Induction of Hypersensitivity to Endotoxin and Tumor Necrosis Factor ...

INFECTION AND IMMUNITY, Apr. 1990,

p.

Vol. 58, No. 4

935-937

0019-9567/90/040935-03$02.00/0 Copyright ©D 1990, American Society for Microbiology

Induction of Hypersensitivity to Endotoxin and Tumor Necrosis Factor by Sublethal Infection with Salmonella typhimurium M. MATSUURA AND C. GALANOS*

Max-Planck-Institut fur Immunbiologie, Stiubeweg 51, 7800 Freiburg, Federal Republic of Germany Received 29 September 1989/Accepted 20 December 1989

The effect of sublethal infection with Salmonella typhimurium on the sensitivity of mice to the lethal activity of lipopolysaccharide (LPS) was studied in C3H/TifF mice. These mice are more resistant to S. typhimurium infection and survive inocula that are lethal for most other strains of mice. Infection of C3H/TifF mice with 2 x 104 CFU of S. typhimurium was without lethal effect. However, administration of LPS at different times after infection revealed that the sensitivity of the animals to the lethal activity of LPS increased exponentially, reaching a maximum by 6 to 8 days after infection. Thereafter, it decreased, reaching preinfection values about 4 weeks after inoculation. At the height of sensitivity, the animals were susceptible to less than 1 ,ug of LPS compared with 100 or 200 ,ug in noninfected mice. The sensitization to LPS by infection was paralleled by a sensitization to tumor necrosis factor. The time course of development of sensitization to tumor necrosis factor, as well as the time course of its decrease and disappearance, was almost identical to that of LPS.

MATERIALS AND METHODS

Endotoxin (lipopolysaccharide [LPS]) induces acute pathophysiological manifestations that are strikingly similar to those seen during gram-negative septicemia (8, 14). Despite these similarities, the role of endotoxin in infection lethality has often been questioned. These doubts arise from quantitative considerations regarding the amounts of LPS calculated to be present in the infected organism at the time lethality is attained. Such amounts are usually far too low compared with those that are known to be lethal in healthy noninfected animals. Support for questioning the role of endotoxin in infection lethality was obtained when it was found that endotoxin-resistant mice (C3H/HeJ) are highly susceptible to lethal infection with gram-negative microorganisms. Sensitivity to endotoxin may be increased under several experimental conditions. Treatment of mice with chemical agents such as dactinomycin (1), lead acetate (12), or Dgalactosamine (6) was shown to increase their susceptibility to endotoxin. Sensitization to endotoxin also increases with live or killed bacteria such as Mycobacterium boi'is bacillus Calmette-Gudrin (13) or Propionibacterium acnes (3). In our laboratory, induction of hypersensitivity to LPS was also shown to proceed during gram-negative infection with Coxiella burnetii (11). Further, in an earlier study, Galanos et al. showed that the sensitivity of mice to the lethal activity of endotoxin increases during lethal infection with Salmonella typhimuriium (5) and that a similar sensitization also occurs in endotoxin-resistant C3H/HeJ mice. These results made it evident that very low levels of endotoxin can be lethal in infected animals. They further demonstrated the important role of endotoxin in the lethality of gram-negative infection in endotoxin-resistant mice. In the present study, we investigated the sensitizing effects of sublethal gram-negative infection on LPS susceptibility and the possible mechanisms involved. It will be shown that sublethal infection with S. typhimurium increases susceptibility to endotoxin. It will be further demonstrated that mice made hypersensitive to LPS by infection are also hypersensitive to tumor necrosis factor (TNF). *

