Neither sequence variation in the IL-10 gene promoter nor presence of IL-10 protein in the cerebral cortex is associated with Alzheimer's disease

Neuroscience Letters 408 (2006) 141–145

Neither sequence variation in the IL-10 gene promoter nor presence of IL-10 protein in the cerebral cortex is associated with Alzheimer’s disease Doris Culpan a,∗ , Jonathan A. Prince b , Sonia Matthews a , Laura Palmer a , Anthony Hughes a , Seth Love a , Patrick G. Kehoe a , Gordon K. Wilcock a a

Dementia Research Group, Institute of Clinical Neurosciences, Department of Clinical Sciences at North Bristol, University of Bristol, John James Buildings, Frenchay Hospital, Frenchay, Bristol, BS16 1LE, United Kingdom b Department of Cell and Molecular Biology, Karolinska Institute, Stockholm, Sweden Received 14 June 2006; received in revised form 22 August 2006; accepted 29 August 2006

Abstract Interleukin 10 (IL-10) is an important anti-inflammatory cytokine produced in response to neuroinflammation and might be involved in modulating the progression of Alzheimer’s disease (AD) through inhibiting the action of pro-inflammatory cytokines. We have used immunohistochemistry, Western blotting, real time-PCR (RT-PCR) on frontal (BA 6/24) and temporal (BA 20–22) neocortex and hippocampus from AD and control brains as well as genetic association analysis to address the possible involvement of IL-10 in AD. Expression of IL-10 in AD and control brains at both protein and mRNA levels were detected. However, the level of expression, particularly of IL-10 protein, varied considerably in individual brains and we did not find a significant difference between AD and controls. Using direct sequencing we examined five single nucleotide polymorphisms (SNPs) (−3538, −1354, −1087, −824, −597) and two microsatellites (IL-10-G, IL-10-R) in the promoter region of the IL-10 gene. None of the identified SNPs were found to be associated with AD either individually or as haplotypes. Levels of IL-10 protein and gene expression examined also did not appear to be related to AD. Despite this being a relatively small sample, these data suggest that IL-10 does not play a major role in the development of AD. © 2006 Elsevier Ireland Ltd. All rights reserved. Keywords: Alzheimer’s disease; Interleukin-10 (IL-10); Single nucleotide polymorphisms; SNPs; IL-10 expression; RNA; RT-PCR; Inflammation; Haplotypes

Alzheimer’s disease (AD) is the most common human neurodegenerative disorder, characterised clinically by progressive loss of memory and cognitive function and pathologically by parenchymal deposits of A␤ (plaques), neurofibrillary tangles, loss of synapses and eventually of neurons. Although the cause of the loss of synapses and neurons is not completely understood, inflammatory mediators, such as cytokines, are thought to play a role. Multiple cytokines are produced in response to A␤ and are over-expressed in activated microglia surrounding plaques in AD [15]. The balance between pro- and antiinflammatory cytokines determines the magnitude of the inflammatory response. Interleukin 10 (IL-10) is one of the main anti-inflammatory cytokines. IL-10 mRNA is detectable in the frontal and parietal lobe of normal brain [22] and has been suggested to play an

∗

Corresponding author. Tel.: +44 117 970 1212x3068; fax: +44 117 957 3955. E-mail address: [email protected] (D. Culpan).

0304-3940/$ – see front matter © 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2006.08.068

important role in neuronal homeostasis and cell survival [21]. IL-10 mediates its biological effects on cells by interacting with specific cell surface receptors (IL-10Rs), present on all the major glial cell populations in the brain [21]. IL-10 limits inflammation by reducing the synthesis of pro-inflammatory cytokines such as IL-1 and TNF-␣, by suppressing cytokine receptor expression and by inhibiting receptor activation in the brain. Like many cytokines, IL-10 signals via the Jak-Stat pathway, Stat3 being essential for all IL-10 downstream effects [10]. Single nucleotide polymorphisms (SNPs) in the regulatory regions of several inflammatory cytokines such as IL-1␣, IL1␤, IL-6 and TNF-␣ have been reported to influence the risk of AD, possibly by influencing the level of expression of the protein [15]. We reported the results of a pilot study demonstrating the lack of association of SNPs in the promoter region of IL-10 with AD [5] our results were in keeping with those reported in two subsequent studies on German [6] and Italian [18] cohorts, but differed from the findings in two studies on Italian and Chinese populations [12,14].

