Immune reactions of lymphocytes and macrophages against PEG-grafted pancreatic islets
Introduction
Insulin-dependent diabetes mellitus (IDDM), which results from an autoimmune destruction of insulin-producing β-cells in pancreatic islet, requires exogenous insulin to maintain normal blood glucose level in the body [1]. Compared to insulin injection or insulin pump, transplantation of pancreatic islets or whole pancreas has emerged as a means to maintain euglycemia because it can provide almost perfect and immediate control of blood glucose level. More specifically, islet transplantation has more advantages than whole pancreatic transplantation; it can reduce immunogenicity by having the graft pretreated, and can also be carried out by a simple portal vein injection of islets owing to their small volume [2], [3], [4]. Therefore, transplantation of pancreatic islets has been proposed as a potential treatment for diabetic patient.
However, transplanted islets are recognized as antigens by host and they trigger the process of recruiting and activating immune cells such as macrophages, fibroblast, granulocytes and lymphocytes. The activated immune cells secrete various cytokines and cytotoxic molecules, which can induce functional and structural damage to islets [5], [6], [7], [8]. Interleukin (IL)-1 activates T-cells, and this results in the increased production and expression of IL-2 and IL-2 receptor (IL-2R), which are considered as the major components in immune rejection. Selective prevention of the IL-2/IL-2R interaction using the IL-2R binding agent prolongs allograft survival [9], [10]. TNF-α increases adhesion molecules on the grafted tissue, thereby stimulating graft rejection [11]. In addition, active nitric oxide (NO) radical generated by activated macrophages also act as a cytotoxic factor [12].
The surface modification by grafting polyethylene glycol (PEG) has been widely used to improve the biocompatibility of implanted medical devices [13], [14], [15]. When PEG was conjugated with a polypeptide or protein, it could shield the protein's surface, thereby preventing the approach of antibody or immune cells and reducing the degradation by proteolytic enzymes [16], [17], [18]. To protect the transplanted islets from host's immune systems in the islet transplantation, the use of PEG such as interfacial photopolymerization using PEG–diacrylate and chemical grafting using protein-reactive PEG–isocyanate [19], [20], [21] were there.
In our previous study, we showed that PEG molecules grafted onto the collagen capsule of islet did not affect the viability and functional activity of islets [22], with the hypothesis that PEG molecules grafted onto the islet collagen capsule might protect the islets from the immune system by inhibiting the activation of immune cells and reducing the release of cytotoxic molecules. In this study, therefore, PEG-grafted islets were cultured with peritoneal macrophages and splenic lymphocytes in order to determine whether PEG-grafted islets could prevent activation of immune cells and to investigate which factors were responsible for the viability of allogeneic islets in vitro.
Section snippets
Synthesis of PEG–SPA
To graft PEG onto islets, monomethoxy-poly(ethylene glycol)–succinimidyl propionate (mPEG–SPA) was synthesized as described in our previous study [22]. Briefly, mPEG (50 g; MW 5,000; Fluka Chemical Co., Switzerland) was reacted with potassium butoxide (3.65 g; Fluka) in toluene at 80°C for 6 h. After cooling down to room temperature, 5 ml of ethylbromo propionate (Aldrich Chemical Co. Milwaukee, WI) was added into the solution and stirred for 20 h. After removing potassium bromide salt, the reacted
Co-culture of islets with splenic lymphocytes
The immunoprotective effect of grafted-PEG on the islet capsule was determined by co-culture of islets with lymphocytes. When free islets were cultured with lymphocytes (5×105 cells), free islets were destroyed after 3 days and they completely lost the integrity of collagen capsule of islet, as shown in Fig. 1. The viability of free islets was 7.7±2.1% after 7 days of the co-culture (Fig. 2). For PEG-grafted islets, however, the islet morphology was not changed by co-culture with lymphocytes.
Discussion
In this study, PEG was covalently coupled with the amine groups of collagen capsule of islets at the physiological pH essential for the viability of the cells, thereby forming a stable amide linkage. PEG was not diffused into the islet but grafted on collagen capsule of islets under the conjugation condition of PEG, and the grafted PEG could not interact with inner cells of islet. PEG-grafted islets were cultured with lymphocytes or macrophages to determine whether the grafted PEG molecules
Acknowledgments
This study was supported by Leading Researchers Project from KRF in Korea.
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Equally qualified as first authors.