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Polymicrobial SepsisPathway Alters Innate Resistance to Modulation of the Phosphoinositide 3-Kinase

Lynne Brooks, Kevin Breuel and John B. SchweitzerOzment-Skelton, John H. Kalbfleisch, Johanna Preiszner, David L. Williams, Chuanfu Li, Tuanzhu Ha, Tammy

http://www.jimmunol.org/content/172/1/449doi: 10.4049/jimmunol.172.1.449

2004; 172:449-456; ;J Immunol 

Referenceshttp://www.jimmunol.org/content/172/1/449.full#ref-list-1

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Modulation of the Phosphoinositide 3-Kinase Pathway AltersInnate Resistance to Polymicrobial Sepsis1

David L. Williams,2* Chuanfu Li,* Tuanzhu Ha,* Tammy Ozment-Skelton,*John H. Kalbfleisch,† Johanna Preiszner,‡ Lynne Brooks,‡ Kevin Breuel,§ andJohn B. Schweitzer‡

We examined the effect of modulating phosphoinositide 3-kinase (PI3K) activity in a murine model of cecal ligation and puncture-induced polymicrobial sepsis. Inhibition of PI3K activity with wortmannin increased serum cytokine levels and decreased survivaltime in septic mice. We have reported that an immunomodulator, glucan phosphate, induces protection in murine polymicrobialsepsis. We observed that glucan stimulated tissue PI3K activity, which positively correlated with increased survival in septic mice.We investigated the effect of PI3K inhibition on survival in septic mice treated with glucan. Treatment of mice with the PI3Kinhibitors, wortmannin and LY294002, completely eliminated the protective effect of glucan, indicating that protection againstseptic mortality was mediated through PI3K. Inhibition of PI3K resulted in increased serum levels of IL1-�, IL-2, IL-6, IL-10,IL-12, and TNF-� in septic mice. Apoptosis is thought to play a central role in the response to septic injury. We observed thatinhibition of PI3K activity in septic mice resulted in increased splenocyte apoptosis and a change in the anatomic distribution ofsplenocyte apoptosis. We conclude that PI3K is a compensatory mechanism that suppresses proinflammatory and apoptoticprocesses in response to sepsis and/or inflammatory injury. Thus, PI3K may play a pivotal role in the maintenance of homeostasisand the integrity of the immune response during sepsis. We also observed that glucan phosphate decreased septic morbidity andmortality through a PI3K-dependent mechanism. This suggests that stimulation of the PI3K pathway may be an effective approachfor preventing or treating sepsis and/or septic shock. The Journal of Immunology, 2004, 172: 449–456.

T he critically ill patient frequently develops a complex dis-ease spectrum that may include adult respiratory distresssyndrome, systemic inflammatory response syndrome

(SIRS),3 sepsis syndrome, and/or septic shock (1). In the UnitedStates �750,000 patients/year develop sepsis syndrome (2). Ofthese patients, 51.1% receive intensive care, and the overall mor-tality rate is 28.6% (�215,000 deaths/year) (2). The annual cost ofsepsis syndrome is �16 billion U.S. dollars/year (2). Current wis-dom implies that after severe injury or infectious challenge somepatients respond by overexpressing inflammatory mediators thatresult in a systemic inflammatory response culminating in severeshock, multiorgan failure, and death (1). At present, we do notunderstand the cellular and molecular mechanisms that are in-volved in the initiation and propagation of septic injury, nor do we

understand the innate physiologic mechanisms that attempt to limitinflammation, maintain homeostasis, and promote survival in theseptic patient. In addition, attempts at developing effective thera-pies for sepsis syndrome have proven to be exceedingly difficult.Consequently, survival outcome in sepsis syndrome has not sig-nificantly improved over the last few decades (1).

The phosphoinositide 3-kinases (PI3K) are a conserved familyof signal transduction enzymes that are involved in regulating cel-lular proliferation and survival (3, 4). Recent data suggest that thePI3K pathway may play an important role as a negative feedbackregulator that limits proinflammatory responses (3, 5, 6). Guha andMackman (5) have reported that activation of the PI3K/Akt sig-naling pathway limits the proinflammatory effects of LPS in cul-tured monocytes. Fukao and Koyasu (6) have speculated that PI3Kmay be a negative feedback mechanism that prevents excessiveinnate immune responses. However, the role of the PI3K pathwayin response to a clinically relevant model of sepsis and inflamma-tion has not been studied. We investigated the effect of inhibitingPI3K activity in a murine model of polymicrobial sepsis (7–9). Wefocused on tissue PI3K activity, serum cytokine levels, splenocyteapoptosis, and survival outcome.

