Chemokine profiles in the cerebrospinal fluid (CSF) during the course of pyogenic and tuberculous meningitis. C. M. MASTROIANNI, L. LANCELLA*, ...
Clin Exp Immunol 1998; 114:210–214
Chemokine profiles in the cerebrospinal fluid (CSF) during the course of pyogenic and tuberculous meningitis C. M. MASTROIANNI, L. LANCELLA*, F. MENGONI, M. LICHTNER, P. SANTOPADRE, C. D’AGOSTINO, F. TICCA* & V. VULLO Department of Infectious and Tropical Diseases, La Sapienza University, and *First Division of Infectious Diseases, Bambin Gesu` Paediatric Hospital, Rome, Italy
(Accepted for publication 9 July 1998)
SUMMARY The concentrations of the chemokines IL-8, monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1a (MIP-1a) were measured in 120 CSF samples from 23 patients with pyogenic meningitis and from 11 patients with tuberculous meningitis (TBM) and in 10 CSF from subjects with non-infectious neurological diseases. The chemokine concentrations in patients with meningitis were significantly higher than in control subjects (P < 0·0001). The highest CSF levels were found for IL-8 (median 2917 pg/ml) and MCP-1 (median 2557 pg/ml), whereas those of MIP-1a were less significantly elevated (median 24 pg/ml) (P < 0·0001). Patients with pyogenic meningitis had higher levels of IL-8 and MCP-1 than those with TBM (P < 0·0001). In serial samples from patients with pyogenic meningitis IL-8 levels declined before MCP-1 and MIP-a. In the case of TBM, IL-8, MCP-1 and MIP-1a decreased more gradually during treatment and were detectable in the CSF for several weeks, without any characteristic time course of elimination. These data indicate that patients with pyogenic meningitis and TBM show different chemokine profiles in CSF. The distinct chemokine pattern could be responsible for a differential attraction and activation of leucocytes in the CSF which is reflected in differences in the inflammatory response and clinical course of pyogenic meningitis and TBM. Keywords
chemokines
leucocytes
cerebrospinal fluid meningitis
INTRODUCTION Despite the development of new broad-spectrum anti-microbial agents, bacterial meningitis continues to be a major cause of morbidity and mortality. Effective anti-microbial defences require the generation of a vigorous inflammatory response that involves the recruitment into the subarachnoid space of phagocytic cells, neutrophils followed by mononuclear cells [1]. The mechanisms whereby neutrophils and monocytes are recruited from the peripheral circulation into the CSF probably involve the generation of early cytokines, such as IL-1 and tumour necrosis factor-alpha (TNF-a), the expression of cell surface adhesion molecules, and the production of chemotactic molecules, such as chemokines [2–4]. Chemokines (or chemotactic cytokines) are a family of structurally related polypeptides with a molecular size between 8 and 13 kD that share the ability to induce migration of specific subsets [5]. Two closely related families of chemotactic cytokines, the CX-C and C-C chemokines, have been increasingly recognized as important mediators in orchestrating inflammation and modulating Correspondence: Claudio M. Mastroianni, Department of Infectious and Tropical Diseases, Policlinico Umberto I, La Sapienza University, Viale Regina Elena 331, 00161 Rome, Italy.
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the innate immune response against invading microorganisms [6–8]. In the C-X-C subfamily two cysteine residues are separated by an intervening amino acid, whereas in the C-C subfamily the cysteine residues are adjacent to each other. The C-X-C chemokines, which include IL-8, have predominant neutrophil stimulatory and chemotactic activities, and the C-C chemokines, which include monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-1a (MIP-1a) and MIP-1b, exert predominant activating and chemotactic effects on mononuclear cells. Because of their ability to induce adhesion, accumulation and activation of leucocytes, chemokines may represent important mediators of the innate immune response against microorganisms in bacterial meningitis [9]. It has recently been shown that in patients with bacterial meningitis C-X-C and C-C chemokines are expressed in the CSF in the acute phase of the disease [10]. There are no data regarding the time course of chemokine levels during the clinical course of meningitis after the beginning of treatment with antibiotics and/or steroids. In the present study, the CSF concentrations of the chemokines IL-8, MCP-1 and MIP-1a are assessed over time in a group of patients with bacterial meningitis, including Mycobacterium tuberculosis infection. q 1998 Blackwell Science
