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The Journal of Immunology, 2005, 175: 1903-1910.
Copyright © 2005 by The American Association of Immunologists

IL-4 and IL-13 Induce Protection of Porcine Endothelial Cells from Killing by Human Complement and from Apoptosis through Activation of a Phosphatidylinositide 3-Kinase/Akt Pathway1

John F. Grehan, Brett K. Levay-Young, Jeremy L. Fogelson, Vanessa François-Bongarçon, Barbara A. Benson and Agustin P. Dalmasso2

Departments of Surgery and Laboratory Medicine and Pathology, University of Minnesota School of Medicine, Minneapolis, MN 55455


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vascular endothelial cells (EC) perform critical functions that require a balance of cell survival and cell death. EC death by apoptosis and EC activation and injury by the membrane attack complex of complement are important mechanisms in atherosclerosis and organ graft rejection. Although the effects of various cytokines on EC apoptosis have been studied, little is known about their effects on complement-mediated EC injury. Therefore, we studied the abilities of various cytokines to induce protection of porcine aortic EC against apoptosis and killing by human complement, a model of pig-to-human xenotransplantation. We found that porcine EC incubated with IL-4 or IL-13, but not with IL-10 or IL-11, became protected from killing by complement and apoptosis induced by TNF-{alpha} plus cycloheximide. Maximal protection required 10 ng/ml IL-4 or IL-13, developed progressively from 12 to 72 h of incubation, and lasted 48–72 h after cytokine removal. Protection from complement was not associated with reduced complement activation, C9 binding, or changes in CD59 expression. Inhibition of PI3K prevented development of protection; however, inhibition of p38 MAPK or p42/44 MAPK had no effect. IL-4 and IL-13 induced rapid phosphorylation of Akt. Although protection was inhibited by an Akt inhibitor and a dominant negative Akt mutant transduced into EC, it was induced by transduction of EC with the constitutively active Akt variant, myristylated Akt. We conclude that IL-4 and IL-13 can induce protection of porcine EC against killing by apoptosis and human complement through activation of the PI3K/Akt signaling pathway.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Vascular endothelial cells (EC)3 perform a multiplicity of critical functions that require a delicate balance in the EC response to stimuli that may promote cell survival, cell growth, or cell death (1). Adjustments in the EC response to these stimuli are required for the EC to perform their multiple functions, such as controlling the transit of molecules and cells through the vessel wall, maintaining the reactivity of the vasculature, regulating inflammation, and contributing to the fluidity of the blood and coagulation systems. If these EC responses become unbalanced, however, the EC may become pivotal in the development of pathologic processes, such as exaggerated or persistent inflammatory states, vascular diseases, or tumor expansion and metastasis. Additionally, the EC response to stimuli that affect cell survival plays a central role in ischemia/reperfusion injury and in allograft and xenograft injury (1, 2). The predominance of cell survival, often measured as protection from apoptosis, may serve to regulate the response to noxious stimuli, so that only severely injured cells are destroyed, as, for example, in inflammation. In other cases, the predominance of cell survival may adversely affect the occurrence or progression of a pathological process, as in remodeling of the myocardium in cardiac hypertrophy or intimal hyperplasia in the vessel wall after vascular injury.

Different cytokines are known to promote the survival of HUVEC through activation of various signaling pathways. Fibroblast growth factor (3), platelet-derived growth factor, RANKL/TNF-related activation-induced cytokine (4), angiopoietin-1 (5), TNF-{alpha}, and IL-1 (6) all use PI3K and Akt, although IL-11 requires MAPK/ERK1 (7). Additionally, IL-4 and IL-13, which are known to promote cell survival through inhibition of apoptosis in various cell types, were found to protect human EC from apoptosis, in association with up-regulation of A1 and A20 expression (8). Moreover, in a rodent model of xenograft accommodation, an infiltrate of predominantly Th2 mononuclear cells that produce protective cytokines, including IL-4 and IL-13, was observed in grafts that became accommodated (9).

