosis than did wild-type mice. CXCR3 expression in CD11b⁺ cells and inducible nitric oxide synthase levels were more attenuated in B6.CCR5^−/− mice. B6.CCR5^−/− mice showed increased arginase-1 and CD206 expression. Macrophage-depleted wild-type mice showed more injury than B6.CCR5^−/− mice after M1 macrophage transfer. Adoptive transfer of LPS-treated RAW 264.7 macrophages reversed the protection against IRI in wild-type, but not B6.CCR5^−/− mice. Upon knocking out CCR5 in macrophages, migration of bone marrow-derived macrophages from wild-type mice towards primary tubular epithelial cells with recombinant CCR5 increased. Phospho-CCR5 expression in renal tissues of patients with acute tubular necrosis was increased, showing a positive correlation with tubular inflammation. In conclusion, CCR5 deficiency favours M2 macrophage activation, and blocking CCR5 might aid in treating acute kidney injury.     

Inflammatory response of tracheobronchial epithelial cells to endotoxin

Respiratory epithelial cells play a crucial role in the inflammatory response in endotoxin-induced lung injury, an experimental model for acute lung injury. To determine the role of epithelial cells in the upper respiratory compartment in the inflammatory response to endotoxin, we exposed tracheobronchial epithelial cells (TBEC) to lipopolysaccharide (LPS). Expression of inflammatory mediators was analyzed, and the biological implications were assessed using chemotaxis and adherence assays. Epithelial cell necrosis and apoptosis were determined to identify LPS-induced cell damage. Treatment of TBEC with LPS induced enhanced protein expression of cytokines and chemokines (increases of 235-654%, P < 0.05), with increased chemotactic activity regarding neutrophil recruitment. Expression of the intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) was enhanced by 52-101% (P < 0.0001). This upregulation led to increased adhesion of neutrophils, with >95% adherence to TBEC after LPS stimulation, which could be blocked by either ICAM-1 (69%) or VCAM-1 antibodies (55%) (P < 0.05). Enhanced neutrophil-induced necrosis of TBEC was observed when TBEC were exposed to LPS. Reduced neutrophil adherence by ICAM-1 or VCAM-1 antibodies resulted in significantly lower TBEC death (52 and 34%, respectively, P < 0.05). Therefore, tight adherence of neutrophils to TBEC appears to promote epithelial cell killing. In addition to indirect effector cell-induced TBEC death, direct LPS-induced cell damage was seen with increased apoptosis rate in LPS-stimulated TBEC (36% increase of caspase-3, P < 0.01). These data provide evidence that LPS induces TBEC killing in a necrosis- and apoptosis-dependent manner.     

TLR-2 and TLR-9 are sensors of apoptosis in a mouse model of doxorubicin-induced acute inflammation

Anthracycline antibiotics are inducers of an immunogenic form of apoptosis that has immunostimulatory properties because of the release of damage-associated molecular patterns. To study the mechanisms used by the innate immune system to sense this immunogenic form of cell death, we established an in vivo model of cell death induced by intraperitoneal injection of doxorubicin, a prototype of anthracyclines. The acute sterile inflammation in this model is characterized by rapid influx of neutrophils and increased levels of IL-6 and monocyte chemotactic protein-1. We demonstrate that acute inflammation induced by doxorubicin is associated with apoptosis of monocytes/macrophages and that it is specific for doxorubicin, an immunogenic chemotherapeutic. Further, the inflammatory response is significantly reduced in mice deficient in myeloid differentiation primary response gene 88 (MyD88), TLR-2 or TLR-9. Importantly, a TLR-9 antagonist reduces the recruitment of neutrophils induced by doxorubicin. By contrast, the acute inflammatory response is not affected in TRIF(Lps2) mutant mice and in TLR-3, TLR-4 and caspase-1 knockout mice, which shows that the inflammasome does not have a major role in doxorubicin-induced acute inflammation. Our findings provide important new insights into how the innate immune system senses immunogenic apoptotic cells and clearly demonstrate that the TLR-2/TLR-9-MyD88 signaling pathways have a central role in initiating the acute inflammatory response to this immunogenic form of apoptosis.     

Secoisolariciresinol and isotaxiresinol inhibit tumor necrosis factor-alpha-dependent hepatic apoptosis in mice

The effects of secoisolariciresinol (1) and isotaxiresinol (2), two major lignans isolated from the wood of Taxus yunnanensis, on tumor necrosis factor-alpha (TNF-alpha)-dependent hepatic apoptosis induced by D-galactosamine (d-GalN)/lipopolysaccharide (LPS) were investigated in mice. Co-administration of d-GalN (700 mg/kg) and LPS (10 microg/kg) resulted in a typical hepatic apoptosis characterized by DNA fragmentation and the formation of apoptotic bodies. Serum glutamic pyruvic transaminase (sGPT) and glutamic oxaloacetic transaminase (sGOT) levels were also raised at 8 h after d-GalN/LPS intoxication due to a severe necrosis of hepatocytes. Pre-administration of 1 or 2 (50, 10 mg/kg, i.p.) 12 and 1 h before d-GalN/LPS significantly reduced DNA fragmentation and prevented chromatin condensation, apoptotic body formation and hepatitis. Pro-inflammatory cytokines such as TNF-alpha and interferon-gamma (IFN-gamma) secreted from LPS-activated macrophages are important mediators of hepatocyte apoptosis in this model. Pre-treatment with 1 or 2 significantly inhibited the elevation of serum TNF-alpha and IFN-gamma levels. In a separate experiment, both lignans had a significant dose-dependent protective effect on d-GalN/TNF-alpha-induced cell death in primary cultured mouse hepatocytes and TNF-alpha-mediated cell death in murine L929 fibrosarcoma cells. These results indicated that 1 and 2 prevent d-GalN/LPS-induced hepatic injury by inhibiting hepatocyte apoptosis through the blocking of TNF-alpha and IFN-gamma production by activated macrophages and direct inhibition of the apoptosis induced by TNF-alpha.