Bidirectional Concentration-Dependent Effects of Tumor Necrosis ...

INFECTION AND IMMUNITY, Apr. 2003, p. 2288–2291 0019-9567/03/$08.00⫹0 DOI: 10.1128/IAI.71.4.2288–2291.2003 Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 4

Bidirectional Concentration-Dependent Effects of Tumor Necrosis Factor Alpha in Shigella dysenteriae-Related Seizures Yael Yuhas,1,2* Abraham Weizman,1,2 and Shai Ashkenazi1,2,3 Felsenstein Medical Research Center1 and Schneider Children’s Medical Center of Israel,3 Beilinson Campus, Petah Tikva, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv,2 Israel Received 26 August 2002/Accepted 3 January 2003

We have previously demonstrated that pretreatment of mice with Shigella dysenteriae sonicate enhanced their susceptibility to pentylenetetrazole-induced seizures and that tumor necrosis factor alpha (TNF-␣) was proconvulsive in this respect. The present study shows that TNF-␣, at high concentrations, may also exert a suppressive effect on Shigella-mediated seizures. This implies that high levels of TNF-␣ may play a protective role in neurologic complications of S. dysenteriae infection. The acute gastrointestinal infections caused by Shigella dysenteriae and enterohemorrhagic Escherichia coli (EHEC) strains are often accompanied by neurologic disturbances, mainly convulsions and encephalopathy (2, 5, 9). The pathogenesis of the neurologic manifestations in shigellosis and EHEC infections is as yet unclear. Accumulating data from animal models and epidemiological studies have linked the family of Shiga toxins (Stx), produced by these strains, to the development of neurologic disturbances (4, 8, 9, 10, 12, 19). However, the Shiga toxins are not classical neurotoxins. It is assumed that their neurotoxicity stems primarily from their ability to inflict cytotoxic damage on the microvascular endothelium of the central nervous system (CNS). The infections caused by S. dysenteriae and EHEC cause a severe inflammation of the bowel which is triggered by extensive production of proinflammatory cytokines (15, 28). An increase in tumor necrosis factor alpha (TNF-␣) and interleukin-1␤ (IL-1␤) has been found in the stool and plasma of patients with acute shigellosis, and the increased levels correlated with the clinical severity of the intestinal disease (18). Recently, our group has developed an animal model to investigate Shigella-related seizures. Administration of the proconvulsant pentylenetetrazole (PTZ) to mice induces clonictonic seizures within minutes of its application, owing to its antagonistic activity at the benzodiazepine/␥-aminobutyric acid receptor complex. Using this model, we found that pretreatment of mice with crude preparations of S. dysenteriae or E. coli H-30 significantly increased their response to PTZ-induced seizures (27). The increased response could be mimicked by coadministration of Shiga toxin and lipopolysaccharide. In the same model, we also demonstrated that TNF-␣, IL-1␤, and nitric oxide (NO) play an important role in the sensitization of the CNS to convulsive activity (3, 26). In the course of these experiments, we noted that pretreatment of mice with high concentrations of S. dysenteriae sonicate was less effective than pretreatment with lower concentrations and sometimes failed completely to enhance the

