Interleukin 4 and Interleukin 10 Levels Are Elevated in the Cerebrospinal Fluid of Patients with Creutzfeldt-Jakob Disease

ORIGINAL CONTRIBUTION Interleukin 4 and Interleukin 10 Levels Are Elevated in the Cerebrospinal Fluid of Patients With Creutzfeldt-Jakob Disease

Katharina Stoeck, MD; Monika Bodemer; Barbara Ciesielczyk; Bettina Meissner, MD; Mario Bartl, MD; Uta Heinemann; Inga Zerr, MD

Background: In neurodegenerative diseases, increas- mentia (n=10), patients with motoneuron disease (n=6), ing attention has been focused on inflammatory media- patients with normal pressure hydrocephalus (n=5), and tors such as pro-inflammatory and anti-inflammatory cy- control subjects (n=20). tokines and their potential influence in the process of neurodegeneration. In prion diseases, much data has been Results: Interleukin 10 levels were significantly el- gained on the cell culture and animal disease models level, evated in the cerebrospinal fluid of CJD patients (me- but only limited information is available on humans af- dian, 9.8 pg/mL). The elevation was significant to other fected by Creutzfeldt-Jakob disease (CJD). dementia (median, 7.9 pg/mL, PϽ.05), motoneuron disease (median, 7.9 pg/mL, PϽ.05), normal pressure hy- Objective: To obtain data on anti-inflammatory cyto- drocephalus (median, 7.0 pg/mL, PϽ.05), and controls kines interleukin 4 and interleukin 10 in the cerebrospi- (median, 1.3 pg/mL, PϽ.001). Levels of interleukin 4 were nal fluid of patients with CJD, patients with other de- significantly elevated in cerebrospinal fluid of patients mentia, and nondemented neurological patients and with CJD (median, 26.4 pg/mL) compared with control controls. subjects (median, 6.2 pg/mL, PϽ.001) and patients with a motoneuron disease (median, 10.5 pg/mL, PϽ.001) Design: Cerebrospinal fluid samples were collected from CJD patients and control subjects, and concentrations of Conclusions: Elevated levels of the anti-inflammatory the anti-inflammatory cytokines interleukin 4 and inter- cytokines interleukin 4 and interleukin 10 in cerebro- leukin 10 were determined using an enzyme-linked im- spinal fluid of patients with CJD are new findings. The munosorbent assay. data of the present study provide a clue toward the possible role of cytokines as immunological modifiers in the Patients: Cerebrospinal fluid samples from 61 pa- neurodegenerative process of CJD. tients were analyzed. The group was composed of patients with CJD (n=20), patients with other forms of de- Arch Neurol. 2005;62:1591-1594

