Introduction

Multiple sclerosis is an inflammatory demyelinating disease of the central nervous system. The inflammation is caused by autoimmune processes especially appearing in the early stages of MS. The neurodegenerative processes appear mainly in later stages of MS. The disease is characterized by many inflammatory infiltrates especially in the white matter, where T-lymphocytes and macrophages are activated (also fewer B-lymphocytes). T-lymphocytes are probably activated in the periphery. After activation these specific lymphocytes proliferate into the final tissue and initiate the inflammatory lesion by the production of many pro-inflammatory cytokines. The damage of myelin and oligodendrocytes is followed in this lesion. The demyelinating fibre loses the ability to spread electric impulses, and the cells of major histocompatibility complex I (MHC I) are also expressed. Through these processes the fibre becomes visible for CD8 cytotoxic lymphocytes, which damage this fibre. The neurodegeneration is caused by microglia’s activation and mediated by oxidative stress and excitotoxicity. The axon loss in white and grey matter is present in early stages of MS and correlates with the cognitive deficit and also with the permanent disability.

Generally, relapses are the clinical equivalent of the acute focal inflammation, and progression is caused by chronic neurodegeneration. The standard diagnostic test in patients with suspected MS is the examination of the intrathecal synthesis of IgG antibodies. However, it is important to discover the more detailed mechanism causing MS. One of these methods is the examination of CSF and levels of inflammatory and neurodegenerative markers in CSF.

The aim of our study was to ascertain levels of some inflammatory markers in MS patients in comparison to the control group. Based on the results of previous studies and the availability of testing in our laboratory, we chose five inflammatory markers (IL-6, IL-8, IL-10, beta-2 microglobulin, orosomucoid) to investigate their CSF levels at the time of first clinical symptoms of MS.

Methods

The study was approved by the Institutional Ethics Committee, and all patients have signed an informed consent to lumbar puncture.

The study was based on CSF and serum examination in patients with MS and the control group. In the control group were patients with non-inflammatory disease of CNS (e.g., headache, vertigo, back pain, etc.). The lumbar puncture was indicated from differential diagnostic reasons, to exclude other pathological processes of CNS. In the MS patients, the lumbar puncture was indicated and performed for diagnostic purposes in cases suggestive of MS, at the time of the first clinical symptoms compatible with MS. The diagnosis of MS was confirmed in all patients included in MS group. All patients included in the MS group fulfilled the McDonald’s revised diagnostic criteria. None of our patients had been treated by corticosteroids before the lumbar puncture.

The lumbar puncture was performed by means of atraumatic needle under aseptic conditions. About 10 ml of CSF was taken along with around 10 ml of blood from each participant. The serum and CSF were the subject of haematological, immunological and biological testing. The protein CSF level and cell count were normal in all patients, without signs of neuroinflammation. There were also no signs of any other inflammation at the time of lumbar puncture.

Biomarker selection and measurement

CSF and serum were analyzed to assess the levels of five inflammatory markers: interleukin-6, interleukin-8, interleukin-10, beta-2-microglobulin, orosomucoid, available for testing in our laboratory at the study onset.

The principle of measurement of IL-6 levels used in our study was ELISA (solid-phase, enzyme-labelled, chemiluminescent sequential immunometric assay, IMMULITE®/IMMULITE® 1000 IL-6, Catalogue Number: LK6PZ, LK6P1). Incubation Cycles: 2 × 30 min. Analytical sensitivity: 2 pg/ml. Reference limits: CSF 0–18 pg/ml, serum 0–5.9 pg/ml.

For measurement of IL-8 ELISA was also used (IMMULITE®/IMMULITE® 1000 IL-8, Catalogue Number LK8P1). Incubation Cycles: 1 × 30 min. Analytical sensitivity: 2 pg/ml. Reference limits: CSF 0–62 pg/ml, serum 0–62 pg/ml.

The same method was used also for measurement levels of IL-10. IMMULITE®/IMMULITE® 1000 IL-10, Catalogue Number: LKXPZ, LKXP1. Incubation Cycles: 1 × 60 min. Analytical sensitivity: 1 pg/ml. Reference limits: CSF 0–39 pg/ml, serum: 0–9.1 pg/ml.

