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The Journal of Immunology, 2006, 177: 4627-4635.
Copyright © 2006 by The American Association of Immunologists, Inc.

Restoration of Natural IgM Production from Liver B Cells by Exogenous IL-18 Improves the Survival of Burn-Injured Mice Infected with Pseudomonas aeruginosa1

Manabu Kinoshita*, Nariyoshi Shinomiya{dagger}, Satoshi Ono{ddagger}, Hironori Tsujimoto{ddagger}, Toshinobu Kawabata{dagger}, Atsushi Matsumoto{dagger}, Hoshio Hiraide* and Shuhji Seki2,{dagger}

* Division of Basic Traumatology, National Defense Medical College Research Institute, Tokorozawa, Japan; and {dagger} Department of Immunology and Microbiology and {ddagger} Department of Surgery I, National Defense Medical College, Tokorozawa, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Pseudomonas aeruginosa is the most common bacterium of postburn infection. In the present study we investigated the immune mechanism of susceptibility to this type of postburn infection and also examined the efficacy of IL-18 treatment. C57BL/6 mice were challenged with P. aeruginosa on day 7 after burn injury. Although the burn-injured mice showed a poor survival rate after bacterial challenge, they retained their IFN-{gamma} production. The burned mice showed lower serum IgM levels and a poor IgM response following P. aeruginosa challenge in comparison with the sham mice, whereas IL-18 treatment after burn injury (alternate day injections for 1 wk) greatly improved the serum IgM levels, which are P. aeruginosa-independent natural IgM before bacterial challenge, thereby increasing the survival rate after the challenge. IL-18 treatment also induced specific IgM to P. aeruginosa in the sera 5 days after bacterial challenge in the burned mice. Interestingly, CD43+CD5–CD23–B220dim cells, namely B-1b cells, increased in the liver after the IL-18 treatment and were found to actively produce IgM in vitro without any additional stimulation. Furthermore, the IL-18 treatment up-regulated the neutrophil count and the C3a levels in the blood as a result of the increased IgM level, which may thus play a critical role in the opsonization and elimination of any invading bacteria. IL-18 treatment for the burned mice and their resultant natural IgM production were thus found to strengthen the host defense against P. aeruginosa infection.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Despite recent advances in patient care, bacterial infections remain a leading cause of death in severely burned patients (1, 2, 3). In our previous study (4, 5) we demonstrated that burn injury suppresses the production of IFN-{gamma} after Escherichia coli challenge in a mouse model whereas IL-18 treatment restores the IFN-{gamma} production, thereby improving the survival of burn-injured mice after infection. However, many clinical studies have described Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus, but not E. coli, as the most common agents of postburn infections (1, 2, 3). P. aeruginosa infection especially has a high mortality rate and remains a major cause of death in burned patients (1, 2). To improve the prognosis of burned patients, the potentiation of postburn defense mechanisms against P. aeruginosa infection is therefore considered to be very important.

In the last decade, the mechanisms of depressed cellular immunity following burn injury have been vigorously studied (4, 5, 6, 7, 8, 9). In particular, an impaired IFN-{gamma} production and its restoration using IFN-{gamma}-inducing cytokines such as IL-12 and IL-18 have been well documented (4, 5, 6, 7, 8, 9). Two to three decades ago, many investigators were already reporting on the changes in the humoral components such as complements and Ig in hosts following burn injury (10, 11, 12, 13, 14). Nevertheless, appropriate treatments for restoring such an impaired humoral immunity are less well-defined and have probably been treated as matters of less significance (15). The effective production of Ab is important not only in the neutralization of bacterial toxins but also in the promotion of efficient opsonization and the initiation of complement activation and bacterial cell lysis (16). In particular, IgM is the first class of Ig and a potent complement activator during the early stage of infections (17). We recently demonstrated that multiple IL-18 injections promote both the Th1 and Th2 responses after a sublethal E. coli infection (18). Interestingly, multiple IL-18 injections also up-regulate IgM production in the liver mononuclear cells (MNC)3 of the E. coli-infected mice (18). Mouse B cells are divided into two phenotypically and functionally distinct populations that are referred to as conventional B (or B-2) and B-1 cells (19, 20, 21). Conventional B cells express CD23 but not CD43 on their surface, whereas B-1 cells express CD43 but not CD23 (19, 20, 21). Mouse IgM can also be divided into two types. Namely, Ag-induced IgM is mostly produced by conventional B cells, whereas natural IgM is mainly secreted by B-1 cells (22, 23). The natural IgM is produced in the absence of external antigenic stimulation and provides immediate, early, and broad protection against pathogens (22, 23). As a result, the induction of natural IgM may thereby be a potent therapeutic tool against bacterial infection.

We herein investigated the effects of IL-18 treatment on postburn P. aeruginosa sepsis. At first, we suspected that burn-injured mice would demonstrate a decreased survival after P. aeruginosa challenge as a result of an impaired IFN-{gamma} production in a manner similar to the case of E. coli challenge and that IL-18-enhanced IFN-{gamma} production would thereby improve any postburn P. aeruginosa sepsis. However, we unexpectedly found that burn-injured mice retain P. aeruginosa–induced IFN-{gamma} production while showing decreased IgM production/response, particularly in the liver. Furthermore, we found that the IL-18 therapy increased the number of CD43+CD5–CD23–B220dim B-1b cells in the liver of the burn-injured mice, thereby enhancing their natural IgM production from B-1b cells before bacterial challenge.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
This study was conducted according to the guidelines of the Institutional Review Board for the Care of Animal Subjects at the National Defense Medical College (Tokorozawa, Japan).

