Elsevier

Biomaterials

Volume 25, Issue 17, August 2004, Pages 3663-3669
Biomaterials

Immune reactions of lymphocytes and macrophages against PEG-grafted pancreatic islets

https://doi.org/10.1016/j.biomaterials.2003.10.062Get rights and content

Abstract

Graft rejection is the major limiting factor in islet transplantation and is closely related with the recruitment and activation of T cells and macrophages against the graft. To reduce the immunogenicity of islets, we have grafted biocompatible polyethylene glycol (PEG) onto the collagen capsule of islets without changing the morphology and function of islets. In this study, we evaluated whether the grafted PEG molecules on the collagen capsule of islet could prevent the activation of immune cells, and investigated factors that are mainly related to the immune reaction in vitro. During the co-culture with lymphocytes, the morphology and viability of PEG-grafted islets were not damaged, and the amounts of IL-2 and TNF-α secreted from lymphocytes co-cultured with PEG-grafted islets were significantly lower than that of free islets. However, when both kinds of islets were cultured with macrophages, there were no significant differences in morphology, viability and the secreted amounts of cytokines and nitric oxide. In conclusion, the grafted PEG could inhibit activation of lymphocytes, which are essential in initiating the graft rejection process. However, the grafted PEG molecules could not completely prevent the infiltration of cytotoxic molecules into the 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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