We have previously reported that the immunomodulator glucanphosphate (GP) induces protection in a murine model of cecalligation and puncture (CLP)-induced polymicrobial sepsis and sep-tic shock (9). We have been studying the cellular and molecularmechanisms associated with GP-induced protection in the CLPmodel (9, 10). An important finding was that GP down-regulatedthe early proinflammatory intracellular signaling pathways in CLPsepsis and that this correlated with improved outcome (9–11). Wehypothesized that the protective effect of GP may be due to acti-vation of early signaling pathways that suppress proinflammatorysignaling and promote survival at the cell, organ, and whole animallevels (9, 10). To test this hypothesis we examined the effect of GP

Departments of *Surgery, †Biometry and Medical Computing, ‡Pathology, and §Ob-stetrics and Gynecology, James H. Quillen College of Medicine, East Tennessee StateUniversity, Johnson City, TN 37614

Received for publication August 11, 2003. Accepted for publication October24, 2003.

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be hereby marked advertisement in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.1 This work was supported in part by U.S. Public Health Service Grant GM53522from the National Institute of General Medical Sciences, Grant AI45829 from theNational Institute of Allergy and Immunology, and Grant AT00501 from the NationalCenter for Complementary and Alternative Medicine (to D.L.W.). This work was alsosupported in part by U.S. Public Health Service Grant HL071837 from the NationalHeart, Lung, and Blood Institute and American Heart Association Grants 0051489Band 0255038B (to C.L.).2 Address correspondence and reprint requests to Dr. David L. Williams, Departmentof Surgery, James H. Quillen College of Medicine, East Tennessee State University,P.O. Box 70575, Johnson City, TN 37614-0575. E-mail address: williamd@etsu.edu3 Abbreviations used in this paper: SIRS, systemic inflammatory response syndrome;CARS, compensatory anti-inflammatory response syndrome; CLP, cecal ligation andpuncture; GP, glucan phosphate; PI3K, phosphoinositide 3-kinase.

The Journal of Immunology

Copyright © 2004 by The American Association of Immunologists, Inc. 0022-1767/04/$02.00

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on tissue PI3K activity in sepsis and whether inhibition of PI3Kactivity altered serum cytokine levels or survival outcome in GP-treated septic mice.

Materials and MethodsMice

Age- and weight-matched male ICR/HSD mice were obtained from HarlanSprague Dawley (Indianapolis, IN). The mice were maintained on standardlaboratory chow and water ad libitum with a 12-h light, 12-h dark cycle.Serologic testing confirmed that the mice were virus free. To examine themortality trend after CLP, groups of operated mice were monitored forsurvival for up to 192 h (8 days). All animal procedures were reviewed andapproved by the institutional review board for animal care at James H.Quillen College of Medicine, East Tennessee State University.

Reagents

We investigated the effect of GP (12) because it has been shown to exertprotection and increase survival in the CLP model (9). Water-soluble GPwas prepared and chemically characterized as previously described (12–16). The final product was stored (�80°C) as a lyophilized powder. It wasdissolved in aqueous media (5% (w/v) dextrose), filter-sterilized (0.45 �mpore size), and screened for endotoxin contamination with the Endospecyassay (Seigakaku, Tokyo, Japan), which is specific for endotoxin but doesnot respond to (133)-�-D-glucans (17, 18). Wortmannin was purchasedfrom Sigma-Aldrich (St. Louis, MO). LY294002 was purchased fromAlomone Laboratories (Jerusalem, Israel). LY294002 has limited solubilityin aqueous medium. We dissolved LY294002 or wortmannin in a smallvolume (50–100 �l) of DMSO (Sigma-Aldrich). The compounds werethen diluted with sterile PBS immediately before injection. Injection ofcontrol or CLP mice with the DMSO/PBS solution (vehicle) had no effecton PI3K activity or survival outcome. Dose-ranging experiments were per-formed with wortmannin and LY294002 to identify doses that would in-hibit in vivo PI3K activity without causing morbidity or mortality (Fig. 1).

CLP polymicrobial sepsis model

CLP was performed as previously described (9, 19, 20). Sham-operated(laparotomy only) mice served as the surgery and anesthesia controls, andmice that were not subjected to anesthesia or surgery served as the negativecontrols. After surgery the mice were injected s.c. with 1 ml of physiologicsaline solution for fluid resuscitation.

