Recovery from an acute systemic and central LPS-inflammation challenge is affected by mouse sex and genetic background.

Genetic and sexual factors influence the prevalence and the pathogenesis of many inflammatory disorders. In this study their relevance on the peripheral and central inflammatory status induced by a peripheral injection of lipopolysaccharide (LPS) was evaluated. BALB/c and CD-1 male and female mice were intraperitoneally injected with LPS. Spleens and brains were collected 2 and 72 hours later to study the levels of IL-6, TNF-α and IL-1ß. Percentage of microglia and astrocytes was determined in the cortex and hippocampus. Locomotor activity was registered before and during the 72 hours after LPS-treatment. Two hours after LPS-injection, a peripheral increase of the three cytokines was found. In brains, LPS increased TNF-α only in males with higher levels in CD-1 than BALB/c. IL-1ß increased only in CD-1 males. IL-6 increased in both strains with lower levels in BALB/c females. Peripheral and central levels of cytokines decline 72 hrs after LPS-treatment whilst a significantly increase of Iba-1 expression was detected. A dramatic drop of the locomotor activity was observed immediately after LPS injection. Our results show that acute systemic administration of LPS leads to peripheral and central increase of pro-inflammatory cytokines and microglia activation, in a strain and sex dependent manner.

Investigation of the expression of irisin and some cachectic factors in mice with experimentally induced gastric cancer.

BACKGROUND: The purpose of this study was to determine whether irisin is secreted by gastric tumor cells experimentally induced in mice, and also if it has any effect on cancer cachexia. DESIGN AND METHODS: 12 out of 60 BALB/c mice were used as a control group, while N-nitroso-N-methylurea (MNU) was administered orally to the remaining 48. After 150 days, the surviving mice were sacrificed by decapitation, blood and stomach, skeletal muscle, brown and white adipose tissue specimens were collected. Following histopathological evaluation of the stomach tissues, it was decided to create four groups, one control group and three consisting of mice administered MNU, no cancer, pre-cancer and cancer. Gene expression analyses of fibronectin type III domain containing protein 5 (FNDC5) and some cachexia-related proteins were performed in tissue samples, while levels of irisin, and various inflammatory and tumor markers together with cachectic factors were determined in serum samples. RESULTS: The levels of inflammatory, tumor markers and cachectic factors in serum samples were significantly higher in the cancer group compared with the control group. No expression of FNDC5 or zinc-α-2 glycoprotein, a cachectic factor, was observed in gastric tissues from the control and MNU groups, whereas significantly increased FNDC5 expression was determined in the both white and brown adipose tissues from the cancer group. CONCLUSION: Increased FNDC5 expression in white and brown adipose tissues may have a cachectic effect in mice with induced cancer. However, it is not possible to explain the mechanism of the relationship between irisin and gastric cancer development on the basis of the results of this study.

Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis.

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4+ cells and a reduction in Th17 (CD4+IL17+) and mature dendritic (MHCII+CD11c+) cells recruitment. Clodronate depletion of macrophages in Ccr6-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.

A Novel Role of Numb as A Regulator of Pro-inflammatory Cytokine Production in Macrophages in Response to Toll-like Receptor 4.

Activation of macrophages triggers the release of pro-inflammatory cytokines leading to inflammation. Numb is a negative regulator of Notch signaling, but the role of Numb in macrophages is not fully understood. In this study, the role of Numb as a regulator of inflammatory responses in macrophages was investigated. Murine bone marrow-derived macrophages, in which expression of Numb was silenced, secreted significantly less TNFα, IL-6 and IL-12 and more IL-10 upon activation by lipopolysaccharide (LPS), a ligand for Toll-like receptor 4 (TLR4), despite increased Notch signaling. The Tnfα mRNA levels both in Numb-deficient and wild-type macrophages were not significantly different, unlike those of Il6 and Il12-p40. In Numb-deficient macrophages, the Tnfα mRNAs were degraded at faster rate, compared to those in control macrophages. Activation of p38 MAPK and NF-κΒ p65 were compromised in activated Numb deficient macrophages. Numb was found to interact with the E3 ubiquitin ligase, Itch, which reportedly regulates p38 MAPK. In addition, blocking the Notch signaling pathway in activated, Numb-deficient macrophages did not further reduce TNFα levels, suggesting a Notch-independent role for Numb. A proteomics approach revealed a novel function for Numb in regulating complex signaling cascades downstream of TLRs, partially involving Akt/NF-κB p65/p38 MAPK in macrophages.