Yet, despite extensive studies of the antiapoptotic properties of these various mediators, limited studies have been performed on the induction of protection against injury in normal EC resulting from processes that may include EC activation and injury mediated by the membrane attack complex of complement (MAC). Although previous work has shown that IL-11 induced protection of HUVEC from apoptosis and killing by rabbit complement (7), it is still unknown whether other cytokines induce protection from complement-mediated activation and injury of normal cells independently of effects on the expression of membrane-associated complement regulators, such as decay-accelerating factor and CD59 (10). Therefore, to address this question, we conducted parallel studies on protection from apoptosis and from complement-mediated killing of porcine EC exposed to several cytokines that are thought to be cytoprotective. Understanding the regulation by cytokines of the response of EC to complement and agents that induce apoptosis is of current interest in studies of the pathophysiology of vascular diseases and graft rejection. In these studies, we investigated the effect of protective cytokines on porcine EC, because apoptosis and complement-mediated injury of the vascular endothelium are critical in the rejection of a porcine organ transplanted into a primate (11, 12, 13). We found that porcine EC exposed to IL-4 or IL-13 become protected from the cytotoxic effects of human complement and from apoptosis induced with TNF-{alpha} plus cycloheximide (TNF-{alpha}/CHX). We also found that induction of protection requires participation of the PI3K/Akt signaling pathway.


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

DMEM, RPMI 1640, HEPES, HBSS with Ca/Mg, L-glutamine, FBS, and antibiotics were purchased from Invitrogen Life Technologies. Pig cytokines TNF-{alpha} and IFN-{gamma} were obtained from BioSource International, pig IL-4 was purchased from R&D Systems, and IL-13 was a gift from M. Murtaugh (University of Minnesota, Minneapolis, MN). Human IL-1{alpha}, TNF-{alpha}, IFN-{gamma}, TGF-{beta}2, IL-4, IL-10, IL-11, and IL-13 were obtained from R&D Systems. Neutral red and general chemicals were purchased from Sigma-Aldrich, and 3,3,3',3'-tetramethylbenzidine was obtained from Pierce. LY294002, SB202190, PD98059, and NL-71-101 were obtained from Calbiochem. Mouse IgG mAbs against human C3bi and C3c were purchased from Quidel, and MEL-2, a mouse IgG mAb against human CD59, was a gift from P. Morgan (University of Wales, Cardiff, U.K.). Ab against human Akt was purchased from Santa Cruz Biotechnology, Abs against caspase 3 and pAkt-Ser473 were obtained from Cell Signaling Technology, and anti-actin was purchased from Chemicon International. Affinity-isolated, HRP-conjugated, goat anti-mouse IgG and Fluoromount-G were purchased from Southern Biotechnology Associates, and affinity-isolated, FITC-conjugated, donkey anti-mouse IgG and HRP-conjugated donkey anti-rabbit IgG were purchased from Jackson ImmunoResearch Laboratories.

Endothelial cells

EC were explanted from pig aorta, cultured, and identified as previously described (14). Cells were maintained in DMEM containing L-glutamine, gentamicin, penicillin/streptomycin, and 10% FBS. The cells were identified as EC by their morphology and cobblestone appearance when confluent and by the ability to take up acetylated low density lipoprotein. Experiments were performed with cells in passages 4–9, 2–3 days after confluence (unless otherwise indicated), in gelatin-coated tissue culture plates as follows: 96-well, flat-bottom plates for ELISA, 48-well plates for cytotoxicity, 24-well plates for apoptosis, and six-well plates for flow cytometry and immunoblotting (Costar). All incubations were conducted at 37°C in a 5% CO2 atmosphere unless otherwise indicated.

Treatment of EC with cytokines

EC were preincubated in DMEM containing 1% FBS (100 µl/well for ELISA studies, 250 µl/well for complement cytotoxicity, 500 µl/well for apoptosis, and 2.5 ml/well for immunoblotting and for complement studies that required flow cytometry) for different time periods, as indicated. In some experiments, the cytokine solution was removed after various time periods, and the cells were washed twice with DMEM-1% FBS. Then the same volume of medium as that used in the previous step was added, and incubation was continued for various time periods. At this point the cells retained a normal capacity to take up the vital dye neutral red, indicating that their viability had not been impaired (15).