PI3K activity assay

Kinase activity was measured as previously described (10, 21). Briefly, 300�g of cytoplasmic proteins were immunoprecipitated with 2 �g of Abs tothe p85 subunit of PI3K (Santa Cruz Biotechnology, Santa Cruz, CA) at

4°C for 1 h, followed by the addition of 15 �l of protein A-agarose beads(Santa Cruz Biotechnology) for 1 h at 4°C. The precipitates were collectedby centrifugation (2500 rpm, 5 min, 4°C), washed twice in lysis buffer (250mM NaCl, 50 mM HEPES (pH 7.4), 1 mM EDTA, 1% Nonidet P-40, 1mM PMSF, 5 �g/ml aprotinin, 5 �g/ml leupeptin, 0.5 �g/ml pepstatin, 5�g/ml bestatin, and 5 �g/ml trypsin inhibitor), twice with solution I (100mM Tris-HCl (pH 7.5), 500 mM LiCl, and 0.1 mM sodium orthovanadateNa3VO4), twice with solution II (10 mM Tris-HCl (pH 7.2), 100 mM NaCl,and 1 mM EDTA) containing 0.1 mM Na3VO4) and twice with kinasebuffer (10 mM HEPES (pH 7.4), 1 mM MnCl2, 5 mM MgCl2, 12.5 mM�-glycero-2-phosphate, 50 �M Na3VO4, 2 �M NaF, 50 �M DTT, and 10�M ATP). The immunoprecipitates were then resuspended in kinase buffer(25 �l) and incubated with 10 �g of sonicated phosphatidylinositol (Sig-ma-Aldrich) and 10 �Ci of [�-32P]ATP (3000 Ci/mmol; Amersham Phar-macia Biotech, Piscataway, NJ) for 15 min at 30°C. The reactions werestopped by addition of 15 �l of 6 N HCl, and phospholipids were extractedwith 120 �l of chloroform-methanol (1/1), followed by centrifugation. Theorganic phase was collected from each sample (50 �l), dried with a Cen-trivap concentrator (Labconco, Kansas City, MO), dissolved in 50 �l ofchloroform/methanol (1:1), and extracted with 10 mM Tris-HCl (pH 7.4),150 mM NaCl, 5 mM EDTA, and 100 mM MgCl2, followed by centrifu-gation. The organic phase was collected and spotted (4 �l) on a silica gelTLC plate pretreated with 1% (w/v) potassium oxalate (Uniplate Silica gelH; Analtech, Newark, DE). Phosphorylated products were separated byTLC in a chloroform (60 ml), methanol (47 ml), water (11.3 ml), andammonium hydroxide (2 ml) developing solvent. The chromatograms werevisualized and quantified on a phosphorimager (Bio-Rad, Hercules, CA).

Serum cytokine measurements

Five mice from each group were euthanized at 12 h after surgery, andserum was harvested and stored in liquid nitrogen until assayed. Serumcytokine levels were assayed with a murine10 Plex cytokine assay (Bio-Source International, Camarillo, CA) on a Luminex 100 instrument (Aus-tin, TX). Specifically, we assayed the serum for IL-1�, IL-2, IL-4, IL-5,IL-6, IL-10, IL-12, TNF-�, GM-CSF, and IFN-�. Cytokine levels wereestablished by comparison with a standard curve according to the manu-facturer’s instructions.

Immunohistochemistry

Mice from each of the experimental groups were overdosed with anestheticand perfused with 2–3 ml of sterile saline through the portal vein. Tissueswere removed and immersion-fixed in 4% buffered paraformaldehyde, em-bedded in paraffin, cut at 5 �m, and stained with an Ab (1/250 dilution)directed against activated caspase-3 (Cell Signaling Technology, Beverly,MA). The sections were counterstained with hematoxylin and examinedwith brightfield microscopy.

Experimental protocol

Mice were pretreated with GP (40 mg/kg i.p.), wortmannin (200 nM), orLY294002 (2 mg/mouse) 1 h before CLP surgery. Mice were followed forsurvival in each group. When an animal became moribund it was humanelyeuthanized, and the time of euthanasia was recorded as the time of death.Sera for cytokine analysis were harvested at 12 h postsurgery. In parallelgroups, mice were sacrificed at 3 and 12 h after surgery. Tissues wereharvested at each time interval. The tissues were subdivided into two por-tions; one was processed for histopathology and caspase-3 immunohisto-chemistry, and the other was used for extraction of protein for the PI3Kassay.

Statistical analysis

Survival trends were compared with the log-rank Wilcoxon nonparametricprocedures and with parametric procedures assuming Weibull, log-normal,and normal survival distributions. Tissue PI3K activity and serum cytokinedata were summarized as the mean and SEM. Group mean responses werecompared by ANOVA and pairwise multiple comparison testing (the leastsignificant difference procedure or Tukey’s procedure for cases whereANOVA was not significant). A value of p � 0.05 was consideredsignificant.

