Peripheral Inflammatory Cytokines as Biomarkers in Alzheimer’s Disease and Mild Cognitive...

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Original Paper

Neurodegenerative Dis 2007;4:406–412 DOI: 10.1159/000107700

Peripheral Inflammatory Cytokines as Biomarkers in Alzheimer’s Disease and Mild Cognitive Impairment

Rita João Guerreiro

a Isabel Santana

b José Miguel Brás

a Beatriz Santiago

b

Artur Paiva

c Catarina Oliveira

a, b

a Center for Neuroscience and Cell Biology, Faculty of Medicine, Coimbra University, b

Neurology Service,Coimbra University Hospital, and c

Histocompatibility Center, Coimbra , Portugal

matory status in AD, reinforcing the hypothesis of a progres-sive impairment of the immune response in this disorder and suggesting that monocytes may be good targets to study the progression from MCI to AD.

Copyright © 2007 S. Karger AG, Basel

Introduction

Alzheimer’s disease (AD) is the most common form of dementia, affecting about 10% of the individuals aged 1 65 years, and doubling its prevalence every 5 years [1] . Although the causes of the disease remain unknown, the extracellular aggregation of amyloid � (A � ) peptides and the accumulation of hyperphosphorylated � proteins are considered the main events involved in the neuronal death leading to dementia. Besides these pathological hallmarks, one can also observe in the brain tissue of AD patients, a diffuse neuronal loss (mainly of cholinergic neurons) and the presence of neuroinflammatory chang-es [1, 2] .

The main cellular event that signals the presence of neuroinflammation is the accumulation of reactive mi-croglia in the degenerated areas. Additionally, A � may stimulate not only these cells but also astrocytes and oli-

Key Words

Alzheimer’s disease � Mild cognitive impairment � Cytokines � Inflammation � Flow cytometry

Abstract

Background: Several lines of evidence in the literature have shown that inflammation is involved in the pathogenesis of Alzheimer’s disease (AD). However, the results from the eval-uation of serum inflammatory markers in AD patients have been controversial. Objective: To determine if any differenc-es exist in the monocytic secretion pattern of IL-1 � , IL-6, IL-12 and TNF- � from mild cognitive impairment (MCI) and AD pa-tients, when compared with healthy age-matched controls. Methods: To evaluate the percentage of peripheral mono-cytes secreting IL-1 � , IL-6, IL-12 and TNF- � along with the rel-ative levels of these proteins, a cytofluorimetric analysis was conducted under basal conditions and after lipopolysaccha-ride-induced cell activation. Results: We found, in AD and MCI patients, a significant raise in the percentage of mono-cytes producing the studied cytokines (under basal condi-tions and after the exposure to an inflammatory stimulus), as well as a decreased competence of these cells to respond to inflammatory challenges, when compared with controls. Conclusions: These results agree with a persistent inflam-

Received: August 23, 2006 Accepted after revision: January 24, 2007 Published online: October 9, 2007D i s e a s e s

Catarina Oliveira Centro de Neurociencias e Biologia Celular Departamento de Zoologia, Universidade de Coimbra PT–3004-517 Coimbra (Portugal) Tel. +351 239 822 751, Fax +351 239 853 409, E-Mail [email protected]

© 2007 S. Karger AG, Basel1660–2854/07/0046–0406$23.50/0

Accessible online at:www.karger.com/ndd

http://dx.doi.org/10.1159%2F000107700

Peripheral Cytokines in AD and MCI Neurodegenerative Dis 2007;4:406–412 407

godendrocytes to produce pro-inflammatory cytokines, chemokines and reactive oxygen species, which can lead to neuronal damage [3, 4] . Chronic neuroinflammation may contribute to the early stages of neuronal dysfunc-tion, leading to a decline in choline acetyltransferase ac-tivity and reduced acetylcholine, thus determining the neuropathologic characteristics of the disease [2] . Sev-eral cytokines, such as IL-1, IL-6, IL-12, TNF- � and TGF- � , have been found to be overexpressed in the brain of AD patients and were associated with the development and progression of this pathology [5–10] .

Numerous studies have tried to detect suitable bio-markers for AD [11–14] . However, as AD is a complex disorder, no biomarker or combination of biomarkers has yet enough sensitivity, specificity, and predictive value to be of clinical utility in screening, diagnosis, or monitor-ing of AD therapy [14] . Even though cytokine plasma lev-els are hard to detect at basal conditions, the role of in-flammation in this pathology highlighted inflammatory cytokines as good targets in the biomarker pursue [15] . Therefore, cytokine release after an immune stimulus has been evaluated, although with resulting controversial data. Nevertheless, an activation of the peripheral im-mune status, probably linked to the inflammatory condi-tion observed in AD brains, has been consistently report-ed [16] .

