of January 21, 2019.This information is current as

PhagocytosisSeptic Melioidosis by Facilitating

Pneumonia-DerivedBacterial Defense against Urokinase Receptor Is Necessary for

Nicholas P. Day, Sharon J. Peacock and Tom van der PollC. M. Meijers, Joris J. Roelofs, Arjen Dondorp, Marcel Levi,Hovius, Gerritje J. W. van der Windt, Alex F. de Vos, Joost W. Joost Wiersinga, Liesbeth M. Kager, Joppe W. R.

http://www.jimmunol.org/content/184/6/3079doi: 10.4049/jimmunol.0901008February 2010;

2010; 184:3079-3086; Prepublished online 8J Immunol 

Referenceshttp://www.jimmunol.org/content/184/6/3079.full#ref-list-1

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2010 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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The Journal of Immunology

Urokinase Receptor Is Necessary for Bacterial Defenseagainst Pneumonia-Derived Septic Melioidosis byFacilitating Phagocytosis

W. Joost Wiersinga,*,† Liesbeth M. Kager,*,† Joppe W. R. Hovius,*,†

Gerritje J. W. van der Windt,*,† Alex F. de Vos,*,† Joost C. M. Meijers,‡

Joris J. Roelofs,x Arjen Dondorp,{ Marcel Levi,* Nicholas P. Day,{,‖ Sharon J. Peacock,{,‖

and Tom van der Poll*,†

Urokinase receptor (urokinase-type plasminogen activator receptor [uPAR],CD87), aGPI-anchoredprotein, is considered to play an

important role in inflammation and fibrinolysis. The Gram-negative bacterium Burkholderia pseudomallei is able to survive and

replicate within leukocytes and causes melioidosis, an important cause of pneumonia-derived community-acquired sepsis in

Southeast Asia. In this study, we investigated the expression and function of uPAR both in patients with septic melioidosis and

in a murine model of experimental melioidosis. uPAR mRNA and surface expression was increased in patients with septic

melioidosis in/on both peripheral blood monocytes and granulocytes as well as in the pulmonary compartment during experimental

pneumonia-derived melioidosis in mice. uPAR-deficient mice intranasally infected with B. pseudomallei showed an enhanced

growth and dissemination of B. pseudomallei when compared with wild-type mice, corresponding with increased pulmonary and

hepatic inflammation. uPAR knockout mice demonstrated significantly reduced neutrophil migration toward the pulmonary

compartment after inoculation with B. pseudomallei. Further in vitro experiments showed that uPAR-deficient macrophages

and granulocytes display a markedly impaired phagocytosis of B. pseudomallei. Additional studies showed that uPAR deficiency

did not influence hemostatic and fibrinolytic responses during severe melioidosis. These data suggest that uPAR is crucially involved

in the host defense against sepsis caused by B. pseudomallei by facilitating the migration of neutrophils toward the primary site of

infection and subsequently facilitating the phagocytosis of B. pseudomallei. The Journal of Immunology, 2010, 184: 3079–3086.

Urokinase receptor (urokinase-type plasminogen activatorreceptor [uPAR], CD87) is a GPI-anchored protein thatfunctions as the receptor for uPA (1). uPAR, which is

expressed by a wide variety of cells, including monocytes, macro-phages, and neutrophils (1, 2), has important roles in both physio-logical and pathological processes; in addition to its regulatory rolein fibrinolysis and inflammation, it has been implicated in tumorinvasion, metastasis, urinary protein loss, and the development ofprotective immunity in infections (1, 3–6). Importantly, uPAR hasbeen shown to contribute to activation and mobilization of leuko-cytes (3, 7) and to play a protective role in murine pulmonary in-fection (7, 8). In humans, i.v. injection of LPSwas associatedwith an

upregulation of uPAR on circulating monocytes and granulocytes(9, 10). However, the precise role of uPAR in human sepsis is stilllargely undefined.Melioidosis, caused by the Gram-negative bacillus Burkholderia

pseudomallei, is an important cause of community-acquired sepsisin Southeast Asia and Northern Australia (11, 12). We recently re-ported that patients with melioidosis demonstrate evidence for bothactivation and inhibition of fibrinolysis, as reflected by concurrentlyelevated concentrations of tissue-type plasminogen activator (tPA)and plasminogen activator inhibitor-1 (PAI-1), accompanied bya net increase in plasmin generation as indicated by elevated plasmaconcentrations of plasmin-a2–antiplasmin complexes and D-dimer(13). More than half of the cases of melioidosis habitually presentwith pneumonia, frequently associated with bacterial disseminationto distant sites (12, 14). Pulmonary exposure to B. pseudomalleirapidly elicits the massive recruitment of activated neutrophils butnot monocytes (15, 16). In vitro models indicate that B. pseudo-mallei survives and replicates within neutrophils and monocytes(12, 17–19). B. pseudomallei is very capable of escaping fromphagocytosis and consequent killing (12, 20). There are no data yet,however, on ways used by the host to enable effective phagocytosisof invading B. pseudomallei.With the aim of clarifying the role of uPAR in pneumonia-derived

melioidosis, we investigate in this study the expression and functionof uPARboth in patients with septicmelioidosis and amurinemodelof experimental melioidosis. Our results show that uPAR is upreg-ulated in melioidosis, and, although its in vivo role in fibrinolysis islimited, it does play a key role in bacterial clearance. This isexplained by both invitro and in vivo experiments inwhichwe showthat neutrophil recruitment and phagocytosis are uPAR-dependent

