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Lymphocyte subset markers and cytokine production in peripheral blood
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     Lymphocyte subset markers and cytokine production in peripheral blood of patients with Alzheimer’s disease

    Ebiotec Biotechnology Division, EuroEspes Biotechnology, 15165-Bergondo, La Corua, Spain

    Correspondence to Dr. Valter R.M. Lombardi,EuroEspes Biotechnology,Polígono Industrial de Bergondo,C/. Parroquia de Guísamo,Parcela A-6, Nave F,15 165 Bergondo (A Corua),Spain

    Tel:+34 981 784848,Fax:+34 981 784842

    E-mail:biotecnologia@ebiotec.com

    [Abstract] A comprehensive peripheral blood immunophenotype analysis of 88 Alzheimer’s disease(AD) patients was performed by double-color flow cytometric analysis. The cell subsets quantified included total T cells (CD3+), B cells (CD19+), NK cells (CD56+), CD69+, CD25+, CD4+ and CD8+ T cells, cytotoxic (CD28+) and suppressor precursor (CD28-) CD8+ T cells, CD45RA+ and CD45RO+ T cells (CD4+ and CD8+). In agreement with results obtained by other groups, it was found that AD patients had an increased CD4/CD8 ratio, due to both a decrease in CD8+ T cells and to an increase of CD4+ T cells. AD patients were found to have a significantly decreased level of suppressor precursor (CD28-) CD8+ T cells, normal levels of cytotoxic (CD28+) CD8+ T cells, and increased levels of CD69+ T lymphocytes compared with that of controls. These data indicate that AD patients do not have a general decrease in CD8+ T cells but that they have a specific decrease in the suppressor precursor subset only and normal levels of cytotoxic CD8+ T cells and that elevation of CD69+ T lymphocytes may represent a manifestation in the peripheral blood of some of the pathological events that occur in the brain.

    [Key words] Alzheimer’s disease; lymphocyte markers; cytokines

     INTRODUCTION

    During the past century, humans have gained more years of average life expectancy than in the last 10,000 years; humans are now leaving in a rapidly ageing world, and during their lifespan they are exposed to a multiplicity of potential infectious and pathogen agents. During evolution all multicellular organisms have developed a number of mechanisms to defend themselves against such assaults, and collectively these defense-related mechanisms constitute part of the innate immune system, an ancient system that seems to prevail in all multicellular organisms [1,2].

    Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by loss of memory and other cognitive functions [3,4]. Evidence based on the identification in post-mortem brains of AD [5,6] and on epidemiological studies [7~9] obtained over the past two decades suggests that inflammatory processes may occur in AD and possibly in other chronic neurodegenerative diseases, contributing to neuronal damage. Markers of activation provide important clues to immune function since any immune response involves lymphocyte activation, cell-to-cell interaction, cytokine release and finally inflammation. Immune deficiency, predominantly associated with progressive decline shared by both CD4+ and CD8+ T lymphocytes, and chronic inflammation, evidenced by increased levels of pro-inflammatory cytokines (IL-6, TNF-α, IL-1β), acute phase proteins including C-reactive protein and serum amyloid A, are both present at increased frequency in AD, Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) [10,11].

    Although T lymphocytes are seldom detected in the normal central nervous system (CNS), these cells do appear during the course of CNS inflammation in multiple sclerosis (MS), experimental allergic encephalomyelitis, HIV-induced encephalomyelitis in humans, and simian immunodeficiency virus (SIV) infection of the CNS [12~15]. An important question with regard to lymphocyte traffic into the CNS of diseased vs. non-diseased animals has to do with the characteristics mediating such lymphocyte entry.To approach the mechanism of immune dysfunction in AD, we focused our studies on the patients’ peripheral blood in which the immunological brain processes are thought to be reflected. Taking into account the well-documented immunoregulatory abnormalities in different neurodegenerative diseases [16~20], we have performed a comprehensive peripheral blood immunophenotype survey of AD patients and healthy controls, as well as proliferative response to different mitogens. Specifically, the levels of T cells, B cells, monocytes, CD4+ and CD8+ T cells, memory and nave CD4+ and CD8+ T cells, suppressor precursor and cytotoxic CD8+ T-cell subsets based on CD28 expression, and early (CD69+)-activated and late (HLA-DR+)-activated T cells were determined.

