MIP-2 Gene Expression Research Articles

GL13K-modified titanium regulates osteogenic differentiation via the NF-κB pathway

The osteoimmune response plays a crucial regulatory role in the osseointegration of dental implants. Previous studies found the antimicrobial peptide coating (GL13K) could activate the immunomodulatory potential of macrophages (Raw 264.7) and promote osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). To further investigate the mechanism of interaction between immunomodulation and differentiation, a co-culture model of the representative cells (Raw 264.7 and BMSCs) was constructed to mimic the immune microenvironment. In this system, GL13K coating of titanium implant effectively inhibited the polarization of the inflammatory M1 type and promoted the polarization of the anti-inflammatory M2 type. Furthermore, the inhibited NF-κB signaling pathway and Mip-2 gene expression were found and validated by bioinformatics analysis and virus-induced gene silencing, which significantly affected the tissue repair process. It can be concluded that the GL13K coating had the potential to establish a localized immune microenvironment conducive to osteogenic differentiation through cellular interactions. Subsequent investigations would be dedicated to a thorough examination of the osseointegration effects of GL13K coating.

Estradiol Modulates Local Gut Injury Induced by Intestinal Ischemia-Reperfusion in Male Rats.

Intestinal ischemia and reperfusion (I/R) triggers a systemic inflammatory response characterized by leukocyte mobilization from the bone marrow, release of cytokines to the circulation, and increased microvascular permeability, leading to high mortality. Females have shown attenuated inflammatory response to trauma when compared with males, indicating a role for female sex hormones in this process. Here, we have evaluated the effect of estradiol on the local gut injury induced by I/R in male rats. I/R was induced by the clamping of the superior mesenteric artery for 45 min, followed by 2 h of reperfusion. A group received 17β-estradiol (280 μg/kg, i.v., single dose) at 30 min of ischemia. Morphometric analysis of the gut showed I/R induced a reduction of villous height that was prevented by estradiol. White blood cells, notably granulocytes, were mobilized from the circulation to the intestine by I/R, which was also prevented by estradiol treatment. Groups had the intestine wrapped in a plastic bag to collect intestinal fluid, where leukocytes count, TNF-α, and IL-10 levels were increased by I/R. Serum chemokines (CINC-1, MIP-1α, MIP-2), ICAM-1 expression in the mesenteric tissue, and neutrophils spontaneous migration measured in vitro were also increased after I/R. Estradiol treatment reduced leukocytes numbers and TNF-α on intestinal fluid, serum chemokine release and also downregulated MIP-1α, MIP-2 gene expression, and spontaneous in vitro neutrophil migration. In conclusion, estradiol blunts intestinal injury induced by I/R by modulating chemokines release and leukocyte trafficking.

The Kaposi's Sarcoma-Associated Herpesvirus (KSHV)-Induced 5-Lipoxygenase-Leukotriene B4 Cascade Plays Key Roles in KSHV Latency, Monocyte Recruitment, and Lipogenesis

Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL). KS lesions are characterized by endothelial cells with multiple copies of the latent KSHV episomal genome, lytic replication in a low percentage of infiltrating monocytes, and inflammatory cytokines plus growth factors. We demonstrated that KSHV utilizes inflammatory cyclooxygenase 2/prostaglandin E2 to establish and maintain latency (Sharma-Walia, N., A. G. Paul, V. Bottero, S. Sadagopan, M. V. Veettil, N. Kerur, and B. Chandran, PLoS Pathog 6:e1000777, 2010 [doi:10.1371/journal.ppat.1000777]). Here, we evaluated the role of 5-lipoxygenase (5LO) and its chemotactic metabolite leukotriene B4 (LTB4) in KSHV biology. Abundant staining of 5LO was detected in human KS tissue sections. We observed elevated levels of 5LO and high levels of secretion of LTB4 during primary KSHV infection of endothelial cells and in PEL B cells (BCBL-1 and BC-3 cells). Blocking the 5LO/LTB4 cascade inhibited viral latent ORF73, immunomodulatory K5, viral macrophage inflammatory protein 1 (MIP-1), and viral MIP-2 gene expression, without much effect on lytic switch ORF50, immediate early lytic K8, and viral interferon-regulatory factor 2 gene expression. 5LO inhibition significantly downregulated latent viral Cyclin and latency-associated nuclear antigen 2 levels in PEL cells. 5LO/LTB4 inhibition downregulated TH2-related cytokine secretion, elevated TH1-related cytokine secretion, and reduced human monocyte recruitment, adhesion, and transendothelial migration. 5LO/LTB4 inhibition reduced fatty acid synthase (FASN) promoter activity and its expression. Since FASN, a key enzyme required in lipogenesis, is important in KSHV latency, these findings collectively suggest that 5LO/LTB4 play important roles in KSHV biology and that effective inhibition of the 5LO/LTB4 pathway could potentially be used in treatment to control KS/PEL.

