Multiple sclerosis (MS) is a progressive immune-mediated disease caused by demyelination of the central nervous system (CNS), with perivascular infiltrates of autoreactive T-cells that recognize and react against autoantigens, including myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocytic glycoprotein (MOG) and myelin-associated glycoprotein (1,2). Cytokines and their receptors are important in the evolution of MS lesions, and pro- and anti-inflammatory cytokine levels have been found to correlate with changes in MS disease activity (3).

MS is considered a T helper (Th) 1 lymphocyte-mediated disorder as levels of Th1-related molecules are increased in MS, however, the causes of the disease remain unclear. Th1 lymphocyte cells producing interferon (IFN)-γ and tumor necrosis factor (TNF)-α have been found in the perivascular infiltrates of MS patients (4). IFN-γ and interleukin (IL)-12 are increased in brain, cerebrospinal fluid (CSF) or peripheral blood of MS patients, particularly during acute exacerbations (5). By contrast, Th2 lymphocyte-related molecules, including IL-10 and -4, which downregulate Th1 cells and exert anti-inflammatory functions, are increased during the remission phases of MS (6). Th17, a subset of T cells that secrete IL-17, has been associated with the pathogenesis of several inflammatory diseases, including MS and encephalomyelitis autoimmune experimental (EAE) (7,8). In EAE, Th1 and Th17 cells have been shown to contribute to disease pathogenesis, however, no absolute requirement for IL-17 or IFN-γ alone has been reported (9).

Cytokines have been identified as major regulators of the immune system and attempts have been made to correlate cytokine levels with MS disease activity, assessed using magnetic resonance imaging (MRI). There is evidence that acute dysregulation of the balance of cytokines is one of the key factors during acute relapse, leading to acute inflammatory lesions in MS patients. Elevated serum and CSF TNF-α and -β levels have been associated with onset of MS relapse (10). Disease progression in MS patients has also been correlated with high CSF TNF-α levels (11).

The clinical disability of MS patients is evaluated using the Expanded Disability Status Scale (EDSS) (12), while the disease activity is evaluated using MRI with gadolinium (Gd)-enhancing lesions, providing the most objective and sensitive tool for assessing the progression and activity of the disease in MS patients. MRI revealed disease activity 5–10 times more frequently than clinical measurements (13). Positive Gd-enhancing lesions are the earliest event in the formation of new MS lesions and are predictive of the course of disability in MS patients (14).

Cytokine changes have been investigated in MS patients from various populations worldwide. The majority of the studies enrolled a limited number of MS patients with various clinical forms of the disease during the remission or relapse phase (15–20). The aims of the present study were to evaluate the serum cytokine profile in RR-MS patients from a Southern Brazilian population and to evaluate the association between the inflammatory and anti-inflammatory cytokine profiles with clinical disability and disease activity of the patients.

The present study evaluated the serum cytokine profile exhibited by RR-MS patients in a remission clinical phase from a population from South Brazil. Theoretically, CSF may reflect the relevant inflammatory process to a higher degree compared with other samples, due to its proximity to inflammatory lesions in the CNS. However, due to the flow pattern of CSF, it is unlikely that CSF in the lumbar cistern accurately reflects the production of inflammatory markers in the supratentorial region, where the majority of MS-related inflammation occurs (29). In addition, CSF collection is an invasive procedure, the intraparenchymal extracellular space may not necessarily communicate with the free CSF space (30) and it remains unclear whether CSF markers possess definite advantages over markers collected from blood and/or urine (29). Therefore, in the current study the cytokine profile was measured in serum samples.

To obtain more homogeneity of the cohort, only patients with the RR-MS clinical form were included in this study. The main result obtained in this study was elevated serum IFN-γ and IL-6, -12 and -4 levels in RR-MS patients compared with controls. MS patients with mild EDSS presented elevated IL-4 levels. RR-MS patients with inactive disease exhibited elevated levels of TNF-α and IL-10 and -17. Logistic regression analysis revealed that low TNF-α and high IFN-γ levels were associated with MS and disease activity.

