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The Journal of Immunology, 2007, 179, 505 -513
Copyright © 2007 by The American Association of Immunologists, Inc.
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Pseudomonas aeruginosa Delays Kupffer Cell Death via Stabilization of the X-Chromosome-Linked Inhibitor of Apoptosis Protein1

Alix Ashare2,*, Martha M. Monick*, Amanda B. Nymon*, John M. Morrison*, Matthew Noble*, Linda S. Powers*, Timur O. Yarovinsky*, Timothy L. Yahr{dagger} and Gary W. Hunninghake*,{ddagger}

* Division of Pulmonary, Critical Care, and Occupational Medicine, and {dagger} Department of Microbiology, University of Iowa Roy J. and Lucille A. Carver College of Medicine and {ddagger} Veterans Administration Medical Center, Iowa City, IA 52242


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Kupffer cells are important for bacterial clearance and cytokine production during infection. We have previously shown that severe infection with Pseudomonas aeruginosa ultimately results in loss of Kupffer cells and hepatic bacterial clearance. This was associated with prolonged hepatic inflammation. However, there is a period of time during which there is both preserved hepatic bacterial clearance and increased circulating TNF-{alpha}. We hypothesized that early during infection, Kupffer cells are protected against TNF-{alpha}-induced cell death via activation of survival pathways. KC13-2 cells (a clonal Kupffer cell line) were treated with P. aeruginosa (strain PA103), TNF-{alpha}, or both. At early time points, TNF-{alpha} induced caspase-mediated cell death, but PA103 did not. When we combined the two exposures, PA103 protected KC13-2 cells from TNF-{alpha}-induced cell death. PA103, in the setting of TNF exposure, stabilized the X-chromosome-linked inhibitor of apoptosis protein (XIAP). Stabilization of XIAP can occur via PI3K and Akt. We found that PA103 activated Akt and that pretreatment with the PI3K inhibitor, LY294002, prevented PA103-induced protection against TNF-{alpha}-induced cell death. The effects of LY294002 included decreased levels of XIAP and increased amounts of cleaved caspase-3. Overexpression of Akt mimicked the effects of PA103 by protecting cells from TNF-{alpha}-induced cell death and XIAP cleavage. Transfection with a stable, nondegradable XIAP mutant also protected cells against TNF-{alpha}-induced cell death. These studies demonstrate that P. aeruginosa delays TNF-{alpha}-induced Kupffer cell death via stabilization of XIAP.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The hepatic reticuloendothelial system (RES)3 plays an important role in removing bacteria and endotoxin from the blood stream. Kupffer cells, the resident macrophages of the hepatic RES, provide a crucial means of defense against microorganisms. Although they were once thought to be uniform, Kupffer cells are now known to be a heterogeneous group of macrophages. They are 2-fold more abundant in the periportal region, where blood enters the liver (1). Periportal Kupffer cells are larger and have greater phagocytic function but appear to be less involved in inflammatory reactions. The smaller centrilobular Kupffer cells appear to be more important in generating inflammatory responses. Recently, a clonal cell line of murine periportal Kupffer cells (KC13-2 cells) has been generated (2). These cells actively phagocytose and kill bacteria but do not produce a large inflammatory response.

We have previously shown that hepatic bacterial clearance by Kupffer cells is decreased during severe bacteremia with Pseudomonas aeruginosa (3). This loss of hepatic bacterial clearance could be exacerbated by Kupffer cell ablation with gadolinium chloride and prevented by pretreatment with the nonspecific caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. A notable difference between mild bacteremia (where hepatic bacterial clearance was preserved) and severe bacteremia was that severe bacteremia was associated with a prolonged increase in hepatic TNF-{alpha}, suggesting that hepatic inflammation may be involved in the eventual loss of bacterial clearance. Interestingly, there was a period of time during severe P. aeruginosa bacteremia during which hepatic bacterial clearance persisted despite significant inflammation. This lead to the hypothesis of these studies that early in infection, P. aeruginosa protects Kupffer cells from TNF-{alpha}-induced cell death via activation of survival pathways.

P. aeruginosa has previously been shown to prevent apoptosis in corneal epithelial cells by activation of the epidermal growth factor (EGF) receptor (4). More recently, Pseudomonas syringae, a plant pathogen, has been shown to prevent apoptosis by translocation of an ubiquitin ligase protein (5). Our data demonstrate that P. aeruginosa promotes KC13-2 cell survival in the setting of inflammation via stabilization of the X-chromosome-linked inhibitor of apoptosis protein (XIAP).