*Center for Infection and Immunity Amsterdam, †Center for Experimental and Mo-lecular Medicine, ‡Department of Vascular Medicine, and xDepartment of Pathology,Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands;{Mahidol-Oxford Tropical Medicine Research Unit, Faculty of Tropical Medicine,Mahidol University, Bangkok, Thailand; and ‖Center for Clinical Vaccinology andTropical Medicine, Nuffield Department of Clinical Medicine, University of Oxford,Oxford, United Kingdom

Received for publication March 30, 2009. Accepted for publication January 12, 2010.

This work was supported by the Dutch Foundation for Tropical Research.

Address correspondence and reprint requests to Dr. W. J. Wiersinga, Academic Med-ical Center, Center for Experimental and Molecular Medicine, Meibergdreef 9, RoomG2-132, 1105 AZ Amsterdam, The Netherlands. E-mail address: w.j.wiersinga@amc.uva.nl

Abbreviations used in this paper: hB2M, human b2-microglobulin; KO, knockout;MFI, mean fluorescence intensity; MPO, myeloperoxidase; PAA, plasminogen acti-vator activity; PAI-1, plasminogen activator inhibitor-1; TATc, thrombin-antithrombincomplex; tPA, tissue-type plasminogen activator; uPAR, urokinase-type plasminogenactivator receptor; WT, wild-type.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0901008

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mechanisms in melioidosis. Activation of uPAR and its favorableeffects on antibacterial host defense represent a new host defensemechanism in melioidosis.

Materials and MethodsHuman subjects

Thirty-four patients (meanage, 52 y; range, 18–86y)with sepsis caused byB.pseudomallei and 32 healthy controls (mean age, 41 y; range, 21–59 y) fromthe same area were studied. All subjects were recruited prospectively atSapprasithiprasong Hospital, Ubon Ratchathani, Thailand, in 2004. Sepsisdue tomelioidosis was defined as culture positivity forB. pseudomallei fromany clinical sample plus a systemic inflammatory response syndrome (21).B.pseudomallei was cultured from body material from all patients: bloodcultures were positive for B. pseudomallei in 21 patients (61.7%), throatswab or tracheal suction in 13patients (38.0%), sputum in 7patients (21.0%),pus from abscess in 7 patients (21.0%), and urine in 5 patients (14.7%). Theoverall patient mortality was 44%. Study design and subjects have beendescribed in detail (22). The study was approved by both the Ministry ofPublic Health, Royal Government of Thailand, and the Oxford TropicalResearch Ethics Committee, University of Oxford, Oxford, U.K. We ob-tained written informed consent from all subjects before the study.

FACS analysis

In humans, expression of uPAR and CD14 on monocytes and neutrophils inwhole blood was determined with an FACSCalibur (BD Biosciences, SanJose, CA) using fluorochrome-conjugated mouse anti-human uPAR (BDPharmingen, San Diego, CA) and CD14 Abs (BD Biosciences) in combi-nationwith the appropriate isotype controlAbs.Granulocyteswere identifiedaccording to their scatter pattern and monocytes according to their scatterpattern and CD14 positivity. In mice, blood and whole lung cell suspensionswere obtained as described previously (16, 22). Immunostaining was per-formed using directly labeled Abs against GR-1 (GR-1 FITC; BD Phar-mingen), F4/80 (Serotec,Oxfordshire,U.K.), and a biotin-labeledAb againstuPAR (R&D Systems, Minneapolis, MN) in combination with streptavidin-conjugated PerCP. After staining, cells were fixed in 2% paraformaldehyde.uPAR mean fluorescence intensity (MFI) was measured in the Gr-1 high(granulocytes), sidescatter low and F4/80 positive (monocytes), and side-scatter high and F4/80 positive (macrophages) gated populations. Abs wereused in concentrations recommended by the manufacturer.

Quantitative real-timePCR

Leukocytes were isolated from heparinized blood using erythrocyte lysisbuffer. Monocyte and granulocyte enriched populations were isolated usingPolymorphprep (Axis-Shield, Dundee, U.K.) as described (22). Monocyteand granulocyte fractions were .98% pure as determined by their scatterpattern on flow cytometry. After isolation, leukocytes, monocytes, andgranulocytes were dissolved in Trizol (Invitrogen, Carlsbad, CA) and storedat 280˚C until used for RNA isolation. Real-time RT-PCR was performedusing the LightCycler (Roche, Woerden, The Netherlands) apparatus asdescribed (16). Gene expression is presented as a ratio of the housekeepinggene b2-microglobulin expression (23). Primers, purchased from Euro-gentec, Seraing, Belgium, used for human uPAR were S606 59-AATCCT-GGAGCTTGAAAATCT-39 and 59-AS875 CCACTTTTAGTACAGCAGG-AGA-39.