REUTZFELDT-JAKOB DIS- phoid organs and B-cells as well as fol- ease (CJD) is a rare and licular dendritic cells are necessary for fatal neurodegenerative prion replication and propagation.2-4 disease that occurs Research concerning the involvement of worldwide. Creutzfeldt- the immune system in the CNS in prion dis- JakobC disease belongs to the human eases has focused mainly on infectivity of prion diseases which are characterized microglia and the release of pro- by the accumulation of an infectious inflammatory cytokines.5-7 Expression of protein, termed prion (PrPSc). This rep- pro-inflammatory cytokines was de- resents the pathological isoform of the scribed earlier in scrapie-infected mice.8 protein PrPc, expressed physiologically Also, elevated levels of the pro-inflamma- and predominantly in the central ner- tory cytokines interleukin (IL) 1␤ and tu- vous system (CNS).1 mor necrosis factor ␣ have been found in Recently, detailed analysis of the func- the CSF of patients with CJD.9 Author Affiliations: tion of the immune system in the periph- The question of anti-inflammatory cy- Department of Neurology, eral prion invasion has been performed. tokines has not been addressed so far. We University Hospital Göttingen, It revealed that major components of the used the CSF as a testing platform to de- Göttingen, Germany. immune system, such as secondary lym- termine levels of anti-inflammatory cyto-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 CSF findings, and normal results from a neurological exami- Table. Levels of IL-4 and IL-10 in CSF of Patients nation, neurophysiological tests, and brain magnetic reso- With CJD and Other Neurological Diseases nance imaging. The diagnoses in this group of patients were determined on the basis of medical records. The following Median Median diagnoses were made: psychiatric diseases (eg, depression), (Minimum-Maximum) (Minimum-Maximum) pain syndromes (eg, headache), and vertigo (eg, benign par- Levels of IL-4, pg/mL Levels of IL-10, pg/mL oxysmal positional vertigo). In all cases, an inflammatory CJD 26.4 (18.2-39.3) 9.8 (6.1-21.5) CNS disease was excluded. Dementia 25.7 (15.3-34.0) 7.9 (4.6-11.1) NPH 23.4 (17.0-25.3) 7.0 (6.8-9.2) CSF SAMPLES MND 10.5 (7.8-19.7) 6.2 (3.7-14.4) Controls 6.2 (4.3-37.1) 1.3 (0.9-7.5) Cerebrospinal fluid samples from patients with CJD were collected at the respective external hospitals. These samples were Abbreviations: CJD, Creutzfeldt-Jakob disease; CSF, cerebrospinal fluid; sent to the laboratory of the CJD surveillance unit at the De- IL-4, interleukin 4; IL-10, interleukin 10; NPH, normal pressure partment of Neurology, University Hospital Göttingen, for de- hydrocephalus; MND, motoneuron disease. termination of protein 14-3-3. Only CSF samples that had been sent on dry ice and that had been immediately stored at−80°C were used for this study. kines in CSF in patients with CJD, since in many neu- Cerebrospinal fluid samples from patients of the other test rological diseases, changes in the CSF protein pattern are groups were obtained from the Neurochemical Laboratory of closely linked to pathological processes of the brain. The the University Göttingen and immediately frozen at−80°C. These detection of elevated levels of 14-3-3 and other neuro- patients had either been admitted to the Neurological Clinic nal proteins in the CSF in patients with CJD exemplifies of the University Hospital Göttingen for inpatient treatment or how the rapid brain damage and release of neuronal pro- had received treatment at the outpatient clinic for clarification teins into the CSF reflect the changes of affected brain of their diagnoses by lumbar puncture. tissue.10 We intended to first obtain information on CSF levels of IL-4 and IL-10 in patients with CJD, since there ENZYME-LINKED IMMUNOSORBENT ASSAY are no data available on this subject in the literature. Commercially available enzyme-linked immunosorbent assay test kits (Bender MedSystems; MedSystems Diagnostics GmbH, Vi- METHODS enna, Austria) were used for quantitative analysis of the anti- inflammatory cytokines IL-4 and IL-10. Tests were performed in CLINICAL CHARACTERISTICS OF CJD PATIENTS accordance with the manufacturer’s instructions. The concen- tration of the first standard was 7.8 pg/mL for IL-4 and 3.2 pg/mL This group consisted of 20 patients who were reported as sus- for IL-10. pected CJD cases to the German CJD surveillance unit. These patients, 11 men and 9 women, were classified as sporadic CJD cases STATISTICS based on established clinical criteria (probable sporadic CJD, n=15) and post-mortem examination (definite sporadic CJD, n=5). The Statistical evaluation of the data was performed using Statistica average age was 64 years (age range, 54-79 years). version 6.0 (Statsoft Inc, Tulsa, Okla). The Mann-Whitney U test was used to determine the statistical significance of the differences between the 2 independent data sets. Differences were con- PATIENTS WITH OTHER FORMS OF DEMENTIA sidered significant at an error probability of PϽ.05. This group consisted of 10 patients, 8 men and 2 women with the following diagnoses: Alzheimer disease, vascular demen- RESULTS tia, and dementia of unknown etiology. The average age was 65 years (age range, 35-79 years). INTERLEUKIN 4 IN CSF