Levels of beta-2-microglobulin were measured by particle-enhanced immunonephelometry using the BN* II and BN ProSpec® System. Reference limits: CSF 0.2–2 mg/l, serum 1–3 mg/l. Coefficients of variation (CV): CSF 2.7 %, serum 2.7 %.

Levels of orosomucoid were also measured by particle-enhanced immunonephelometry using the BN* II and BN ProSpec® System. Reference limits: CSF 1.8–4.5 mg/l, serum 0.3–1.3 g/l. Coefficients of variation (CV): CSF 3.3 %, serum 3.8 %.

Statistical analysis

Then, the results were statistically processed using SPSS software version 15 (SPSS Inc., Chicago, USA). The categorical data were compared by Fisher’s exact test.

The Mann–Whitney U test was used to compare quantitative parameters. The age was compared by the Student’s t test. The normality of data was checked by the Shapiro–Wilk test. Tests were made at the significance level of 0.05.

Results

We investigated a group of 66 patients. CSF and serum examinations were performed in 38 patients with newly diagnosed MS meeting McDonald’s revised diagnostic criteria (25 females, 13 males; mean age 35.8 ± 9.1 years) and in 28 control group patients (20 females, 8 males; mean age 39.7 ± 13.0 years). No statistically significant differences in demographic data between patients and control group were found. The demographic data are shown in Table 1.

Table 1 Characteristic of patients
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Significantly higher CSF levels of IL-8 (52.8 (41.6–68.3); p < 0.001, Mann–Whitney U test) and beta-2-microglobulin (1.26 (1.03–1.37); p = 0.007, Mann–Whitney U test) in MS patients group were found. No differences in other CSF markers (IL-6, IL-10 and orosomucoid) and in serum levels of all markers between both groups were found (Tables 2, 3; Figs. 1, 2).

Table 2 Levels of investigated markers in CSF
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Table 3 Levels of investigated markers in serum
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Fig. 1
figure 1

Box graph showing statistically significantly higher levels of interleukin-8 in MS patients in comparison to controls

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Fig. 2
figure 2

Box graph showing statistically significantly higher levels of beta-2-microglobulin in MS patients in comparison to controls

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Discussion

We identified increased levels of IL-8 and beta-2 microglobulin in CSF. These results confirm the presence of inflammatory processes at time of the first clinical symptoms of MS.

There have been multiple studies investigating the levels of inflammatory markers in the cerebrospinal fluid in MS. Their results are not entirely consistent, which probably results from the great clinical diversity of the studied populations. There are studies which examine a wider range of biomarkers, which are not included in our research and have interesting results. Martins et al. (2011) analyzed the serum concentrations of 13 proinflammatory and anti-inflammatory markers. Significant increases between patients and control subjects were found in seven of them. Another frequently discussed marker is the cytokine interleukin-23 (IL-23). IL-23 was found significant elevated in study by Wen et al. (2012). The study by Vaknin-Dembinsky et al. (2006) reported an increase in the levels of IL-23 secreted from monocyte-derived dendritic cells as well as increased mRNA production of both IL-23p19 in MS patients. There is also a more comprehensive study by Modvig et al. (2013) dealing with the interrelation among biomarkers of inflammation, demyelination and neurodegeneration in acute optic neuritis and to assess their association with MS risk. In that study, it was found that the CSF biomarkers of leukocyte infiltration (CXCL13, MMP-9, CXCL10) are associated strongly with MS-risk parameters, whereas CHI3L1 and MBP correlated with MRI findings (dissemination in space), but not with CSF MS-risk parameters and osteopontin and NF-L do not correlate with any MS-risk parameters. However, in the following discussion, we will focus on the markers we have included in our study.

Beta-2 microglobulin is the part of histocompatibility antigens on cell surfaces. It plays an important role in intercellular interactions and is used as a cell turnover marker. It is mostly found on white blood cells (especially B lymphocytes). Beta-2- microglobulin has been found to correlate with disease activity in several autoimmune disorders. It is also used as a pharmacodynamic marker of interferon beta, which is used for treating MS patients at the present time (Bagnato et al. 2003; Bjerrum et al. 1988). This outcome correlates with our conclusion. However, no other studies showed differences in serum and CSF levels of beta-2 microglobulin in MS patients in comparison to controls (Sladkova et al. 2011; Carrieri et al. 1992). As a result, there are inconsistent results concerning beta-2-microglobulin in MS patients today. Additional studies of beta-2-microglobulin are needed.

