Cutaneous Wound Healing By Antibodies to Injured Tissue

Jameson Frankel^*
*Correspondence: Jameson Frankel, Department of Anesthesiology and Critical Care Medicine, Freie Universität Berlin, Berlin, Germany, Email:

Keywords

Wound healing • Autoantibodies • B cells • Skin

Introduction

The process of healing a wound involves an extremely Vwell-ordered series of actions, including hemostasis, inflammatory cell infiltration, tissue regeneration, and remodelling. First, platelets form a clot that stops the bleeding, acts as a transient barrier and a source of chemotactic chemicals, and it also functions as a temporary barrier. A further inflammatory reaction is then started by attracted leucocytes, followed by the migration of fibroblasts and endothelial cells to the wound to produce tissue that tightens the wound boundaries [1]. Epithelial cells cover the denuded wound surface to finish the repair process.

However, it has not yet been investigated whether antibodies to damaged tissues aid in wound healing. We splenectomized experimentally injured mice to examine the function of acquired immunity in wound healing. Unexpectedly, splenectomy significantly slowed down wound healing. B cell transplantation into splenectomized mice brought back the ability to heal wounds. Additional research revealed the significance of immunoglobulin tailored for damaged cutaneous tissues.

Materials and Methods

KSN nude mice and two-month-old C57BL/6 (C57BL/6J) female mice were bought from Japan SLC, Inc. in Hamamatsu, Japan. With Dr. Okabe's approval, green mice (C57BL/6-Tg(CAG-EGFP) C14-Y01-FM131Osb) were kindly provided by Riken in Tsukuba, Japan. These mice were kept in the Nagoya University Graduate School of Medicine's Animal Research Facility under particular pathogen-free conditions and utilized in accordance with institutional policies. Mice under anesthesia were given either a fake procedure or a splenectomy [2].

Macroscopic inspection and punch biopsy injuring

The dorsal skin was taken up at the midline and two layers of skin were punched through with a sterile disposable biopsy punch after shaving and thorough cleaning with 70% ethanol. This is a model of an aseptic wound, which is quite distinct from a pressure ulcer. Two excision full-thickness wounds were produced by this technique, one on either side of the midline. The identical process was carried out four times, leaving each animal with eight wounds. Each wound site was digitally photographed at the specified intervals, and the areas of each wound were identified on the photos using Photoshop (version 7; Adobe Systems), and the totals were computed using Excel's "area calculated" function [3].

Wound site histopathological analyses

2% formaldehyde buffered with phosphate-buffered saline (PBS; pH 72) was used to fix wound specimens before they were embedded in OCT compound. Five-mm frozen portions were colored. The sections underwent additional processing for immunohistochemical examinations of the antigen at the site of the wound. Fluorescein isothiocyanate (FITC)-labeled anti-mouse immunoglobulin G1 (IgG1), FITC-labeled anti-mouse CD4, and phycoerythrinlabeled anti-mouse B220 were applied to fixed slides.

After being homogenized in buffer (50 mm Tris-HCl pH 6-8, 2% sodium dodecyl sulphate (SDS), 2 mm sodium ethylenediaminetetraacetic acid (EDTA), the tissues were lysed in sample buffer (50 mm Tris-HCl pH 6-8, 2% SDS, 25% glycerol, 5% 2-mercaptoethanol, and 005% bromophenol blue). SDS-polyacrylamide gel electrophoresis (PAGE) was used to separate one milligram of total protein per milliliter, and the resolved protein was then transferred to polyvinylidene difluoride membranes. The 1/100 diluted serum was used to react with blotted membranes. By using an improved chemiluminescence technique, bound antibodies were found [4].

B cell separation

Depletion of magnetically tagged cells allows for the isolation of extremely pure B cells from spleen. Non-B cell contamination, i.e. By suspending spleen cells in a solution containing the B Cell Isolation kit (Miltenyi Biotec, Tokyo, Japan) and a combination of biotin-conjugated antibodies against CD43 (Ly- 48), CD4 (L3T4), and Ter-119, as well as Anti-Biotin MicroBeads, T cells, B cells, natural killer cells, dendritic cells, macrophages, granulocytes, and By keeping the magnetically labeled non-B cells on a MACS® Column (LS column) in the magnetic field of a MACS Separator while the unlabeled B cells pass through the column, the non-B cells with magnetic labels were reduced in number.

Macrophage phagocytosis

Three days after injecting 1 ml of 3% thioglycollate media into groups of three mice, peritoneal macrophages were extracted by adhering the cells to tissue plates for an overnight period at 37° in 5% CO₂. As previously mentioned, Green mice were given a 0–5 mg intraperitoneal injection of Zymosan to collect their peritoneal neutrophils. Heat treatment at 100° for 10 seconds caused harm to them. At a ratio of one macrophage to one target cell, injured neutrophils (5 105) and macrophages (5 105) were plated. After one hour at 37 degrees in 5% CO₂, the mixture was rinsed with PBS to eliminate any unincorporated target cells. Trypsin/EDTA caused them to separate. A flow cytometer (FACS Calibur, BD, Tokyo, Japan) was used to analyze the cells [5].

