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Chin Med J (Taipei) 1997;59:248-53.
Department of Pediatrics, Veterans General Hospital-Taipei; and National Yang-Ming University School of Medicine, Taipei, Taiwan, R.O.C.
Background. Certain cytokines may contribute to the sequence of events that lead to meningeal inflammation in bacterial meningitis. However, their role in viral meningitis is not so less well defined. We determined the cytokines levels in the cerebrospinal fluid (CSF) of children with aseptic meningitis and discussed their relationship with clinical and laboratory findings.
Methods. We determined the concentrations of interleukin-l beta (IL-1b), interferon-gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the CSF of 62 patients with aseptic meningitis including 17 patients with culture-proved enteroviral meningitis, and from 19 control acute febrile patients without meningitis.
Results. The GM-CSF in the cerebrospinal fluid was detected from one of the 62 patients with aseptic meningitis and none of the 19 controls. Fourteen (23%) of the 62 patients with aseptic meningitis and 2 (10.5%) of 19 controls had detectable IL-1beta. There was no significant difference in IL-1beta levels between patients with aseptic meningitis (4.4 +/- 11.4pg/ ml) and control group (2.4 +/- 7.7pg/ml). The CSF IFN-gamma level was detectable in 40 (65%) of 62 patients and 6 (31.6%) of 19 controls. The mean CSF IFN-gamma concentration was significantly higher in patients with aseptic meningitis when compared with that in control group (37.9 +/- 48.8pg/ml vs 17.5 +/- 29.7pg/ml; p=0.007).
Conclusions. IFN-gamma was detectable in the CSF in 65% of patients with aseptic meningitis and the role of interferon-gamma remains to be determined.
[Chin Med J (Taipei) 1997;59:248-53.]
Keywords: aseptic meningitis, cerebrospinal fluid, granulocyte-macrophage colony-stimulating factor, interferon-gamma, interleukin-1beta
Received: July 23, 1996.
Accepted: January 10, 1997.
Address reprint requests to: Ren-Ben Tang, Department of Pediatrics, Veterans General Hospital-Taipei, 201, Sec. 2, Shih-Pai Road, Taipei, Taiwan, R.O.C.
Acute aseptic meningitis, a common and significant disease in children, often toxic appear and can be difficult to distinguish from bacterial meningitis, especially in young infants [1]. Recent attention has been directed toward the study of the role of cytokines in the regulation of inflammation and host responses to infection [2-6].
Recent studies in bacterial meningitis, both in animal models and clinical studies, suggest that release of certain cytokines, such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha), may be responsible for the severe inflammatory response in meningitis and may correlate with morbidity and mortality [7-10]. However, the role of cytokines in viral meningitis remains poorly defined. Except for some general agreements, several reports on a limited number of patients have presented inconsistent results [9-17].
In this study, we determined the concentrations of interleukin Ibeta (IL-1beta), interferon-gamma ( IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the cerebrospinal fluid (CSF) derived from 62 children with aseptic meningitis and 19 controls and evaluated their relationship with clinical and laboratory findings.
From February 1993 through June 1993, acute aseptic meningitis was diagnosed in 62 patients between one month and 14 years of age at the Veterans General Hospital-Taipei. Upon admission to hospital, all patients had clinical signs and symptoms consistent with the diagnosis of meningitis and all had CSF examinations and cultures for bacteria and viruses.The clinical data were obtained by personal interview and / or by review of the medical records. In 17 patients (27 %), the diagnosis was confirmed by positive CSF culture for enteroviruses, whereas in the remaining 45 patients, the diagnosis was based on pleocytosis (> 10/ mm3), negative CSF bacteriologic studies (CSF and blood cultures, CSF gram-stained smears, and bacterial antigen detection tests), no history of prior antibiotic treatment, and an enteroviral isolation from other sources such as throat and rectal swab.
The CSF specimens were analyzed for white blood cell (WBC) count with differential cell count and for glucose and protein concentrations. All CSF specimens were submitted for viral cultures using HEp-2, MK-2, MDCK and human embroyonic lung cell lines. A positive result was reported when enteroviral cytopathic effect in cell cultures became apparent. The viral isolates were identified by neutralization test using the intersecting pools of antisera supplied by the American Type Culture Collection Laboratory( Rockville, Maryland, USA). The remaining CSF samples were stored at -70OC for later assays for cytokines.
A second group of 19 acute febrile patients without meningitis served as controls. These patients were admitted for acute febrile illness with CNS symptoms requiring a lumbar puncture to rule out CNS infection. All CSF specimens were submitted for studies as outlined above. All control patients revealed no pleocytosis (< 5/mm3), a normal glucose and protein and negative culture for bacteria and viruses.
For the following cytokine assays all CSF samples were measured in duplicate (100ul each). The IL-1beta levels were determined by enzyme immunoassay (Cayman, Ann Arbor, USA). The minimum detectable concentration for IL-1beta was 3.9pg/ml. The IFN-gamma levels were determined by immunoassay (Biosource International, Camarillo, CA) and the minimum detectable levels was 15.6pg/ml. The GM-CSF levels were determined by enzyme immunoassay (Innogenetics N.V. Belgium) and the low detectable level was 20pg/ ml. We followed the methods and procedures for the above cytokines assays as described in the respective instruction booklets.
The Wilcoxon scores (rank sums) tests were used for two group comparisons. The correlation between CSF cytokines and CSF cell count or protein content were analyzed by Pearson correlation. For all tests, a p value below 0.05 was considered significant.
