Ron receptor deficient alveolar myeloid cells exacerbate LPS-induced acute lung injury in the murine lung

Previous studies have shown that the Ron receptor tyrosine kinase is an important regulator of the acute lung inflammatory response induced by intranasal administration of bacterial LPS. Compared to wild-type mice, complete loss of the Ron receptor in all cell types in?vivo was associated with increased lung damage as determined by histological analyses and several markers of lung injury including increases in pro-inflammatory cytokines such as TNF-α. Tumor-necrosis factor-α is a multifunctional cytokine secreted by macrophages, which plays a major role in inflammation and is a central mediator of several disease states including rheumatoid arthritis and sepsis. Based on increased TNF-α production observed in the Ron-deficient mice, we hypothesized that Ron receptor function in the inflammatory cell compartment is essential for the regulating lung injury in?vivo. To test this hypothesis, we generated myeloid lineage-specific Ron-deficient mice. In this study, we report that loss of Ron signaling selectively in myeloid cells results in increased lung injury following intranasal administration of LPS as measured by increases in TNF-α production, ensuing neutrophil accumulation and increased lung histopathology. These findings corroborate the role of Ron receptor tyrosine kinase as a negative regulator of inflammation and further demonstrate the in?vivo significance of Ron signaling selectively in myeloid cells as a major regulator of this response in?vivo. These data authenticate Ron as a potential target in innate immunity and TNF-α-mediated pathologies.     

Cytokine-induced inflammatory liver injuries

IL-18 is a pleiotropic cytokine and is produced by various types of cells including activated macrophages, particularly Kupffer cells. IL-18 has potential to activate inflammatory responses through induction of IFN-gamma production in collaboration with IL-12. Somewhat paradoxically, IL-18 also has the capacity to induce allergic responses via induction of IL-4 production by T helper cells and to activate mast cells and basophils to release atopic effector molecules such as histamine. Indeed, IL-18 is involved in inflammatory tissue injuries, such as Crohn's disease and atherosclerosis, and also in hyper IgE and atopic dermatitis. IL-18 is particularly important for induction of experimental liver diseases. Endotoxin-induced liver injury or Fas ligand-induced hepatitis is caused by endogenous IL-18 in mice. Moreover, patients with liver diseases such as fulminant hepatitis, liver cirrhosis due to hepatitis virus infection and primary biliary cirrhosis show elevation of serum levels of IL-18, that correlates with the corresponding disease severity. Therefore, endogenous IL-18 plays a major role in induction of some types of liver injuries in mice and human. NKT cells that express both T cell receptor and NK cell marker are abundant in the liver of mice and human. Recent studies have revealed that NKT cells participate in some types of liver injuries, such as concanavalin A-induced T cell-mediated hepatitis and malaria hepatitis. In this review article, we focus on IL-18-involving liver damages and NKT-cell-mediated liver injuries.     

Acute phase mediated change in glycosylation of rat alpha 1-acid glycoprotein in transgenic mice.

Transgenic mouse lines carrying the gene for rat alpha 1-acid glycoprotein (AGP) express the protein in the plasma at concentrations equal to or exceeding that of acute phase rats. Owing to the high basal level, these transgenic mice represent a unique experimental system for defining the largely unknown function of AGP. Since the carbohydrate moiety of AGP has been found to be changed during acute phase and the oligosaccharide structure to be important for immunomodulating activity of the protein, the rat AGP in transgenic mice was characterized by lectin-affinity immuno-electrophoresis. Unlike in the rat, the AGP in the transgenic mouse plasma consisted primarily of strongly concanavalin A-reactive forms. Acute phase mediated a several-fold increase in the total plasma concentration of AGP concomitant with a shift toward moderately concanavalin A-reactive forms. A similar change in concanavalin A-reactive forms was observed for the endogenous acute phase plasma protein haptoglobin. To define the role of inflammatory factors in AGP production, primary cultures of hepatocytes were prepared. In contrast to in vivo, the AGP recovered from tissue culture medium represented primarily the concanavalin A-non-reactive form. Treatment of the cells with recombinant human interleukin-1, interleukin-6 and dexamethasone stimulated the production of concanavalin A-reactive AGP forms. The data indicate that the glycosylation pattern of plasma-resident AGP is modulated by acute phase, but that the profile of AGP forms does not coincide with that secreted by hepatocytes in tissue culture. This finding demands an assessment of which of the possible glycosylated forms of AGP is functionally significant in vivo.     