Mice and burn injury

Male C57BL/6 mice were studied (8 wk old, weight 20 g; Japan SLC). As previously described (4, 5), the mice were anesthetized using an i.p. injection with pentobarbital (1 mg/mouse; Abbott Laboratories). Once the mice were fully anesthetized, the dorsa were shaved and the mice were placed in a plastic mold that exposed 20% of their total body surface area. The mice were then subjected to full-thickness burn injury by pressing a heated brass blade to the skin. Immediately after burn injury, PBS (1 ml/mouse) was i.p. administered for fluid resuscitation. The unburned sham mice only had their backs shaved without any burn injury.

Reagents

P. aeruginosa (strain PAK) was grown in a brain-heart infusion broth (Difco Laboratories). Mouse rIL-18 (Medical & Biological Laboratories), mouse rIFN-{gamma} (PeproTech), and mouse IgM (PP50; Chemicon International), which was collected from normal mouse sera, were used for the experiments.

Viable or heat-killed P. aeruginosa challenge and the collection of blood samples

Viable P. aeruginosa were heat killed by boiling for 30 min. The mice were then i.v. challenged with 1 x 108 CFU of viable or heat-killed P. aeruginosa on day 7 after either the burn or sham injury. Blood samples were thereafter obtained from the retro-orbital plexus of mice and then the serum or plasma was stocked at –80°C until being assayed.

IL-18 treatment (alternate day injections of IL-18) and IFN-{gamma} or IgM injection

IL-18 treatment was performed by i.p. injections of IL-18 (0.2 µg/0.5 ml/body) on alternate days for 7 days (1, 3, 5, and 7 days after injury). Sham treatment was injection with PBS (0.5 ml) in the same way as the IL-18 treatment. IFN-{gamma} injection (5 µg/0.5 ml/body) into the burned mice was performed i.p. 1 h after P. aeruginosa challenge. IgM injection (350 µg/0.5 ml/body) to burned mice was performed intraperitoneally 1 h before P. aeruginosa challenge. A sham injection of PBS (0.5 ml) was performed in the same way as the IFN-{gamma} or IgM injection.

Isolation of the liver, spleen, and bone marrow MNC

Under deep anesthesia with ether, the mice were euthanized to remove their livers, spleens, and femurs. The liver and spleen MNC were obtained as previously described (4, 5). Briefly, the liver was minced and passed through a 200-gauge stainless steel mesh. The cells were washed, suspended in 33% Percoll solution, and centrifuged at 500 x g for 20 min at room temperature. The pellet was resuspended in RBC lysing solution and then was washed twice in 10% FBS-RPMI 1640. Similarly, the splenocytes were passed through a stainless steel mesh, treated then with the RBC lysing solution, and finally washed twice in 10% FBS-RPMI 1640. Bone marrow cells were obtained by injecting 1% FBS-RPMI 1640 into the femurs using a 1-ml syringe with a 26-gauge needle, treated then with the RBC lysing solution, and finally washed twice in 10% FBS-RPMI 1640.

Phenotypical analysis of liver and spleen MNC

The liver and spleen MNC were incubated for 10 min at 4°C with Fc blocker (2.4 G2; BD Pharmingen) to prevent any nonspecific bindings before staining. The cells were then stained with FITC-conjugated anti-mouse CD43 mAb (eBioscience), PE-conjugated anti-CD5, CD23, and IgM mAbs (eBioscience) and PE-Cy5-conjugated anti-mouse B220 mAb (eBioscience). The percentage of fluorescence-positive cells was then analyzed using EPICS XL (Beckman Coulter).

In vivo depletion of NK or NK/NKT cells

Anti-asialo GM1 (AGM1) Ab (Wako) (50 mg/mouse) or anti-NK1.1 Ab (PK136) (200 mg/mouse) was injected i.v. into the mice twice at 3 days before and after burn injury. Anti-AGM1 Ab depletes NK cells, and anti-NK1.1 Ab depletes both NK and NKT cells for ~7 days as we previously reported (24, 25). However, the treatment for burned mice with AGM1 Ab or anti-NK1.1 Ab could not effectively deplete the NK/NKT cells, presumably because burn injury has been reported to induce a depletion/inactivation of complements (11, 12, 13, 14), and therefore NK/NKT cells could not be depleted by the combination of Ab and complements. We then injected the mice with these Abs before and after burn injury.

Neutralization of IL-18

Anti-IL-18 Ab (200 mg/mouse) (Medical & Biological Laboratories) was i.v. injected into the mice at 1 h before P. aeruginosa challenge. Rat IgG1 (200 mg/mice; Sigma-Aldrich) was also injected into the mice as an isotype control.

Cell cultures

Extracted MNC from the liver and spleen were stained with Turk solution (Wako) and then counted using a microscope. After counting the cells, 5 x 105 of the liver, spleen, or bone marrow MNC in 200 µl of 10% FBS-RPMI 1640 medium were cultured in 96-well flat-bottom plates in 5% CO2 at 37°C for 24 h, and the culture supernatants were then stored at –80°C until the assays were performed.

