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Oncomodulin and Macrophages Derived Factors in Pancreas Injury and Development Paradigms

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Oncomodulin and Macrophages Derived Factors in Pancreas Injury and Development Paradigms Vol.2, No.1, 1-8 (2013) Modern Research in Inflammation http://dx.doi.org/10.4236/mri.2013.21001 Oncomodulin and macrophages derived factors in pancreas injury and development paradigms Joël Fleury Djoba Siawaya1,2*, Carmen Capito1, Raphael Scharfmann1 1Centre de Recherche “Croissance et Signalisation”, Inserm Unité-845, Faculté de Médecine Paris 5, Paris, France; *Corresponding Author: [email protected], [email protected] 2Laboratoire National de Santé Publique, Unité de Recherche, Libreville, Gabon Received 6 December 2012; revised 14 January 2013; accepted 5 February 2013 ABSTRACT against beta cells appears to be the main cause for the loss of the insulin-producing beta cells leading to insulin Prior studies in the optic nerve injury paradigm deficiency whereas, in type 2 diabetes obesity related showed conflicting data regarding production pro-inflammatory response is involved in the develop- and significance of the Ca2+-binding protein on- ment of insulin resistance. comodulin (OCM). Some have shown its potent Although treatment options for the type 1 diabetes re- axon-regenerative or-growth attribute, where other main principally limited to insulin replacement therapy, showed little to no effect. We show here that today new avenues such as islet cell regeneration, re- pancreas inflammation lead to macrophages in- growth of host own islet cells or islet transplantation are filtration that produce OCM and inflamed tissues being explored. The principal roadblock to the wide- that express OCM receptors in vivo. In culture spread application of these new therapeutic approaches OCM has a cytostatic effect on embryonic pan- creas explants. Secretory products of zymosan- are the molecular mechanisms and relevant growth fac- activated macrophages are cytotoxic and fac- tors implicated in endocrine pancreas development which tors derived from non-activated macrophages are yet to be fully elucidated and which are required seem to promote pancreas development. It is to devise techniques to generate insulin-producing beta our view that OCM is involved in protective in- cells. jury response that allows through metabolism Macrophages secrete a variety of biologically active slowing sub-lethally injured cells to undergo molecules that can have both positive and negative ef- recovery. fects on tissue depending of the circumstances. Macro- phages derived factors have been shown to play and in- Keywords: Macrophages; Oncomodulin; fluence the mechanism leading to injury healing and tis- Inflammation; Pancreas sues regeneration. Recently, it has been shown that fac- tors secreted by tissue-infiltrated macrophages such as oncomodulin (OCM), a small, ~12 kDa calcium-binding 1. INTRODUCTION protein in the parvalbumin family had a regenerative and The pancreas is a gland, with both endocrine and exo- growth promoting effect on retinal ganglion cell axon crine features. The endocrine parts of the pancreas con- [2-4]. Furthermore, the regeneration of beta cells after sist of cell clusters called islets of langerhans. There are injury has lead to the concept that factors derived from four main cell types in the islets: α cells (glucagons se- injury related immune response (or inflammation) might creting cells), β cells (insulin secreting cells), δ cells be involved beta cells progeny differentiation and matu- (somatostatin secreting cells), and PP cells (pancreatic ration [5]. polypeptide secreting cells) [1]. In contrast to the endo- We therefore investigated pancreas injury and the ef- crine pancreas, which secretes hormones the exocrine fect of the macrophages derived factors including Ca2+ pancreas through acinar cells produces alkaline fluid and binding protein oncomodulin (OCM) on pancreas devel- digestive enzymes including trypsin chymotrypsin, pan- opment. Pancreas duodenum homeobox-1 (Pdx-1) that creatic lipase and pancreatic amylase. regulates pancreas development during embryogenesis The immune system is in the core of the mechanisms [6], neurogenin3 (Ngn3) required for the development of leading to the development of both form of diabetes. In endocrine pancreas [7], insulin and amylase were used as type one diabetes T-cell–mediated autoimmune response indicators of effects. Copyright © 2013 SciRes. OPEN ACCESS 2 J. F. Djoba Siawaya et al. / Modern Research in Inflammation 2 (2013) 1-8 2. METHODS 2.3. Macrophage Culture and Production of Macrophages Conditioned Medium 2.1. Partial Duct Ligation (PDL) of Adult Mice Pancreas Macrophages conditioned medium was produced as described by Y. Q. Yin et al. [11] with some adjustment. Pancreas inflammation was induced by partial duct ligation (PDL) of adult BALB/C