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The Journal of Immunology, 2002, 169: 2536-2544.
Copyright © 2002 by The American Association of Immunologists

IL-18 Levels and the Outcome of Innate Immune Response to Lipopolysaccharide: Importance of a Positive Feedback Loop with Caspase-1 in IL-18 Expression1

Vishwas D. Joshi*, Dhananjaya V. Kalvakolanu{dagger},¶, Jeffrey D. Hasday{ddagger}, Richard J. Hebel§ and Alan S. Cross2,*,¶

Departments of * Medicine, Division of Infectious Diseases, {dagger} Microbiology and Immunology, {ddagger} Medicine, Division of Pulmonary and Critical Care Medicine, and § Epidemiology, and Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
LPS enhanced antibacterial host defenses (ABHD) when given at low (75 µg) doses (16 of 19 mice survived 3x LD50 Escherichia coli vs 3 of 19 LPS-naive mice; p = 0.0001), but induced lethal inflammation at high (500 µg) doses (5 of 5 died). Differences in the cytokine profiles induced by these LPS doses may provide insight into the mechanism(s) of transition from beneficial to lethal LPS responses. The 75 µg LPS induced 5.9 ± 0.9 ng/ml serum IL-18 at 8 h, which decreased to 2.3 ± 0.4 ng/ml by 24 h, whereas 500 µg LPS induced 11.1 ± 1.6 ng/ml serum IL-18 levels at 8 h, which increased until death. Compared with 75 µg, higher but sublethal (150 µg) doses of LPS induced greater serum IL-18 levels and less effectively induced ABHD (3 of 8 survived). Reduction of serum IL-18 with neutralizing Ab improved the ABHD induced by 150 µg, but reduced that produced by 75 µg LPS, suggesting an optimal range of serum IL-18 level was essential for efficient ABHD. Increased expression of caspase-1 mRNA in response to the higher IL-18 levels induced at the 150 and 500 µg, but not at the 75 µg doses of LPS may represent a positive feedback regulatory loop leading to sustained serum IL-18 levels. We conclude that the regulation of serum IL-18 expression is critical to the outcome of innate immune responses to LPS.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Gram-negative bacterial sepsis is a result of a poorly regulated inflammatory response to bacterial LPS, leading to release of proinflammatory cytokines such as TNF-{alpha}, IL-1{beta}, and IFN-{gamma} (1, 2, 3, 4). IFN-{gamma} plays an important role in the pathogenesis of sepsis (3), LPS-induced lethality (5, 6, 7), as well as antibacterial host defenses (ABHD)3 (8). As expected, cytokines such as IL-12 and IL-18 that stimulate IFN-{gamma} synthesis also play a central role in LPS-induced lethality (9, 10, 11) and ABHD (10, 12, 13, 14, 15).

IL-12 and IL-18 share several proinflammatory activities, including stimulation of IFN-{gamma} synthesis (16, 17); enhancement of NK cell cytotoxicity (18, 19, 20, 21, 22); induction of NO synthesis (23); and stimulation of IL-1{beta}, TNF-{alpha}, and IL-8 with subsequent local neutrophil accumulation (23, 24). Cooperation between IL-12 and IL-18 is essential for IFN-{gamma} synthesis. Up-regulation of the IL-18R by IL-12 is required for IL-18-induced IFN-{gamma} production (16, 25). In turn, IL-18-dependent up-regulation of IL-12R is also critical for Ag-dependent IFN-{gamma} synthesis by T cells (26). The simultaneous expression of IL-12 and IL-18 not only may be necessary for optimal physiologic responses to inflammatory stimuli, but also these cytokines may synergistically amplify inflammation and cause toxic effects. Consequently, a delicate regulatory mechanism(s) may be required to insure optimum cooperation between IL-12 and IL-18 while minimizing toxicity.

At different doses, bacterial endotoxin stimulates different immune responses in mice. At low doses (protective), LPS stimulates an enhanced innate immune response whereby mice are protected from increased levels of bacterial challenge (27). High doses of LPS induce lethal inflammatory reactions. IL-12 and IL-18 play important roles in such protective (10, 12, 13, 14) and lethal (11, 28) responses. Examination of the differences in the expression of IL-12 and IL-18 during these immune responses might provide insights into their relative role(s) in the outcomes of LPS treatment.

Caspase-1 (Cas-1), also known as IL-1-converting enzyme, is an intracellular enzyme that is essential for proteolytic maturation of IL-18 and IL-1{beta} (29, 30). Present in the cytoplasm as an inactive precursor, it must be activated itself before acting on the cytokine precursors. We have shown earlier that cas-1 expression increased significantly in mice treated with lethal, but not with a protective dose of LPS.4 IFN-{gamma} is known to induce cas-1 expression at the transcriptional and translational levels (31, 32) through NF-{kappa}B activation (33) and IFN regulatory factor-1 mobilization (32, 34). We speculate that factor(s) such as IL-18, which induces IFN-{gamma} synthesis (35) and activates NF-{kappa}B (36), may also affect cas-1 expression.