ResultsWortmannin treatment increases susceptibility to CLP-inducedseptic mortality

Survival experiments were repeated twice. As similar results wereobtained in both experiments, the data were pooled. Pretreatmentwith wortmannin decreased ( p � 0.01) the median survival time

FIGURE 1. Pretreatment with wortmannin increased susceptibility topolymicrobial sepsis in CLP mice. The median survival time and the timeto 100% mortality were significantly reduced in wortmannin-treated mice.Wortmannin (200 nM) was administered i.p. 1 h before CLP. Administra-tion of wortmannin to control, no-surgery mice or sham-surgery mice didnot result in mortality. n � 18–20/group.

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and time to 100% mortality in polymicrobial sepsis (Fig. 1). Themedian survival time in the wortmannin-treated mice was �18 h( p � 0.01) compared with 26 h in the control mice (Fig. 1). Thetime to 100% mortality was 20 h ( p � 0.01) in the wortmannin-treated group vs 60 h in the control mice (Fig. 1). Administrationof wortmannin to control, no-surgery mice or sham-surgery micedid not induce mortality. Thus, the mortality observed is not due towortmannin administration.

Wortmannin or LY294002 treatment eliminated the protectiveeffect of GP in CLP-induced polymicrobial sepsis

In accordance with previous results (9), we observed that pretreat-ment of ICR/HSD mice with GP (40 mg/kg i.p.) increased ( p �0.01) long term survival (�60%) in mice with CLP-inducedpolymicrobial sepsis (Fig. 2, A and B). In contrast, wortmannin orLY294002 treatment completely eliminated the protective effect ofglucan in the septic model (Fig. 2, A and B). Mice treated withwortmannin and glucan showed a median survival time of 20 h and100% mortality at 36 h (Fig. 2A). Mice treated with LY294002 andglucan showed a median survival time of 24 h and 100% mortalityat 36 h (Fig. 2B).

Effect of PI3K inhibition on serum cytokine levels in CLP sepsis

There was no significant difference between the effects of wort-mannin and LY294002 on survival in the sepsis model. We em-

ployed these two PI3K inhibitors, which work via different mech-anisms (22, 23), to be certain that the effect we observed was dueto PI3K inhibition and was not an artifact specific to one of thepharmacologic agents. However, LY294002 is expensive and dif-ficult to work with due to solubility problems (24). Therefore, weelected to employ wortmannin for the studies of serum cytokinelevels, tissue PI3K activity, and splenic apoptosis.

Inhibition of PI3K activity with wortmannin resulted in in-creased ( p � 0.05) serum levels of the proinflammatory cytokinesIL-1�, TNF-�, and IL-6 12 h after induction of sepsis (Fig. 3).PI3K inhibition also resulted in increased serum IL-2, IL-10, andIL-12 in CLP mice 12 h after induction of sepsis (Fig. 4). Wort-mannin treatment did not significantly alter serum levels of IL-4,IL-5, GM-CSF, or IFN-� in CLP mice (data not shown).

Wortmannin administration to CLP mice treated with glucanresulted in increased serum IL-1� and IL-12 levels compared withthe CLP plus glucan controls (Figs. 3 and 4). The same trend wasobserved for IL-2, IL-6, IL-10, and TNF-�, although the increasesobserved in these cytokines was not statistically significant. Therewas no statistically significant difference between the serum cyto-kine levels in the CLP plus wortmannin group vs the CLP, GP, andwortmannin group (Figs. 3 and 4). We also observed that there wasno significant difference in serum cytokine levels in CLP micecompared with CLP mice treated with glucan (Figs. 3 and 4).

In striking contrast to the effect of wortmannin treatment oncytokine levels in septic mice, we observed that wortmannin ad-ministration to control mice (no surgery or anesthesia) resulted indecreased ( p � 0.05) serum IL-2, IL-10, IL-12, and TNF-� levelscompared with control mice (no surgery or anesthesia) that did notreceive wortmannin (Figs. 3 and 4).

