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Previous Article | Next Article 
Infection and Immunity, December 2002, p. 6896-6903, Vol. 70, No. 12
0019-9567/02/$04.00+0 DOI: 10.1128/IAI.70.12.6896-6903.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Division of Infectious Diseases,1 Pulmonary and Critical Care Medicine,5 Department of Microbiology and Immunology,2 Department of Epidemiology,4 Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, Maryland 212013
Received 9 July 2002/ Accepted 15 August 2002
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ABSTRACT |
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INTRODUCTION |
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Proinflammatory cytokines, such as interleukin-1ß (IL-1ß) and gamma interferon (IFN-
), are involved in antimicrobial defenses (15, 16, 20, 40). The recently discovered IFN--inducing factor IL-18 exerts pleotropic activities (7) and plays an important role in defense against a variety of gram-positive and -negative bacterial pathogens (3, 8, 9, 12, 25, 27, 31, 34, 39). Excessive synthesis of IL-1ß and IL-18, however, is also associated with septic death (17, 24, 28, 29, 36). This suggests that a controlled expression of IL-18 expression may be critical to the lethal outcome of LPS-induced innate immune response.
Synthesis of IL-1ß and IL-18 depends upon the proteolytic cleavage of their precursor polypeptides (pro-IL-1ß and pro-IL-18) by the cysteine protease caspase 1 (Cas-1), also called IL-1-converting enzyme (13, 14). Like IL-18-/- mice, cas-1-/- mice do not produce bioactive IL-18 nor do they produce IL-1ß and are resistant to LPS toxicity (21, 22). Also, Cas-1 inhibitors suppress IL-1ß and IL-18 synthesis and protect mice from endotoxin shock (14, 23). Thus, Cas-1 is considered essential for the toxic effects of LPS. Given its involvement in IL-18 maturation, Cas-1 can also be expected to play an important role in gram-negative antibacterial host defenses (ABHD). However, this is not well characterized.
In the present study, we show that Cas-1 is important in innate host defenses against Escherichia coli infection. However, unlike the case with a nonlethal dose of LPS, high doses of LPS dramatically upregulate cas-1 expression leading to lethality. This association of different levels of expression of one component of the innate immune response to LPS, Cas-1, with both ABHD and lethal inflammation lends support to the hypothesis that sepsis is a dysregulation of innate immune response.
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MATERIALS AND METHODS |
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Mice. Seven- to twelve-week-old female C3H/HeN (National Cancer Institute, Frederick, Md.) and cas-1-/- mice were housed under a 12-h daylight cycle in controlled humidity and temperature conditions and supplied with food and water ad libitum. One male and three female cas-1+/- mice (with a C3H background [kindly supplied by Tariq Ghayur, Abbott Bioresearch Center, Worcester, Mass., and Paul Jolicoeur, Clinical Research Institute of Montreal, Montreal, Quebec, Canada]) were mixed and allowed to breed naturally and pregnant females were separated from the male immediately. Tail-snip DNA prepared (DNeasy Tissue Kit; Qiagen, Inc., Valencia, Calif.) from 4-week-old pups was genotyped by using cas-1-specific PCR. The primers were (i) forward (5'-GAA GAG ATG TTA CAG AAG CC-3') and (ii) reverse (for the wild-type gene, 5'-CAT GCC TGA ATA ATG ATC AC-3', and for the mutated gene, 5'-GCG CCT CCC CTA CCC GG-3'), and the PCR conditions were 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min for 30 cycles.
To determine the cytokine levels in serum, C3H/HeN mice were treated with a predetermined nonlethal (75 µg/mouse) and lethal (500 µg/mouse) doses of LPS in normal saline by the intraperitoneal (i.p.) route, euthanized at different time points, and bled by cardiac puncture. Serum prepared from freshly drawn blood was diluted in phosphate-buffered saline (PBS)-bovine serum albumin and used for cytokine determination. Control mice were treated with normal saline.
Bacteria. For all live infection experiments, E. coli O18:K1:H7 strain Bort was used as the challenge organism. This clinical isolate, originally isolated from the cerebrospinal fluid of a neonate, has been previously employed in the murine peritonitis model. This isolate is serum resistant and cannot be killed by neutrophils in the absence of type-specific antibody but rather requires activated macrophages (4). A single colony of E. coli, grown overnight on tryptic soy agar, was inoculated into tryptic soy broth and grown to log phase. Bacteria were washed and resuspended in PBS to an approximate concentration of 108 CFU/ml. This cell suspension was diluted further in PBS to the desired dose of bacteria for administration to mice i.p.