Complement-mediated cytotoxicity

Pretreated cells, in triplicate wells, were washed twice with RPMI 1640–0.5% BSA or 1 mM HEPES in HBSS containing 0.5% BSA. After washing, the EC were incubated for 2 h with 150 µl of a human serum pool in RPMI 1640 as a source of anti-pig Abs and complement or 50 µM H2O2 in 1 mM HEPES-HBSS. The serum pool, which was prepared from normal donors and stored at –70°C, had a normal complement level and a high titer of anti-pig EC Abs (16) and was used at a final concentration of 20–25%. After exposure to serum or H2O2, EC cytotoxicity was measured with neutral red as described previously (14). The percent specific cytotoxicity was calculated after correction for the OD of solubilized cells not treated with the dye, as follows: percent cytotoxicity = (1 – (test OD/control OD)) x 100. Values are given as the mean ± SE of triplicate samples. Neutral red is a vital dye and, as such, is not taken up by dead cells regardless of whether the cells died from necrosis or apoptosis. However, we have previously established the validity of this assay to measure necrotic cell death from human complement at serum concentrations and incubation times similar to those we used in this study; the percent killing was the same whether killing was measured with 51Cr release, a standard method to measure necrosis, or with neutral red (14). We have now confirmed the validity of the neutral red assay for the complement studies by comparing the degree of killing measured with lactate dehydrogenase (LDH) release (Roche), a second standard method to measure necrosis, and neutral red. Using 20% human serum and 2-h incubation at 37°C, LDH release was 74.5 ± 7.5%, and killing measured with neutral red was 74.0 ± 3.6% (one representative experiment of three, performed in triplicate wells). These data imply that at the conditions used in our studies, complement kills porcine EC by necrosis, not by apoptosis. Moreover, we analyzed complement-treated EC by flow cytometry for the presence of apoptosis and found none (see Results, Fig. 3A).



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  		FIGURE 3. Porcine EC incubated with human complement at conditions that cause necrotic killing do not exhibit apoptosis, and IL-4 and IL-13 induce protection of EC from apoptosis induced with TNF-{alpha}/CHX. A, EC were incubated in medium alone or in medium containing 20% human serum for 2 h at 37°C. The EC were tested for apoptosis by flow cytometry to determine the population distribution of EC DNA content. No evidence of significant apoptosis is seen. B and C, EC were incubated at 37°C for 72 h in medium alone or in medium containing 10 ng/ml IL-4 (B) or 20 ng/ml IL-13 (C). After washing, the EC were tested for sensitivity to apoptosis induced by incubation with TNF-{alpha}/CHX for 4 h. Analyses by flow cytometry showed induction of protection from apoptosis by IL-4 (B), and Hoechst staining demonstrated induction of protection from apoptosis by IL-13 (C; the percent apoptosis is given in each panel). D, Immunoblot analysis of extracts from EC that were pretreated with 20 ng of IL-13 for 48 h showed abrogation of caspase 3 cleavage after incubation with TNF-{alpha}/CHX for 3 h. E, Protection from apoptosis was induced in a dose-dependent manner after continuous incubation of EC with IL-4 and IL-13 for 48 h. IL-10 did not induce protection. Apoptosis was assessed by flow cytometry, and results were normalized to 50% apoptosis of EC incubated with medium without cytokine. F, Time of incubation with IL-4 influenced the degree of protection from apoptosis in EC that were incubated with 20 ng of IL-4. Apoptosis was assessed by flow cytometry. G, Duration of protection from apoptosis after incubation with 20 ng of IL-4 for 24 h; sensitivity to apoptosis was tested immediately after removal of IL-4 and at various times thereafter. Apoptosis was assessed by flow cytometry. Results are representative of three independent experiments.

 
Binding of complement components and expression of CD59

Pretreated cells, in duplicate, were washed twice with RPMI 1640-0.5% BSA and incubated for 30 min with 1.5 ml of 20% serum or 20% heat-inactivated serum in RPMI 1640, and then binding of complement components C3 and C9 was measured by flow cytometry. The cells were detached with 0.05% trypsin, washed, and suspended at 1 x 106/ml in PBS-2% FBS-0.1% NaN3; then 100 µl was incubated with 0.5 µg of mouse IgG1 mAb against C3c or 2.5 µg of mAb against C9 neoantigen (17) for 30 min on ice, and cells were washed twice. C3 was detected by incubation with FITC-donkey anti-mouse IgG, and C9 was detected with biotin rat anti-mouse IgG, followed by avidin-PE. After two washes, the cells were fixed with 1% paraformaldehyde and analyzed using a FACSCalibur flow cytometer (BD Biosciences). In some experiments deposition of C3bi and C9 on the EC and expression of CD59 were assessed by ELISA as previously described (18).

Induction and measurement of apoptosis

Pretreated EC in 6- or 24-well plates were exposed to porcine rTNF-{alpha} (20 ng/ml) and CHX (3 µg/ml), as previously described, to induce apoptosis (19). Apoptosis was measured by Hoechst staining for microscopy or by flow cytometry to analyze the population distribution of EC DNA content after propidium iodide staining (19). Total cell lysate was prepared as described previously (6). Activation of caspase 3 was assessed by immunoblotting.