In this study, we show that protective and lethal doses of LPS stimulate similar patterns of serum IL-12 (p70) expression, but distinct profiles of serum IL-18 levels. The circulating level of IL-18 influences the outcome of bacterial infection and LPS lethality. The presence of an optimal, but reversible (i.e., regulated) increase in IL-18 level is essential for protection from bacterial infections in LPS-treated mice. We speculate that high levels of LPS-induced IL-18 may stimulate expression of cas-1, resulting in the progressive synthesis of IL-18 until death.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

LPS from Escherichia coli O111:B4 (lot 60K4078; Sigma-Aldrich, St. Louis, MO) was suspended in normal saline (NS) and sonicated at 30°C for 15 min before use. Murine rIL-18 and anti-IL-18 mAb (clone 93-10C) were purchased from R&D Systems (Minneapolis, MN). Cas-1 substrate (Ac-YVAD-pNA) and inhibitor (Ac-YVAD-CHO) were purchased from Alexis Biochemicals (San Diego, CA).

Mice

Seven- to 12-wk-old female mice (C3H/HeN (National Cancer Institute, Frederick MD), C57BL/6J (The Jackson Laboratory, Bar Harbor, ME), and CD1/ICR (Charles River Laboratories, Wilmington, MA)) were used in these studies. Mice were given water and food ad libitum and housed under a 12-h day/light cycle with controlled humidity and temperature.

Serum prepared from freshly collected heart blood from mice treated i.p. with different doses of LPS in saline was used for determination of cytokine levels.

For protection experiments, the lethality of E. coli O18:K1:H7 was determined from the percentage of mortality induced by different doses of exponentially growing E. coli injected i.p., in naive C3H/HeN and C57BL/6J mice in 0.1 ml saline. The LD50 of E. coli in these two mouse species was found to be 12,000 CFU/mouse for C3H/HeN and 8,000 CFU/mouse for C57BL/6J. Three times this dose (3x LD50) for each strain of mouse was used to infect LPS-treated mice in protection experiments. To study the effect of anti-IL-18 Ab, 1 µg anti-IL-18 mAb was diluted in normal saline and administered i.p., 2 h before treatment with LPS. Serum or tissue samples were harvested at specified time points after LPS treatment and used for cytokine or cas-1 enzyme activity determinations. For studying the effect of LPS or anti-IL-18 Ab on ABHD, mice were infected 48 h after LPS treatment.

Cytokine measurements

Serum IFN-{gamma} and IL-1{beta} concentrations were determined by ELISA using R4-6A2/XMG1.1 (BD PharMingen, San Diego, CA; IFN-{gamma}) and PM425B/MM 425B (Endogen, Woburn, MA; IL-1{beta}) Ab pairs, as described (37). IL-18 was measured using a mouse IL-18 detection ELISA kit (Medical and Biological Laboratories, Naka-ku Nagoya, Japan), according to the manufacturer’s instructions, while ELISA for IL-12 (p70) was performed using Duoset system (R&D Systems), according to the manufacturer’s instructions.

Cas-1 activity

Cas-1 activity was determined according to the method of Thornberry (38) using the colorimetric assay with murine liver and spleen extracts as the enzyme source. Briefly, the liver and spleen from LPS-treated mice were homogenized in lysis buffer (25 mM HEPES, pH 7.5, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 2 mM dithiothrietol) at 5 ml/100 mg liver tissue and 2 ml/spleen. 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 L8 M ultracentrifuge (Beckman Coulter, Fullerton, CA). Cytosol was then dialyzed against homogenization buffer containing 10% sucrose (Sigma-Aldrich), 0.1% Nonidet P-40 (Sigma-Aldrich), and 2 mM DTT (Sigma-Aldrich) overnight at 4°C. The dialysate was used for cas-1 activity measurements. Reactions with enzyme preparation alone, enzyme mixed with cas-1 substrate (Ac-YVAD-pNA) or inhibitor (Ac-YVAD-CHO), and substrate alone were also run as controls. The total increase in OD405 over enzyme-alone wells was determined. The cas-1 activity was determined in terms of arbitrary units, defined as the maximum OD405 obtained with 100-µl tissue extracts divided by total protein in the same amount of sample and multiplied by 10,000.

Cas-1 mRNA levels

Total cellular RNA was prepared using the RNAstat-60 reagent (Tel-Test, Friendswood, TX) from 1) RAW 264 cells, which were treated with different concentrations of IL-18 for 24 h, and 2) spleens of C3H/HeN mice, which were treated with 75, 150, and 500 µg LPS for 3 h. Two micrograms of RNA were used to set up an RT-PCR using GeneAMP RT-PCR kit (PerkinElmer, Norwalk, CT). The primers used were 1) cas-1, forward, 5'-GAA GAG ATG TTA CAG AAG CC-3'; reverse, 5'-CAT GCC TGA ATA ATG ATC AC-3'; 2) GAPDH, forward, 5'-TGA AGG TCG GTG TGA ACG GAT TTG GC-3'; reverse, 5'-CAT GTA GGC CAT GAG GTC CAC CAC-3'. The PCR were run as follows: 1) Cas-1, 94°C for 30 s, 56°C for 30 s, and 72°C for 2 min, 30 cycles; 2) GAPDH, 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, 35 cycles.

Bacteria

For all live infection experiments, E. coli O18:K1:H7, strain Bort, originally isolated from the cerebrospinal fluid of a neonate and used in the murine peritonitis model (39), was used as the challenge organism. A single colony of E. coli, grown overnight on tryptic soy agar (TSA) was inoculated into tryptic soy broth and grown to log phase. Bacteria were washed, resuspended in PBS to an approximate concentration of 1 x 108 CFU/ml. This cell suspension was diluted further in PBS to the desired dose of bacteria for administration to mice i.p.