mice (Centre d’élevage Succinctly, rat alveolar macrophages (NR8383) from René Janvier, Le Genest, France) pancreas as described American Type Cell Culture, (Manassas, VA) were main- in previous reports [5]. tained for few days in 15% fetal calf serum enriched F-12K medium (Life Technologies, Gaithersburg, MD) 2.2. Immunohistochemistry with 1% penicillin-streptomycin at 37˚C in a humidified 5% CO2 incubator. Macrophages were then washed in Immunohistochemistry and quantification—tissues were serum-free F-12K medium and centrifuged down to re- fixed in 10% formalin, pre-embedded in agarose gel (4% move serum components. Resuspended macrophages (in of type VII low gelling temperature agarose (Sigma- F-12K medium) were either were treated by with Zymo- Aldrich)) and embedded in paraffin. Sections (4 μm thick) san (1.25 mg/ml, final concentration incubated over night were collected and processed for immunohistochemistry, at 37˚C in 5% CO2) or were left untreated. Culture su- as described previously [8,9]. Antibodies were used at pernatants were collected, centrifuged (1500 × g) for 10 the following dilutions: rat monoclonal to macrophage min and put through a 0.2 µm low-protein binding filter (Abcam; 1/200), rabbit polyclonal to oncomodulin (Ab- (Pall-Gelman Laboratory, Ann Arbor, MI) before adding- cam; 1/1000), mouse anti-human insulin (Sigma-Aldrich, protease inhibitors (Complete, Roche, Indianapolis, IN). 1/2000), rabbit anti-amylase (Sigma-Aldrich, 1/300), Some of the Zymosan Macrophage-conditioned medium guinea pig anti-insulin (Dako, Trappes, France; 1/500), was fractioned by means of a 3 kDa molecular weight mouse anti-BrdUrd (Amersham Biosciences, Bucking- cutoff ultrafiltration membrane (Amicon/Millipore, Bed- hamshire, UK; 1/2). Enzyme-conjugated secondary an- ford, MA) and stored at −80˚C until it was use. tibody was: HRP-conjugated anti-rat anti-body. The fluo- rescent secondary antibodies were: fluorescein isothio- 2.4. Pancreas Explants Procurement and cyanate anti-rabbit antibody (Jackson Immuno research, Culture Baltimore, MD; 1/200), Texas Red anti-mouse antibody (Jackson ImmunoResearch; 1/200) and Alexa Fluor anti- Pregnant Wistar rats were obtained from Janvier rabbit antibody (Biogenex, San Ramon, CA; 1/400). Nu- breeding center (Centre d’élevage René Janvier, Le clei were stained in blue using Hoechst 33342 (0.3 μg/ml, Genest, France) and embryos harvested at E13. Embryos Invitrogen). The diaminobenzidine (DAB) was used as were then dissected and embryonic dorsal pancreas re- substrate for HRP-conjugated anti-body. Photographs were trieved as previously described [8]. Dorsal pancreatic taken using a fluorescence microscope (Leica, Leitz explants were laid on white 0.45 M FHLC membrane DMRB, Reuil-Malmaison, France) and digitized using a (Millipore, St-Quentin-en-Yve-lines, France) at the air- Hamamatsu (Middlesex, NJ) C5810 cooled 3CCD camera. liquid interface in sterile multi-wells tissue culture plate Oncomodulin ligand binding assay (adapted from- (Becton Dickinson, Le Pont de Calais, France), contain- Caroline Brennan and Jez Fabes protocol, Science’s ing 2 ml of 1) control medium (F-12K modified medium STKE 2003) [10]. enriched 15% of fetal calf serum and supplemented with Slides of snap freeze dissected adult mice pancreas penicillin-streptomycin (1%), Hepes (0.01 M), non-es- (inflamed and non-inflamed) were rehydated for ten sential amino acids (1 X)); 2) control medium + OCM minutes and incubated for with alkaline phosphatase on- (10 nM); 3) control medium + forskolin (10 μM); 4) comodulin fusion protein (AP-OCM), alkaline phos- control medium + OCM (10 nM) + Forskolin; 5) non- phatase (AP) or with an OCM receptor ligand and OCM activated macrophages conditioned medium; 6) Zymosan competitor (PEP) was used to verify the specificity of activated macrophages conditioned medium. For culture AP-OCM to OCM receptor binding. After washing (with macrophages conditioned mediums were supplemented HBS), tissues were fixed for 90 seconds (acetone-for- as we did control for control medium. Cultures were maldehyde fixative), washed again and incubated at 65˚C maintained at 37˚C in humidified 95% air, 5% CO2. Fol- for one hour to reduce endogenous AP activity. Slides lowing culture, dorsal pancreatic explants were harvested were then rinsed in AP reaction buffer (Tris-HCL: 100 for RNA extraction. mM; NaCl: 100 mM and MgCl2: 50 mM in distilled wa- ter) and incubated with AP staining buffer (nitro blue 2.5. RT-PCR and Q-PCR tetrazolium chloride/5-bromo-4-chloro-3-indoyl phos- phate toluidine salt (BCIP/NBT)). To determine the effect of macrophages derived fac- Copyright © 2013 SciRes. OPEN ACCESS J. F. Djoba Siawaya et al. / Modern Research in Inflammation 2 (2013) 1-8 3 tors on dorsal pancreas growth, transcript levels of Ngn3+, parison Test. A p-value of 0.05 was judged significant. Pdx-1 insulin1, amylase and DNA
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