Murine melioidosis

The Animal Care and Use of Committee of the University of Amsterdamapproved all experiments. All mice were on a C57BL/6 background.Pathogen-free 8–10-wk-old wild-type (WT) mice were purchased fromHarlan Sprague Dawley (Horst, The Netherlands). uPAR knockout (KO)mice were purchased from The Jackson Laboratory (Bar Harbor, ME) (24).Age- and sex-matched animals were used in each experiment. For theinoculum, B. pseudomallei strain 1026b, kindly provided by Dr. DonWoods (25, 26), was used and prepared as described (16, 22). Pneumoniawas induced by intranasal inoculation of a 50-ml (5 3 102 CFU/50 ml)bacterial suspension. Twenty-four, 48, and 72 h postinfection, mice weresacrificed by bleeding from the inferior vena cava. Pulmonary homoge-nates, cell suspensions, peritoneal lavage, and bronchoalveolar lavage fluidwere obtained from infected mice as described previously (16, 22, 27).

Assays

TNF-a, IFN-g, IL-6, IL-10, and IL-12p70 were determined using a cyto-metric bead array multiplex assay (BD Biosciences). Myeloperoxidase(MPO; HyCult Biotechnology, Uden, The Netherlands), thrombin-anti-

thrombin complexes (TATc; Dade Behring, Marburg, Germany), andD-dimer (Diagnostica Stago, Asnieres-sur-Seine, France) were measuredwith commercially available ELISA kits. Plasminogen activator activity(PAA) was measured by an amidolytic assay. Briefly, diluted euglobulin-precipitated fractions of plasma were incubated with 0.30 mmol/l S-2251,0.13 mol/l plasminogen, and 0.12 mg/ml CNBr fragments of fibrinogen (allobtained from Chromogenix, Molndal, Sweden). Conversion of plasmin-ogen to plasmin was detected by subsequent conversion of the chromo-genic substrate S-2251 and was detected with a spectrophotometer. Resultsare expressed in percentage increase as compared with baseline values ofnormal plasma (28). Aspartate aminotransferase, alanine aminotransferase,blood urea nitrogen (urea), and creatinin were determined with commer-cially available kits (Sigma-Aldrich, St. Louis, MO) using a Hittachi an-alyzer (Boehringer Mannheim, Mannheim, Germany).

Histologic examination

Organs were harvested at indicated time points, fixed in 10% formalin, andembedded in paraffin. Four-micrometer sections were stained with H&Eand analyzed by a pathologist blinded for groups. To score inflammationand damage, the entire organ surface was analyzed regarding the presenceof the following: necrosis/abscess formation, interstitial inflammation,endothelialitis, bronchitis, edema, thrombus formation, and, when appli-cable, pleuritis (22, 29). Neutrophils were counted in six randomly chosenfields (3100 magnification) as described (30). Granulocyte staining wasdone as described earlier (8). Fibrin(ogen) stainings were performed asearlier described (31), after which digital images were captured of threenonoverlapping areas (320 objective) using a DFC500 digital cameramounted on a DM5000B microscope (both from Leica Microsystems,Wetzlar, Germany). The area for positive for fibrin(ogen) was determinedwith Image Pro Plus software (Media Cybernetics, Silver Spring, MD) andexpressed as the percentage of the total surface area.

Cell culture and stimulation

Whole blood and peritoneal macrophages from untreated uPARKO andWTmice (n = 5–8/strain) were harvested as described (16, 22, 27). Cells andheparinized whole blood were stimulated with LPS from B. pseudomallei1026b (22) (500 ng/ml), mitomycin C-treated (0.2 mg/ml) (Sigma-Aldrich)growth-arrested B. pseudomallei (53 106 CFU/ml), or RPMI 1640 mediumfor 16 h. Supernatants were collected and stored at220˚C until assayed.