Effects of nanoparticulate saponin-platinum conjugates on 2,4-dinitrofluorobenzene-induced macrophage inflammatory protein-2 gene expression via reactive oxygen species production in RAW 264.7 cells

Nanoparticulate platinum (II) (nano Pt) is a powerful antioxidant that is widely used to scavenge reactive oxygen species (ROS). The antioxidant activity of nano Pt has gained attention as a potentially useful therapeutic for a variety of diseases including cancer and aging. In the present study, we prepared nanoparticulate saponin-Pt (II) (nano saponin-Pt) conjugates using the ethanol reduction method to enhance the permeability and retention effect of Pt. The nano saponin-Pt conjugates were found to restore the viability of approximately 40% of 2,4-dinitrofluorobenzene (DNFB)-treated RAW 264.7 cells. In addition, we found that nano saponin-Pt conjugates acted as a potent antioxidant that reduced the production of ROS and inhibited activation of the MAP kinase pathway and MIP-2 gene expression in response to DNFB. These results provide insight into the potential usefulness of nano saponin-Pt conjugates as a treatment for contact hypersensitivity.

M1633 Regulation of Gastric Inflammation By Bone Morphogenetic Protein Signaling

BACKGROUND/AIMS: The bonemorphogenetic proteins (BMP)s are regulators of gastrointestinal physiology. The BMPs can inhibit inflammation in the gut. BMP-2 and BMP-4 are expressed in epithelial and mesenchymal cells of the stomach, respectively. We tested the hypothesis that 1) inhibition of BMP signaling In Vivo, by means of the BMP-signaling inhibitor noggin, activates inflammatory mechanisms and that 2) inflammatory stimuli regulate BMP expression. METHODS: The parietal cell-specific H+/K+-ATPase beta-subunit gene promoter was used to drive expression of noggin in the gastric epithelium. Morphology of the fundus was analyzed in sections stained with H&E. Expression of the MIP-2, TNF-alfa, INF-gamma, BMP-4 and BMP-2 genes was measured by QRT-PCR. Western blots were used to assess the phosphorylation and activation of Smad1-5-8, JNK and ERK and to measure the expression of BMP-4. Canine mesenchymal cells isolated from the gastric mucosa, were characterized by staining with antibodies against vimentin and cytokeratin 18, mesenchymal and epithelial cell markers, respectively. Mesenchymal cell proliferation was assessed by BrdU incorporation. RESULTS: The mucosa of the TG-mice exhibited decreased phosphorylation of Smad1-58, confirming inhibition of BMP signaling, and it displayed increased height, dilated glands and a modest increase in inflammatory cells. The TG-mice also exhibited enhanced ERK and JNK activation and a 2to 3-fold increase in MIP-2, INF-gamma and TNF-alfa gene expression. Infection of TG-mice for 4 months with Helicobacter pylori (HP), led to a greater then 100-fold increase in the expression of these cytokines, to a robust influx of inflammatory cells, and to the development of numerous lymphoid follicles and severe dysplasia. Infection of non-TG mice with HP led to inflammatory changes that were less severe then those seen in HP-infected TG-mice. In the non-TG mice, HP also caused a 5-fold increase in cytokine gene expression and it stimulated more then 5-fold the expression of BMP-4 but not that of BMP-2. INF-gamma induced BMP-4 gene and protein expression and it induced cell proliferation in cultures of gastric mesenchymal cells. CONCLUSIONS: The BMPs inhibit gastric inflammation since inhibition of BMP signaling activates pro-inflammatory mechanisms and it leads to an enhanced response to inflammatory stimuli. Moreover, cytokines stimulate BMP-4 expression, an event that might represent a self-safe mechanism that limits the intensity of the inflammatory response. These findings might contribute to a better understanding of the mechanisms that lead to the development of both dysplastic and neoplastic lesions in the stomach.