MIGRAs: are they the new IGRAs? Development of monokine-amplified IFN-γ release assays.

IFN-γ release by antigen-specific T cells can be used to track immune responses to infections and vaccines. In recent years, there have been substantial advances in the techniques available to measure IFN-γ release and a generation of such assays are now available for clinical use, as well as in a research setting. Interferon release leads to subsequent release of interferon-responsive chemokines such as MIG and IP-10, thus amplifying the original signal. A number of investigators have assessed whether measurement of these chemokines might provide a sensitive platform for detection of infection and antigen-specific T-cell responses. In this article, we assess the potential of these new approaches. We have termed the new antigen-specific T-cell assays monokine-amplified IFN-γ release assays (MIGRAs). Overall, it seems likely that improvements in the detection threshold could be made by analysis of antigen-triggered chemokines and potentially of other molecules in the future, although whether MIGRAs will provide additional clinical utility still remains to be determined.

Upregulations of glucocorticoid-induced leucine zipper by hypoxia and glucocorticoid inhibit proinflammatory cytokines under hypoxic conditions in macrophages.

Hypoxia and inflammation often develop concurrently in numerous diseases, and the influence of hypoxia on natural evolution of inflammatory responses is widely accepted. Glucocorticoid-induced leucine zipper (GILZ) is thought to be an important mediator of anti-inflammatory and immune-suppressive actions of glucocorticoid (GC). However, whether GILZ is involved in hypoxic response is still unclear. In this study, we investigated the effects of hypoxic exposure and/or the administration of dexamethasone (Dex), a synthetic GC on GILZ expression both in vitro and in vivo, and further explored the relationship between GILZ and proinflammatory cytokines IL-1ß, IL-6, and TNF-α under normoxic and hypoxic conditions. We found that hypoxia not only remarkably upregulated the expression of GILZ, but also significantly enhanced Dex-induced expression of GILZ in macrophages and the spleen of rats. ERK activity is found involved in the upregulation of GILZ induced by hypoxia. Inhibiting the expression of GILZ in RAW264.7 cells using specific GILZ small interfering RNA led to a significant increase in mRNA production and protein secretion of IL-1ß and IL-6 in hypoxia and abrogated the inhibitory effect of Dex on expression of IL-1ß and IL-6 in hypoxia. We also found that adrenal hormones played pivotal roles in upregulation of GILZ expression in vivo. Altogether, data presented in this study suggest that GILZ has an important role not only in adjusting adaptive responses to hypoxia by negatively regulating the activation of macrophages and the expression of proinflammatory cytokines, but also in mediating the anti-inflammatory action of GC under hypoxic conditions.

Monokine induced by interferon-gamma (MIG/CXCL9) is derived from both donor and recipient sources during rejection of class II major histocompatibility complex disparate skin allografts.

