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Original Articles Indian Pediatrics 2000;37: 608-614 |
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CSF
Interleukin-1ß, Tumor Necrosis Factor-a and Free Radicals Production |
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Key words:
Acute bacterial meningitis, Cerebrospinal fluid, Interleukin–Ib,
Free radicals, Meningitis in infants and children is associated with significant mortality rate and adverse neurological outcome in survivors despite the introduction of newer antibiotics and advances in intensive care technology. Extensive clinical and experimental data generated in recent years has revealed that live meningeal pathogens are not by themselves alone responsible for the harmful effects on the central nervous system(1,2). Indeed, clinical expression of meningitis arises largely from the host response to the inciting organism in the subarachnoid space. Data from experimental meningitis studies have shown that endotoxin, bacterial products, immune complexes stimu-late brain microvessel endothelial cells and pericytes to produce interleukin-1b (IL–1b) and tumor necrosis factor–a (TNF–a). These proinflammatory cytokines initiate leukocyte endothelial interaction resulting in generation of superoxide and other toxic oxygen radicals. The secretory products of polymorphonuclear neutrophils particularly reactive oxygen intermedia can be a major source of damage to the inflamed tissue. The free radicals are highly reactive and initiate a chain reaction which form new free radicals. The interaction of all these pathophysiologic events can eventually lead to cerebral edema, increased intracranial pressure (ICP), loss of cerebrovascular autoregulation, alteration in cerebral blood flow and ultimately to irreversible focal or diffuse brain damage. The induction and amplification of these host inflammatory responses, to control locally the infectious process, may actually exert a destructive effect on the CNS and thus may contribute to the morbidity and mortality of meningitis. The increased levels of cytokines in bacterial meningitis have been well docu-mented(3,4). However, there is relative lack of work on the relationship between cytokines and free radicals in cerebrospinal fluid (CSF) and their relation to clinical outcome. This study was conducted to find out diagnostic and prognostic implications of cytokines and free radicals in acute bacterial meningitis and to study their correlation with other indices of meningeal inflammation and ultimate clinical outcome.
A total of 52 cases aged 3 months to 12 years were enrolled for the study. Group I consisted of 32 patients of proven acute bacterial meningitis, i.e., suggestive clinical picture and CSF parameters (poly-morphonuclear pleocytosis >5/cu mm; CSF protein >50 mg/dl, CSF hypogly-corrachia) along with presence of bacterial pathogen identified by positive Gram stain/CSF culture or Latex agglutination test. Group 2 consisted of 20 controls with febrile seizures in whom CSF study was normal. Exclusion criteria were (i) Children less than 3 months of age; (ii) Children with partially treated meningitis: (iii) Prior neurodevelopmental delay; (iv) Severe malnutrition; and (v) Presence of papilledema. All the cases were subjected to detailed history and neurological examination was done on admission. In all the patients standard treatment protocol was followed at the time of diagnosis for acute bacterial meningitis. Corticosteroids were not included in the treatment protocol. Neurological follow up was done during hospital stay and till 3 months after discharge from the hospital. In follow up, patients were assessed for the presence of seizures, focal neurological signs, hearing and visual impairment. The CSF specimens were taken after informed consent of the parents within 6 hours of admission. The CSF samples were analyzed for white blood cell, differential cell count, glucose and protein concentrations. Gram staining, culture and latex agglutination tests were done to identify the causative organism.
Cytokines Determination CSF Free Radicals and Enzymes: 1.5 ml of CSF was collected in Ependroff’s tubes for estimation of free radicals and enzymes.
Statistical Methods:
The clinical and bacteriological profile in 32 cases of acute bacterial meningitis is shown in Table I. The median age of cases was 21 months and that of controls was 12 months. There was male preponderance in both groups (cases 26 males and 6 females, controls 15 males and 5 females). The mean concentration of IL–1b, TNF–a, superoxide anion, H2O2, SOD and CPK in CSF of children with ABM was much higher as compared to controls (Table II). There was direct correlation between CSF superoxide and hydrogen peroxide production and levels of CSF cytokines. Levels of CSF IL–1b, TNF–a also correlated directly with CSF CPK concentration (Table III). The levels of CSF IL–1b, TNF–a , CPK and free radicals viz.,superoxide and H2O2 correlated directly with CSF cell count and protein concentration and inversely with CSF glucose concentration. However, enzyme SOD did not show any correlation with CSF proteins and sugar. Of these routinely measured parameters CSF protein showed the best correlation with CSF TNF–a (r = .7383, p <0.001), IL–1b (r = 0.73, p <0.001), CSF superoxide (r = 0.53, p = 0.002) and CSF hydrogen peroxide production (r = 0.40, p = 0.026). Eighteen patients were discharged of which five had sequelae in form of motor deficit (n = 2), recurrent seizures (n = 2) and hydro-cephalus (n = 1) profound hearing loss (n = 2). Of the patients who expired 71.4% were infants and all had acute onset fulminant menin-gitis.There was significant difference between the mean CSF concentrations of cytokines, superoxide and hydrogen peroxide production in patients with ABM who expired and patients with neurological sequelae as compared to those who had normal survival (Table IV). All the patients who had IL–1b Ievels > 500 pg ml and TNF–a levels > 1500 pg/ml expired. High levels of IL–1b, TNF–a and superoxide anion emerged as important predictors of mortality in ABM using logistic regression analysis.
