of May 10, 2010 This information is current as

doi:10.4049/jimmunol.0904160 published online Apr 30, 2010; J. Immunol.

 Leiner, Michael L. Dustin and Eric G. Pamer Chao Shi, Peter Velázquez, Tobias M. Hohl, Ingrid 

ExpressionIntercellular Adhesion Molecule-1 Independent and Directed by Focalof Bacterial Infection Is Chemokine Monocyte Trafficking to Hepatic Sites

DataSupplementary

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright ©2010 by The American Association ofRockville Pike, Bethesda, MD 20814-3994.The American Association of Immunologists, Inc., 9650

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The Journal of Immunology

Monocyte Trafficking to Hepatic Sites of Bacterial Infection IsChemokine Independent and Directed by Focal IntercellularAdhesion Molecule-1 Expression

Chao Shi,*,† Peter Velazquez,‡ Tobias M. Hohl,*,1 Ingrid Leiner,* Michael L. Dustin,‡ and

Eric G. Pamer*,†

Recruitment of CCR2+Ly6Chigh monocytes to sites of infection is essential for efficient clearance of microbial pathogens. Although

CCR2-mediated signals promote monocyte emigration from bone marrow, the contribution of CCR2 to later stages of monocyte

recruitment remains unresolved. In this article, we show that CCR2 deficiency markedly worsens hepatic Listeria monocytogenes

infection because Ly6Chigh monocytes are retained in the bone marrow. Intravenously transferred, CCR2-deficient Ly6Chigh

monocytes traffic normally to hepatic foci of infection and contribute to bacterial clearance. Pertussis toxin treatment of adop-

tively transferred monocytes does not impair their intrahepatic trafficking, suggesting that chemokine signaling, once CCR2+

Ly6Chigh monocytes emigrate from the bone marrow, is not required for monocyte localization to sites of bacterial infection in the

liver. Expression of ICAM-1 is induced in close proximity to foci of bacterial infection in the liver, including on CD31+ endothelial

cells, and blockade of CD11b and CD44 diminishes monocyte localization to these hepatic foci. Our studies demonstrated that

Ly6Chigh monocyte recruitment from the bloodstream to the L. monocytogenes-infected liver does not require chemokine receptor-

mediated signals but instead is principally dependent on integrin- and extracellular matrix-mediated monocyte adhesion. The

Journal of Immunology, 2010, 184: 000–000.

Monocytes are myeloid leukocytes that reside in thebloodstream, bone marrow, and spleen. Murine bloodmonocytes are divided into two major subsets based on

their differential expression of surface markers and traffickingbehaviors. One subset expresses high levels of the chemokinereceptor CX3CR1 and patrols the luminal surface of blood vessels.The other subset, also referred to as inflammatory monocytes,expresses lower levels of CX3CR1 and high levels of CCR2 andLy6C and is recruited to sites of inflammation (1). InflammatoryLy6Chigh monocytes can differentiate into mucosal dendritic cells(DCs) after their depletion from gut and lung (2–5) and contributeto inflammatory diseases, such as atherosclerosis and multiplesclerosis (6, 7). Ly6Chigh monocytes make essential contributionsto immune defense against microbial pathogens, including Lis-teria monocytogenes (8, 9), Toxoplasma gondii (10, 11), Brucellamelitensis (12), and Mycobacterium tuberculosis (13).

L. monocytogenes, a Gram-positive bacterium, infects humansvia the gastrointestinal tract (14) and is recognized by the innateimmune system with TLR- and Nod-like receptor-mediated mecha-nisms (15–18). Intravenous inoculation of micewith a sublethal doseofL.monocytogenes results in systemic infection involving the spleenand liver and induces innate and adaptive immune responses that clearthe infection.More than60%ofListeria is takenupby the liverwithin10 min of i.v. inoculation (19, 20). Ab-mediated blockade ofCD11b dramatically reduces the recruitment of myeloid cells to theliver and results in markedly increased growth of L. monocytogenes(21). Administration of the RB6-8C5 Ab, which binds to an epitopeshared by Ly6C and Ly6G, depletes granulocytes and Ly6Chigh

