Interleukin-5 and interleukin-10 are major cytokines in cerebrospinal fluid from patients with active neurocysticercosis

Neurocysticercosis (NCC) is a common neurological disorder especially in developing countries, caused by infection of the brain with encysted larvae of the tapeworm Taenia solium. Seizures are a common finding associated with this disease. The objective of the present study was to evaluate the correlation between the levels of various cytokines present in the cerebrospinal fluid (CSF) of patients with NCC and the severity of the disease. The levels of the cytokines IL-1ß, TNF-a, IL-5, IL-10 and IFN-g were determined in the CSF of 22 patients with active NCC, 13 patients with inactive NCC and 15 control subjects. CSF from patients with active NCC presented significantly higher IL-5 levels compared to control subjects. IL-5 and IL-10 levels in CSF from NCC patients with inflammatory CSF were significantly higher than those detected in non-inflammatory CSF. These results show a predominant Th2 lymphocyte activation in human NCC and also indicate the possible use of cytokines in the CSF as a marker for the differential diagnosis between inactive disease and the active form of NCC. Correspondence


Introduction
Neurocysticercosis (NCC) is a common neurological disorder in several developed and developing countries.It is endemic in Latin America, Asia and Central and South Africa and is relatively frequent in Portugal, Spain and Eastern European countries (1)(2)(3).In other developed countries NCC can be frequent among immigrants.The disease is caused by the encysted larvae of the tape-worm Taenia solium lodged in the central nervous system (CNS).In the same communities, teniasis is a public health problem with a frequency of family antecedents reaching 34.2% in some regions (4).
NCC may be asymptomatic or may cause a variety of clinical manifestations depending on the number, location and stage of development of the cysticercus-associated lesions.Epileptic seizures, hydrocephaly and intracranial hypertension, in this order, are the most frequent clinical features.This pleomorphic disease is a result of the presence of the parasite itself (cysticerci), the inflammatory process that surrounds the larvae, and the residual fibrosis and calcification (5,6).
The cysticercus contains a large number of antigens that can elicit a host immuneinflammatory reaction.The inflammatory cellular infiltrate, if present, may be discrete with the presence of lymphocytes and eosinophils in the vesicular stage, or may be a more intense lymphocyte infiltrate with giant multinuclear foamy macrophages in the necrotic stage, with parasites in different stages of desintegration (7).
Most of the previous studies involving cerebrospinal fluid (CSF) were limited to an analysis of the presence of specific antibodies and nonspecific mediators of the inflammatory reaction such as parasite-specific antibodies, increased protein levels and eosinophilia (8).Eosinophilia provides indirect evidence for the active form of NCC.Recently, several reports on cysticercosis involving studies of the cellular infiltrate in humans and in animal models have indicated that a Th1 immune response predominates over a Th2 response (9,10), while studies of the CSF have revealed a predominant Th2 response (11,12).
Cytokines are pleotropic hormones that regulate many aspects of the immune response and of inflammatory reactions.Interleukin-5 (IL-5) is produced mainly by Th2 lymphocytes and plays a role in eosinophil maturation, migration and activation and also induces immunoglobulin A switch in B lymphocytes (13).Interleukin-10 is produced by Th2 lymphocytes, B cells, and macrophages and has strong anti-inflammatory properties (14).Interferon-g (IFN-g) is produced by Th1 lymphocytes and natural killer (NK) cells and is involved in macrophage activation and in immunoglobulin class switch (15).Other cytokines such as IL-1ß and tumor necrosis factor alpha (TNF-a) are produced mainly by macrophages and by glial cells in the CNS, and are involved in the acute phase of inflammatory reactions.
In the present study, we determined the presence of IL-5, IL-10, IFN-g, TNF-a and IL-1ß in CSF from patients with active and inactive NCC and compared their levels with those found in the CSF of unaffected controls.The results indicate that high levels of IL-5 are significantly associated with active NCC and that IL-10 reaches higher levels in inflammatory CSF.

Patients
CSF samples collected from 22 patients with active NCC, 13 with inactive NCC and 15 controls (patients with chronic headache, normal topography and standard parameters of CSF analysis within normal values) were analyzed.All patients and controls were submitted to neurological investigation including clinical examination, brain tomography and standard CSF analysis.CSF analyses consisted of serological tests for anti-cysticercus and anti-treponema antibodies, protein concentration, sodium, potassium and chloride measurements, and standard blood cell counts.Patients with active and inactive NCC were classified on the basis of a brain tomography scan (16).All of them exhibited anti-cysticercus antibodies in CSF and were negative for anti-treponema antibodies.This study was approved by the Ethics Committee of the Faculdade de Medicina do Triângulo Mineiro.

Cytokines in neurocysticercosis
Nonspecific binding was prevented by incubating the plates with 3% BSA (Sigma Chemical Co., St. Louis, MO, USA) in PBS.Plates were incubated overnight with 100 µl of 1:2 dilution of CFS in PBS, 2% BSA and standard cytokines (Pharmingen and R&D, Minneapolis, MN, USA).Plates were then washed 4 times with PBS and 0.05% Tween and incubated with rabbit anti-IL-1ß antibody (Genzyme), rabbit anti-TNF-a antibody (Genzyme), biotinylated anti-IL-5 mAb (Pharmingen), biotinylated anti-IL-10 mAb (Pharmingen) or biotinylated anti-IFNg mAb (Mabtec) for 4 h.Plates were then washed and incubated for 2 h with alkaline phosphatase-conjugated goat anti-rabbit IgG (Immunotech, Marseille, France) or alkaline phosphatase conjugated to streptavidin.Finally, plates were washed 4 times and enzymatic activity was developed by incubating the plates with p-nitrophenyl phosphate (Sigma).Absorbance was read at 405 nm in a microplate reader apparatus (Biorad, Hercules, CA, USA).The sensitivity of the tests was 20 pg/ml.