The demographic and clinical characteristics exhibited by the RR-MS patients are consistent with those of previous studies (18,24,31,34). The majority of RR-MS patients exhibited mild clinical disability, predominance of lesions in brain white matter and low disease activity. These results were expected considering all the RR-MS patients were in clinical remission and the majority of them were treated with IFN-β (1a or b) which has been demonstrated to decrease clinical disability and disease activity (18,35–37).

RR-MS patients and controls were age-, gender-, ethnicity- and BMI-matched for cytokine analysis. These parameters were controlled to minimize their effect of them on serum cytokine levels (38,39). IL-1 levels were higher among controls compared with RR-MS patients and may be explained by the hypothesis that IFN-β therapy decreases IL-1β expression in the dendritic cells and monocytes of RR-MS patients (40). This pro-inflammatory cytokine is considered an important mediator for neuronal and axonal damage of the CNS in MS patients. One of the actions of IL-1β in the brain is the induction of astrogliosis by astrocyte activation and IL-6 production, an effect that is markedly augmented in the presence of TNF-α, IFN-γ, IL-6 and the IL-6 receptor (41). However, IL-1 has also been revealed to induce proliferation of the nerve growth factor in vitro and may exhibit protective effects in MS patients (42).

The increased IL-6 levels identified among RR-MS patients compared with controls are consistent with previous studies. One study demonstrated that the number of IL-6 mRNA expressing mononuclear cells was enriched in CSF of MS patients (43). Increased IL-6 production was also observed in peripheral blood monocytes and macrophages stimulated with LPS from MS patients (44). IL-6 deficient mice were found to be resistant to induction of EAE (17) and also revealed a reduction of anti-MOG antibody titers, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) molecule expression, which may affect the entry of Th1 cells into the CNS (45). By contrast, IL-6 also presented beneficial effects, including anti-inflammatory properties. IL-6 inhibits IL-1β and TNF-α secretion in vitro(46) and induces circulating IL-1 receptor antagonist in vivo(47). Results of the current study are consistent with those of previous studies considering that elevated IL-6 levels were found in RR-MS patients, IL-1 levels were higher in controls and no difference was observed in TNF-α levels.

Navikas et al(48) demonstrated that MS patients present with high numbers of MBP-reactive cells in their blood which, following culture with MBP and myelin PLP, respond to production and secretion of additional cytokines, including IFN-γ, IL-4, transforming growth factor β (TGF-β), IL-10 and TNF-α. The authors of that study demonstrated that MS patients presented elevated MBP- and PLP-reactive IL-6 mRNA expressing cells in the peripheral blood. Cytokines are known to modulate production of other cytokines (49). Therefore, IL-6 functions with additional cytokines within the cytokine network.

TNF-α levels were lower in RR-MS patients than the controls, as demonstrated by the multivariate analysis. This result is consistent with previous studies reporting lower TNF-α levels in MS patients compared with controls (11,19). Decreased production of TNF-α by stimulated PBMCs from MS patients compared with controls was also demonstrated (3).

In the present study, increased IFN-γ levels obtained among MS patients compared with controls were also consistent with previous studies. Lymphocytes producing IFN-γ were identified in the perivascular infiltrates of MS patients (4). A previous study evaluated mRNA serum transcript in patients receiving various doses of IFN-β1b and confirmed that IFN-β decreases the expression of Th1 inflammatory molecules, including IFN-γ and TNF-α. In addition, IFN-β increases the Th2-related molecule IL-10 (3).

Although IL-17 expression has been demonstrated to generate a highly pro-inflammatory environment and to induce severe pathological conditions (50), in the present study no difference was observed in IL-17 levels between MS patients and controls. A previous study (51) revealed that the proportion of Th17 cells in patients with relapses were significantly higher compared with patients during remission, in clinically isolated syndrome (CIS) and RR-MS clinical forms of MS. Another study (52) also revealed that IFN-β inhibits IL-23 which is involved in the differentiation of Th0 (naïve) to Th17 cells and, consequently, IL-17 production. In addition, IFN-β therapy inhibits the IL-27 cytokine which, in turn, inhibits Th17 cells directly and through the inhibition of IL-23, promotes development of these pathogenic T cells (8). IL-23 and -12 exert antagonistic effects. While IL-23 supports IL-17 production, IL-12 inhibits this production (53). IL-17, in turn, stimulates astrocyte IL-6 production and macrophage production of IL-6, TNF-α and IL-1β, cytokines that are involved in MS pathology (54).