Chemokines, including monokine induced by interferon-gamma (Mig/CXCL9), are produced both in allografts and during the direct T-cell infiltration that mediates graft rejection. Neither the specific production nor contribution of allograft donor versus recipient Mig in allograft rejection is currently known. C57BL/6 mice with a targeted deletion in the Mig gene were used as both skin allograft donors and recipients in a class II major histocompatibility complex-mismatched graft model to test the requirement for donor- versus recipient-derived Mig for acute rejection. B6.Mig(-/-) allografts had a 10-day prolonged survival in B6.H-2(bm12) recipients when compared with wild-type C57BL/6 allograft donors, and B6.H-2(bm12) skin allografts had a 5-day prolonged survival in B6.Mig(-/-) versus wild-type recipients. Transplantation of B6.Mig(-/-) skin grafts onto B6.H-2(bm12).Mig(-/-) recipients resulted in further prolonged allograft survival with more than 30% of the grafts surviving longer than 60 days. Prolonged allograft survival was also associated with delayed cellular infiltration into grafts but not with altered T-cell proliferative responses to donor stimulators. Immunohistochemical staining of allograft sections indicated that Mig is produced by both donor- and recipient-derived sources, but Mig from each of these sources appeared in different areas of the allograft tissue. These results therefore demonstrate the synergy of donor- and recipient-derived Mig in promoting T-cell infiltration into allografts.

Ecstasy (3,4-methylenedioxymethamphetamine) limits murine gammaherpesvirus-68 induced monokine expression.

While Ecstasy (3,4-methylenedioxymethamphetamine, MDMA) has been shown to modulate immune responses, no studies have addressed drug-induced alterations to viral infection. In this study, bone marrow-derived macrophages were exposed to MDMA, then infected with murine gammaherpesvirus-68, and the expression of monokines assessed. MDMA-induced reductions in virus-stimulated monokine mRNA expression were observed in a dose-dependent manner. In particular, IL-6 mRNA expression and secretion was significantly decreased in gammaherpesvirus-infected macrophages exposed to MDMA. Concentrations of MDMA capable of reducing monokine production did not induce significant cell death and allowed normal viral gene expression. These studies represent the first to demonstrate the ability of this drug of abuse to alter a viral-induced macrophage response.

Expression and purification of bioactive high-purity mouse monokine induced by IFN-gamma in Escherichia coli.

MIG (monokine induced by IFN-gamma) is a CXC-chemokine (CXCL9). It plays important roles in regulation of immune activities, and knowledge of the protein in areas of allograft transplants, autoimmune diseases, and cancer therapy is evolving quickly. The non-tagged recombinant murine MIG (rMuMIG) is therefore required to facilitate the functional studies of this important chemokine. Here we present the use of a bacteria expression system to produce non-tagged rMuMIG. The coding sequence for MIG was cloned into the pET28a (+) vector that was transformed into Escherichia coli BL21 (DE3). Expression of rMuMIG was induced by IPTG. Bacteria inclusion bodies containing the protein were isolated and washed to remove contaminated bacteria proteins, and resolved in Urea buffer. Renaturation of the denatured protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose cation exchange chromatography. The final preparation of the rMuMIG was more than 99% pure as measured by capillary electrophoresis and SDS-PAGE analysis. The biological activity of rMuMIG was demonstrated in a murine spleen cell chemotaxis assay with ED50 30 ng/ml. Further experiments showed that rMuMIG could inhibit proliferation of mouse bone marrow cells in vivo.

High-cholesterol diet facilitates Anaplasma phagocytophilum infection and up-regulates macrophage inflammatory protein-2 and CXCR2 expression in apolipoprotein E-deficient mice.

BACKGROUND: Anaplasma phagocytophilum is an obligatory intracellular bacterium that infects granulocytes and causes human granulocytic anaplasmosis (HGA). This bacterium requires cholesterol for host cell infection in vitro and incorporates exogenous cholesterol into its membrane. METHODS: To understand the role of host cholesterol in A. phagocytophilum infection in vivo, we analyzed the effects of a high-cholesterol diet and reduced apolipoprotein E (apoE) activity on A. phagocytophilum infection in mice. RESULTS: A high-cholesterol diet significantly facilitated A. phagocytophilum infection in the spleen, liver, and blood of apoE-deficient (apoE(-/-)) mice, compared with the level of infection in apoE(-/-) mice fed a normal-cholesterol diet or wild-type (WT) mice fed a high- or normal-cholesterol diet. A. phagocytophilum infection induced a significant elevation in the mRNA expression of macrophage inflammatory protein (MIP)-2 and an MIP-2 receptor, CXCR2, in the spleen in apoE(-/-) mice fed a high-cholesterol diet, compared with the other 3 groups. CONCLUSION: Our results suggest that high blood cholesterol levels resulting from an interaction between dietary and genetic factors facilitate A. phagocytophilum infection and up-regulate a proinflammatory chemokine and its receptor, which may contribute to HGA pathogenesis.