Susceptibility of mice of to E. coli O18:K1:H7. C3H/HeN+/+ (wild type), C3H/cas-1+/-, or C3H/cas-1-/- mice were injected with different doses of freshly grown and diluted culture of E. coli O18:K1 i.p. and observed for mortality. The 50% lethal dose (LD50) of E. coli O18:K1 (Bort) was determined by plotting the percent mortality against the dose by using GraphPad Prizm software (GraphPad, San Diego, Calif.). The effect of Cas-1 inhibition on bacterial infection was studied by treating C3H/HeN+/+ mice with 5 mg of Ac-YVAD-CHO/kg i.p. 2 h prior to infecting with different doses of E. coli.
Supplementation of cas-1-/- mice with IL-18 and IL-1ß. cas-1-/- mice were treated i.p. with different doses of rmIL-18 (10 to 200 ng/mouse) or IL-1ß (500 U/mouse). These doses of rmIL-18 were previously found to induce circulating IL-18 levels of at least 5 ng/ml (data not shown) at 3 h, which corresponded to the circulating IL-18 concentration induced in mice treated with a nonlethal dose (75 µg) of LPS at that same time. Similarly, 500 IU of IL-1ß was shown to be protective against infection with E. coli Bort in C3H/HeJ mice (5). At 3 h after cytokine administration, the mice were infected with 3x LD50 (12,000 CFU of E. coli Bort/mouse) i.p. and then observed for mortality. Untreated cas-1+/- littermate mice were infected with the same dose of E. coli Bort as controls.
Cytokine measurements.
The concentrations of IFN- and IL-1ß in mouse serum were determined by enzyme-linked immunosorbent assays (ELISAs) with R4-6A2/XMG1.1 (IFN-; PharMingen) and PM425B/MM425B (IL-1ß; Endogen) antibody pairs as described previously (6). IL-18 was measured by using a mouse IL-18 detection ELISA kit (Medical and Biological Laboratories Co., Ltd., Nagoya, Japan) according to the manufacturer's instructions.
RT-PCR. The total RNA was prepared from liver and spleen cells of LPS-treated C3H/HeN mice by using the RNAstat-60 reagent (Tel-Test, Inc., Friendswood, Tex.) and was used to set up an reverse transcription-PCR (RT-PCR) by using GeneAMP RNA PCR kit (Perkin-Elmer, Norwalk, Conn.). The primers used were as follows: (i) for IL-18, forward (5'-ACT GTA CAA CCG CAG TAA TAC GG-3') and reverse (5'-AGT GAA CAT TAC AGA TTT ATC CC-3'), (ii) for cas-1, forward (5'-GAA GAG ATG TTA CAG AAG CC-3') and reverse (5'-CAT GCC TGA ATA ATG ATC AC-3'), and (iii) for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), forward (5'-TGA AGG TCG GTG TGA ACG GAT TTG GC-3') and reverse (5'-CAT GTA GGC CAT GAG GTC CAC CAC-3').
Cas-1 assay. Cas-1 activity was determined by a colorimetric assay using liver and spleen extracts, prepared according to the method of Thornberry (35), as the enzyme source. Briefly, the liver and spleen from LPS-treated mice were homogenized in a lysis buffer (25 mM HEPES [pH 7.5], 1 mM EDTA, 10 µg of aprotinin/ml, 10 µg of leupeptin/ml, 2 mM dithiothreitol) at 5 ml/100 mg of liver tissue and 2 ml/100 mg of spleen tissue. Extracts were centrifuged at 15,000 x g for 30 min at 4°C, and the supernatant was centrifuged again at 200,000 x g for 1 h at 4°C in a Beckman L8M ultracentrifuge. The cytosol was then dialyzed against homogenization buffer containing 10% sucrose (Sigma), 0.1% NP-40 (Sigma), and 2 mM dithiothreitol (Sigma) overnight at 4°C. The dialysate was used for Cas-1 activity measurements. Reactions with enzyme preparation alone, with enzyme mixed with Cas-1 substrate (Ac-YVAD-pNA) or inhibitor (Ac-YVAD-CHO), and with substrate alone were also run as controls. The total increase in the optical density at 405 nm (OD405) versus that of the enzyme-alone wells was calculated.
