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Laboratory of Veterinary Medicine, Istituto Superiore di Sanità, 00161 Rome, Italy,1 Immunology and Disease Resistance Laboratory, Animal and Natural Resources Institute, Agricultural Research Service, United States Department of Agriculture, Beltsville, Maryland 207052
Received 13 August 2001/ Returned for modification 6 November 2001/ Accepted 28 November 2001
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) is a prominent mediator in conferring protection both in vitro (1, 2) and in vivo (7, 9, 12).
Interleukin-12 (IL-12) is pivotal for the development of Th1 responses (10), and it is involved in the outcome of resistance against many infections (8). Mice depleted of IL-12 have been shown to be more susceptible to infection with B. abortus, and this susceptibility has been correlated with decreased IFN- production (13, 14). This suggests that IL-12 contributes to resistance to Brucella infection mainly via an IFN--dependent pathway. However, mice treated with a single administration of recombinant murine IL-12 did not clear more efficiently an infection with the vaccine strain B. abortus RB51. In addition, treated mice did not display augmented cellular immune responses to Brucella antigen, nor did they respond more efficiently to a challenge infection (3).
IL-18 is a newly cloned cytokine synthesized mainly by activated macrophages. IL-18 is able to stimulate IFN- production (5, 11) and can synergistically act with IL-12 on T cells (4, 6). In this study, we explored the effect of exogenous IL-18 given alone or in combination with IL-12 on B. abortus 2308 infection in an attempt to better understand the immune mechanisms that control Brucella infections.
Female BALB/c mice were purchased from Charles River (Milan, Italy) and used at 10 to 12 weeks of age. Mice were infected intraperitoneally with 5 x 103 CFU of B. abortus 2308 in 0.2 ml of phosphate-buffered saline (PBS). Mice were injected intraperitoneally with 500 ng of cytokines (R&D Systems, Minneapolis, Minn.) in 0.2 ml of PBS 1 day before and on the day of the infection. Equal amounts of PBS were injected into control mice. At 1 and 5 days after infection, the mice were sacrificed and their spleens were collected and then dispersed in RPMI 1640 (GIBCO Laboratories, Grand Island, N.Y.) containing 2 mM L-glutamine, 25 mM HEPES, and 5 x 10-5 M 2-mercaptoethanol. An aliquot of the resulting cell suspension was plated to determine the number of CFU. To evaluate cytokine production, spleen cells (2 x 106) were cultured in 0.5 ml of RPMI 1640 and stimulated with 0.5 ml of heat-inactivated B. abortus 2308 at 108 CFU/ml. Supernatants were collected 72 h after culture for measurement of tumor necrosis factor alpha (TNF-
), IFN-, and IL-10 production. Mouse cytokines were detected by enzyme-linked immunosorbent assay (ELISA) according to the manufacturer's instructions (R&D Systems).
As shown in Table 1, combined inoculation of IL-12 and IL-18 reduced the number of bacterial cells in the spleens of treated mice compared to the number in untreated controls at 1 and 5 days after infection (P = 0.07 and P < 0.05, respectively). IL-18 alone induced a decrease in the bacterial count in the spleens of injected and infected mice compared to that in the spleens of untreated infected mice. However, this reduction was not statistically significant. In contrast, a single inoculation of IL-12 did not exert any effect on the course of the infection. The combined treatment also induced a marked change in spleen weight. Treated animals showed an enlargement of their spleens as early as 1 day after infection. In addition, there was a significant reduction in spleen weight in IL-18-treated infected animals compared to that in untreated infected mice at 5 days after infection. The reason for this difference was not investigated.
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production as early as 1 day after infection. The levels of IFN- were similar in treated and untreated mice 5 days after infection, suggesting that the effects of the cytokines are transient and are more prominent in the early phases of infection. Levels of TNF- were not statistically different between treated and untreated mice throughout the experiment. However, there was a slight increase of TNF- 1 day after infection and a slight decrease 5 days after infection in treated mice compared to the levels in untreated mice. Finally, IL-10 levels were not different between treated and untreated mice at either 1 or 5 days after infection.
Overall, our results demonstrate that the combined administration of IL-12 and IL-18 induces protection against a B. abortus 2308 infection in mice, while treatment with either IL-12 or IL-18 alone resulted in little or no effect. It is interesting to note that the treatment induced an increased production of IFN- during the early phases of infection. These findings are consistent with recent observations demonstrating that IFN- is involved early in Brucella infections (7). In addition, these results support the concepts that IL-18 has effects similar to those of IL-12 regarding the induction of IFN- production by Th1 and NK cells (5) and that IL-18 may act synergistically with IL-12 in defense mechanisms against infectious agents (6). These results show that exogenous treatment with IL-12 at the tested dose does not significantly induce resistance to a B. abortus 2308 infection, confirming the reports of others (3). The reason why exogenous IL-12 is not able to confer protection against Brucella infection has not been ascertained. It is possible that IL-12 alone does not promote increased IFN- production at a level that is effective in influencing the outcome of the infection.
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production by IL-12 and IL-18. Curr. Opin. Immunol. 10:259-264.[CrossRef][Medline]
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