Clearance assay

Outbred mice were pretreated with saline or 5 ng IL-18 in saline (5/group) 2 h before challenge with 1 x 107 CFU (1x LD50 i.v.) E. coli into the lateral tail vein in 0.1 ml vol. The rate of removal of E. coli from the bloodstream was determined by bleeding sequentially by tail snips at 1, 20, and 60 min after i.v. injection of E. coli. A total of 10 µl of blood was collected in heparinized capillary tubes, plated directly on TSA, or diluted in PBS to determine the CFU/ml (18, 19). The 1-min sample was defined as zero time.

Statistical analysis

The p values for the comparisons of IL-18 levels were derived from the two-way ANOVA, and those for survival rates were determined by Fisher’s exact test. The significance of the cas-1 mRNA levels was studied using the unpaired t test utilizing GraphPad Prizm software.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
LPS induces protective and lethal immune responses

Gram-negative bacterial sepsis is considered to be a result of a poorly regulated inflammatory response to bacterial LPS (1, 2, 3, 4). Relatively low doses of LPS induce a controlled inflammatory response and enhance ABHD of mice (27). Protective and lethal doses of LPS were determined for C3H/HeN and C57BL/6J mice (Table IGo). In C3H/HeN mice, 75 µg LPS enhanced host defenses against lethal bacterial infection (i.e., protective effect against 3x LD50 E. coli). At that dose of LPS pretreatment, 3 of 19 (16%) mice died vs 16 of 19 (84%) without LPS pretreatment (p < 0.0001 vs untreated). Similarly, in C57BL/6J mice, pretreatment with 25 µg LPS enhanced ABHD such that only 1 of 12 LPS-treated mice compared with 17 of 19 saline-treated mice died following lethal (3x LD50) infection with E. coli (p < 0.0001 vs untreated). In contrast, 500 µg LPS was uniformly lethal to both C3H/HeN and C57BL/6J mice before any bacterial infection. Thus, LPS stimulated both protective and lethal immune responses in mice. Understanding the differences in the IL-18 response to these two doses of LPS might provide clues regarding regulatory mechanisms central to the pathogenesis of sepsis.


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  		Table I. Protective and lethal doses of LPSa

 
Differential synthesis of IL-18 in mice receiving protective and lethal doses of LPS

IFN-{gamma} has an important role in host resistance response to LPS (3, 5, 6, 7). LPS-naive C3H/HeN mice were treated i.p., with 75 or 500 µg LPS. Serum was harvested at different time points and tested for the presence of this cytokine. In mice receiving the protective (75 µg) LPS dose, serum IFN-{gamma} increased to 8.7 ± 1.8 ng/ml at 8 h and returned to near basal levels by 15 h (Fig. 1GoA). By contrast, in mice receiving a lethal dose (500 µg) of LPS, serum IFN-{gamma} levels increased to 28.2 ± 1.8 ng/ml by 8 h and remained elevated at 23.7 ± 2.7 ng/ml until death at 20 h (p < 0.0001 by two-way ANOVA analysis between 75 vs 500 µg over 24 h).



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  		FIGURE 1. Serum cytokine levels in LPS-treated mice. C3H/HeN mice were treated with protective (75 µg; {blacktriangleup}) and lethal (500 µg; {blacksquare}) LPS, and cytokine levels in serum prepared from blood drawn at different time points after LPS treatment were measured using commercial ELISA reagents. IFN-{gamma} (A) and IL-18 during the first 6 h after LPS (B); IL-18 (C) and IL-12 (p70) (D). Data represent mean ± SE of three to five mice per dose per time point.

 
This difference in IFN-{gamma} expression could have been due to differences in the expression of IFN-{gamma}-stimulating cytokines, IL-12 and IL-18. We therefore tested the serum levels of the two cytokines in mice treated with these doses of LPS.

Serum IL-18 levels increased within 1 h after administration of either dose of LPS (Fig. 1GoB). After administration of 75 µg LPS, IL-18 levels increased to 5.9 ± 0.9 ng/ml by 8 h and decreased to 1.7 ± 0.2 ng/ml by 24 h (Fig. 1GoC). In mice receiving 500 µg LPS, serum IL-18 levels increased to 11.1 ± 1.6 ng/ml at 8 h and continued increasing to 18.8 ± 1.2 ng/ml until the death of the mice, usually around 20 h post-LPS (p = 0.025 and 0.002 at 8 and 24 h for 75 vs 500 µg, respectively). Changes in IL-18 levels preceded, but mirrored the changes in serum IFN-{gamma} levels, reflecting the possible involvement of IL-18 in stimulating IFN-{gamma} synthesis in LPS-challenged mice (Fig. 1Go, A and B).

In marked contrast to the differences in LPS-induced IL-18 expression, serum IL-12 (p70) increased until 6 h and then decreased to near basal levels by 15 h after treatment with both doses of LPS (Fig. 1GoD). However, at all time points, the lethal dose of LPS stimulated greater levels of IL-12 (p70) than the protective dose. IL-12 was detected in the serum 2 h after LPS treatment and increased to 390 ± 277 pg/ml and 928 ± 235 pg/ml at 6 h after treatment with 75 and 500 µg LPS, respectively. The IL-12 levels returned to basal levels by 15 h after LPS administration, in both treatment groups (Fig. 1GoD). The levels of the p40 subunit of IL-12, however, increased to 3553 ± 1190 pg/ml at 3 h and remained elevated at 15 h after treatment with 500 µg LPS (Fig. 1GoD inset). By contrast, in 75 µg LPS-treated mice, the serum p40 subunit concentrations increased to 1822 ± 360 pg/ml at 3 h, but decreased to 621 ± 190 pg/ml by 15 h after LPS administration (p = NS, n = 2/each time point).