M1634 Enterococcal Infection of Gastric Cells Induce Expression Cytokines and Suppress DNA Damage Repair Genes

BACKGROUND/AIMS: The bonemorphogenetic proteins (BMP)s are regulators of gastrointestinal physiology. The BMPs can inhibit inflammation in the gut. BMP-2 and BMP-4 are expressed in epithelial and mesenchymal cells of the stomach, respectively. We tested the hypothesis that 1) inhibition of BMP signaling In Vivo, by means of the BMP-signaling inhibitor noggin, activates inflammatory mechanisms and that 2) inflammatory stimuli regulate BMP expression. METHODS: The parietal cell-specific H+/K+-ATPase beta-subunit gene promoter was used to drive expression of noggin in the gastric epithelium. Morphology of the fundus was analyzed in sections stained with H&E. Expression of the MIP-2, TNF-alfa, INF-gamma, BMP-4 and BMP-2 genes was measured by QRT-PCR. Western blots were used to assess the phosphorylation and activation of Smad1-5-8, JNK and ERK and to measure the expression of BMP-4. Canine mesenchymal cells isolated from the gastric mucosa, were characterized by staining with antibodies against vimentin and cytokeratin 18, mesenchymal and epithelial cell markers, respectively. Mesenchymal cell proliferation was assessed by BrdU incorporation. RESULTS: The mucosa of the TG-mice exhibited decreased phosphorylation of Smad1-58, confirming inhibition of BMP signaling, and it displayed increased height, dilated glands and a modest increase in inflammatory cells. The TG-mice also exhibited enhanced ERK and JNK activation and a 2to 3-fold increase in MIP-2, INF-gamma and TNF-alfa gene expression. Infection of TG-mice for 4 months with Helicobacter pylori (HP), led to a greater then 100-fold increase in the expression of these cytokines, to a robust influx of inflammatory cells, and to the development of numerous lymphoid follicles and severe dysplasia. Infection of non-TG mice with HP led to inflammatory changes that were less severe then those seen in HP-infected TG-mice. In the non-TG mice, HP also caused a 5-fold increase in cytokine gene expression and it stimulated more then 5-fold the expression of BMP-4 but not that of BMP-2. INF-gamma induced BMP-4 gene and protein expression and it induced cell proliferation in cultures of gastric mesenchymal cells. CONCLUSIONS: The BMPs inhibit gastric inflammation since inhibition of BMP signaling activates pro-inflammatory mechanisms and it leads to an enhanced response to inflammatory stimuli. Moreover, cytokines stimulate BMP-4 expression, an event that might represent a self-safe mechanism that limits the intensity of the inflammatory response. These findings might contribute to a better understanding of the mechanisms that lead to the development of both dysplastic and neoplastic lesions in the stomach.

