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. Author manuscript; available in PMC: 2009 Apr 3.
Published in final edited form as: Int Psychogeriatr. 2008 Dec 1;21(2):413–414. doi: 10.1017/S1041610208008107

IL6 Release by LPS-stimulated peripheral blood mononuclear cells as a potential biomarker in Alzheimer Disease

Paul B Rosenberg 1,*, Katherine AL Carroll 2, Jenn Cheng 3, Rameeza Allie 4, Constantine G Lyketsos 1, Peter Calabresi 4, Adam Kaplin 1
PMCID: PMC2664968  NIHMSID: NIHMS102061  PMID: 19040786

Abstract

BACKGROUND:

In Alzheimer's disease (AD), microglia are activated by amyloid-beta (Aβ) to release IL6 among other cytokines, which in turn may be neurotoxic. Prior studies suggest that the brain inflammatory response to various antigens can be modeled by measuring the release of IL6 from peripheral blood mononuclear cells stimulated by lipopolysaccharide (LPS). We sought to replicate these results and extend to an AD-specific stimulus (Aβ).

METHOD:

PBMCs were purified from 5 AD and 5 age-gender matched cognitively healthy controls and exposed to LPS at two concentrations (20 and 100 ng/ml) and Aβ1-42(20 ug/ml). IL6 release was measured with standard ELISA kits, and the ratio of “IL6 release” with and without LPS stimulation was reported as the “IL6 release ratio.” Correlations were performed with Pearson's r2.

RESULTS:

IL6 release ratios were increased in AD participants as compared to cognitively normal age-gender matched controls with LPS 100 ng/ml exposure at a trend level (p=.07). Aβ1-42 increased the IL6 release ratio at a trend level (P=.07) with LPS 20 ng/ml exposure. IL6 release ratio was significantly correlated with worse performance on a verbal category fluency test (p=.03, r2=.49)and higher scores on the Neuropsychiatric Inventory (p=.01, r2=.63). There were trend level correlations of Il6 release with worse ratings on the CDR and MMSE.

CONCLUSIONS:

The IL6 release ratio derived from peripheral blood has potential as a biomarker of AD disease severity, both for cognitive outcomes and neuropsychiatric symptoms of AD.

Keywords: Cytokines, Inflammation, Neuropsychiatric symptoms

INTRODUCTION

Microglia are the resident immune cells of the brain [Banati. 2002]. There is increasing evidence for the role of activated microglia in the neurotoxic pathways of Alzheimer's disease (AD). Activated microglia are found abundantly in the vicinity of senile plaques in the AD brain [Rogers, et al. 2001]. Amyloid-beta1-42 (Aβ1-42) is a potent activator of microglia [Benveniste, et al. 2001], causing them to release pro-inflammatory cytokines including IL6, IL1β, TNFα among others. These cytokines in turn may mediate neurotoxicity by decreasing synaptic plasticity, inhibiting long-term potentiation [Bellinger, et al. 1993] or by inhibiting hippocampal neurogenesis [Vallieres, et al. 2002]. Given interest in anti-inflammatory strategies to treat AD, these processes may prove to be treatment targets.

A peripheral blood marker reflecting CNS inflammatory processes would be useful for treatment development, but the search for such a in vivo marker has been elusive. Peripheral blood & CSF levels of pro-inflammatory cytokines do not consistently differ in AD and controls (reviewed in [Rosenberg. 2005]. Both microglia and peripheral blood mononuclear cells (PBMCs) release pro-inflammatory cytokines when exposed to immunologic stimuli including lipopolysaccharide (LPS) and phytohemagglutinin (PHA). There are recent data suggesting that PBMCs from AD patients release more cytokines in this paradigm than normal controls [Reale, et al. 2004]. If replicated, these data suggest either that peripheral inflammatory processes are directly affected by AD, or that PBMCs can be used to model physiologic processes of CNS microglia.

We sought to replicate and extend these studies by exposing PBMCs both to the non-specific microglial activator LPS and to Aβ1-42 as an AD-specific microglial activator. In addition, given the potential links between inflammation, AD, and mood symptoms [Rosenberg. 2005, Miller, et al. 2006] we examined associations between this biomarker and neuropsychiatric symptoms as well as performance on standard cognitive tests. Although there are several potential pro-inflammatory cytokines of interest we focused on IL6 because increased release of IL6 from AD PBMCs has been the most replicable finding to date [Rosenberg. 2005]. Our hypotheses were that 1) PBMCs from AD participants would release more IL6 with LPS stimulation than PBMCs from control participants; 2) Aβ1-42 exposure would magnify IL6 release disproportionately for AD cases as compared to controls.