The inhibitor of apoptosis protein (IAP) are a family of eight proteins that block the activity of caspases-3, -7, and -9 (6, 7, 8). XIAP is the most potent IAP, blocking caspase activity with nanomolar affinity (7). TNF-{alpha} induces cleavage of XIAP to an inactive fragment (9). Recently, XIAP was found to be phosphorylated by Akt, a kinase downstream of PI3K, rendering it resistant to cleavage (10). We demonstrate that inhibition of PI3K blocked the protective effect of P. aeruginosa on KC13-2 cell death. Conversely, overexpression of Akt protected cells against TNF-{alpha}-induced cell death. Finally, transfection with a mutant XIAP that is resistant to cleavage resulted in protection of KC13-2 cells against TNF-{alpha}. These data illustrate that P. aeruginosa protects Kupffer cells against caspase-mediated cell death early via stabilization of XIAP. Furthermore, there may be a window of opportunity in P. aeruginosa infection during which Kupffer cells are protected, preventing the eventual loss of hepatic bacterial clearance.


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

Chemicals were obtained from Sigma-Aldrich. The PI3K inhibitor LY294002 was obtained from Calbiochem. LY294002 was resuspended in ethanol and used at a final concentration of 20 µM. Abs were from various sources: anti-XIAP Abs were from AbCam and Cell-Signaling; Abs to cleaved caspase-3 and total and phosphorylated Akt were also from Cell Signaling; the Ab to beta-actin was from Sigma-Aldrich. Secondary Abs for Western analysis were from Santa Cruz Biotechnology. Recombinant murine TNF-{alpha} was obtained from R&D Systems and had <1.0 endotoxin U per µg of cytokine. TNF-{alpha} was dissolved in PBS and used at a final concentration of 40 ng/ml. Escherichia coli LPS was obtained from List Biological Laboratories. E. coli LPS was used at a final concentration of 600 ng/ml. ELISA kit for IL-6 was obtained from R&D Systems and was performed according to the manufacturer’s instructions. The caspase-3 inhibitor, Z-D(OMe)-E(OMe)-V-D(OMe)-FMK (Z-DEVD-FMK) was obtained from R&D Systems. A stock solution of 2 mM concentration in DMSO was diluted in PBS before use in cell culture. The final concentration in cell culture was 20 µM.

Cell culture

KC13-2 cells were a gift from Professor R. Landman (University Hospital, Basel, Basel, Switzerland; see Ref. 2). These cells were cultured as previously described in conditioned RPMI 1640 medium containing 1% Glutamax, 1% nonessential amino acids, 1% sodium pyruvate, 0.15% HEPES, 5% heat-inactivated FCS, and 10% volume of supernatants from the human hepatocyte cell line HepG2 and the human endothelial cell line EAhy926 (2). P. aeruginosa strains PA103, PA103 ExsA{Omega}, PAO smooth (AK857), and PAO rough (AK1012) were provided by Dr. T. Yahr (Department of Microbiology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, IA). For studies involving live strains of P. aeruginosa, bacteria were cultured overnight at 37°C in Luria-Bertani broth. Bacteria were subcultured the following morning in Luria-Bertani broth, grown to log phase, quantified based on OD600, and confirmed by standard plating. The multiplicity of infection in all experiments was 0.5. For experiments using cell-free supernatants of P. aeruginosa, bacteria were cultured overnight in KC13-2 medium at 37°C and subcultured in KC13-2 medium the following morning. Bacteria were grown to the late stationary phase and centrifuged at low speed; culture supernatants were sterile filtered. The final supernatant was added to the cell culture in 25% of the total volume. For experiments using heat-killed supernatants or LPS, the supernatants or LPS were boiled at 100°C before use.

Cell survival assays

Dead cells were visualized by adding ethidium homodimer (Invitrogen Life Technologies) to cells in culture to a final dilution of 1/250 and evaluated using a fluorescent inverted microscope after 5 min. A total of 500 cells were counted in 5 fields of view. Cellular ATP was measured using the CellTiter-Glo Luminescent Viability Assay (Promega) and was performed according to the manufacturer’s instructions.

Cleaved caspase-3 assay

Caspase-3 activity was measured using the CaspGLOW Red Active Caspase-3 Staining kit (BioVision Research Products) and was performed according to the manufacturer’s instructions using Guava PCA flow cytometer (Guava Technologies).

Western blot analysis

Western blot analysis for specific proteins was performed on total cellular protein isolated from KC13-2 cells. Protein concentrations in lysates were measured using the Bradford assay. Twenty micrograms of protein were mixed 1:1 with 2x sample buffer (20% glycerol, 4% SDS, 10% 2-ME, 0.05% bromphenol blue, and 1.25 M Tris, pH 6.8) and separated using SDS-PAGE. Cell proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad). Equal loading of proteins was evaluated using Ponceau S dye staining (Sigma-Aldrich). The polyvinylidene difluoride membrane was saturated with methanol, washed, and then incubated with primary Ab overnight at 4°C. Blots were washed four times and incubated with HRP-conjugated anti-IgG Ab (1/5,000–1/40,000). Immunoreactive bands were developed using a chemiluminescent substrate, ECL, and ECL Plus (Amersham Biosciences) and detected by autoradiography. Protein levels were quantified using densitometry via a FluorS scanner and Quantity One software for analysis (Bio-Rad). Densitometry is expressed as mean and SE of three separate Western blots.