Animals. Female C3H/TifF mice (6 to 8 weeks old) and C57BL/6 mice (8 weeks old) were obtained from the breeding stock of this institute. Materials. The LPS used was the uniform triethylamine salt of Salmonella abortus subsp. equi, prepared as previously described (7). Human recombinant TNF was a kind gift from Knoll AG, Ludwigshafen, Federal Republic of Germany. The specific activity of the preparation was 6.6 x 106 U of TNF per mg of protein. For animal application, all materials were dissolved in pyrogen-free phosphate-buffered saline. Infection. A culture of S. typhimuriium C5 was obtained from S. Schlecht of this institute. The cells were grown overnight at 37°C on Loeb agar composed of 1% tryptose (Difco Laboratories, Detroit, Mich.), 0.1% yeast extract, 0.1% glucose, 0.8% NaCl, and 1% Bacto-Agar (pH 7.1). The cells were suspended in sterile 0.85% NaCl (saline). The approximate number of bacteria was estimated by measuring the turbidity of the bacterial suspension. The suspension was appropriately diluted with saline, and the desired number of bacteria was administered in mice intraperitoneally in 0.4 ml of saline. The precise number of CFU in the suspension used for infection was determined by plating appropriate dilutions on Loeb agar and counting the number of colonies after overnight culture at 37°C.

RESULTS Survival pattern of mice after S. typhimurium C5 infection. C57BL/6 and C3H/TifF mice were infected intraperitoneally with S. typhimurium C5, and the survival of the infected mice was observed. In the case of the C57BL/6 mice, lethality commenced on day 6 after infection with 80 CFU and was 100% by day 10 (Fig. 1). In this strain of mouse, infection with S. typhimurium always resulted in lethality for some or all mice, even when 30 CFU or less was used for inoculation. In contrast, C3H/TifF mice were more resistant to this level of infection. Lethality of 100% was obtained with a dose of 2 x 105 CFU. A lower dose (2 x 104) was usually without lethal effect; mice infected with the lower dose survived the 28-day observation period (Fig. 1).

Corresponding author. 935

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FIG. 1. Deaths of C57BL/6 and C3H/TifF mice after S. typhimurium CS infection. Survival percentages over time were determined for C3H/TifF mice infected with 2.1 x 104 CFU ( ) or 2.3 x 105 CFU (--) and for C57BL/6 mice infected with 82 CFU (-----).

Induction of hypersensitivity to lethal toxicity of LPS in C3H/TifF mice by infection with S. typhimurium. C3H/TifF mice were infected with 2 x 104 CFU of S. typhimurium. On different days thereafter, groups of four to six mice were administered different amounts of LPS intravenously, and the resulting deaths were recorded. In four independent experiments, similar results were obtained; these results are summarized in Fig. 2. Following infection, the sensitivity of the animals to LPS increased exponentially, reaching a maximum about 1 week later. Thereafter, it gradually decreased, reaching preinfection levels approximately 4 weeks later. At the height of sensitization, the 50% lethal dose of LPS was 0.5 ,ug, which represents a sensitization factor of 300, compared with a 50% lethal dose of 150 ,ug for noninfected mice. The infected animals not only were susceptible to smaller amounts of LPS but also died much faster after LPS challenge with deaths usually occurring on the same day the LPS was administered. In noninfected mice (controls) receiving lethal amounts of LPS (200 jLg), death occurred 48 to 72 h after LPS injection. Sensitization of C3H/TifF mice to lethal activity of TNF by

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FIG. 2. Sensitivity of C3H/Tif mice to LPS or TNF lethality during infection with S. typhimurium. C3H/TifF mice were infected with S. typhimurium (2 x 104 CFU). On different days after infection, groups of six animals received different amounts of LPS ( ) or TNF (-----) intraperitoneally and the resulting deaths were recorded. Fifty percent lethal doses were calculated by the method of Karber (9).