monocytes and results in uncontrolled hepatic growth of L. mono-cytogenes (22). The relative contribution of granulocytes and Ly6-Chigh monocytes to hepatic clearance of L. monocytogenes remainsunresolved.The innate immune response during L. monocytogenes infection

depends on rapid monocyte recruitment, a process that has beenstudiedmost extensively in the spleen (23, 24). Ly6Chigh monocytesare recruited from bone marrow to spleen during L. monocytogenesinfection, and they differentiate into TNF- and inducible NO syn-thase (iNOS)-producing DCs (TipDCs) that enhance bacterialclearance (23). The recruitment of Ly6Chigh monocytes requiresCCR2, a chemokine receptor that responds to MCP-1 and -3.CCR22/2 mice have decreased numbers of Ly6Chigh monocytes inthe periphery because monocytes are retained in the bone marrow,markedly increasing susceptibility to L. monocytogenes infection(24, 25). CCR2 deficiency also increases the severity of L. mono-cytogenes infection in the liver (8), but it remains unclear whetherthis results from defective recruitment of monocytes or otherCCR2-expressing cell populations.Although the role of CCR2 in monocyte egress from the bone

marrow is established (24–29), it remains unresolved whetherCCR2 also plays a role in monocyte trafficking into and withinperipheral tissues. Some studies indicated that CCR2-mediated

*Infectious Disease Service, Department of Medicine, Memorial Sloan-KetteringCancer Center; †Program in Immunology and Microbial Pathogenesis, Weill CornellGraduate School of Medical Sciences, New York, NY 10021; and ‡Skirball Instituteof Biomolecular Medicine, New York University Langone School of Medicine, NewYork, NY 10016

1Current address: Vaccine and Infectious Disease Institute, Fred Hutchinson CancerResearch Center, Seattle, WA.

Received for publication December 28, 2009. Accepted for publication March 17,2010.

This work was supported by National Institutes of Health Grants 5P01CA023766 and5R37AI039031-14 (to E.G.P.) and K08 AI071998 (to T.M.H.).

Address correspondence and reprint requests to Dr. Eric G. Pamer, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, Box 9, New York, NY 10021. E-mailaddress: pamere@mskcc.org

The online version of this article contains supplemental material.

Abbreviations used in this paper: CMTMR, 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine; DC, dendritic cell; EGFP, enhanced GFP; HA, hyaluronan; iNOS,inducible NO synthase; TipDC, TNF- and inducible NO synthase-producing dendritic cell.

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

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0904160

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signaling is required for monocyte entry into the CNS (7), athero-sclerotic plaques (6), ischemic myocardium (30), and the peritonealcavity (26) in the setting of tissue inflammation. However, it wasalso shown that the migration of monocytes from the bloodstreaminto the white pulp of L. monocytogenes-infected spleen is CCR2independent (24). In the current study, we demonstrated that neitherthe targeting of Ly6Chigh monocytes from the bloodstream to he-patic foci of infection nor their bactericidal function in the liverrequires CCR2-mediated signals. Our study demonstrated thattrafficking of Ly6Chigh monocytes within the liver is not mediatedby pertussis toxin-sensitive chemokine receptors but depends onintegrin- and extracellular matrix-mediated adhesion.

Materials and MethodsMice and infections

All mice used in this study were bred at Memorial Sloan-Kettering ResearchAnimal Resources Center. The CCR2 reporter transgenic strain was pre-viously generated in our laboratory using bacterial artificial chromosomecarrying the gene encoding enhanced GFP (EGFP) under the control ofCCR2 promoter (31). The CCR22/2Tg(CCR2-EGFP) strain was generatedby crossing the CCR2-reporter transgenic strain to a CCR22/2 back-ground. Bone marrow chimeras were generated according to the standardprotocol. Briefly, C57BL/6 mice were subjected to irradiation at a dose of1000 cGy, followed by i.v. injection of 1 3 106 bone marrow cells bearingdifferential CD45.1/2 congenic markers. Mice were infected i.v. with 3 3103 or 1 3 104 L. monocytogenes strain 10403S, as described previously(32). At indicated times following infection, livers were harvested anddissociated in PBS containing 0.05% Triton X-100, and bacterial CFUwere determined by plating on brain-heart infusion agar plates.