METHOD

Participants

Study participants were enrolled and had been characterized in the Johns Hopkins University Alzheimer's Disease Research Center. AD was diagnosed by NINCDS-ADRDA criteria [McKhann, et al. 1984], while control participants had no cognitive diagnosis. Diagnoses were made by an interdisciplinary research treatment team including geriatric psychiatrists, neurologists, neuropsychologists, and nurses. All AD participants had a knowledgeable informant for clinical measures. Each participant signed separate informed consents for the clinical measures and for the IL6 determinations; in the cases where participants lacked cognitive capacity to consent, the consent process followed the Alzheimer's Association procedures [Alzheimer's Association. 2004]. All human subjects procedures were approved by a Johns Hopkins Institutional Review Board.

AD and control participants were matched for age within 5 years and matched for gender.

Clinical Measures

Severity of dementia was assessed on the Clinical Dementia Rating Scale (CDR) [Morris. 1993]. The CDR uses a 5-point anchored ordinal scale to characterize six domains of cognitive and functional performance: memory, orientation, judgment, community, hobbies, and personal care. CDR is rated after a semi-structured interview and has excellent reliability and validity. Mini-Mental State Exam (MMSE) is a 30-point assessment of global cognition, including tests of orientation, verbal episodic recall, attention, calculation, language, ideomotor praxis, and visuoconstructional praxis [Folstein, et al. 1975]. Category verbal fluency was measured for both animals and vegetables over a 60-second time interval. The Neuropsychiatric Inventory (NPI) is a standard assessment of NPS in older and cognitively impaired persons and populations [Cummings, et al. 1994], evaluating 12 domains: delusions, hallucinations, dysphoria, anxiety, agitation/aggression, euphoria, disinhibition, irritability/lability, apathy, aberrant motor activity, sleep and appetite. A score for frequency (scored 1-4) and severity (scored 1-3) is derived for each domain, and the sum of frequency*severity for all domains is termed “NPI Total” below.

IL6 levels

60 cc of blood was drawn into heparinized tubes and PBMCs purified by standard Ficoll gradient methods, then cryopreserved. PBMCs were subsequently thawed and exposed to T-cell medium or to LPS in concentrations of 20 or 100 ng/ml; similar contrasts were made between exposure to T-cell medium vs. Aβ1-42 20 ug/ml. Concentrations chosen by prior experiments and extrapolations from the literature. Supernatants taken at 48 hours and IL6 measured by standard ELISA kit (R&D Biosystems).

Data analysis

The primary outcome was the ratio of IL6 release with LPS exposure to IL6 release without LPS exposure (“Il6 release ratio”). Paired Students' t-tests were used to assess individual differences in IL6 release ratio between AD and control participants. Linear regression was used to correlate IL6 release ratio with clinical measures. Statistical significance was set a priori at an alpha=0.05. Data analysis was performed with Stata 8 statistical software (College Station, TX).

Results

Ten age-gender matched participants with AD (n=5, mean age 78 +/− 2.0) and cognitively normal controls (n=5, mean age 79 +/− 1.8) were enrolled. One pair was female (20%). Only one participant (normal control) was taking an NSAID, and none were taking immunosuppressive agents including oral corticosteroids. AD participants had lower mean MMSE scores of 21.4 (S.D. 2.4) vs. 29.2 (S.D. 0.6) for the controls. NPI mean scores were higher in AD participants (9.5, S.D. 3.4) vs. controls (1.8, S.D. 1.2). AD participants performed worse on category fluency (data not shown).

