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The immunological response in pulmonary and pleural tuberculosis has been extensively studied. However, the response in tuberculous meningitis has not been well documented.1 In pulmonary disease, exposure to tuberculous antigens results in a T cell and natural killer cellular response, elaborating various cytokines, mainly of T helper type 1 (Th1) origin. Stimulated macrophages elaborate tumour necrosis factor (TNF) α, interleukin (IL) 12, and IL 1, promoting further recruitment and activation of macrophages and lymphocytes.
TNF α correlates with disease severity and may contribute to tissue necrosis; however, TNFα has also contributed to survival in mouse studies.2 Transforming growth factor β (Th3 cytokine) suppresses macrophage activation. IL 2 may be beneficial in promoting an immune response in HIV seropositive patients. Th1 and Th2 cytokine responses have been observed in cerebrospinal fluid (CSF) of HIV seronegative patients with tuberculous meningitis.3,4. Whether the response is similar in HIV seropositive patients with tuberculous meningitis is unknown.
We studied the cytokine response and its correlation with disease severity in HIV seropositive and HIV seronegative patients with tuberculous meningitis.
Tuberculous meningitis was diagnosed on clinical and CSF examination after exclusion of viral, acute bacterial, and other causes of aseptic meningitis. Disease severity was assessed according to the Medical Research Council stages 1 to 3. HIV ELISA was done on all patients. CSF samples were subjected to microscopy, culture, protein and glucose analysis, Venereal Disease Research Laboratory test, fluorescent treponemal antibody analysis, cryptococcal antigen analysis, viral studies, cysticercus ELISA, CD4 counts, and determination of concentrations of adenosine deaminase (ADA), CSF IgG, and albumin.
For cytokine assays, CSF was centrifuged at 3000 g, and supernatant was aliquoted and stored at –70°C. TNF α, interferon (IFN) γ, and IL 10 concentrations were measured by ELISA kits (Genzyme Diagnostics, Cambridge, Massachusetts, USA) with detection limits of 3 pg/ml, 3 pg/ml, and 5 pg/ml, respectively.
Data were summarised as medians and ranges. Non-parametric Wilcoxon rank sum tests were used to compare HIV seropositive groups with HIV seronegative groups, tuberculous meningitis severity groups, and groups derived according to the blood brain barrier index for cytokine concentrations. Spearman’s rank correlation was used to derive correlations of cytokine concentration, ADA concentrations, and CD4 counts in CSF.
There were 27 patients: 18 (67%) women and 9 (33%) men. Seventeen were HIV seropositive and 10 HIV seronegative. The average interval between onset of symptoms and the first clinical assessment was 17 days (range 5–90 days) in 18 patients where this was recorded. The mean (SD) age was 26.8 (11.6) years. There was one patient aged 10 and one aged 60, and the rest were between 25 and 40. The cytokine concentrations were not analysed according to age, as this would make the categories too small and of little value. The IgG index was calculated for 23 patients. There was no significant difference between the HIV seropositive and HIV seronegative groups for ADA (p = 0.4) and CD4 counts (p = 0.19) in CSF and cytokine concentrations (table 1).
Ten patients (37%) were classified as having grade 1 tuberculous meningitis. Sixteen (59%) had grade 2 and one (4%) grade 3, which for analysis was considered to be grade 2. Table 1 summarises the cytokine concentrations for patients in stages 1 and 2.
Patients with stage 2 disease had significantly stronger Th1 responses. There was no difference in the IL 10 concentrations. The two patients with stage 2 disease who died had very high IFN γ concentrations, both greater than 2048 pg/ml.
IL 10 concentrations were moderately positively correlated with IFN γ concentrations (r = 0.53). The correlation coefficients were –0.18 for IFN γ, −0.33 for TNF α, and −0.34 for IL 10. Correlation coefficients between ADA and cytokine concentrations were 0.34 for IFN γ, 0.47 for TNF α, and 0.22 for IL 10. Cytokine concentrations correlated poorly with CD4 counts in CSF.
It is postulated that in HIV infection a predominant Th2 response accounts for extrapulmonary disease.5 This study does not favour a predominance of either Th1 or Th2 in the CSF. It is possible that a Th0 response, which is a non-differentiated response seen early on in immune activation, was seen in our patients, as they were examined untreated and relatively early in the disease. Other investigators have also documented this phenomenon.3 The positive correlation between IFN γ and IL 10 suggests that these were produced concurrently. This may reflect a control mechanism regulating Th1 and Th2 responses.
There was no difference in cytokine and ADA concentrations and CD4 counts between HIV seropositive and HIV seronegative patients. It is known that the clinical response to antituberculous treatment in both groups is similar.5 Perhaps this similarity correlates with similar immune responses in both groups. The size of each group is small and a type 1 statistical error has to be considered. Further studies to confirm our findings would be of value.
The significantly greater TNF α and IFN γ concentrations in the severe group of tuberculous meningitis is confirmed by other studies6 and suggests that disease severity results mainly from the immune response rather than the organism itself.
The lack of correlation between CD4 and cytokine concentrations may be explained by the fact that there are other sources of cytokines in the CSF, namely macrophages and natural killer cells. Concentrations of ADA, which are derived from lymphocytes, are consistent with other reports, where they were correlated with cytokine concentrations.
There was no correlation between the IgG index and cytokine concentrations, suggesting that the blood brain barrier did not significantly influence concentrations. Unfortunately, corresponding serum concentrations were not available. This would have been valuable. This is the first study correlating CSF cytokine responses to severity of tuberculous meningitis and comparing HIV positive with HIV negative groups. Further studies should be done to confirm these findings, perhaps to define their relevance to complications and to explore the possibility of IL 2 treatment in HIV positive patients.
This study was sponsored by the Glaxo TB initiative.
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