Western blot analysis of IL-18 and Cas-1 protein. Equal amounts of tissue protein were separated on a 7.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel. The proteins were transferred to an Immobilon-P membrane by using a semiblot transfer procedure and immunostained with polyclonal antibodies reactive to the mature and precursor forms of murine IL-18 and Cas-1 (Santa Cruz Biologicals, Inc., Santa Cruz, Calif.), according to the manufacturer's protocol. The blots were developed by using TMB membrane substrate (Kirkegaard & Perry Laboratories, Gaithersburg, Md.).
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RESULTS |
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12,000 CFU/mouse) and then observed for mortality. The administration of 500 U of IL-1ß completely protected most of the cas-1-/- mice from E. coli infection (one of seven died), whereas 250 U did not (data not shown). Similarly, treatment with 10 ng of IL-18 also protected cas-1-/- mice from E. coli infection (none of three died) (Table 2). Thus, the administration of both products of Cas-1, IL-1ß, or IL-18 alone protected mice against lethal bacterial infection.
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transiently, whereas LPS-induced lethality was associated with a sustained induction of high levels of these cytokines (Fig. 2). The differences between these nonlethal and lethal immune responses may provide insights into the immune dysregulation observed during lethal shock.
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stimulated by 75 and 500 µg of LPS was compared. No IFN- was detected for up to 3 h after LPS administration at either dose. At 8 h after LPS treatment, IFN- levels increased to 8.7 ± 1.8 ng/ml (75 µg of LPS) and 19.3 ± 5.6 ng/ml (500 µg). However, by 20 h, when lethally endotoxic mice were generally moribund, no IFN- was detected in mice treated with 75 µg of LPS, whereas 23.7 ± 2.7 ng of IFN-/ml was present in 500 µg of LPS-treated mice (P < 0.0001 by two-way analysis of variance [ANOVA] analysis) (Fig. 2A and D).
Since IFN- synthesis is induced by IL-18, we next measured the IL-18 levels in these mice. Similar changes in IL-18 levels were induced by nonlethal and lethal doses of LPS (Fig. 2B and E), as were seen with IFN-, suggesting that the IL-18 levels measured with this ELISA detected biologically active IL-18. IL-18 synthesis started soon after LPS treatment, increased up to 5.86 ± 0.91 ng/ml at 8 h and decreased to 1.7 ± 0.2 ng/ml by 24 h in mice treated with 75 µg of LPS (Fig. 2B). In contrast, in mice treated with 500 µg of LPS, IL-18 levels increased to 9.1 ± 1.6 ng/ml at 8 h and continued to increase until death (Fig. 2E). Serum from moribund mice (ca. 20 h post-LPS) contained 18.8 ± 1.2 ng of IL-18/ml (P < 0.0001 by two-way ANOVA analysis of IL-18 levels in mice treated with 75 and 500 µg of LPS for 24 h.).
Expression of bioactive IL-18 requires proteolytic cleavage of its precursor form by Cas-1, which also catalyzes proteolytic maturation of IL-1ß (13). Therefore, the IL-1ß levels in serum were also measured in these mice. Figure 1C shows that IL-1ß levels in serum increased after treatment with 75 µg of LPS to 0.8 ± 0.25 ng/ml at 3 h, decreased to 0.45 ± 0.09 ng/ml at 8 h, and returned to basal levels at 24 h. In contrast, in mice given lethal doses, IL-1ß levels in serum increased to 0.88 ± 0.07 ng/ml at 3 h and 1.53 ± 0.04 ng/ml at 8 h and persisted at that level till death (Fig. 2C and F). Thus, unlike the nonlethal dose, lethal doses of LPS induced a sustained increase of Cas-1 products, IL-1ß, and IL-18.
