Protective effect of milk fat globule-epidermal growth factor-factor VIII after renal ischemia-reperfusion injury in mice.

OBJECTIVES: Renal ischemia-reperfusion injury causes acute renal failure, and the hallmarks of renal ischemia-reperfusion injury are inflammation, apoptosis, necrosis, and capillary dysfunction. Milk fat globule-epidermal growth factor-factor VIII (MFG-E8), a membrane-associated secretory glycoprotein, is produced by immune cells and reported to participate in multiple physiologic processes associated with tissue remodeling. We have recently shown that MFG-E8 treatment attenuates organ injury, inflammatory responses, and survival after sepsis through the enhancement of phagocytosis of apoptotic cells. The purpose of this study was to determine whether administration of MFG-E8 attenuates renal ischemia-reperfusion injury. DESIGN: Prospective, controlled, and randomized animal study. SETTING: : A research institute laboratory. SUBJECTS: Male C57BL/6J mice (20-25 g). INTERVENTIONS: : Renal ischemia-reperfusion injury with bilateral renal pedicle clamping for 45 mins, followed by reperfusion. A recombinant murine MFG-E8 (0.4 µg/20 g) was given intraperitoneally at the beginning of reperfusion. MEASUREMENTS AND MAIN RESULTS: MFG-E8 levels, organ injury variables, inflammatory responses, histology, apoptosis, and capillary functions were assessed at 1.5 and 20 hrs after reperfusion. A 60-hr survival study was conducted in MFG-E8 and recombinant murine MFG-E8-treated wild-type mice. After renal ischemia-reperfusion injury, MFG-E8 mRNA and protein expressions were significantly decreased in the kidneys and spleen. Treatment with recombinant murine MFG-E8 recovered renal dysfunction, significantly suppressed inflammatory responses, apoptosis, necrosis, and improved capillary functions in the kidneys. In the survival study, MFG-E8 mice showed a significant deterioration and, in contrast, recombinant murine MFG-E8-treated wild-type mice showed a significant improvement of survival compared with vehicle-treated wild-type mice. CONCLUSIONS: MFG-E8 can be developed as novel treatment for renal ischemia-reperfusion injury. This protective effect appears to be mediated through the enhancement of apoptotic cell clearance and improvement of capillary functions in the kidneys.

MFG-E8 attenuates intestinal inflammation in murine experimental colitis by modulating osteopontin-dependent alphavbeta3 integrin signaling.

MFG-E8 (milk fat globule-epidermal growth factor 8) deficiency is strongly associated with acquisition of immune-mediated disorders due to the loss of tissue homeostasis. However, comparatively little is known regarding its functions in gastrointestinal tract disorders, in which immune homeostasis is a major concern. Herein, we report altered MFG-E8 expression in inflamed colons during the acute phase of murine experimental colitis and found that treatment with recombinant MFG-E8, but not its arginine-glycine-aspartate mutant counterpart, ameliorated colitis by reducing inflammation and improving disease parameters. To reveal the MFG-E8-mediated antiinflammatory mechanism, we employed an in vitro system, which showed the down-regulation of NF-kappaB in an LPS-dependent manner. Additionally, MFG-E8 altered alpha(v)beta(3) integrin-mediated focal adhesion kinase phosphorylation by impeding the binding of one of its potent ligands osteopontin, which becomes activated during colitis. Taken together, our results indicated that MFG-E8 has a novel therapeutic potential for treatment of colitis.

Decreased production of interleukin-10 and transforming growth factor-ß in Toll-like receptor-activated intestinal B cells in SAMP1/Yit mice.

A unique subset of B cells expressing interleukin-10 (IL-10) and transforming growth factor-ß (TGF-ß) plays an essential role in preventing inflammation and autoimmunity. We investigated the presence of this cell subset in intestines and its role in the pathogenesis of ileitis using SAMP1/Yit and age-matched control AKR/J mice. Mononuclear cells were isolated from mesenteric lymph nodes (MLNs) and the expressions of B220, CD1d, CD5, Toll-like receptor 4 (TLR4) and TLR9 in isolated cells were analysed. Purified B cells were stimulated with lipopolysaccharide (LPS) or CpG-DNA, then IL-10 and TGF-ß(1) expressions were examined by enzyme immunoassay and flow cytometry. Production of IL-1ß by TLR-mediated macrophages co-cultured with or without purified MLN B cells from SAMP1/Yit and AKR/J mice was evaluated. In addition, interferon-γ (IFN-γ) production in intestinal T cells co-cultured with MLN B cells were also assessed in SAMP1/Yit and AKR/J strains. The production levels of IL-10 and TGF-ß(1) stimulated by LPS and CpG-DNA were significantly lower in B cells separated from MLNs from the SAMP1/Yit strain. B cells expressing IL-10 and TGF-ß(1) were mainly located in a population characterized by the cell surface marker CD1d(+) . Interleukin-1ß production by TLR-activated macrophages co-cultured with MLN B cells from SAMP1/Yit mice was significantly higher than that of those from AKR/J mice. Interestingly, IFN-γ production by T cells was noted only when they were co-cultured with SAMP1/Yit but not the AKR/J B cells. These results are the first to show that disorders of regulatory B-cell function under innate immune activation may cause disease pathogenesis in a murine model of Crohn's disease.

