Factors influencing the anti-inflammatory effect of dexamethasone therapy in experimental pneumococcal meningitis

I. Lutsar*, I. R. Friedland, H. S. Jafri, L. Wubbel, A. Ahmed, M. Trujillo, C. C. McCoig and G. H. McCracken Jr

Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA

Received 11 January 2003; returned 24 February 2003; revised 14 July 2003; accepted 15 July 2003


    Abstract
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Dexamethasone (DXM) interferes with the production of tumour necrosis factor-{alpha} (TNF-{alpha}) and interleukin-1 (IL-1) and can thereby diminish the secondary inflammatory response that follows initiation of antibacterial therapy. A beneficial effect on the outcome of Haemophilus meningitis in children has been proven, but until recently the effect of DXM therapy in pneumococcal meningitis was uncertain. The aim of the present study was to evaluate factors that might influence the modulatory effect of DXM on the antibiotic-induced inflammatory response in a rabbit model of pneumococcal meningitis. DXM (1 mg/kg) was given intravenously 30 min before or 1 h after administration of a pneumococcal cell wall extract, or the first dose of ampicillin. In meningitis induced by cell wall extract, DXM therapy prevented the increase in cerebrospinal fluid (CSF) leucocyte and lactate concentrations, but only if given 30 min before the cell wall extract. In meningitis caused by live organisms, initiation of ampicillin therapy resulted in an increase in CSF TNF-{alpha} and lactate concentrations only in animals with initial CSF bacterial concentrations >=5.6 log10 cfu/mL. In those animals, DXM therapy prevented significant elevations in CSF TNF-{alpha} [median change –184 pg/mL, –114 pg/mL versus +683 pg/mL with DXM (30 min before or 1 h after ampicillin) versus controls (no DXM), respectively, P = 0.02] and lactate concentrations [median change –10.6 mmol/L, –1.5 mmol/L versus +14.3 mmol/L with DXM (30 min before or 1 h after ampicillin) versus controls (no DXM), respectively, P = 0.01]. These effects were independent of the timing of DXM administration. In this model of experimental pneumococcal meningitis, an antibiotic-induced secondary inflammatory response in the CSF was demonstrated only in animals with high initial CSF bacterial concentrations (>=5.6 log10 cfu/mL). These effects were modulated by DXM therapy whether it was given 30 min before or 1 h after the first dose of ampicillin.

Keywords: animal models, CSF, experimental meningitis, inflammatory response, S. pneumoniae


    Introduction
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Pneumococcal meningitis remains a significant cause of morbidity and mortality. It is the host’s own inflammatory response that is responsible for the central nervous system injury characteristic of the disease.1This inflammatory response can be exacerbated by antibacterial therapy that increases rapidly the release of proinflammatory pneumococcal cell wall products.25

Currently, dexamethasone (DXM) is the only anti-inflammatory agent that has been documented to improve the outcome of bacterial meningitis in clinical trials, most convincingly in children with Haemophilus influenzae meningitis.6 DXM inhibits production of TNF-{alpha} and IL-1, reverses development of brain oedema and limits the increase in cerebrospinal fluid (CSF) lactate and leucocyte concentrations.1,7,8 Studies have suggested that in H. influenzae meningitis the timing of DXM in relation to the first antibiotic injection is critical. The antibiotic-induced inflammatory response is prevented only if DXM therapy is given simultaneously with ceftriaxone; DXM given 1 h later failed to modulate the antibiotic-induced inflammatory response.3 A meta-analysis of 10 clinical trials in children suggested that the timing of DXM therapy might also be critical in pneumococcal meningitis. DXM therapy prevented the development of severe hearing loss only when it was given before or at the same time as the first antibiotic injection (OR = 0.09; 95% CI: 0.00–0.71).6 Recently de Gans & van de Beek9 in a prospective, randomized, placebo-controlled, multicentre trial demonstrated the beneficial effect of adjunctive DXM therapy on the outcome of pneumococcal meningitis in adults.

The present study was conducted to evaluate the modulatory effect of DXM therapy given 30 min before, compared with 1 h after, intracisternal injection of pneumococcal cell walls, or after the first injection of ampicillin, in experimental meningitis caused by live pneumococci.


