Tumour Necrosis Factor-α Production and Immune Cell Activation in Tuberculous Meningitis

The local production of tumour necrosis factor-α (TNFα) was evaluated in the cerebrospinal fluid (CSF) from ten patients with tuberculous meningitis (TBM). The degree of intrathecal immune activation was also studied by assessing the CSF levels of β2-microglobulin (β2-M) and adenosine deaminase activity (ADA). Results indicate that elevated CSF concentrations of TNFα, β2-M and ADA were found in all TBM patients. Moreover, TNFα is produced and selectively concentrated for a long period of time, while β2-M and ADA values progressively decline during the course of TBM. Our findings suggest that in TBM patients, after an early activation of immune cells, there is an enhanced and continuous production of TNFα at the site of infection.


Introduction
Tumour necrosis factor-o (TNFo 0 is a cytokine mainly released by cells of the monocytemacrophage lineage in response to lipopolysaccharide (LPS) and other immune and inflammatory stimuli. It has been recognized as a primary mediator in the pathogenesis of infection, injury and inflammation and in the beneficial processes of host response. In particular, TNFe is an important macrophage-activating factor for antibacterial resistance against infections caused by facultative intracellular organisms, such as mycobacteria. 2 In experimental models it has been reported that TNF enhances macrophage phagocitic capacity and mycobacterial killing by human macrophages. In addition, recent lines of evidence indicated that TNF is synthesized in large amounts in pulmonary tuberculosis and it is locally concentrated at the site of disease activity. 4,s To date, little is known about the intrathecal production of TNF during the course of tuberculous meningitis (TBM). 6 Therefore, it appears to be of interest to evaluate the local production and release of TNFo in the cerebrospinal fluid (CSF) of patients with TBM.
In the present study, TNFo concentrations were measured in CSF specimens obtained from hospital patients on admission and during the course of TBM. In order to determine the extent to which cell-mediated immunity is involved in the disease, the CSF levels of two markers of immune activation, such as fl2-microglobulin (/2-M) and adenosine deaminase activity (ADA), were also measured.

Materials and Methods
Patients: Ten patients with TBM (five males, five females) admitted to the Institute of Infectious Diseases of the University of Rome 'La Sapienza', were enrolled in this study. The mean age was 34.4 years (range, 12-55 years). TBM was diagnosed on the basis of findings in the CSF. In six cases CSF smears were positive by acid-fast staining and fluid cultures grew Mycobacterium tuberculosis; in the remaining patients, diagnosis was made by compatible cytological and biochemical findings, supported by clinical features and symptomatic improvement with antituberculous therapy. All patients were sero-negative for human immunodeficiency virus (HIV) and did not  In all patients CSF levels of TNF were low on admission, then progressively increased and remained detectable for a long period of time. Antibiotic treatment did not appear to influence the TNF levels. Similarly, there was no correlation between the cytokine concentrations and the clinical course of the disease.

Discussion
Cetl-mediated immunity plays an important role in the control of infections by M. tuberculosis. It is widely assumed that T-lymphocytes, through lymphokine-mediated macrophage activation, are the major immune cells involved both in the pathogenesis and protective mechanisms in human tuberculosis. 1 5'0 In the case of TBM, it is conceivable that the involvement of cellular immune system mainly occurs in the central nervous system (CNS). In this respect, the dosage in the CSF of two markers of immune activation, such as fl2-M and ADA, represents a useful tool to investigate the degree to which cell-mediated immunity is stimulated in the CNS. /2-M is a portion of the major histocompatibility complex class I antigen (MHC-1) and is expressed on the surface of lymphocytes and macrophages. With regard to the adenosine deaminase, the activity of this enzyme is increased in lymphoid cells, especially during T-cell proliferation and differentiation.
Previous investigations have already demon-$0 strated a close correlation between elevated CSF ADA values and TBM, 1'12 while an increase in the CSF concentrations of/2-M has been reported only in viral and pyogenic meningitis. 13 In the present Mediators of Inflammation. Vol 3.1994 59 study, both /2-M and ADA levels were found to be elevated in the CSF of all TBM patients, thus indicating a marked stimulation of immune system within the CNS. The increased CSF levels of/2-M were possibly a consequence of cell-mediated cytotoxicity directed against macrophages infected by M. tuberculosis. 14 The elevation of CSF ADA values was most likely related to the local immune response as the result of proliferating lymphocytes in response to mycobacterial antigen.
Intrathecal immune activation appears to be also associated with increased cytokine expression in the CNS. Indeed, our results showed that TBM patients had significantly higher CSF TNFo concentrations than those found in control subjects with non-infectious neurological diseases. The local production of TNF is low in the initial phase of TBM, while the peak in the CSF concentration was obtained later. Moreover, the CSF levels of this cytokine did not decrease during the course of the disease, but they were elevated for a long time in all patients, irrespective of the antibiotic therapy and the clinical course of TBM. This pattern is quite different from that observed in the case of/2-M and ADA, whose levels declined rapidly during the course of the disease. Only two patients who developed neurological complications showed a further increase in the CSF/2-M and ADA values.
Taken together, our findings suggest that a great stimulation of immune T-cells primarily occurs in the acute phase of TBM and may account for the early increase in the CSF /2-M and ADA values.
On the contrary, the enhancement of cytokine expression related to TNF production is a later but more prolonged phenomenon. It is likely that brain macrophages activated by T-cell-mediated pathway are the source of TNFo in TBM patients. This hypothesis is also supported by previous investigations that have demonstrated that M. tuberculosis cell wall components can trigger the release of TNFo from human and murine macrophages, as well as from pleural fluid mononuclear cells of patients with pulmonary tuberculosis. 5,15,16 In conclusion, our data provide direct evidence that TNFo is produced and selectively concentrated for a long period of time in the CSF from patients with TBM. The chronic release of TNFo at the site of the infection suggests that this cytokine may be involved in the complex immunoregulatory mechanism that contributes to mycobacterial containment and elimination.