Bacterial killing, phagocytosis, and oxidative burst

Bacterial killingofmacrophageswasdeterminedasdescribedpreviously (27,32). In brief, B. pseudomallei was spun onto a monolayer of peritonealmacrophages (derived from five different mice per group), after which plateswere placed at 37˚C for 10min.Afterwashingfive timeswith ice-coldPBS toremove extracellular bacteria, bacterial uptake after 10 min was determinedby lysing the wells with sterile distilled H2O. This was designated as t = 0.RPMI 1640was added to remainingwells, and plates were placed at 37˚C for5 and 30 min, after which cells were washed and lysed with distilled H2O.Cell lysates were plated on blood agar plates,and bacterial counts wereenumerated after 16 h. Bacterial killing ofmacrophageswas expressed as thepercentage of killed bacteria in relation to t = 0. Phagocytosis was evaluatedessentially as described before (27, 33). Growth-arrested B. pseudomalleiwas labeled with CFSE dye (Invitrogen). Peritoneal macrophages (derivedfrom five different mice per group) were incubated with growth-arrestedCFSE-labeled B. pseudomallei (2.5 3 107 CFU/ml) for 0, 15, and 60 min.Phagocytosis was stopped by placing cells on ice; thereafter, cells werewashed in PBS and suspended in quenching solution (Orpegen, Heidelberg,Germany). To determine the neutrophil phagocytosis capacity, 50 ml wholeblood was incubated with bacteria, after which cells were suspended inquenching solution and incubated in FACS lysis/fix solution (BD Bio-sciences), and neutrophils were labeled using anti–Gr-1-PE (BD Pharmin-gen). Phagocytosis was determined using FACS. Phagocytosis index of eachsample was calculated: (MFI 3 % positive cells at 37˚C) 2 (MFI 3 %positive cells at 4˚C). The oxidative burst response of macrophageswas determined using anAmplexRed kit (Invitrogen).Macrophages (1.253106 cells/ml) were plated in a black 96-well plate with a clear bottom(Greiner, Alphen a/d Rijn, The Netherlands) and allowed to adhere for 2 h.Macrophages were stimulated with buffer, 1 mg/ml serum-treated zymosan(Sigma-Aldrich), or 1253 106 heat-killedB. pseudomallei bacteria/ml in thepresence of Amplex Red (0.5 mm) and HRP (1 U/ml). Fluorescence wasmeasured with an HT Synergy platereader (Bio-Tek Instruments, WatfordHerts, U.K.) using excitation/emission wavelengths of 530/580 nm. Meas-urements (n = 8 for both WT and KO macrophages) were performed every2 min for 120 min. Results are depicted as relative fluorescence units.

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Statistical analysis

Values are expressed as means 6 SEM. Differences between groups wereanalyzed by Mann-Whitney U test or Kruskal-Wallis analysis with Dunn’spost hoc test where appropriate. For survival analysis, Kaplan-Meieranalysis followed by log-rank test was performed. These analyses wereperformed using GraphPad Prism version 4.03 (GraphPad, San Diego,CA). Values of p , 0.05 were considered statistically significant.

ResultsIncreased uPAR expression in patients with severe melioidosis

We quantified uPAR mRNA and surface expression on both pe-ripheral blood monocytes and granulocytes in 34 individuals withculture-proven severe melioidosis and 32 healthy controls. Patientsshowed profoundly elevated levels of both uPAR mRNA and cellsurface expression compared with controls (Fig. 1). In addition, anoverall increase in the percentage of monocytes expressing uPARon their cell membranes was seen in patients compared withcontrols (97.7% 6 2.3 versus 80.1 6 3.1%; p , 0.0001), togetherwith a modest decline in the percentage of uPAR-positive gran-ulocytes (74.6 6 4.6% versus 89.9 6 2.3%; p , 0.05). In thiscohort of patients, in which the mortality rate was 44%, the levelsof either cell-associated uPAR or uPAR mRNA did not differbetween survivors and nonsurvivors (data not shown).

Increased uPAR expression in the pulmonary compartmentduring experimental pneumonia-derived melioidosis

Because the majority of severe melioidosis cases present withpneumoniawith bacterial dissemination to distant body sites (11, 12,14), and considering the fact that it is not feasible to study uPARexpression at tissue level in patients with melioidosis, we useda murine model of pneumonia-derived melioidosis in which miceare intranasally infected with B. pseudomallei (16, 22, 34). In linewith the data obtained in patients with melioidosis, 48 h post-infection mice showed an upregulation of uPAR expression on theirgranulocytes, monocytes (Fig. 2), and alveolar macrophages (datanot shown). The increase in uPAR expression was much morepronounced at the primary site of infection, the pulmonary com-partment, when compared with the systemic compartment, whereonly a trend toward increased uPAR expression was seen (Fig. 2).

uPAR-deficient mice show an enhanced growth anddissemination of B. pseudomallei in vivo

To obtain insights into the functional role of uPAR in melioidosis,we infected uPAR KO and WT mice with B. pseudomallei and

performed quantitative cultures of lung, liver, and blood at varioustime points thereafter. Relative to WT mice, uPAR KO micedisplayed strongly increased bacterial loads in the lungs, liver, andblood at 48 and, most markedly, 72 h post intranasal infection witha lethal dose of B. pseudomallei (Fig. 3).

uPAR KO mice display increased late lung inflammation butdecreased early neutrophil migration during melioidosis

We have previously shown that intranasal inoculation with B. pseu-domallei causes significant and rapid inflammation and granulocyte,but notmonocyte, recruitment toward the lung (16). To further evaluatethe role of uPAR in antibacterial defense against B. pseudomallei,pulmonary inflammation and granulocyte recruitment into lung tissuewere assessed. Consistent with the observed enhanced growth of B.pseudomallei in uPARKOmice, uPARKOmice showed increased latepulmonary inflammation, which was characterized by significantlymore inflammation, pleuritis, peribronchial inflammation, edema, andendothelialitis when compared with control mice (Fig. 4). Strikingly,however, early pulmonary neutrophil recruitment was impaired inuPARKOmice in response to intranasal infectionwithB.pseudomalleias visualized by Ly-6 staining and confirmed by lower MPO concen-trations in lung homogenates at 24 h postinfection (Fig. 5).