The RR-MS patients enrolled in this study presented high levels of IL-12 which, in turn, antagonized IL-17 production and by this effect, reduced IL-1β- and TNF-α-induced production by IL-17. In addition to the consumption of TNF-α in CNS lesions, the reduced production may explain, in part, why TNF-α levels did not differ among the patients and controls (54).

The higher IL-12 levels exhibited by RR-MS patients compared with controls enrolled in the present study are also consistent with previous observations that IL-12 levels were upregulated in MS patients (55). Overexpression of IL-12 in the CNS resulted in increased inflammation and cellular infiltrates (56). Although several studies have suggested a key pro-inflammatory role for IL-12 in the development of autoimmune responses, controversial results have been reported with respect to IL-12 levels obtained from MS patients and MS patients treated with IFN-β. A previous study (40) demonstrated that treatment with IFN-β1a significantly induces IL-12p35 gene expression in dendritic cells from RR-MS patients. By contrast, it was demonstrated that treatment with IFN-β inhibits IL-12 and decreases C-X-C motif chemokine 10, IL-18, IFN-γ and TNF-α (57). However, this therapy increases IL-10 and TGF-β levels (58).

In the Brazilian cohort enrolled in the present study, higher levels of IL-4 were found in MS patients compared with controls, a result consistent with previous studies (59,60). IL-4 mRNA was previously revealed to be undetectable until disease remission in SJL mice immunized with PLP (61). IL-4 is a key cytokine produced by Th2 cells and inhibits Th1 cells by decreasing IL-1 and TNF-α cytokines. Administration of IL-4 in EAE exerts an ameliorating and protective effect. Studies demonstrated that IL-4 upregulation may reduce the severity of EAE, while its absence does not alter the course of the disease, possibly since in the absence of IL-4, an additional Th2 cytokine may substitute its functions and contribute to the tolerance of EAE (62).

Although IL-10 levels were higher in MS patients compared with controls, no statistically significant difference was found. It was previously suggested that IL-10 is important for recovery of EAE (63). In contrast to other Th2 cytokines (IL-4), the absence of IL-10 results in a more severe EAE, indicating that its function cannot be replaced by an additional Th2 cytokine.

The cytokine profile was also evaluated according to the EDSS of the RR-MS patients. Patients with mild disability were younger than those with moderate and severe disability, a result that was expected since at the beginning of the disease, low EDSS scores are detected among RR-MS patients and disability increases during disease progression (64). However, logistic regression analysis revealed no effect of age in the cytokine profile according to the EDSS. Although higher serum IL-6, TNF-α, IFN-γ and IL-12 levels were found in RR-MS patients with moderate and severe disability than those with mild disability, no significant differences were identified. As observed, the results of TNF-α levels are contradictory in various clinical disabilities. While low levels of TNF-α have been reported in specific MS patients (11,19), another study reported that TNF-β is the principal mediator of inflammatory response and is increased among MS patients, indicating that this cytokine is an important molecule in the pathogenesis and progression of MS (65).

In addition, serum IL-17 and -10 did not differ between EDSS categories. Several studies have reported that differential production of IL-10 may be a factor in the severity of MS (31,63). With regards to serum IL-4 levels, results demonstrate a significant increase of this cytokine among RR-MS patients suffering from mild disability compared with those with moderate or severe disability. This result confirms the important role of IL-4 as an anti-inflammatory modulator of MS lesion formation (6,19).

It was not possible to control age of RR-MS patients with regards to disease activity. However, logistic regression analysis revealed that age did not affect cytokine levels. There is evidence that an acute deregulation in the balance of cytokines is one of the key factors during an acute relapse leading to acute inflammatory lesions. RR-MS patients with active disease presented with lower TNF-α and higher IFN-γ levels compared with RR-MS patients without active disease, as demonstrated by multivariate analysis. IFN-γ and IL-12 levels were increased in brain, CSF or peripheral blood from MS patients, particularly during acute exacerbation phases (5). Previously, Begolka and Miller (61) observed that IFN-γ expression increases during the peak of the disease and decreases during the remission phase. Clinical attacks correlate with increased IFN-γ production (66) and patients experiencing relapses have significantly increased IFN-γ production by PBMCs following stimulation with PHA compared with patients in remission. However, this production was reduced following treatment with IFN-β (67). IFN-γ is produced within MS lesions by infiltrating lymphocytes and induces apoptosis in human oligodendrocytes (68).

