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The Journal of Immunology, 2007, 178, 5940 -5948
Copyright © 2007 by The American Association of Immunologists, Inc.
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*Sepsis

The Phosphatidylinositol 3-Kinase Signaling Pathway Exerts Protective Effects during Sepsis by Controlling C5a-Mediated Activation of Innate Immune Functions1

Christiane D. Wrann2,*, Navid A. Tabriz2,*, Tanja Barkhausen*, Andreas Klos{ddagger}, Martijn van Griensven{dagger}, Hans C. Pape§, Daniel O. Kendoff*, Renfeng Guo, Peter A. Ward, Christian Krettek* and Niels C. Riedemann3,*

* Department of Trauma Surgery, Hannover Medical School, Hannover, Germany; {dagger} Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Vienna, Austria; {ddagger} Department of Medical Microbiology, Hannover Medical School, Hannover, Germany; § Department of Orthopedic Surgery, Division Orthopedic Traumatology, University of Pittsburgh, Pittsburgh, PA 15213; and Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The PI3K/Akt signaling pathway has been recently suggested to have controversial functions in models of acute and chronic inflammation. Our group and others have reported previously that the complement split product C5a alters neutrophil innate immunity and cell signaling during the onset of sepsis and is involved in PI3K activation. We report in this study that in vivo inhibition of the PI3K pathway resulted in increased mortality in septic mice accompanied by strongly elevated serum levels of TNF-{alpha}, IL-6, MCP-1, and IL-10 during sepsis as well as decreased oxidative burst activity in blood phagocytes. PI3K inhibition in vitro resulted in significant increases in TLR-4-mediated generation of various proinflammatory cytokines in neutrophils, whereas the opposite effect was observed in PBMC. Oxidative burst and phagocytosis activity was significantly attenuated in both neutrophils and monocytes when PI3K activation was blocked. In addition, PI3K inhibition resulted in strongly elevated TLR-4-mediated generation of IL-1beta and IL-8 in neutrophils when these cells were costimulated with C5a. C5a-induced priming effects on neutrophil and monocyte oxidative burst activity as well as C5a-induced phagocytosis in neutrophils were strongly reduced when PI3K activation was blocked. Our data suggest that the PI3K/Akt signaling pathway controls various C5a-mediated effects on neutrophil and monocyte innate immunity and exerts an overall protective effect during experimental sepsis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
During the early inflammatory response to invading microorganisms, crucial innate immune players such as neutrophils and monocytes are set into place to defend the host. During experimental sepsis, neutrophils are activated very early in the onset phase of the inflammatory response as first line of defense, contributing significantly to mediator generation, pathogen phagocytosis, and O2– radical production. In various diseases related to acute inflammation, neutrophils are also thought to be responsible for host tissue damage and organ failure. However, during sepsis it is a well-described phenomenon that neutrophils undergo a status of immune paralysis with regard to their ability to fight invading microorganisms (1), setting the stage for super infection and for high lethality during sepsis (2), whereas the overall t1/2 in the serum is prolonged. The latter observation could be explained by activation of the PI3K pathway in neutrophils (3, 4), which, during sepsis, could at least partially be due to generation of the complement split product C5a (5, 6).

During the onset of experimental sepsis, the complement system is activated via three well-known pathways, leading to generation of the potent inflammatory split product C5a. There is growing evidence for various harmful effects of C5a and C5aR activation during the onset of sepsis (1, 7, 8). Blockade of either C5a or C5aR leads to greatly improved survival in septic rodents (9, 10, 11). Earlier work suggested that C5a generation during sepsis plays a critical role for suppression of neutrophil innate immune functions (11, 12, 13, 14). We recently demonstrated that C5a has a key function for altering intracellular signaling pathways in neutrophils in vitro and during the onset of sepsis in vivo (2, 15, 16), offering an explanation for the above-mentioned suppression of innate immune functions.

The PI3K signaling pathway, including the downstream Akt kinase, has been described as important inhibitory regulator of neutrophil apoptosis (3, 4). Recent work pointed out an important role of this signaling pathway for neutrophil respiratory burst (17, 18) as well as chemotaxis of neutrophils in response to fMLP (17, 19, 20, 21). Various studies then reported the importance of PI3K activation for neutrophil sequestration in inflamed tissue in different animal models (22, 23, 24, 25). These results implicated an important role of this signaling pathway for the innate immune response during acute inflammation. We thought to investigate the regulatory potential of this pathway for TLR-4- and C5a-mediated activation of neutrophils and monocytes and for outcome in experimental sepsis.


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

Human rC5a and other reagents were purchased from Sigma-Aldrich, if not otherwise indicated.

Neutrophil and PBMC isolation from whole blood and in vitro stimulation

EDTA/heparin was used as an anticoagulant for the isolation of human neutrophils and PBMC from blood. After Ficoll-Paque gradient centrifugation (Biocoll; Biochrom), PBMC were collected from the buffy coat and neutrophils were separated from the pellet by dextrane (Roth) sedimentation. Hypotonic RBC lysis was achieved, using sterile H2O. Neutrophils were resuspended in DMEM containing 10% FCS (Biochrom). A final concentration of 6 x 106 cells/ml was used for stimulation at 37°C and 5% CO2 for the times indicated with C5a (50 or 200 ng/ml) or LPS (20, 50, or 100 ng/ml), or both. Supernatant fluids were collected after pelleting of the cells and frozen at –80°C until used for ELISA analysis. For certain experiments, neutrophils were preincubated for 30 min with 50 µM PI3K inhibitor LY294002 (New Englands Biolabs), which inhibits downstream phosphorylation of the Akt pathway.