Different serum IL-18 levels are associated with differences in the efficacy of LPS-enhanced ABHD

These data suggested that a dose of LPS that stimulated reversible expression of IFN-{gamma} and IL-18 expression enhanced ABHD, while that which stimulated a progressive, irreversible increase in IFN-{gamma} and IL-18 levels induced lethal inflammation. Because changes in LPS-induced IL-18 levels preceded changes in IFN-{gamma} concentrations (Fig. 1Go, A vs B), and IL-18 is known to be a major stimulatory of IFN-{gamma}, we focused on the regulation of IL-18 levels as an important determinant of outcome after exposure to LPS. To confirm the hypothesis that the optimal enhancement of ABHD requires the up-regulation of serum IL-18 to an optimal range, serum IL-18 levels were monitored in two different strains of mice treated with protective, lethal, and intermediate doses of LPS. Protective doses of LPS varied with the two different mouse strains examined (75 µg in C3H/HeN and 25 µg in C57BL/6J mice). Eight hours after treatment with this dose of LPS, 5855 ± 906 and 3268 ± 154 pg/ml IL-18 were detected in serum of C3H/HeN and C57BL/6J mice, respectively, which decreased to 2551 ± 466 pg/ml and 3030 ± 248 pg/ml, respectively, by 24 h (Table IIGo). Furthermore, pretreatment with such a dose of LPS enhanced the ABHD from subsequent lethal bacterial infection (p < 0.0001 for both mouse strains vs no LPS treatment).


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  		Table II. IL-18 levels correlate with LPS dose and outcome of infectiona

 
Higher, but sublethal doses of LPS induced the expression of higher IL-18 levels. In C3H/HeN mice, 150 µg LPS stimulated 7705 ± 3085 pg/ml IL-18 at 8 h, which decreased to 3674 ± 462 pg/ml by 24 h. In contrast, in C57BL/6J mice, the 4968 ± 664 pg/ml IL-18 stimulated by 75 µg LPS was maintained at that level for 24 h. Although they induced more IL-18, these intermediate doses of LPS did not cause any visible toxicity. However, they were less effective in stimulating the ABHD. Only 3 of 8 (37%) C3H/HeN and 9 of 19 (47%) C57BL/6J mice treated with these doses of LPS survived lethal infection E. coli (p = NS vs untreated control). In contrast, 500 µg LPS induced the greatest levels of IL-18 and was uniformly lethal to both mouse strains before any bacteria could be administered (Table IIGo).

It therefore appeared that the level of LPS-induced serum IL-18 corresponded to the enhancement of ABHD, and stimulation of IL-18 beyond an optimal level was associated with reduced survival following bacterial infection.

Optimal IL-18 levels are essential for protective antibacterial response

To more directly assess the role of IL-18 in ABHD, we administered neutralizing anti-IL-18 mAb 2 h before treatment with optimal and intermediate doses of LPS (Table IIIGo). In both C3H/HeN and C57BL/6J mice, administration of 1 µg IL-18 mAb before LPS treatment reduced IL-18 levels at 15 h; however, there was no further reduction in IL-18 levels by increasing the dose of anti-IL-18 Ab to 10 µg (data not shown). Therefore, in all additional experiments, 1 µg anti-IL-18 mAb/mouse was used.


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  		Table III. Effect of anti-IL-18 Abs on LPS-induced ABHDa

 
If an optimal level of serum IL-18 was important to the LPS-enhanced ABHD, reducing IL-18 activity by anti-IL-18 Ab might decrease the survival observed after treatment with protective dose of LPS, but improve the survival following treatment with the intermediate dose of LPS. The 75 µg dose of LPS induced accumulation of 2551 ± 466 pg/ml IL-18 in C3H/HeN mice serum and protected 84% (16 of 19) mice from subsequent infection with 3x LD50 E. coli (p < 0.0001 vs untreated). Administration of neutralizing anti-IL-18 mAb (1 µg/mouse) before LPS treatment of C3H/HeN mice reduced circulating IL-18 levels to 1561 ± 83 pg/ml, but increased the mortality following E. coli infection from 16 to 39% (5 of 13; p = 0.021 vs no Ab treatment). In contrast, anti-IL-18 mAb treatment of the same mice treated with an intermediate dose of LPS had an opposite effect. By itself, the intermediate dose (150 µg) of LPS did not cause any visible toxicity in C3H/HeN mice and stimulated the accumulation of circulating IL-18 to 3674 ± 462 pg/ml by 24 h. However, infection of these mice with E. coli resulted in a 63% (5 of 8) mortality (p = NS vs untreated mice). Administration of anti-IL-18 Ab before 150 µg LPS treatment reduced the serum IL-18 levels to 2112 ± 319 pg/ml (i.e., approximately the levels induced by 75 µg LPS) and also reduced the mortality after E. coli infection to 20% (p = 0.032 vs no Ab treatment). A similar effect of anti-IL-18 Abs was also observed in C57BL/6J mice treated with protective (25 µg) (p = NS, vs no Ab treatment) and intermediate (75 µg) (p = 0.005 vs no Ab treatment) LPS. Thus, decreasing the circulating IL-18 levels below the levels induced by protective dose LPS compromised the LPS-enhanced antibacterial resistance, while reducing the excess serum IL-18 stimulated by the intermediate LPS dose-enhanced ABHD. These results suggest that an optimal range of serum IL-18 is essential for maximal ABHD (Fig. 2Go).