Cytokines only function when bound to membrane receptors and the TNF-α receptor is present in almost all cells (6,61,69). In MS patients, TNF-α is produced and consumed at sites of inflammation, including lesions in brain white matter, spinal cord and optic nerve (70,71). TNF-α and -β cytokines were detected in astrocytes from demyelinating plaques (71), contributing to white matter destruction at the onset of MS (16). Results obtained in the present study are consistent with these previous findings. However, serum TNF-α levels measured in RR-MS patient samples of the present study may be the remaining expressed cytokines, neither acting at lesions on the CNS, nor binding to transmembrane or soluble receptors. Additional studies also demonstrated lower serum TNF-α levels in MS patients in remission compared with those in the relapse clinical phase (19,20).

Although IL-6 levels were higher in RR-MS patients with inactive disease, this difference was not identified to be statistically significant. This result is in agreement with a previous study (72) which demonstrated that macrophages within the MS lesion expressed IL-6 and the highest number of IL-6 expressing cells were found in inactive demyelination lesions. We hypothesize that IL-6 also has anti-inflammatory properties (46) that may affect disease activity.

A previous study has demonstrated that serum IL-12 levels correlate with active lesions (73), however, no significant difference was observed in serum IL-12 levels obtained from RR-MS patients with positive and negative Gd-enhancing lesions enrolled in the present study.

Increased levels of IL-17 were found in RR-MS patients with inactive disease. Th17 cell differentiation is orchestrated by multiple cytokines, including TGF-β and IL-6, -1-β, -21 and -23 (stimulating) and IFN-γ and IL-4, -12, -10 and -27 (inhibiting). The Th17 cell is a recently identified cell lineage that has been hypothesized to be critical for the development of the autoimmune response, in addition to Th1 cells (74). Studies from MS patients indicated that IL-17 mRNA is elevated in active MS brain lesions. IL-17 was also detected at lesions (75).

Serum IL-4 levels did not differ between RR-MS patients with active and inactive disease and this result was not consistent with previous studies that demonstrated high IL-4 levels in acute and chronic lesions (62). However, in the present study, serum IL-10 levels were high among RR-MS patients with inactive lesions. This result is consistent with previous observations of increased production of IL-10 and TGF-β by isolated lymphocyte clones of MS patients in remission (76). Decreased IL-10 levels were found prior to onset of exacerbation in RR-MS patients (15). IL-10 levels were also significantly decreased in MS patients with the secondary progressive form of MS compared with patients at four weeks prior to detection of MS by MRI and six weeks prior to clinical relapse (73).

Previously, Cannella and Raine (62) reported that the expression of adhesion molecules, including VCAM-1, ICAM-1 and their receptors on leukocytes, very late activation antigen-4 (VLA-4) and lymphocyte function-associated antigen-1 (LFA-1), respectively, together with a selection of pro-inflammatory and immunomodulatory cytokines including, IL-1, -2, -4 and -10, TNF-α, TGF-β and IFN-γ, are important for lymphocytic infiltration and interactions during tissue inflammation. In general, these molecules are not expressed by CNS cells. The authors identified high levels of these molecules in MS patients, particularly in those with chronic active lesions and also found a significantly increased expression of TNF-α and IL-4 in MS compared with non-inflammatory neurological diseases (62).

The present study demonstrates that RR-MS patients exhibit a complex system of cytokines which interact to modulate disease activity and the disabilities associated with it. Even in the remission phase of the disease and under immunomodulatory therapy, RR-MS patients present with an imbalance between pro-inflammatory, Th1, -2 and -17 cytokines. TNF-α was observed to be consumed in CNS lesions and activation of the cellular immune response was mediated by high levels of IFN-γ. These results are likely to contribute to an improved understanding of the role of the cytokine profile in RR-MS and may aid the development of new neuroprotective and neurorestorative therapeutic strategies.