S. typhimurium infection. It was shown recently that TNF is a mediator that initiates the lethal activity of endotoxin (2, 4, 10). In different experimental models, mice sensitized to the lethal effects of LPS were found to be concurrently hypersensitive to the lethal activity of TNF. For this reason, the effect of S. typhimurium infection on TNF susceptibility was investigated. Groups of four to six mice were injected with 2 x 104 CFU of S. typhimurium and challenged with various amounts of TNF on different days thereafter. The results obtained from three independent experiments are depicted in Fig. 2. It can be seen that sensitivity to TNF increased after infection, reaching a maximum on days 5 and 6. Thereafter, sensitization slowly subsided, and the sensitivity of the animals became normal about 4 weeks later. Thus, the pattern of development and disappearance of sensitization to TNF by infection with S. typhimurium was almost identical to that of LPS. The kinetics of TNF-induced death in the infected mice were extremely fast, commencing as early as 4 h after TNF injection. In the noninfected controls, the kinetics of death caused by TNF were very similar to those of LPS, with deaths occurring over a period of 72 h. DISCUSSION In an earlier study by Galanos et al. (5), it was shown that lethal infection of C57BL/6 mice with S. typhimurium increases the sensitivity of the animals to the lethal effects of LPS. In the same study, it was shown that such sensitization also occurred in C3H/HeJ mice, indicating that endotoxin becomes a factor of pathogenicity for endotoxin-resistant mice during infection. A limitation of the above-described model was that infection with S. typhimurium of this mouse strain was always lethal, even when very small numbers (10 to 30 cells) of infecting microorganisms were administered. S. typhimurium is also highly virulent in most other strains of mice. Consequently, the sensitivity of the animals to LPS may be studied for only a short time between days 2 and 5 (at the latest) after infection. After this time, deaths caused by the LPS overlap with deaths due to infection, which usually begin between days 5 and 6. In the present study, the use of C3H/TifF mice, which are more resistant to S. typhimurium, allowed the effect of sublethal infection on LPS sensitivity to be studied and the timecourse of the development and disappearance of sensitization to be determined. The results show that sublethal infection (with 2 x 104 CFU) of these mice increases their sensitivity to the lethal activity of LPS. The maximum sensitivity was measured on days 6 to 8 after infection; thereafter, sensitization gradually decreased, disappearing about 4 weeks later. The increase in sensitivity up to day 6 followed an exponential pattern. At the time of maximum sensitization, the animals were susceptible to 0.5 ,ug of LPS. The higher sensitivity of the infected mice to LPS may be explained by a higher sensitivity to the endogenous mediator TNF. This is suggested by the fact that the increase in sensitivity of the infected mice to LPS was paralleled by an increased sensitivity to TNF, with the patterns of sensitization to LPS and TNF being almost identical. TNF was shown in a previous study to cause death in normal mice and to mediate the lethal activity of LPS in D-galactosaminesensitized mice (4, 10). Further, its toxicity was found to be enhanced by a number of models which originally were designed for enhancing the toxicity of LPS (4). The finding that sublethal infection increases sensitivity to the toxic properties of TNF suggests that subclinical infections can, in principle, enhance the toxicity of any agent

SENSITIZATION TO LPS AND TNF BY INFECTION

VOL. 58, 1990

(bacterial, viral, parasitic, etc.) potentially capable of inducing TNF formation. If this is so, a cofactor role for gramnegative microorganisms in different pathogenetic situations not attributable primarily to gram-negative infections may then be envisaged. Even though the hypersensitivity of the infected mice to LPS may be explained simply by a higher susceptibility to the endogenous mediator TNF, the contribution of other mechanisms to the overall hypersensitization of the animals cannot be excluded. One such likely mechanism is the generation of LPS-hyperresponsive macrophages by the sublethal infection. Macrophages were shown to mediate the lethal activity of LPS, and any increase in their responsiveness to this agent would express itself as a hypersensitivity of the infected organism to endotoxin. In view of the multiplicity of biologically active substances present in gram-negative microorganisms, such a hypersensitization of macrophages seems very likely and is currently being investigated. So far, the development of hypersensitivity to endotoxin during gram-negative infection has been demonstrated in infection models with different types of gramnegative microorganisms. Exceptions have not yet been found. Infections with C. burnettii, Klebsiella pneumoniae, Escherichia coli, and S. typhimurium were all shown to increase the sensitivity of the infected animals to endotoxin. Sensitization to the lethal effects of endotoxin therefore seems to be a property widely shared by pathogenic gramnegative bacteria. We believe that the development of endotoxin shock during gram-negative sepsis probably always takes place in a state of hypersensitivity to endotoxin. The results so far support this hypothesis. Hypersensitivity to endotoxin may also occur naturally. It is encountered frequently in apparently healthy experimental animals (mice, rabbits, etc.), but for reasons which are usually unknown. In view of the present findings, the cause of natural hypersensitivity to endotoxin may be explained by asymptomatic infection with gram-negative microorganisms. Such a mechanism appears not only probable but also very likely, considering the abundance of gram-negative microorganisms in the environment. We therefore propose that latent infections with gram-negative bacteria, proceeding without clinical symptoms, represent a cause of natural sensitization to endotoxin. ACKNOWLEDGMENTS