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  		FIGURE 2. Optimum IL-18 levels are necessary for LPS-induced protection against bacterial infection in C3H/HeN and C57BL/6J mice. The percentage of survival of C3H/HeN (A) and C57BL/6J mice (B) was plotted against serum IL-18 levels from Tables IIGo and IIIGo. Serum IL-18 levels were dependent upon the LPS dose, which is depicted on the upper x-axis so that IL-18 levels correlate with the corresponding stimulating dose of LPS.

 
Effect of IL-18 on ABHD

If IL-18 played an important role in the outcome of ABHD, pretreatment with exogenous IL-18 before lethal E. coli infection would enhance ABHD in a manner similar to LPS. First, we ascertained that i.p. administered rIL-18 was rapidly absorbed circulation within 2 h of treatment (data not shown).

C3H/HeN mice were then pretreated with IL-18 doses corresponding to the levels stimulated by various doses of LPS, and their survival following infection with 3x LD50 dose of E. coli (30,000 CFU/mouse) was studied. IL-18 alone was nontoxic up to a dose of 50 ng; however, different doses of IL-18 affected ABHD differently. Administration of 2.5, 5, and 10 ng IL-18 increased the ABHD and protected 2 of 8 (25%), 4 of 11 (36%), and 3 of 11 (27%) naive C3H/HeN mice from subsequent lethal infection, respectively. However, mice pretreated with 25 or 50 ng IL-18 were not protected from subsequent lethal infection (Fig. 3GoA). Thus, pretreatment with IL-18 in the absence of LPS stimulation exerted a direct protective effect against lethal infection.



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  		FIGURE 3. Effect of IL-18 on bacterial clearance and ABHD. LPS-naive C3H/HeN mice were treated with IL-18 (at least 5/group) and infected 2 h later with 3x LD50 E. coli i.p. and observed for mortality. The results were compared with saline-treated control using Fisher’s exact test; p = 0.037 (A). CD1/ICR mice were treated with saline or 5 ng/mouse IL-18 1 h before infection with 1x LD50 E. coli i.v., and bacterial load was determined at 0, 20, and 60 min postinfection. Assuming 0-min bacterial load as 100% (2.7 ± 0.7 x 106 CFU/ml), the percentage of bacterial load at 20- and 60-min time points was calculated (n = 5/group; p = 0.02 by two-way ANOVA analysis) (B). Spleen (open bars) and liver (filled bars), isolated 24 h after infection of IL-18-pretreated CD1/ICR mice with 1 x 107 CFU E. coli (i.v.), were homogenized and used for determination of total bacterial load by dilution plating on TSA plates (n = 3); *, p = 0.01 for 5 ng vs saline treatment; **, p < 0.05 for 5 ng vs other dose group using unpaired t test (C).

 
IL-18 enhanced bacterial clearance

To determine the possible mechanism by which IL-18 enhanced ABHD, the effect of IL-18 pretreatment on bacterial clearance was studied (Fig. 3GoB). Outbred mice were treated with saline or 5 ng/ml IL-18 in saline i.p. and, 1 h later, were infected with 1 x 107 CFU E. coli i.v. The number of bacteria was determined in the blood collected from the lateral tail vein at 0, 20, and 60 min postinfection. Immediately after infection (0 min), 2.7 ± 0.7 x 106 CFU/ml (100%) E. coli was detected. This decreased to 85 ± 4.5% of initial inoculum at 20 min and 70 ± 9% of initial inoculum at 60 min in saline-treated mice. In contrast, in 5 ng IL-18-treated mice, the circulating bacteria decreased to 62.5 ± 11% at 20 min and 7.7 ± 2% of initial inoculum at 60 min postinfection (p = 0.02 by two-way ANOVA) (Fig. 3GoB). Mice treated with 25 ng IL-18 were equally effective at clearing bacteria from circulation (data not shown). Thus, IL-18 promotes the clearance of bacteria from the circulation.

Measurement of organ bacterial load at 24 h, however, showed important differences in IL-18-treated mice (Fig. 3GoC). Saline-treated mice contained 8.1 x 109 and 2.8 x 109 CFU E. coli/100 mg spleen and liver tissue, respectively, whereas 5 ng IL-18-treated mice showed evidence of almost complete elimination of infecting bacteria by 24 h. Two of three mice contained no bacteria in the spleen or liver, while the third contained 1.1 x 105 and 3.9 x 105 CFU E. coli/100 mg spleen and liver tissue, respectively. By contrast, consistently greater numbers of bacteria were detected in the spleen and liver of mice pretreated with 10, 25, and 50 ng IL-18. Therefore, although higher dose IL-18 enhanced bacterial uptake as effectively as 5 ng IL-18, it was associated with impairment of bactericidal activity.

Potential role of cas-1 and serum IL-18 expression in response to different doses of LPS

The activation of existing intracellular pools of cas-1 in turn cleaves the inactive forms of IL-1{beta} and IL-18 to their active forms. We previously showed that: 1) LPS induced cas-1 activity, and 2) lethal, but not protective, doses of LPS stimulated the expression of cas-1 mRNA and protein in the spleen, leading to a sustained cas-1 activity.4 We hypothesized that the induction of cas-1 mRNA may be associated with the sustained levels of serum IL-18. We therefore examined whether intermediate doses of LPS that also stimulated sustained IL-18 levels also induced cas-1 mRNA. In agreement with the observations of Lin et al. (40), splenic cas-1 mRNA levels were unaffected in mice treated with protective dose LPS compared with those treated with normal saline (vehicle). However, our observation of increased splenic cas-1 mRNA levels in mice treated with intermediate and lethal doses of LPS suggests cas-1 induction as a possible mechanism contributing to the sustained serum IL-18 levels (Fig. 4Go).