Rapid conditional targeted ablation of cells expressing human CD59 in transgenic mice by intermedilysin

Conditional targeted cell ablation is a powerful approach for investigating the pathogenesis of human diseases and in vivo cellular functions. Intermedilysin (ILY) is a cytolytic pore-forming toxin secreted by Streptococcus intermedius that lyses human cells exclusively, owing to its receptor specificity for human CD59. We generated two transgenic mouse strains that express human CD59 either on erythrocytes (strain ThCD59(RBC)) or on endothelia (strain ThCD59(END)). Intravenous injection of ILY in ThCD59(RBC) mice induced acute intravascular hemolysis, leading to reduced nitric oxide bioavailability, increased platelet activation and rapid death. In ThCD59(END) mice, ILY induced rapid endothelial damage, leading to acute death and disseminated intravascular coagulation. Additionally, we show that human serum contains ILY-specific neutralizing antibodies not found in any other animal species. Together, these results suggest that this new rapid conditional targeted ILY-mediated cell ablation technique can be used in combination with any available transgenic expression system to study the physiologic role of specific cell populations.     

TP-58, a novel thienopyridine derivative, protects mice from concanavalinA-induced hepatitis by suppressing inflammation

Hepatitis represents a ubiquitous human health problem but effective therapies with limited side effects are still lacking. In this study, we investigated the effect and mechanism of TP-58, a novel thienopyridine derivative, on a murine fulminant hepatitis model induced by concanavalin A (ConA). We found TP-58 markedly alleviated ConA-caused liver injury and increased survival ratio of mice injected with a lethal dose of ConA. Oral administration of TP-58 significantly alleviated ConA-caused liver injury in mice by the reduction of serum aminotransferases and liver necrosis.The analysis of proinflammatory cytokines showed that TP-58 decreased both hepatic mRNA expressions and serum protein levels of TNF-α and IL-6. And the result from LPS-stimulated RAW 264.7 cells showed TP-58 suppressed the production of TNF-α, IL-6, and Nitro Oxide (NO) in the supernatant of LPS-stimulated RAW 264.7 cells. The study of activation of nuclear factor-κB (NF-κB) by electrophoretic mobility shift assay (EMSA) showed that TP-58 inhibited the activation of NF-κB both in vivo and in vitro. The inhibitory effect was also accompanied by a parallel reduction of IκB phosphorylation. These results indicate that TP-58 protects against liver injury by inhibition of the NF-κB-mediated inflammation and suggest a potential role of TP-58 against acute liver injury and other inflammatory diseases.     

CpG DNA-mediated induction of acute liver injury in D-galactosamine-sensitized mice: the mitochondrial apoptotic pathway-dependent death of hepatocytes

Unmethylated CpG motifs present in bacterial DNA (CpG DNA) induce innate inflammatory responses, including rapid induction of proinflammatory cytokines. Although innate inflammatory responses induced by CpG DNA and other pathogen-associated molecular patterns are essential for the eradication of infectious microorganisms, excessive activation of innate immunity is detrimental to the host. In this study, we demonstrate that CpG DNA, but not control non-CpG DNA, induces a fulminant liver failure with subsequent shock-mediated death by promoting massive apoptotic death of hepatocytes in D-galactosamine (D-GalN)-sensitized mice. Inhibition of mitochondrial membrane permeability transition pore opening or caspase 9 activity in vivo protects D-GalN-sensitized mice from the CpG DNA-mediated liver injury and death. CpG DNA enhanced production of proinflammatory cytokines in D-GalN-sensitized mice via a TLR9/MyD88-dependent pathway. In addition, CpG DNA failed to induce massive hepatocyte apoptosis and subsequent fulminant liver failure and death in D-GalN-sensitized mice that lack TLR9, MyD88, tumor necrosis factor (TNF)-alpha, or TNF receptor I but not interleukin-6 or -12p40. Taken together, our results provide direct evidence that CpG DNA induces a severe acute liver injury and shock-mediated death through the mitochondrial apoptotic pathway-dependent death of hepatocytes caused by an enhanced production of TNF-alpha through a TLR9/MyD88 signaling pathway in D-GalN-sensitized mice.     