Western blot analysis

The pretreated EC were washed twice with 5 ml of PBS containing 100 µM NaVO4, then each well received 100 µl of a lysing buffer containing 50 mM TBS (pH 6.8), 1% Triton X-100, 5 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.5 mM PMSF. The cells were incubated at 4°C for 20 min, collected, passed through a 27-gauge needle to shear the DNA, and placed into a 1.5-ml conical microfuge tube on ice for 15–30 min. Insoluble materials were removed by centrifugation, and the cell extracts were stored at –80°C. Cell extracts (20 µg) were electrophoresed under reducing conditions in 10% NuPage gels (Invitrogen Life Technologies). Proteins were transferred onto Immobilon-P membranes (Millipore) and blocked with 5% nonfat milk in TBS-0.1% Tween 20, then incubated with rabbit anti-phospho-Akt-Ser473, followed by HRP-conjugated donkey anti-rabbit IgG. Bands were visualized by chemiluminescence using Lumi-glo (Cell Signaling Technology). The blots were stripped with ReBlot Plus (Chemicon International), incubated with anti-Akt, and visualized as described above.

Adenoviral transfection

EC were transduced with adenovirus (provided by K. Walsh, St. Elizabeth’s Medical Center, Boston, MA) expressing either constitutively active myristylated Akt (myr-Akt) or dominant negative, inactive Akt as previously described (20, 21). Recombinant control adenoviruses lacked the insert (Imgenix) or expressed {beta}-galactosidase (LacZ; BD Biosciences). Adenovirus was propagated on 293 cells and used as unpurified culture lysates. Adenovirus titer was determined on 293 cells using supernatant from 2Hx-2 hybridoma (American Type Culture Collection), followed by anti-mouse second Ab. EC at 70% confluence were transduced at multiplicity of infection (MOI) of 100–250 PFU/cell in 10% FBS-DMEM for 16 h at 37°C in 5% CO2. The supernatants were removed, fresh medium was added, and the EC were incubated for 24 h to confluence before use. The EC were washed, incubated with medium or cytokine, and then tested for sensitivity to complement. Transduction efficiency on EC was determined with LacZ adenovirus; 48 h after infection, the EC were fixed and stained for {beta}-galactosidase. Efficiency was ~80% at 100 MOI and >95% at 250 MOI.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
IL-4 and IL-13 induce protection of porcine EC from killing by human complement without interfering with complement activation, the amount of C9 that binds to EC, or EC expression of CD59

EC that were preincubated with human IL-4 or IL-13 for 24 or 72 h were protected from cytotoxicity by human complement in a dose-dependent manner; maximal protection was usually obtained with 10 ng/ml of either cytokine (Fig. 1, A and B). Porcine IL-4 and IL-13 were equally efficient as the corresponding human cytokines at inducing protection from complement (results not shown). In contrast to IL-4 and IL-13, no protection from complement was achieved with IL-10, IL-11, or TGF-{beta}2 over a wide range of cytokine concentrations (Fig. 1, A and B). To ensure maximal response, in subsequent experiments we generally used a cytokine concentration of 20 ng/ml. After 12 h of incubation with 20 ng/ml IL-4, the EC exhibited significant protection from complement; this protection increased progressively through the 72 h of incubation with IL-4 (Fig. 1C). EC that were incubated with 20 ng of IL-4 for 24 h maintained protection from complement after incubation for 48–72 h in medium without IL-4 (Fig. 1D). In both cases similar results were obtained with IL-13 (results not shown). IL-4 and IL-13, each at 2.5 ng/ml, when used together, induced the same degree of protection from complement killing as each cytokine alone at 5 ng/ml (Fig. 1E). Not shown are similar results that were obtained with cytokine concentrations of 1, 10, or 20 ng/ml. Thus, the protective effect of both cytokines is additive, not synergistic. EC pretreated with 20 ng of IL-4 for 24 h showed no modification in the sensitivity to killing by 50–75 µM H2O2 (results not shown).