Western blot analysis

Neutrophils were isolated from human blood and stimulated at 37°C in vitro with human rC5a (10–100 ng/ml) or LPS (50 ng/ml), or both. Approximately 2 x 106 cells per condition were then used for whole cell lysis using Laemmli buffer containing 5% 2-ME. Lysates were separated on a NuPAGE 4–12% Bis-Tris gel (Invitrogen Life Technologies), and proteins were then transferred to a nitrocellulose membrane. Membranes were incubated overnight with Abs to phosphorylated and nonphosphorylated human/rat Akt, FKHR,4 glycogen synthase kinase (GSK)-3beta, phosphatase and tensin homolog deleted on chromosome 10, and phosphoinositide-dependent protein kinase 1 (New Englands Biolabs). For detection of the protein, ECL plus was used (Amersham Biosciences), according to the manufacturer’s instructions.

Quantitation of IL-6, TNF-{alpha}, IL-1beta, IL-12, IL-10, and IL-8 in cell supernatants

Neutrophils were isolated from human whole blood, as outlined above, and stimulated at 37°C in vitro with human rC5a (50 or 200 ng/ml) or LPS (50 ng/ml), or both for 6 h in an incubator with 5% CO2 under sterile conditions. Cell supernatants were than isolated and frozen at –80°C until analyzed for various mediators using a commercially available flow cytometric bead assay, according to the manufacturer’s instructions (BD Biosciences). For IL-8 ELISA experiments were conducted using a commercially available IL-8 ELISA kit, according to the manufacturer’s instructions (BioSource International).

Cecum ligation and puncture (CLP) in mice and inhibition of the PI3K/Akt pathway in vivo

Specific pathogen-free C57BL/6 mice (Own Laboratories, Zentrales Tierlabor Medizinische Hochschule Hannover) were used for all CLP studies. Anesthesia was achieved by i.p. injection of ketamine (Ketanest; Pfizer) and xylazine (Rompun; Bayer). In the CLP model, approximately two-thirds of the cecum was ligated through a 3-cm abdominal midline incision. The ligated part of the cecum was punctured through and through with a 21-gauge needle. After repositioning of the bowel, the abdomen was closed in layers, using a 4.0 surgical suture (Ethicon) and metallic clips. For inhibition of the PI3K/Akt signaling pathway in vivo, a specific inhibitor (LY294002; New Englands Biolabs) was diluted in 200 µl of Dulbecco’s PBS (DPBS) solution and injected into the penal vein of mice immediately following CLP to achieve a total blood concentration of 50 µM. Control animals received 200 µl of DPBS, including equal amounts of DMSO, also immediately after CLP. All animal studies were reviewed and approved by the local ethic committee of the state of Lower Saxony, Germany.

Collection of serum samples in mice

After induction of CLP, animals were sacrificed at the indicated time points and blood was drawn from the inferior caval vein. Blood samples were allowed to clot at 5°C for 6 h before centrifugation at 4000 rpm for 15 min at 4°C. Serum was collected and immediately frozen at –80°C until used for ELISA analysis. For experiments using the flow cytometric analysis of oxidative burst in neutrophils and monocytes from whole blood in mice, animals were treated with LY294002, as outlined earlier, and were then sacrificed 90 min thereafter. Next, blood was drawn from the inferior caval vein, and 100 µl of mouse whole blood was used for further flow cytometric analysis of oxidative burst activity, as outlined in the section below. Heparin was used as anticoagulant.

Quantitation of IL-6, TNF-{alpha}, IL-10, MCP-1, IL-12, IFN-{gamma}, and keratinocyte-derived chemokine (KC) in serum samples

Serum samples were collected, as outlined above. For quantification of various mediators, a commercially available flow cytometric bead assay was performed, according to the manufacturer’s instructions (BD Biosciences). For quantification of mouse KC ELISA experiments were conducted using a commercially available mouse KC ELISA kit, according to the manufacturer’s instructions.

Quantitation of oxidative burst and phagocytosis in whole blood cells

To determine the ability of blood neutrophils and monocytes to generate oxygen radicals and to conduct phagocytosis, commercially available flow cytometry-based assays were used, according to the manufacturer's instructions (Phagoburst, Phagotest; ORPEGEN Pharma). The Phagoburst assays use dihydrorhodamine 123 as a fluorogenic substrate and determine the percentage of active cells and their enzymatic activity/degree of activity. For Phagotest analysis, whole blood samples were incubated with FITC-labeled Escherichia coli bacteria (3.3 x 107 bacteria/ml) for 10 min in a 37°C warm water bath. Leukocyte surface-bound bacteria were neutralized using quenching solution. Cells were analyzed in a FACSCalibur flow cytometer (BD Biosciences). In a forward/side scatter dot plot, gates were set on granulocytes and monocytes to analyze each population with regard to mean fluorescence intensity (MFI). For oxidative burst measurement in mouse whole blood, samples were collected after inhibitor treatment, as described earlier, and then stimulated with PMA (1.4 µM) for 10 min in a 37°C water bath and processed according to the same protocol explained in this paragraph.