Flow cytometry

At various times postinfection, livers were perfused, excised, and digested incollagenase/DNase, as previously described (33). Nonparenchymal cellswere further purified using 40%/80% Percoll gradient. Cells at the interfaceof the upper and lower layer of Percoll solution were collected and subjectedto Ab staining. The following Abs were purchased from BD Pharmingen(San Diego, CA): anti–CD11b-PerCP–Cy5.5 (M1/70), anti–Ly6C-FITC(AL-21), anti–Ly6C-biotin (AL-21), streptavidin-allophycocyanin-Cy7,anti–Ly6G-PE (1A8), anti–CD45.1-allophycocyanin (A20), anti–TNF-allophycocyanin (MP6-XT22), and anti–CD11c-allophycocyanin (HL3).Anti-MHC class II-PE (M5/114.15.2) was purchased from eBioscience (SanDiego, CA). Anti–CD45-Pacific Blue (30-F11) was purchased from Bio-Legend (San Diego, CA). Rabbit anti-iNOS Ab was purchased from Mil-lipore (Billerica, MA). Alexa633-F(ab9)2 of anti-rabbit IgG was purchasedfrom Invitrogen (Carlsbad, CA). For intracellular TNF staining, liver non-parenchymal cells were incubated in RPMI 1640 medium in the presence ofbrefeldin A for 4 h without stimulation and processed according to themanufacturer’s protocol for Cytofix/Cytoperm (BD Pharmingen).

Histology and immunofluorescence

The left liver lobe was collected and fixed in 4% paraformaldehyde; 10-mmcryosections were prepared using Leica CM1900UV microtome (LeicaMicrosystems, Wetzlar, Germany). Infection foci were visualized bystaining with L. monocytogenes antiserum (BD Biosciences, San Jose, CA),followed by staining with Alexa633-F(ab9)2-anti-rabbit IgG (Invitrogen).iNOS primary staining was performed using anti-iNOS Ab purchased fromMillipore. ICAM-1 was stained by anti–ICAM-1–biotin (3E2) Ab pur-chased from BD Biosciences. CD31 was stained by anti-CD31 (MEC 13.3)Ab purchased from BD Biosciences. Nuclei were visualized with DAPI(Vector Laboratories, Burlingame, CA). Images were acquired with LeicaTCS SP2 AOBS laser scanning confocal microscope (Leica Microsystems)with a 203 0.7 NA objective lens. Acquired images were processed andanalyzed with Volocity software (PerkinElmer Improvision,Waltham,MA).

Cell sorting and transfer

Bone marrow cells were collected from femurs and tibias. EGFP+ cells weresorted by Memorial Sloan-Kettering Cancer Center’s flow cytometry corefacility using a BD ARIA flow cytometer (BD Biosciences). NK1.1+ cellswere depleted using NK1.1-biotin (clone PK136 from eBioscience), fol-lowing the MACS sorting protocol (Miltenyi Biotec, Auburn, CA). Forlabeling, cells were incubated with 2 mM CFSE in PBS or 4 mM 5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine (CMTMR; both

from Invitrogen) in RPMI 1640 for 15 min at 37˚C and washed twice inPBS. CMTMR-labeled cells were incubated in RPMI 1640 for an addi-tional 20 min at 37˚C. Cells were counted and mixed to give a 1:1 ratio ofCFSE- or CMTMR-labeled cells. The cell suspension was transferred intoa recipient mouse by i.v. injection. For pertussis toxin treatment, dye-labeled cells were incubated with 200 ng/ml Bordetella pertussis toxin(Calbiochem, San Diego, CA) in RPMI 1640 with 2% FBS at 37˚C for20 min and washed three times with PBS before transfer.

Calcium influx assay

A calcium influx assay was performed, as previously described (34), toconfirm the inhibition of chemokine signaling by pertussis toxin treatment.Cells were labeled with indo-1 (Invitrogen), according to the productmanual, and stimulated at 37˚C with 10 ng/ml rMCP-1 that was preparedin our laboratory. Calcium influx was detected with a BD LSRII flowcytometer (BD Biosciences). One micromolar ionomycin was used asa positive control to induce calcium influx.