     METHODS

    Patients 88 patients (46 male and 42 female;age range,64 to 78 years) with diagnosed AD were included in the study.At the time of recruitment,none was receiving inhaled or oral corticosteroids,sodium chromoglycate,or theophylline.No patients smoked or had had an upper respiratory tract infection in the 8 weeks before the study.24 age-and sex-matched normal individuals served as controls.No control had a history of dementia.

     Flow Cytometry Reagents and Analyses

    Immunophenotyping was carried out on peripheral blood T cells and monocytes from patients and controls by using the following monoclonal antibodies in various combinations: phycoerythrin-conjugated anti-CD3 (Becton Dickinson, Mountain View, California), which recognizes up to 90% of T cells bearing the αβ γδ T-cell receptor heterodimer; anti-CD4, anti-CD8, and anti-CD19 (Becton Dickinson), which stain helper/inducer, cytotoxic T-cell, and B cell subsets; anti-CD25 (anti-interleukin-2 receptor, Becton-Dickinson) and anti-HLA-DR, which recognize, respectively, the interleukin-2 receptor a chain and the major histocompatibility complex class II molecule (both referring to activated T cells); anti-CD45R0 (Becton Dickinson), a common leukocyte antigen present on activated/memory T cells and fluorescein-conjugated anti-CD69 (Becton Dickinson). For staining, PBMCs were labeled with monoclonal antibodies (20 μl/100 ml blood), incubated at 4 ℃ for 30 minutes, lysed, centrifuged, washed twice, resuspended in 50 μl of saline, and analyzed by flow cytometry (FACScan, Becton-Dickinson). For analysis of two-color cytofluorometric data, an electronic gate was set on the lymphocyte population based on the forward-angle versus the right-angle light scatter histogram. Quadrant markers in fluorescence histograms were set by using matched isotype controls. The CellQuest program (Becton-Dickinson) was used to optimize gating of lymphocytes and to provide an objective way to exclude both debris (noncellular events due to particulate matter) and other cells from the lymphocyte gate and for analysis of the results.

    Blastogenic Response

    Blastogenic response of peripheral lymphocytes to phyto-hemagglutinin (PHA; Sigma Chemical Co., St. Louis, MO), concanavalin A (CONA; Sigma Chemical Co.), and pokeweed mitogen (PWM; Sigma Chemical Co.) was measured using the MTT Cell Proliferation Assay (LGC Promochem, Spain), a colorimetric assay system which measures the reduction of a tetrazolium component (MTT) into an insoluble formazan product by the mitochondria of viable cells. After incubation of the cells (200,000 cells/well) with the MTT reagent for approximately 2 to 4 hours, a detergent solution was added to lyse the cells and solubilize the colored crystals. The samples were read using an ELISA plate reader at a wavelength of 570 nm. The amount of color produced is directly proportional to the number of viable cells. Triplicate cultures from each sample with each mitogen were supplemented with 25 μl of fetal bovine serum, using the following concentrations of mitogens: 25 μg/ml of ConA, 40 μg/ml of PHA, and 15 μg/ml of PWM. Lymphocyte proliferation was calculated as a stimulation index: (SI)=(OD experimental/OD background unstimulated)

     Cytokine Measurement

    IL-1β, IL-6 and TNF-α were measured in supernatants of peripheral blood activated with LPS and cultured for 72 hours at 37 ℃. 100 μl of individual supernatants were assayed for cytokine concentrations using a commercially available ELISA (BioSource International, Inc., Camarillo, CA). IL-1β, IL-6 and TNF-α were sensitive at 2 pg/ml, 2 pg/ml and 15 pg/ml, respectively. Inter-and intra-assay coefficients of variation were <10%.