Tissue PI3K activity in CLP mice treated with glucan and/orwortmannin

Fig. 5, a–c, shows PI3K activity in the liver, lung, and heart at 3 hpost-CLP. Although there were differences among the tissues, theoverall trend was that PI3K activity increased in response to glu-can, sham surgery, or CLP (Fig. 5). When glucan was administeredto CLP mice, tissue PI3K increased relative to all the appropriatecontrols (Fig. 5). Wortmannin inhibited ( p � 0.05) tissue PI3Kactivity in the presence of CLP or CLP plus glucan (Fig. 5). Wort-mannin suppressed lung and heart PI3K activity in the presence ofCLP and glucan by 58 and 62% ( p � 0.05), respectively. HepaticPI3K activity was decreased by a nonsignificant 32.4% comparedwith that in the CLP plus glucan group (Fig. 5a). There was nosignificant difference between heart and lung PI3K activity in theCLP plus wortmannin vs CLP, wortmannin, and glucan groups.However, there was a significant increase in liver PI3K activitybetween these two groups (Fig. 5). By 12 h postsurgery there wasno significant difference in PI3K activity among the various groups(data not shown).

Effect of glucan and/or wortmannin on splenocyte apoptosis inpolymicrobial sepsis

There was no difference in splenocyte apoptosis at 3 h postsurgery.However, we observed dramatic differences at 12 h postsurgery.Immunohistochemistry with Ab to activated caspase-3 revealedonly an occasional positive cell in the spleen of mice given vehiclealone (Fig. 6a). Administration of wortmannin to control (no sur-gery) mice did not alter splenic apoptotic profiles (data not shown).Sham surgery increased the incidence of positivity (Fig. 6b). CLPtreatment plus vehicle further increased the number of caspase-positive cells (Fig. 6c). CLP plus treatment with glucan (Fig. 6d)produced a pattern of caspase positivity qualitatively similar to that

FIGURE 2. Inhibition of the PI3K pathway by wortmannin (A) orLY294004 (B) treatment completely eliminated the protective effect ofglucan in mice with CLP sepsis. Glucan (40 mg/kg) was administered 1 hbefore CLP. Wortmannin (200 nM) or Ly294004 (2 mg/mouse) was ad-ministered 1 h before CLP. n � 10–11/group.

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with sham surgery alone (Fig. 6b). We noted that caspase positiv-ity largely resided in the white pulp. The red pulp was more evi-dent as a pallid zone in septic animals than nonseptic controls,presumably due to a stress response. Treatment of CLP animalswith wortmannin caused a large increase in caspase-positive cellsand fragments (Fig. 6e) compared with the appropriate controls(Fig. 6, a–c). Caspase-3-positive cells were scattered throughoutall areas of the spleen, (i.e., both red and white pulp; Fig. 6e).

Treatment of CLP plus glucan mice with wortmannin (Fig. 6f)resulted in less caspase-3 positivity compared with the CLP pluswortmannin group (Fig. 6e). However, caspase positivity was stillqualitatively greater than with CLP alone and involved all areas ofthe spleen. We compared sham surgery, CLP alone, and CLP pluswortmannin groups (Fig. 6, g–i) using high magnification. Theincreased numbers of positive cells and the more advanced frag-mentation of apoptotic profiles was evident in the CLP (Fig. 6g)and the CLP plus wortmannin (Fig. 6h) groups. Fragments wereparticularly numerous in the CLP plus wortmannin group (Fig. 6i).

FIGURE 3. Treatment of CLP mice with wortmannin resulted in in-creased serum levels of IL-1�, TNF-�, and IL-6. Mice were subjected toCLP at time zero. Glucan (40 mg/kg) was administered 1 h before CLP.Wortmannin (200 nM) was administered i.p. 1 h before CLP. Serum forcytokine analysis was harvested at 12 h postsurgery. n � 5/group. �,p � 0.05.

FIGURE 4. Treatment of CLP mice with wortmannin resulted in in-creased serum levels of IL-2, IL-10, and IL-12. Mice were subjected toCLP at time zero. Glucan (40 mg/kg) was administered 1 h before CLP.Wortmannin (200 nM) was administered i.p. 1 h before CLP. Serum forcytokine analysis was harvested at 12 h postsurgery. n � 5/group. �,p � 0.05.

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DiscussionWe observed that inhibition of PI3K activity increased morbidityand mortality in experimental polymicrobial sepsis. In strikingcontrast, stimulation of PI3K activity strongly correlated with de-creased morbidity and improved survival outcome. Guha andMackman (5) have reported that the “PI3K-Akt pathway imposesa braking mechanism to limit the expression” of proinflammatorymediators in LPS-treated monocytes. Our data support the conclu-sion that the PI3K pathway ameliorates inflammatory disease. Ourresults also extend these observations to a clinically relevant ani-mal model of fulminating polymicrobial sepsis, which has a sig-nificant inflammatory component. Fukao and colleagues (25) havereported that p85� knockout mice showed impaired clearance ofenterobacteria injected into the peritoneal cavity; however, thisstudy did not examine morbidity or mortality in the p85� knockoutmodel. Our data extend this observation by demonstrating that