Quantitative real-time PCR

Cells were treated as described. Bacterial DNA was isolated using the Bugs N' Beads kit (Genpoint) according to the manufacturer’s instructions. Quantitative real-time PCR with primers specific for P. aeruginosa was performed as previously described (3).

Adenovirus vectors

First-generation recombinant adenovirus vectors were generated by the University of Iowa Gene Transfer Vector Core (11) with the exception of the myristylated constitutively active Akt vector (Ad-myr-Akt), which was provided by Dr. K. Walsh (Boston University, Boston, MA). The construction (12) and activity (12, 13) of the Ad-myr-Akt vector have been described previously. The particle titers of the adenoviral stocks were typically 1012 DNA particles/ml; functional titers were ~2 x 1010 PFU/ml. Adenovirus vectors expressing the transgene for GFP (AdGFP) driven by the cytomegalovirus promoter, Ad-myr-Akt, or an empty vector containing no transgene (Ad-EV), were used to infect the cells at a multiplicity of infection of 100. These vectors were free of wild-type virus contamination by both plaque assay and PCR. KC13-2 cells were plated, and virus was added in serum-free medium. The cells were incubated at 37°C for 2 h, and then medium containing serum was added. The cells were incubated overnight and stimulated the following morning according to each individual experimental protocol. Either cells were evaluated for viability or protein was harvested. Efficiency of infection was determined in each experiment by examining green fluorescence of the AdGFP-infected cells using an inverted fluorescent microscope and was >75% in all experiments. Similar infection efficiencies were assumed with the other adenovirus vectors.

Plasmid transfections

XIAP-S87D was a generous gift from Dr. Jin Cheng (University of South Florida, Tampa, FL) (10). Plasmids were transfected using FuGENE 6 reagent (Roche Molecular Systems) according to the manufacturer’s instructions with a 3:2 ratio of FuGENE 6 reagent to DNA.

Flow cytometry

KC13-2 cells were grown to 70% confluency and cotransfected with a GFP plasmid and either XIAP-S87D or an empty plasmid. 48 h after transfection, cells were stimulated as described in the specific experimental protocol. At set time points, cells were harvested, fixed, permeabilized, and stained with PE-labeled Ab against cleaved caspase-3 (Cell Technology). Cytometric analysis looking at GFP- and PE-stained cells was performed using an LSR flow cytometer (BD Biosciences).

Statistical analysis

Statistical analyses were performed on cellular ATP data using ANOVA followed by Bonferroni’s test for multiple comparisons and represent the mean ± SEM. These methods were performed using GraphPad Prism (Prism).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
P. aeruginosa protects Kupffer cells against early cell death

To evaluate the relative effects of P. aeruginosa and TNF-{alpha} on Kupffer cell survival, KC13-2 cells were treated with P. aeruginosa (strain PA103), TNF-{alpha}, or both. This strain is a known human pathogen and produces epithelial cell injury via the elaboration of type III secreted toxins (14). We used a final concentration of 40 ng/ml TNF-{alpha} in all experiments. This is the peak concentration of serum TNF-{alpha} in a murine model of severe P. aeruginosa sepsis (3). Because ATP amounts have been shown to correlate with metabolically active (viable) cells (15), we measured cellular ATP as a marker of viability. We found that at 90 min, PA103 protected KC13-2 cells against TNF-{alpha}-induced cell death (Fig. 1A, left). This was manifested as preservation of cellular ATP levels. Use of ethidium homodimers to assess plasma membrane permeability confirmed the early effect of PA103 on KC13-2 cell survival (Fig. 1A, right). TNF-{alpha} was dissolved in PBS and studies looking at PBS alone showed no effect on KC13-2 cell survival (data not shown).


Figure 1
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  		FIGURE 1. PA103 protects Kupffer cells from early TNF-{alpha}-induced cell death. A, ATP viability assay was performed after incubation of KC13-2 cells with PA103, TNF-{alpha}, or both (left). PA103 was protective against TNF-{alpha}-induced cell death. *, p < 0.05, TNF-{alpha} alone compared with TNF-{alpha} with PA103. Values reflect mean ± SE of three separate experiments. Staining KC13-2 cells with ethidium homodimers after incubation with PA103, TNF-{alpha}, or both confirms the protective effect of PA103 (right). The number of cells positive for ethidium per 100 cells counted is expressed as percent of cell death. B, ATP viability assay comparing PA103 ExsA{Omega}, TNF-{alpha}, or both shows protection by PA103 ExsA{Omega}. *, p < 0.05, TNF-{alpha} alone compared with TNF-{alpha} with PA103 ExsA{Omega}. Values reflect mean ± SE of three separate experiments. C, ATP viability assay comparing LPS (600 ng/ml), TNF-{alpha}, or both shows no protection against TNF-{alpha}-induced cell death by LPS (left). Values reflect mean ± SE of three separate experiments. ATP viability assay comparing TNF-{alpha}, smooth PAO, rough PAO, or smooth or rough PAO in combination with TNF-{alpha} shows improved survival in cells treated with rough PAO and TNF-{alpha} or smooth PAO and TNF-{alpha} compared with TNF-{alpha} alone. *, p < 0.05) (right). Values reflect mean ± SE of three separate experiments.