Cell sorting for CD43+B220 dim or CD43–B220+ cells

The liver MNC were obtained from the IL-18-treated burned mice and then stained with anti-CD43 Ab and anti-B220 Ab to sort out the CD43+B220dim cells or CD43– B220+ cells using EPICS Elite (Beckman Coulter). After sorting, 2.5 x 105 of cells in 100 µl of 10% FBS-RPMI 1640 medium were cultured in 96-well flat-bottom plates (half area) in 5% CO2 at 37°C for 24 h.

Measurements of cytokine, Ig, alanine aminotransferase (ALT), or the creatinine levels using sera or culture supernatants

The serum levels of TNF, IL-4, IL-10, total IL-12, and IFN-{gamma} were measured using cytokine-specific ELISA kits (Endogen). The serum IL-18 levels were also measured using a mouse IL-18 ELISA kit (Medical & Biological Laboratories). The sera were usually diluted 10-fold by the assay buffer included in the respective ELISA kit and used to perform the measurements. The IgM, IgG1, and IgG2a levels of the culture supernatants or the sera were measured using a mouse IgM, IgG1, and IgG2a ELISA quantitation kit (Bethyl Laboratories), respectively. The sera were usually diluted 200- to 50,000-fold by the assay buffer and then used to perform the measurements. The serum ALT and creatinine levels were measured using the Fuji Dri-Chem system (Fuji Film).

Determination of specific IgM directed against P. aeruginosa

Specific IgM directed against P. aeruginosa in the mouse serum was determined by ELISA as previously described (26). A 96-well polystyrene plate (Nunc) was coated with 100 µl of heat-killed P. aeruginosa suspension and incubated overnight at 37°C. For coating, a 1 x 108/ml suspension of washed, heat-killed P. aeruginosa was diluted 1/20 in 0.05 M carbonate-coating buffer (pH 9.6) with 0.2% (w/v) sodium azide. The mouse sera were diluted 1/10 in PBS containing 0.05% Tween 20. After washing three times with PBS containing 0.05% Tween 20, 100 µl of the diluted serum sample was added in triplicate to the inner wells and then incubated at 37°C for 90 min. The plates were washed three times, and 100 µl of a 1/1000 dilution of goat anti-mouse IgM (µ-chain specific) alkaline phosphatase conjugate (Sigma-Aldrich) was then added. The plates were incubated again at 37°C for 90 min and washed, and 100 µl of the enzyme substrate (alkaline phosphatase yellow (p-nitrophenylphosphate) liquid substrate from Sigma-Aldrich) was added. After the enzyme reaction reached an OD of at least 1.0 in the positive control well, it was inhibited by the addition of 50 µl of 3 M NaOH. Absorbance was measured at 405 nm using a plate reader. On each plate the same negative and positive controls were used. The serum from a nontreated C57BL/6 mouse was used for negative control. The serum from a P. aeruginosa (1 x 108 CFU)-challenged C57BL/6 mouse taken on day 5 after challenge was also used for positive control. The serum for positive control was diluted 1/10, 1/12.5, 1/25, 1/50, 1/100, 1/125, 1/250, and 1/1000 with PBS containing 0.05% Tween 20 to compare the OD with those of the obtained serum samples. When a serum sample (diluted 10 times) showed a higher OD than that of the 10 times-diluted positive control, the sample was further diluted 1/100 and then determined again. Next, the relative titer of each sample was counted, with the titer of positive control as 1.

Measurements of neutrophil count in the blood and plasma C3a levels

Blood leukocytes were counted using a PEC-170 hematology analyzer (Beckman Coulter). The neutrophil percentage in the leukocytes was also determined using a smear stained by the Wright-Giemsa stain. The neutrophil count in the blood was then determined by the leukocyte count multiplied by the neutrophil percentage. Plasma C3a levels were measured using a mouse C3a ELISA kit (Cedarlane Laboratories).

Viable bacterial counts in the liver, lung, and blood

The livers and lungs (bilateral) were aseptically removed to produce a homogenized PBS suspension. The blood samples (1 ml) were also aseptically withdrawn from the abdominal vena cava. The bacterial suspensions were serially diluted 10-fold by PBS, placed by a spiral platter on brain heart infusion agar plates, and incubated at 37°C for 24 h. The number of viable bacteria in the liver, lung, and blood was then counted according to the observed colonies on the agar plates.

Pathological examinations

The mice were euthanized to remove the lungs, livers, and kidneys. The livers and kidneys were then immersed in 20% formalin for 2 days. The lungs were also immersed in 20% formalin for 2 days after the gentle intratracheal instillation of 20% formalin with a pressure of ~10 cm of H2O. From the specimens, slides were prepared and stained with H&E.