inhibition of PI3K in surgically induced peritonitis correlates withincreased disease severity, increased disease progression, and de-creased survival outcome. Of potentially greater significance, ourdata demonstrate that stimulation of the PI3K pathway correlateswith improved outcome. Although it is not clear that pharmaco-logic inhibition of PI3K (26) and genetic depletion of p85� havethe same effects, it is clear that both observations support a role forPI3K in innate host resistance to peritoneal infections. Fukao andKoyasu (6) have recently reviewed the role of PI3K in the regu-lation of Toll-like receptor-mediated inflammatory responses.They concluded that PI3K may be a negative feedback regulatorthat is crucial to the maintenance and integrity of the immunesystem (6). They also concluded that PI3K was important in main-taining the balance between Th1 vs Th2 responses in vivo (6). Ourdata also support a role for the PI3K pathway as a compensatoryand/or feedback mechanism that plays a role in innate host resis-tance by limiting inflammatory responses and promoting survivalin polymicrobial sepsis and septic shock. Fukao and Koyasu (6)have speculated that the “PI3K-mediated machinery could be anideal therapeutic target.” Our data support that contention by dem-onstrating that stimulation of the PI3K pathway may be an effec-tive approach to prevent and/or treat septic/inflammatory sequelaerelated to polymicrobial sepsis. However, not all reports have dem-onstrated a negative regulatory role for PI3K. Yum and colleagues(27) have reported that administration of endotoxin to PI3K�knockout mice resulted in decreased acute lung injury, suggestingthat the PI3K pathway played a role in the pathophysiology ofendotoxic pulmonary injury. The difference between our resultsand those of Yum et al. (27) may be due to the fact that we em-ployed a model of fulminating polymicrobial sepsis that is quitedifferent from the endotoxin lung injury model.

We observed that inhibition of PI3K by wortmannin orLY294002 completely eliminated the protective effect of GP inCLP sepsis. Previous data demonstrate that glucans will alter mor-bidity and mortality in various models of sepsis (9, 28). However,the precise mechanisms by which glucans altered the septic statewere unknown. The beneficial effect of glucans in sepsis has beenattributed to stimulation of innate immunity, increased bacterialclearance, increased bactericidal activity, and other nonspecific ef-fects (28, 29). Although any or all of these conclusions may bevalid, they also presented a paradox, in that glucans are wellknown immune stimulators (30). There is a mild inflammatoryresponse associated with glucan-induced immune stimulation (31,32). The pathophysiology of sepsis/septic shock suggests that aftersevere injury or infectious challenge the host responds by overex-pressing inflammatory mediators resulting in a systemic inflam-matory response that may produce severe shock, multiorgan fail-ure, and death (1). An important question is how aproinflammatory agent, such as glucan, could ameliorate a diseasethat has a significant inflammatory component? The present datashed new light on this paradox and, more importantly, suggest anew and unexpected mechanism of action for these immunomodu-lators in sepsis. Our data indicate that GP activates the PI3K path-way, which, in turn, results in decreased morbidity and increasedlong term survival in the sepsis model. We employed wortmanninand LY294002 to inhibit PI3K activity because these pharmaco-logic agents have been widely used to study the role of PI3K, andthey work by different mechanisms of action (26, 33). The fact thatthe protective effect of glucan could be completely abolished bytwo different PI3K inhibitors strengthens our contention that thePI3K pathway is responsible for the protection. To the best of ourknowledge, this is the first report of glucan stimulating PI3K ac-tivity and the first report that stimulation of PI3K results in animproved outcome in a clinically relevant model of septic injury.

FIGURE 5. Modulation of tissue PI3K activity in response to CLP-in-duced polymicrobial sepsis, glucan treatment, and/or wortmannin treat-ment. We observed that glucan treatment increased PI3K activity in thepresence of septic challenge. Wortmannin decreased tissue PI3K activity.Changes in tissue PI3K activity correlated with survival outcome (see Figs.2 and 3). n � 4/group. �, p � 0.05 vs no-surgery control; ��, p � 0.05 vsCLP plus glucan group; #, p � 0.05 vs CLP plus wortmannin group.