In vivo Ab blocking

F(ab9)2 fragment of anti-CD11b, anti-CD44 Abs were prepared accordingto the manufacturer’s protocol for the F(ab9)2 Preparation Kit (Pierce,Rockford, IL). A dose (4 mg/kg body weight) of CD11b (5C6), CD44(IM7), ICAM-1 (YN1/1.7.4), CD11a (M17/4) blocking Ab or IgG controlwas injected i.v. into infected C57BL/6 mice at 12 h postinfection. Livermonocytes were analyzed by FACS at 6 h after treatment.

Intravital microscopy

Liver intravital microscopy was conducted, as previously described (35), ona Zeiss LSM 710 single-photon microscope (Oberkochen, Germany) witha 253 1.0 NA lens. In brief, animals were anesthetized; an incision wasmade along the peritoneum to expose a single lobe of the liver, and themouse was placed on an inverted microscope platform. Animals wereprovided with additional anesthesia, as needed, and oxygen during imaging.Blood flow was visualized by injecting 10-kDa fluorescent dextran duringthe imaging. L. monocytogenes was visualized by using a 10403S strainexpressing red fluorescent protein. Image analysis was conducted withVolocity Software (PerkinElmer Improvision).

Statistics

Statistical validation of data was done with the Student t test when thesample size was greater than five per group; the nonparametric Mann-Whitney U test was used when the sample size was three to five per group.All data are presented as the arithmetic mean 6 SEM.

ResultsLy6Chigh monocytes are recruited to foci of L. monocytogenesinfection in the liver

Hepatocytes constitute the major cell type in the liver parenchyma.Resident immune cells, including Kupffer cells, DCs, NK cells, NKT cells, and T cells, reside in the lumen of portal tracts and sinusoids(36). During steady state, Ly6Chigh monocytes represent ∼1–3% oftotal CD45+ cells in the liver. Systemic L. monocytogenes infectioninduces the influx of leukocytes into the liver, and increasednumbers of Ly6Chigh monocytes are detectable within 1 d of in-fection (Fig. 1A). The number of Ly6Chigh monocytes in the liverincreases as the infection progresses and peaks 3 to 4 d after bac-terial inoculation, accounting for ∼30% of CD45+ cells in the liver(Fig. 1B). Upon bacterial clearance 10 d following infection, thenumber of Ly6Chigh monocytes in the liver decreases, eventuallyreturning to steady-state levels 2 wk postinfection (Fig. 1B, 1C).Using a CCR2-reporter mouse strain (31, 37), in which 95% of

EGFP-expressing cells are Ly6Chigh monocytes and ∼99% of Ly6-Chigh monocytes express EGFP (Supplemental Fig. 1), we trackedLy6Chigh monocytes. EGFP+ cells that are not Ly6Chigh monocytesexpress NK1.1 and account for ∼5% of total EGFP+ cells. In un-infected mice, a small number of EGFP+ cells are seen within thelumen of presinusoid venules, and very few are found within si-nusoids. During L. monocytogenes infection, EGFP+ cells surroundfoci of infection and form lesions within the liver (Fig. 1D).

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However, the majority of EGFP+ monocytes are not infected, asdetermined by quantitative culture of FACS-sorted EGFP+ mono-cytes from infected livers. One million sorted monocytes yieldedonly 100–200 CFU L. monocytogenes, indicating that only 1 in5,000–10,000 monocytes harbors living bacteria.

Ly6Chigh monocytes differentiate into TipDCs at foci ofinfection

Our previous studies showed that Ly6Chigh monocytes differentiateinto TipDCs in the spleen and contribute to immune defense (9). Todetermine whether recruited monocytes undergo similar differen-tiation in the liver, we isolated leukocytes from infected liver andanalyzed the expression of characteristic markers of TipDCs. In-tracellular cytokine staining revealed high levels of TNF expres-sion in CD11bhighLy6Chigh monocytes (Fig. 2A). Increased levelsof iNOS expression were also detected in these monocytes by in-tracellular staining (Fig. 2B), and histologic analysis demonstratedthat monocytes located within foci of infection express iNOS,suggesting that in vivo proximity to bacteria induces iNOS ex-pression (Fig. 2D). These CD11bhighLy6Chigh monocytes expressCD11c and MHC class II, albeit at various levels (Fig. 2C).