The IL6 levels for all reaction conditions are listed in Table 1. At an LPS concentration of 20 ng/ml with no Aβ1–42, the mean IL6 release ratio was 53 (S.D. 26) for controls and 61 (S.D. 15) for AD (p=0.75, Student's t); the corresponding ratios with Aβ1–42 20 υg/ml were 60 (S.D. 29) for controls and 71 (S.D. 12) for AD (p=0.75, Student's t). At an LPS concentration of 100 ng/ml with no Aβ1–42, the IL6 release ratio was 45 (S.D. 22) for controls and 94 (S.D. 32) for AD (p=.07, Student's t); the corresponding ratios with Aβ1–42 20 υg/ml were 59 (S.D. 30) for controls and 101 (S.D. 12) for AD (p=0.12, Student's t). The ratio of IL6 release ratio with Aβ1–42 20 υg/ml to no Aβ1–42 exposure was 2.09 (S.D. 1.05) for AD vs. 1.1 (S.D. .12) for controls at LPS 20 ng/ml (p=0.43, Student's t); similar results were seen with LPS 100 ng/ml (data not shown). There were significant correlations between IL6 release ratio and both cognitive and NPS measures. IL6 release ratio was significantly correlated with worse animal category fluency (figure 1a) (p=0.03, r2=0.49)and higher NPI-total scores (figure 2) (p=0.01, r2=0.63). IL6 release ratio correlated significantly (p<.05) with and higher NPI Total (figure 1 b). Similarly, There were trend level correlations of Il6 release with worse ratings on the CDR (p=0.08, r2=0.33) and MMSE (p=0.05, r2=0.39) (data not shown).

Table 1. IL6 levels with and without LPS and Aβ42.

PBMCs were prepared as described in the Methods and exposed to T-cell medium or to LPS in concentrations of 20 or 100 ng/ml; similar contrasts were made between exposure to T-cell medium vs. Aβ1-42 20 ug/ml. Supernatants taken at 48 hours and IL6 measured by standard ELISA kits. Reported as mean (standard deviation).

Exposure No Aβ42 Aβ42 20 υg/ml
Controls (N=5) AD (N=5) Controls N=5) AD (N=5)
No LPS 2323 (3924) 645 (1098) 2716 (4901) 207 (149)
LPS 20 ng/ml 15216 (3639) 12566 (6379) 14715 (4347) 12918 (6182)
LPS 100 ng/ml 14884 (5443) 15954 (5628) 16483 (7604) 16141 (7114)
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Figure 1a.

Figure 1a

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Ratio of IL6 release with LPS stimulation to IL6 release without LPS stimulation, +/− Aβ1-42 20 υg/ml, vs. category fluency (animals).

Figure 1b.

Figure 1b

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Ratio of IL6 release with LPS stimulation to IL6 release without LPS stimulation, +/− Aβ1-42 20 υg/ml, vs. NPI Total.

Discussion

In this pilot project, we sought to replicate prior findings suggesting that IL6 release from LPS-stimulated PBMCs is higher in AD than cognitively normal controls. We chose the outcome of IL6 release ratio (the ratio of IL6 release with LPS stimulation vs. no LPS stimulation) to allow for ready comparison with other studies. We found increased IL6 release ratios in AD, with trend level significance at the higher LPS concentration only. This is not surprising given the small sample; since the results are in the predicted direction they offer support for hypothesis 1. We sought to extend prior results by adding an AD-specific stimulus (Aβ1-42), in an attempt to more closely model the neuroinflammatory processes of AD. Modest support was seen for hypothesis 2 with Aβ1–42 increasing the IL6 release ratio by a factor of 2 in AD patients but not controls, although this was not significant; these results are in the direction predicted by hypothesis 2.

Additionally we found a significant or trend correlation between IL6 release ratio and several clinical variables, including verbal category fluency (animals), NPI, CDR, and MMSE. A higher IL6 release ratio was associated with worse illness severity (higher NPI Total and CDR, lower MMSE and category fluency). This suggests that the IL6 release ratio may have value as a marker of AD disease severity, especially severity of neuropsychiatric symptoms (NPS). The latter appears to be a unique finding, and may reflects the involvement of inflammatory mechanisms (peripheral and central) in the development of NPS.

This study is limited by small sample size, assessment of only one candidate cytokine, and limited capacity to assess multiple reaction conditions. The latter is of relevance to the Aβ1-42 concentration, where we extrapolated concentrations from studies of AD mouse models but were lacking preliminary data in human PBMCs. The strengths of the study include a well-characterized cohort of AD and control participants and the extension of prior results to neuropsychiatric and functional as well as cognitive findings.

These preliminary results suggest that IL6 release from PBMCs may be a useful marker of disease severity and NPS severity in AD, thus meriting further study.

ACKNOWLEDGEMENTS

Supported by grants 1U01MH 66136 (Depression in Alzheimer's Disease) from the National Institute of Mental Health, P50AG005146 (Johns Hopkins University Alzheimer's Disease Research Center) from the National Institute of Aging,, and by donor funds from the Division of Geriatric Psychiatry and Neuropsychiatry, Johns Hopkins University.