 
PA103 has been shown to induce macrophage apoptosis via the type III secretion (T3S) system (16). To examine whether the T3S system was playing a role in the prosurvival effect of PA103 in this model, we infected cells with a mutant strain of PA103 (PA103 ExsA{Omega}) that is devoid of T3S capabilities (17). We found that the PA103 ExsA{Omega} strain had the same protective effect of KC13-2 cells as the wild-type strain (Fig. 1B). This suggests that the T3S system is not required for this effect.

To evaluate whether this was an effect of LPS, we performed the same experiment using E. coli LPS in place of PA103. We found that E. coli LPS did not protect KC13-2 cells against TNF-{alpha}-induced cell death (Fig. 1C, left). To ensure that E. coli LPS was functional and able to activate KC13-2 cells, we measured the amount of IL-6 in the cell supernatant after incubation with E. coli LPS. We found that E. coli LPS caused an increase in secreted IL-6 as measured by ELISA (data not shown), indicating that E. coli LPS is able to activate KC13-2 cells and induce cytokine production. However, E. coli LPS does not protect Kupffer cells from TNF-{alpha}-induced death.

Because E. coli LPS may not exert the same effect as P. aeruginosa LPS on KC13-2 cells, we treated cells with rough and smooth variants of P. aeruginosa strain PAO. The smooth strain PAO is a common laboratory stain of P. aeruginosa. Similar to PA103, we found that PAO protects KC13-2 cells from TNF-{alpha}-induced cell death at 90 min (Fig. 1C, right). The rough variant of PAO lacks the O Ag and has a poorly functional LPS (18). We found that the rough variant of PAO also protects KC13-2 cells from TNF-{alpha}-induced cell death. These data suggest that the protective effect of P. aeruginosa on TNF-induced cell death is not unique to the PA103 strain of P. aeruginosa and is likely not related to the secretion of LPS.

To evaluate whether the protective effect of PA103 was mediated by a secreted product, we incubated KC13-2 cells with a cell-free supernatant of PA103, TNF-{alpha}, or both. We found that the cell-free supernatant was able to protect KC13-2 cells from TNF-{alpha}-induced cell death (data not shown). This suggests that live replicating bacteria are not required for the protective effect of P. aeruginosa on KC13-2 cells. To further evaluate whether the secreted product responsible for the protective effect of PA103 was LPS, we boiled cell-free supernatants at 100°C to denature protein. It is widely accepted that LPS is heat resistant (19, 20). In fact, a recent study showed that concentrations of LPS as low as 10 ng/ml were effective after boiling for 15 min (21). We found that PA103 cell-free supernatants that were heat inactivated had no protection against TNF-{alpha}-induced cell death (data not shown). As a whole, these data suggest that P. aeruginosa protects KC13-2 cells from early TNF-{alpha}-induced cell death via a secreted product that is neither LPS nor a member of the T3S family.

PA103 prevents early TNF-{alpha}-induced KC13-2 cell death via increased Akt and stabilization of XIAP

Because TNF-{alpha} signaling can result in cleavage of XIAP (9), we examined XIAP protein using Western blot. Using an XIAP Ab designed to evaluate for the presence of the inactive cleavage product, we found that as early as 1 h after treatment with TNF-{alpha}, there was evidence of cleavage of XIAP to its inactive 30kD fragment (data not shown). This XIAP cleavage was prevented by PA103. We next examined the effect of TNF-{alpha} and PA103 on XIAP protein at time points just before and just after 1 h. We found that at 30 min, XIAP levels were stable in all groups (data not shown). However, at 90 min, there was a decrease in XIAP in the cells treated with TNF-{alpha}. This was prevented by infection with PA103 (Fig. 2A).


Figure 2
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  		FIGURE 2. PA103 stabilizes XIAP and prevents TNF-{alpha}-induced Kupffer cell death at early time points. A, Western blot for XIAP shows that PA103 prevents the TNF-{alpha}-induced reduction in XIAP protein levels at 90 min. beta-Actin is included as a loading control. Values representative of three experiments and densitometry comparing all three Western blots show that TNF-{alpha} results in a significant reduction in XIAP protein. *, p < 0.05, TNF-{alpha} compared with PA103 and TNF-{alpha}. B, Western blots demonstrate the time course of Akt activation following PA103, TNF-{alpha}, or both. These Western blots demonstrate that PA103 prolongs the activation of Akt following TNF-{alpha}. Values are representative of three experiments. C, Western blot for cleaved caspase-3 shows that PA103 prevents the TNF-{alpha}-induced increase in cleaved caspase-3 at 90 min (left). Active caspase-3 assay via flow cytometry using a fluorescent substrate confirms that PA103 protects against TNF-{alpha}-induced caspase-3 activation at 90 min (right). *, p < 0.05, TNF-{alpha} alone compared with TNF-{alpha} with PA103.