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FIGURE 6. Increased splenocyte apoptosis in CLP mice treated with wortmannin. Apoptosis was assessed by immunohistochemical staining foractivated caspase-3 at 12 h after CLP. Cell cytoplasm or cellular fragments that have activated caspase-3 stain brown. a, No-surgery, vehicle-treated miceshowed occasional positive cells. b, Sham-surgery, vehicle-treated mice showed a mild increase in positivity. c, CLP plus vehicle-treated mice showedscattered cells and cell fragments that were caspase positive; these appear mainly in germinal center-type regions of white pulp. d, CLP plus glucan produceda pattern of caspase positivity similar to that in the sham surgery group (b). e, In CLP plus wortmannin group, positive cells and fragments of cells weregreatly increased in number throughout the spleen. There was particular accentuation of apoptotic cells in regions of white pulp peripheral to germinal centerareas. Many positive cells appeared in red pulp and subcapsular areas, whereas such areas in the CLP plus vehicle-treated group (c) showed virtually nopositive cells or fragments. f, In the CLP, wortmannin, and glucan group, the total number of positive cells was decreased compared with that in the CLPplus wortmannin group (e), but the distribution of positive cells was approximately the same. However, the number of positive cells or fragments was stillgreater than in the CLP plus vehicle (c) or CLP plus glucan (d) groups. Magnification, �200. Using higher magnification (�400), the increased numbersof positive cells and the more advance fragmentation were evident in CLP plus wortmannin (h and i) vs CLP (g) groups.

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We are currently investigating the cellular and molecular mecha-nisms by which glucan stimulates PI3K activity. Such informationmay result in the identification of potential targets for rationaledrug design.

We observed that in vivo inhibition of PI3K in polymicrobialsepsis resulted in significant increases in circulating IL-1�, IL-2,IL-6, IL-10, IL-12, and TNF-�. These data are consistent with thein vitro studies by Guha and Mackman (5) regarding the brakingeffect of the PI3K/Akt pathway on mediator release. Our data arealso consistent with the findings of Fukao and colleagues (34), whohave reported increased IL-12 expression in wortmannin-treateddendritic cells. Fukao and Koyasu (6) have concluded that PI3Kplays a crucial role in balancing the Th1 vs the Th2 response. Theirdata indicate a negative regulatory role for PI3K via inhibition ofIL-12 expression during induction of Th1 responses (6, 34). Ourresults tend to support the conclusions of Fukao and colleagues (6,34). Our data extend those observations by demonstrating that inresponse to sepsis, PI3K serves as a negative regulatory mecha-nism for several important immunoregulatory and proinflamma-tory cytokines. It should also be noted that the precise role ofcirculating cytokines in the pathophysiology of sepsis/septic shockis controversial, and there is no definitive cause-and-effect rela-tionship between systemic cytokine levels and survival outcome insepsis (35). Nevertheless, it is clear that inhibition of PI3K resultsin increased serum cytokine levels during sepsis and increasedsusceptibility to septic mortality, but it is not clear that these twoevents are causally related.

We also noted that inhibition of PI3K had a differential effect onin vivo cytokine expression. In control mice, which were not ex-posed to anesthesia, surgery, or sepsis, PI3K inhibition with wort-mannin resulted in significantly lower serum levels of IL-2, IL-4,IL-10, IL-12, GM-CSF, and TNF-� (Figs. 3 and 4). This is instriking contrast to the effect of PI3K inhibition on cytokine ex-pression in the presence of sepsis. The significance of this differ-ential regulation of in vivo cytokine expression after PI3K inhibi-tion is not clear. However, the PI3K pathway may play a role inmaintaining basal levels of cytokine expression in the normal host,further supporting the role for this pathway as a physiologic mech-anism. Another interesting observation emerged from this study.There is considerable evidence that glucans will modulate innateimmunity and increase resistance to a variety of experimental in-fections (9). Some of the reports attribute the protective effect ofglucan to changes in cytokine levels (36, 37). However, we ob-served that glucan treatment in CLP mice (in the absence of wort-mannin) did not result in significant changes in serum cytokinelevels even though survival outcome was increased.

We examined PI3K activity in the liver, lung, and heart becauseit is well established that these organs play a pivotal role in theresponse to septic injury, shock, and multiorgan failure (7, 8, 38).Although there were differences among the tissues, the overalltrend was very similar, in that PI3K activity increased in responseto sham surgery, glucan, or CLP. Tissue PI3K activity was furtherincreased when glucan was administered before CLP. Wortmannintreatment blunted tissue PI3K activity in the presence of CLP orCLP plus glucan. However, tissue PI3K activity in the CLP, glu-can, and wortmannin group was not reduced to the level observedin the CLP plus wortmannin group. Nevertheless, inhibition ofPI3K activity correlated with increased susceptibility to septicmortality, whereas increased PI3K activity correlated with im-proved outcome. We also observed that the effect of glucan onPI3K activity occurred early (3 h) and was not observed by 12 h.Thus, PI3K appears to act early in sepsis.