PATIENTS WITH Table summarizes the levels of IL-4 in CSF. The level NORMAL PRESSURE HYDROCEPHALUS of IL-4 was significantly increased in the CSF of patients with CJD vs normal controls (PϽ.001), patients Here we included a group of patients with normal pressure hy- with normal pressure hydrocephalus (PϽ.02), and pa- drocephalus (n=5, median age, 70 years, age range, 63-78 years). tients with motoneuron disease (PϽ.001) (Figure 1). There was no statistical difference between patients with PATIENTS WITH MOTONEURON DISEASE CJD and patients with other forms of dementia (P=.4). This group included 6 patients (median age, 52 years, age range, 35-80 years). INTERLEUKIN 10 IN CSF Levels of IL-10 are summarized in Table. The level of IL-10 CONTROL PATIENTS was significantly elevated in the CSF of patients with CJD compared with controls (PϽ.001), patients with normal- This group included 20 patients, 15 women and 5 men. The Ͻ average age was 39 years and the age range was 16 to 78 pressure hydrocephalus (P .05), patients with moto- years. These patients received inpatient treatment at the neuron disease (PϽ.05), and—most interestingly— Neurological Clinic of the University Hospital Göttingen, compared with patients with other forms of dementia Göttingen, Germany, had a lumbar puncture and normal (PϽ.05) (Figure 2).

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Level of Interleukin 4, pg/mL 6 Level of Interleukin 10, pg/mL

10 Median 4 25%-75% Range Without Extreme 5 ∗ Extreme 2

0 0 Control MND NPH Dementia CJD Control MND NPH Dementia CJD

Figure 1. Level of interleukin 4 in cerebrospinal fluid in patients with Figure 2. Level of interleukin 10 in cerebrospinal fluid of patients with Creutzfeldt-Jakob disease (CJD), various other neurological diseases, and Creutzfeldt-Jakob disease (CJD), various other neurological diseases, and controls. MND indicates motoneuron disease; NPH, normal pressure controls. MND indicates motoneuron disease; NPH, normal pressure hydrocephalus. hydrocephalus.

/ COMMENT been investigated in IL-10− −mice. These animals de- veloped disease earlier in comparison to wild type mice, The exact mechanisms leading to the neurodegenera- suggesting an important modifying function for anti- tion in prion diseases are not known in detail and the inflammatory cytokines in the development of prion involvement of microglia is subject for discussion.11 Vari- disease.13 ous studies have recently been performed to identify im- The main purpose of this study has been to investi- munological mechanisms. These have contributed mainly gate the anti-inflammatory cytokines IL-4 and IL-10 in to an understanding of the involvement of the immune CSF of patients with CJD. Interleukin 4 and IL-10 are system during the peripheral prion invasion in experi- potent anti-inflammatory cytokines which are pro- mental models, in which the lymphoreticular organs are duced by a variety of cells, specifically TH2 cells, and by necessary for accumulating and replicating prions. How- macrophages in the periphery. In the CNS, they are pro- ever, it still remains unclear to what extent the CNS is duced by microglial and astroglial cells under various con- immunologically involved in CJD. ditions, eg, multiple sclerosis, ischemia, and also in neu- Cerebrospinal fluid changes as seen in other infectious rodegenerative diseases such as Alzheimer disease.14,15 We and inflammatory diseases are absent in CJD. In histo- show in our study that IL-4 and IL-10 are significantly pathological examination, inflammatory infiltrates are ab- elevated in the CSF of patients with CJD compared with sent. One important histological finding relates to acti- controls and patients with other neurodegenerative CNS vated microglia, which represent resident CNS macrophages diseases. To date there are no studies in the literature de- that can react as immunologically potent cells in case of scribing levels of IL-4 or IL-10 in the CSF or brain tissue stress to the CNS. In their activated state, they are sup- of patients with CJD. posed to play a predominant role in the so-called atypical In the literature, a similar investigation has been car- inflammatory process in prion diseases with expression of ried out on the ventricular CSF of patients with juvenile major histocompatibility complex class II molecules and parkinsonism and Parkinson disease. The authors have de- release of inflammatory and tissue-toxic mediators such as tected elevated levels of IL-4 (among other interleukins) pro-inflammatory and anti-inflammatory cytokines. How- in patients, but not in controls, and speculate that alter- ever, it remains unclear whether the neurodegenerative pro- ations of cytokines in the CSF may reflect changes of cy- cess in CJD is primarily caused by prion accumulation alone tokines in the brain during the process of neurodegenera- or whether tissue-toxic mediators released by activated mi- tion.16 They also speculated that the increase in cytokines croglia may contribute to the neurotoxicity—and subse- might not only be due to a compensatory response, but quently to neurodegeneration. Another hypothesis posits might itself trigger neurodegeneration. Another study has that even neurons may release cytokines, especially anti- shown that IL-4 and IL-10 differentially regulate microg- inflammatory cytokines after damage. In CJD and also in lial responses to A␤ and may play a role in the observed Alzheimer disease, the anti-inflammatory cytokine trans- pathology surrounding senile plaques in Alzheimer dis- forming growth factor ␤2 could be localized intraneuro- ease.17 Unfortunately, no data on the levels of these cyto- nally in degenerative neurons.12 kines in CSF in Alzheimer disease were given. The potential role of anti-inflammatory cytokines Our own data are in line with these observations, (IL-4, IL-10, and IL-13) in prion disease has recently since we could also detect elevated levels of IL-4 and