Expression and function of endothelial monocyte-activating polypeptide-II in acute lung inflammation.

OBJECTIVE AND DESIGN: We tested the hypothesis that total endothelial monocyte-activating polypeptide-II (EMAP-II) expression (proEMAP/p43 and mature EMAP-II) is up-regulated in lipopolysaccharide (LPS)-induced acute lung inflammation (ALI) and that mature EMAP-II induces monocyte/macrophage and granulocyte recruitment in vivo. MATERIALS: Thirty-five 10 week old, male Sprague-Dawley rats. TREATMENT: Animals were instilled intratracheally with 250 microg of E. coli LPS (N = 15) or saline (N = 5) or 20 microg of mature EMAP-II (N = 5). METHODS: Total EMAP-II was quantified using ELISA and the protein was localized with light and electron microscopic immunocytochemistry in lungs of rats at 1, 3 and 12 h (n = 5/group). RESULTS: ELISA showed increased total EMAP-II concentrations (p < 0.05) in lungs from LPS-treated rats compared to control animals. Compared to the controls, light and electron microscopic imunocytochemistry localized total EMAP-II in monocytes/macrophages and alveolar septa at 1 and 3 h and in vascular smooth muscles at 12 h post-LPS treatment. Instillation of mature EMAP-II increased lung monocytes/macrophages and granulocytes compared with control animals (p < 0.05). However, compared to the LPS treatment, mature EMAP-II instillation did not induce expression of IL-1beta and MIP-2 (p < 0.05) and provoked less vigorous recruitment of monocytes/macrophages. CONCLUSION: EMAP-II expression is increased in LPS-induced ALI, and that intra-tracheal instillation of mature EMAP-II induces recruitment of monocytes/macrophages and granulocytes into the lungs without stimulating IL-1beta or MIP-2 expression.

Regulation of human LZIP expression by NF-kappaB and its involvement in monocyte cell migration induced by Lkn-1.

Human LZIP is a transcription factor that is involved in leukocyte cell mobility. Expression of LZIP is known to differentially regulate monocyte cell migration induced by CCR1-dependent chemokines. However, its transcriptional regulation has not been characterized. Our results indicate that Lkn-1 induces LZIP expression in a time- and dose-dependent manner, and the induction of LZIP shows an immediate early response to Lkn-1. We identified and cloned approximately 1.4 kb of the LZIP promoter from a human genomic DNA. To identify regulatory elements controlling restricted expression of LZIP, deletion mutants were constructed from the 1469-bp LZIP promoter region (-1219/+251) linked to the luciferase reporter gene. Maximal promoter activity was contained within 613 bp from the tentative transcription initiation site and was sharply reduced in a truncated construct (-338/+251). This promoter sequence contained consensus NF-kappaB- and Sp-1-binding sites. Results from an inhibitor assay showed that NF-kappaB is involved in Lkn-1-induced LZIP expression, but Sp-1 is not. We also demonstrated that NF-kappaB binds to the LZIP promoter and that the binding is specific, as revealed by an electrophoretic mobility shift assay and a mutation analysis. Chemotaxis analysis showed that LZIP expression because of the NF-kappaB subfamily is specifically involved in Lkn-1-induced chemotaxis. Our findings suggest that transcription factor NF-kappaB plays an important role in regulation of LZIP expression, and LZIP expression regulates the monocyte cell migration induced by Lkn-1.

Macrolide antibiotics promote the LPS-induced upregulation of prostaglandin E receptor EP2 and thus attenuate macrolide suppression of IL-6 production.