Expression of IL-18 protein but not mRNA is affected in LPS-treated mice. IL-18 is usually expressed as a precursor polypeptide present in the cytoplasmic pool, which, upon cleavage by Cas-1, becomes biologically active IL-18 (13). Increased transcription of the IL-18 gene may not be essential after nonlethal doses of LPS. However, the continuous synthesis of IL-18 triggered by lethal doses of LPS may demand a larger pool of IL-18 message. RT-PCR analysis of the total RNA prepared from liver and spleen tissue of mice treated with nonlethal and lethal doses of LPS revealed no significant differences in the expression of IL-18 mRNA in response to nonlethal or lethal doses of LPS (Fig. 3A and B). This is in agreement with the report that IL-18 mRNA is constitutively expressed and lacks the commonly observed RNA-destabilizing features of other cytokine mRNAs resulting in high constitutive levels of inactive pro-IL-18 precursor protein (7).
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Cas-1 activity differs in mice treated with nonlethal and lethal doses of LPS. Since LPS treatment did not increase the transcription of IL-18 gene (Fig. 3A and B) but induced different levels of IL-18 and its precursor protein in spleen (Fig. 3C), changes in Cas-1 activity may have been responsible for the differences in LPS-induced IL-18 expression. Cas-1 activity was therefore measured in spleen extracts prepared from LPS-treated mice. As shown in Fig. 4A, Cas-1 activity increased by 3 h after LPS treatment in the spleens of mice receiving both doses of LPS (75 and 500 µg). However, it decreased at 8 and 15 h after LPS treatment in the 75-µg group. In contrast, in mice treated with 500 µg of LPS, the Cas-1 activity increased by 3 h and remained elevated even at 8 and 15 h post-LPS (Fig. 4A). No significant differences in the Cas-1 activity were observed in the livers of mice treated with 75 and 500 µg of LPS (data not shown).
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In agreement with the mRNA levels, moderate levels of mature and precursor forms of Cas-1 protein were detected in spleen extracts from mice treated with 75 µg of LPS (Fig. 4D, lanes 3 to 8, two mice per condition). In contrast, severalfold-higher levels of both these forms of Cas-1 were detected by 3 h in the spleen extracts of mice treated with 500 µg of LPS (Fig. 4D, lanes 9 and 10). Compared to the 3-h levels, these levels decreased at later time points; high levels of Cas-1 and pro-Cas-1 proteins persisted in the spleens of mice treated with 500 µg of LPS for up to 15 h, particularly compared to the nonlethal dose of LPS (Fig. 4D, lanes 11 to 14). These differences in the patterns of cas-1 expression and activity could contribute to the observed differences in IL-18 (and, ultimately, IFN-) levels in serum and may be responsible for different outcomes after LPS treatment.
Cas-1 inhibitor protects against LPS lethality. To determine whether the increased level of Cas-1 expression plays a role in lethal endotoxemia, we treated mice with Cas-1 inhibitor 2 h before and after administration of lethal and nonlethal doses of LPS. Figure 5 shows that treatment with 75 µg of LPS alone did not cause death (none of three died) in C3H/HeN mice; however, treatment with 500 µg of LPS caused the death of C3H/HeN mice in less than 24 h (three of three died). However, treatment with 10 mg of Cas-1 inhibitor (Ac-YVAD-CHO)/kg at 2 h before and after LPS treatment, protected all mice from endotoxic death (none of three died; P < 0.0001). This shows that Cas-1, a critical enzyme in ABHD, is also of pivotal importance in the lethal immune responses to LPS.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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IL-18 was originally discovered as an IFN--stimulating factor and has been shown to play an important role in antibacterial defense (3, 39). Another cytokine, IL-1ß, also participates in murine antibacterial defenses (5) (20, 40). However, these cytokines have also been associated with endotoxin-induced liver injury and death (17, 28, 30, 33). Since their expression requires Cas-1-mediated maturation of their inactive precursor proteins, we hypothesized that understanding the expression of Cas-1 may help to explain the dual involvement of IL-18 and IL-1ß in host defense and lethal endotoxemia.
We observed that cas-1-/- mice were three- to fourfold more sensitive to bacterial infection than wild-type controls and treatment with Cas-1 inhibitor, Ac-YVAD-CHO rendered wild-type mice susceptible to infection. These observations differ from those of Sansonetti et al., who showed that Shigella spp. utilize host Cas-1 for invasion (34). Here we show that Cas-1 is essential for defense against infections with E. coli. Our findings are in contrast to the report that Cas-1 deficiency does not affect host defense against primary infections with Candida albicans (26). The differences in these observations may represent basic differences between immune mechanisms involved in host defense against infections with prokaryotic and eukaryotic microbes.
