Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro apoptosis in healthy and Alzheimer's Disease (AD) individuals

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Abstract

In order to investigate the possibility of whether or not the lymphocytes of patients with Alzheimer's Disease (AD) are in an activated state, blood mononuclear cells from 45 AD patients and 45 healthy age matched controls were immunophenotyped by measuring the expression of CD3, CD4, CD7, CD8, CD25, CD28, CD56 and HLA-DR by flow cytometry. Circulating and in-vitro-produced cytokines were also measured by ELISA tests. CD7 and CD8 were significantly decreased in AD patients (48.3% and 18.2%, respectively) when compared to healthy subjects ((63.2% and 28.3%, respectively). A significant increase in the CD4, CD25 and CD28 antigen expression was also observed in the AD group (55.3% 24.8% and 65.1%) with respect to healthy subjects (44.5%, 10.3% and 54.3%). In addition there was a significant difference in the extent of apoptosis in lymphocyte culture, as measured by mean fluorescence intensity (MFI) of Fas antigen (CD95) expression on CD4+ T cells in 6 AD patients (MFI=36% and 43%, by anti-CD3 and hyperthermia mediated-apoptosis, respectively) with respect to 6 healthy individuals (MFI=24% and 31%, by anti-CD3 and hyperthermia mediated-apoptosis, respectively), as well as in T-cell proliferation assay. A decline of Fas antigen expression on CD8+ subset was observed in the AD group with both stimuli (19% and 28%) comparing to the control group (29% and 39%). No differences were observed on circulating cytokines and spontaneous in vitro production of proinflammatory interleukin 1β (IL-1β), Tumor Necrosis Factor-α (TNF-α), IL-6 and IL-10 cytokines. Lipopolysaccharide (LPS)-stimulated in vitro production of IL-1β, TNF-α, IL-6 and IL-10 measured by a whole blood culture system was significantly higher in AD patients comparing to controls. Furthermore, the observed differences were more evident at late stages of disease. These findings suggest that immunological tests, based on lymphocyte immunophenotyping combined with pro-inflammatory cytokine determinations and measurement of apoptosis in peripheral blood might represent a useful tool to obtain more insight into the pathogenesis of AD and into the level of immune activation which could characterize the pathological state of lymphocytes from individual AD patients.

Introduction

Several theories have been postulated to explain the complex and multifactorial process of aging (Burns and Goodwin, 1997; Grubeck Loebenstein, 1997; Heuser and Adler, 1997; Hodes, 1997; Voets et al., 1997; Wick and Grubeck Loebenstein, 1997). A decline in immunological function, which involves biological changes in many organ systems, is one of hallmark of aging (Pawlec et al., 1997; Albright and Albright, 1998; McNerlan et al., 1998). The activity life and death of each cell are dictated by its responses to a wide variety of stimuli derived from its matrix, diffusible chemical signals, and its interactions with its neighbours (Thoman, 1997) and in some cases under genetic control (Doria et al., 1997; Effros, 1998). Recent advances in availability and reproducibility of monoclonal antibody reagents specific for a wide range of cell types, coupled with lower costs for increasingly automated flow cytometers, have made flow cytometry the method of choice for immunophenotyping in the clinical laboratory (Cossarizza et al., 1997) and in in vitro cell function studies (Flaherty et al., 1997). Monoclonal antibodies selectively reactive with T cell, B cell, myelomonocytic, and natural killer cell populations have proven useful for monitoring changes in primary and acquired immunodeficiency diseases (Adler et al., 1997), leukemias (Glasova et al., 1998), lymphomas (Heslop et al., 1998), following transplantation (Pei et al., 1998) and during cancer therapy (Hirokawa, 1997; Banker et al., 1998).