2,4-Dinitrofluorobenzene Modifies Cellular Proteins and Induces Macrophage Inflammatory Protein-2 Gene Expression via Reactive Oxygen Species Production in RAW 264.7 Cells

The skin sensitizer 2,4-dinitrofluorobenzene (DNFB) provokes delayed hypersensitivity responses as a result of topical application to the skin. Here, we demonstrate that DNFB modifies proteins in RAW 264.7 cells and skin tissues in NC/Nga mice; we also show the functional involvement of DNFB-induced modification of cellular proteins in the DNFB-induced macrophage inflammatory protein (MIP)-2 gene expression in RAW 264.7 cells. In addition, we demonstrate that DNFB strongly induces reactive oxygen species (ROS) production. Our RT-PCR analysis and reporter gene assays reveal that the DNFB-induced intracellular ROS production is necessary for MIP-2 gene expression by DNFB. We observed that the vitamin C and chemical oxidant scavenger N-acetyl-cysteine have an inhibitory effect on the generation of ROS, the activation of MAP kinase pathways, and the MIP-2 gene expression in DNFB-treated RAW 264.7 cells. These results provide insight into the mechanisms involved in DNFB-induced contact hypersensitivity.

Rubratoxin B induces interleukin-6 secretion in mouse white adipose tissues and 3T3-L1 adipocytes

Rubratoxin B is a mycotoxin that causes hepatic fatty changes. We examined whether white adipose tissue (WAT) contributes to rubratoxin B toxicity through effects on interleukin (IL)-6. Rubratoxin B was intraperitoneally injected into mice at 1.5 mg/kg. Urinary albumin and macrophage inflammatory protein (MIP)-2 secretion were increased 24 h after treatment with rubratoxin B. Rubratoxin B was previously reported to induce IL-6 secretion, although the secreting tissue was unknown. Here, rubratoxin B prominently augmented IL-6 transcription in epididymal WAT and to a lesser extent in perirenal WAT and liver. Rubratoxin B may thus exert its toxicity partly through IL-6 secretion from WATs. In contrast, MIP-2 gene expression increased only in liver. To examine the specific involvement of adipocytes, we used mouse 3T3-L1 cells, an in vitro differentiation model of adipocytes. Expression of IL-6 and MIP-2 mRNA in 3T3-L1 adipocytes after 24 h of rubratoxin B treatment increased dose-dependently. Rubratoxin B also increased IL-6 and MIP-2 secretion from 3T3-L1 adipocytes. The increase in IL-6 secretion was markedly higher than the increase in IL-6 gene transcription, indicating that rubratoxin B-induced secretion of IL-6 from 3T3-L1 adipocytes is chiefly controlled post-transcriptionally. Rubratoxin B is thus the first mycotoxin known to exert its toxicity through effects on WATs.

Both the NADPH oxidase and the mitochondrial respiratory chain regulate chemokine gene expression.

Alveolar macrophages are involved in the pathogenesis of immune and inflammatory disorders of the lung. One hallmark of the cellular response to inflammation is the recruitment of neutrophils to sites of inflammation. The generation of chemotactic proteins, such as MIP-2, triggers neutrophil recruitment. We have previously shown that alveolar macrophages spontaneously generate high levels of ROS, especially O2.−-, and their high catalase and glutathione peroxidase activity limits the effectiveness of H2O2 to act as a signaling mediator. In this study, we ask if O2.− generation in alveolar macrophages had a role in the expression of inflammatory genes. Specifically, we hypothesized that O2.− generation is necessary for MIP-2 gene expression in alveolar macrophages after TNF-α stimulation. We found that TNF-α increased O2.− generation by activating both NADPH oxidase and the mitochondrial respiratory chain in alveolar macrophages. TNF-α-induced O2.− generation was necessary for MIP-2 gene expression because inhibition of NADPH oxidase, the mitochondrial respiratory chain, or over expression of SOD significantly inhibited expression of MIP-2. In addition, TNF-α-induced NADPH oxidase and mitochondrial respiratory chain activity were necessary for ERK MAP kinase activation, and ERK activity was essential for MIP-2 gene expression. Furthermore, over expression of a constitutive active MEK1, the upstream kinase that activates ERK, significantly increased TNF-α-induced chemokine gene expression. These results suggest that in alveolar macrophages, O2.− generation from both NADPH oxidase and the mitochondrial respiratory chain mediates MIP-2 gene expression after TNF-α stimulation in an ERK-dependent manner.