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  		FIGURE 1. IL-4 and IL-13 induce protection of porcine EC from killing by human complement. EC were incubated at 37°C in medium alone or in medium containing various doses of cytokines. After washing, the EC were tested for sensitivity to complement by incubation for 2 h at 37°C with human serum as a source of anti-pig Ab and complement, as described in Materials and Methods. A and B, Protection was induced in a dose-dependent manner after continuous EC incubation with IL-4 and IL-13 for 24 h (A) or 72 h (B); IL-10, IL-11, and TGF-{beta}2 did not induce protection. Results were normalized to 50% cytotoxicity of EC incubated with medium without cytokine. C, Time of incubation with IL-4 influenced the degree of protection from complement in EC that were incubated with 20 ng IL-4. D, Duration of protection from complement after incubation with 20 ng of IL-4 for 24 h; sensitivity to complement was tested immediately after removal of IL-4 and at various times thereafter. E, Induction of protection from complement killing by IL-4 and IL-13 together. EC were incubated with 5 ng/ml IL-4, 5 ng/ml IL-13, or 2.5 ng/ml IL-4 plus 2.5 ng/ml IL-13 for 48 h, then tested for sensitivity to complement. *, Medium vs IL-4, medium vs IL-13, and medium vs IL-4 plus IL-13: p < 0.001. **, IL-4 vs IL-13, IL-4 vs IL-4 plus IL-13, and IL-13 vs IL-4 plus IL-13: p > 0.05. All experiments were performed with 20–25% human serum. Results are representative of at least three independent experiments.

 
The protection from complement-mediated cytotoxicity induced in EC by pretreatment with IL-4 was not associated with decreased binding of complement components C3 or C9 to the EC, assessed by flow cytometry (Fig. 2) or ELISA (results not shown). Expression of membrane-associated complement inhibitor CD59 by EC after incubation with IL-4 was similar to that of EC exposed to medium alone (results not shown). These experiments indicate that protection results from inhibition of the cytotoxic effects of the MAC of complement, not from a reduction in complement activation or binding or from up-regulated expression of CD59.



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  		FIGURE 2. IL-4 pretreatment does not reduce C3 and C9 binding to EC incubated with human serum. EC were incubated in medium (A and C) or in medium containing 20 ng of IL-4 (B and D) for 24 h at 37°C. After washing, the cells were incubated for 30 min at 37°C with medium (dotted line), 20% heat-inactivated human serum (continuous line), or 20% human serum as a source of anti-pig Ab and complement (gray histogram). Bound C3 (A and B) and C9 (C and D) were assessed by flow cytometry.

 
Porcine EC incubated with human complement at conditions that cause necrotic killing do not exhibit apoptosis

The conditions used in this study to examine protection from complement killing of porcine EC are thought to result in necrotic cell death. Using conditions that caused little necrotic cell death, however, Nauta et al. (22) reported that complement induced apoptotic cell death of rat mesangial cells if incubation was conducted for 14 h after treatment with complement at sublytic doses for 1 h. Therefore, it was important to establish whether in our complement experiments there was any contribution of apoptosis to killing, because we assessed the degree of killing and cytokine-induced protection with the vital dye neutral red, which does not distinguish between necrotic and apoptotic cell death. As described in Materials and Methods, we established that with the conditions used in this study, the degree of EC complement killing measured with neutral red, 51Cr, and LDH was the same, which suggests that apoptosis was not a cause of cell death in these experiments. We also examined EC that had been incubated with human complement (20% serum, 2 h) for the possible presence of apoptosis using flow cytometry and demonstrated that few of these cells died by apoptosis (Fig. 3A). Thus, the assessment of protection from complement killing reported in this study represents protection from necrotic cell death. Our results with porcine EC are in agreement with the findings of Nauta et al. (22) that under conditions of complement treatment that result in degrees of lysis similar to those obtained in this study, rat mesangial cells are killed by necrosis, not by apoptosis.

IL-4 and IL-13 induce protection of porcine EC from apoptosis elicited by TNF-{alpha}/CHX

Flow cytometric analyses showed that EC that were preincubated with IL-4 for 72 h were partially protected from apoptosis elicited by incubation with TNF-{alpha}/CHX (Fig. 3B). EC that were preincubated with IL-13 for 72 h were also partially protected from apoptosis elicited by incubation with TNF-{alpha}/CHX, as shown by Hoechst staining visualized by microscopy (Fig. 3C). Moreover, pretreatment with IL-13 resulted in inhibition of caspase 3 activation (Fig. 3D). EC that were preincubated with human IL-4 or IL-13 for 48 h were protected from apoptosis in a dose-dependent manner (Fig. 3E). Maximal protection was usually achieved with 10 ng/ml of either cytokine. Similar results were obtained with porcine IL-4 and IL-13 (results not shown). In contrast to IL-4 and IL-13, no protection from apoptosis was achieved with IL-10 (Fig. 3E) or IL-11 (not shown). After 12 h of incubation with 20 ng/ml IL-4, the EC showed some protection from apoptosis, which increased as incubation was prolonged (Fig. 3F). EC that were incubated with 20 ng of IL-4 (or IL-13, not shown) for 24 h maintained the protection from apoptosis for 48 h of incubation in medium without IL-4 (Fig. 3G). Thus, IL-4 and IL-13 induced protection of EC from apoptosis with similar characteristics to protection from complement, albeit usually somewhat less pronounced.