 
XIAP is known to be phosphorylated by Akt at Ser87 (10). The phosphorylated state of XIAP renders it resistant to TNF-{alpha}-induced cleavage. To evaluate whether PA103-induced stabilization of XIAP was due to increased Akt, we measured phospho-Akt in cell lysates following a time course of incubation with PA103, TNF-{alpha}, or both (Fig. 2B). After infection with PA103, Akt activity increases at 30 min and remains activated as long as 2 h. In contrast, TNF-{alpha} exposure results in an early activation of Akt that starts to decrease by 60 min. Treatment of KC13-2 cells with both TNF-{alpha} and PA103 results in a more prolonged increase in activated Akt compared with TNF-{alpha} alone. These data are consistent with PA103 stabilizing XIAP via activation of Akt.

To evaluate whether XIAP cleavage was associated with caspase activation, we measured levels of cleaved caspase-3 protein. We found that cleaved caspase-3 was present in the cells treated with TNF-{alpha} for 90 min (Fig. 2C, left). This was prevented by infection with PA103. To confirm that the cleaved caspase-3 protein levels represent activity, we measured caspase-3 activity via flow cytometry using a fluorescent substrate. We found that TNF-{alpha} alone resulted in increased cleaved caspase-3 activity, whereas infection with PA103 prevented this effect (Fig. 2C, right).

Because our prior study demonstrated eventual Kupffer cell death after PA103 bacteremia, we evaluated whether XIAP stabilization by PA103 was transient. We found that at later time points, PA103 was no longer protective against TNF-{alpha}-induced XIAP cleavage (data not shown). This corresponded with lack of protection against TNF-{alpha}-induced cell death (data not shown). These data indicate that PA103 delays Kupffer cell death following TNF-{alpha} via increased Akt and stabilization of XIAP.

Inhibition of caspase-3 increases Kupffer cell survival and enhances clearance of bacteria

We have previously shown that Kupffer cell survival is important for bacterial clearance and that the loss of hepatic bacterial is associated with increase caspase-3 activity in vivo (3). To test whether inhibition of caspase-3 would protect Kupffer cells in vitro, we pretreated KC13-2 cells with the caspase-3 inhibitor Z-DVED-FMK before incubation with TNF-{alpha} (Fig. 3A). After 6 h of incubation with TNF-{alpha}, there was a 40% reduction in cellular ATP compared with control cells, suggesting significant cell death. In contrast, pretreatment with Z-DEVD-FMK significantly improved survival of KC13-2 after TNF-{alpha}. DMSO alone had no effect on Kupffer cell survival (data not shown).


Figure 3
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  		FIGURE 3. Inhibition of caspase-3 improves Kupffer cell survival and enhances bacterial clearance. A, KC13-2 cells were treated with TNF-{alpha}, the caspase-3 inhibitor Z-DEVD-FMK 20 µM, or both (with Z-DEVD-FMK pretreatment initiated 30 min before incubation with TNF-{alpha}). ATP viability assay was performed 6 h after treatment. Treatment with the caspase-3 inhibitor with TNF-{alpha} decreased the amount of ATP loss compared with cells treated with TNF-{alpha} alone. *, p < 0.01). Values reflect mean ± SE of three separate experiments. B, KC13-2 cells were pretreated with either 20 µM Z-DEVD-FMK or no inhibitor 30 min before incubation with PA103, TNF-{alpha}, or both. After 6 h, cells and supernatants were harvested, and bacterial DNA was isolated. Quantitative real-time PCR with primers specific for P. aeruginosa was performed to determine residual bacterial load. Pretreatment with Z-DEVD-FMK before PA013 and TNF-{alpha} decreased the bacterial burden compared with cells treated with PA103 and TNF-{alpha} (*, p < 0.05).

 
To determine whether improved Kupffer cell survival was associated with enhanced clearance of bacteria, we treated cells with either no inhibitor or Z-DEVD-FMK before treatment with PA103, TNF-{alpha}, or both. Cells were incubated for 6 h, and then bacterial load was measured using quantitative real-time PCR with primers specific for P. aeruginosa. We found that treatment with Z-DEVD-FMK before PA103 and TNF-{alpha} resulted in a decreased bacterial load of P. aeruginosa compared with cells treated with PA103 and TNF-{alpha} alone (Fig. 3B). This is consistent with our prior work showing that survival of Kupffer cells is critical for maintenance of bacterial clearance.