In vitro stimulation of oxidative burst and phagocytosis in blood phagocytes

Heparinized human whole blood was preincubated for 30 min with 50 µM PI3K inhibitor LY294002 (New England Biolabs) or with an equal amount of the vehicle (DMSO) at 37°C and 5% CO2. Human rC5a was then added at different concentrations (1–10,000 ng/ml), and the blood was further incubated for different time periods (10, 20, and 60 min). In one set of experiments, the cells were then stimulated with either unlabeled opsonized bacteria (E. coli) (6.7 x 108 bacteria/ml), PMA (1.4 µM), or the chemotactic peptide fMLP (0.8 µM) for 10 min in a 37°C water bath and processed afterward, according to the protocol explained above. In another set of experiments, the cells were processed immediately after C5a incubation with no further stimulation.

Assessment of bacterial growth in the peritoneal cavity during CLP-induced sepsis in mice

Sepsis was induced with CLP, and animals were treated with or without LY294002, according to the explained protocol, before being sacrificed at 6 h after CLP. The peritoneal cavity was lavaged with 10 ml of sterile 0.9% NaCl. Samples were diluted serially in 0.9% NaCl and incubated in parallel on Columbia 5% sheep blood plates, McConkey plates, and Slanetz-Bartley Enterococci-specific indicator plates for 24 h in aerobic atmosphere. The resulting bacterial colonies were further analyzed by biochemical assays, counted, and expressed as CFU per sample.

Statistical analysis

All values were expressed as the mean ± SEM. Significance was assigned where p < 0.05. Data sets were analyzed using Student’s t test or using one-way ANOVA, with individual group means being compared with the Tukey multiple comparison test. Statistical analysis for survival studies was performed using proportional hazards modeling. The software used was GraphPad Prism 3.0 (GraphPad).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Impact of PI3K/Akt inhibition on outcome during sepsis in mice

To determine whether inhibition of the PI3K/Akt pathway had effects on outcome during experimental sepsis in rodents, we conducted CLP experiments in mice receiving either 200 µl of DPBS as control or 200 µl of DPBS containing the PI3K inhibitor LY294002 to achieve a serum concentration of 50 µM. Mice were followed up for 7 days and monitored every 6 h for various signs of sickness. The group of mice receiving LY294002 demonstrated significantly reduced survival as well as earlier and more severe onset of sepsis (Fig. 1) when compared with the control group. These results demonstrated an overall protective effect mediated by the PI3K/Akt signaling pathway during the onset of sepsis.


Figure 1
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  		FIGURE 1. Effect of PI3K inhibition on outcome in experimental sepsis CLP study in C57BL/6 mice. CLP was induced with a 21-gauge needle and a two-thirds ligation of the cecum. CLP was induced and mice were treated with LY294002 directly after CLP by penal vein injection with 200 µl of DPBS containing body weight-adjusted concentrations of LY294002 to reach a calculated serum concentration of ~50 µM. Control groups were treated equally with 200 µl of DPBS containing no inhibitor. Experiments were conducted with n = 12 animals per group.

 
Impact of in vivo PI3K/Akt pathway inhibition on cytokine generation during sepsis and on burst activity in phagocytes

We conducted CLP experiments in mice receiving either 200 µl of DPBS as control or 200 µl of DPBS containing the PI3K inhibitor LY294002 to achieve an intravascular concentration of 50 µM, as described in Materials and Methods. Six hours after induction of sepsis, blood was drawn from the caval vein, followed by exsanguinations of the animals. Serum samples were prepared and analyzed for presence of various mediators using a flow cytometric bead assay. As depicted in Fig. 2A, significantly higher serum levels were found in LY294002-treated animals for TNF-{alpha}, IL-6, IL-10, and MCP-1, when compared with control mice. For KC, a tendency toward higher levels in LY294002-treated mice (mean value 33,833 pg/ml) was observed when compared with control mice (mean value 26,998 pg/ml), which was not found to be statistically significant. No differences were found for IL-12 and IFN-{gamma} (data not shown). These results demonstrated an inhibitory function of the PI3K/Akt pathway for generation of crucial inflammatory mediators such as TNF-{alpha} and IL-6 during the onset phase of sepsis, suggesting an overall negative feedback mechanism on inflammation in this model. We further investigated the effect of in vivo inhibition of the PI3K pathway on oxidative burst in neutrophils and monocytes in a whole blood assay. Mice were treated with 200 µl of DPBS containing LY294002 (50 µM serum concentration) or no inhibitor. Heparinized whole blood samples were drawn 90 min after treatment and stimulated with PMA (1.4 µM) for 10 min. The inhibitor treatment resulted in a significant decrease of burst activity in both cell populations, neutrophils and monocytes (Fig. 2B), demonstrating in vivo activity of the administered PI3K inhibitor. The number of bursting cells was not affected (data not shown).