Limited effect of uPAR deficiency on cytokine response, distantorgan injury, and survival

Because the localized production of cytokines is an important part ofhost defense against infection (35), we measured the concentrationsof these mediators in the pulmonary and systemic compartment.Overall, uPAR deficiency did not have a major impact on cytokineconcentrations postinfection with B. pseudomallei (Table I). Themost notable difference was a mean 50% reduction in TNF-a levelsin lung homogenates of uPAR KO mice at 24 h postinfection (p ,0.05 versusWTmice), but not in plasma and/or later time points. At72 h postinfection, cytokine levels tended to be higher in uPAR KOmice, most likely reflecting the increased bacterial loads and lunginflammation, but probably due to a relatively large interindividualvariation, the difference with WT mice only reached statisticalsignificance for lung IL-6 and plasma IL-10 concentrations. Fur-thermore, in line with the observed increased bacterial loads in theliver, uPAR KO mice showed significantly more hepatic in-flammation after 72 h comparedwithWTmice postinoculationwithB. pseudomallei (meanhistological score 4.76 0.5 versus 2.160.4;p, 0.01). Consistent with these pathology data, the plasma levels of

FIGURE 1. uPAR is upregulated in

patients with severe melioidosis. Both

uPAR mRNA (A, D) and uPAR cell sur-

face (B, E) expression were strongly in-

creased in/on both monocytes (A–C) and

granulocytes (D–F) of patients (n = 34)

with sepsis caused by B. pseudomallei

compared with healthy controls (n = 32).

C and F show representative histograms

of monocyte and granulocyte uPAR ex-

pression, respectively, in a healthy con-

trol (bold line) and in a patient (filled gray

area). CD87 is synonymous for uPAR.

hB2M, human b2-microglobulin. ppp ,0.01; pppp, 0.001.

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aspartate aminotransferase (12846 457 versus 1836 45; p, 0.05)and alanine aminotransferase (8146 351 versus 866 21; p, 0.05)were higher in uPAR KO mice 72 h postinfection compared withWT mice, reflecting increased hepatocellular injury in these ani-mals. Additionally, all mice showed evidence of renal failure, asindicated by elevated plasma concentrations of urea (12.5 6 4.4versus 7.56 0.6; not significant) and creatinine (19.26 9.3 versus

8.5 6 0.5; not significant); however, no differences were seen be-tween uPAR KO and WT mice. As a last part of our in vivo ex-periments, we performed a survival experiment for which weinoculated WT and uPAR KO mice with B. pseudomallei andmonitored them for 14 d. Infection with the bacterial dose alsoemployed in the experiments described above caused lethality in 12out of 12 WT mice and 11 out of 12 uPAR KO mice (Fig. 6). Re-markably, mortality was slightly but statistically significantly de-layed in uPAR KO mice (p , 0.05; Fig. 6).

Contribution of uPAR toward cellular responsiveness to B.pseudomallei in vitro

To obtain additional insights into the function of uPAR in the hostdefense against B. pseudomallei, we started to analyze the re-quirement of uPAR signaling upon first encounter between the bac-terium and the host. Therefore, we tested the cytokine productioncapacity of macrophages and whole blood harvested from WT anduPAR KO mice upon stimulation with B. pseudomallei LPS orgrowth-arrested B. pseudomallei (E:T ratio 1:10). Whole blood ob-tained fromuPARKOmice released equal amounts ofTNF-a, IFN-g,

FIGURE 4. Increased late lung inflammation in uPAR KO mice infected

with B. pseudomallei. Representative lung histology ofWT (A) and uPARKO

mice (B) showing significantly more inflammation, pleuritis, peribronchial

inflammation, edema, and necrosis 72 h after intranasal infectionwith 53 102

CFUB.pseudomallei in theuPARKOmicecomparedwithcomparedwithWT

controls (H&E, original magnification 3100). C shows the corresponding

pathology scores (means 6 SEM) calculated as described in Materials and

Methods.Whitebars representWTmice; graybars represent uPARKOmice (n

= 8 per group at each time point). pp, 0.05.

FIGURE 2. uPAR is upregulated in murine melioidosis. Cell-surface

uPAR expression on blood monocytes (A), blood granulocytes (B), pul-

monary monocytes (C), and pulmonary granulocytes (D) 48 h after in-

tranasal inoculation with B. pseudomallei (gray bars) compared with saline

treated control mice (white bars). n = 8 per group. pp , 0.05.

FIGURE 3. uPAR deficiency results in an enhanced growth and dissem-

ination of B. pseudomallei in vivo. uPAR KO mice (gray bars) demonstrate

strongly increased bacterial loads at 48 and 72 h postinfection in their lungs

(A), liver (B), and blood (C) compared with WT mice (white bars; n = 8 per

group per time point). pp, 0.05; ppp, 0.01; pppp , 0.001.