Inhibition of PI3 kinase prevents PA103-induced protection of Kupffer cells

We next evaluated whether inhibition of PI3 kinase with the inhibitor LY294002 was sufficient to prevent PA103-induced protection of Kupffer cells. We found that pretreatment with LY294002 completely ablated the protective effect of PA103 on TNF-{alpha}-induced cell death in KC13-2 cells (Fig. 4A). To examine whether this effect was associated with a decrease in XIAP, we evaluated levels of XIAP protein in whole cell lysates using Western blot. We found that pretreatment with LY294002 resulted in an expected loss of Akt activity and a loss of XIAP protein (Fig. 4B).


Figure 4
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  		FIGURE 4. Inhibition of PI3K blocks PA103 protection against TNF-{alpha}-induced Kupffer cell apoptosis. A, ATP viability assay with and without the PI3 kinase inhibitor LY294002 (20 µM) shows that the PI3 kinase inhibitor negates the protective effect of PA103 at 90 min. *, p < 0.05, LY + PA103 + TNF-{alpha} compared with PA103 + TNF-{alpha}. Values reflect mean ± SE of three separate experiments. Ethanol control had no effect (data not shown). B, Western blot for phospho-Akt confirms effect of the inhibitor. Western blot for XIAP illustrates that LY294002 prevents the stabilization of XIAP by PA103 in the setting of TNF-{alpha}. Western blot for cleaved caspase-3 shows inhibition of PI3 kinase prevents PA103-induced protection against apoptosis. Values are representative of three experiments. C, Active caspase-3 assay via flow cytometry with a fluorescent substrate confirms the results demonstrated in Fig. 3C. *, p < 0.05, LY + PA103 + TNF-{alpha} compared with PA103 + TNF-{alpha}. Values reflect mean ± SE of three separate experiments.

 
Subsequently, we examined activated caspase-3 as a marker of apoptosis in this model. We found that pretreatment with LY294002 resulted in increased cleaved caspase-3 in the cells treated with TNF-{alpha} and PA103 compared with those cells treated with either PA103 or TNF-{alpha} alone (Fig. 4B). To confirm this, we evaluated caspase-3 activity using a PE-labeled substrate and found increased caspase-3 activity in cells treated with LY294002 along with PA103 and TNF-{alpha} compared with cells treated with PA103 and TNF-{alpha} alone (Fig. 4C).

Overexpression of Akt prevents TNF-{alpha}-induced Kupffer cell apoptosis

To examine the effect of active Akt overexpression on TNF-{alpha}-induced Kupffer cell death, we infected cells with an adenovirus expressing a myristylated form of Akt which is constitutively active. We found that cells treated with Ad-myr-Akt and TNF-{alpha} had improved survival compared with cells treated with TNF-{alpha} alone or empty vector with TNF-{alpha} (Fig. 5A). To evaluate the efficacy of the viral vector, we measured protein levels of total and active Akt. We found that treatment with Ad-myr-Akt increased levels of total and active Akt in KC13-2 cells with or without TNF-{alpha} (Fig. 5B) compared with control cells or cells treated with the empty vector. We next examined the effect of Ad-myr-Akt on XIAP and cleaved caspase-3 in KC13-2 cells. We found that Ad-myr-Akt stabilized XIAP protein in KC13-2 cells and that this effect was not overcome by treatment with TNF-{alpha} (Fig. 5C). Furthermore, Ad-myr-Akt prevented caspase-3 activation compared with cells treated with TNF-{alpha} alone or empty vector and TNF-{alpha}.


Figure 5
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  		FIGURE 5. Overexpression of Akt prevents TNF-{alpha}-induced Kupffer cell apoptosis. A, KC13-2 cells were infected with Ad-EV, Ad-myr-Akt, or no vector. After 24 h, cells were treated with or without TNF-{alpha} for 90 min. ATP viability assay shows that pretreatment with the Ad-myr-Akt prevented TNF-{alpha}-induced cell death. *, p < 0.05 compared with Ad-EV + TNF and control + TNF. Values reflect mean ± SE of three separate experiments. B, Western blot for total and phospho-Akt demonstrates that the Ad-myr-Akt vector increases levels of Akt protein with or without the presence of TNF-{alpha}. Values are representative of three experiments. C, Western blots for XIAP and cleaved caspase-3 demonstrate that infection with Ad-myr-Akt results in stabilization of XIAP and prevents activation of caspase-3. Values are representative of three experiments.