Pyrrolidine dithiocarbamate-induced macrophage inflammatory protein-2 gene expression is NF-κB-independent but c-Jun-dependent in macrophage cell line RAW 264.7

Pyrrolidine dithiocarbamate (PDTC) is a stable compound that acts as antioxidant or prooxidant, and is widely used to inhibit the activation of NF-κB. PDTC was also reported to activate NF-κB depending on its dose and metal ions in PC12 cells. In this work, we demonstrated a working mechanism of PDTC and its effects on the proinflammatory cytokine gene expression in a mouse macrophage cell line, RAW 264.7. PDTC alone induced NF-κB-independent MIP-2 promoter activation that can be assessed by transient transfection and confocal image analysis. The involvement of AP-1 transcription factor was noticed by promoter deletion/site-specific mutation analysis and electrophoretic mobility shift assay (EMSA). Among three different mitogen-activated protein kinase (MAPK) pathways tested, only the stress-activated protein kinase (SAPK)/Jun N-terminal kinase (JNK) pathway was significantly activated in RAW 264.7 cells after the stimulation with PDTC. Using pathway-specific inhibitors, we found that the SAPK/JNK pathway is clearly associated with PDTC-induced MIP-2 gene expression. Our experimental results indicate that PDTC-induced proinflammatory cytokine expressions are mediated by SAPK/JNK pathway, which activates AP-1.

Transcriptional and Translational Regulation of Inflammatory Mediator Production by Endogenous TGF-β in Macrophages That Have Ingested Apoptotic Cells

Abstract We recently reported that phagocytosis of apoptotic cells inhibits the release of inflammatory cytokines by human macrophages. In this paper we show that apoptotic cell uptake by mouse J774 macrophages also inhibits the synthesis and secretion of the chemokines, macrophage inflammatory protein-2 (Mip-2), KC, and Mip-1α (but not that of monocyte chemoattractant protein-1 (MCP-1)/JE), and increases TGF-β formation. Anti-TGF-β neutralizing Abs largely reversed the inhibitory effect of apoptotic cell uptake, and accordingly, exogenous TGF-β down-regulated the synthesis of the same mediators. Apoptotic cell ingestion or TGF-β also inhibited Mip-2 and Mip-1α gene expression in LPS-treated J774 cells, whereas TNF-α mRNA levels were unaffected. Importantly, TGF-β pretreatment of J774 cells did not significantly alter chemokine and TNF mRNA stability. Finally, we found that apoptotic cell uptake and TGF-β did not modulate NF-κB or AP-1 DNA binding in J774 cells. We conclude that the decreased production of chemokines and TNF resulting from apoptotic cell ingestion is largely mediated by a common event, i.e., feedback inhibition by endogenous TGF-β, but involves different mechanisms. Whereas TNF-α production appears to be translationally down-regulated, the suppression of most chemokines investigated appears to reflect transcriptional inhibition. In a broader context, the impairment of chemokine and TNF generation by apoptotic cell uptake might represent an important mechanism contributing to the resolution of inflammation. An additional consequence could be the selective recruitment of monocytes into inflammatory sites, as MCP-1/JE production by mouse macrophages was unaffected by apoptotic cell uptake, in contrast to other chemokines.

Regulation of macrophage inflammatory protein-2 gene expression by oxidative stress in rat alveolar macrophages.