We studied the influence of the inhibitory effect of clarithromycin (CAM) and erythromycin (EM) on the production of macrophage inflammatory protein (MIP)-2, interleukin-6 (IL-6), and prostaglandin E(2) (PGE(2)), as well as PGE(2) receptor (EP(2)) expression, by LPS-stimulated RAW264.7 cells. Production of IL-6 was significantly decreased by treatment with CAM or EM in a dose-dependent manner, but the inhibitory effect of CAM was significantly weaker than that of EM. In contrast, the production of MIP-2 and PGE(2) was inhibited to the same extent by CAM and EM. LPS induced the expression of EP(2) mRNA and its expression was promoted further by treatment with CAM or EM. In particular, CAM significantly upregulated EP(2) mRNA expression compared with that after stimulation by LPS alone. After treatment with a nonselective cyclooxygenase (COX) inhibitor (indomethacin), a selective COX-2 inhibitor (NS398), or an EP(2)/EP(4) receptor antagonist (AH6809), the inhibitory effect of CAM and EM on LPS-induced IL-6 production was equalized. These results indicate that macrolide antibiotics upregulate the expression of EP(2), which then attenuates the suppressive effect on IL-6 production of these antibiotics, suggesting that these drugs have a variable anti-inflammatory effect that could influence host defenses.

Hyperoxia increases ventilator-induced lung injury via mitogen-activated protein kinases: a prospective, controlled animal experiment.

INTRODUCTION: Large-tidal volume (VT) mechanical ventilation and hyperoxia used in patients with acute respiratory distress syndrome can damage pulmonary epithelial cells through lung inflammation and apoptotic cell death. Hyperoxia has been shown to increase ventilator-induced lung injury, but the mechanisms regulating interaction between large VT and hyperoxia are unclear. We hypothesized that the addition of hyperoxia to large-VT ventilation would increase neutrophil infiltration by upregulation of the cytokine macrophage inflammatory protein-2 (MIP-2) and would increase apoptosis via the mitogen-activated protein kinase pathways. METHODS: C57BL/6 mice were exposed to high-VT (30 ml/kg) mechanical ventilation with room air or hyperoxia for one to five hours. RESULTS: The addition of hyperoxia to high-VT ventilation augmented lung injury, as demonstrated by increased apoptotic cell death, neutrophil migration into the lung, MIP-2 production, MIP-2 mRNA expression, increased DNA binding activity of activator protein-1, increased microvascular permeability, and c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) 1/2 activation. Hyperoxia-induced augmentation of high-VT-induced lung injury was attenuated in JNK-deficient mice and in mice with pharmacologic inhibition of ERK activity by PD98059. However, only JNK-deficient mice, and not mice with ERK activity inhibition by PD98059, were protected from high-VT-induced lung injury without hyperoxia. CONCLUSION: We conclude that hyperoxia increased high-VT-induced cytokine production, neutrophil influx, and apoptotic cell death through activation of the JNK and ERK1/2 pathways.

Theiler's virus induces the MIP-2 chemokine (CXCL2) in astrocytes from genetically susceptible but not from resistant mouse strains.

The murine encephalomyelitis virus of Theiler (TMEV) induces demyelination in susceptible strains of mice by a CD4(+) Th1 T cell mediated immunopathologic process. We focused on the production of one chemokine, the macrophage inflammatory protein-2 (MIP-2 or CXCL2), by cultured mouse astrocytes infected with the BeAn strain of TMEV. Analysis of a murine genome DNA hybridized with cRNA from mock- and TMEV-infected astrocytes, revealed up-regulation of three sequences encoding MIP-2. Northern blot analysis indicated increased MIP-2 mRNA expression. Levels of MIP-2 in the supernatants of infected cells as detected by ELISA, varied directly with the multiplicity of infection used. This secreted CXCL2 was biologically active inducing chemoattraction of neutrophils but not of lymphocytes. CXCL2 was specifically induced by TMEV infection, since induction was inhibited by anti TMEV antibodies. The inflammatory cytokines, IL-1alpha and TNF-alpha, which are also induced in astrocytes by TMEV, were very potent inducers of CXCL2. Nevertheless, both mechanisms of induction follows different pathways as antibodies to both cytokines fails to inhibit TMEV-induced CXCL2 up-regulation. Sera from TMEV-infected SJL/J mice with chronic demyelination, but not from BALB/c TMEV-resistant mice, revealed CXCL2 at the peak of clinical disease. Our main novel finding is the strain-dependent differences in CXCL2 expression both in vitro and in vivo. This suggest an role for this chemokine in attracting immune cells within the CNS, which in turn, might trigger demyelination in this experimental model of MS.