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The human IL-10 gene is located on chromosome 1(1q31–q32) and consists of five exons [9]. The IL-10 promoter region is highly polymorphic. Three haplotypes (GCC, ACC, ATA) derived from SNPs at position −1082 (G/A) (rs1800896), −819 (C/T) (rs1800871) and −592 (C/A) (rs1800872) [23] have been described in the Caucasian population. In addition, two microsatellite polymorphisms lie 1.2 kb (IL-10G) and 4.0 kb (IL-10R) upstream of the transcription start site [7]. An additional SNP has been reported immediately upstream of the IL10G microsatellite (−1354 (G/A) (rs1800893)) and another downstream from the IL10R microsatellite (−3538 (T/A) (rs1800890)). The IL-10 microsatellite alleles and SNPs have been reported to influence IL-10 synthesis after in vitro stimulation [23,7] and have been linked to several diseases [3]. Here, we have investigated whether sequence variation in the promoter region of the IL-10 gene is associated with AD and have augmented our analysis to address whether IL-10 protein and mRNA levels in brain differ between the disease and normal states. This retrospective case-control study was carried out with Local Research Ethics Committee approval. Brain tissue from 160 cases of neuropathologically confirmed AD (mean age 81.2 years, S.D. = 7.3, range 65–105 years, male:female 56:104) and 92 neuropathologically-normal non-demented elderly controls (mean age 79.7 years, S.D. = 7.5, range 65–93 years, male:female 47:45) were obtained from South West Dementia Brain Bank, Bristol. Genotyping was performed on genomic DNA extracted from the frozen brain tissue (Nucleon ST Extraction kit, Nucleon Biosciences). We sequenced five SNPs (−3538, −1354, −1082, −819, −592) and two microsatellites (IL-10G, IL-10-R). DNA was amplified using following primers: IL-10R, IL-10(−3538) (Tm:59C) IL-10G, IL-10(−1354, −1082, −819, −592) (Tm:65 C) IL-10 (−1082, −819, −592) sequencing only

for:5 -CCCTCCAAAATCTATTTGCA-3 rev:5 -CTCACACTGTGAGCTTCTTG-3 for:5 -GATGAGTGATTTGCCCTGAC-3 rev:5 -CAAGCAGCCCTTCCATTTTA-3 for:5 -AATCCAAGACAACACTAC-3

In all reactions standard PCR conditions were used to amplify target sequences. PCR products were sequenced using the BigDye Terminator Cycle Sequencing Ready Reaction Kit (PE Biosystems) according to the manufacturer’s protocols. Samples were electrophoresed on the ABI Prism 310 Capillary Genetic Analyzer (PE Biosystems). Paraffin sections of formalin-fixed tissue from the frontal and temporal lobes of 5 of the control and 18 of the AD brains were immunostained for IL-10 (sc8438 mouse monoclonal diluted 1:100; Santa Cruz Biotechnology, CA) by a standard biotin–avidin–peroxidase technique using Vectastain Biotinylated Universal antibody and VectaElite ABC complex (Vector Laboratories, Peterborough) and lightly counterstained with haematoxylin. To confirm the presence and expression of IL-10 in brain tissue, we extracted protein and mRNA from homogenates of frozen tissue (collected in RNAlater, Ambion) from three different regions of brain – frontal cortex (BA 6/24), temporal cortex

