HUMAN IMMUNOLOGY doi: 10.1111/sji.12131 ..................................................................................................................................................................

Increased Levels of Cytokines in Cerebrospinal Fluid of Children with Aseptic Meningitis Caused by Mumps Virus and Echovirus 30 A. Sulik*, A. Kroten*, M. Wojtkowska† & E. Oldak*

Abstract *Department of Pediatric Infectious Diseases, Medical University of Bialystok, Bialystok, Poland; and †Department of Pediatric Laboratory Diagnostics, Medical University of Bialystok, Bialystok, Poland

Received 20 August 2013; Accepted in revised form 24 October 2013 Correspondence to: A. Sulik, MD, PhD, Department of Pediatric Infectious Diseases, Medical University of Bialystok, Waszyngtona 17, 15-274 Bialystok, Poland. E-mail: artur. [email protected]

We measured levels of pro-inflammatory cytokines in the cerebrospinal fluid (CSF) of patients with mumps meningitis, enteroviral echovirus 30 meningitis and children without central nervous system infection to investigate whether these molecules were involved in the pathogenesis of viral meningitis. The CSF was obtained from 62 children suspected with meningitis. These patients were classified to the mumps meningitis (n = 19), echovirus 30 meningitis (n = 22) and non-meningitis (n = 21) groups. The concentrations of interleukin-1 (IL-1), interleukin-1 soluble receptor type 2 (IL-1R2), interleukin-8 (IL-8), human interferon gamma (IFN-c) and human tumour necrosis factor alpha (TNF-a) were determined by immunoassay. A significant increase was noted in the levels of IL-8, TNF-a and IL-1R2 in the CSF of both meningitis groups as compared to controls. The concentrations of IFN-c and IL-1 differed significantly only between the mumps group and control. The levels of IL-1, IFN-c and TNF-a were significantly higher in mumps meningitis when compared to the echovirus 30 group. Of all cytokines examined, only IFN-c correlated with pleocytosis (r = 0.58) in the mumps meningitis group. The increased CSF cytokine levels are markers of meningeal inflammation, and each virus may cause a specific profile of the cytokine pattern.

Introduction Aseptic meningitis is the most common cause of inflammation of the central nervous system (CNS) in children worldwide. Most cases of aseptic meningitis are of viral aetiology. Before the wide introduction of epidemic parotitis vaccine, mumps virus was a common cause of viral meningitis. Currently, mumps vaccine is offered to children in more than a hundred countries throughout the world. At present, the overwhelming majority of meningitis cases are caused by enteroviruses. Although enteroviral meningitis is usually a benign disease, it may exceptionally lead to serious complications or death [1]. Recent attention has been directed towards the role of cytokines in the regulation of inflammation and host responses to CNS infection [2–4]. The mechanisms by which viruses cross the blood–brain barrier to stimulate transmigration of leucocytes are unclear and need more profound investigations. The literature data suggest that the release of certain cytokines, such as interleukin-1 (IL-1), interleukin-8 (IL-8), tumour necrosis factor-a (TNF-a) and interferon-c (IFN-c), may be responsible for meningeal inflammatory infiltration

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in purulent and aseptic meningitis and may correlate with the disease outcome [5–7]. Interleukin-1 has a number of functions in the CNS system and is regulated at many levels [8–10]. IL-1 is the name that designates two proteins, IL-1a and IL-1b, which are the products of distinct genes, but which recognize the same cell surface receptors. Two distinct IL-1 receptor types have been isolated, both structurally similar but functionally diverse. IL-1 type 1 receptor (IL-1R1) binds IL-1a and IL-1b, whereas IL-1 type 2 receptor (IL-1R2) has much higher affinity for IL-1b than IL-1a [11–13]. Interestingly, IL-1R1 is the primary receptor responsible for IL-1 signal transduction while IL-1R2 possesses the ability to inhibit IL-1 signalling. Soluble form of IL-1R2 has been implicated in human disease pathogenesis [14, 15]. IL-8 plays a key role in neutrophil infiltration in infectious CNS inflammation and contributes to extensive spinal cord lesion in multiple sclerosis [16]. Cytokines, such as IFN-c and TNF-a, play a crucial role in the immune response. TNF-a is an important proinflammatory cytokine and apoptotic molecule involved in the pathogenesis of neuroinfection. It has been suggested