Figure 2
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  		FIGURE 2. Impact of in vivo PI3K/Akt pathway inhibition on cytokine generation during sepsis and on burst activity in phagocytes. A, Flow cytometric bead ELISA of serum samples from septic animals. Animals underwent CLP and were treated with 200 µl of DPBS containing LY294002 (50 µM serum concentration) or no inhibitor directly after CLP. Blood serum samples were drawn 6 h after induction of CLP. The symbol * indicates statistical significant difference from the control group. Data are representative for five to seven animals per group. B, Flow cytometric analysis of oxidative burst in whole blood samples gated for neutrophil and monocyte cell populations. Animals were treated with 200 µl of DPBS containing LY294002 (50 µM serum concentration) or no inhibitor. Heparinized whole blood samples were drawn 90 min after treatment and stimulated with PMA (1.4 µM) for 10 min. Fifty thousand cells were analyzed for each experimental point. Oxidative burst activity per single cell is depicted as MFI. Neutrophils are shown in the left panel and monocytes in the right panel. *, Statistical significant difference from the control group. Data are representative for three to five animals per group.

 
Effect of PI3K/Akt inhibition on bacterial growth in the peritoneal cavity during sepsis

Six hours after induction of sepsis and inhibitor treatment with or without LY294002, peritoneal lavage fluids were collected from the mice and cultured and bacterial counts were determined as CFU after 24 h of incubation. The dominating bacteria found in all samples were Enterococci spp.; Staphylococci and Lactobacillus spp. were also detected to a lesser extent in most samples. No statistical significant difference in CFU counts of Enterococci spp. could be detected in the LY294002-treated group when compared with the vehicle control group (data not shown). Also, no significant differences in the variety of other bacteria were detected. These results suggested that PI3K inhibition resulted in adverse effects on outcome in septic mice most likely triggered by impaired innate immune functions rather than by direct effects on bacterial growth.

Role of the PI3K/Akt pathway on LPS-induced cytokine generation in neutrophils and PBMC

An earlier study has described the involvement of the PI3K pathway in LPS/TLR-4-dependent TNF-{alpha} generation in PBMC, demonstrating reduction of LPS-induced TNF-{alpha} generation after PI3K inhibition (26), suggesting a promoting effect of PI3K for TNF-{alpha} generation. Because we found in vivo strongly elevated mediator levels 6 h after CLP in serum samples of mice, we thought to investigate LPS-induced in vitro generation of various mediators in neutrophils and PBMC after 6 h of stimulation. Interestingly, we observed that in neutrophils the proinflammatory mediators TNF-{alpha}, IL-6, and IL-1beta were significantly increased when PI3K activity was inhibited (Fig. 3, A, C, E, and G), whereas the opposite effect could be seen in PBMC (Fig. 3, B, D, F, and H), suggesting an inhibitory potential of this signaling pathway for LPS-induced neutrophil mediator generation and a stimulatory function in PBMC. However, under in vivo conditions as described above, an overall inhibitory function of the PI3K pathway was dominating, with respect to TNF-{alpha}, IL-6, but also IL-10 and MCP-1 generation, as described earlier.


Figure 3
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  		FIGURE 3. Effects of PI3K inhibition on LPS-induced mediator generation in human neutrophils (A, C, E, G) and PBMC (B, D, F, H) in vitro. Flow cytometric bead ELISA of neutrophil and PBMC supernatant fluids. Neutrophils and PBMC were isolated from whole blood and incubated in vitro with LPS (50 ng/ml) or medium control for 6 h at 37°C at a concentration of 6 x 106 cells/ml. Cells were stimulated in the absence or presence of LY294002 after preincubation for 30 min at a concentration of 50 µM. *, Statistical significant difference from the control group; **, statistical significant difference from the LPS-treated group with no inhibitor. Data are representative of three to four independent experiments, with incubation and analysis being conducted in separate triplicate or quadruplicate samples.

 
Effects of PI3K inhibition on oxidative burst in neutrophils and monocytes in vitro

We investigated the role of the PI3K pathway on oxidative burst in neutrophils and monocytes in a whole blood assay, as described in Materials and Methods. Whole blood samples were preincubated with 50 µM LY294002 for 30 min, and oxidative burst was then stimulated with PMA, E. coli, as well as fMLP. PI3K inhibition resulted in a strong reduction of PMA- and E. coli-induced oxidative burst in neutrophils and in monocytes (Fig. 4, A and B), as well as in reduction of the number of cells initiating oxidative burst (Fig. 4, C and D). The latter effect was more apparent in PMA-induced oxidative burst when compared with E. coli as stimulus, especially in neutrophils (Fig. 4C). PMA appeared to be the strongest stimulus for neutrophils, whereas in monocytes, E. coli achieved a stronger stimulation of oxidative burst. The induction of oxidative burst by fMLP resulted in only minor initiation of oxidative burst in neutrophils and monocytes. Also, no inhibitory effects could be seen after PI3K inhibition when cells were stimulated with fMLP. Earlier studies have suggested a stimulatory effect of PI3K for fMLP-induced oxidative burst in isolated neutrophils in vitro (17), which could not be confirmed in this whole blood assay (Fig. 4A). Our data suggest an overall promoting effect of the PI3K pathway on PMA- and E. coli-induced oxidative burst in neutrophils and monocytes.