(BA 20–22) and hippocampus – from some of the immunostained cases, for Western blotting and real time-PCR (RT-PCR). Protein was loaded onto 4–12% pre-cast PAGE-SDS gel (BioRad), electrophoresed for 60 min at 150 V and electrophoretically transferred (overnight) onto nitrocellulose paper in a BioRad Mini-Trans-Blot chamber (30 V) at 4 ◦ C. Non-specific binding was blocked using TBS/Tween (0.05%) (TTBS) containing 10% non-fat milk, for 60 min at room temperature. IL-10 antibody was diluted 1:100 in 5% non-fat milk/TTBS and applied to the membranes for 60 min at room temperature. Bound antibody was detected by incubation with peroxidase labelled rabbit antimouse antibody (ECL, Amersham Biosciences) and enhanced chemiluminescence (ECL, Amersham Biosciences). The membranes were exposed to film (Kodak) and developed. RNA was extracted from homogenates of the tissue using the ABI Prism 6100 Nucleic Acid PrepStation and TransPrep Chemistry as described by the manufacturer (Applied Biosystems). cDNA was produced with the High Capacity C-DNA Archive Kit from Applied Biosystems according to manufacturer’s protocol. RT-PCR was performed by the ABI 7000 sequencing detection system (ABI Prism) with Assay-on-Demand gene Expression Products for IL-10 and Cyclophorin (Cyclo) (Housekeeping gene) (TaqMan MGB probes, FAM dye-labelled) and TaqMan Universal PCR Master Mix. All samples were prepared twice and each preparation set up in triplicate. Data were analysed using the 2−ΔΔCt method [13], according to the formula: Ct = (CtIL-10 − CtCyclo )AD − (CtIL-10 − CtCyclo )Control , where Ct = threshold cycle. Statistical analysis of individual SNPs (genotype distribution and allele frequencies) was performed by a chi-square (χ2 ) test (Statistical Package for Social Sciences (SPSS for WindowsRelease 11.0)). The level of significance in all tests was set at p < 0.05, two-tailed, with a Bonferroni correction applied where multiple testing was carried out in the univariate phase of analysis. The Hardy-Weinberg equilibrium (HWE) was also tested using the chi-square (χ2 ) method. All 5 SNPs were detected in our AD (160) and control (92) cohorts (Table 1). Marginally significant deviations from Hardy-Weinberg equilibrium were identified in cases (but not controls) for −1354 (p = 0.042) and −3538 (p = 0.040) but these are rendered non-significant when adjustments for multiple testing are considered. Three predominant haplotypes of the IL-10 promoter region have previously been described, GCC, ACC, ATA, comprising combinations of three SNPs, −1082 G/A, −819 C/T, −592 C/A. We examined the haplotypes (Table 2) generated from the five SNPs we studied using PHASE v2.1 which implements a Bayesian method of haplotype estimation for unrelated individuals [19,20]. For estimation of empirical p-values in case-control tests, 100 permutations were used with all other settings at default. We found six haplotypes which were present in over 1% of subjects and the three most common were those previously reported: GCC (27.1% AD cases versus 27.5% controls); ATA (18.3% AD cases versus 19.6% controls) and ACC (15.9% AD cases versus 15.1% controls). No haplotypes were found to be significantly associated with AD (Table 2). The haplotype structures were also examined in pair-wise manner using the

D. Culpan et al. / Neuroscience Letters 408 (2006) 141–145

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Table 1 Genotype and allele distribution of IL-10 promoter SNPs SNPs

AD (n = 160)

Control (n = 92)

AD

Control

−3538*

TT TA AA

65 (0.41) 64 (0.40) 31 (0.19)

39 (0.42) 36 (0.39) 17 (0.19)

Allele T Allele A

129 (0.58) 95 (0.42)

75 (0.59) 53 (0.41)

−1354*

GG GA AA

45 (0.28) 67 (0.42) 48 (0.30)

27 (0.29) 44 (0.48) 21 (0.23)

Allele G Allele A

112 (0.49) 115 (0.51)

71 (0.52) 65 (0.48)

−1082

GG GA AA

41 (0.26) 79 (0.49) 40 (0.25)