The technical assistance of M.-L. Gundelach and H. Stubig is gratefully acknowledged.

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The study was supported in part by the DAAD and by the Bundesminister fur Forschung und Technologie (grant 01 Ki 8809). LITERATURE CITED 1. Berry, L. J., and D. S. Smythe. 1964. Effects of bacterial endotoxins on metabolism. IV. Enzyme induction and cortisone protection. J. Exp. Med. 120:721-731. 2. Beutler, B., I. W. Milsark, and A. C. Cerami. 1985. Passive immunization against cachectin/tumor necrosis factor protects mice from lethal effect of endotoxin. Science 229:869-871. 3. Cluff, L. E. 1971. Effects of lipopolysaccharides (endotoxins) on susceptibility to infections, p. 399-413. In S. Kadis, G. Weinbaum, and S. J. Ajl (ed.), Microbial toxins. vol. 5. Academic Press, Inc. New York. 4. Galanos, C., M. A. Freudenberg, A. Coumbos, M. Matsuura, V. Lehman, and J. Bartholeyns. 1988. Induction of lethality and tolerance by endotoxin are mediated by macrophages and tumour necrosis factor, p. 114-127. In B. Bonavida et al. (ed.), Tumor necrosis factor, cachectin and related cytokines. S.

Karger AG, Basel. 5. Galanos, C., M. A. Freudenberg, D. Krajewska, D. Takada, G. Georgiev, and J. Bartholeyns. 1986. Endotoxin: structural aspects and immunobiology of host-responses. EOS-J. Immunol. Immunopharmacol. 6:78-86. 6. Galanos, C., M. A. Freudenberg, and W. Reutter. 1979. Galactosamine-induced sensitization to the lethal effects of endotoxins. Proc. NatI. Acad. Sci. USA 76:5939-5943. 7. Galanos, C., 0. Luderitz, and 0. Westphal. 1979. Preparation and properties of a standardized lipopolysaccharide from Salmonella abortus equi. Zentralbl. Bakteriol. Hyg. Abt. I Orig. Reihe A 243:226-244. 8. Kadis, S., G. Weinbaum, and S. J. AjI (ed.). 1971. Microbial toxins, vol. 5. Academic Press Inc., New York. 9. Karber, G. 1931. Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Arch. Exp. Pathol. Pharmakol. 162:480-484. 10. Lehman, V., M. A. Freudenberg, and C. Galanos. 1987. Lipopolysaccharide and TNF express similar lethal toxicity in Dgalactosamine-treated mice. J. Exp. Med. 165:657-663. 11. Schramek, S., J. Kazar, Z. Sekeyova, M. A. Freudenberg, and C. Galanos. 1984. Induction of hypersensitivity to endotoxin in

mice by Coxiella burnetii. Infect. Immun. 45:713-717. 12. Selye, H., B. Tuchweber, and L. Bertok. 1966. Effect of lead acetate on the susceptibility of rats to bacterial endotoxins. J. Bacteriol. 91:884-890. 13. Suter, E., G. E. Ullman, and R. G. Hoffman. 1958. Sensitivity of mice to endotoxin after vaccination with BCG (Bacillus Calmette-Gudrin). Proc. Soc. Exp. Biol. Med. 99:167-169. 14. Weinbaum, G., S. Kadis, and S. J. Ajl (ed.). 1971. Microbial toxins, vol. 4. Academic Press Inc., New York.