Figure 4
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  		FIGURE 4. Effects of PI3K inhibition on oxidative burst in neutrophils and monocytes in human whole blood. Flow cytometric analysis of oxidative burst in whole blood samples gated for neutrophil and monocyte cell populations. Fifty thousand cells were analyzed for each experimental point. Human whole blood samples were preincubated with LY294002 (50 µM) or equal amounts of vehicle control and then stimulated with either PMA (1.4 µM), E. coli bacteria (6.7 x 108/ml), or fMLP (0.8 µM) for 10 min. Oxidative burst activity per single cell is depicted as MFI (A and B), and numbers of bursting cells are shown in percentages (C and D). Data are representative of two to three independent experiments per graph.

 
Effects of PI3K inhibition on phagocytosis activity in neutrophils and monocytes in vitro

We further thought to investigate whether PI3K inhibition would also affect phagocytosis in neutrophils and monocytes. Whole blood samples were collected from healthy donors and subjected to PI3K inhibition by preincubation for 30 min with 50 µM LY249002 or control containing PBS and equal amounts of DMSO. PI3K inhibition clearly attenuated E. coli-induced phagocytosis activity in both neutrophils and monocytes (Fig. 5A), as well as the total number of phagocytosing cells (Fig. 5B), with the latter effect being less prominent in monocytes. Our data suggest a promoting effect of the PI3K signaling pathway for neutrophil and monocyte phagocytosis activity.


Figure 5
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  		FIGURE 5. Effects of PI3K inhibition on E. coli-induced phagocytosis in neutrophils and monocytes in human whole blood. Human whole blood samples were preincubated with LY294002 (50 µM) or equal amounts of vehicle control and then stimulated with opsonized FITC-conjugated E. coli bacteria (3.3 x 107/ml) for 10 min. Fifty thousand cells were analyzed for each experimental point. Phagocytosis activity per single cell is depicted as MFI (A), and numbers of bursting cells are shown in percentages (B). Data are representative of three independent experiments.

 
Activation of the PI3K/Akt signaling pathway in neutrophils by C5a

It has been described recently that C5a activates the PI3K/Akt signaling pathway in neutrophils in vitro, which could be linked to inhibition of apoptosis in neutrophils (4, 5). In monocytes, LPS-induced phosphorylation of Akt, Raf-1, and GSK-3beta has been described before (26). The c-Raf kinase is known to initiate MEK1/2 phosphorylation, thereby initiating activation of the ERK1/2 MAPK pathway. The c-Raf kinase therefore represents an important link between Akt and ERK1/2 MAPK signaling pathways. Another group has described recently that, in macrophages, C5a induces PI3K/Akt activation, leading to downstream activation of ERK1/2 (27). Our group demonstrated earlier rapid phosphorylation of Akt in neutrophils upon stimulation with C5a (2). We thought to investigate in further detail the activation patterns of C5a within the PI3K/Akt signaling pathway in neutrophils. We conducted in vitro studies stimulating isolated human neutrophils with C5a at various time points and various concentrations (Fig. 6). We found that C5a was capable of inducing Akt phosphorylation within 2–15 min, peaking at 5-min activation (data not shown). Concentrations of 50–100 ng/ml C5a demonstrated the strongest Akt phosphorylation potential (Fig. 6A). Blockade of the PI3K with LY294002 resulted in complete inhibition of Akt phosphorylation (Fig. 6B). We investigated upstream and downstream activation within the Akt signaling pathway induced by C5a. We found that C5a induced phosphorylation of GSK-3beta as well as the forkhead kinase family member FKHR in a time-dependent manner (Fig. 6C). Induction of the known negative regulator of Akt activation, the so-called phosphatase and tensin homolog deleted on chromosome 10, could also not be detected (data not shown).


Figure 6
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  		FIGURE 6. Activation of the PI3K pathway in human neutrophils by C5a. Western blot analysis of Akt activation (phosphorylation) in whole cell lysates of human neutrophils. A, Cells were stimulated at 37°C for 5 min with various concentrations of C5a, as depicted. B, Cells were stimulated for 5 min at 37°C in the absence or presence of 50 µm LY294002. C, Western blot analysis of Akt, GSK-3beta, and FKHR activation in whole cell lysates of isolated human neutrophils. Cells were stimulated at 37°C with C5a (50 ng/ml) for various time points, as depicted. The blots are representative of two to three independent experiments.