24 (0.26) 50 (0.54) 18 (0.20)

Allele G Allele A

120 (0.50) 119 (0.50)

74 (0.52) 68 (0.48)

−819

CC CT TT

94 (0.59) 56 (0.35) 10 (0.06)

49 (0.53) 38 (0.41) 5 (0.06)

Allele C Allele T

150 (0.69) 66 (0.31)

87 (0.67) 43 (0.33)

−592

CC CA AA

103 (0.65) 47 (0.29) 10 (0.06)

55 (0.60) 31 (0.34) 6 (0.06)

Allele C Allele A

150 (0.72) 57 (0.28)

86 (0.70) 37 (0.30)

Differences in genotype and allele frequencies in our case-control population were evaluated using the chi-square (χ2 ) statistic (significance set at p < 0.05). HardyWeinberg equilibrium (HWE) was also tested using the χ2 method. (*) Denotes marginally significant deviation from HWE of AD case genotypes for −1354 (p = 0.042) and −3538 (p = 0.040). Table 2 Haplotype frequencies of IL-10 promoter Haplotype

Control (%)

Case (%)

−592 (rs1800872)

−819 (rs1800871)

−1082 (rs1800896)

−1354 (rs1800893)

−3538 (rs1800890)

H1 H2 H3 H4 H5 H6 H7 H6

27.5 19.6 15.1 10.3 11.0 2.0 3.7 3.5

27.1 18.3 15.9 10.5 9.6 6.2 4.0 2.1

C A C C C C C C

C T C C C C C C

G A A G G A A G

A G G A G A A G

A T T T T A T A

Haplotype frequencies as estimated using PHASE v2.1 in cases and controls. The global empirical p-value was not significant (p = 0.61 using 100 permutations). Only haplotypes above 1% are shown. SNPs and alleles highlighted in bold are those previously reported in which significant association with AD was found.

r2 metric (Table 3) and we found, like Eskdale et al. (1999), that the −1082 locus was in strong linkage disequilibrium (LD) with −1354 and −3538 although the strongest LD observed was between −592 and −819. Generally there was strong LD (r2 > 0.1) across all five SNPs studied. Two previously reported IL-10 microsatellites were also examined and no significant differences in the levels of variation were observed between cases and controls (data not shown). Immunolabelling showed IL-10 to be confined to nonneuronal cells (Fig. 1). Some of these cells had the appearance

of microglia or macrophages—cells with small, oval nuclei, either sparsely scattered within the cortex or located adjacent to small blood vessels. Others had round or oval nuclei and were located adjacent to neuronal perikarya and probably included satellite cells. Labelled cells were detected in both AD and control tissue in all regions of cortex examined (Fig. 1). They tended to be more prominent in AD cases but the intensity of labelling was variable and in many cases too weak to allow meaningful quantitative comparison between the AD and control groups.

Table 3 Pairwise linkage disequilibrium (LD) structure of the IL-10 promoter locus

−592 −819 −1082 −1354 −3538

−592 (rs1800872)

−819 (rs1800871)

−1082 (rs1800896)

−1354 (rs1800893)

−3538 (rs1800890)

*

0.853 *

0.113 0.136 *

0.176 0.185 0.302 *

0.138 0.147 0.307 0.518 *

LD between markers has been estimated using the r-squared metric with PHASE v2.1. Markers in bold and underlined are those previously reported by Eskdale et al. (1999) to be in tight LD and which our data support. (*) Denotes pairwise LD analyses of SNPs with themselves.

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Fig. 1. (A and B) Immunolabelling of IL-10 within sections of frontal lobe from a case of AD. Fixed paraffin-embedded sections from the frontal lobe of control and neuropathologically confirmed AD brains were immunostained with sc8438 mouse monoclonal antibody (1:100) for IL-10 by a standard biotin–avidin–peroxidase technique and lightly counterstained with haematoxylin. IL-10 is present within scattered non-neuronal cells, some adjacent to small blood vessels (arrow in A) and some adjacent to neurons (arrows in B).