response to PTZ. We hypothesized that, in high concentrations, mediators induced by Shigella sonicate may have an inhibitory effect on enhancement of seizures. This was supported by evidence that cytokines, and specifically TNF-␣, may exert dual effects in the CNS (20, 23). The aim of the present study was to investigate the concentration-dependent effects of TNF-␣ in Shigella-related seizures. Pretreatment with different doses of S. dysenteriae sonicate. Mice (ICR) were preinjected (intraperitoneally [i.p.]) with different doses of S. dysenteriae 60R sonicate (26) and treated with PTZ (50 mg/kg of body weight, i.p.) at 7 and 24 h after bacterial injection. The reaction to PTZ included several phases: unresponsiveness, mild contraction, clonic seizures, and tonic seizures, occasionally followed by death. Mean convulsion score and rate of seizures were calculated for each group, as previously described (26). The administration of S. dysenteriae sonicate increased the susceptibility of the mice to PTZ. This was reflected in the significantly higher mean convulsion score and the greater number of mice reaching clonic-tonic seizures compared to control (saline-pretreated) mice. The ability of S. dysenteriae sonicate to sensitize mice to PTZ depended on its concentration (Fig. 1). The most pronounced seizure enhancement was achieved with a dose of 2 50% lethal doses (LD50). Pretreatment with a very high dose of 8 LD50 did not change the mice’s response to PTZ, whereas 4 LD50 caused a significant increase in seizures, albeit a less potent one than that caused by 2 LD50, as indicated by a reduction in the power of significance. The percentages of mice responding with clonic-tonic seizures in groups treated with saline, 2 LD50 of Shigella, and 8 LD50 of Shigella were 19, 47, and 24%, respectively (P ⫽ 0.0017 for 2 LD50 versus saline, P ⫽ 0.4 for 8 LD50 versus saline). Similar results were obtained when the response to PTZ was examined 24 h after Shigella injection. Serum TNF-␣ levels were determined after Shigella sonicate administration by an enzyme-linked immunosorbent assay kit (R & D Systems Inc.). S. dysenteriae 60R sonicate induced an elevation in circulatory TNF-␣ levels in mice, as early as 1 h after injection. This effect was dose dependent, with the highest levels occurring after administration of 8 LD50 of the sonicate (Fig. 2). Mice injected with saline did not have detectable levels of TNF-␣.

* Corresponding author. Mailing address: Unit of Infectious Diseases, Schneider Children’s Medical Center of Israel, P.O. Box 8145, Petah Tikva 49181, Israel. Phone: 972-3-9253680/3792. Fax: 972-39253056. E-mail: [email protected]. 2288

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FIG. 2. Serum TNF-␣ production in response to different doses of S. dysenteriae 60R sonicate (60R). The values are the means of determinations from five animals ⫾ standard errors (R2 ⫽ 0.75, P ⫽ 0.05).

suggest that, at higher doses of Shigella sonicates, TNF-␣ may play a protective role in limiting convulsions. Treatment with exogenous TNF-␣. The enhancement of seizures by anti-mTNF-␣ in the mice given a dose of S. dysenteriae sonicate that failed to increase their convulsive response to PTZ prompted us to investigate if high concentrations of TNF-␣ may have an inhibitory effect on enhancement of seizures by S. dysenteriae. Mice were treated with an effective dose (2 LD50) of S. dysenteriae 60R sonicate, with S. dysenteriae sonicate (2 LD50) followed 30 min later by TNF-␣ (30 ␮g/ mouse, i.p.), or with saline alone. Recombinant human TNF-␣ (specific activity, 2.5 ⫻ 107 U/mg of protein) was obtained from ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel). As shown in Fig. 4, administration of TNF-␣ abolished the enhanced response to PTZ. The incidence of clonic-tonic seizures was also lower in mice pretreated with TNF-␣ and S. dysenteriae than in mice pretreated with S. dysenteriae alone: 17% (2 of 12), 63% (7 of 11), and 8% (1 of 12) after injection of saline, Shigella, or Shigella with TNF-␣, respectively (P ⫽ 0.029 for FIG. 1. Effect of pretreatment with different doses of S. dysenteriae 60R sonicate (60R) on PTZ-induced seizures. Data are presented as means ⫾ standard errors of the means. (A) Mice treated 7 h before PTZ. P ⫽ 0.013 for saline (n ⫽ 36) versus 1 LD50 (n ⫽ 23), P ⫽ 0.008 for saline versus 2 LD50 (n ⫽ 36), P ⫽ 0.05 for saline versus 4 LD50 (n ⫽ 24), and P ⫽ 0.22 for saline versus 8 LD50 (n ⫽ 21). (B) Mice treated 24 h before PTZ. P ⫽ 0.003 for saline versus 60R at 1 LD50 (n ⫽ 12), P ⫽ 0.001 for saline versus 60R at 2 LD50 (n ⫽ 23), P ⫽ 0.046 for saline versus 60R at 4 LD50 (n ⫽ 15), and P ⫽ 0.196 for saline versus 60R at 8 LD50 (n ⫽ 15).