[Expression of Crg-2 by a bacteria recombinant system of adenovirus].

AIM: To express an immunochemotactic and angiostatic anti-tumor molecule, CXC chemokine Crg-2, by E. coli recombinant system of adenovirus. METHODS: By E. coli recombination system of adenovirus, the shuttle vector pShuttle-cmv/crg-2 was co-transfected into BJ5183 E. coli with adenovirus backbone plasmid pAdEasy-1. The recombinants were transfected into 293 packaging cells and adenovirus was packaged and amplified. The molecular weight and bioactivity of recombinant protein were determined by Western blot and chemotaxis assay, respectively. RESULTS: Recombinant adenovirus genome backbone bearing crg-2 gene, pAd/crg-2, was successfully obtained as evidenced by endonucleases digestion and PCR. Adenovirus was packaged and amplified to a titre of 4x10(9) TCID50/L. The cultured supernatant of the infected 293 cells contented a protein of nearly 10,000 and presented a significantly chemotatic effect towards activated spleen lymphoblasts. CONCLUSION: Recombinant adenovirus Ad/crg-2 obtained by E. coli recombinant system of adenovirus can efficiently express bioactive Crg-2 chemokine.

Role of metallothionein in coagulatory disturbance and systemic inflammation induced by lipopolysaccharide in mice.

Although metallothionein (MT) can be induced by inflammatory mediators, its roles in coagulatory disturbance during inflammation are poorly defined. We determined whether MT protects against coagulatory and fibrinolytic disturbance and systemic inflammation induced by intraperitoneal administration of lipopolysaccharide (LPS) in MT-I/II null (-/-) and wild-type (WT) mice. As compared with WT mice, MT (-/-) mice revealed significant prolongation of prothrombin and activated partial thromboplastin time, a significant increase in the levels of fibrinogen and fibrinogen/fibrin degradation products, and a significant decrease in activated protein C, after LPS treatment. LPS induced inflammatory organ damages in the lung, kidney, and liver in both genotypes of mice. The damages, including neutrophil infiltration, were more prominent in MT (-/-) mice than in WT mice after LPS treatment. In both genotypes of mice, LPS enhanced protein expression of interleukin (IL)-1beta, IL-6, granulocyte/macrophage-colony-stimulating factor, macrophage inflammatory protein (MIP)-1alpha, MIP-2, macrophage chemoattractant protein-1, and keratinocyte chemoattractant in the lung, kidney, and liver and circulatory levels of IL-1beta, IL-6, MIP-2, and KC. In overall trends, however, the levels of these proinflammatory proteins were greater in MT (-/-) mice than in WT mice after LPS challenge. Our results suggest that MT protects against coagulatory and fibrinolytic disturbance and multiple organ damages induced by LPS, at least partly, via the inhibition of the expression of proinflammatory proteins.

Early growth response factor-1 is critical for cholestatic liver injury.

