(b) Expression of chemokines and cytokines in wounded tissues

(b) Expression of chemokines and cytokines in wounded tissues. Using immunoprecipitation with anti-IgG bound to protein G we found that the intensity of several bands was lower in the serum from splenectomized mice than in that from sham-operated mice. These bands were matched to myosin IIA, carbamoyl-phosphate synthase, argininosuccinate synthase, actin and -actinin-4 by liquid chromatography tandem mass spectrometry analysis. Keywords: wound healing, autoantibodies, rodent, B cells, skin Introduction The wound repair process is a highly ordered sequence of events that encompasses haemostasis, inflammatory cell infiltration, and tissue regrowth and remodelling. First, platelets generate a clot, which stops the bleeding, and serves as a temporary barrier and a source of chemotactic factors. Subsequently, attracted leucocytes initiate an inflammatory response before fibroblasts and endothelial cells migrate to the wound to generate tissue that contracts the wound margins. Finally, epithelial cells complete the repair process by covering the denuded wound surface.1 The function of granulocytes and macrophages in wound healing has been extensively studied. These cells produce cytokines, chemokines, matrix proteins, matrix metalloproteinases and growth factors.2C8 In contrast, there are few reports that describe the effects of T or B cells Isobutyryl-L-carnitine in wound healing except T cells.9,10 In aseptic wound healing, Isobutyryl-L-carnitine very few T or B cells migrate to the wound site. However, immunoglobulin secreted by B cells can reach wound sites. In the case of bacterial infections, immunoglobulin specific for the bacteria bind to the surface and induce bacterial killing by phagocytosis mediated by Fc receptors. In addition to antibodies specific for micro-organisms, antibodies to tissue antigens also reach wound sites. However, until now it has not been Rabbit polyclonal to Cyclin B1.a member of the highly conserved cyclin family, whose members are characterized by a dramatic periodicity in protein abundance through the cell cycle.Cyclins function as regulators of CDK kinases. studied whether antibodies to damaged tissues work to repair wounds. To explore the role of acquired immunity in wound repair, we splenectomized experimentally wounded mice. Interestingly, splenectomy greatly delayed wound healing. Transfer of B cells into splenectomized mice restored the wound repair ability. Further studies indicated the importance of immunoglobulin specific for injured dermal tissues. Materials and methods Mice and splenectomy Two-month-old C57BL/6 (C57BL/6J) female mice and KSN nude mice were purchased from Japan SLC, Inc., Hamamatsu, Japan. Green mice [C57BL/6-Tg(CAG-EGFP) C14-Y01-FM131Osb] were kindly supplied from Riken (Tsukuba, Japan) with the permission of Dr Okabe.11 These mice were maintained in the Animal Research Facility at the Nagoya University Graduate School of Medicine under specific pathogen-free conditions and used according to Isobutyryl-L-carnitine institutional guidelines. Anaesthetized mice were subjected to either a sham operation or splenectomy. Punch biopsy wounding and macroscopic examination After shaving and extensive cleaning with 70% ethanol, the dorsal skin was picked up at the midline and two layers of skin were punched through with a sterile disposable biopsy punch (diameter 3 mm; Kai Industries, Tokyo, Japan). This is an aseptic wound model, which is completely different from pressure ulcers. This procedure generated two excision full-thickness wounds with one on each side of the midline. The same procedure was repeated four times, generating eight wounds on each animal. Each wound site was digitally photographed at the indicated time intervals, and wound areas were determined on photographs using photoshop (version 70; Adobe Systems) and calculated using the area calculated software on Excel. Changes in the area of wound sites are expressed as the proportion of the initial wound areas. In some experiments, wounds and their surrounding areas (including the scab and epithelial margins) were cut for further analyses with a sterile disposable biopsy punch with a diameter of 6 mm (Kai Industries) at the indicated time-points. Histopathological analyses of wound sites Wound specimens were fixed in 2% formaldehyde buffered with phosphate-buffered saline (PBS; pH 72), and then embedded in OCT compound (Sakura Finetechnical Co., Ltd., Tokyo, Japan). Frozen 5-mm sections were stained. The sections were further processed for immunohistochemical analyses to the antigen at the wound area. Fixed slides Isobutyryl-L-carnitine were treated with fluorescein isothiocyanate (FITC)-labelled anti-mouse immunoglobulin G1 (IgG1), FITC-labelled anti-mouse CD4 and phycoerythrin-labelled anti-mouse B220 (BD). Western blotting Tissues were homogenized in buffer [50 mm TrisCHCl pH 68, 2% sodium dodecyl sulphate (SDS), 2 mm sodium ethylenediaminetetraacetic acid (EDTA)], and then lysed in sample buffer (50 mm TrisCHCl pH 68, 2% SDS, 25% glycerol, 5% 2-mercaptoethanol and 005% bromophenol blue). One milligram per millilitre of total protein was resolved by SDSCpolyacrylamide gel electrophoresis (PAGE) and then transferred to polyvinylidene difluoride membranes (Millipore, Tokyo, Japan). Blotted membranes were reacted Isobutyryl-L-carnitine with 1/100 diluted serum. Bound antibodies were detected by the enhanced chemiluminescence system12 (GE Healthcare UK Ltd., Buckinghamshire, UK). Isolation of B cells Isolation of highly pure B cells.