The CSF and blood laboratory findings of the 62 patients with aseptic meningitis and 19 patients with control group are listed in Table 1. The CSF concentrations of glucose, and serum C-reactive protein were not significantly different between aseptic meningitis patients and the control group. GM-CSF in CSF was detected in 1 of 62 patients with aseptic meningitis and none from the control group. Fourteen (23%) of 62 patients with aseptic meningitis had detectable IL-1beta levels and 2 (10.5%) of 19 controls had detectable IL-1beta concentration. No significant difference was found between patients with aseptic meningitis (4.4 +/- 11.4pg/ml) and those in control group (2.4 +/- 7.7pg/ml). The IFN-gamma level in CSF was detectable in 40 (65%) of 62 patients and 6 (31.6%) of 19 controls. The mean CSF IFN-gamma concentration was significantly higher in patients with aseptic meningitis than in controls (37.9 +/- 48.8pg/ml vs 17.5 +/- 29.7pg/ml; p=0.007).
The CSF findings and cytokine levels from 62 patients with aseptic meningitis are listed in Table 2. Echovirus 30 was isolated from CSF specimens in 17 of them. In comparing the CSF features between patients with positive or negative enteroviral cultures, there was no significant difference in WBC, glucose, protein, IL-1beta, IFN-gamma and GM-CSF.
Correlation of IL-1beta, IFN-gamma and GM-CSF in CSF with other inflammatory parameters (i.e., WBC and protein) in patients with aseptic meningitis was also investigated (Table 3). There was no significant finding in this respect.
The clinical and laboratory findings between patients with detectable IFN-gamma level (positive group) and with undetectable IFN-gamma (negative group) were compared (Table 4) to make no significant difference in the duration of illness or fever between both groups. In addition, there was no significant difference in the maximal body temperature. Comparison of the CNS findings of the IFN-gamma positive and IFN-gamma negative groups revealed no significant difference in CSF leukocyte, protein or sugar.
Recent studies on the relationship between cytokines and inflammation have yielded new information about the potential role of TNF-alpha, IL-1beta, IL-6, IL-8, INF-gamma and GM-CSF in the pathogenesis of bacterial meningitis [7-10]. Data derived from both animal models and clinical studies have suggested that certain cytokines may participate in the acute inflammatory responses of the central nervous system [7-17].
The inflammatory responses in the subarachoid space and brain tissues in viral meningitis are generally less intense than those of bacterial meningitis. In recent years, several cytokines, occurring at varying frequency and concentrations, have been detected in the cerebrospinal fluids of patients with aseptic meningitis. These cytokines include IL-1beta [14], IL-6 [ 9,12,13], IL-8 [9,17], TNF-alpha [9], INF [10,11,15] and GM-CSF [16]. In view of lack of consistent findings from these and other reports, significance of cytokines in the cerebrospinal fluids is largely unknown and the role of cytokines in the pathogenesis of viral meningitis remains to be elucidated.
Ramilo et al. [l4] have detected IL-1beta in the cerebrospinal fluids from 86% of 36 patients with aseptic maningitis, but at a lower concentration than that in patients with bacterial meningitis, They also found a significant correlation between IL-1beta concentrations and WBC count in the CSF of patients with aseptic meningitis. In contrast, in the present study, we found IL-1beta in the cerebrospinal fluids of only 23% of 62 patients with aseptic meningitis. In addition, the correlation between IL-1beta concentrations and WBC count was not established. Shimoda et al [16] recently demonstrated detectable GM-CSF in CSF of 6 of 9 patients with aseptic meningitis in the concentration ranging from 49 to 114 pg/ml. In contrast, we found only one of the 62 patients with aseptic meningitis with detectable GM-CSF in the cerebrospinal fluids. Factors affecting the presence or absence of IL-1beta and GM-CSF from patients with aseptic meningitis remain not known. Variations in host factor, viral agent, and method for measurement of cytokine may have contributed to the discrepancy between both studies. Further investigations in this respect are needed.
Interferon response during acute bacterial or viral meningitis has been extensively studied. A literature review indicated that 68% of 196 patients with aseptic meningitis contained interferon (alpha, beta, and gamma types not distinguished) in their cerebrospinal fluids [10]. Recently, it has been suggested that IFN- gamma, acting as one of the endogenous pyogens, is produced in substantial quantities in response to acute viral infection [18]. Two recent studies [11,15] have reported elevated levels of IFN- gamma in the CSF from patients with aseptic meningitis. Abbott et al. [11] have observed 7 patients with viral meningitis having concentrations of IFN- gamma in the CSF ranging from 2 to 8 micron/ml. Minamishima et al. [15] have reported high levels of IFN- gamma in the CSF from 19 (70%) of 27 patients in acute phase of aseptic meningitis. In the present study, we found 65% of 62 patients with aseptic meningitis having elevated levels of IFN- gamma. Minamishima et al. [15] found that the CSF IFN- gamma levels correlated with body temperature, CSF cell count and protein level; however, we found no clear correlation between CSF IFN- gamma levels and leukocyte count or protein.
Overall, data from previous reports [10,11,15] and from our present study indicated that IFN- gamma is produced in the CSF in response to viral meningitis and that the majority (65 to 70%) of patients with aseptic meningitis contain IFN-gamma in the cerebrospinal fluids. The factors affecting the production of IFN-gamma and the role of IFN-gamma in the pathogeneisis of acute viral meningitis remain to be defined and deserve further investigation.
The study was supported by grants from the Veterans General Hospital-Taipei and in part by the Infectious Disease Training Program, Institute of Biomedical Sciences of Republic of China.
Copyright: 1997, Chinese Medical Association (Taipei)