Chemokines in bacterial meningitis PATIENTS AND METHODS Patients The patient population consisted of 34 patients with bacterial meningitis who were admitted to the Bambin Gesu` Paediatric Hospital and the Department of Infectious and Tropical Diseases of the La Sapienza University, both in Rome. Diagnosis of bacterial meningitis was based on the presence of a positive bacterial culture from CSF or the presence of a positive blood culture in combination with clinical evidence of meningitis and CSF leucocyte count > 1000/ml. The diagnosis of tuberculous meningitis (TBM) was confirmed by isolation of M. tuberculosis from the CSF or was established on the basis of clinical findings plus one or more of following criteria: CSF parameters (pleocytosis, elevated protein levels, low glucose levels, and raised adenosine deaminase activity), neuroimaging showing hydrocephalus and basilar enhancement, or evidence of concomitant extraneurological tuberculous localization. The staging of TBM was established by the Medical Research Council (MRC) [11]. All patients underwent routine audiometric assessment. Brain stem evoked potentials were performed in infants. Before the hospital discharge, electroencephalogram, computed tomography of the head and neurological evaluation were performed. A total of 120 CSF samples was collected on admission before antibiotic treatment and subsequently over time during the course of the disease. Serum samples were also collected from the 34 patients with bacterial meningitis at the time of hospitalization. As controls, CSF specimens were obtained from 10 patients with non-infectious neurological diseases, including cerebrovascular disease, hydrocephalus, encephalomyelitis and epilepsy. Institutional approval of the study was given by the local responsible committee, and fully informed consent was obtained from the patients’ parents. Laboratory studies CSF specimens, upon collection, were examined for leucocyte count, levels of glucose and protein. Adenosine deaminase activity was also measured as previously reported [12]. An aliquot was immediately placed in a refrigerated centrifuge (48C) and spun at 1500 g for 10 min. Thereafter cell supernatants were removed and stored at –808C before chemokine assay. The CSF concentrations of IL-8, MCP-1 and MIP-1a were measured by a quantitative ELISA (Quantikine; R&D Systems, Minneapolis, MN) according to the manufacturer’s instructions, with the following detection limits: IL-8, 10 pg/ml; MCP-1, 5 pg/ml; MIP-1a, 3 pg/ml. For statistical analysis, samples below the detection limit were arbitrarily assigned the value for the detection limit in the assays.
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Statistical analysis Non-parametric statistical methods were used. Mann–Whitney Utest was used for the comparison of two groups, since values were not normally distributed. Values were correlated with the Spearman rank correlation test. P < 0·05 was regarded as significant. RESULTS Patients Twenty-two patients were children (median age 30 months; range 2–88 months). The ages of the 12 adult patients ranged from 27 to 58 years (median 41 years). Pyogenic meningitis was diagnosed in 23 patients, while the remaining 11 patients had TBM. The causative pathogens of pyogenic meningitis were Neisseria meningitidis (n ¼ 5), Streptococcus pneumoniae (n ¼ 7), and Haemophilus influenzae (n ¼ 11). The initial CSF biochemical data of patients are reported in Table 1. The mean duration of illness previous to the first lumbar puncture was 1·3 days (range 1–4 days) for pyogenic meningitis and 20·7 days (range 13–30 days) for TBM. All CSF samples from the control subjects with non-infectious neurological disease were sterile and had normal CSF characteristics. All patients with pyogenic meningitis received dexamethasone therapy in conjunction with antibiotics (ceftriaxone or cefotaxime). Ten subjects developed neurological sequelae, including hearing loss and cerebral atrophy. Of the patients with TBM, three were classified as stage 1 (fully conscious without any focal neurological signs); six as stage 2 (depressed level of consciousness and hemiparesis or a single cranial nerve palsy); two as stage 3 (patients were comatose or complete hemiplegia or paraplegia). Two TBM patients had HIV infection. Steroid treatment was given in seven cases, two with stage 3 TBM and five with stage 2 TBM. Two patients died of progressive neurological deterioration. Of the remaining nine TBM patients, five developed neurological sequelae (hydrocephalus, n ¼ 3; hemiparesis, n ¼ 2; diplopia, n ¼ 1). Chemokine levels on admission The chemokine levels measured in the CSF at the onset of meningitis in comparison with the control group are shown in Fig. 1. The CSF concentrations of the three chemokines in patients with meningitis were significantly higher than those in control subjects with non-infectious neurological diseases (P < 0·0001). All 34 patients with meningitis had CSF IL-8 and MCP-1 above the detection limit, while MIP-1a was detectable in 28/34 (82%) cases. The highest CSF concentrations were found for IL-8 (median 2917 pg/ml; range 123–7642 pg/ml) and MCP-1 (median 2557 pg/ml;
Table 1. CSF biochemical data in patients with pyogenic and tuberculous meningitis
Patients (n)
Leucocyte count (× 109/l)
Neutrophils (× 109/l)
Mononuclear cells (× 109/l)
Protein (g/l)
Glucose (nmol/l)
Pyogenic meningitis (23) Tuberculous meningitis (11) Controls (10)
4285 (1120–121 000) 356 (31–723) 1 (0–2)
3001 (785–85 000) 89 (8–180) –
1285 (335–36 000) 267 (23–542) 1 (0–2)
1·52 (0·2–8·3) 2 (0·75–3·5) 0·2 (0·2–0·4)
1·5 (< 0·1–3·2) 1·4 (< 0·1–2·2) 3 (2·6–3·8)
Data are expressed as median (range). q 1998 Blackwell Science Ltd, Clinical and Experimental Immunology, 114:210–214
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10 000
pg/ml
1000
100
10
1 IL-8
MCP-1
MIP-1α
Fig. 1. CSF concentrations of IL-8, monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1a (MIP-1a) in 34 patients at the onset of bacterial meningitis (D) and 10 subjects with non-infectious neurological diseases (O). Solid lines, detection limit of each chemokine.