Immunoprecipitation

For standard preparations, 50 ml of mouse serum was added to 100 ml of protein G beads (GE Healthcare UK Ltd) in RIPA buffer (25 ml of Tris- HCl pH 8-0, 150 ml of sodium chloride, 10% glycerol, 2 ml of EDTA, 5 ml of magnesium chloride, 0 ml of nonidet-P40, and 1 ml of phenylmethylsulphonyl flu Unbound serum was removed using 1 ml of 015 m sodium borate twice after 1 hour of incubation with rotation at room temperature. Dimethylphosphate (DMP) at a concentration of 20 millimoles was applied to serum-bound protein G beads. The mixture was centrifuged at 400 grams at 4 degrees after 30 minutes of spinning at room temperature. 500 ml of 0 2 m ethanolamine was added after the supernatant was discarded. It was centrifuged once and spun at ambient temperature for two hours. he precipitate was added to 100 μl PBS with 0·05% azide. The tissue sample was taken and homogenized using a Potter homogenizer in RIPA buffer. Non-specific binding of serum to protein G was obtained by the following procedure [6]. Two hundred millilitres of sample was mixed with 50 μl protein G beads in RIPA buffer. After 2 hr incubation with rotation at room temperature for 2 hr, the supernatant was taken by centrifugation. The 200 μl of sample (supernatant) was mixed with 50 μl of protein G beads, which gives bound serum. Immunoprecipitation was carried out at 4° for 16 hr, and beads were washed five times in the immunoprecipitation buffer.

Protein identification

Protein spots were removed from the gel for liquid chromatography tandem mass spectrometry (LC-MS/MS) ion search analysis. The gel fragments were vacuum centrifuged, destained, and dried. The dried gel fragments were rehydrated in 100 mm of ammonium bicarbonate that contained 10 mm of dithiothreitol for carbamidomethyl modification. The gel fragments were alkylated after the solution was removed, and they were subsequently rehydrated in a trypsin digest solution using Trypsin Gold, Mass Spectrometry Grade (Promega Co., Madison, WI)]. Thermo Fisher Scientific, Inc., Waltham, MA's LCQ Advantage nanospray ionization ion-trap mass spectrometer was used in the LC-MS/MS ion search study together with a Michrom BioResources, Inc., Auburn, CA's MAGIC2002TM HPLC System with a MonoCap^® column with a 0-1-mm diameter and 50-mm length [7].

Analysis using the reverse transcription-polymerase chain reaction

TRIzol reagent (Invitrogen, Japan K.K., Tokyo, Japan) was used to isolate total RNA in accordance with the manufacturer's instructions. DNase I treatment (Roche Diagnostics K.K., Tokyo, Japan) was used to digest and extract remaining genomic DNA. For the reverse transcription-polymerase chain reaction (RT-PCR), first-strand complementary DNA (cDNA) was created using the Superscript First-Strand Synthesis System (Invitrogen) and oligo-dT(12-18) primers. With DNase-free water, the cDNA was diluted to a concentration of 10 ng/l. The Ex-Taq PCR kit (TAKARA BIO INC., Shiga, Japan) was used for the RT-PCR in accordance with the manufacturer's instructions [8]. The following primers were used: actin(f) 5′-AGTGTGACGTTGACATCCGT; -actin(r) 5′-GCAGCTCAGTAACAGTCCGC; monocyte chemotactic protein-1 (MCP-1)(f) 5′-TGAATGTGAAGTT GACCCGT; MCP-1(r) 5′-AAGGCATCACAGTCCGAGTC; CCL3(f) 5′-CC.

Discussion

In this research, we have demonstrated how splenectomy delays wound healing. In splenectomized mice, adoptive transfer of spleen cells dramatically recovered the typical kinetics of wound healing. Mice with high cholesterol have been used in similar studies. Splenectomy significantly worsened atherosclerosis in hypercholesterolemic, apoE-deficient animals. Young apoEmice with illness were considerably less likely to develop it after receiving spleen cells from atherosclerotic apoE- mice. Our research and the research by Caligiuri et al. imply that immune responses are not always harmful when atherosclerosis develops, just as they are not always harmful to tissue when wounds are mending.

B lymphocytes' contribution to wound healing has not been thoroughly studied. According to these studies, B lymphocytes are unlikely to have a major impact on the control of wound healing. In all of our experiments, we never found a sizable B cell population in the injured tissue. However, we did see antibodies clinging to injured tissues. These antibodies were produced by B cells found in secondary lymphoid organs including the spleen and lymph nodes. B cells were found to have the ability to heal wounds in experiments with naked mice and concentrated spleen-derived B cells. Atherosclerosis has also been studied in terms of the B-cell protective effects.

To prevent atherosclerosis, T15 anti-phosphorylcholine (anti-PC) antibodies attach to apoptotic cells that exhibit oxidized phospholipid epitopes like OxLDL. However, some anti-OxLDL antibodies have been found to interfere with macrophage scavenging and worsen atherosclerosis. It is technically challenging to determine whether opsonized damaged tissues were adequately phagocytosed and cleared of macrophages. T15 is released from the spleen.The antibodies linked to damaged tissues may stimulate phagocytosis by Fc receptors found on neutrophils and macrophages. Therefore, as phagocytes, we used 3% thioglycollate-induced peritoneal macrophages. The target cells were Green mouse peritoneal zymosaninduced neutrophils. We discovered that macrophages successfully absorbed heated-damaged neutrophils.

Acknowledgments

None.

Conflict of Interest

The authors have no financial conflict of interest.

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