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IL-6, IL-10, and IL-12p70 upon stimulation with B. pseudomalleiinvitro as comparedwithWTmice (Table II). These data suggest thatuPAR does not contribute to cellular responsiveness to B. pseudo-mallei in whole blood in vitro. Of interest, uPAR-deficient macro-phages released more TNF-a, IL-6, and IL-10 compared withWT cells upon exposure to B. pseudomallei (Table II).

uPAR deficiency does not influence hemostatic and fibrinolyticresponses during severe melioidosis

BecauseuPARis thought toplay a regulatory role infibrinolysis (1, 6),we measured pulmonary TATc, D-dimer, PAA, and fibrin levels inboth WT and uPAR KO mice post intranasal inoculation with B.pseudomallei. No differences in thrombin generation, as reflected byTATc plasma levels betweenWTand uPARKOmice, were seen (Fig.7). To investigate whether the upregulated uPAR expression influ-enced the fibrinolytic activity, we measured D-dimer and PAA levels.No differences were observed in D-dimer and PAA levels betweenuPARKOandWTmice (Fig. 7).Finally,measurement of theextent offibrin deposition in lung tissue showed increased fibrin accumulationpostinfection, but again, no differences between uPAR KO and WTmice (Fig. 7).Together, thesedatadonot support amajor role of uPARin fibrinolysis and coagulation during melioidosis.

Impaired phagocytosis of B. pseudomallei in uPAR-deficientmacrophages and granulocytes

The experiments described above established that uPAR KO dis-play a diminished antibacterial defense toward B. pseudomalleiinfection, characterized by increased bacterial loads and accom-panied by diminished early recruitment of neutrophils to the pri-mary site of infection and reduced neutrophil MPO levels. Theearly host response to infection is characterized by a coordinatedseries of effector functions that include the generation of reactiveoxygen species, such as reactive oxygen species and phagocytosis.We found that the basal production of oxidative products in un-challenged macrophages is similar in WT and uPAR-deficient cells(Fig. 8). Similarly, upon activation with B. pseudomallei, uPAR-deficient macrophages displayed an unaltered ability to produce anoxidative burst when compared with WT cells (Fig. 8). We nextwished to determine whether uPAR contributes to phagocytosisand/or killing of B. pseudomallei. No difference in the killingcapacity between WT and uPAR KO peritoneal macrophages wasobserved (Fig. 8). However, both uPAR KO macrophages anduPAR KO granulocytes demonstrated a markedly diminished ca-pacity to phagocytose B. pseudomallei (Fig. 8). Taken together,the observed impairment of bacterial clearance in uPAR KO mice

FIGURE 5. Decreased early granulocyte migration

in uPAR KO mice infected with B. pseudomallei.

Representative of Ly6G-immunostaining (original

magnification3400) for granulocytes of WT (A) and

uPAR KO mice (B) showing significantly less neu-

trophil influx at 24 h postinoculation in uPAR KO

mice when compared with WT mice (C), corre-

sponding with decreased early MPO activity levels in

lung tissues in uPAR KO mice (D). White bars rep-

resent WT mice; gray bars represent uPAR KO mice

(n = 8 per group at each time point). Number of

granulocytes expressed as mean number of gran-

ulocytes per field6 SEM. pp, 0.05; ppp, 0.01.

Table I. Cytokine response in lung homogenates and plasma of WT and uPAR KO mice during melioidosis

24 h 48 h 72 h

WT uPAR KO WT uPAR KO WT uPAR KO

Lung homogenate (pg/ml)TNF-a 850 6 133 418 6 107* 1326 6 241 757 6 83 538 6 39 749 6 191IL-6 1472 6 128 1484 6 343 2269 6 395 1738 6 140 532 6 138 4465 6 1078**IL-10 77 6 18 120 6 25 102 6 21 122 6 12 159 6 58 194 6 60IL-12p70 32 6 7 26 6 7 38 6 10 47 6 4 58 6 11 25 6 6*IFN-g ND ND ND ND 34 6 2 54 6 10

Plasma (pg/ml)TNF-a 20 6 3 13 6 3 125 6 32 137 6 18 48 6 7 65 6 16IL-6 410 6 82 372 6 72 2016 6 524 2003 6 188 285 6 56 750 6 370IL-10 15 6 7 16 6 9 38 6 15 28 6 14 20 6 6 56 6 12***IL-12p70 40 6 10 25 6 9 51 6 10 22 6 8* 25 6 2 28 6 6IFN-g ND ND 1389 6 542 675 6 168 127 6 12 1431 6 1082

Pulmonary and systemic cytokine levels after intranasal infection with 5 3 102 CFU B. pseudomallei. WT and uPAR KO mice were sacrificed 24, 48, or 72 h postinfection.Data are means 6 SEM of eight mice per group per time point.

pp , 0.05; ppp , 0.001; pppp , 0.01.ND, not detectable.

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can be explained by both a reduction of early neutrophil re-cruitment and a diminished phagocytosis capacity of these re-cruited uPAR-deficient immune cells.