CCR2 deficiency results in impaired monocyte recruitment tothe liver and greater bacterial burdens

Recruitment of Ly6Chigh monocytes to the liver was greatly di-minished in CCR2-deficient mice during the steady state and afterListeria infection (Fig. 3A). Impaired monocyte recruitment re-sulted in a greater bacterial burden in the liver as early as 1 dfollowing infection. Three days following inoculation with a nor-mally sublethal dose of bacteria, CCR22/2 mice had 100-foldmore L. monocytogenes in the liver than did wild type mice (Fig.

3B) and died of infection by day 5. Recruitment of CD11bhigh

Ly6CintLy6G+ neutrophils was not diminished by CCR2 de-ficiency (Supplemental Fig. 2). The absolute number of neu-trophils in the liver of infected CCR2-deficient mice was twicethat of their wild type counterparts, most likely in response toincreased hepatic infection in CCR2-deficient mice. Of note,splenic infection in CCR22/2 and wild type mice did not differuntil day 3 of infection (23), whereas we detected a 5-fold dif-ference in the liver 1 d after infection. This may result froma relative paucity of resident Ly6Chigh monocytes in liver com-pared with spleen prior to and during the early stages of infection.Althoughprevious studies demonstrated that Ly6Chighmonocytes

accumulate in the bone marrow of CCR22/2 mice during L. mon-ocytogenes infection, it remains unclear whether cell-autonomousexpression of CCR2 is essential for egress from the bone marrow orwhether emigration of a few CCR2-expressing monocytes is suffi-cient to promote the emigration of other monocytes. To address thisissue, we generated bone marrow chimerical mice using irradiatedwild type C57BL/6 mice as recipients of CCR2+/+ and CCR22/2

donor bonemarrowmixed in different ratios. Infection of thesemicewith L. monocytogenes confirmed a cell-intrinsic and essential rolefor CCR2 in the egress of Ly6Chigh monocytes from bone marrow(Fig. 3C). By changing the ratio of donorCCR2+/+ toCCR22/2 bonemarrow cells, we also demonstrated a correlation between bacterialclearance from the liver and the number of recruited CCR2+/+

Ly6Chigh monocytes (Fig. 3D).

CCR2 is dispensable for Ly6Chigh monocyte trafficking from thebloodstream to hepatic foci of infection

Ly6Chigh monocytes circulating in the bloodstream express CCR2and, thus, are presumed to be responsive to CCR2 ligands expressed

FIGURE 1. Ly6Chigh monocytes are recruited to

the liver during L. monocytogenes infection. A,

C57BL/6micewere infected with L. monocytogenes,

and CD45+ leukocytes from liverswere characterized

for Ly6C and CD11b expression by flow cytometry 1,

2, and 3 d later.B, The number of Ly6Chighmonocytes

was determined for 14 d following infection with

L. monocytogenes. C, The mean number of bacteria

per liver, from five mice per time point, was de-

termined by culturing organ homogenates on brain-

heart infusion plates. D, Livers were harvested from

naive CCR2-reporter mice and CCR2-reporter mice

infected for 2 or 3 d with L. monocytogenes, sec-

tioned, and stained for L. monocytogenes with

a polyclonal antiserum and with DAPI to detect nu-

clei. Monocytes were detected by EGFP fluorescence

(original magnification320; scale bar, 70mm.).

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by peripheral tissues. The expression of MCP-1 and -3, two che-mokine ligands of CCR2, is readily detected in peripheral tissues,including spleen and liver (25). However, it remains unclearwhether CCR2-mediated signals are required for monocyte traf-

ficking into and within L. monocytogenes-infected organs. To di-rectly compare recruitment of CCR2+/+ and CCR22/2 Ly6Chigh

monocytes from the circulation to the liver, we injected mixturesof wild type and CCR22/2 monocytes into the bloodstream of