Apoptosis of immune-competent and/or proinflammatory cellsis a feature of the sepsis syndrome (1, 39). However, the role of

apoptosis in the pathophysiology of sepsis, SIRS, and shock iscontroversial. In general, there are two schools of thought regard-ing the role of apoptosis in sepsis. First, apoptosis of immune-competent cells may be a mechanism of immunosuppression in thecritically ill and/or septic host (1, 39). Alternately, neutrophil ap-optosis may represent a beneficial anti-inflammatory mechanismthat serves to down-regulate the proinflammatory response thatoccurs in conditions such as adult respiratory distress syndromeand SIRS by eliminating inflammatory cells (1, 39). The PI3Ksignaling pathway plays a prominent role in cellular survival, inpart by exerting an antiapoptotic effect (4, 40). By way of example,Yang et al. (40) have reported that neutrophil apoptosis is in-creased in response to LPS administration in PI3K� knockoutmice. We examined the effect of PI3K modulation on apoptosis inthe CLP model. The relationship between PI3K modulation,splenocyte apoptosis, and mortality in polymicrobial sepsis ap-pears to be complex. We observed that CLP alone increasedsplenocyte apoptosis. When wortmannin was introduced into theCLP model, there was a dramatic increase in apoptosis and ananatomic change in the distribution of splenocyte apoptotic pro-files. Wortmannin caused widespread apoptosis in peripheral whitepulp, red pulp, and/or subcapsular regions of the spleen under con-ditions of sepsis, but this did not occur when wortmannin wasadministered to nonseptic (no surgery control) animals. This mayrepresent recruitment of a different population of splenocytes to theapoptotic pathway, it may also be a consequence of the sequestra-tion properties of the reticulo-endothelial system of the spleen inan environment of generalized activation of apoptosis, or it mayinvolve some combination of the two. Fragmentation of apoptoticbodies appeared further advanced in the wortmannin-treated septicanimals, indicating an acceleration of the apoptotic program in thiscondition. The increased splenocyte apoptosis observed in wort-mannin plus CLP mice correlated with increased susceptibility toseptic mortality. When wortmannin was administered to glucanplus CLP mice, the degree of splenic apoptosis was reduced com-pared with that in wortmannin- plus CLP-treated mice. This isconsistent with the tissue PI3K data. However, the kinetics of mor-tality in the wortmannin, glucan, and CLP group were essentiallyidentical with those in the wortmannin plus CLP group. It is pos-sible that the reduction in splenocyte apoptosis in the glucan, wort-mannin, and CLP group was not of sufficient magnitude to influ-ence survival outcome. It is also possible that splenocyte apoptosisdoes not play a major role in the mortality of CLP sepsis. We alsoexamined liver and lung tissue for apoptosis at the same time in-tervals. We saw no evidence of hepatocyte or pneumocyte apo-ptosis, although there was some increased apoptotic debris asso-ciated with either vascular spaces or cells of the reticulo-endothelial system. We conclude that PI3K suppresses splenocyteapoptosis in response to septic injury, thus preserving immune-competent cells. This may be important for the maintenance andintegrity of the immune response during septic injury. We are at-tempting to identify the phenotype of the apoptotic cells in thespleen. This may provide additional insights into the roles of PI3Kand apoptosis in sepsis.

Our data indicate that modulation of the PI3K pathway dramat-ically alters disease progression, severity, and survival outcome ina clinically relevant model of polymicrobial sepsis and septicshock. In 1996, Bone (41) coined the term compensatory anti-inflammatory response syndrome (CARS) to describe compensa-tory physiologic responses to persistent or uncontrolled inflamma-tion/sepsis in critically ill patients. The intracellular pathways thatmediate the CARS response have not been delineated. The PI3Kpathway appears to meet the criteria for a CARS response, i.e.,inhibiting proinflammatory cytokine expression, limiting apoptosis

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of immune-competent cells, and promoting survival. Our data areconsistent with the hypothesis that the PI3K pathway is a physi-ologic process that serves as a compensatory or feedback mecha-nism to negatively regulate proinflammatory responses in the faceof fulminating sepsis. Thus, we speculate that the PI3K pathwayplays an important role in the integrity of the immune response andthe maintenance of homeostasis. Our data also indicate that stim-ulation of the PI3K pathway may be an effective approach for theprevention and/or treatment of septic and inflammatory sequelae.

AcknowledgmentsWe thank Joel Norman, a medical student at East Tennessee State Univer-sity, for his assistance with compiling the apoptosis figure.

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