IL-10 protein was also readily detected in both control (n = 4) and AD (n = 9) brain homogenates (Fig. 2). Again the amount of IL-10 protein appeared to vary considerably in individual control and AD brains; in general, those cases with weak immunohistochemical labelling also had weak bands in the western blots.

RT-PCR using cDNA from a limited number of the same brains (six AD cases versus two controls) that were used for Western blotting showed the levels of transcription of the IL-10 gene to be lower than those of the cyclophorin (housekeeping) gene. Transcription of the IL-10 gene did not differ significantly between AD and control brains in any of the regions examined (2−Ct frontal 0.62, temporal 0.76, hippocampal 0.66) but we acknowledge that the number of samples studied was very small. There is evidence that IL-10 has an anti-inflammatory role in the brain [11,21] and therefore might be important in downregulating inflammatory processes associated with AD pathology [16]. IL-10 secretion induced by stimulation of human blood cultures with LPS (lipopolysaccharide) varied markedly between individuals, according to IL-10 promoter haplotypes [7]. Polymorphisms in the promoter region of the IL-10 gene were suggested to be risk factors for AD in some populations [12,14] but not others [6], [18]. We have found no evidence in our limited sample of association between individual IL-10 promoter SNPs and/or haplotypes and AD. However, larger studies will likely be required to provide enough power to detect statistically significant differences should they exist although this data like many others lend themselves to future meta-analyses such as that available on AlzGene (available at: http://www.alzgene.org) [2]. Our immunolabelling, Western blotting and RT-PCR all confirmed the presence and expression of IL-10 in human postmortem brain tissue, although only at a relatively low level. However, we were not able to demonstrate significant differences between AD and control tissues. A␤ is a potent activator of glial cells and induces the release of a number of inflammatory factors, such as IL-1, IL-6, TNF␣, and related signal transduction factors [1]. A␤ does not seem to stimulate IL-10 production by glial cells in vitro [8]. Preexposure of glial cells to IL-10 inhibits A␤- or LPS-induced production of pro-inflammatory cytokines [22], suggesting that IL-10 receptors are present on cultured glial cells [11]. Binding of IL-10 to its receptor activates the Jak-Stat pathway, with Stat3 being essential for all IL-10 downstream effects [10]. Knockout mouse studies have demonstrated that Jak1 and Stat3 are essential requirements for IL-10-induced inhibition of cytokine production and for its auto-regulation [17]. IL-10 is a key mediator for the termination of inflammatory responses and is important in regulating the immune system. Our findings indicate that promoter variation in the IL-10 gene may not have a major effect upon AD and that there appears to

Fig. 2. Western blot of IL-10 in hippocampus from AD and control brains. Western blotting was performed by loading protein (30 ␮g) onto 4–12% pre-cast PAGESDS gels (Bio-Rad) and IL-10 protein detected using a combination of sc8438 mouse IL-10 monoclonal antibody (1:100 dil) and incubation with peroxidase labelled rabbit anti-mouse antibody. The membranes were exposed to film (Kodak) and developed as shown whereby the levels of IL-10 appear to be higher in the AD subjects than the control tissue but this was not a consistent finding.

D. Culpan et al. / Neuroscience Letters 408 (2006) 141–145

be large individual variation of IL-10 protein expression in controls and cases. The low level of IL-10 gene expression in AD we observed is potentially interesting and the reported lack of responsiveness of the IL-10-Jak-Stat pathway to A␤ and other pro-inflammatory stimuli supports a suggestion that dysregulation of the immune system may lead to the persistence of a chronic inflammatory state within the AD brain although this would need further examination in larger study populations.

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Acknowledgements We thank Dr. Katy Chalmers for helpful suggestions with the manuscript. This study was supported through salary support grants and an endowment fund provided by Bristol Research into Alzheimer’s and Care of the Elderly (BRACE) as well as a Gestetner Research Fellowship to Dr. Patrick Kehoe from the Sigmund Gestetner Foundation.

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