     Statistical Analysis

    Patients were stratified according to age. Pairwise comparisons for measuring the significance of the differences among mean values (± SD) calculated in the various groups (patients with AD compared with controls) were done by using the Mann-Whitney U test. P values less than 0.05 were chosen for rejection of the null hypothesis. Statistical computations were done by using SPSS, version 11.0 (SPSS, Chicago, Illinois).

     RESULTS

    The mean percentage of CD3+ and CD4+ T cells in the lymphocyte population was higher in AD patients than in the healthy group P<0.01 and P<0.005, respectively). However, CD8+ subset was lower in the AD group and the CD4/CD8 ratio was therefore significantly higher in the AD group in comparison with the controls (data not shown). We investigated in the two groups three different markers indicative of recent activation. A significantly higher surface expression of both activation or nave markers, such as CD25+ (24.8% ± 5.1%), CD19+ (9.3% ± 2.5%), and CD45RA+ (44.7% ±7.3%), was found in AD patients in comparison with matched healthy subjects (CD25+, 8.4% ± 2.6% [P<0.001]; CD19+, 5.6% ± 2.9% [P<0.03]; CD45RA+, 31.5% ± 3.8% [P<0.05]) (Table 1). However, the percentage of CD45RO, which is present on a subpopulation of resting (memory) T cells within both CD4 and CD8 subsets, was significantly reduced (P<0.05) in the AD group (33.8±3.6) with respect to the HS group (45.6%±2.9%).

    Table 1 Phenotypic Analysis of Peripheral Blood Lympho-Cytes in AD Patients and Healthy Subjects (HS).(Data are the mean of 88 patients in the AD group and the mean of 24 samples in the HS group)

    TP1

    Note:*P<0.01;**P<0.005

    Blastogenic responses to ConA, PHA and PWM and NK cytotoxicity assessed in blood drawn from all AD patients, revealed a diminished proliferation response to ConA (SI: 19.8), PWM (SI: 20.8) and PHA (SI:23.0) mitogen stimulation as compared to controls (ConA: 22.8; PWM: 25.5; PHA: 26.3) (Table 2) as well as a lower percentage of NK cell cytotoxicity in the AD group with respect to controls (data not shown).

    Table 2 Lymphocyte Blastogenic Response in AD Patients and Healthy Subjects Expressed as the Mean of Triplicate OD Values and Stimulation Index (SI).(200,000 lymphocytes/well were incubated with MTT reagent during 4 hours and read using an ELISA plate reader at a wavelength of 570 nm)

    Note:*P<0.05

    When the presence of the activation marker, CD69, on monocytes derived from AD patients was compared to CD69 expression on monocytes derived from healthy controls, a significant difference was observed. Flow analysis showed that AD patients had an elevated percentage of CD69+ cells (P=0.005) over age matched controls (Figure 1). In addition, when the expression of CD69+ was analyzed on gated lymphocytes, a clear increase of the expression of this early activation marker was observed (AD group: 37%±4.5%; HS: 27%±3.8%).

    To determine if the increase of activation markers observed in the lymphocyte population was associated with increased inflammatory cytokines, supernatants from LPS activated peripheral blood from all studied population were assayed for IL-1β, IL-6 and TNF-α (Figure 2). The LPS-induced increase in all cytokines was greater in the AD group (IL-1β, 128 pg/ml,P<0.05; IL-6, 88 pg/ml,P<0.01; TNF-α, 95 pg/ml,P<0.01) with respect to the HS group (IL-1β, 77 pg/ml; IL-6, 55 pg/ml; TNF-α, 62 pg/ml).