FIGURE 3. CCR2 deficiency results in impaired monocyte recruitment to the liver and higher bacterial burdens. A, Representative FACS plots of liver cells

gated on CD45+ nonparenchymal cells and stained for CD11b and Ly6C. Cells were collected from C57BL/6 or CCR22/2 mice that were uninfected or mice

that were infected with L. monocytogenes for 3 d. B, CCR2 deficiency results in greater bacterial burdens in the liver. Liver CFU of C57BL/6 and CCR22/2

mice were quantified 1 and 3 d following L. monocytogenes infection. C, CCR2 is required for the egress of Ly6Chigh monocytes from bone marrow. C57BL/6

mice were lethally irradiated and reconstituted with a mixture of bone marrow cells from CCR2+/+ mice (CD45.1+CD45.2+) and CCR22/2 mice (CD45.12

CD45.2+). Cells from bone marrow and liver of uninfected and L. monocytogenes-infected chimerical mice, gated on Ly6Chigh monocytes, were characterized

by flow cytometry. D, Lethally irradiated C57BL/6 mice were reconstituted with different mixtures of CCR2+/+ and CCR22/2 bone marrow cells. The ratios

following reconstitution were confirmed by staining for congenic markers. Mice were infected with L. monocytogenes 6 wk following bone marrow

transplantation, and the numbers of live bacteria in livers were determined 3 d following infection.

FIGURE 2. Ly6Chigh monocytes differentiate into TipDCs at foci of infection. Liver nonparenchymal cells were collected from C57BL/6 mice 3 d

following L. monocytogenes infection and incubated with brefeldin A for 4 h. A, CD11b expression and TNF production by cells obtained from uninfected

and infected mice were detected by flow cytometry. B, iNOS expression by Ly6Chigh monocytes was determined by intracellular staining in uninfected and

L. monocytogenes-infected mice. C, CD11bhighLy6Chigh monocytes express CD11c and MHC class II. Dot plot is gated on CD45+ cells (gray) and

CD11bhighLy6Chigh cells (green), showing the increased expression of CD11c and MHC class II by monocytes relative to that of other cell populations. D,

Immunofluorescence staining for iNOS was performed on liver sections of infected CCR2-reporter mice. DAPI staining identifies hepatocyte nuclei, and

EGFP expression identifies Ly6Chigh monocytes (original magnification 320; scale bar, 70mm.).

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L. monocytogenes-infected recipient mice and monitored theirtargeting to the liver. First, CCR2-reporter mice were crossed tothe CCR22/2 background to obtain the CCR22/2Tg(CCR2-EGFP)strain. We sorted EGFP+ bone marrow cells, in which NK1.1+ cellshad been depleted, from CCR2+/+ Tg(CCR2-EGFP) and CCR22/2

Tg(CCR2-EGFP) mice that had been infected 6 h previously withL. monocytogenes, differentially labeled them with fluorescentdyes in vitro, and then mixed cells at a 1:1 ratio prior to transferinto infected C57BL/6J recipient mice (Fig. 4A). We retrievedcomparable numbers of transferred CCR2+/+ or CCR22/2 Ly6Chigh

monocytes from the liver 12 h after cell transfer (Fig. 4B). Histo-logic analysis indicated CCR2+/+ and CCR22/2 monocytes local-ized equivalently to foci of infection (Fig. 4C).We monitored trafficking of monocytes in the livers of infected

CCR2+/+ and CCR22/2 CCR2-reporter mice by intravital mi-croscopy. EGFP+ cells accumulated at foci of infection in bothmice, although the density of EGFP+ cells was greater in CCR2+/+

mice, reflecting markedly decreased emigration of EGFP+ cellsfrom the bone marrow of CCR22/2 mice. The movement of

EGFP+ cells in areas proximal to foci of infection was similar inCCR2+/+ and CCR22/2 mice, suggesting that CCR2 is not re-quired for movement within the liver (Supplemental Videos 1, 2).Of note, monocytes that entered the center of foci of infectionbecame less mobile, potentially as a result of decreased bloodperfusion to these regions. To further characterize the relativetrafficking of CCR2+/+ and CCR22/2 monocytes in the L. mono-cytogenes-infected liver, we adoptively transferred bone marrowmonocytes from CCR2+/+ or CCR22/2 donor mice, each ex-pressing EGFP under the CCR2 promoter, into the bloodstreamof infected wild type recipient mice and recorded their traffickingin the regions adjacent to infected hepatic foci (SupplementalVideos 3, 4). Quantitative analysis of EGFP+ donor-cell traf-ficking showed no difference in the speed, arrest, or linearity(meandering index) between monocytes sufficient or deficient forCCR2 (p = 0.57, p = 0.37, and p = 0.84, respectively) (Fig. 4D).These results indicated that CCR2-mediated signals do not con-tribute to trafficking of monocytes within the L. monocytogenes-infected liver.

