Chapter 2 Introduction
2.3 Cell signaling pathways
All processes within a cell are tightly regulated by a complex network of different signaling pathways. Every pathway consists of many interacting molecules and finally leads to activation or suppression of specific processes on transcriptional level.
Cross-talk between pathways occurs very often. Because it is possible to block single pathways by specific inhibitors of key regulating signaling molecules these cell signaling pathways have become of great interest for the development of new targets for pharmacotherapy.
In this study we concentrated on cell signaling pathways that are well known to play an important role in regulating innate immunity (for details refer to chapter 3 and 4):
the PI3K/Akt signaling pathway, the MAPK signaling pathway with its three major key players, ERK1/2, p38 MAPK, and JNK, as well as the PKC signaling pathway.
2.3.1 PI3K/Akt pathway
The PI3K/Akt pathway can be activated by various survival factors, such as growth factors and cytokines, as well as insulin (MEISENBERG u. SIMMONS 2006). The PI3K (Figure 2-2) binds either to an autophosphorylated growth factor receptor or to tyrosine-phosphorylated IRS-1 (insulin receptor substrate-1) thereby becoming allosterically activated. In addition, the small GTPase Ras when bound to guanosine triphosphate (Ras-GTP) can activate PI3K. Activated PI3K phosphorylates inositol lipids from the cell membrane at position 3 thereby generating phosphatidyl-inositol-3,4,5-trisphosphate (PIP3) from phosphatidyl-inositol-4,5-bisphosphate (PIP2). Now Akt, a member of the serine/threonine-specific protein kinase family, also known as protein kinase B can be recruited to the plasma membrane by binding with its pleckstring homology domain to the 3-phosphorylated-inositol lipids in the membrane. Akt contains two amino acid residues that have to be phosphorylated for activation: Thr308 and Ser473. Thr308 is phosphorylated by the phosphoinositide-dependent kinase (PDK) which is activated by either PIP2 or PIP3. Ser473 is phosphorylated by the integrin-linked kinase (ILK) which is found in focal adhesion (YOGANATHAN et al. 2000). Known downstream targets of activated Akt are Bad (Bcl-xL/Bcl-2 associated death promoter) (CARDONE et al. 1998), forkhead
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transcription factors (FKHR) (BRUNET et al. 1999), c-Raf (c-rapidly growing fibrosarcoma) (ZIMMERMANN u. MOELLING 1999) and caspase-9 thereby inhibiting apoptosis and promoting cell survival. Akt is also involved in the regulation of glycogen synthesis through phosphorylation and inactivation of GSK-3α and β (CROSS et al. 1995; HAJDUCH et al. 2001). In addition to its role in survival and glycogen synthesis, Akt plays a role in the regulation of the cell cycle (GESBERT et al. 2000; ZHOU et al. 2001) and cell growth (NAVE et al. 1999). Recent research has also suggested controversial functions in models of acute and chronic inflammation (refer to chapter3).
Figure 2-2: The PI3K/AKT signaling pathway.
The autophosphorylated receptor (R) recruits the phosphatidylinositol-3 kinase (PI3K). Alternatively, PI3K can be recruited by the small GTPase bound to GTP (Ras-GTP). After binding of PI3K substrates Akt travels to the membrane and can be phosphorylated by either an integrin-activated or PIP3-activated protein kinase on two amino acid residues. Active Akt phosphorylates serine and threonine side chains in its substrates. GTP, guanosine triphosphate; PIP3, phosphatidyl-inositol-3,4,5-trisphosphate (MEISENBERG u. SIMMONS 2006).
2.3.2 MAPK pathways
The MAPK also belongs to the serine/threonine-specific protein kinase family and three major types of MAP kinase cascades have been identified that are key regulating three separate signaling pathways: the ERK1/2, p38 MAPK, and JNK (DAVIS 1994). These pathway play an important role in cell proliferation and differentiation (WASKIEWICZ u. COOPER 1995; MORIGUCHI et al. 1996; SU u.
KARIN 1996; PEARSON et al. 2001). Dysregulated activity of these MAPKs has been found to be involved in cancer (SIVARAMAN et al. 1997; WU 2007). Although the different MAPK pathways can only be activated by specific stimuli, for example cell stress leads to the activation of the JNK pathway without activation of ERK, cross-talk between the different pathways has been reported. However, finally on
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transcriptional level an unique activation pattern will be achieved allowing for a appropriate cellular response to a particular stimulus (Figure 2-3).
Figure 2-3: The MAPK signaling cascades: ERK1/2, p38MAPK, and JNK.
Different kind of stimuli, such asgrowth factors, inflammatory cytokines or UV radiation activate MAPK cascade that leads to changes at transcriptional level generating an unique pattern of action a appropriate cellular response to each different stimulus. a-/b-/c-Raf, a-/b-/c-rapidly growing fibrosarcoma; MLK3, mixed-lineage kinase 3; DLK, dual leucine zipper bearing kinase, TAK, Tat-associated kinase; MEKK1,3, MEK kinase 1,3; MEK1/2 mitogen-activated protein kinase and ERK kinase 1/2; MKK3/6, MAPK kinase 3/6; MKK4/7, MAPK kinase 4/7; ERK1/2, extracellular regulated kinase1/2; p38 MAPK, p38 mitogen-activated protein kinase; JNK1/2, c-Jun N-terminal kinase.