 
Role of PI3K inhibition for C5a-mediated effects on LPS-induced generation of IL-1beta and IL-8 in neutrophils

A recent report described C5a as negative regulator for IL-12 generation in human monocytes, with this effect being dependent on C5a-induced PI3K/Akt activation (28). Our group described various regulatory effects of C5a on neutrophil mediator generation in concert with LPS-induced TLR-4 stimulation (2, 15, 16). We thought to investigate in greater detail the involvement of PI3K activation for LPS-induced mediator generation in neutrophils in vitro. Human neutrophils were isolated from blood and incubated for 6 h in vitro with C5a (50 ng/ml), LPS (50 ng/ml), or both in the absence or presence of the PI3K inhibitor LY294002 (Fig. 7). Stimulation of neutrophils with C5a alone did not result in significant generation of IL-1beta (Fig. 7A). LPS stimulation resulted in minor generation of IL-1beta, which was increased when PI3K signaling was absent. Costimulation of neutrophils with C5a and LPS per se did not result in significant increase of IL-1beta generation when compared with LPS-only stimulated cells. This changed dramatically, under conditions of PI3K inhibition. A similar phenomenon could be observed for generation of IL-8 (Fig. 7B). PI3K inhibition attenuated baseline IL-8 generation completely. This effect was counteracted by stimulation with C5a or LPS. The latter stimulation led to small, but significant increases in IL-8 generation after PI3K signaling was inhibited, when compared with C5a- or LPS-stimulated neutrophils undergoing no PI3K inhibition. Costimulation of neutrophils with LPS and C5a after PI3K blockade resulted in significantly enhanced IL-8 generation when compared with cells with intact PI3K signaling. These results suggested that the PI3K signaling pathway exerts an important inhibitory control function for C5a-mediated boost of TLR-4-dependent generation of the proinflammatory mediator IL-1beta and the potent chemoattractant IL-8.


Figure 7
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  		FIGURE 7. Effects of PI3K inhibition on C5a-mediated modulation of TLR-4-dependent generation of IL-1beta and IL-8 in human neutrophils. Flow cytometric bead ELISA of neutrophil supernatant fluids for IL-1beta (A) and IL-8 (B). Neutrophils were isolated from whole blood and incubated in vitro with C5a (50 ng/ml), LPS (50 ng/ml), or both for 6 h at 37°C at a concentration of 6 x 106 cells/ml. Cells were stimulated in the absence or presence of LY294002 after preincubation for 30 min at a concentration of 50 µM. *, Statistical significant difference from the corresponding single treatment group (LPS and C5a, respectively) without LY294002. **, Statistical significant difference from the LPS + C5a-costimulated treatment group with no inhibitor. Data are representative of two to three independent experiments, with incubation and analysis being conducted in separate triplicate or quadruplicate samples.

 
Role of PI3K inhibition for C5a-mediated effects on oxidative burst generation in neutrophils and monocytes

Generation of oxidative burst represents a crucial function of the innate immune response to invading microorganisms. We thought to investigate the effect of PI3K inhibition on C5a-mediated oxidative burst and on C5a-mediated priming effects in human neutrophils and monocytes. We found that C5a induced oxidative burst in neutrophils and monocytes with regard to the intensity of burst activity per cell (Fig. 8, A and B), whereas only small effects could be observed on the total number of bursting cells (data not shown). When PI3K activation was inhibited, the observed increase was almost completely abolished in neutrophils and strongly reduced in monocytes, demonstrating involvement of this signaling pathway in C5a-mediated oxidative burst. We further investigated whether C5a-mediated priming of oxidative burst was dependent on PI3K activation (Fig. 8, C–F). Whole blood samples were preincubated with or without LY294002 for 30 min and than treated with various concentrations of C5a for 20 min before being stimulated with either PMA or E. coli. PMA-induced oxidative burst was strongly primed by preincubation with C5a in both neutrophils and monocytes (Fig. 8, C and D). This effect was strongly reduced in both cell populations when PI3K activation was inhibited, suggesting PI3K dependency of this C5a-mediated priming effect for PMA-induced oxidative burst. Similar experiments were conducted using E. coli as stimulus for oxidative burst. Low concentrations of C5a (1 ng/ml) achieved a noticeable priming effect for E. coli-induced oxidative burst in neutrophils and monocytes (Fig. 8, E and F). This effect was clearly reduced when PI3K activation was inhibited. However, in neutrophils, preincubation with high doses of C5a (1000 ng/ml) resulted in strongly increased E. coli-induced oxidative burst even after inhibition of PI3K activation (Fig. 8D), suggesting another pathway being responsible for priming effects induced by high-dose C5a preincubation before E. coli stimulation. Similarly, in monocytes, the C5a-induced priming effect for higher doses of C5a (200–1000 ng/ml) was only slightly inhibited when PI3K activation was blocked (Fig. 8F).


Figure 8
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  		FIGURE 8. Effects of PI3K inhibition on C5a-mediated modulation of oxidative burst in neutrophils and monocytes in human whole blood. Flow cytometric analysis of oxidative burst in whole blood samples gated for neutrophil and monocyte cell populations. Fifty thousand cells were analyzed for each experimental point. A and B, Human whole blood samples were preincubated with LY294002 (50 µM) or equal amounts of vehicle. Samples were then incubated with or without C5a (100 ng/ml) for 60 min, and oxidative burst was measured with no further stimulation. C–F, Whole blood samples were preincubated with or without LY294002 (50 µM) for 30 min and then incubated with different concentrations of C5a (0–10,000 ng/ml) for 20 min before final stimulation with either PMA (1.4 µM) (C and D) or E. coli bacteria (6.7 x 108/ml) (E and F) for 10 min. Oxidative burst activity per single cell is depicted as MFI. Percentages of the maximum increase in oxidative burst activity are calculated compared with control values (0 C5a) and depicted on the right panels of the curves. Data are representative of two to three independent experiments.