Chemokines are important mediators in the development of inflammation. Our previous work demonstrated that an oxidative stress can up-regulate mRNA expression of a CC chemokine macrophage inflammatory protein (MIP)-1alpha in rat alveolar macrophages. In the present study, we further investigate whether an oxidative stress can regulate the gene expression of a related CXC chemokine MIP-2, involved in both neutrophil chemotaxis and activation. A rat alveolar macrophage cell line (NR8383) was exposed to 10 microg/ml bacterial lipopolysaccharide (LPS) and MIP-2 mRNA levels dramatically increased after 4 hr of stimulation. This increase by LPS was attenuated by co-treatment with the antioxidants N-acetylcysteine and dimethylsulphoxide, suggesting that the induction of MIP-2 mRNA is mediated via the generation of reactive oxygen species. To assess directly the role of oxidative stress on regulation of MIP-2 mRNA expression, macrophages were exposed to H2O2. MIP-2 mRNA levels had significantly increased after 1 hr exposure to 0.5 mm H2O2, were maximally increased after 4 hr and decreased after 6 hr. Co-treatment of macrophages with the transcriptional inhibitor actinomycin D eliminated the H2O2-induction of MIP-2 mRNA, implicating a role for transcriptional activation in increased expression of MIP-2. Genomic cloning of the rat MIP-2 gene 5'-flanking region has identified a consensus nuclear factor-kappaB (NF-kappaB) binding site. Gel-mobility shift assays revealed NF-kappaB binding to the MIP-2 promoter/enhancer sequence was induced by H2O2. LPS treatment for 4 hr also significantly activated NF-kappaB binding, which could also be attenuated by pretreatment with N-acetylcysteine at the doses that reduced MIP-2 mRNA expression. The half-life of MIP-2 mRNA transcripts was also increased by H2O2 treatment. These observations indicate that MIP-2 gene expression is subject to both transcriptional and post-transcriptional control in response to an H2O2 oxidative stress.

Crocidolite activates NF-kappa B and MIP-2 gene expression in rat alveolar epithelial cells. Role of mitochondrial-derived oxidants.

Nuclear factor kappa B (NF-kappa B) is a transcription factor that regulates expression of several genes coding for inflammatory and immunoregulatory proteins including the neutrophil chemotactic cytokine MIP-2. In previous studies we found that crocidolite asbestos activates the nuclear translocation of NF-kappa B as well as MIP-2 gene expression in rat alveolar type II cells. Here we report that both crocidolite-induced NF-kappa B activation of MIP-2 gene expression can be attenuated by the antioxidant tetramethylthiourea, suggesting the dependence of these responses on oxidative stress. Crocidolite exposure of RLE-TN cells also increased production of H2O2, a response that was inhibited by the mitochondrial respiratory chain inhibitor thenoyltrifluoroacetone (TTFA). TTFA treatment of RLE-6TN cells also inhibited crocidolite-induced nuclear translocation of NF-kappa B and MIP-2 gene expression. These results indicate crocidolite exposure of rat alveolar type II cells results in increased production of mitochondrial-derived hydrogen peroxide and that mitochondrial-derived oxidants contribute to crocidolite activation of NF-kappa B and increases in MIP-2 gene expression.

Differential expression of macrophage inflammatory protein-2 and monocyte chemoattractant protein-1 in experimental glomerulonephritis

We examined the relation between glomerular expression of chemokines from alpha-subfamily (macrophage inflammatory protein-2, MIP-2) and beta-subfamily (monocyte chemoattractant protein-1, MCP-1) and infiltration of neutrophils and monocytes in antibody mediated glomerulonephritis in rats. In the accelerated model of nephrotoxic nephritis (NTN), glomerular expression of MIP-2 and MCP-1 genes correlated with the sequential migration of neutrophil and monocyte influx, respectively. These relationships were investigated further in the heterologous phase of NTN by applying various treatments known to modulate the severity of injury. Pretreatment with bacterial lipopolysaccharide resulted in greater injury, MIP-2 expression increased 25- to 50-fold, and the glomerular neutrophil count increased two- to fourfold. Both MIP-2 mRNA levels and neutrophil infiltration were reduced by additional pretreatment with IL-6, IL-1 receptor antagonist, soluble IL-1 receptor or soluble TNF receptor (Spearman correlation coefficient r = 0.897, P < 0.005). In the heterologous phase of NTN, different pre-treatments only resulted in trivial changes in MCP-1 expression and monocyte infiltration. In conclusion, glomerular MIP-2 gene expression correlates with neutrophil infiltration both temporally during the evolution of nephritis, and when glomerular injury is modified by treatment. Glomerular MCP-1 gene expression correlates with monocyte influx. The data show chemokines of alpha- and beta-subfamilies co-operative to cause selective and sequential migration of different leukocyte subsets during development of antibody mediated glomerulonephritis.