Regulation of lung injury and repair by Toll-like receptors and hyaluronan

Mechanisms that regulate inflammation and repair after acute lung injury are incompletely understood. The extracellular matrix glycosaminoglycan hyaluronan is produced after tissue injury and impaired clearance results in unremitting inflammation. Here we report that hyaluronan degradation products require MyD88 and both Toll-like receptor (TLR)4 and TLR2 in vitro and in vivo to initiate inflammatory responses in acute lung injury. Hyaluronan fragments isolated from serum of individuals with acute lung injury stimulated macrophage chemokine production in a TLR4- and TLR2-dependent manner. Myd88(-/-) and Tlr4(-/-)Tlr2(-/-) mice showed impaired transepithelial migration of inflammatory cells but decreased survival and enhanced epithelial cell apoptosis after lung injury. Lung epithelial cell-specific overexpression of high-molecular-mass hyaluronan was protective against acute lung injury. Furthermore, epithelial cell-surface hyaluronan was protective against apoptosis, in part, through TLR-dependent basal activation of NF-kappaB. Hyaluronan-TLR2 and hyaluronan-TLR4 interactions provide signals that initiate inflammatory responses, maintain epithelial cell integrity and promote recovery from acute lung injury.     

Genetic deficiency of NADPH oxidase does not diminish,but rather enhances,LPS-induced acute inflammatory responses in vivo

Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in the etiology of cell dysfunction and tissue damage in sepsis. However, there is limited and controversial evidence from in vivo studies that ROS mediate cell signaling processes that elicit acute inflammatory responses during sepsis. Because NADPH oxidase is one of the main cellular sources of ROS, we investigated the role of this enzyme in lipopolysaccharide (LPS)-induced acute inflammation in vivo, utilizing mice deficient in the gp91^phox or p47^phox subunits of NADPH oxidase. Age-and body weight-matched C57BL/6J wild-type (WT) and gp91^phox?/? and p47^phox?/? mice were injected ip with 50 μg LPS or saline vehicle and sacrificed at various time points up to 24 h. We found that LPS-induced acute inflammatory responses in serum and tissues were not significantly diminished in gp91^phox?/? and p47^phox?/? mice compared to WT mice. Rather, genetic deficiency of NADPH oxidase was associated with enhanced gene expression of inflammatory mediators and increased neutrophil recruitment to lung and heart. Furthermore, no protection from LPS-induced septic death was observed in either knockout strain. Our findings suggest that NADPH oxidase-mediated ROS production and cellular redox signaling do not promote, but instead limit, LPS-induced acute inflammatory responses in vivo.     

Constitutive expression of cyclo-oxygenase 2 transgene in hepatocytes protects against liver injury

The effect of COX (cyclo-oxygenase)-2-dependent PGs (prostaglandins) in acute liver injury has been investigated in transgenic mice that express human COX-2 in hepatocytes. We have used three well-established models of liver injury: in LPS (lipopolysaccharide) injury in D-GalN (D-galactosamine)-preconditioned mice; in the hepatitis induced by ConA (concanavalin A); and in the proliferation of hepatocytes in regenerating liver after PH (partial hepatectomy). The results from the present study demonstrate that PG synthesis in hepatocytes decreases the susceptibility to LPS/D-GalN or ConA-induced liver injury as deduced by significantly lower levels of the pro-inflammatory profile and plasmatic aminotransferases in transgenic mice, an effect suppressed by COX-2-selective inhibitors. These Tg (transgenic) animals express higher levels of anti-apoptotic proteins and exhibit activation of proteins implicated in cell survival, such as Akt and AMP kinase after injury. The resistance to LPS/D-GalN-induced liver apoptosis involves an impairment of procaspase 3 and 8 activation. Protection against ConA-induced injury implies a significant reduction in necrosis. Moreover, hepatocyte commitment to start replication is anticipated in Tg mice after PH, due to the expression of PCNA (proliferating cell nuclear antigen), cyclin D1 and E. These results show, in a genetic model, that tissue-specific COX-2-dependent PGs exert an efficient protection against acute liver injury by an antiapoptotic/antinecrotic effect and by accelerated early hepatocyte proliferation.     