 
Role of PI3K inhibition for C5a-mediated effects on phagocytosis in neutrophils and monocytes

The ability of neutrophils and monocytes to conduct phagocytosis represents another crucial function of the innate immune response, which has also been demonstrated to be strongly dependent on C5a/C5aR (29), as well as on C5a and activating Fc{gamma}R in Kupffer cells (30). We thought to investigate whether C5a effects on E. coli-induced phagocytosis were dependent on PI3K signaling in neutrophils and monocytes. Preincubation of neutrophils with C5a resulted in dose-dependent enhancement of the phagocytic process induced by attenuated E. coli bacteria. This effect was completely abolished, when the PI3K signaling pathway was blocked, suggesting a strong dependency of this C5a-mediated priming effect on intact PI3K signaling (Fig. 9A). These findings could not be seen in monocytes applying the same experimental setting and assay (Fig. 9B). The number of phagocytosing cells was not considerably affected by C5a in both neutrophils and monocytes (data not shown).


Figure 9
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  		FIGURE 9. Effects of PI3K inhibition on C5a-mediated priming of E. coli-induced phagocytosis in neutrophils and monocytes in human whole blood. Flow cytometric analysis of phagocytosis in whole blood samples gated for neutrophil and monocyte cell populations. Fifty thousand cells were analyzed for each experimental point. Samples were preincubated with LY294002 (50 µM) or equal amounts of vehicle control, and then incubated with different concentrations of C5a (0–1000 ng/ml) for 60 min before being stimulated for 10 min with opsonized FITC-conjugated E. coli bacteria (3.3 x 106/ml). The phagocytosis activity per single cell of neutrophils (A) and monocytes (B) is given as MFI. Percentages of the maximum increase in phagocytosis activity are calculated compared with control values (0 C5a) and depicted on the right panels of the curves. Data are representative of two to three independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The PI3K/Akt signaling pathway has been suggested before to exert protective effects in models of acute inflammation (20, 31, 32), but rather harmful effects in models of chronic inflammation such as systemic lupus (33) or rheumatoid arthritis (34). It has also been described as important inhibitory pathway for apoptosis in neutrophils (5, 6, 35) and as important signaling pathway for LPS-induced B cell activation (36). Overexpression of Akt in lymphocytes prolonged their survival and also improved outcome in experimental sepsis (31), suggesting an important role of this signaling pathway during inflammation. However, until now, only little is known about the function of this pathway for innate immunity during acute inflammation. We demonstrate in this study an overall protective role of this pathway in our acute model of polymicrobial sepsis (Fig. 1) and a controlling inhibitory function for generation of various inflammatory mediators during the onset phase of sepsis (Fig. 2A). We were able to demonstrate that in vivo injection of LY294002 resulted in significant reduction of oxidative burst activity in blood monocytes and neutrophils in mice, offering a possible explanation for observed adverse effects for outcome during sepsis. Recent data demonstrated that inhibition of PI3K with wortmannin reversed the protective effects of glucan phosphate during experimental sepsis, demonstrating a protective effect of this pathway during acute inflammation (32). Our observations are in line with such earlier findings.

We compared the role of the PI3K pathway for TLR4-dependent generation of cytokines in vitro in neutrophils and monocytes. Interestingly, we were able to demonstrate opposing effects in these two cell populations. In neutrophils, PI3K activation appeared to limit the LPS response for generation of TNF-{alpha}, IL-6, IL-1beta, and IL-8 (Fig. 3, A, C, E, and G), whereas in monocytes PI3K activation appeared to have a significantly promoting function for LPS-dependent generation of these mediators (Fig. 3, B, D, F, and H). Similar findings in monocytes have been reported regarding the role of PI3K for LPS-induced TNF-{alpha} generation (26). In bone marrow mouse neutrophils, a recent study reported that TLR-2-dependent activation of PI3K occurs, and that inhibitions of this pathway resulted in reduced generation of TNF-{alpha} and MIP2. The latter observations are somewhat in contrast to our findings, even though it cannot be excluded that TLR-2-dependent generation of TNF-{alpha} in neutrophils is controlled by PI3K differently from the TLR-4-dependent one. Also, another study recently reported crucial differences between mouse and human neutrophils with respect to the importance of various PI3K subunits, making observation regarding PI3K activation in mouse neutrophils difficult to compare with those in humans (18). The same may or may not apply to monocytes.

In the context of mediator generation, the findings in vivo during sepsis in mice (Fig. 2A) clearly demonstrate an inhibitory potential of the PI3K pathway. Our in vitro studies reflect these findings only in neutrophils, but not in PBMC (Fig. 3). The question as to which of these two cell populations is responsible for the cytokine storm in vivo during sepsis is puzzling. Complex interactions between various blood phagocytes and also endothelial cells further complicate this matter. Earlier studies of our own group demonstrated that neutrophil depletion clearly diminished the IL-6 generation in the serum of septic mice (15), suggesting a leading role of neutrophils. However, the question outlined above cannot be finally answered as of yet.