Induction of protection of porcine EC with IL-4 and IL-13 requires protein synthesis and participation of PI3K

Protection from complement killing did not develop when EC were incubated with IL-4 or IL-13 in the presence of 2.5 µM CHX (Fig. 4A). This experiment suggests that protection is due to an active process that requires protein synthesis and that protection is not related to preformed molecules that are routed to new compartments in the cell, nor is it due to inhibition of metabolism. In addition, residual or carryover effects of IL-4/IL-13 signaling are not manifest, because the cells are provided a recovery period of 24 h in the absence of CHX and cytokine. The presence of the PI3K-specific inhibitor LY294002 during incubation of EC with IL-4 or IL-13 reduced the development of protection from complement (Fig. 4, B and C). Inhibition of PI3K also reduced the protection from apoptosis, although to a lesser extent (Fig. 4D). In contrast, the p38 MAPK inhibitor SB202190 and the p42/44 MAPK inhibitor PD98059 had no effect on the development of IL-4-induced protection from complement (Fig. 4, E and F). The almost complete inhibition of complement-mediated killing induced by IL-4 in the data shown in Fig. 4, B and E, was characteristic of the EC line used in these experiments; however, with most other lines, partial protection was observed.



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  		FIGURE 4. Induction of protection of porcine EC with IL-4 and IL-13 requires protein synthesis and the participation of PI3K, but not of p38 MAPK or p42/44 MAPK. A, EC were incubated with 20 ng/ml IL-4 or IL-13 for 20 h with or without 2.5 µM CHX and washed, medium was added, and the incubation was continued for 24 h. The EC were then washed and tested for sensitivity to killing by complement. B–F, EC were incubated at 37°C for 1 h with medium containing the inhibitor concentrations indicated: the PI3K inhibitor LY294002, the p38 MAPK inhibitor SB202190, or the p42/44 MAPK inhibitor PD98059. The supernatants were then replaced with medium or medium containing 20 ng/ml IL-4 or IL-13; both contained the same concentration of the inhibitor that was used in the initial incubation. Incubation was continued for either 24 h (B and E) or 48 h (C, D, and F). EC were then washed and tested for sensitivity to killing by complement (20–25% human serum; A–C, E, and F) or apoptosis induced with TNF-{alpha}/CHX (D). Results are representative of at least three independent experiments.

 
Stimulation of porcine EC with IL-4 and IL-13 induces Akt phosphorylation

Western blot analyses of cell extracts of EC that were exposed to increasing amounts of IL-4 (or IL-13; results not shown) for 60 min showed phosphorylation of Akt occurring in a dose-dependent manner (Fig. 5A). Akt phosphorylation was maximal at 20 ng/ml IL-4 or 5–10 ng/ml IL-13, and there was no effect of IL-4 or IL-13 on the expression of total Akt. Akt phosphorylation progressed in a time-dependent manner in EC stimulated with 20 ng/ml IL-4 (Fig. 5B) or IL-13 (not shown), with maximum phosphorylation at 30 min. Akt phosphorylation was abrogated by the presence of low concentrations of the PI3K inhibitor LY294002 at the time EC were incubated for 60 min with 20 ng/ml IL-4 (Fig. 5C) or IL-13 (not shown). These experiments indicate that EC protection induced by IL-4 and IL-13 is associated with Akt phosphorylation.



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  		FIGURE 5. IL-4 induces PI3K-dependent phosphorylation of Akt in porcine EC. Cell extracts (20 µg of protein/lane) were resolved on SDS-PAGE and immunoblotted with specific Ab against either phospho-Akt-Ser473 (p-Akt) or total Akt (Akt). A, Dose-dependent Akt phosphorylation in EC exposed to IL-4 for 30 min at 37°C. B, Time-dependent Akt phosphorylation in EC exposed to 20 ng of IL-4. C, Inhibition of Akt phosphorylation by LY294002 in EC exposed to IL-4. EC were incubated at 37°C for 30 min with medium or medium containing 20 ng/ml IL-4 plus various doses of LY294002. A and B, Results of one of three independent experiments; C, results of one of two independent experiments.