    Material and methods
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Inocula

Pneumococcal cell wall (1 cm3 of this product was obtained from 2.5 x 109 live organisms) was produced and provided by Dr Elaine Tuomanen.10 Streptococcus pneumoniae (MIC and MBC of ampicillin, 0.01 mg/L) was originally isolated from a patient with bacterial meningitis. To induce meningitis, 0.2 mL pneumococcal cell wall product, or 104–105 cfu/mL of live organisms, were inoculated intracisternally.

Meningitis model and treatment

A rabbit meningitis model originally described by Dacey & Sande11 was used. In the first set of experiments, DXM (1 mg/kg) was given intravenously 30 min before or 1 h after the administration of pneumococcal cell walls to six and seven animals, respectively. In the second set of experiments, 16–18 h after inoculation of live organisms, animals were treated with ampicillin alone (75 mg/kg every 12 h) for 24 h, or with the combination of ampicillin and intravenous DXM (1 mg/kg) given 30 min before or 1 h after the first ampicillin dose. Each treatment group consisted of 12–13 animals. No treatment was given to control animals.

CSF sample collection and analysis

CSF was collected directly from the cisterna magna under acepromazine and ketamine anaesthesia before and 2, 4, 6, 12 and 24 h after start of therapy. For the first four CSF collections, animals were restrained under anaesthesia in stereotactic frames. Leucocytes were counted in a Neubauer haemocytometer. Bacterial concentrations in CSF were measured by plating undiluted and serial dilutions of CSF on sheep blood agar and incubating in 5% CO2 at 35°C for 24 h. The lower limit of detection was 10 cfu/mL. The remaining CSF was centrifuged and the supernatant stored at –70°C. CSF lactate concentrations were measured by a photocolorimetric assay (Behring Diagnostics Inc, Milton Keynes, UK). TNF-{alpha} concentrations were measured by cytotoxic assay using L929 tumorigenic murine fibroblasts.12 The standard curve for the TNF-{alpha} assay was linear from 40–2500 pg/mL.

Statistical analysis

Normally distributed data are presented as mean ± S.D. and non-normally distributed data as median and range. Student’s t-test was used for comparison of parametric data and the Kruskall–Wallis analysis of variance for non-parametric variables.


    Results
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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
Meningitis induced by pneumococcal cell walls

Administration of pneumococcal cell walls resulted in the release of TNF-{alpha}, an influx of leucocytes and increased lactate concentrations in CSF (Figure 1). The elevation in TNF-{alpha} concentrations was prevented by DXM therapy when given 30 min before or 1 h after pneumococcal cell walls. The increase in CSF leucocyte and lactate concentrations, however, was inhibited only when DXM therapy was given 30 min before the cell wall products.



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Figure 1. Concentrations of WBC (cells/mL; left), TNF-{alpha} (pg/mL; middle) and lactate (mmol/L; right) in the CSF. Meningitis was induced by the intracisternal administration of pneumococcal cell walls. Animals were treated with DXM given 30 min before (open squares) or 1 h after (solid squares) administration of cell walls. No treatment was given to the control animals (solid triangles). *P = 0.02 versus controls or those treated with DXM 1 h after introduction of cell walls.

 
Meningitis induced by live organisms

The addition of DXM to ampicillin therapy resulted in a lower initial bacterial killing rate compared with ampicillin therapy alone (0.39 ± 0.1 cfu/mL/h versus 0.57 ± 0.12 cfu/mL/h; P = 0.04). The changes in CSF inflammatory indices were similar in all study groups and were not influenced by the co-administration of DXM (Figure 2).



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Figure 2. Concentrations of WBC (cells/mL), TNF-{alpha} (pg/mL), lactate (mmol/L) and S. pneumoniae (log10 cfu/mL) in CSF in animals with pneumococcal meningitis. Animals were treated with ampicillin alone (solid triangles), with DXM given 30 min before (open squares) or 1 h after ampicillin therapy (solid squares). No treatment was given to the control (open triangles) animals, and they died after 6–10 h. Error bars demonstrate lower and upper quartile. *P = 0.04 versus those not receiving DXM therapy.