Statistical analysis

The data are presented as the mean values ± SE. Statistical analyses were performed using an iMac computer (Apple) and the StatView 4.02J software package (Abacus Concepts). The survival rates were compared using the Wilcoxon rank test, and any other statistical evaluations were compared using the standard one-way ANOVA followed by the Bonferroni post hoc test. A value of p < 0.05 was considered as indicating a significant difference.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-18 treatment improves the survival of burn-injured mice after P. aeruginosa challenge

Burn-injured mice, sham-injured mice, and IL-18-treated burn-injured mice were i.v. injected with P. aeruginosa on day 7 after burn or sham injury. Burn injury significantly decreased the mouse survival after P. aeruginosa challenge, whereas IL-18 treatment dramatically improved the survival of burn-injured mice (Fig. 1A). Consistently, IL-18 treatment significantly decreased the number of viable bacteria in the livers, lungs, and blood of the burned mice 24 h after P. aeruginosa challenge (Fig. 1, B–D) and also significantly decreased the serum ALT and creatinine levels during the same time period in the burned mice (Fig. 1, E and F). According to pathological examinations, although the burn-injured mice showed coagulation necrosis in the liver (Fig. 2Ab, as indicated by the arrows), acute tubular necrosis in the kidney (Fig. 2Bb, as indicated by the arrows), and septum thickness and alveolar edema in the lung (Fig. 2 Cb) 24 h after P. aeruginosa challenge, IL-18 treatment apparently improved the pathological findings in the burned mice (Fig. 2, Ac, Bc, and Cc) as well as those in the unburned mice (Fig. 2, Aa, Ba, and Ca).


Figure 1
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  		FIGURE 1. The effect of IL-18 treatment of burn-injured mice on the survival rate (A), the number of viable bacteria in the liver (B), lung (C), and blood (D), the serum ALT level (E), and the creatinine level (F) after P. aeruginosa challenge. The mice were i.p. injected with IL-18 (0.2 µg/body) or PBS on days 1, 3, 5, and 7 after burn injury. The unburned control mice were similarly injected with PBS after sham injury. Thereafter, the mice were i.v. injected with P. aeruginosa (1 x 108 CFU) on day 7 after injury. The mouse survival rates after bacterial challenge were monitored using 10 mice in each group (A). The livers (B), lungs (C), and blood (D) were obtained from the mice 24 h after P. aeruginosa challenge to count the number of bacteria. The blood samples were also obtained from the mice 24 h after P. aeruginosa challenge to measure the serum ALT (E) and creatinine (F) levels. The data are the mean ± SE from seven mice in each group. *, p < 0.01; {dagger}, p < 0.05 vs other groups.

 

Figure 2
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  		FIGURE 2. The pathological findings of the burn-injured mice, IL-18-treated burned mice, and unburned mice 24 h after P. aeruginosa challenge. The liver is shown as A (x200 magnification; H&E staining), the kidney is B (x400 magnification; H&E staining), and the lung is C (x200 magnification; H&E staining). The PBS-treated unburned mice (n = 5) are labeled as a, PBS-treated burned mice (n = 5) are labeled as b, and IL-18-treated burned mice (n = 5) are labeled as c in each column.

 
Burn-injured mice retain the serum IFN-{gamma} level after P. aeruginosa challenge, and extrinsic IFN-{gamma} does not improve the survival of burned mice after P. aeruginosa challenge

In our previous study (4, 5) we showed that IL-18-enhanced IFN-{gamma} improves the survival of burn-injured mice after E. coli challenge. We therefore examined the serum IFN-{gamma} levels in the P. aeruginosa-challenged mice. As expected, IL-18 treatment enhanced the serum IFN-{gamma} level after P. aeruginosa challenge in the burned mice (Fig. 3A). However, the burned mice unexpectedly retained the IFN-{gamma} peak at as high a level as that observed in the unburned mice (Fig. 3A), thus suggesting that the burned mice can induce a sufficient amount of IFN-{gamma} after P. aeruginosa challenge. We then examined the effect of increased IFN-{gamma} on the survival after P. aeruginosa challenge in the burned mice. Mouse recombinant IFN-{gamma} was i.p. injected into the burned mice 1 h after P. aeruginosa challenge. Although the injection of IFN-{gamma} exhibited a serum IFN-{gamma} peak similar to that from the IL-18 treatment in the burned mice, extrinsic IFN-{gamma} did not improve the survival after P. aeruginosa challenge in the burned mice (Fig. 3, B and C). Although some P. aeruginosa strains produce proteolytic enzymes that inactivate exogenous or endogenous IFN-{gamma} (27, 28), we confirmed by coculture study that the culture supernatant of the P. aeruginosa strain PAK in the current study does not have a degradation activity for recombinant mouse IFN-{gamma} (data not shown). These results suggest that IL-18 induced a certain protective factor other than IFN-{gamma} that may play an important role in the survival of postburn P. aeruginosa sepsis.


Figure 3
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  		FIGURE 3. A, The effect of IL-18 treatment on the serum IFN-{gamma} levels after P. aeruginosa challenge in the burn-injured mice. The mice were i.p. injected with IL-18 or PBS after burn or sham injury as described in Fig. 1. Thereafter, they were i.v. injected with P. aeruginosa on day 7 after injury. The serum IFN-{gamma} level was measured at the indicated times after bacterial challenge (n = 7 in each group). B and C, The effect of extrinsic IFN-{gamma} on the survival after a P. aeruginosa challenge in the burned mice. The mice were i.v. injected with P. aeruginosa 7 days after burn injury and subsequently, after 1 h, they were i.p. injected with 5 µg of mouse recombinant IFN-{gamma} or PBS. The mouse survivals after bacterial challenge were monitored using 10 mice in each group (B), and their serum IFN-{gamma} levels were measured at the indicated times (C). *, p < 0.05 vs other groups.

