Activated T lymphocytes from patients with high risk of type I diabetes mellitus have different ability to produce interferon-γ, interleukin-6 and interleukin-10 and undergo anti-CD95 induced apoptosis after insulin stimulation

Abstract

Type I Diabetes mellitus (DM1) is the effect of T cell dependent autoimmune destruction of insulin producing beta cells in the pancreas islet. T cells are activated in response to islet dominant autoantigens, the result being the development of DM1. Insulin is one of the islet autoantigens responsible for activation of T lymphocyte functions, inflammatory cytokine production and development of DM1. The experiments reported in this study have shown the spontaneous increase of CD95 molecule expression on lymphocytes of the first-degree relatives of DM1 patients. The autoantigen insulin is responsible for stimulation in vitro of potentially hazardous ‘memory’ lymphocytes to produce interleukin-6 (IL-6) and interleukin-10 (IL-10) interleukins. Insulin induced stimulation of lymphocytes in vitro was observed in patients at high risk of developing diabetes mellitus (prediabetics). Phytohaemagglutinin (PHA) stimulates lymphocytes of all groups in the same way. Stimulated lymphocytes in second cultures undergo apoptosis induced with anti-Fas specific antibodies. The deletion in vitro of resting peripheral lymphocytes is nonfunctional. Insulin activated T lymphocytes, which undergo apoptosis were not observed in peripheral blood of healthy people and in patients with DM1. This observation suggests that insulin is involved as autoantigen in DM1 progression in patients with high risk of diabetes type I. The autoreactive T lymphocytes may persist in peripheral blood of patients with high risk DM1. Defective elimination of autoreactive T cells may result in autodestructive damage of islets β cells in the prediabetic stage and disease progression to DM1.

Introduction

Apoptosis has been recognized as a basic phenomenon that ensures proper differentiation of lymphocytes and elimination of autoreactive cells. This mechanism is thought to maintain tolerance and limit ongoing immune response [1]. Persistence of autoreactive T cells may be responsible for autoimmune diseases. Type I diabetes is an organ specific chronic autoimmune disease resulting from pleiotropic T cell mediated destruction of pancreatic cells. The infiltration of the islets of pancreas (insulitis) with mononuclear cells leads to the damage of β cells and causes insulin deficiency. Clinically this type of diabetes is defined as type I diabetes mellitus (DM1) or traditionally insulin dependent diabetes mellitus [1], [2]. Pancreatic β cells (which are especially prone to this form of injury) may be destroyed as an effect of IL-1 mediated Fas expression on the islet cells and thus FasL mediated apoptosis [2]. The resulting activation of islet resident macrophages and local nitric oxide production as well as reactive oxygen intermediates production is also responsible for nonimmunological cytotoxic damage of target islet cells [3]. The transgenic expression of interferon-γ (IFN-γ) in the islets of mice can generate an autoreactive T cell repertoire responsible for breaking tolerance against islets antigens in mice that are not the subject of autoimmune diabetes [4].

Islet specific T cells constitute the major components of insulitis infiltrates in humans and NOD mice. Mature T lymphocytes express Fas (CD95/APO-1) molecules. Their expression can be significantly enhanced upon activation of the T cell by the antigen or inflammation and thus the cells become more sensitive to FasL mediated apoptosis [5], [6], [7]. Defective regulation of leukocyte apoptosis may be a factor which contributes to the pathogenic mechanism of autoimmune disease [5]. The major autoantigens in DM1 are proinsulin, insulin and glutamic acid decarboxylase (GAD) [8]. They all may induce antigen dependent clone expansion of antigen specific T cells [9], [10], [11]. In an IL-10 transgenic model, IL-10 enhances the accumulation of CD8 T cells in the pancreas leading to an earlier onset of diabetes in nonobese diabetic mice [12]. The autoantigens can act as effective permanent stimulators and immunomodulators which affect diabetes development. Insulin administration increases circulating TGF-β in normal mice and down-regulates the inducible nitric oxide synthetases NO activity and thereby prevents islet cells disruption [13]. Insulin or GAD oral immunotherapy for type I diabetes prevention is now in the course of pre-clinical stage of study [14], [15]. Insulin may have potential therapeutic applications in the prevention of DM1 due to its growth factor-like effects and antiapoptotic efficacy [12]. Evidence has been provided that insulin administration can down-regulate the inducible nitric oxide synthetases and diabetes development in NOD mice [15]. The multicentre randomized controlled clinical trial proved that oral administration of insulin at clinical onset of type I diabetes did not prevent the deterioration of β cell function [16]. It is very difficult to establish immune markers for monitoring the progress of autoimmune disease; for example autoantibody levels are not affected by immunomodulation and therefore could not be used[M1] for monitoring the disease [17]. The inability to discriminate the proliferative responses of lymphocytes to individual islets autoantigens normal controls from onset of type I diabetes patients suggests that proliferation represents an incomplete picture of the immune reaction and results in conflicting variability [18].

Our study demonstrates that both on peripheral blood T lymphocytes and on in vitro cultured T lymphocytes of patients with high risk of DM1 (prediabetes) the increased lymphocyte percentage with CD95 antigen expression was observed. The increased IL-6 and IL-10 production by lymphocytes of pre-diabetic patients stimulated in vitro by insulin as autoantigen was noted. The anti-CD95 IgM antibodies induce apoptosis of PHA preactivated lymphocytes of the controls, prediabetics and DM1 patients, the insulin preactivated lymphocytes undergo apoptosis in prediabetics only. These events may be critical for the early processes of diabetogenesis and the mechanism responsible for disease progression due to IL-10 overproduction and the failure of autoreactive T cells apoptosis. Potential autoantigen activated T cells, cytokine disbalance and different susceptibility to apoptosis are suggested as potential elements for disease activity assessment.