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  		FIGURE 4. Effect of LPS on caspase-1 expression in LPS-treated mice. Total RNA was prepared from spleens at 3 h after treatment of C3H/HeN mice (n = 3/group) with protective (75 µg), intermediate (150 µg), and lethal doses (500 µg) of LPS, and the relative abundance of cas-1 mRNA was measured by RT-PCR analysis. The cas-1 mRNA levels were normalized with GAPDH levels, and the ratio of cas-1 to GAPDH signals was plotted. *, p = 0.048 for 150 µg, and **, p = 0.007 for 500 µg vs no LPS treatment in each case.

 
IL-18-enhanced LPS induced expression of cas-1 mRNA

Recently, IFN-{gamma} was shown to induce expression of cas-1 protein and mRNA through NF-{kappa}B and IFN regulatory factor-mediated transcription (31, 32, 33, 34). Because IL-18 induces IFN-{gamma} and also is known to activate the transcription factor NF-{kappa}B (35, 36), the high serum IL-18 levels observed in mice treated with intermediate or lethal doses of LPS may be expected to stimulate expression of cas-1. If this were to occur, it might establish a positive feedback regulatory loop by which high levels of IL-18 promote the induction of cas-1 mRNA and the further production of IL-18.

The stimulatory effect of IL-18 on expression of cas-1 mRNA was confirmed in a murine macrophage-like cell line (RAW 264.7) that was earlier shown to produce no IFN-{gamma} (data not shown). Stimulation of RAW cells with different concentrations of IL-18 for 24 h induced cas-1 mRNA expression, as detected by RT-PCR (Fig. 5Go). At 100 ng/ml IL-18, the relative cas-1 mRNA accumulation increased to 1.68 ± 0.19 from 1.16 ± 0.12 in unstimulated cells (p = 0.047). Thus, increasing levels of LPS-induced IL-18 may induce further its own synthesis through increasing cas-1 mRNA expression.



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  		FIGURE 5. Effect of IL-18 on expression of cas-1 mRNA in RAW 264.7 cells. Total cellular RNA prepared from RAW 264.7 cells (0.5 x 106 cells/well) stimulated with medium alone (M), or different concentrations of IL-18 (1, 10, and 100 ng/ml) for 24 h, was subjected to RT-PCR analysis to determine the relative abundance of cas-1 mRNA. The cas-1 mRNA levels were normalized with GAPDH levels, and the ratio of cas-1 to GAPDH signals was plotted. *, p = 0.047 between 100 ng/ml IL-18- and medium alone-stimulated cells.

 
IL-18 levels and cas-1 activity are interdependent

If high doses of LPS stimulate increased serum levels of IL-18 that in turn induce cas-1 mRNA expression, then a reduction in either the serum IL-18 level or cas-1 activity should break this positive feedback loop. Neutralizing IL-18 Abs and cas-1 inhibitors are known to protect animals from lethal inflammation (11, 41, 42). In addition, the cas-1-inducing activity (Figs. 4Go and 5Go) of IL-18 and the ability of anti-IL-18 mAbs to protect animals from lethal bacterial infection (Table IIIGo) suggested that anti-IL-18 Abs may act through modulation of cas-1 expression and activity. Therefore, C3H/HeN mice were treated with 75 µg LPS in the presence and absence of anti-IL-18 Ab, and the cas-1 activity in liver and spleen was determined at 15 h after LPS treatment. LPS induced cas-1 activity in the spleen by almost 2-fold (to 17.01 arbitrary U/mg from 8 U/mg) and nearly 6-fold in the liver (38 arbitrary U/mg from 6 U/mg) in the absence of anti-IL-18 mAb. Reducing LPS-induced serum IL-18 levels by pretreatment with IL-18 mAb restricted the cas-1 activity in liver (15.8 U/mg) and in the spleen to nearly basal levels (7 arbitrary U/mg) (Fig. 6GoA). A control well using the same tissue extracts in the presence of the cas-1 inhibitor (Ac-YVAD-CHO) showed no cas-1 activity (data not shown). In another experiment, C3H/HeN mice were treated with 500 µg LPS in presence or absence of the cas-1 inhibitor, Ac-YVAD-CHO. Cas-1 inhibitor treatment reduced LPS-induced serum IL-18 to 4.1 ± 0.7 ng/ml compared with 18.8 ± 2.1 ng/ml in saline-treated mice (Fig. 6GoB). Thus, IL-18 levels and cas-1 activity are interdependent, and inhibition of any one reduces the other, possibly by interrupting a positive feedback mechanism involving IL-18 and cas-1.