The survival of an organism and its host defense mechanisms require, among other processes, a complex but target-oriented interaction and an interdependence between the immune and inflammatory pathways. The immune system is one of many systems that interact in inflammation, host defense, and other vital processes (Horan and Ashcroft, 1997). These interactions may involve direct cellular contact, chemical mediators, or both (Zheng et al., 1997). It has been recognized for many years that immunological responses in the central nervous system (CNS) are different from those that occur at most other sites in the body (Lombardi and Cacabelos, 1998; Maneiro et al., 1998). Among the factors that are thought to contribute to this immunologically privileged status of the CNS are the specialized microvascular endothelial (Cines et al., 1998) cells that are able to hold back most inflammatory cells and the lack of expression of MHC molecules by neurons and most glia. However, quite exceptionally, activated T cells (in contrast to resting ones) are able to cross the BBB, irrespective of their antigen receptor specificity. Once an immune response has been initiated in the CNS, as it occurs in the course of many neurodegenerative disorders (Dickson et al., 1993) it may proceed with devastating effects on neuronal and glial function. A key question in the analysis of these events is the extent to which resident glial cells mediate or regulate the inflammatory response. Glial cells of the CNS, especially microglia and astrocytes, are both the target and source of cytokines in CNS inflammation (Arai et al., 1990). In addition, astrocytes are known to produce inflammatory mediators such as IL-1β and TNF-α. A variety of molecules in the brain may be classified as neurotrophic and/or neurotoxic (Hopkins and Rothwell, 1995). With the advent of new molecular technologies their function and dysfunction in neurodegenerative disorders has become a topic of intense investigation. Some of these cytokines have been proposed as early contributors to a cascade of neurodegenerative events that lead to neuropathophysiological changes that characterize AD, Down syndrome, and AIDS (Merrill and Benveniste, 1996).

This investigation was conducted on Caucasian subjects and analyzed immunophenotypes of peripheral blood mononuclear cells (PBMC) from 45 AD patients and 45 simultaneously studied healthy controls matched for age. We also measured circulating IL-1β, TNF-α, IL-6 and IL-10 plasma levels and in vivo production of these proinflammatory cytokines by using a whole blood culture system that does not require separation of lymphocytes and probably more closely resembles the in vivo situation. The aim of this study was to investigate whether peripheral immunologic abnormalities could be found in a randomly chosen group of patients with Alzheimer, compared with well-matched controls. Our data show that PBMC from AD patients display an increased `active state' than those of patients with other CNS disorders (major depression and schizophrenia) and age-matched controls and suggest that this phenomenon may be due to the fact that these cells are influenced by many inflammatory cytokines and/or by increased levels of spontaneous apoptosis.

Section snippets

Subjects

In this study, we selected 45 AD patients (19 men, 42%, 26 women, 58%) and 45 healthy controls (24 men, 53%; 21 women, 47%) matched for age and neighbourhood, who attended the Institute for CNS Disorders at the EuroEspes Biomedical Research Center, La Coruña, Spain from May 1997 to May 1998. Using the Global Deterioration Scale (GDS), patients were divided into three subgroups: (a) mild dementia (GDS=2; n=23; 9 men and 14 women); (b) moderate dementia (GDS=3; n=12; 6 men and 6 women); and (c)

Immunophenotyping

Data were acquired on a FACScan flow cytometer. Instrument set-up was performed daily using CaliBRITE brand beads and AutoCOMP brand software. A minimum of 20 000 events was acquired to establish an analysis gate that included at least 95% of the lymphocytes and was contaminated by no more than 5% of the monocytes in the samples. A minimum of 2000 lymphocyte events was acquired from each tube. Markers for determining cells positive and negative for any reagent were set by the SimulSet software

Discussion

One in 10 people over the age of 65 suffers from dementia, and the risk rises rapidly with age. Aging, a complex process involving biological changes in many organ systems, is associated with a reduction in most physiological functions and in particular with a decreased ability to maintain homeostasis (Burns and Goodwin, 1997; Ershler et al., 1997). It is generally believed that this inability to maintain homeostasis is at least partially responsible for the increase in morbidity/mortality that

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