Interleukin-8 (IL-8) is an inflammatory chemokine. It attracts and activates neutrophils and a subpopulation of lymphocytes. It also has a strong angiogenic effect. Interleukin-8 is produced by many cells such as monocytes, lymphocytes, granulocytes, fibroblasts, endothelial cells and astrocytes. It is elevated in CSF in inflammatory processes of CNS (Bielekova et al. 2012). Interleukin-8 is also responsible for blood–brain barrier disruption and the migration of immune cells to the CNS. According to one very interesting study on the effect of high-dose corticosteroids on levels of interleukin-8, the blood levels of interleukin-8 decrease after therapy (Mirowska-Guzel et al. 2006). The serum levels are also decreased in MS patients receiving interferon beta 1a therapy in comparison to untreated MS patients (Lund et al. 2004). Two other studies also confirm our results, and their authors found a significant rise of CSF levels of IL-8 in MS during relapse (Bartosik-Psujek and Stelmasiak 2005; Franciotta et al. 2001). The fact that IL-8 is associated with disease activity was used in other research. Neuteboom et al. (2009) researched levels of IL-8 in MS patients during pregnancy. High serum levels of IL-8 during the first trimester were associated with a high risk of postpartum relapses. Another study compared levels of interleukins in two types of MS (opticospinal and conventional). Elevated levels of IL-8 were found in both groups. However, in opticospinal MS the levels were significantly higher than in conventional MS. According to this study, elevated levels of IL-8 also correlate with Expanded Disability Status Scale (EDSS) (Ishizu et al. 2005). All of these studies suggest that IL-8 is an important chemokine in the inflammatory phase of MS. The results of our study also confirm these findings.

We found no statistically significant levels of the other three biomarkers (IL-6, IL-10, orosomucoid) in our study.

Interleukin-6 (IL-6) is the proinflammatory cytokine. It might be responsible for immunoglobulin (Ig) synthesis in the CNS (Maimone et al. 1991). Low levels of IL-6 were found by Maimone et al. (1991) in most sera from MS patients and also in patients with non-inflammatory neurological disease. Other results of this study indicate increased CSF levels of IL-6 in MS patients and also in patients with inflammatory neurological disease, without correlation to intrathecal synthesis of IgG. Some degree of dependence between serum IL-6 concentration and the level of EDSS were ascertained by Stelmasiak et al. (2000).

Interleukin-10 (IL-10) is an anti-inflammatory cytokine. B cells producing IL-10 regulate autoimmune diseases, including MS (Hirotani et al. 2010). There are some studies dealing with therapy of MS by interferons and glatiramer acetate (GA) and their effect on the production of some cytokines, including IL-10. The study by Pul et al. (2011) shows the effect of GA on CNS. Among other things, increased secretion of IL-10 after therapy by GA was found.

The small molecule of orosomucoid is able to transgress the blood–brain barrier (BBB). Its concentration increases in malignant CNS processes (Sladkova et al. 2011). The increased CSF levels can occur in some MS patients (50–60 %) after several years of disease duration (Adam et al. 2003). Decreased levels of orosomucoid in secondary progressive MS were found in the same study.

In the future, we intend to broaden the spectrum of available inflammatory markers including, e.g., IL-23 and also to include comparison with patients suffering from other inflammatory and infectious diseases of the nervous system, such as meningitis, encephalitis and inflammatory demyelinating polyradiculoneuropathy.

Conclusion

The levels of two studied inflammatory markers were found to be increased at the time of first clinical symptoms of MS. The increased levels of IL-8 were also confirmed in other studies. Thus, IL-8 seems to be an important chemokine in the inflammatory phase of MS. The second marker with elevated levels was beta-2-microglobulin, for which, however, other studies in MS patients have yielded inconsistent results. As the aetiology of MS is still unknown, research on inflammatory and neurodegenerative markers in MS should continue.