 
IL-18 treatment increases the serum IgM level, plasma C3a activity, and neutrophil count in the burn-injured mice before and after P. aeruginosa challenge

We recently demonstrated that multiple IL-18 injections increase IgM production in the liver MNC by mouse E. coli infection (18). We next examined the serum IgM level after P. aeruginosa challenge in the burned mice. The burn-injured mice showed a significantly lower serum IgM level 7 days after injury and also showed a significantly poor response of serum IgM after P. aeruginosa challenge (Fig. 4A). Interestingly, IL-18 treatment significantly restored the serum IgM level in the burned mice by day 7 after burn injury and, furthermore, IL-18 treatment improved the IgM response to P. aeruginosa challenge (Fig. 4A). Because IgM is a potent complement activator and enhances neutrophil-involved opsonization (17), we also measured plasma C3a levels and neutrophil counts after bacterial challenge. Although the plasma C3a level was significantly suppressed in the burned mice before and after P. aeruginosa challenge, IL-18 treatment restored these plasma C3a levels in the burned mice (Fig. 4B). One hour after bacterial challenge, there was a significant drop in the neutrophil count as compared with the value before the challenge in the unburned mice (Fig. 4C). This early drop of the neutrophil count in the circulation suggests that activated neutrophils efficiently marginate and/or migrate toward the initial infectious sites from the circulation (29). Interestingly, the burned mice did not exhibit such a drop of neutrophil count within 1 h (Fig. 4C). IL-18 treatment remarkably increased the neutrophil count in the burned mice 7 days after injury (before bacterial challenge), whereas the count rapidly dropped within 3 h after challenge. IL-18 treatment also significantly increased the P. aeruginosa-induced neutrophil count 12 and 24 h after bacterial challenge in the burned mice. These results suggest that IL-18 treatment augments the neutrophil activation via complement-involved opsonization. We further examined IgM specific to P. aeruginosa. Although none of the mice induced IgM specific to P. aeruginosa in the sera before P. aeruginosa challenge, the IL-18 treatment significantly increased the serum IgM specific to P. aeruginosa in the burned mice 5 days after challenge (Fig. 4D), thus suggesting that IL-18 treatment following burn injury might induce not only P. aeruginosa-independent natural IgM before bacterial challenge but also P. aeruginosa-specific IgM after P. aeruginosa challenge. The burn-injured mice did not show a significant suppression of either serum IgG1 or IgG2a levels by day 7 after injury, and IL-18 treatment did not affect the IgG1 or IgG2a levels in the burned mice at day 7 after injury (data not shown). No differences in the change of serum IgG1 or IgG2a levels within 7 days after P. aeruginosa challenge were also observed among the burned, unburned, and IL-18-treated burned mice (data not shown).


Figure 4
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  		FIGURE 4. The effect of IL-18 treatment of the burn-injured mice on the serum IgM levels (A), plasma C3a levels (B), neutrophil count (C), and P. aeruginosa-specific IgM levels in the sera (D) after P. aeruginosa challenge. The mice were i.p. injected with IL-18 or PBS after burn or sham injury as described in Fig. 1. Thereafter, they were i.v. injected with P. aeruginosa 7 days after injury. The blood samples were obtained from the mice at the indicated times to measure the serum IgM levels (A), plasma C3a levels (B), and leukocyte count. The neutrophil percentage of leukocytes was also obtained using a smear stained using the Wright-Giemsa stain. The neutrophil count in the blood was then determined by the leukocyte count multiplied by the neutrophil percentage (C). IgM specific to P. aeruginosa was also measured using blood samples as described in Materials and Methods and was shown as a relative titer. The data are the mean ± SE from 10 mice in each group. *, p < 0.01; {dagger}, p < 0.05 vs other groups; {ddagger}, p < 0.05 vs burn plus PBS group.

 
IgM injection dramatically improves the survival of burn-injured mice in P. aeruginosa infection

IL-18 treatment up-regulated the serum IgM level in the burn-injured mice not only before challenge but also 3~5 days after P. aeruginosa challenge (Fig. 4A). Because half of the burned mice without IL-18 therapy died within 2 days after P. aeruginosa challenge (Fig. 1A), the elevation of IgM before bacterial challenge, which might be a P. aeruginosa-independent natural IgM, may thus play an important role in the protective effect of IL-18 treatment in postburn P. aeruginosa sepsis. We thereby i.p. injected the burn-injured mice with mouse IgM 1 h before P. aeruginosa challenge (on day 7 after burn injury). The IgM injection greatly improved the survival of the burned mice after P. aeruginosa challenge (Fig. 5A). We also measured the plasma C3a levels and the neutrophil counts after challenge. Injecting the burned mice with IgM tended to increase the plasma C3a levels (Fig. 5B) and, interestingly, showed an early drop in the neutrophil count after bacterial challenge (Fig. 5C). In addition, IgM injection remarkably increased the neutrophil count between the 6- and 24-h points after bacterial challenge (Fig. 5C), thus suggesting further neutrophil activation by IgM. In contrast, the injection of mouse IgG (PP54; Chemicon International) (5 mg/body, in the same way as IgM injection) to the burned mice did not show an improved survival after P. aeruginosa challenge (data not shown).