In this study, and considering the relation between the immune and the nervous systems, we further explore the release of IL-1 � , IL-6, IL-12 and TNF- � from blood monocytes, under basal conditions and after lipopolysac-charide (LPS) cell stimulation, from AD and mild cogni-tive impairment (MCI) patients as well as from age-matched controls.

Materials and Methods

Subjects In this study, 105 subjects were evaluated: 25 cognitively

healthy controls (19 females, 6 males) with ages ranging from 51 to 83 years (mean 69.1 8 8.3); 45 individuals with MCI (26 fe-males, 19 males), with ages ranging from 52 to 85 years (mean 70.7 8 8.5) and 35 patients with mild AD (16 females, 19 males) with ages ranging from 61 to 89 years (mean 71 8 7.6). In all individu-als, general medical or other neurological diseases were ruled out by clinical screening and formal neurological assessment. The di-agnosis of probable AD was made in accordance with criteria de-fined by the Diagnostic and Statistical Manual, Revision 4 (DSM-IV) [17] and the guidelines of the National Institute of Neuro-logical Disorders and Stroke and the Alzheimer’s Disease and Related Disorders Association (NINCDS-ADRDA) [18] . All par-ticipants were assessed with the Mini-Mental State Examination

(MMSE) [19] and the Clinical Dementia Rating Scale [20] . The diagnosis of MCI was made in accordance with the criteria de-fined by Petersen et al. [21] . Informed consent was obtained from all participants.

Sample Preparation and Flow Cytometry Analysis A total of 105 peripheral whole blood samples were collected

in heparin-containing tubes and immediately processed for the analysis of cytokine production. Blood samples were stained ac-cording to a whole blood technique, as previously reported [22–24] . In brief, peripheral blood samples were suspended in 500 � l of RPMI-1640 culture medium (BioWhittaker, Inc., Walkersville, Md., USA), and incubated for 6 h at 37 ° C in a sterile environment with 5% CO 2 and 95% humidity. Bacterial LPS from Escherichia coli (serotype 055:B5; 100 ng/ml; Sigma Chemical Co., St. Louis, Mo., USA) was used as a stimulus, in the presence of 10 mg/mlof brefeldin A (Sigma Chemical Co.) to trap produced cytokines into monocytes [25] . Simultaneously, a control was generated which was submitted to the same conditions, but without the ex-posure to LPS. Finally, the cells were recovered by centrifugation at 1,500 g , washed, and stained with suitable volumes of CD33 (APC-conjugated) monoclonal antibodies (Becton Dickinson, San Diego, Calif., USA). Cells were then fixed and, following per-meabilization, cytokine staining was accomplished by using ap-propriate volumes of the below mentioned fluorochrome-labeled monoclonal antibodies (Becton Dickinson, San Diego, Calif., USA): TNF- � (fluorescein isothiocyanate), IL-1 � (phycoeryth-rin – PE), IL-6 (PE), and IL-12 (PE).

The cytofluorimetric analysis was conducted using the Flow Cytometer FACScalibur (Becton Dickinson) and the acquisition process was stopped after 10,000 total events, and after 10,000 monocytes under live gate, on the basis of CD33 bright fluores-cence, for each sample. For data acquisition and analysis, the CellQuest and Paint-A-Gate PRO software programs (Becton Dickinson Biosciences) were used [23] .

Statistical Analysis For all parameters under study, the mean values of measure-

ments, their standard errors and the correlations between values were calculated with the Microsoft � Excel 2002 software pro-gram. To explore the potential existence of statistically significant differences between groups, the ANOVA test with Bonferroni and Games-Howell as post-hoc tests, and the Statistical Package for Social Sciences (SPSS) version 10.0.1 were used. Values of p ! 0.05 were generally considered statistically significant.

Results

As can be seen in figure 1 , the ANOVA for TNF- � , IL-6, IL-1 � and IL-12 established a significant effect of diag-nosis in both the basal and stimulated paradigms, with post-hoc comparisons demonstrating increased levels in AD versus controls. The same was found for stimulated assays when MCI and AD groups were compared. Addi-tionally, the percentage of stimulated monocytes produc-ing IL-1 � and of non-stimulated monocytes producing

Guerreiro /Santana /Brás /Santiago /Paiva /Oliveira

Neurodegenerative Dis 2007;4:406–412408

TNF- � was found to be significantly increased in MCI patients when compared with controls.

Regarding the relative amounts of cytokines, present-ed in figure 2 , a significant diagnosis effect for IL-12 was found after ANOVA analysis comparing stimulated as-says in AD and MCI groups and assays without cell stim-ulation in AD and control groups.