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A. Sulik et al. Cytokines in Mumps and Enteroviral Meningitis 69 ..................................................................................................................................................................

that the levels of TNF-a may be a useful diagnostic tool for distinguishing bacterial from aseptic meningitis. Measurement of this cytokine in bacterial and aseptic meningitis has a sensitivity of 74% and a specificity of 81% [17]. It has been shown using the animal model that blood-derived leucocytes are mainly responsible for the release of IL-1 and TNF-a into the cerebrospinal fluid (CSF) during bacterial meningitis, whereas IL-8 is rather produced by local cells within the CNS [18]. Various serological methods are currently in use for determining the expression of molecules in the CNS [19]. The aim of our study was to measure selected cytokine concentrations in the CSF of children with two different types of viral meningitis and normal controls to investigate their role in inciting CNS inflammation.

Material and methods Subjects. The population of patients consisted of 62 children, aged 1–17 years, suspected with meningitis, who were followed in the Department of Pediatric Infectious Diseases. On admission, all the patients had clinical signs and symptoms of meningitis, and their CSF was examined. According to the diagnosis, the children were divided into three groups: group I consisted of 19 patients with mumps meningitis; group II was made up of 22 children with echovirus 30 meningitis; and the control group included 21 patients without CNS involvement. Aseptic meningitis was diagnosed on the basis of the increased number of cells in CSF > 6 leucocytes/ll and lack of bacteria in culture. The diagnosis of mumps was based on the clinical case definition during mumps outbreak. Meningitis that directly followed epidemic parotitis was assumed to be mumps meningitis. Echovirus type 30 meningitis was diagnosed by enteroviral culture. The control group consisted of children diagnosed with meningitis-like symptoms: headaches, stiff neck, stupor or seizures. In these children, CSF examination disclosed normal leucocyte count and on that basis and clinical follow-up, the diagnosis of meningitis was finally ruled out. The study protocol was approved by the Research Ethical Committee, and informed consent of parents, caregivers and adolescents when required by law was obtained prior to enrolment. Sample collection and measurements. A lumbar puncture was performed urgently upon admission to hospital. All study patients underwent routine CSF collection for analysis including cell count, proportion of mononuclear and polymorphonuclear cells, protein level and any further CSF examination required by diagnostic protocols for children with suspected meningitis. All the CSF samples used were free of erythrocytes contamination. After the routine analysis, the remaining CSF samples were centrifuged and frozen for the purpose of this study. Patients’

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medical data were recorded together with basic CSF findings. Afterwards, CSF samples were simultaneously investigated for five different cytokines, namely: IL-1, IL-1R2, IL-8, IFN-c and TNF-a. All the immunoassays were supplied by R&D Systems, Minneapolis, MN, USA, and tests were performed according to the manufacturer’s instructions. Results were expressed in picograms per millilitre. Whenever the test result was below detection level of assay, a value of 0.01 was assumed for statistical analysis. Statistical analysis. Results of the immunoassay measurements were presented as median, minimum and maximum values, and nonparametric tests were used for statistical analysis. Significance was determined with Mann–Whitney U-test, and Spearman’s rank correlation coefficient was used to assess associations between values. For Table 1, statistics were performed on the mean values for each group. P values of

Increased levels of cytokines in cerebrospinal fluid of children with aseptic meningitis caused by mumps virus and echovirus 30.

We measured levels of pro-inflammatory cytokines in the cerebrospinal fluid (CSF) of patients with mumps meningitis, enteroviral echovirus 30 meningit...
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