Infection induces a positive acute phase apolipoprotein E response from a negative acute phase gene: role of hepatic LDL receptors

Apolipoprotein E (apoE) plays important roles in lipid homeostasis, anti-inflammation, and host defense. Since tissue apoE mRNA levels have been reported to decrease during inflammatory responses, we were surprised to find that plasma apoE levels were significantly elevated during septic infections in both humans and mice. This apparent paradox was also observed during lipopolysaccharide-induced acute inflammation in mice: plasma levels of apoE increased up to 4-fold despite sharply decreased apoE gene expression in the liver, macrophages, and extrahepatic tissues. We hypothesized that apoE levels were augmented by decreased plasma clearance. Our analysis revealed that apoE associated principally with HDL in mice and that apoE was cleared from the circulation principally via LDL receptors. The acute inflammatory response decreased LDL receptor expression in the liver and significantly reduced the rate of apoE clearance. In contrast, the same inflammatory stimuli increased LDL receptor expression in macrophages. Our results define a novel acute phase mechanism that increases circulating apoE levels as apoE production decreases. Diminished hepatic LDL receptor expression may thus cooperate with elevated LDL receptor expression in macrophages to facilitate the forward transport of apoE and its associated lipids to these key defense cells.     

Prostaglandin D2 plays an essential role in chronic allergic inflammation of the skin via CRTH2 receptor

PGD(2) plays roles in allergic inflammation via specific receptors, the PGD receptor designated DP and CRTH2 (chemoattractant receptor homologous molecule expressed on Th2 cells). We generated mutant mice carrying a targeted disruption of the CRTH2 gene to investigate the functional roles of CRTH2 in cutaneous inflammatory responses. CRTH2-deficent mice were fertile and grew normally. Ear-swelling responses induced by hapten-specific IgE were less pronounced in mutant mice, giving 35-55% of the responses of normal mice. Similar results were seen in mice treated with a hemopoietic PGD synthase inhibitor, HQL-79, or a CRTH2 antagonist, ramatroban. The reduction in cutaneous responses was associated with decreased infiltration of lymphocytes, eosinophils, and basophils and decreased production of macrophage-derived chemokine and RANTES at inflammatory sites. In models of chronic contact hypersensitivity induced by repeated hapten application, CRTH2 deficiency resulted in a reduction by approximately half of skin responses and low levels (63% of control) of serum IgE production, although in vivo migration of Langerhans cells and dendritic cells to regional lymph nodes was not impaired in CRTH2-deficient mice. In contrast, delayed-type hypersensitivity to SRBC and irritation dermatitis in mutant mice were the same as in wild-type mice. These findings indicate that the PGD(2)-CRTH2 system plays a significant role in chronic allergic skin inflammation. CRTH2 may represent a novel therapeutic target for treatment of human allergic disorders, including atopic dermatitis.     

Growth differentiation factor 15 protects against the aging‐mediated systemic inflammatory response in humans and mice