 
Induction of protection of porcine EC from complement by preincubation with IL-4 requires the participation of Akt

We addressed the question of whether Akt activation is required for IL-4-induced protection from complement in three ways. First, we showed that the presence of a low concentration of the Akt inhibitor, NL-71-101, at the time EC were incubated with IL-4 interfered with the induction of protection (Fig. 6A). Second, we tested whether inhibition of Akt phosphorylation in EC that were transduced with dominant negative Akt would show impairment of the induction of protection. The results showed that EC transduced with dominant negative Akt failed to develop protection when incubated with IL-4 (Fig. 6B). Finally, we asked whether protection could be directly induced by transduction of EC with constitutively active myr-Akt. We found that EC transduced with this Akt variant exhibited significant protection from complement when tested 2 days after infection (Fig. 6C). These experiments demonstrate that Akt activation plays a fundamental role in IL-4-induced protection of EC from the cytotoxic effects of complement.



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  		FIGURE 6. Akt is required for induction of protection of porcine EC with IL-4 from killing by complement. A, EC were incubated at 37°C for 1 h with medium or medium containing 5 µM of the Akt inhibitor NL-71-101. The supernatants were then replaced with medium, medium containing 20 ng/ml IL-4, or medium containing 20 ng/ml IL-4 plus 5 µM NL-71–101, and incubation was continued for 24 h. EC were washed and exposed to human serum to determine the sensitivity to complement, as described in Materials and Methods. B, EC were transduced with 250 MOI of adenovirus control or dominant negative adenovirus-expressing Akt (DN Akt), washed, incubated with medium or cytokine for 48 h, and tested for sensitivity to complement. C, EC were incubated at 37°C for 16 h with medium or medium containing 100 MOI of myr-Akt (Myr Akt) or adenovirus expressing LacZ (Control). The cells were washed, medium was added, and incubation was continued for 2 days. EC were then tested for sensitivity to complement. All experiments were performed with 20–25% human serum. A and B, Results of one of three independent experiments; C, results of one of two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
From these studies we report the following new findings. First, porcine EC exposed to IL-4 or IL-13 become protected from the cytotoxic effects of human complement, and this protection requires participation of the PI3K/Akt signaling pathway. Second, after stimulation with either cytokine, the characteristics of the protection from complement and from apoptosis induced with TNF-{alpha}/CHX are similar; protection develops with the same time course, requires a similar cytokine dose, and persists for 2–3 days after removal of the cytokine. These findings suggest that similar mechanisms are involved in the protection from two distinct types of cell death: apoptosis caused by TNF-{alpha}/CHX and necrosis caused by complement. We also found that the cytokines IL-4 and IL-13 generally had similar potency to induce cytoprotection in porcine EC, and the characteristics of this protection were similar for both cytokines. These cytokines often have similar effects on target cells due to sharing of receptors (23). The IL-4R in EC, as in other cells of nonhemopoietic lineage, is a dimer of the IL-4R {alpha}-chain and the IL-13R {alpha}1-chain; although there are two types of IL-13R, one is the same as the IL-4R, and the other is a dimer of the IL-13R {alpha}1-chain and the decoy receptor IL-13R{alpha}2 (23). Our results show that by inducing protection from complement, when added together these cytokines have an additive effect and exhibit no synergy.

PI3K/Akt activation has previously been reported to be the signaling pathway that is activated by several growth factors and cytokines that may cause survival of HUVEC through inhibition of apoptosis (3, 4, 5, 6). This pathway has also been shown to be involved in protection of oligodendrocytes from apoptosis (24). In our current studies, LY294002, a specific PI3K inhibitor (25), strongly prevented Akt phosphorylation and impaired the development of protection. Akt activation is required for induction of protection, because both the Akt inhibitor NL-71-101 (26) and transduction of EC with dominant negative Akt abrogated IL-4-induced protection from complement. Moreover, EC transduced with constitutively active myr-Akt spontaneously exhibited protection from complement. We are currently studying mechanisms downstream of Akt that may participate in the induction of protection. Although previous findings with HUVEC showed that protection from apoptosis induced with IL-13 was associated with up-regulation of A20 mRNA (8), our unpublished results with porcine EC stimulated with IL-4 or IL-13 showed no change in the expression of either A20 or porcine inhibitor of apoptosis, Bad, or HO-1 proteins. Unexpectedly, however, these EC showed a marked and progressive reduction in Bcl-2 protein expression.