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  		FIGURE 6. Effect of anti-IL-18 Ab on cas-1 activity in LPS-treated mice. C3H/HeN mice (3/group) were treated with 1 µg anti-IL-18 Ab and 2 h later with 75 µg LPS i.p. Extracts were prepared from the spleen (open bars) and liver (filled bars) tissue isolated from these mice 15 h after LPS treatment and evaluated for cas-1 activity using the colorimetric method of Thornberry (38 ) (A). C3H/HeN mice were treated with 10 mg/kg Ac-YVAD-CHO (filled bars) or saline (open bars) at -1 h, followed by challenge with 500 µg LPS at 0 h and again with 10 mg/kg Ac-YVAD-CHO or saline at +3 h. Serum IL-18 was determined at 6 and 24 h post-LPS (20 h in case of saline-treated control), using ELISA (n = 3) (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study, we show that different doses of LPS stimulate different profiles of serum IL-18 expression in mice. Doses of LPS that induced enhanced ABHD stimulated a lower peak increase in serum IL-18 levels, whereas higher LPS doses caused greater and sustained increases in serum IL-18 levels, but poor enhancement of ABHD. In mice treated with lethal quantities of LPS, IL-18 expression continued unabated until the death of the animals. In contrast, serum IL-12 (p70) profile differed minimally in response to various doses of LPS. An optimal level of IL-18 was essential for maximal enhancement of ABHD. At higher circulating levels, IL-18 initiated a positive feedback loop by stimulating the expression of cas-1. Neutralizing IL-18 by Abs as well as cas-1 inhibition by specific inhibitor (Ac-YVAD-CHO) reduced LPS-inducedserum IL-18. This suggests the important role of IL-18 and cas-1 in maintaining the sustained, high serum IL-18 levels during lethal endotoxemia.

LPS is an important bacterial product that stimulates innate immunity. Small quantities of LPS stimulate a well-regulated inflammatory response comprised of an orderly and sequential expression of various inflammatory mediators and enhance ABHD without inducing LPS hyporeactivity (tolerance). However, this highly integrated response became dysregulated at higher doses of LPS and was first manifest in our studies as loss of enhanced innate immunity and later as death. We hypothesized that a better understanding of how different doses of this same microbial component can lead to such disparate responses may provide a clue as to which regulatory mechanism(s) becomes altered during the development of sepsis.

We initially observed that in mice treated with a protective dose of LPS, the levels of IFN-{gamma} were up-regulated reversibly (Fig. 1GoA), whereas in mice treated with a lethal dose of LPS, the levels of IFN-{gamma} continued increasing until the death of the animal. We asked whether these differences may have resulted from a variation in the expression of the major IFN-{gamma}-stimulating cytokines, IL-12 and/or IL-18.

On the basis of differing IL-18 profiles, the LPS treatment doses could be classified into three groups: protective, intermediate, and lethal. The optimal dose of LPS that enhanced antibacterial immunity was the smallest dose of LPS that rendered animals resistant to more lethal (3x LD50) bacterial infection and stimulated a reversible expression of IL-18. It varied with mouse strain (75 µg/mouse in C3H/HeN and 25 µg/mouse in C57BL/6J mice). Serum IL-18 levels became detectable at 2 h following the protective LPS treatment, peaked by 8 h, and decreased to near basal levels by 24 h. Intermediate levels of LPS induced a higher and sustained elevation of IL-18, but were not lethal. In contrast, a lethal dose of LPS induced still higher and continuously increasing levels of serum IL-18 and was by itself lethal.

An optimal level of IL-18 was essential for the protective effect of LPS, which may enhance host defenses by any one of the various mechanisms, including stimulation of IFN-{gamma} synthesis or enhancement of NK cell activity. The optimum IL-18 levels were very similar in the two species of mice tested (2.6–3.03 ng/ml in C57BL/6J and 2.3–2.9 ng/ml in C3H/HeN mice), even though the protective doses of LPS differed in these species of mice. Reducing IL-18 levels below these optimal levels by neutralizing mAb compromised the LPS-induced enhancement of host defenses. Intermediate doses of LPS (150 µg in C3H/HeN and 75 µg in C57BL/6J) induced a higher and sustained increase in serum IL-18 levels, but stimulated poorer antibacterial resistance compared with protective dose LPS. Pretreatment of these mice with neutralizing anti-IL-18 mAb enhanced the protective effect of this less protective intermediate LPS dose (Table IIGo). Thus, a strong correlation was observed between the level of LPS-induced IL-18 and LPS-induced enhancement of ABHD in mice. The association of a narrow range of IL-18 with enhanced ABHD emphasizes the highly regulated nature of this innate immune response to LPS and also that the immune dysregulation underlying lethal inflammation may be initiated well before lethal levels of LPS are achieved. The ABHD-enhancing effect of IL-18 at 10 ng, but not at higher or lower doses, suggests its importance in the differential enhancement of ABHD by LPS. This may further be supported by the previously reported association of high IL-18 levels with 1) IFN-{gamma}-mediated lethal liver injury induced in heat-killed Propionibacterium acnes-challenged cas-1 transgenic mice (43); 2) LPS-induced lethality in P. acnes-primed mice (35, 44); 3) lethal endotoxemia (11, 28); and 4) the excessive inflammation observed during acute experimental shigellosis (45). However, our studies do not rule out other mechanism(s) involved in LPS-induced ABHD.