 
For further analysis, animals were divided in two groups based on CSF bacterial concentrations at the start of therapy (<=5.5 log10 or >=5.6 log10 cfu/mL) (Figure 3). After the first dose of ampicillin, animals with high initial bacterial concentrations demonstrated significantly greater changes in CSF TNF-{alpha} [median {Delta}+683 pg/mL (quartiles +246 to +758) versus {Delta}–16.3 pg/mL (quartiles –6 to –29)], white blood cells (WBC) [median {Delta}+2175 cells/mL (quartiles 437 to 6987) versus {Delta}–325 cells/mL (quartiles –787 to +25)] and lactate [median {Delta}+14.3 mmol/L (quartiles –7.6 to –14.6) versus {Delta}–8.5 mmol/L (quartiles –5.1 to –16.4)] concentrations compared with animals with lower initial bacterial concentrations.



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Figure 3. Changes in CSF concentrations of WBC (cells/mL), TNF-{alpha} (pg/mL), lactate (mmol/L) and S. pneumoniae (log10 cfu/mL) in pneumococcal meningitis after introduction of ampicillin therapy. The CSF bacterial concentrations at the introduction of ampicillin therapy were <=5.5 log10 cfu/mL (open squares) or >=5.6 log10 cfu/mL (solid squares). Error bars demonstrate lower and upper quartile. *P < 0.05

 
In those with high initial bacterial concentrations (>=5.6 log10 cfu/mL), DXM therapy prevented elevations in CSF TNF-{alpha} [median {Delta}–184 pg/mL (quartiles –116 to –258) or {Delta}–114 pg/mL (quartiles –39 to –175) versus {Delta}+683 pg/mL (quartiles 246 to +758) with DXM given 30 min before or 1 h after ampicillin versus without DXM, respectively; P = 0.02] and lactate concentrations [median {Delta}–10.6 mmol/L (quartiles 7.6 to 17.4) or {Delta}–1.5 mmol/L (quartiles –19.7 to –1.3) versus {Delta}+14.3 mmol/L (quartiles –7.6 to –14.6) with DXM given 30 min before or 1 h after ampicillin and without DXM, respectively; P = 0.01]. These effects were not significantly different as a result of the timing of DXM administration, although there was a trend indicating that changes in TNF-{alpha} and lactate values were lower in animals given DXM before ampicillin therapy (Figure 4). The changes in leucocyte concentrations were not affected by DXM therapy. In animals with low CSF bacterial concentrations (<=5.5 log10 cfu/mL), there were no differences in TNF-{alpha}, WBC or lactate concentrations between those treated with or without DXM (data not shown). There was no correlation between changes in TNF-{alpha}, leucocyte and lactate concentrations in CSF, and the degree of bacterial killing after introduction of ampicillin therapy (r = 0.06; r = 0.47 and r = 0.46, respectively; P > 0.05).



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Figure 4. Concentrations of WBC (cells/mL, left), TNF-{alpha} (pg/mL, middle), lactate (mmol/L, right) in CSF in animals with initial CSF bacterial concentrations >=5.6 log10 cfu/mL. DXM therapy was given 30 min before (open squares) or 1 h after the first dose of ampicillin (solid squares). Control animals (solid triangles) were treated with ampicillin only. Error bars demonstrate lower and upper quartiles. *P < 0.05 versus those treated with ampicillin and DXM.

 

    Discussion
 Top
 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
In this model of pneumococcal meningitis, we demonstrated that the antibiotic-induced secondary inflammatory response, as evidenced by an elevation of CSF TNF-{alpha} and lactate concentrations, occurred only in animals with high bacterial concentrations before the start of ampicillin therapy. The increase in CSF lactate and TNF-{alpha} concentrations were inhibited by adjunctive DXM therapy regardless of whether it was given 30 min before or 1 h after the first dose of ampicillin.

Liberation of free endotoxin or cell-wall components by cell-wall active antibiotics has been demonstrated in vitro and in experimental meningitis.3,4,5,13 This is associated with enhanced inflammation in the subarachnoid space, as evidenced by an increase in leucocyte, TNF-{alpha} and lactate concentrations.3,14 Our study showed that the antibiotic-induced inflammatory burst did not occur in all animals and was seen only in those with greater CSF bacterial concentrations (>=5.6 log10 cfu/mL). Although this has been intimated previously, data were not provided.14 In experimental meningitis, the magnitude of the inflammatory response in CSF depends on the concentration of inoculated cell walls10 and thus it is not surprising that animals with higher bacterial counts demonstrate a more pronounced host inflammatory response after antibiotic therapy.