    Figure 1 FACS analysis of CD69 expression on peripheral blood monocytes and lymphocytes from AD patients and healthy subjects (HS)

    Data are represented as the mean ± SD of 88 AD blood samples and 24 HS blood samples. Asterisk indicates a statistically significant difference (P<0.05)

    Figure 2 IL-1β, IL-6 and TNF-α were higher in supernatants of peripheral blood from AD patients in comparison to HS group after stimulation with LPS

    All cytokines were measured after 72 hours in culture. Bars are means ± SD (AD: n=88; HS: n=24). Asterisks indicate significant difference (*P<0.05;**P<0.01)

     DISCUSSION

    The immune system is responsible for the recognition and elimination of pathogenic bacteria and viruses. Highly specialized cell types have evolved to provide these critical functions and ensure both immediate and long-term defense against infection. A predominant focus of flow cytometric analysis in immunology has been to correlate antigenic phenotype with discrete immune functions. While this approach has been extremely helpful in understanding diversity within the immune system, it must be stressed that in most cases the findings are strictly correlations and not absolute values. There is often a tendency to extrapolate from these functional correlations demonstrated in very limited in vitro experimental systems to in vivo situations. For example, many investigators will observe an increase in CD8 T-lymphocytes in blood and conclude that there is an increase in “suppressor” or “cytotoxic” T-cells, implying a parallel augmentation in these functions. Likewise, it is concluded that a patient expressing a lower frequency of NK-cells or CD4+.T-cells must have lower “NK activity” or “helper ” function, respectively. Such conclusions are completely inappropriate and likely wrong. In order to conclude that a biological function has been affected, it is necessary to directly measure that function. One important consideration is that function is usually a reflection of cellular activation status, rather than the number of frequency of cells.

    To avoid misleading interpretations of antigenic phenotype data obtained from patients affected with AD, we decided to combine phenotype analysis, lymphocyte proliferation, and IL-1β, IL-6 and TNF-α production which have been shown to be implicated in the pathogenesis of AD [21,22]. The elderly posses more or less the same numbers of peripheral T cells as the young (data not shown), but as it is shown in Table 1, a very different subset and clonal composition with respect to AD can be observed in age-matched controls. The mean percentage of CD3+ and CD4+ T cells in the lymphocyte population was higher in AD patients than in the healthy group. However, CD8+ nave subset, in correlation with increased levels of TNF-α, was lower in the AD group, suggesting that these alterations may be due to a differential sensitivity to apoptosis and contribute to cell immune dysfunction associated with AD. In addition, abnormal phagocytosis of apoptotic body and failure to generate anti-inflammatory response by dendritic cells may explain the paradoxical increased inflammation associated with the increase of CD25+, CD19+, CD69+ and CD45RA+ activation markers. It is important to emphasize that the ability of the immune system to mount a protective CD4+ effector response is not only dependent on the number of ag-specific memory T cells present, but also on the functional phenotype of these cells.

    In a previous study it has been demonstrated [23] that circulating immune cells may recognize metabolic products of the APP molecule and that PBLs from healthy old and young persons are able to react upon stimulation with amyloid precursor protein (APP) peptides with the expression of CD25 and proliferation. Conversely, patients with AD were unable to proliferate. Lymphocyte proliferation measures the ability of immune cells placed in short-term tissue culture to undergo a clonal proliferation when stimulated in vitro by a foreign molecule or mitogen. Although these compounds provide strong stimuli that are not antigen specific, and usually do not discriminate as well as antigens in reflecting different levels of immunodeficiency, they give important clues on the capacity of T lymphocyte to proliferate non-specifically. Blastogenic responses to ConA, PHA and PWM and NK cytotoxicity assessed in blood drawn from all AD patients, revealed a diminished proliferation response to all mitogens used. Although CD25 expression is unimpaired upon stimulation in patients with AD, the reduced level of proliferation suggests a situation of peripheral T cell anergy.

    In the healthy adult brain, there is a balance between pro-inflammatory and anti-inflammatory cytokines, with microglial cells remaining quiescent. However, in chronic neurodegenerative disorders, microglial cell change their morphology, start to express MHC class II antigens and complement receptors [24,25] and become activated [26]. The present studies indicate that peripheral blood cells from AD patients produced more IL-1β, IL-6 and TNF-α compared with healthy subjects when treated with LPS. It has frequently been suggested that inflammatory and immune mechanisms play a major role in the pathophysiology of AD [27].Acute phase proteins such as α-1 antichymotrypsin are frequently elevated in the serum and cerebrospinal fluid of AD patients [28] and may become part of the amyloid deposit of senile plaques, the histopathological hallmark of this illness. Acute phase proteins are mediated by cytokines, particularly IL-1β, IL-6 and TNF-α. Previous studies have shown that there is a significant increase of the level of IL-1β, IL-6 and TNF-α both in the serum and brain extracts in patients with AD [29,30].