 
Overexpression of an XIAP mutant decreases Kupffer cell apoptosis

Because Akt expression induces several known survival pathways (22), we examined whether XIAP was necessary to prevent apoptosis in KC13-2 cells. XIAP can be phosphorylated by Akt at Ser87 to generate a phosphorylated state resistant to cleavage and degradation (10). To determine whether increased XIAP was sufficient to prevent TNF-{alpha}-induced cell death associated with caspase activation in KC13-2 cells, we used a mutant XIAP with an aspartic acid substituted at Ser87 (S87D-XIAP). This mutant mimics the phosphorylated state and is resistant to cleavage. We cotransfected KC13-2 cells with GFP and S87D-XIAP or an empty plasmid followed by treatment with TNF-{alpha}. We evaluated caspase activation by staining cells with a PE-labeled cleaved caspase-3 and analyzing cells with flow cytometry (Fig. 6). We found that transfection with the empty plasmid resulted in 17.1% of cells cleaved caspase-3 positive, whereas transfection with S87D-XIAP resulted in only 4.7% of cells cleaved caspase-3 positive. These data demonstrate that stabilization of XIAP increased Kupffer cell survival after TNF-{alpha} exposure.


Figure 6
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  		FIGURE 6. Stabilization of XIAP protects Kupffer cells against TNF-{alpha}-induced apoptosis. KC13-2 cells were cotransfected with GFP and either an empty plasmid or S87D-XIAP. After 48 h, cells were treated with or without TNF-{alpha}. After 90 min, cells were harvested, fixed, permeabilized, and stained with PE-labeled Ab against cleaved caspase-3. Flow cytometry was performed to analyze the percentage of cells that were both GFP and PE positive. The panel to the left demonstrates equivalent transfection rates in both groups. This panel also shows that, without TNF-{alpha}, very few cells are PE-positive. In contrast, the panel to the right demonstrates, in the presence of TNF-{alpha}, the cells transfected with the empty plasmid have 17.1% positive for cleaved caspase-3, whereas the cells transfected with S87D-XIAP have only 4.7% positive for cleaved caspase-3. Values are representative of three experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The hepatic RES is critical for both bacterial clearance and cytokine production in response to systemic infection (23, 24). In severe bacteremia, endotoxin and other bacterial products activate subpopulations of Kupffer cells, resulting in liberation of TNF-{alpha} and other inflammatory mediators (23). These inflammatory cytokines, whereas necessary to help clear infection, promote tissue injury and likely contribute to hepatic apoptosis and necrosis. We and others have shown that hepatic RES function is impaired during severe bacteremia (3, 25). Here we show that, unlike many other populations of macrophages, periportal Kupffer cells are susceptible to TNF-{alpha}-induced cell death. Furthermore, we report the novel finding that infection with P. aeruginosa delays TNF-{alpha}-induced Kupffer cell death by stabilizing XIAP.

Kupffer cells represent the largest population of tissue macrophages. Similar to alveolar macrophages, Kupffer cells are derived from peripheral blood monocytes that migrate to the liver and differentiate into at least two different Kupffer cell populations (1). Prior studies have shown that both peripheral blood monocytes and alveolar macrophages are resistant to TNF-{alpha}-induced apoptosis (26, 27). In fact, TNF-{alpha} has been shown to prevent monocyte apoptosis in vitro (26). Our data indicate that the large periportal Kupffer cells are susceptible to TNF-{alpha}-induced cell death. To our knowledge, this is the first report of TNF-{alpha}-induced Kupffer cell death in the literature. Taken together with our prior studying showing loss of hepatic bacterial clearance in the setting of prolonged TNF-{alpha} elevation (3), these data suggest a potential role of TNF-{alpha} in the development of impaired bacterial clearance in sepsis.

P. aeruginosa is a virulent human pathogen associated with a high mortality. The strain PA103 is particularly virulent due to the presence of a T3S system (28). The T3S system is cytotoxic to epithelial cells (14). Prior studies have implicated P. aeruginosa in the induction of macrophage apoptosis. The T3S systems of both P. aeruginosa and Yersinia spp. have been shown to mediate macrophage apoptosis (16, 29). A variety of P. aeruginosa secreted products have been shown to induce macrophage apoptosis in culture (30, 31, 32, 33). However, these studies did not examine the effect of P. aeruginosa on macrophages as early as 1 h after infection. To our knowledge, this is the first report of PA103 exerting a protective effect on macrophages.

P. aeruginosa, strain PA01, has been shown to prevent human corneal epithelial cell apoptosis via EGF receptor activation (4). This is consistent with our observation, given that the EGF receptor is upstream of PI3K and Akt. The plant pathogen, P syringae, prevents apoptosis in tomato plants via the elaboration of a protein that mimics the host E3 ubiquitin ligase (5). Our results illustrate that early protection of Kupffer cells by P. aeruginosa is not unique to strain PA103. Furthermore, a key finding in this study is that P. aeruginosa protects Kupffer cells early by a mechanism that is not mediated by the T3S system or the ability to secrete a fully functional LPS. The mechanism of P. aeruginosa protection of Kupffer cells involves a heat-labile secreted product. Potential candidates would include both phospholipase and exotoxin A. However, further studies are needed to determine the specific factor(s) responsible for this effect.