There is concern that rapid bacterial killing by antibiotics could result in an enhanced inflammatory response and a worsening of the clinical outcome in meningitis.15,16 The results of this and previous studies, however, do not support these speculations. On the contrary, clinical and experimental studies have demonstrated that rapid effective bactericidal therapy protects against the development of deafness and other neurological disabilities.17,18 Moreover, this and previous studies4 found no correlation between CSF inflammatory markers and the magnitude of bacterial killing. Uncontrolled bacterial growth eventually results in a much greater release of cell wall components—and consequent enhancement of the inflammatory response—than that induced by antibacterial therapy.4,13 These findings suggest that early antibacterial therapy and rapid clearance of bacteria from CSF outweigh the potential adverse effects caused by the antibiotic-induced inflammatory burst.

DXM, as an adjunct to antibacterial therapy in bacterial meningitis, has been shown to be beneficial in H. influenzae meningitis in children.6 In pneumococcal meningitis, however, its modulatory effect was uncertain because of the relatively small numbers of children enrolled in prospective controlled trials.19 In adults with pneumococcal meningitis, an unfavourable outcome was seen in 26% of patients receiving adjunctive DXM compared with 52% among those receiving placebo.9 The results of our study clearly supported previous findings by Tuomanen et al.8 and showed that, similar to H. influenzae meningitis, DXM therapy prevents the antibiotic-induced release of TNF-{alpha} and lactate concentrations in CSF in pneumococcal meningitis.3

Early institution of DXM therapy has been suggested because of its delayed onset of action.20 In our study, the timing of DXM appeared to be critical only in meningitis induced by the pneumococcal cell wall. In meningitis induced by live microorganisms, however, once CSF inflammation was established, administration of DXM before the dose of ampicillin did not appear to have a significantly greater salutary effect than 1 h after ampicillin; DXM administration that was delayed further was not assessed. These results contrast with those obtained in experimental H. influenzae meningitis, where the antibiotic-induced inflammatory response was modulated only if DXM was given before or simultaneously with ceftriaxone therapy.3 The effectiveness of early DXM administration was also highlighted in a recent meta-analysis of 10 randomized controlled clinical trials.6

At the time of diagnosis, ~40% of patients with pneumococcal meningitis have CSF bacterial concentrations <106 cfu/mL21, and our findings suggest that antibiotic-induced enhanced inflammation may not occur in such patients. Whether these patients benefit from DXM therapy, and how they can be identified at diagnosis, requires further clarification. Detection of bacteria in Gram-stained specimens of CSF may be useful in identifying patients with large bacterial loads.22

One possible detrimental effect of DXM therapy, also demonstrated in this study, is decreased bacterial clearance from CSF.23,24 DXM decreases the permeability of the blood–brain barrier, resulting in decreased concentrations of hydrophilic antibiotics in the CSF.25 Also, high concentrations of DXM (400 µg/mL) inhibit phagocytosis by CSF leucocytes.26The clinical relevance of these findings, however, has not been demonstrated.6,9

In this model of pneumococcal meningitis, CSF bacterial concentrations at the start of therapy appeared to be more important than the timing of DXM therapy in influencing the antibiotic-induced inflammatory response. It is likely that there is a time beyond which DXM loses its effectiveness, but this point has not been clearly defined.


    Acknowledgements
 
I. Lutsar was a recipient of a fellowship award from the European Society for Paediatric Infectious Diseases, supported by Lederle-Praxis Biologicals. Part of this study was presented at the 1997 annual meeting of Infectious Diseases Society of America (IDSA), San Francisco. The study has followed animal experimentation guidelines and was approved by the Institutional Animal Care and Research Advisory Committee of the University of Texas.


    Footnotes
 
* *Correspondence address. Clinical Development, Pfizer Ltd., Ramsgate Road ,CT13 9NJ, UK. Tel: +44-1304-645173; Fax:+44-1304-655669; E-mail: irja_lutsar{at}sandwich.pfizer.com Back


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 Abstract
 Introduction
 Material and methods
 Results
 Discussion
 References
 
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