    The results presented in this work suggest that also peripheral immune cells may participate to a cytokine up-regulation, and possibly contribute to brain tissue destruction. Ageing of the immune system is part of a continuum of developmental process, encompassing complex events and both qualitative and quantitative alterations in the correct function of several systems and organs. Among those systems that undergo major changes during ageing, the immune system plays a major role [31,32]. Aged peripheral immune pool is characterized by the accumulation of T-cell capable of limited replication capability and since an efficient immune response is based upon the expansion of antigen-specific clones, the consequence of a qualitative and quantitative impairment of the system is an increased susceptibility to infections or cancer.

    An important goal of future research into the mechanisms of inflammation of the brain and role(s) of preventive anti-inflammatory treatment in AD will be to determine if and when the peripheral immune system begins to upregulate the synthesis of markers of activation, as well as complement and cytokine receptors. These considerations have fundamental implications for the design of targeted pharmacological interventions in AD.

     REFERENCES

    1. Ausubel FM. Are innate immune signaling pathways in plants and animals conserved? Nat Immunol,2005, 6: 973-979.

    2. Nurnberger T, Brunner F, Kemmerling B, et al. Innate immunity in plants and animals: striking similarities and obvious differences. Immunol Rev, 2004, 198: 249-266.

    3. Arnaiz E, Almkvist O. Neuropsychological features of mild cognitive impairment and preclinical Alzheimer’s disease. Acta Neurol Scand Suppl,2003,179: 34-41.

    4. Twamley EW, Ropacki SA, Bondi MW. Neuropsychological and neuroimageing changes in preclinical Alzheimer’s disease. J Int Neuropsychol Soc,2006,12: 707-735.

    5. Korolainen MA, Auriola S, Nyman TA, et al. Proteomic analysis of glial fibrillary acidic protein in Alzheimer’s disease and ageing brain. Neurobiol Dis,2005,20: 858-870.

    6. Yu WF, Guan ZZ, Bogdanovic N, et al. High selective expression of alpha7 nicotinic receptors on astrocytes in the brains of patients with sporadic Alzheimer’s disease and patients carrying Swedish APP 670/671 mutation: a possible association with neuritic plaques. Exp Neurol,2005, 192: 215-225.

    7. McGeer EG, McGeer PL. Inflammatory processes in Alzheimer’s disease. Prog Neuropsychopharmacol Biol Psychiatry,2003,27:741-749.

    8. Pasinetti GM. From epidemiology to therapeutic trials with anti-inflammatory drugs in Alzheimer’s disease: the role of NSAIDs and cyclooxygenase in beta-amyloidosis and clinical dementia. J Alzheimers Dis, 2002, 5:435-445.

    9. Townsend KP, Pratico D. Novel therapeutic opportunities for Alzheimer’s disease: focus on nonsteroidal anti-inflammatory drugs. FASEB J,2005, 19: 1592-1601.

    10. McGeer PL, McGeer EG. Inflammation and the degenerative diseases of ageing. Ann N Y Acad Sci,2004, 1035: 104-116.

    11. McGeer EG, Klegeris A, McGeer PL. Inflammation, the complement system and the diseases of ageing. Neurobiol Ageing, 2005,(Suppl1): 94-97.

    12. Becher B, Bechmann I, Greter M. Antigen presentation in autoimmunity and CNS inflammation: how T lymphocytes recognize the brain. J Mol Med, 2006,84: 532-543.

    13. Linsen L, Somers V, Stinissen P. Immunoregulation of autoimmunity by natural killer T cells. Hum Immunol,2005, 66: 1193-1202.