XIAP is a potent inhibitor of apoptosis. It directly binds to caspases-3, -7, and -9, rendering them inactive (7, 34, 35). IAPs are characterized by the presence of one or more baculovirus IAP repeat domains and a caspase activation recruitment domain. It is generally accepted that XIAP is the most potent inhibitor of apoptosis protein (7). In fact, a recent study suggests that two other members of the IAP family, cIAP1 and cIAP2, bind to caspases but fail to inhibit their activity (36). XIAP, however, binds and inhibits caspases-3, -7, and -9 with nanomolar affinity. Although the antiapoptotic properties of XIAP have been extensively studied in malignancy (37, 38, 39), very few studies exist evaluating the role of XIAP in sepsis. One study demonstrated that increased XIAP was associated with delayed neutrophil apoptosis in septic patients (40). However, to our knowledge, ours is the first study to look at the role of XIAP in macrophage survival during bacterial infection.

We found that prevention of KC13-2 cell apoptosis by PA103 was mediated by prevention of XIAP cleavage. TNF-{alpha} and its counterpart, TRAIL, are known to induce cleavage and degradation of XIAP although the exact mechanism remains unclear (9, 41, 42). XIAP is regulated by several proteins. In response to mitochondrial injury, smac/Diablo and Omi/HtrA2 are released into the cytosol where they bind and inactivate XIAP (8, 43). This results in polyubiquitination and degradation of XIAP. Recently, XIAP was found to have an Akt phosphorylation site (10). Phosphorylation at Ser87 stabilizes XIAP, protecting it from degradation. Here we have shown that phosphorylation of XIAP is sufficient to promote survival of Kupffer cells treated with TNF-{alpha}.

KC13-2 cells lack expression of several membrane receptors found in primary cells, which may affect their response to bacterial infection (2). In addition, cell death pathways in clonal cell lines do not always mirror those of primary cells. We recognize the use of a clonal cell line as a potential weakness of our studies. However, Kupffer cells are morphologically and functionally heterogeneous (1). The large periportal Kupffer cells are responsible for clearance of bacteria, whereas the smaller centrilobular Kupffer cells are involved in the immune response. Using standard Kupffer cell isolation protocols, it is difficult to separate these cell populations without contamination (2). However, we were interested in evaluating the effect of TNF-{alpha} and PA103 on the large periportal Kupffer cells because these are the cells responsible for bacterial clearance during bacteremia. In fact, contamination with the small Kupffer cell population would confound our experiments because these cells would produce more inflammatory mediators. Use of the clonal Kupffer cell line allowed us to concentrate our studies on the large periportal Kupffer cells to determine the mechanism of early survival in these cells. Future studies will develop effective isolation procedures for the large periportal Kupffer cells.

The ability of the body to clear a bloodstream infection is critical to survival. Kupffer cells represent the most abundant tissue macrophage population in the body. Our prior work has shown that Kupffer cells are necessary for survival in bacteremia. Furthermore, we have shown that, over time, bacteremia results in a loss of hepatic bacterial clearance. Here we show that TNF-{alpha} causes Kupffer cell death and that early Kupffer cell survival, even in the presence of TNF-{alpha}, during P. aeruginosa infection is mediated by stabilization of XIAP. Even though Pseudomonas ultimately contributes to cell death at later time points, our data suggest that there is a period of time during which Kupffer cell survival is maintained. This may be clinically relevant because early antibiotic therapy directed against P. aeruginosa has been shown to improve survival in a murine model (44). Furthermore, our data clearly demonstrate that prevention of Kupffer cell apoptosis by inhibition of casapase-3 results in enhanced clearance of bacteria. This may provide a window of opportunity to protect Kupffer cells from death and potentially prevent the loss of hepatic bacterial clearance seen during sepsis. Future studies using an animal model are necessary to determine the clinical applicability of modulating XIAP during bacteremia.


   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 by a Veterans Affairs Merit Review Grant, by National Institutes of Health Grants HL-60316, HL-077431, and HL079901-01A1 (to G.W.H.); K12 RR017700 and K08 DK073519-01A1 (to A.A.); and RR00059 from the General Clinical Research Centers Program, National Center for Research Resources, National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Alix Ashare, Division of Pulmonary, Critical Care and Occupational Medicine, University of Iowa, 200 Hawkins Drive, C33GH, Iowa City, IA 52242. E-mail address: alix-ashare{at}uiowa.edu Back

3 Abbreviations used in this paper: RES, reticuloendothelial system; EGF, epidermal growth factor; IAP, inhibitor of apoptosis protein; XIAP, X-chromosome-linked IAP; Z-DEVD-FMK, Z-D(OMe)-E(OMe)-V-D(OMe)-FMK; T3S, type III secretions. Back

Received for publication February 5, 2007. Accepted for publication April 14, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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