DiscussionIn this study, we show that uPAR is upregulated in severe melioidosisand plays a major role in the antibacterial innate immune response.Duringmelioidosis,uPARcontributestotherecruitmentofneutrophilsto the primary site of infection and the capacity of neutrophils tophagocytose B. pseudomallei. As a consequence, uPAR KO micedisplayed a markedly impaired clearance of B. pseudomallei in thepulmonary and systemic compartment upon intranasal infection to-gether with increased lung and liver inflammation. uPAR did notimpact on the fibrinolytic response to infection withB. pseudomallei.These data are the first to describe a role for uPAR inmelioidosis andfurther add to our understanding of how infectionwith this facultativeintracellular organism can lead to a full-blown septic illness.Our study is the first to provide insights into the expression of both

mRNA and protein cell surface uPAR expression in a cohort ofpatientswithsepsis. Inbloodsamplesobtainedfrom34prospectivelyenrolled patients with sepsis caused by B. pseudomallei, we showedthat the increased uPAR mRNA expression is accompanied by en-hanced uPAR surface expression on both monocytes and gran-ulocytes. Becausewewere also interested in uPAR expression at theprimary infection site and given the fact that pneumonia with bac-terial dissemination to distant body sites is a common presentationof humanmelioidosis (11, 12, 14), wemade use of amousemodel ofmelioidosis in which mice are intranasally infected with a lethaldose of B. pseudomallei (16, 22, 34). By doing so we were able to

demonstrate increased expression of uPAR on both monocytes andgranulocytes at the primary place of infection. These data are inaccordance with previous data showing an increased release ofuPAR in both plasma and urine of patients with urosepsis (36) andincreased uPAR cell surface expression on monocytes of healthyvolunteers injected with endotoxin (9, 10).Oneof themost importantcomponentsof theinitial innate immune

response in the lung against bacterial infection is the vigorous re-cruitmentofneutrophils intopulmonarytissueandairspaces(37,38).uPARhas been shown to play a key role in both neutrophilmigrationand activation (3, 7, 39). uPAR can facilitate cell migration in twoways: first, after binding to uPA, it facilitates the generation ofplasmin at the cell surface, resulting in degradation of the extra-cellular matrix and induction of cell migration (1, 3); second, uPARcauses the activation and mobilization of leukocytes through in-teractionwithb2-integrins,most notably CD11b/CD18 (3, 7). In theevent of critical illness due to invasion of pathogens, uPAR has beenshown to be important in the recruitment of leukocytes toward theprimary site of infection in pneumococcal meningitis (40) and bothPseudomonas aeruginosa and pneumococcal pneumonia (7, 8). Wenowunderwrite these earlier reports by showing that uPARKOmicehave an impaired host defense against B. pseudomallei as indicatedby increased bacterial outgrowth and increased organ inflammationaccompanied by a reduced early neutrophil migration toward theprimary site of infection. This fully underscores the emerging in-sight that neutrophils play a vital role in host defense against B.pseudomallei (15). One has to recall, however, that epithelial andcertain serosal cells are also known to express uPAR; therefore, inthis context, one cannot rule out a potential important role for thesecells during the initial immune response againstB. pseudomallei (1).This notion is underscored by the finding that the epithelium isa known site of replication for B. pseudomallei (12, 41).Our results showing a markedly impaired host defense in mice

lacking uPAR after inoculation with B. pseudomallei can be ex-plained not only by a diminished neutrophil recruitment toward thepulmonary compartment but also by the finding that uPAR is cru-cially involved in phagocytosis of B. pseudomallei. Because B.pseudomallei is a very virulent and facultative intracellular organ-ism (12, 17–19), effective killing and phagocytosis are of paramountimportant during melioidosis. We and others (39, 42) have pre-viously reported on the potential role of uPAR in the phagocytosis ofEscherichia coli and P. aeruginosa. We now extend these findingsby showing that uPAR is necessary for effective phagocytosis of B.

FIGURE 6. Effect of uPAR deficiency on survival of mice infected with

B. pseudomallei. WT and uPAR KO mice were inoculated with 5 3 102

CFU of B. pseudomallei intranasally. n = 12 mice per group. p , 0.05.

Table II. Cytokine production from blood and peritoneal macrophages harvested from WT and uPAR KO upon in vitro stimulationwith B. pseudomallei

Medium B. pseudomallei LPS B. pseudomallei

WT uPAR KO WT uPAR KO WT uPAR KO

Whole blood (pg/ml)TNF-a 108 6 31 348 6 144 532 6 115 585 6 141 5452 6 1720 6790 6 2030IL-6 249 6 140 212 6 74 427 6 195 446 6 123 1785 6 645 1543 6 497IL-10 51 6 16 157 6 100 65 6 25 105 6 37 1066 6 250 1508 6 470IL-12p70 14 6 5 36 6 27 11 6 1 16 6 3 68 6 22 25 6 4IFN-g ND ND ND ND ND ND

Peritoneal macrophages (pg/ml)TNF-a 283 6 134 124 6 72 1624 6 135 8238 6 1116* 3038 6 250 8333 6 1667*IL-6 176 6 83 46 6 27 2777 6 284 3687 6 89* 2490 6 130 2903 6 125*IL-10 60 6 22 18 6 11 87 6 7 79 6 13 253 6 18 347 6 26*IL-12p70 ND ND ND ND ND NDIFN-g ND ND ND ND ND ND

WT and uPAR KO-derived whole blood and isolated peritoneal macrophages were stimulated with B. pseudomallei LPS or growth-arrested B.pseudomallei (E:T ratio 1:10; see Materials and Methods). Data are means 6 SEM of 5–8/group per time point.

pp , 0.05.ND, not detectable.