Figure 5
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  		FIGURE 5. The effect of IgM injection to the burned mice on the survival rate (A), the plasma C3a levels (B), and the neutrophil count (C) after P. aeruginosa challenge. The mice were i.v. injected with P. aeruginosa 7 days after burn or sham injury. The burned mice were i.p. injected with 350 mg of IgM or PBS 1 h before bacterial challenge. The unburned mice were similarly i.p. injected with PBS 1 h before bacterial challenge. The blood samples were obtained from the mice at the indicated times to measure the plasma C3a levels (B) and neutrophil count (C). The neutrophil count in the blood was obtained in the same way as in Fig. 3C. The data are the mean ± SE from 10 mice in each group. *, p < 0.01; {dagger}, p < 0.05 vs burn plus PBS group; {ddagger}, p < 0.05 vs other group.

 
IL-18 treatment following burn injury increases IgM production in the mouse liver MNC

We next examined which organ MNC produce IgM by the IL-18 treatment in the burn-injured mice. The liver, spleen, and bone marrow MNC were separated from the mice on day 7 after burn or sham injury. IgM production in the liver and bone marrow MNC was significantly suppressed after burn injury, but that in the spleen MNC was not. IL-18 treatment given to the burned mice significantly restored IgM production of the liver MNC, whereas the enhancing effect of IL-18 treatment on IgM production was not statistically significant in the spleen or bone marrow MNC of the burned mice (Table I). In contrast, IgG1 production in the spleen MNC was significantly suppressed in the burned mice, but there was no reduction of IgG1 in the liver or bone marrow MNC. IL-18 treatment did not however restore the IgG1 production of the spleen MNC from the burned mice. Instead, it significantly enhanced the IgG1 production of the liver MNC (Table I). Regarding the IgG2a production of the organ MNC, no significant differences were seen among the mouse groups (data not shown). IL-18 treatment also enhanced the elevation of serum IgM levels in the burned mice 3~5 days after P. aeruginosa infection (Fig. 3A). By day 3 after infection, IL-18 treatment significantly increased IgM production in the liver MNC of the burned mice but not in their spleen or bone marrow MNC (data not shown).


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  		Table I. The effect of IL-18 treatment on IgM production from the organ MNC in mice with and without burn injurya

 
IL-18 treatment following burn injury increases the proportion of CD43+B220dim cells that produce a large amount of IgM

Because IL-18 treatment up-regulated the IgM production in the liver MNC of the burn-injured mice (Table I), we examined the effect of IL-18 treatment on the mouse liver B cells. IL-18 treatment significantly increased the proportion of CD43+B220dim cells in the liver MNC of the burned mice by day 7 after injury (Fig. 6A). IL-18 treatment also significantly increased the CD43+B220dim cells in the unburned mice, whereas their proportion of CD43+B220dim cells was significantly lower than that of the IL-18-treated burned mice (Fig. 6A). There was no difference in the proportion of CD43–B220+ cells, namely conventional B cells, after IL-18 treatment. These IL-18-induced CD43+B220dim cells showed a CD5–CD23– phenotype but were IgM+ (data not shown), suggesting the characteristics of B-1b cells. We then sorted CD43+B220dim cells and CD43–B220+ cells from the liver MNC of the IL-18-treated burned mice and compared their ex vivo IgM-producing capacity. The CD43+B220dim cells showed a significantly higher IgM production than the CD43–B220+ cells (Fig. 6B). In contrast, in vivo treatment of IL-18 did not show any remarkable effects on the spleen or bone marrow MNC in both the burned and unburned mice (data not shown).


Figure 6
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  		FIGURE 6. A, The effect of IL-18 treatment on the proportion of CD43+B220dim cells in the mouse liver MNC. The mice received a burn or a sham injury and were subsequently i.p. injected with IL-18 (0.2 µg/body) or PBS 1, 3, 5, and 7 days after injury. The liver MNC were obtained from the mice 2 h after the last injection (7 days after the injury). The cells were stained with FITC-conjugated anti-CD43 Ab and PE-Cy5-conjugated anti-B220 Ab to analyze the proportion of CD43+B220dim or CD43–B220+cells using a flow cytometer. The percentages in the upper left quadrants are for the CD43–B220+cells, and the percentages in the upper right quadrants are for the CD43+B220dim cells (square area, mean ± SE; n = 7 in each group). Data shown are representative of each group with similar results. *, p < 0.01 vs other groups; {dagger}, p < 0.01 vs unburned plus IL-18 group. B, IgM production of the CD43+B220dim cells in the liver of the IL-18-treated burned mice. CD43+B220dim cells or CD43–B220+cells were sorted from the liver MNC of the IL-18-treated burned mice 7 days after injury using a cell sorter and were then cultured for 24 h. Culture supernatants were subjected to ELISA. The data are pooled from three individual experiments. The data are the mean ± SE. *, p < 0.01 vs another group.

 
Burn-injured mice retain the IFN-{gamma} production from NK/NKT cells by up-regulating the endogenous IL-18 level in P. aeruginosa infection.