Fibroblast-derived HB-EGF promotes Cdx2 expression in esophageal squamous cells.

The molecular basis of attaining columnar phenotype in Barrett's esophagus is poorly understood. One hypothesis states that factors locally produced by cells of mesenchymal origin in chronic reflux esophagitis induce metaplastic transformation. This study was performed to elucidate the factors secreted from fibroblasts that cause columnar phenotype in adjacent squamous epithelium. Human fibroblast cells were exposed to acidified medium for 20 min, followed by medium neutralization for 2 h, and then total RNA was hybridized to Sentrix Human-6 Expression BeadChips. Furthermore, esophageal mucosal biopsy specimens from reflux esophagitis patients were examined for HB-EGF expression using immunohistochemistry. In addition, cells from the human esophageal squamous epithelial cell line HET1A were treated with recombinant HB-EGF, and changes in expressions of Cdx2 and columnar markers were analyzed. The gene expression profile revealed significant upregulation of a variety of growth factors and inflammatory chemokines in response to acid exposure. Among them, HB-EGF was upregulated more than 10-fold. Biopsy specimens from reflux esophagitis patients showed a strong expression of HB-EGF in fibroblast cells underlying the damaged epithelium. Furthermore, in vitro stimulation of HET1A cells with HB-EGF increased Cdx2 in dose-dependent manners. Functional analysis of human Cdx2 promoter also revealed its upregulation by HB-EGF stimulation, showing the role of potential responsive elements (AP-1 and NF-kappaB) for its transcriptional activation. Moreover, the columnar markers cytokeratin 7 and villin were also upregulated by HB-EGF stimulation. HB-EGF induces several genes characteristics of columnar phenotypes of esophageal squamous epithelium in a paracrine manner.

Exacerbation of indomethacin-induced small intestinal injuries in Reg I-knockout mice.

Nonsteroidal anti-inflammatory drug (NSAID)-induced small intestinal injuries are serious clinical events and a successful therapeutic strategy is difficult. Regenerating gene (Reg) I protein functions as a regulator of cell proliferation and maintains intercellular integrity in the small intestine. The aim of this study was to evaluate the role of Reg I in NSAID-induced small intestinal injuries. First, to examine the effect of Reg I deficiency on such injuries, indomethacin, a widely used NSAID, was injected subcutaneously into 10-wk-old male Reg I-knockout (Reg I(-/-)) and wild-type (Reg I(+/+)) mice twice with an interval of 24 h, after which the mice were euthanized. Small intestinal injuries were assessed by gross findings, histopathology, and contents of IL-1beta and MPO in the experimental tissues. Next, we investigated the therapeutic potential of Reg I in indomethacin-induced small intestinal injuries. Recombinant Reg I protein (rReg I) was administered to 10-wk-old male ICR mice, then indomethacin was administered 6 h using the same protocol as noted above, after which small intestinal injuries were assessed after euthanasia. Our results showed that Reg I(-/-) mice had a greater number of severe small intestinal lesions after indomethacin administration. Histological examinations of the small intestines from those mice revealed deep ulcers with prominent inflammatory cell infiltration, whereas the mucosal content of proinflammatory agents was also significantly increased. In addition, rReg I administration inhibited indomethacin-induced small intestinal injuries in ICR mice. In conclusion, Reg I may be useful as a therapeutic agent in NSAID-induced small intestinal injuries.

Heparin-binding EGF-like factor augments esophageal epithelial cell proliferation, migration and inhibits TRAIL-mediated apoptosis via EGFR/MAPK signaling.