Neutrophils are generally regarded as driving force for acute inflammation, whereas monocytes are thought to be more important in chronic inflammation. In this context, our observations may partially explain why PI3K activation appears to be beneficial in acute inflammatory processes, e.g., by limiting neutrophil activation, whereas being harmful in more chronic inflammatory processes, e.g., by promoting monocyte activation.

As outlined earlier, PI3K activation has been reported before to be involved in regulating oxidative burst and phagocytosis in neutrophils and monocytes (17, 18, 20, 37). However, no studies have been conducted to compare these cell populations directly. We report in this study that PI3K inhibition resulted in impaired oxidative burst and phagocytosis activity in both neutrophils and monocytes (Figs. 4 and 5). In neutrophils, this effect was especially strong for PMA-induced oxidative burst, whereas in monocytes the inhibitory effect was more prominent for E. coli-induced oxidative burst. These findings again suggest different regulatory functions of this pathway in neutrophils when compared with monocytes.

The concept of C5a/C5aR interception during sepsis has evolved in recent years, and C5a has become an interesting target for potential clinical interventions in the field of acute inflammation and especially sepsis (1). Our and other groups have suggested that large amounts of C5a are generated early during the onset of sepsis, but the intracellular regulatory mechanisms controlling C5a-induced effects on innate immune functions are not understood in detail. We have found recently that C5a negatively regulates LPS-induced production of TNF-{alpha} by rat neutrophils in vitro, but has the opposite effect on macrophages (16). In this study, TNF-{alpha} generation in neutrophils was strongly dependent on NF-{kappa}B activation, and treatment of CLP mice with anti-C5a caused enhanced serum levels of TNF-{alpha}, apparently by protecting the signaling pathways from C5a-induced dysfunction. We also recently demonstrated involvement of p38 and ERK1/2 activation in generation of IL-6 in neutrophils (15) and reported that the PI3K signaling pathway is involved in LPS-mediated macrophage migration inhibitory factor generation in neutrophils (2). A recent report from another group described C5a as negative regulator for IL-12 generation in human monocytes, with this effect being dependent on C5a-induced PI3K/Akt activation (28). We thought to investigate in greater detail the role of the PI3K/Akt signaling pathway for C5a-induced alterations of mediator generation upon TLR4 stimulation in neutrophils. We found that C5a-mediated effects on LPS-induced IL-1beta and IL-8 generation were controlled in an inhibitory fashion by the PI3K pathway (Fig. 7), offering a partial explanation for the observed rise of serum inflammatory mediators in CLP mice after PI3K inhibition.

Oxidative burst and phagocytosis represent key players of the innate immune response to invading microorganisms. Recent work has demonstrated the importance of C5a for an intact oxidative burst and phagocytosis in neutrophils and monocytes in response to E. coli in a new whole blood assay applying flow cytometric analysis (29). The PI3K/Akt pathway has also been described to be an important mediator of neutrophil oxidative burst in other assays using isolated human neutrophils (17). The ability of Kupffer cells to conduct phagocytosis has been described to be regulated by C5a as well as activating Fc{gamma}R in recent studies (30). The PI3K pathway appears to be important late in the process of phagocytosis in neutrophils and monocytes, namely during the phagosome closure after cup formation (37, 38). We thought to investigate in how far C5a-dependent regulation of oxidative burst and phagocytosis was dependent on PI3K activation in neutrophils and peripheral blood monocytes. Our results demonstrated that C5a-mediated priming of E. coli- and PMA-induced oxidative burst was largely dependent on intact PI3K signaling in both neutrophils and monocytes. C5a-mediated enhancement of phagocytosis appeared also to be dependent on PI3K activation in neutrophils.

Taken together, our results suggest that the PI3K signaling pathway exerts an overall protective role during the onset of sepsis in rodents by limiting C5a-mediated effects on neutrophil cytokine generation, and promoting oxidative burst and phagocytosis. We further propose that this pathway has opposing regulatory mechanisms in TLR-4-mediated cytokine generation in neutrophils when compared with monocytic cells. Inducing the activation of this pathway may represent a therapeutic approach for limiting acute inflammatory diseases such as sepsis.


   Acknowledgment
 
We thank Gerda Bartling for excellent technical assistance with the microbiological analyses of the peritoneal lavage fluids.


   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 Hochschulinterne Leistungsfoerderung-1 Grant (Hannover Medical School, Hannover, Germany) and by Deutsche Forschungsgemeinschaft Grant RI 1216/4-1 (German Research Council (Deutsche Forschungsgemeinschaft)). Back

2 C.D.W. and N.A.T. contributed equally to this work. Back

3 Address correspondence and reprint requests to Dr. Niels C. Riedemann, Department of Trauma Surgery, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, 30625 Hannover, Germany. E-mail address: nriedeman{at}yahoo.com Back

4 Abbreviations used in this paper: FKHR, forkhead-related transcription factor; CLP, cecum ligation and puncture; DPBS, Dulbecco’s PBS; GSK, glycogen synthase kinase; KC, keratinocyte-derived chemokine; MFI, mean fluorescence intensity. Back

Received for publication October 27, 2006. Accepted for publication February 20, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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