Although more susceptible to bacterial infection, cas-1-/- mice did not appear to be as susceptible to infection with E. coli as LPS-hyporesponsive mice such as C3H/HeJ, which can die after infection with as few as 10 bacteria (4). This may be due to the role of other LPS-induced, Cas-1-independent cytokines in antibacterial defense. For example, IL-12 confers protection against yersiniosis in BALB/c mice (2, 26). Alternatively, other caspases and proteases may mediate the synthesis of IL-1ß (1, 18) and IL-18 (1, 10, 14) in cas-1-/- mice and maintain basal antibacterial resistance (11, 26). In agreement with the previously reported involvement of IL-18 or IL-1ß in ABHD (3, 39), exogenous IL-18 and IL-1ß rendered cas-1-/- mice resistant to bacterial infection (Table 2).
Administration of excessive IL-18 to cas-1-/- mice, however, led to mortality after bacterial infection (data not shown). This was consistent with our earlier observation that the presence of insufficient or an excess amount (i.e., greater than an "optimal" level) of circulating IL-18 caused mortality after bacterial infection. Supra-optimal IL-18 levels induced cas-1 mRNA expression, thus initiating a positive regulatory loop, which led to the synthesis synthesis of even higher IL-18 levels (and mortality) after treatment with a high dose of LPS (17a). Although LPS presumably induced these levels by its effect on Cas-1 activity, we wanted to study the effect of Cas-1 upregulation on immune responses more directly.
We observed that the patterns of Cas-1 activity after the different doses of LPS paralleled that of IL-18 expression. Both lethal and nonlethal doses of LPS induced Cas-1 enzymatic activity at 3 h. However, Cas-1 activity decreased by 8 h after treatment with 75 µg of LPS, whereas it remained elevated for 15 h in animals given lethal doses. Thus, the ability to downregulate Cas-1 activity appears critical for a well-regulated response to LPS.
Cas-1 is usually expressed constitutively in an inactive (pro-Cas-1) form that requires proteolytic activation through Cas-11 (37). This posttranslational processing alone appeared responsible for increased Cas-1 activity in animals treated with 75 µg of LPS. In contrast, increased transcription, translation, and proteolytic activation of Cas-1 were all found to contribute to the sustained enhancement of Cas-1 expression and activity in mice treated with 500 µg of LPS.
The mechanism(s) responsible for this reduction in Cas-1 activity and consequently the levels of IL-1ß, IL-18, and IFN- in serum after treatment with nonlethal doses of LPS may have been overwhelmed by the lethal dose of LPS. The increased cas-1 mRNA and protein expression observed in the spleens of mice given lethal doses of LPS at 3 h (Fig. 3C and D) was not reflected in a proportional increase in splenic Cas-1 enzyme activity (Fig. 3A). This low enzyme activity despite high level of protein expression may be due to the presence of endogenous inhibitors of Cas-1 activity such as nitric oxide (19) or the serpin proteinase inhibitor 9 (42). If true, this may suggest that in mice challenged with a lethal dose of LPS, the endogenous Cas-1 inhibitor(s) is exhausted, resulting in a late, unopposed Cas-1 activity. We are investigating this possibility further.
Sustained activity and protein levels of Cas-1 may be critical for the development of LPS-induced lethality. We showed that high levels of IL-18 in serum obtained after a lethal dose of LPS induced Cas-1 expression, thus initiating a novel positive regulatory feedback loop and resulting in synthesis of even greater levels of circulating IL-18 (17a). Cas-1 activation can inhibit apoptosis of neutrophils (38), thereby promoting neutrophil mediated-tissue injury, a process that may be important in lethal endotoxemia (28). Excess Cas-1 protein may also cause inflammatory injury through the deleterious accumulation of proinflammatory cytokines (IL-18, IL-1ß, and consequently IFN-) (41). Indeed, Cas-1-deficient mice are resistant to LPS-induced shock (14, 21, 22)
In conclusion, we have shown that Cas-1 is a component of innate ABHD; however, excessive Cas-1 expression is accompanied by LPS lethality in the absence of infection. A better understanding of the mechanisms that regulate Cas-1 expression may provide early clues to the development of sepsis and suggest additional targets for therapeutic intervention.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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, without Cas-1 inhibitor; 
, with Cas-1 inhibitor.
) and 500 µg (
, P < 0.0001 (Fisher exact test).