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/30/2021 IL-10 in the CSF of patients with dementia other than REFERENCES CJD. In our article we focused on CJD, but our group of patients with dementia also included patients with Alz- 1. Prusiner SB. Novel proteinaceous infectious particles cause scrapie. Science. 1982; heimer disease and elevated levels were observed in this 216:136-144. subgroup as well. 2. Aguzzi A, Heppner FL, Heikenwalder M, et al. Immune system and peripheral nerves Cerebrospinal fluid changes may reflect changes of the in propagation of prions to CNS. Br Med Bull. 2003;66:141-159. 3. Klein MA, Frigg R, Flechsig E, et al. A crucial role for B cells in neuroinvasive CNS to a major degree. Cerebrospinal fluid analysis is scrapie. Nature. 1997;390:687-690. performed routinely for the purpose of diagnosis in a ma- 4. Prinz M, Heikenwalder M, Junt T, et al. 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Analysis of EEG and CSF 14-3-3 proteins as Correspondence: Inga Zerr, MD, Department of Neu- aids to the diagnosis of Creutzfeldt-Jakob disease. Neurology. 2000;55: rology, Georg-August University, Robert-Koch-Str 40, 811-815. 37075 Göttingen, Germany ([email protected] 11. Giese A, Kretzschmar HA. Prion-induced neuronal damage–the mechanisms of .de). neuronal destruction in the subacute spongiform encephalopathies. Curr Top Mi- Author Contributions: Study concept and design: Stoeck crobiol Immunol. 2001;253:203-217. 12. Tashiro H, Dohura K, Iwaki T. Differential expression of transforming growth factor- and Zerr. Acquisition of data: Stoeck, Bodemer, Ciesiel- beta isoforms in human prion diseases. Neuropathol Appl Neurobiol. 1998; czyk, Meissner, Bartl, and Heinemann. Analysis and in- 24:284-292. terpretation of data: Stoeck, Meissner, and Zerr. Drafting 13. Thackray AM, McKenzie AN, Klein MA, Lauder A, Bujdoso R. Accelerated prion of the manuscript: Stoeck and Zerr. Critical revision of the disease in the absence of interleukin-10. J Virol. 2004;78:13697-13707. 14. Hulshof S, Montagne L, De Groot CJ, Van Der Valk P. Cellular localization and manuscript for important intellectual content: Stoeck, expression patterns of interleukin-10, interleukin-4, and their receptors in mul- Bodemer, Ciesielczyk, Meissner, Bartl, Heinemann, and tiple sclerosis lesions. Glia. 2002;38:24-35. Zerr. Statistical analysis: Stoeck and Zerr. Obtained 15. Suzumura A, Sawada M, Itoh Y, Marunouchi T. Interleukin-4 induces prolifera- funding: Stoeck and Zerr. Administrative, technical, and tion and activation of microglia but suppresses their induction of class II major material support: Stoeck, Bodemer, Ciesielczyk, Meiss- histocompatibility complex antigen expression. J Neuroimmunol. 1994; 53:209-218. ner, Bartl, Heinemann, and Zerr. Study supervision: 16. Mogi M, Harada M, Narabayashi H, Inagaki H, Minami M, Nagatsu T. Interleukin Stoeck and Zerr. 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