OBJECTIVE: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) has been shown to stimulate the growth and migration of human keratinocytes in an autocrine or paracrine manner. Bearing in mind the preceding narratives, present study was designed to explore the role of HB-EGF on esophageal epithelial cell growth, migration and anti-apoptosis. MATERIAL AND METHODS: HET-1A and TTn cells were treated with recombinant HB-EGF, and cell proliferation and migration were assessed by MTT and Boyden chamber assays, respectively. Anti-apoptotic effects of HB-EGF was studied by Bcl-2/Bcl-xL gene expression and utilizing a TNF-related death apoptosis inducing ligand (TRAIL). RESULTS: Recombinant HB-EGF promotes human esophageal epithelial cell proliferation in a dose dependent manner, where 1 and 10 ng/ml doses were found to be most effective. HB-EGF induced cell migration was noted in TTn, but not in HET-1A cells. Recombinant HB-EGF induced the Bcl-2, Bcl-xL mRNA/protein expression in HET-1A and TTn cells. TRAIL induced the apoptosis in TTn, whereas it was significantly inhibited in HB-EGF treated conditions. Finally, we also revealed HB-EGF induced phosphorylation of EGFR and p38 MAPK in those cell lines, while all cellular functions were repressed by EGFR inhibitor AG1478. CONCLUSION: HB-EGF promotes esophageal epithelial cell proliferation, migration and induces anti-apoptotic gene expression via EGFR/p38 MAPK phosphorylation.

Prolactin induces MFG-E8 production in macrophages via transcription factor C/EBPbeta-dependent pathway.

The lactogenic hormone prolactin (PRL) regulates milk protein gene expression in mammary glands. To maintain homeostatic balance in the body, milk fat globule epidermal growth factor 8 (MFG-E8) is vital for phagocytic clearance of apoptotic cells. We investigated the effects of PRL on MFG-E8 expression in macrophages by evaluating its promoter function. Macrophages were stimulated with PRL, and the expression of MFG-E8 was determined using real-time PCR and Western blotting. The role of MFG-E8 on phagocytosis of apoptotic cells in PRL-treated macrophages was assessed using microscopy, while the response of PRL to MFG-E8 expression was evaluated using luciferase assay. Following treatment with PRL, significant up-regulations of the PRL receptor and MFG-E8 were observed in macrophages, though PRL-treated macrophages more efficiently engulfed apoptotic cells. The results of MFG-E8 promoter analysis showed considerable up-regulation of promoter activity in macrophages following PRL treatment and results from mutation analysis of the MFG-E8 promoter suggested that the C/EBPbeta binding site was responsible for PRL-induced activation of the MFG-E8 promoter. C/EBPbeta activity was found to be up-regulated in PRL-treated cells as revealed by an electrophoretic mobility shift assay (EMSA). In conclusion, PRL is a potent inducer of MFG-E8 expression in macrophages, while its effect is mediated by the presence of a responsive element in the MFG-E8 promoter.

Intrarectal administration of milk fat globule epidermal growth factor-8 protein ameliorates murine experimental colitis.

Milk fat globule epidermal growth factor-8 (MFG-E8) promotes phagocytic clearance of apoptotic cells to maintain normal tissue homeostasis. However, its functions in intestinal inflammatory disorders are unknown. Since the pathogenesis of those disorders are due to abnormal interactions between intestinal epithelial cells (IECs) and microbial pathogens, we analyzed the effects of MFG-E8 on IECs to determine its protective role in murine experimental colitis. Expression of αvß3-integrin in Colon-26 cells was examined by RT-PCR and immunostaining. Colon-26 cells were pretreated with recombinant wild-type and mutant MFG-E8 proteins, following stimulation with flagellin as an inducer of innate immunity, and the effects of the recombinant proteins on inhibition of nuclear factor-κB (NF-κB) and inflammatory cytokine production in flagellin-stimulated Colon-26 cells were determined using a luciferase assay and EIA, respectively. Experimental colitis was induced in mice by intrarectal administration of trinitrobenzene sulfonic acid (TNBS). Recombinant proteins were then intrarectally administered into TNBS-induced colitic mice, after which disease activity parameters (body weight, colon length, histological score), as well as interleukin (IL)-6 and MIP-2 levels were determined in inflamed tissues. Flagellin-induced inflammatory cytokine production in vitro was significantly downregulated via αvß3-integrin following pretreatment with wild-type MFG-E8 due to inhibition of NF-κB activation. In vivo, intrarectal treatment with wild-type MFG-E8, but not its mutant counterpart, significantly inhibited body weight loss, colon shortening and histological inflammation induced by TNBS administration. Our findings suggest that MFG-E8 has anti-inflammatory effects on flagellin-induced inflamed intestinal epithelial cells and may be a useful therapeutic agent for colitis.