Pretreatment with anti-mTNF-␣. In a previous study, we found that pretreatment of mice (intravenously) with antimurine TNF-␣ (anti-mTNF-␣) at 30 min before administration of S. dysenteriae 60R sonicate abolished the enhanced response to PTZ due to Shigella pretreatment (26). However, in the present study there was an opposite effect in the mice that received higher doses (8 LD50) of S. dysenteriae 60R sonicate, which did not enhance seizures: the pretreatment with antimTNF-␣ significantly increased their response to PTZ, compared to that of saline-pretreated mice (Fig. 3). These data

FIG. 3. Enhancement of PTZ-induced seizures by anti-mTNF-␣ in mice pretreated with a high dose of S. dysenteriae 60R sonicate (60R). PTZ (50 mg/ml) was given i.p. at 24 h. P ⫽ 0.09 for 60R (n ⫽ 24) versus saline (n ⫽ 26), P ⫽ 0.054 for 60R versus 60R with anti-mTNF-␣ (n ⫽ 26), and P ⫽ 0.048 for 60R with anti-mTNF-␣ versus saline.

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and to potentiate glutamate neurotoxicity (14). In contrast to this study, others have shown that TNF-␣ prevented the excitotoxic effects of the excitatory amino acids glutamate, N-methyl-D-aspartate, and DL-␣-amino-3-hydroxy-5-methylisoxazolepropionic acid in cultured neurons (7). In addition, studies of mice with deficient TNF-␣ receptor expression showed greater neuronal damage after KA injection (11). To the best of our knowledge, ours is the first study linking TNF-␣ concentration and its bidirectional effect (attenuating or aggravating) in convulsive activity. The mechanism by which TNF-␣ exerts opposite effects in Shigella-mediated seizures is still an enigma. The enhanced response to PTZ is a result of the mutual action of Stx and lipopolysaccharide and is mediated by TNF-␣, IL-1␤, and NO (3, 26, 27). Both TNF-␣ and IL-1␤ upregulate the expression of Stx receptor in endothelial cells, thereby augmenting Stx cytotoxicity (17, 13, 24, 25). Moreover, TNF-␣ and IL-1␤ act synergistically to exert a neurotoxic effect in cell culture (6). They are also potent inducers of NO, which itself can exert both anticonvulsive and proconvulsive effects on epileptic activity in different models (21). It is plausible, therefore, that the effects of TNF-␣ in the CNS are mediated on one hand by its cytotoxicity, either direct or indirect, and on the other hand through the induction of NO. Interestingly, in cerebral malaria, high concentrations of NO are associated with better prognosis (1). In conclusion, our findings imply that at high levels TNF-␣ may play a protective role in the neurologic sequelae of human shigellosis and EHEC infections. The effect of TNF-␣ concentration on neurologic manifestations of other infectious diseases merits further investigation on the pathophysiologic and therapeutic levels. REFERENCES

FIG. 4. Effect of exogenous TNF-␣ on the enhancement of PTZinduced seizures by S. dysenteriae 60R sonicate (60R). (A) Mice pretreated with S. dysenteriae 60R sonicate 7 h before PTZ. P ⫽ 0.04 for 60R versus saline, and P ⫽ 0.003 for 60R versus 60R followed by TNF-␣. n ⫽ 12 in all groups. (B) Mice pretreated with S. dysenteriae 60R sonicate 24 h before PTZ. P ⫽ 0.003 for 60R versus saline, and P ⫽ 0.002 for 60R versus 60R followed by TNF-␣. n ⫽ 12 in all groups.

Shigella versus saline; P ⫽ 008 for Shigella versus Shigella with TNF-␣). TNF-␣ is a well-recognized modulator in the brain, involved in multiple activities, including modulation of neurotransmitter release, synaptic activity, and the expression of other cytokines (14). In several neuropathologies, such as brain trauma or demyelination disorders, TNF-␣ can play either a detrimental or a beneficial role (14, 20). The mode of TNF-␣ activity may be influenced by its concentration, the time of exposure, and the presence of other modulators (23). TNF-␣ has been also implicated in convulsive activity. Studies have shown that TNF-␣ expression and activity are markedly elevated in the brain following kainic acid (KA) injection and in the hippocampus after KA-induced seizures (11). Neurologic disorders, including seizures, have been reported elsewhere to occur during cancer therapy with TNF-␣ and in transgenic mice that overexpress TNF-␣ in their neurons (16, 22). In an in vitro study, TNF-␣ was shown to provoke synaptic activity

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