Few studies have investigated the induction of protection from the effects of MAC that are manifested despite uninhibited MAC assembly on the cell membrane. Most studies of protection from complement addressed mechanisms of up-regulation of expression of membrane-associated regulators of complement activation, such as decay-accelerating factor, MCP, and CD59 (10), or elimination of MAC complexes soon after their formation on the cell membrane (27, 28). However, studies by Fishelson et al. (29, 30) uncovered a modality of induced protection from cytotoxicity by the MAC distinct from those mentioned above, in which protection was induced in tumor cell lines by exposure to a sublytic dose of complement; protection was induced rapidly and required protein kinase C-mediated ERK activation. Sublytic complement was also found to induce protection from the lytic effects of the MAC in one type of normal cells, rat glomerular epithelial cells, through activation of p38 MAPK, a mechanism of protection that may be involved in a complement-dependent rat model of membranous nephropathy (31). In vitro studies have also shown that under certain conditions, including prolonged incubation, sublytic MAC may induce apoptosis of rat mesangial cells instead of protection (22). These studies suggest that the MAC is potentially capable of inducing a large variety of cellular responses, which are dependent on complement dose and target cell type: proliferation, inflammatory responses, protection from lytic complement, apoptosis, and, finally, necrotic cell death (22, 32, 33, 34). Many of these responses have been implicated in injury to organ allo- and xenografts (34, 35).

Porcine EC can develop cytoprotection by ligation of Gal{alpha}(1, 2, 3)Gal (abbreviated {alpha}Gal) epitopes with Abs or {alpha}Gal-binding lectins (14, 16) or by prolonged incubation with human i.v. Ig (36). Low concentrations of IL-11 were found to induce protection of HUVEC from complement killing through activation of STAT3 and p42/44 MAPK (7). Our findings that exposure of porcine EC to IL-4 and IL-13 induces protection from MAC indicate that these cytokines are able to elicit concomitant protection from two forms of cell death, apoptosis and complement-mediated necrosis, by sharing an induction process that involves PI3K/Akt activation. Moreover, the inability of inhibitors of p38 MAPK and ERK/MAPK to block cytoprotection shows that these pathways are not required.

One potential application of our in vitro finding that IL-4 and IL-13 can induce cytoprotection may be to protect an organ from the injury of ischemia/reperfusion and other types of injury occurring with organ transplantation. This application is supported by previous observations. For example, activation of Akt with insulin-like growth factor-I resulted in protection from ischemia/reperfusion injury in a mouse model (37), suggesting that IL-4 and IL-13 may ultimately be used in a similar manner. Additional support for this argument comes from the findings in a rodent model of xenograft accommodation where the presence of a predominantly Th2 mononuclear cell infiltrate was thought to play a role in the development of graft protection secondary to the production of protective cytokines, including IL-4 and IL-13 (9).

Our results with these cytokines on protection of pig EC from human complement and apoptosis suggest that it may be possible to improve the fate of a pig organ transplanted into a primate by using transgenic donor pigs that selectively express inducers of protection from immunologic injury, such as IL-4 or IL-13, in the vasculature of that organ. This approach may be more useful than other methods to induce protection such as exposure to i.v. Ig (36) or {alpha}Gal ligation with Abs or lectins (14, 16). Our results (unpublished) also suggest that pretreatment of porcine EC with IL-4 or IL-13 inhibits the expression of E-selectin, as shown previously with HUVEC (8). However, it has been shown that incubation of porcine EC with IL-4 causes a slow increase in P-selectin expression, which may contribute to leukocyte adhesion to the endothelium and promote inflammation (38). Regulated expression of protection in EC from complement, as described in this study with IL-4 and IL-13, may also be of interest in studies of various diseases, as, for example, in atherosclerosis, in which MAC may play an important pathogenic role due to its proinflammatory and mitogenic effects on EC (32, 33, 34).


   Acknowledgments
 
We acknowledge the excellent technical assistance of Robert Konz. We thank Dr. Kenneth Walsh for generously providing adenovirus expressing constitutively active Akt (myr-Akt) and dominant negative, inactive Akt.


   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 National Institutes of Health Grant RO1HL62195 and a grant from the Lillehei Heart Institute, University of Minnesota. J.F.G. was supported by National Research Service Award 1F32DK10006 and Training Grant T32HL07934 from the National Institutes of Health. Back

2 Address correspondence and reprint requests to Dr. Agustin P. Dalmasso, Department of Surgery, University of Minnesota, MMC 220, 420 Delaware Street S.E., Minneapolis, MN 55455. E-mail address: dalma001{at}umn.edu Back

3 Abbreviations used in this paper: EC, endothelial cell; CHX, cycloheximide; {alpha}Gal, Gal{alpha}(1–3)Gal; LDH, lactate dehydrogenase; MAC, membrane attack complex of complement; MOI, multiplicity of infection; myr-Akt, myristylated Akt. Back

Received for publication November 19, 2004. Accepted for publication May 16, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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