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pseudomallei by both macrophages and neutrophils. This newlydescribed mechanism provides new insights into how the immunesystem is capable of mounting an effective host response against B.pseudomallei. We did not observe any difference in the killing ca-

pacity between WTand uPAR-deficient macrophages. A limitationof our current study is the fact thatwe do currently not have a reliableand reproducible neutrophil killing assay. Furthermore, it remains tobe studied if the impaired phagocytosis observed in uPAR-deficientcells is caused by signaling deficits in the uPAR KO leukocytes. Inthis respect, it is of interest that uPAR is upstream of myeloid Scrkinases, which are known to play a pivotal role in phagocytosis (1,43, 44). Interestingly, uPAR does not play a direct role in the killingof B. pseudomallei nor in the induction of oxidative burst. Fur-thermore, in preliminary experiments, we were not able to showa direct interaction between uPAR and B. pseudomallei (data notshown), suggesting the existence of additional important mediatorsin this uPAR phagocytosis pathway. Of interest, the observedmodest survival advantage of uPARKOmice could indicate that therole of uPAR changes in time during the host defense againstB. pseudomallei from an early protective role toward a possibledetrimental role later on during the overwhelming septic response.Further studies are warranted to assess whether uPAR plays a pro-tective role in less severe models of melioidosis (i.e., using in-fectious doses that are not associated with almost 100% lethality).Our study not only demonstrates the important role of uPAR in

neutrophil migration and phagocytosis during melioidosis but alsoreveals the relative unimportance of uPAR in the fibrinolytic re-sponse during sepsis caused by B. pseudomallei. In patients withmelioidosis, we have recently shown that the fibrinolytic system isboth activated and inhibited as reflected by elevated concentrationsof tPA, PAI-1, plasmin-antiplasmin complex, and D-dimer (13). Ourcurrent data argue against a major role of uPAR in fibrinolysisbecause there were no differences in D-dimer and PAA expressionlevels between uPAR KO and WT mice during experimental me-lioidosis. Moreover, fibrin deposition was equal in both mice strainspostinfection. Interestingly, these data are in line with previousinvestigations (3). For instance, fibrin deposits were only found inthe livers of adult mice with a dual deficiency in uPAR and tPA butnot in uPAR KO mice (45). In animal models of lung injury and

FIGURE 7. uPAR deficiency does not influence pulmonary fibrinolytic activity during melioidosis. No differences were seen in pulmonary TATc (A), D-

dimer (B), and PAA (C) levels between WT (white bars) and uPAR KO (gray bars) mice 72 h after intranasal inoculation with 5 3 102 CFU B. pseu-

domallei. Representative fibrin(ogen) immunostaining of lung tissue of infected WT (D) and uPAR KO (E) mice. Original magnification 320. Graphical

representation of the percentage of the total area with positive fibrin(ogen) staining (F) shows no difference between WT and uPAR KO mice. Data

represent mean 6 SEM. n = 8 per group.

FIGURE 8. Impaired phagocytosis of B. pseudomallei in uPAR-deficient

cells. A, Killing capacity of macrophages are shown as percentage of killed

B. pseudomallei compared with t = 0. B, Respiratory burst, depicted as

relative fluorescence units, upon activation with B. pseudomallei of WT

and uPAR-deficient macrophages. Macrophages (C) and peripheral blood

neutrophils (D) were incubated at 37˚C with CFSE-labeled growth-arrested

B. pseudomallei (1 3 107 CFU/ml), after which time-dependent phago-

cytosis was quantified (see Materials and Methods). Data are mean 6SEM. n = 5–8 per mouse strain. Open rounds represent WT cells; black

squares represent uPAR KO mice. pp , 0.05; ppp , 0.01.

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septic shock, reduced uPA-mediated proteolysis correlated withexcessive fibrin deposition, suggesting that uPA facilitates fibri-nolysis by a uPAR-independent mechanism (3, 46, 47). Clearly, therole of the fibrinolytic system in the host defense against B. pseu-domallei remains to be elucidated. Studies making use of uPA-,tPA-, and PAI-1–deficient animals are underway in our laboratory.In conclusion, our data suggest that uPAR is crucially involved in

the host defense against B. pseudomallei by facilitating the mi-gration of neutrophils toward the primary site of infection andsubsequently facilitating the phagocytosis of B. pseudomallei.Activation of uPAR and its favorable effects on antibacterial hostdefense represent a new host defense mechanism in melioidosis.Manipulation of uPAR expression or function may be a potentialtarget for immunomodulation in septic melioidosis.

AcknowledgmentsWe are grateful to Joost Daalhuisen, Marieke ten Brink, and Jennie Pater for

expert technical assistance.

DisclosuresThe authors have no financial conflicts of interest.

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