Mitochondrial dysfunction is associated with aging‐mediated inflammatory responses, leading to metabolic deterioration, development of insulin resistance, and type 2 diabetes. Growth differentiation factor 15 (GDF15) is an important mitokine generated in response to mitochondrial stress and dysfunction; however, the implications of GDF15 to the aging process are poorly understood in mammals. In this study, we identified a link between mitochondrial stress‐induced GDF15 production and protection from tissue inflammation on aging in humans and mice. We observed an increase in serum levels and hepatic expression of GDF15 as well as pro‐inflammatory cytokines in elderly subjects. Circulating levels of cell‐free mitochondrial DNA were significantly higher in elderly subjects with elevated serum levels of GDF15. In the BXD mouse reference population, mice with metabolic impairments and shorter survival were found to exhibit higher hepatic Gdf15 expression. Mendelian randomization links reduced GDF15 expression in human blood to increased body weight and inflammation. GDF15 deficiency promotes tissue inflammation by increasing the activation of resident immune cells in metabolic organs, such as in the liver and adipose tissues of 20‐month‐old mice. Aging also results in more severe liver injury and hepatic fat deposition in Gdf15‐deficient mice. Although GDF15 is not required for Th17 cell differentiation and IL‐17 production in Th17 cells, GDF15 contributes to regulatory T‐cell‐mediated suppression of conventional T‐cell activation and inflammatory cytokines. Taken together, these data reveal that GDF15 is indispensable for attenuating aging‐mediated local and systemic inflammation, thereby maintaining glucose homeostasis and insulin sensitivity in humans and mice.     

The use of synthetic analogues of Arg-Gly-Asp (RGD) and soluble receptor of tumor necrosis factor to prevent acute and chronic experimental liver injury.

In chronic viral hepatitis, autoimmune hepatitis, and some chronic cholestatic liver diseases, T-lymphocytes serve as effector cells of the immunostimulatory processes. Cellular interactions of immune cells with extracellular matrix (ECM) components are regulated primarily via the beta 1 subfamily of integrin receptors. The target epitope of several such integrin receptors is the Arg-Gly-Asp (RGD) sequence, a cell adhesion motif shared by several matrix-associated adhesive glycoproteins. We review the use of synthetic nonpeptidic analogues of RGD and of soluble receptor of tumor necrosis factor (TNF)-alpha in the prevention of immune-mediated, concanavalin A-induced liver damage in mice and of RGD analogues in inhibiting the development of liver cirrhosis in rats. The concanavalin A-induced elevation of serum transaminases and TNF-alpha, and the infiltration of liver tissue by inflammatory cells, were inhibited by pretreatment of the mice with the synthetic RGD mimetics and soluble TNF receptor. In rats, the progression of thioacetamide-induced liver cirrhosis was markedly inhibited by the coadministration of the RGD mimetic SF-6,5. The compounds described here may be examined therapeutically for pathological conditions in the liver, manifested as necroinflammation, cholestasis and fibrosis.     

Monocyte chemoattractant protein-1 and CCR2 interactions are required for IFN-alpha/beta-induced inflammatory responses and antiviral defense in liver

IFN-alpha/beta-mediated functions promote production of MIP-1alpha (or CCL3) by mediating the recruitment of MIP-1alpha-producing macrophages to the liver during early infection with murine CMV. These responses are essential for induction of NK cell inflammation and IFN-gamma delivery to support effective control of local infection. Nevertheless, it remains to be established if additional chemokine functions are regulated by IFN-alpha/beta and/or play intermediary roles in supporting macrophage trafficking. The chemokine MCP-1 (or CCL2) plays a distinctive role in the recruitment of macrophages by predominantly stimulating the CCR2 chemokine receptor. Here, we examine the roles of MCP-1 and CCR2 during murine CMV infection in liver. MCP-1 production preceded that of MIP-1alpha during infection and was dependent on IFN-alpha/beta effects for induction. Resident F4/80(+) liver leukocytes were identified as primary IFN-alpha/beta responders and major producers of MCP-1. Moreover, MCP-1 deficiency was associated with a dramatic reduction in the accumulation of macrophages and NK cells, as well as decreased production of MIP-1alpha and IFN-gamma in liver. These responses were also markedly impaired in mice with a targeted disruption of CCR2. Furthermore, MCP-1- and CCR2-deficient mice exhibited increased viral titers and elevated expression of the liver enzyme alanine aminotransferase in serum. These mice also had widespread virus-induced liver pathology and succumbed to infection. Collectively, these results establish MCP-1 and CCR2 interactions as factors promoting early liver inflammatory responses and define a mechanism for innate cytokines in regulation of chemokine functions critical for effective localized antiviral defenses.     