Footnotes

Disclosures: none

CONFLICT OF INTEREST

The authors have no conflicts of interest to disclose regarding the procedures and data presented in this paper. The sponsors of the research (NIMH, NIA, and Johns Hopkins University) have had no involvement in the design, data collection, or authorship of this paper.

REFERENCES

  1. BANATI RB. Visualising microglial activation in vivo. Glia. 2002;40:206–217. doi: 10.1002/glia.10144. [DOI] [PubMed] [Google Scholar]
  2. ROGERS J, LUE LF. Microglial chemotaxis, activation, and phagocytosis of amyloid beta-peptide as linked phenomena in Alzheimer's disease. Neurochemistry international. 2001;39:333–340. doi: 10.1016/s0197-0186(01)00040-7. [DOI] [PubMed] [Google Scholar]
  3. BENVENISTE EN, NGUYEN VT, O'KEEFE GM. Immunological aspects of microglia: relevance to Alzheimer's disease. Neurochemistry international. 2001;39:381–391. doi: 10.1016/s0197-0186(01)00045-6. [DOI] [PubMed] [Google Scholar]
  4. BELLINGER FP, MADAMBA S, SIGGINS GR. Interleukin 1 beta inhibits synaptic strength and long-term potentiation in the rat CA1 hippocampus. Brain research. 1993;628:227–234. doi: 10.1016/0006-8993(93)90959-q. [DOI] [PubMed] [Google Scholar]
  5. VALLIERES L, CAMPBELL IL, GAGE FH, SAWCHENKO PE. Reduced hippocampal neurogenesis in adult transgenic mice with chronic astrocytic production of interleukin-6. The Journal of neuroscience : the official journal of the Society for Neuroscience. 2002;22:486–492. doi: 10.1523/JNEUROSCI.22-02-00486.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. ROSENBERG PB. Clinical aspects of inflammation in Alzheimer's disease. International review of psychiatry (Abingdon, England) 2005;17:503–514. doi: 10.1080/02646830500382037. [DOI] [PubMed] [Google Scholar]
  7. REALE M, IARLORI C, GAMBI F, FELICIANI C, SALONE A, TOMA L, DELUCA G, SALVATORE M, CONTI P, GAMBI D. Treatment with an acetylcholinesterase inhibitor in Alzheimer patients modulates the expression and production of the pro-inflammatory and anti-inflammatory cytokines. Journal of neuroimmunology. 2004;148:162–171. doi: 10.1016/j.jneuroim.2003.11.003. [DOI] [PubMed] [Google Scholar]
  8. MILLER AH, RAISON CL. Cytokines, p38 MAP kinase and the pathophysiology of depression. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology. 2006;31:2089–2090. doi: 10.1038/sj.npp.1301032. [DOI] [PubMed] [Google Scholar]
  9. MCKHANN G, DRACHMAN D, FOLSTEIN M, KATZMAN R, PRICE D, STADLAN EM. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology. 1984;34:939–944. doi: 10.1212/wnl.34.7.939. [DOI] [PubMed] [Google Scholar]
  10. ALZHEIMER'S ASSOCIATION Research consent for cognitively impaired adults: recommendations for institutional review boards and investigators. Alzheimer Disease and Associated Disorders. 2004;18:171–175. doi: 10.1097/01.wad.0000137520.23370.56. [DOI] [PubMed] [Google Scholar]
  11. MORRIS JC. The Clinical Dementia Rating (CDR): current version and scoring rules. Neurology. 1993;43:2412–2414. doi: 10.1212/wnl.43.11.2412-a. [DOI] [PubMed] [Google Scholar]
  12. FOLSTEIN MF, FOLSTEIN SE, MCHUGH PR. “Mini-mental state”. A practical method for grading the cognitive state of patients for the clinician. Journal of psychiatric research. 1975;12:189–198. doi: 10.1016/0022-3956(75)90026-6. [DOI] [PubMed] [Google Scholar]
  13. CUMMINGS JL, MEGA M, GRAY K, ROSENBERG-THOMPSON S, CARUSI DA, GORNBEIN J. The Neuropsychiatric Inventory: comprehensive assessment of psychopathology in dementia. Neurology. 1994;44:2308–2314. doi: 10.1212/wnl.44.12.2308. [DOI] [PubMed] [Google Scholar]

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