The ability of blood monocytes to respond to an in-flammatory stimulus (exposure to LPS) was assessed by analyzing the difference in the percentage of cells pro-ducing each cytokine in the absence and in the presence of this stimulus. As can be seen in table 1 and figure 3 , the inflammatory stimulus induced a smaller increase in the release of all the evaluated cytokines in AD patients,

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Fig. 1. Results are presented as means 8 SDs for TNF- � ( a ), IL-6 ( b ), IL-12 ( c ) and IL-1 � ( d ). White bars correspond to assays without cell stimulation and black bars to assays with cell stimulation (exposure to LPS 100 ng/ml). % = Percentage; Ctrl = control group; MCI = mild cognitive im-pairment group; AD = Alzheimer’s disease group. * p ! 0.05 and * * p ! 0.005 when the MCI and AD groups are compared; j

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the groups MCI and controls are com-pared.

Group TNF-� IL-6 IL-12 IL-1�

% MFI % MFI % MFI % MFI

Ctrl 50.82 379.85 37.59 23.71 14.77 5.09 58.32 48.75MCI 43.39 277.18 34.40 31.62 17.49 0.34 59.14 19.08AD 23.46 265.66 19.01 28.75 1.79 1.24 41.99 9.33

These data were achieved by subtracting the results from the assays with no cell stim-ulation from that obtained in the assays with cell stimulation. % = Percentage; MFI = mean fluorescence intensity; Ctrl = control group; MCI = mild cognitive impairment group; AD = Alzheimer’s disease group.

Table 1. Differences between the means (percentage of cells and MFI) obtained for assays with and without cell stimulation

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Fig. 2. Results are presented as means 8 SDs for TNF- � ( a ), IL-6 ( b ), IL-12 ( c ) and IL-1 � ( d ). White bars correspond to assays without cell stimulation and black bars to assays with cell stimulation (exposure to LPS 100 ng/ml). MFI = Mean fluorescence intensity; Ctrl = control group; MCI = mild cognitive impairment group; AD = Alzheimer’s disease group. * p ! 0.05 when the MCI and AD groups are compared; jj p ! 0.005 when the groups AD and con-trols are compared.


than in healthy age-matched controls. These results prob-ably unveil the compromised capacity of these cells to respond to an extrainflammatory challenge, found in AD and MCI patients.

In the MCI group, under basal conditions, statistically significant correlations were observed between the per-centage of monocytes producing TNF- � , the subjects age, and the age at onset of the disease (r = –0.44, p ! 0.01 and r = –0.41, p ! 0.01, respectively).

In the AD group, after cell stimulation, a statistically significant correlation was observed between the per-centage of monocytes producing TNF- � (r = 0.40, p ! 0.05), IL-6 (r = 0.60, p ! 0.01) and IL-12 (r = 0.42, p ! 0.05) and the age of the patients. In the same way, a statisti-cally significant correlation between the percentage of monocytes producing IL-6 (r = 0.37, p ! 0.05) and the age of the patients was observed without cell stimulation. The secretion of IL-12, in the absence or following cell stimu-lation, also correlates (r = 0.45, p ! 0.05 and r = 0.50, p ! 0.01, respectively) with the age of the patients.

In the same group, a statistically significant correla-tion was also observed between the percentage of mono-cytes producing IL-6 (r = 0.52, p ! 0.01 and r = 0.40, p ! 0.05, respectively) and IL-12 (r = 0.50, p ! 0.01 and r = 0.41, p ! 0.05, respectively), both in the absence or follow-ing cell stimulation, and the age-at-onset of the disease. A similar correlation was observed for IL-6 secretion (r = 0.44, p ! 0.05) and IL-1 � (r = 0.36, p ! 0.05) in the assays without cell stimulation, and for the secretion of IL-12(r = 0.55, p ! 0.01) following stimulation.

The MMSE score correlates significantly with the percentage of monocytes in the AD group (r = 0.40, p ! 0.05), but not in the MCI group. No correlation wasfound between the age of controls and the studied pa-rameters.

Discussion

Previous studies have reported high levels of several inflammatory cytokines in serum or plasma of AD pa-tients [5, 26–28] . Indeed, increased levels of IL-1 � , IL-6, and TNF- � have been found both in peripheral blood [26, 27, 29–32] and in autopsy specimens [33, 34] of patients with mild to moderate late-onset AD. Likewise, after cell stimulation with LPS, AD patients continued to produce higher levels of IL-1 � , IL-6, IL-10, and TNF- � than healthy controls [35] . This is consistent with the hypoth-esis of the existence of an activated inflammatory state in demented patients; however, some reports presented con-troversial data regarding increased cytokine levels in blood and cerebrospinal fluid of AD patients [36–38] .

Aiming to overcome these shortfalls, in our study we used a specific and sensible method, flow cytometry, to detect cytokines produced by a particular blood cell pop-ulation – peripheral monocytes. This technique allows a clear picture of cytokine production at the cellular level, since it is possible to distinguish between cellular subsets and determine the relative amount of cytokines produced by large populations of cells.