Finally, we examined the mechanisms by which burn-injured mice retained the serum IFN-{gamma} level in P. aeruginosa infection in contrast to the case of E. coli infection. Because NK cells mainly produce IFN-{gamma} after bacterial infection (4, 24), we depleted NK cells or NK/NKT cells in the burned mice using anti-AGM1 Ab or anti-NK1.1 Ab, respectively, and then i.v. challenged them with P. aeruginosa. Depletion of either NK or NK/NKT cells remarkably suppressed the serum IFN-{gamma} level in the burned mice (Fig. 7A), thus suggesting the importance of the NK cell fraction in the IFN-{gamma} production after postburn Pseudomonas infection. Interestingly, when the mice were injected with heat-killed P. aeruginosa, substantial IFN-{gamma} production was observed only in the unburned mice (Fig. 7B). The result was almost the same when P. aeruginosa-derived LPS was used for challenge (data not shown). These results suggest that viable P. aeruginosa bacteria, but not bacterial Ag itself, can stimulate the mouse NK cells to produce a sufficient amount of IFN-{gamma} after burn injury. Because previous reports have indicated that IL-12 and IL-18 stimulate mouse NK/NKT cells to produce IFN-{gamma} (30, 31), we measured these cytokines in the burn- or sham-injured mice after Pseudomonas challenge. There was a significant increase in the serum IL-18 levels of the burn-injured mice at the 3- and 6-h points after viable P. aeruginosa challenge, though no difference in the serum IL-12 levels between burn-injured and unburned mice was observed (Fig. 7, C and D). Although the serum TNF or IL-10 levels tended to increase in the burn-injured mice after infection, there was no statistical significance in comparison to the sham mice (data not shown). We next examined the effect of P. aeruginosa-enhanced IL-18 on the IFN-{gamma} production in the burn-injured mice. The neutralization of IL-18 by the Ab injection remarkably suppressed the elevation of serum IFN-{gamma} after bacterial challenge in the burned mice (Fig. 7E). These results suggest that an enhanced production of IL-18 after viable P. aeruginosa challenge may account for the retained serum IFN-{gamma} levels in the burn-injured mice.


Figure 7
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  		FIGURE 7. A, The effect of depletion of NK or NK/NKT cells on the serum IFN-{gamma} levels after P. aeruginosa challenge in the burned mice. The mice were i.v. injected with AGM1 Ab (50 mg/body) or anti-NK1.1 Ab (200 mg/body) twice 3 days before and after burn injury to deplete NK or NK/NKT cells, respectively. Subsequently, they and the nontreated control burned mice were i.v. injected with P. aeruginosa (1 x 108 CFU) 7 days after the burn injury. The serum IFN-{gamma} levels were measured at the indicated times (n = 7 in each group). B, The serum IFN-{gamma} levels after heat-killed P. aeruginosa challenge in the burned mice. The mice were i.v. injected with P. aeruginosa bacteria (1 x 108 CFU) that were heat-killed by boiling 7 days after burn or sham injury. The serum IFN-{gamma} levels were measured at the indicated times (n = 7 in each group). C and D, The serum IL-12 (C) and IL-18 levels (D) after P. aeruginosa challenge in the burned mice. The mice were i.v. injected with P. aeruginosa (1 x 108 CFU) 7 days after burn or sham injury. The serum IL-12 and IL-18 levels were measured at the indicated times (n = 7 in each group). E, The effect of neutralizing IL-18 on the serum IFN-{gamma} levels after P. aeruginosa challenge in the burned mice. The mice were i.v. injected with P. aeruginosa (1 x 108 CFU) 7 days after burn injury. Beforehand, the mice were i.v. injected with anti-IL-18 Ab (200 mg/body) or rat IgG1 1 h before bacterial challenge. Subsequently, the serum IFN-{gamma} levels were measured (n = 5 in each group). The data are the mean ± SE. *, p < 0.01; {dagger}, p < 0.05 vs other groups.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Although the burn-injured mice showed a significantly decreased survival after P. aeruginosa challenge, IL-18 treatment following burn injury greatly improved the survival after challenge. Unexpectedly, the burn-injured mice retained their IFN-{gamma} production in response to P. aeruginosa in a manner similar to that for the unburned mice, and exogenous IFN-{gamma} also did not affect the mouse survival in the postburn sepsis. However, the mice had decreased IgM production after burn injury, particularly in the liver, and showed a poor IgM response following infection. IL-18 treatment after burn injury induced not only P. aeruginosa-independent natural IgM before bacterial challenge but also P. aeruginosa-specific IgM after P. aeruginosa challenge. Interestingly, IL-18 treatment increased the CD43+CD5–CD23–B220dim cells (namely B-1b cells) in the liver of burned mice and thereby restored natural IgM production in the liver. In addition, IL-18 also increased C3a and neutrophils in the postburn P. aeruginosa sepsis. IgM injection into the burned mice also greatly improved their survival after P. aeruginosa challenge and was accompanied by an increase of C3a and neutrophils. Finally, the burn-injured mice were found to up-regulate the serum IL-18 level more than the unburned mice after P. aeruginosa challenge and thereby retained IFN-{gamma} production from NK/NKT cells.

The suppression of Ig production in a particular IgM after burn injury might be a key issue in the postburn P. aeruginosa sepsis. Natural IgM induced by IL-18 therapy before P. aeruginosa challenge and the subsequent induction of complement and neutrophil during the infection may be crucial for both the opsonization of bacteria and for achieving an improvement in mouse survival. The important role of nonspecific natural IgM and neutrophil recruitment induced by natural IgM against severe bacterial infections has been recently recognized (23). Several investigators reported that IL-18 activates mouse neutrophils and promotes their accumulation in the inflammatory lesions (32, 33). We also found that IL-18 treatment enhanced the opsonizing capacity (C3a levels) and increased the neutrophil counts, which is presumably due to the action of induced IgM. IL-18 is also reported to possess inflammatory effects by up-regulating the synthesis of proinflammatory cytokines such as TNF, IL-1beta, and the chemokines like IL-8, MIP-1{alpha}, and MIP-2 (34, 35). In addition to its IFN-{gamma} inducing capacity, IL-18 is thus a potent cytokine with many different proinflammatory activities and may play a larger role in the innate immunity than was originally recognized (31, 36, 37).