Hepatocyte injury during cholestasis depends in part on the release of proinflammatory mediators that cause neutrophils to accumulate in the liver and become activated to damage hepatocytes. The mechanism by which cholestasis stimulates production of proinflammatory mediators in the liver is not completely understood. The studies presented here tested the hypothesis that the transcription factor early growth response factor-1 (Egr-1) is required for inflammation to occur in the liver during cholestasis. The results of these studies show that Egr-1 was rapidly upregulated, primarily in hepatocytes, in mice subjected to bile duct ligation, an animal model of cholestasis. To determine whether Egr-1 was required for inflammation and hepatocyte injury during cholestasis, bile duct ligation was performed in wild-type and Egr-1 knockout mice. Hepatocyte injury, neutrophil accumulation, and upregulation of macrophage inflammatory protein-2 (MIP-2) and intercellular adhesion molecule-1 (ICAM-1) in the liver were significantly reduced in Egr-1 knockouts. By contrast, levels of tumor necrosis factor-alpha (TNF-alpha) and collagen (i.e., a biomarker of liver fibrosis) were not different between wild-types and Egr-1 knockouts subjected to bile duct ligation. Because hepatocytes are exposed to elevated concentrations of bile acids during cholestasis, it was determined that bile acids upregulate Egr-1 in primary mouse hepatocytes. Deoxycholic acid dose-dependently increased Egr-1 protein in hepatocytes. Results from these studies suggest a scenario in which elevated concentrations of bile acids during cholestasis increase expression of Egr-1 in hepatocytes. Egr-1 then upregulates proinflammatory mediators that cause neutrophils to accumulate in the liver and become activated to damage hepatocytes.

Immunosuppressive activity of the immunophilin-binding drug Sanglifehrin A in human whole blood: potent inhibition of interleukin-6 produced by lymphocytes and monocytes.

The novel immunosuppressant Sanglifehrin A (SFA) is an immunophilin-binding metabolite with a yet unidentified mechanism of action. Several reports demonstrated the effects of SFA on proliferation and cytokine production of purified T cells with in part different results. However, less is known about the impact of SFA on the regulation of innate immune responses. We used a whole blood assay to investigate the impact of SFA on monocyte responses and T-lymphocyte activity/proliferation upon lipopolysaccharide (LPS) stimulation and anti-CD3/anti-CD28 costimulation, respectively. SFA was found to inhibit interleukin (IL)-2 protein expression of T lymphocytes. Whereas IL-2 mRNA expression was significantly reduced after 4 h of costimulation, the mRNA expression of IL-4 and IL-6 but not tumour necrosis factor (TNF)-alpha was inhibited by SFA both after 4 and 24 h of costimulation. The production of IL-2 and IL-6 protein in T lymphocytes was even strongly affected by SFA than the mRNA expression of the respective cytokine. Unlike other immunophilin-binding immunosuppressants, SFA also inhibited LPS-induced IL-6 and TNF-alpha mRNA and protein expression. At the single cell level, SFA was demonstrated to block the intracellular production of IL-6 in CD14+ monocytes but not the expression of other proinflammatory cytokines such as IL-8 and TNF-alpha. On the basis of these data, we propose that SFA may have a significant effect on the initiation and direction of immune responses. Considering the pleiotropic role of bioactive IL-6 production at the interface of innate and acquired immunity in a variety of disease conditions, it was found that these novel aspects of the unique immunosuppressive action could strongly impact on future clinical application of SFA.

Down-regulation of tristetraprolin expression results in enhanced IL-12 and MIP-2 production and reduced MIP-3alpha synthesis in activated macrophages.

In inflammation, the post-transcriptional regulation of transiently expressed genes provides a potential therapeutic target. Tristetraprolin (TTP) is of the factors regulating decay of cytokine mRNAs. The aim of the present study was to identify cytokines whose expression is regulated by TTP. We established a TTP knock-down cell line by expressing shRNA against TTP (shTTP cell line). A cytokine antibody array was used to measure cytokine production in macrophages exposed to lipopolysaccharide (LPS). Cytokines IL-6, IL-12, TNF-alpha, and MIP-2 (a homologue to human IL-8) were expressed at higher levels whereas MIP-3alpha was produced at lower levels in LPS-treated shTTP cells than in control cells suggesting that the expression of these cytokines is regulated by TTP. The present data provide IL-12, MIP-2, and MIP-3alpha as novel inflammatory cytokine targets for TTP-mediated mRNA decay and stress the role of TTP in the regulation of the inflammatory process.