range 235–6337 pg/ml). These levels were significantly more elevated than those obtained for MIP-1a (median 24 pg/ml; range < 3–56 pg/ml) (P < 0·0001). CSF levels of MIP-1a were below the detection limit in all 10 subjects with non-infectious neurological diseases. One CSF sample had low concentrations of IL-8 (15 pg/ml), and 8/10 control CSFs contained small amounts of MCP-1 (median 7 pg/ml; range < 5–25 pg/ml). IL-8, MCP-1 and MIP-1a were not detectable in the patients’ blood, thus indicating a local release of chemokines within the central nervous system (CNS). The pathogen-dependent CSF concentrations of chemokines are shown in Table 2. The CSF levels of IL-8 and MCP-1 were much higher in pyogenic than in tuberculous meningitis, but did not correlate with the species of pyogenic bacterium. The levels of MIP-1a were similar for all four species. No age- or antibiotic-related differences in chemokine levels were found. As shown in Table 3, a positive highly significant correlation between all measured chemokines was found. On the other hand, there was no correlation between the chemokine levels and the total number of leucocytes in the CSF. Also, when comparing either CSF concentrations of IL-8 with neutrophil counts or MCP-1 and MIP-1a with mononuclear cells in the CSF, no correlation was observed (data not shown). Time course of chemokine levels during meningitis The dynamics of CSF chemokine levels in patients with pyogenic meningitis was evaluated for 4 weeks following the initiation of
antibiotic treatment. As shown in Fig. 2, IL-8 decreased rapidly within the first week of admission to hospital and institution of treatment. In contrast, levels of both MCP-1 and MIP-1a decreased steadily, reaching a minimum at 4 and 3 weeks, respectively. The kinetics of production of chemokines during the course of TBM was different. In fact, the concentrations of IL-8, MCP-1 and MIP1a were elevated for a long period of time, being still detectable in the CSF 32 weeks after the beginning of specific treatment. No significant differences were found in the chemokine concentrations in TBM patients who received steroid treatment compared with those who did not receive steroids. Moreover, no differences were observed in the expression of chemokines in patients with pyogenic meningitis or TBM who developed neurological sequelae compared with those who did not develop sequelae. DISCUSSION The appearance of an infiltrative cellular response in the subarachnoid space is an important hallmark of bacterial meningitis. The recruitment into the CNS of neutrophils and mononuclear cells is crucial for recognition and elimination of pathogens. Nevertheless, the presence of these leucocyte populations in the CSF may also be deleterious to the host, by contributing to the development of neurologic damage [13]. Chemokines are candidate mediators of leucocyte migration from circulation into the CSF along a chemotactic gradient at the site of inflammation. In the present study we prospectively evaluated CSFs from 34 patients with bacterial meningitis in order to investigate the CSF concentrations of CX-C and C-C chemokines in these CNS infections. Our results demonstrate that in meningitis the initial CSF samples collected before antibiotic and/or steroid treatment contained significantly higher concentrations of IL-8, MCP-1 and MIP-1a than CSFs from control subjects with non-infectious neurological diseases. The highest concentrations were obtained for C-X-C chemokine IL-8 and C-C chemokine MCP-1, which represent the most important chemotactic cytokines for neutrophils and monocytes, respectively. Augmented production of C-X-C and C-C chemokines in the CSF of patients with bacterial and viral meningitis has been previously reported [14–18]. Sprenger et al. found high levels of IL-8, growth-related gene product (GRO)-a and MCP-1 in the CSF of patients with bacterial and viral meningitis, whereas no significant intrathecal production of MIP-1a and RANTES (regulated upon activation, normal T cell expressed and secreted) was observed [19]. In addition, these authors showed that the levels of IL-8 and GRO-a significantly correlated with the number of
Table 2. Pathogen-dependent levels of IL-8, monocyte chemotactic protein-1 (MCP-1) and macrophage inflammatory protein-1a (MIP-1a) in the CSF of patients with bacterial meningitis
Organism(s) Neisseria meningitidis Haemophilus influenzae Streptococcus pneumoniae Mycobacterium tuberculosis Controls
IL-8 (pg/ml)
MCP-1 (pg/ml)
MIP-1a (pg/ml)
4525 (2569–6360) 4258 (1233–7142) 4454 (1546–6000) 963 (123–1236) < 10
4604 (2145–4724) 3569 (1091–6337) 3145 (1808–4135) 808 (235–1375) 7 (< 5–25)
25 (3–28·3) 19·1 (3–53) 23 (3–42) 26·6 (16·3–48)