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Fig. 3. Representation of the mean differ-ences in the percentage of cells producing TNF- � , IL-6, IL-12 and IL-1 � before and after cell stimulation. % = Percentage;Ctrl = control group, MCI = mild cognitive impairment group, AD = Alzheimer’s dis-ease group. * p ! 0.05 and * * p ! 0.005.



From all the peripheral blood cell populations, mono-cytes are the ones most similar to microglia cells – the immune defenses in the brain. These are considered to be the resident macrophages in the central nervous system (CNS) [39] . They produce the same cytokines that mono-cytes do after activation and originate from the mono-cytes present in the CNS after its vascularization [40] . Furthermore, the important role of these cells in the pro-gression of AD is evidenced by the presence of great amounts of activated microglia in the brain tissue of AD patients, particularly the presence of reactive microglia aggregates encircling senile plaques [41] . Besides all these facts, lymphocytes were also assessed in our study (data not shown), presenting no significant differences be-tween the three studied groups.

Even though the blood-brain barrier prevents a free access of peptides, such as cytokines, from the brain to the periphery, the subsistence of proficient communica-tion pathways, involving humoral, sympathetic, and neu-roendocrine connections has been clearly demonstrated [42–44] . Additionally, it was observed that the intracere-broventricular injection of Abeta 1–42 in mice caused not only an increase in IL-6 in the brain but also in the plas-ma levels of this cytokine. These data showed undeniably that the central administration of Abeta 1–42 is able to induce a systemic response, linking peripheral altera-tions to the deposition of Abeta 1–42 within the CNS [10] . Therefore, although the actual mechanism is poorly un-derstood, there is some relation between the central and peripheral immune systems.

To clarify the origin of circulating cytokines, plus to assess the ability of blood mononuclear cells of demented patients to respond to an inflammatory stimulus, De Lu-igi et al. [45] measured cytokine release triggered by ex-posure to LPS. The results showed that TNF- � and IL-6 release after cell stimulation with LPS were significantly decreased in moderate AD and multi-infarct dementia (MID) and that IL-10 was decreased only in MID pa-tients. However, they further found in the plasma of AD and MID patients, significantly higher levels of circulat-ing IL-1 � and raised levels of TNF- � only in the later subset of patients. They inferred that these high levels of cytokines in plasma could not be explained by an in-creased responsiveness of blood mononuclear cells.

Since we found statistically significant differences in the percentage of monocytes producing IL-1 � , IL-6, IL-12 and TNF- � between the controls and the patients groups ( fig. 1 ), our results point towards a contrasting hy-pothesis – the source of elevated circulating inflamma-tory cytokines levels are the blood cells. These differenc-

es are present in the form of a continuous increase from healthy controls to MCI and from these to AD, both in the assays with and without cell stimulation. This can probably be due to a persistent inflammatory condition present in AD, leading to a lower capacity of response to additional inflammatory stimuli, by these cells ( table 1 ).

In MCI, our data show that this can clearly be consid-ered as an intermediate state between healthy elderly in-dividuals and AD patients. This obviously reinforces two different assumptions: first, that MCI is an early and pro-dromal stage leading to dementia, mainly AD; second, that inflammatory response is an early event in dementia, which agrees with the observation of higher efficiency levels of non-steroidal anti-inflammatory therapies in the period before the setting up of dementia symptoms [46] .

We found no correlations between the age of control subjects and the studied parameters. However, in the MCI group the age of the individuals and age at onset of cognitive impairment were significantly correlated with the percentage of monocytes producing TNF- � in the ab-sence of cell stimulation, whereas in the AD group the age of the patients and age at onset of the disease were sig-nificantly correlated with many of the inflammatory pa-rameters studied. The absence of significant correlations between the studied parameters and the MMSE score can be explained by the homogeneity observed in each group (MMSE scores: MCI group: 22–30; AD group: 12–28). This is a good characteristic to compare MCI and AD but prevents the establishment of a relationship with the se-verity of the disease.

The aptitude of blood monocytes to respond to an in-flammatory stimulus was assessed by analyzing the dif-ference in the percentage of cells producing each cytokine in the absence and in the presence of this stimulus. We found that the inflammatory stimulus induced a smaller increase in the release of all the evaluated cytokines in AD patients than in healthy age-matched controls. Al-though the percentage of cells that express cytokines increases less in AD and does not necessarily imply low-er immune competence – it can be an artifact of the scal-ing – these results probably unveil the compromised ca-pacity of these cells to respond to an extrainflammatory challenge found in AD and MCI patients.

In conclusion, our results show that in AD there is a significant increase of the percentage of monocytes pro-ducing inflammatory cytokines, reinforcing the sugges-tion of the occurrence of a chronic inflammatory status in this disease. Although our results should be validated in a larger prospective, well-controlled clinical study,


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Acknowledgment

The authors thank all patients for their participation in this study.



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