Results
Patients with NCC were classified according to a computer tomography scan and the presence of anti-cysticercus antibodies (14).As shown in Figure 1, CSF from patients with inactive NCC did not present relevant alterations in cellularity or protein concentration.Nevertheless, 8 patients with active NCC presented elevation of cells and proteins in CSF, with eosinophils ranging from 4 to 9%.
Levels of IL-1ß, TNF-a, IL-5, IL-10 and IFN-g were measured in CSF by ELISA.CSF from the majority of control patients did not present measurable levels of the tested cytokines.The levels of TNF-a and IFN-g were below the limit of detection in the study group.CSF from patients with active NCC showed an elevation of IL-1, IL-5, and IL-10 levels.IL-5 levels were significantly higher (t-test) than those found in the CSF from patients with inactive NCC and from control subjects (Figure 2).These findings were particularly clear when patients with alterations in terms of both cellular and biochemical criteria were compared with the other group.In the CSF from patients with active NCC with protein concentration above 50 mg% and more than 20 cells/ml, IL-5 levels ranged from 57 to 792 pg/ml and the levels of IL-10 levels ranged from 38 to 398 pg/ml.In contrast, the levels of IL-5 and IL-10 were significantly lower in CSF from patients with active NCC with a non-inflammatory reaction (Figure 3).

Discussion
The clinical manifestations of human cysticercosis depend on various factors including number of cysts, their location, and host reaction.When the cysticerci are lodged in the CNS, the disease is known as neurocysticercosis, and the involvement of the CNS represents the most dangerous clinical form of this parasitosis.NCC is usually asymptomatic for several years and during this period, a discrete circumscribed inflammatory reaction is observed (17).Conversely, symptomatic disease is associated with a prominent inflammatory response, consisting of lymphocytes, eosinophils, granulocytes and plasma cells (18).
The mechanisms by which cysticercal lesions develop in the CNS depend on a combination of host immune-inflammatory response, mainly mediated by cytokines produced by resident and infiltrating cells activated by cysticercal antigens.The lesions containing viable cysticerci have a small inflammatory infiltrate surrounding the worm, which is usually composed of mono-nuclear cells and eosinophils (7).These cyst antigens stimulate the production of antibodies which are inefficient in killing mature metacestodes.However, the anti-cysticercus antibodies can be detected in CSF of patients with active and inactive NCC and are the basis for the immune diagnosis of this disease.The inflammatory reaction of NCC was demonstrated in CSF (19) and confirmed by some studies on experimental models and on human tissues.These studies showed an immune response predominantly consisting of Th1 lymphocytes (9,10) and the later development of a Th2 response associated with parasite destruction (11).
In the present study, we observed that CSF from patients with NCC contains elevated concentrations of IL-1ß, IL-5, and IL-10.The levels of these cytokines in the CSF from the majority of uninfected controls was below ELISA sensitivity.However, statistical analysis demonstrated that IL-5 levels were significantly higher in patients with active NCC than in patients with inactive NCC and in control subjects.Moreover, IL-10 and IL-5 levels were higher in active NCC patient with elevated concentrations of proteins and increased cellularity.
Our results are significant because they can help us to understand some of the cellular alterations present in the CSF of NCC patients, i.e., a marked eosinophilia.This alteration may be the result of the intense production of IL-5 during the immune response to the cysticercal antigens, because one of the main functions of IL-5 is to induce and activate eosinophils to mediate an antibody-dependent helminthotoxic effect (20).We suggest that the eosinophilia present in the CSF of patients with active NCC is, at least in part, mediated by the IL-5 produced by the host in response to the infectious process.Similarly, the presence of IL-10 and the absence of IFN-g in CSF from patients with NCC confirm a Th2 response elicited by helminthic infection.Moreover, IL-10, as an anti-inflammatory cytokine, may be in- Finally, our results show a predominant activation of Th2 lymphocytes in human NCC, with the presence of increased levels of IL-1ß, IL-5, and IL-10 in the absence of IFN-g and TNF-a.These results show that the determination of cytokines in CSF may be an important asset by providing an addi-tional parameter for the differential diagnosis between inactive NCC and active disease.

Figure 1 -
Figure 1 -Protein concentration and cell counts in CSF from patients with active (circles, N = 22) and inactive (squares, N = 13) NCC and from control subjects (X, N = 15).

Figure 2 -
Figure 2 -Cytokine levels in CSF from patients with active (filled bars, N = 22) and inactive (striped bars, N = 13) NCC and from control subjects (open bars, N = 15).The bar represents the mean and the vertical lines the standard error of the mean.Statistical analysis was performed on log-transformed cytokine levels.*P<0.05 compared to control subjects (Student t-test).

Figure 3 -
Figure 3 -Cytokine levels in CSF from 8 patients with active NCC with inflammatory CSF (filled bars) and from 14 NCC patients with non-inflammatory (striped bars) CSF based on protein concentrations and cell counts.Statistical analysis was performed on log-transformed cytokine levels.*P<0.05 compared to NCC patients with non-inflammatory CSF (Student t-test).