Endogenous alphaCGRP protects against concanavalin A-induced hepatitis in mice

To evaluate hepatoprotective effect of alpha-calcitonin gene-related peptide (alphaCGRP), we compared the susceptibilities of alphaCGRP-/- and wild-type mice to concanavalin A (Con A)-induced hepatitis. Twelve hours after Con A administration, serum transaminases were markedly higher in alphaCGRP-/- than wild-type mice, and much more extensive TUNEL-positive lesions and DNA fragmentation were detected in the livers of alphaCGRP-/- mice. Notably, expression of IL-6 was selectively diminished in alphaCGRP-/- mice, suggesting that induction of IL-6 during acute inflammatory responses is blunted in alphaCGRP-/- mice. In addition, primary cultured alphaCGRP-/- hepatocytes were more susceptible to IFN-gamma-induced cell death than hepatocytes from wild-type mice. Administration of exogenous alphaCGRP reduced the incidence of apoptosis among hepatocytes and endothelial cells. It thus appears that alphaCGRP exerts a hepatoprotective effect by modulating cytokine expression and preventing apoptosis.     

Critical role for CXC chemokine ligand 16 (SR-PSOX) in Th1 response mediated by NKT cells

The transmembrane chemokine CXCL 16 (CXCL16), which is the same molecule as the scavenger receptor that binds phosphatidylserine and oxidized lipoprotein (SR-PSOX), has been shown to mediate chemotaxis and adhesion of CXC chemokine receptor 6-expressing cells such as NKT and activated Th1 cells. We generated SR-PSOX/CXCL16-deficient mice and examined the role of this chemokine in vivo. The mutant mice showed a reduced number of liver NKT cells, and decreased production of IFN-gamma and IL-4 by administration of alpha-galactosylceramide (alphaGalCer). Of note, the alphaGalCer-induced production of IFN-gamma was more severely impaired than the production of IL-4 in SR-PSOX-deficient mice. In this context, SR-PSOX-deficient mice showed impaired sensitivity to alphaGalCer-induced anti-tumor effect mediated by IFN-gamma from NKT cells. NKT cells from wild-type mice showed impaired production of IFN-gamma, but not IL-4, after their culture with alphaGalCer and APCs from mutant mice. Moreover, Propionibacterium acnes-induced in vivo Th1 responses were severely impaired in SR-PSOX-deficient as well as NKT KO mice. Taken together, SR-PSOX/CXCL16 plays an important role in not only the production of IFN-gamma by NKT cells, but also promotion of Th1-inclined immune responses mediated by NKT cells.     

Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice

The sequence of the hepatitis B virus (HBV) major envelope (Env) protein (ayw subtype) was scanned for the presence of H-2(d,b) motifs. Following binding and immunogenicity testing, two new H-2(d)-restricted epitopes (Env.362 and Env.364) were identified. These epitopes induced CTLs capable of recognizing naturally processed HBV-Env, but were apparently generated with lower efficiency than the previously defined dominant Env.28 epitope. Next, HBV-transgenic mice that express all of the HBV proteins and produce fully infectious particles were immunized with a mixture of lipopeptides encompassing the Env.28, Env.362, and Env.364 epitopes. Significant CTL responses were obtained, but they had no effect on viral replication in the liver, nor did they induce an inflammatory liver disease. However, in adoptive transfer experiments, CTL lines generated from the HBV-transgenic mice following immunization were able to inhibit viral replication in vivo without causing hepatitis. This is in contrast to CTL lines derived from nontransgenic mice that displayed both antiviral and cytopathic effects, presumably because they displayed higher avidity for the viral epitopes than the transgenic CTLs. These results suggest that T cell tolerance to HBV can be broken with appropriate immunization but the magnitude and characteristics of the resultant T cell response are significantly different from the response in HBV-naive individuals since their antiviral potential is stronger than their cytotoxic potential. This has obvious implications for immunotherapy of chronic HBV infection.