The prophylactic use of i.v. Ig (IVIG) in severely burned patients was previously reported (38). The patients who received a replacement therapy of polyclonal and polyspecific Ig showed a significant improvement of the parameters related to infections. However, there was no significant improvement in the mortality rate or in the incidence of positive blood cultures in patients with severe bacterial infections. In other clinical studies, IVIG therapy did not reduce the mortality of the patient with sepsis either, although a moderate improvement of the morbidity of sepsis or the amelioration of multiple organ dysfunctions was reported (39, 40, 41). We suggest that one reason for such equivocal results may be that the major component of IVIG was not IgM but IgG (42, 43). Each IgG is a major contributor to the acquired humoral immunity against specific pathogens, whereas natural IgM plays a critical role in the nonspecific innate immunity before specific immunity is produced. We and several other investigators (43, 44, 45, 46) have previously pointed out that IVIG therapy suppresses the production of proinflammatory cytokines, including IL-12 and IFN-{gamma}, which may help to improve the clinical parameters while likely not having a significant effect on bacteria clearance or prognosis.

Although the liver MNC contain a considerable number of B cells, little is known about the hepatic B cells (47, 48). In the present study, extrinsic IL-18 increased the CD43+CD5–CD23–IgM+B220dim cell fraction in the liver of burned mice, which is suggested to consist of B-1b-like cells. B-1 cells were reported to differ from conventional B (B-2) cells by their characteristic localization in the pleural and peritoneal cavities of normal mice (20, 49). B-1b cells comprise a specific fraction in the B-1 cells that can secrete high levels of IgM, although their precise function remains to be investigated. Interestingly, in our experiments the stimulation of extrinsic IL-18 increased this B-1b cell fraction in the liver of the burn-injured mice and enhanced the production of natural IgM. As a result of its natural presence, polyreactivities with high avidities, and a strong ability to activate the complement system, natural IgM provides a first line defense against bacterial infections (23, 50). It may bind to the invading pathogens immediately after their entry and then promptly activate the complements to kill the bacteria.

The production and role of IFN-{gamma} are quite different in the case of postburn P. aeruginosa infection from those in the case of E. coli infection. The serum IL-18 level of the burn-injured mice in response to viable P. aeruginosa stimulation was higher than that of the unburned mice, whereas the burned mice after infection with E. coli did not show a similar enhancement of IL-18 (5). The IL-18 response after bacterial infection might be closely related to IFN-{gamma} production and thereby to the host defense mechanism, particularly regarding cellular immunity against postburn bacterial infections. Toliver-Kinsky et al. (51, 52) studied the mechanism of immune suppression after burn injury and claimed that IFN-{gamma} production was suppressed after P. aeruginosa infection; but they used heat-killed P. aeruginosa, and we have also shown similar results using heat-killed P. aeruginosa. Horvat et al. (27) and Parmely et al. (28) have demonstrated that some P. aeruginosa strains secrete alkaline protease and elastase and thereby inactivate recombinant IFN-{gamma}. The proteolytic inactivation of cytokines by P. aeruginosa might be one of the reasons why P. aeruginosa is an opportunistic pathogen that can cause serious and lethal infections in some compromised hosts. Pierangeli et al. (53) also demonstrated that exogenous IFN-{gamma} does not protect burn-injured mice from the infection with P. aeruginosa strain KU6, because this strain produces metalloproteases that inactivate recombinant IFN-{gamma}. In contrast, however, the P. aeruginosa strain PAK in this study reportedly does not show an obvious proteolytic activity, including elastase activity (54, 55). We also confirmed by coculture experiments that this PAK strain does not have a degradation activity for recombinant mouse IFN-{gamma} (data not shown). However, we do not rule out the possibility that multiple injections of IFN-{gamma} before and after infection may improve mouse survival after PAK strain infection, partly because IFN-{gamma} has a short half-life time (56).

Natural IgM may be crucial for the general host defense by various bacterial insults. In contrast, specific bacterial Ag-induced IgM is important for an efficient elimination of corresponding bacteria. However, natural IgM might more effectively protect burn-injured mice from P. aeruginosa infection than P. aeruginosa-specific IgM, because a majority of mice died within 2 days after infection when specific IgM did not increase. Taken together, IL-18 treatment and resultant natural IgM could therefore be a powerful and effective immunomodulation therapy for bacterial infections even under immunosuppressive conditions such as severe burn injury.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported in part by a Grant-in-Aid for Special Research (Host Stress Responses to Internal and External Factors) from the National Defense Medical College (to N.S. and S.S.). Back

2 Address correspondence and reprint requests to Dr. Shuhji Seki, Department of Immunology and Microbiology, National Defense Medical College, Namiki 3-2, Tokorozawa 359-8513, Japan. E-mail address: btraums{at}res.ndmc.ac.jp Back

3 Abbreviations used in this paper: MNC, mononuclear cell; AGM1, anti-asialo GM1; ALT, alanine aminotransferase; IVIG, intravenous Ig. Back

Received for publication April 3, 2006. Accepted for publication June 21, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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