* Staph. aureus (2), E. coli (1), Salmonella typhimurium (1), Klebsiella (1), Pseudomonas aeruginosa (1)
Values represent Mean ± SD. Table III: Correlation Between CSF Cytokines and Free Radicals in ABM
r - Correlation coefficient
Table IV: Relationship
of CSF Cytokines, Proteins and Free Radicals with Clinical Outcome in
ABM
Discussion
In the present study all children with ABM had
elevated concentrations of cytokines in CSF obtained at time of
diagnosis, as compared to the relatively lower detection rates in
previous studies(8,9). The relatively lower detection rates in other
studies may be due to the fact that partially treated cases were
also included and there was difference in time point during patient’s
illness when CSF was sampled. In the present study, CSF for
cytokines determination was sampled within few hours of admission
and partially treated cases were excluded. This study found high
cytokines concentration not only in patients with Gram negative but
also in those with Gram positive meningitis. This is in contrast to
earlier findings of slightly high proportions of elevated TNF–a
levels in CSF with meningitis due to S. pneumoniae and N.
meningitidis than in H. influenzae group(10). However,
the sample size in the present study is not large enough to detect
difference between the two groups. In the present study a significant correlation
was found between levels of both the IL–1b
and TNF–a
with clinical outcome. Patients who expired or had neurological
sequelae had significantly higher levels of both cytokines as
compared to patients with normal survival. Various authors have
found correlation of clinical outcome with either of the two
cytokines but none of them have found correlation of outcome with
both the cytokines together. Sharief et al. found higher
incidences of death and neurological sequelae in patients with
detectable TNF–a in
CSF(9). Mustafa et al. found that patients with CSF IL–1b
> 500 pg/m were more likely to develop neurologic sequelae(11).
But other authors did not find any correlation between CSF cytokines
levels and outcome of disease(8,12). Synergy between TNF–a
and IL–1b has
been documented(9). These workers did not find correlation between
IL–1b
level and blood brain barrier damage although such damage correlated
with TNF–a concentrations.
These findings suggest that effects of TNF–a on cerebral endothelium can be
dissociated from that of IL–1b.
However, in the present study the levels of IL–1b directly correlated with TNF–a
(r = 0.89) and level of both cytokines had direct relation with
outcome. Since we did not study the kinetics, it is difficult to say
whether production of one cytokine is induced by the other or both
are produced simul-taneously by continuous exposure of the
macrophage equivalent cells to bacterial cell wall components. All analytes were correlated to cytokines, total
protein and white cell count suggesting that cytokines have a direct
effect on the blood brain barrier (BBB) causing increasing
permeability which allows protein leakage into the CSF and
facilitates leukocyte migration. This was consistent with previous
studies where positive correlation was detected between cytokines,
CSF bacterial density, degree of BBB disruption, CSF proteins and
lactate(9,11). There was direct correlation between cytokines and
creatine kinase activity which is the marker of tissue injury. Other
workers have also noted positive correlation between levels of CSF
IL–1b and
TNF–a and
severity of clinical complications of menin-gitis(13,14). An
association of inflammatory mediators and cerebral blood flow has
been demonstrated in patients with bacterial meningitis(15).
Patients with high blood flow velocities had significantly increased
concentration of IL–1b and
raised cell count in CSF. Thus, exaggerated inflammatory response is
also responsible for alteration in cerebral blood flow in bacterial
meningitis which may contribute to mortality. The present study detected significantly higher
levels of free radicals, antioxidant enzymes and CPK concentration
in children with ABM indicating their increased synthesis and
release by leukocytes which have migrated into subarachnoid space
following BBB disruption. This study demonstrated a direct
correlation between CSF cytokines concen-trations and superoxide,
hydrogen peroxide production in ABM. There was no correlation
between CSF cytokines and SOD in this study. Due to difficulty in
obtaining CSF and the risk of infection in serial sampling, kinetics
of cytokines and free radicals were not evaluated. It may be
possible that enzymes rise later in course of disease after the
cytokines cause liberation of free oxygen radicals from migrated
neutrophils. There is only one study which showed that
cytokines in ABM reached peak values in disease day before or when
MnSOD reached peak levels(16). Immunohistochemically, a large number
of glial cells stained positive for Mn SOD in cerebral cortex from a
patient with bacterial meningitis suggeting the role of cytokines in
the induction of Mn SOD in nervous tissues(17). It may be possible
that if we had obtained CSF later in the course of disease, SOD
levels would have been much more elevated. On the other hand failure
of simultaneous increase in SOD activity with free radicals and
cytokines production may explain the high mortality in this group
due to failure to scavenge free radicals resulting in increased
tissue damage. IL–1b,
TNF–a and
superoxide emerged to be good predictors of mortality in ABM and
these inflammatory mediators had good correlation with CSF protein
which is routinely measured parameter and thus can be used
indirectly to predict adverse outcome. The detection of cytokines
and free radicals in CSF may also provide additional help in
diagnosis and prognosis of ABM.
Contributors:
SA coordinated and supervised the study and drafted the paper; she
will act as the guarantor for the paper. MJ collected the data and
compiled it. GM and VSR performed microbiological tests and
estimation of cytokines. GNR and SB estimated free radicals. Funding: None. References
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