In general the ERK 1/2 pathway is activated by growth factors, e.g. epidermal growth factor (STURGILL et al. 1991) that bind to a receptor tyrosine kinase leading to activation of one of the Raf serine-threonine kinases (a-Raf, b-Raf, or c-Raf) (AVRUCH et al. 1994). Next the dual specificity kinase MEK 1/2 (mitogen-activated protein kinase and ERK kinase 1/2) is either phosphorylated by active Raf kinase on serine218 and serine222 or by MEKK1 (MEK kinase 1). The activated MEK phosphorylates ERK1/ERK2 on threonine183 and tyrosine185. This dual-phosphorylated form represents the high active form of these kinases (COBB et al.
1996). The p38 MAPK shows a fifty percent homology to the ERKs and is mostly involved in response to inflammatory cytokines, endotoxins, and osmotic stress (HAN et al. 1994; RAINGEAUD et al. 1995). Mixed-lineage kinase 3 (MLK3) that also can activate the JNK pathway, TAK1 (Tat-associated kinase), or DLK (dual leucine zipper
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bearing kinase) can activate MKK3/6 (MAPK kinase 3/6) that acts directly upstream of p38 MAPK. The JNK pathway is also known as the stress activated protein kinase (SAPK) pathway because it becomes activated upon cell stress exposure, such as UV radiation, heat shock, or inflammatory cytokines (WINSTON u. HUNTER 1996).
JNK/SAPK shares a forty to forty-five percent homology to the classical MAP kinases. MEKK1,4, MLK3, or ASK1 (apoptosis signal-regulating kinase 1) can activate MKK4/6 that in turn JNK/SAPK. The active JNK/SAPK can phosphorylate c-Jun at serine63 and serine73.
2.3.3 PKC pathway
The PKC is a cyclic nucleotide-independent but phospholipide-dependent enzyme that phosphorylates serine and threonine residues in many target proteins. It plays an important role in the regulation of cell proliferation, differentiation, and apoptosis (NEWTON 1997; MELLOR u. PARKER 1998; WEBB et al. 2000; YOSHIDA 2007).
PKC seems also to be involved in the pathogenesis of different types of cancer, such as breast cancer, adenomatous pituitaries, leukemia, lung cancer, and colon adenocarcinomas (MACKAY u. TWELVES 2007).
So far eleven closely related PKC isozymes have been identified and divided into conventional (α, β1, β2, γ), novel (δ, ε, η, θ, µ), and atypical (ζ, λ) isozymes depending on their requirement for cofactors: conventional isozymes require both, calcium and diacylglycerol (DAG) for activation, the novel isozymes require only DAG and the atypical isoforms require neither calcium or DAG for activation. These isoforms also differ in their structure, biochemical properties, tissue distribution, subcellular localization, and substrate specificity, but all PKCs can interact with the plasma membrane by a phospholipid-binding domain. In resting state most PKCs are localized in the cytosol. If an effector molecule binds to a specific subtype to a G-protein coupled-receptor Gq at the cell membrane phospholipase C (PLC) or phospholipase A2 (PLA2) is activated generating DAG and inositol-1,4,5-trisphosphate (IP3) by hydrolysis of phosphatidylinositol-4, 5-bisphosphate (PIP2) in the cell membrane (Figure 2-4). IP3 results in a release of endogenous Ca2+ from the endoplasmatic reticulum. Ca2+ and DAG lead to activation of classical PKCs and
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activated classical PKCs are translocated to the cell membrane. Phorbol esters, such as phorbol 12-myristate 13-acetate (PMA), are experimentally used as DAG analogues facilitating activation of conventional and novel PKC isoforms (GOEL et al.
2007).
Figure 2-4: The PKC signaling pathway.
R3, receptor 3; R4, receptor 4; R6, receptor 6; Gq, G-Protein q; PLC, phospholipase C; PKC, protein kinase C; ER, endoplasmatic reticulum; PIP2, phosphatidylinositol-4, 5-bisphosphate; IP3, inositol-1,4,5-trisphosphate; DAG, diacylglycerol; IP3-R, IP3-receptor (MEISENBERG u. SIMMONS 2006).
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Chapter 3
The Phosphatidylinositol 3-Kinase Signaling Pathway Exerts Protective Effects during Sepsis by Controlling
C5a-Mediated Activation of Innate Immune Functions
Christiane D. Wrann, Navid A. Tabriz, Tanja Barkhausen, Andreas Klos, A., Martijn van Griensven, Hans C. Pape, Daniel O. Kendoff, Rengfeng Guo, Peter A. Ward,
Christian Krettek and Niels C. Riedemann
Journal of Immunology, 2007 May 1;178(9):5940-8
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The extent of Christiane D. Wrann’s contribution to the article is evaluated according to the following scale:
A: has contributed to collaboration (0-33%) B: has contributed significantly (34-66%)
C has essentially performed this study independently (67-100%)
Design of the project including design of individual experiments: B
Performing of the experiments of the study: C
Analysis of the experiments: C
Presentation and discussion of the study in article form: B
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Abstract
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-α, 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-1β 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.
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Introduction
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 –11). Earlier work suggested that C5a generation during sepsis plays a critical role for suppression of neutrophil innate immune functions (11–14). 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, 19–21).
Various studies then reported the importance of PI3K activation for neutrophil sequestration in inflamed tissue in different animal models (22–25). These results implicated an important role of this signaling pathway for the innate immune
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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 Reagents
Human recombinant C5a 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 interphase 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 6x106 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 2x106 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)-3β, phosphatise and tensin homolog deleted on chromosome 10, and
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phosphoinositidedependent 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-α, IL-1β, 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 (50ng/ml), or both for 6 h in an incubator with 5% CO2 under sterile conditions. Cell supernatants were then 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 twothirds of the cecum were 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.
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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-α, IL-10, MCP-1, IL-12, IFN-γ, 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 (3.3x107 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
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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 opsonised bacteria (E. coli) (6.7x108 bacteria/ml), PMA (1.4µM), 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.
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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
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
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