    14. Roberts ES, Huitron-Resendiz S, Taffe MA, et al. Host response and dysfunction in the CNS during chronic simian immunodeficiency virus infection. J Neurosci,2006, 26: 4577-4585.

    15. Roberts ES, Burudi EM, Flynn C, et al. Acute SIV infection of the brain leads to upregulation of IL6 and interferon-regulated genes: expression patterns throughout disease progression and impact on neuroAIDS. J Neuroimmunol,2004, 157: 81-92.

    16. Monsonego A, Zota V, Karni A, et al. Increased T cell reactivity to amyloid beta protein in older humans and patients with Alzheimer disease. J Clin Invest,2003, 112: 415-422.

    17. Blasko I, Grubeck-Loebenstein B. Role of the immune system in the pathogenesis, prevention and treatment of Alzheimer’s disease. Drugs Ageing,2003, 20: 101-113.

    18. Vehmas AK, Kawas CH, Stewart WF, et al. Immune reactive cells in senile plaques and cognitive decline in Alzheimer’s disease. Neurobiol Ageing, 2003, 24: 321-331.

    19. Lombardi VRM, Garcia M, Rey L, et al. Characterization of cytokine production, screening of lymphocyte subset patterns and in vitro apoptosis in healthy and Alzheimer’s Disease (AD) individuals. J Neuroimmunol,1999, 97: 163-171.

    20. Lombardi VRM, Fernández-Novoa L, Etcheverría I,et al. Association between APOE e4 allele and increased expression of CD95 on T cells from patients with Alzheimer’s disease (AD). Methods Find Exp Clin Pharmacol, 2004, 26: 523-529.

    21. Singh VK, Guthikonda P. Circulating cytokines in Alzheimer’s disease.J Psychiatr Res, 1997, 31: 657-660.

    22. Kovaiou RD, Grubeck-Loebenstein B. Age-associated changes within CD4(+) T cells. Immunol Lett,2006, 107: 8-14.

    23. Trieb K, Ransmayr G, Sgonc H. APP peptides stimulate lymphocyte proliferation in normals, but not in patients with Alzheimer’s disease. Neurobiol Aging, 1996, 4: 541-547.

    24. Sheffield LG, Berman NE. Microglial expression of MHC class II increases in normal aging of nonhuman primates. Neurobiol Aging, 1998,19: 47-55.

    25. Morgan TE, Xie Z, Goldsmith S, et al. The mosaic of brain glial hyperactivity during normal ageing and its attenuation by food restriction. Neuroscience,1999,89: 687-699.

    26. Perry VH, Newman TA, Cunningham C. The impact of systemic infection on the progression of neurodegenerative disease. Nat Rev Neurosci,2003,4: 103-112.

    27. Aisen PS, Davis KL. Inflammatory mechanisms in Alzheimer’s disease: implications for therapy. Am J Psychiatry,1994,151: 1105-1113.

    28. Abraham CR, Selkor DJ, Potter H. Immunohistochemicalm identification of the serum protein inhibitor alpha-1-antichynotripsin in the brain amyloid deposits of Alzheimer’s disease. Cell, 1988, 52: 487-501.

    29. Griffin WS, Stanley LC, Ling C, et al. Interleukin 1 and S-100 immunoreactivity are levated in Down syndrome and Alzheimer’s disease. Proc Natl Acad Sci USA, 1989, 86: 7611-7615.

    30. Bauer J, Gauter U, Strauss S, et al. The participation of interleukin-6 in the pathogenesis of Alzheimer′s disease. Res Immunol, 1992, 54: 135-142.

    31. Zanni F, Vescovini R, Biasini C,et al. Marked increase with age of type 1 cytokines within memory and effector/cytotoxic CD8(+) T cells in humans: a contribution to understand the relationship between inflammation and immunosenescence. Exp Gerontol, 2003,38: 981-987.

    32. Pawelec G, Koch S, Franceschi C, et al. Human immunosenescence: does it have an infectious component? Ann N Y Acad Sci,2006, 1067: 56-65.

    (Editor LEE)(Valter RM Lombardi, Ignacio Etcheverría,)