Multiple mechanisms may be responsible for the lethality observed at supraoptimal levels of IL-18. Cytokine-mediated dysregulation may account for the lethality. Our studies show cas-1 mRNA up-regulation in presence of intermediate LPS (animals) and IL-18 (RAW cells). Up-regulation of cas-1 expression by Shigella and Salmonella sp. is known to induce macrophage apoptosis (46). Similar activation of apoptosis by cas-1-inducing doses of LPS or IL-18 may allow bacteria to multiply unchecked. Alternatively, induction of LPS tolerance/hyporeactivity at higher doses, but not protective dose, could debilitate the immune defenses of mice and allow uncontrolled replication of bacteria and death. Indeed, although mice treated with 5 or 25 ng IL-18 showed equally enhanced capacity of bacterial clearance at 60 min (Fig. 3GoB), more bacterial regrowth was observed in mice treated with higher IL-18 doses, but not 5 ng IL-18, suggesting impairment of bactericidal mechanisms at the higher doses. Given our previous finding of impaired bactericidal activity following seemingly small changes in cytokine balance (47), it is attractive to speculate that the inflammatory network induced by LPS requires precise regulation such that changes in any one component may compromise the ability to defend against bacterial infection. We are currently studying the mechanism by which high doses of IL-18 impair the bactericidal activity.

Like IFN-{gamma}, high levels of IL-18 stimulated the expression of cas-1 mRNA in RAW cells (which do not make IFN-{gamma}) in vitro. A similar mechanism may not be activated at physiologic levels of IL-18 induced by protective doses of LPS. Consistent with this notion, cas-1 mRNA synthesis was increased within 3 h of LPS treatment in spleen of intermediate and lethal, but not protective dose LPS-treated mice. The pivotal role of IL-18 in regulating this cas-1 activity following LPS treatment in vivo was confirmed when treatment with neutralizing IL-18 mAb as well as cas-1 inhibitor Ac-YVAD-CHO reduced LPS-induced cas-1 expression.

Together, these data suggest the presence of a positive feedback mechanism that may explain the different patterns of IL-18 expression. At lower doses of LPS, active IL-18 is generated through the activity of pre-existing levels of cas-1, and no cas-1 mRNA expression is detected. Once these pools are reduced, little further pro-IL-18 is processed, and no cas1 mRNA expression is detectable. In contrast, a feedback mechanism may be established at higher (i.e., intermediate and lethal) doses of LPS, which leads to increased processing of IL-18 from its inactive form by the induction of cas-1 mRNA, which may replace exhausted preformed intracellular pools. The prolonged presence of circulating LPS at these higher doses may also act in synergism with IL-18 to induce cas-1 mRNA expression.

Due to its involvement in maturation of IL-1{beta} and IL-18, cas-1 is recognized as an important regulator of inflammatory reactions. Anti-inflammatory therapies directed at cas-1 inhibition have been attempted. However, cas-1-directed therapies have found limited success. Extraordinarily high levels of specificity are required to target a single caspase enzyme from the cytoplasmic pool of multiple caspases. Our observation that IL-18 modulation influences cas-1 activity may provide new approach to controlling cas-1 activity and inflammatory reactions.

In contrast to IL-18 expression, expression of IL-12 (p70) did not appear to vary significantly in response to protective and lethal doses of LPS. This was unexpected because overexpression of IL-12 has been reported during LPS-induced shock and neutralizing IL-12 protected mice from LPS-induced shock (9). IL-12 is a heterodimeric molecule comprised of a p35 35-kDa and p40 40-kDa subunit whose expression is independently regulated. The p35 subunit transcripts are constitutively expressed, while p40 expression is transcriptionally controlled (48, 49). As a result, the levels of p40 subunit have been assumed to represent the IL-12 (50). Our observations of elevated serum p40 levels at 24 h after LPS treatment are similar to earlier observations of LPS-induced IL-12 (p40) synthesis. However, p40 subunit can also exist as a homodimeric anti-inflammatory molecule (10). Furthermore, it was recently reported that expression of p35, but not p40, was a determinant of synthesis of bioactive IL-12 (51). Therefore, estimation of the p70 form of IL-12 would be expected to provide an accurate picture of IL-12 expression in inflammatory reactions. Our results suggest that IL-12 expression is similarly increased and restored to basal levels by both protective and lethal LPS doses. This suggests that the mechanisms regulating the expression and activity of IL-12 are unaffected by the doses of LPS and, by itself, IL-12 may not mediate LPS-induced lethality. We are currently exploring this possibility further.

In conclusion, LPS induced dose-related increases in serum IL-18 whose levels correspond to disparate outcomes: enhanced innate immune protection from lethal bacterial infection at low doses to a lethal inflammatory response at much higher ones. The mechanism for this difference may in part be related to the further induction of cas-1 mRNA at the higher LPS doses leading to sustained levels of circulating IL-18. These findings may have important implications for the prophylactic or therapeutic treatment of sepsis.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI 40568 (to A.S.C.), CA 71401, and CA 78282 (to D.V.K.). Back

2 Address correspondence and reprint requests to Dr. Alan S. Cross, MSTF 9-11, Division of Infectious Diseases, University of Maryland School of Medicine, 10 South Pine Street, Baltimore, MD 21201. E-mail address: across{at}umm.edu Back

3 Abbreviations used in this paper: ABHD, antibacterial host defense; cas-1, caspase-1; TSA, tryptic soy agar. Back

4 V. D. Joshi, D. V. Kalvakolanu, R. J. Hebel, J. D. Hasday, and A. S. Cross. Caspase 1 is essential for host defense against Gram-negative bacterial infections. Submitted for publication. Back

Received for publication January 25, 2002. Accepted for publication June 20, 2002.


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V. D. Joshi, D. V. Kalvakolanu, J. R. Hebel, J. D. Hasday, and A. S. Cross
Role of Caspase 1 in Murine Antibacterial Host Defenses and Lethal Endotoxemia
Infect. Immun., December 1, 2002; 70(12): 6896 - 6903.
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