FIGURE 4. CCR2 is dispensable for the recruitment

of Ly6Chigh monocytes from the circulation to foci of

infection in the liver. A, Ly6Chigh monocytes were sorted

from the bone marrow of infected CCR2+/+ Tg(CCR2-

EGFP) and CCR22/2 Tg(CCR2-EGFP) mice based on

their expression of EGFP, and NK1.1+ cells were de-

pleted by MACS sorting. Monocytes were differentially

labeled with CFSE or CMTMRfluorescent dyes in vitro,

mixed at a 1:1 ratio, and transferred into infected

C57BL/6J recipient mice. Livers of recipients were

harvested 12 h later. B, Representative FACS plot gated

on liver Ly6Chigh monocytes, showing the percentage of

transferred CCR2+/+ and CCR22/2 cells. C, Immuno-

fluorescence analysis of liver sections of recipients.

CCR2+/+ monocytes were labeled with CFSE (green),

whereas CCR22/2 monocytes were labeled with

CMTMR (red) (original magnification 320; scale bar,

70mm.). D, EGFP+ monocytes sorted from the bone

marrow of infected CCR2+/+ Tg(CCR2-EGFP) or

CCR22/2 Tg(CCR2-EGFP) mice were injected into

infected C57BL/6J recipient mice. Liver intravital mi-

croscopy was conducted following cell transfer. In-

dividual cells were plotted based on trafficking-behavior

indices from image analysis.

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CCR2 is dispensable for the bactericidal function of Ly6Chigh

monocytes in the liver

To test whether monocytes lacking CCR2 control infection inthe liver, we transferred sorted EGFP+ bone marrow cells fromCCR22/2 or CCR2+/+ Tg(CCR2-EGFP) donors into infectedCCR22/2 recipients and measured the intensity of infection 24 h

later. We observed significant and similar reduction of liver bac-terial counts in recipients receiving CCR2+/+ or CCR22/2 mono-cytes compared with mice receiving no transferred monocytes(Fig. 5). This indicated that monocytes that emigrated from thebone marrow and entered the bloodstream traffic to hepatic sitesof infection provide protection against L. monocytogenes in aCCR2-independent manner.

Chemokine-independent adhesion mechanisms mediate thetargeting of monocytes to foci of infection

Because CCR2 is not required for the targeting of Ly6Chigh mon-ocytes to foci of hepatic infection, we asked whether chemokinereceptor-mediated signals in general are required. To address this,we treated EGFP+ bone marrow cells from CCR2-reporter micein vitro with pertussis toxin, which blocks G protein-coupled re-ceptors, prior to transfer into a L. monocytogenes-infected recipientmouse (Fig. 6A, 6B). Retention and targeting of pertussis toxin-treated and untreated monocytes to the liver and to foci of infectionwere comparable, as determined by flow cytometry and histologicanalysis (Fig. 6C).Chemokine receptor-independent targeting of monocytes from

the bloodstream to the liver suggested that monocytes might betargeted to foci of infection by other mechanisms. Stimulatedmonocytes express high levels of surface Mac-1 integrin (38),which consists of CD11b and CD18 subunits and can serve asa receptor for ICAM-1 (39). To determine whether L. mono-cytogenes infection induces ICAM-1 expression in the liver andwhether induction in the liver is localized to foci of infection, we

FIGURE 6. Chemokine-independent targeting

of monocytes to the foci of infection. A, Ly6Chigh

monocytes were sorted from the bone marrow of

infected CCR2+/+ Tg(CCR2-EGFP) mice. Mono-

cytes were treated with 200 ng/ml pertussis toxin

or PBS and differentially labeled with CFSE or

CMTMR fluorescent dyes in vitro. A mixture of

toxin- and control-treated monocytes (1:1 ratio)

was transferred into infected C57BL/6J recipient

mice. Livers of recipients were harvested 12 h after

cell transfer. B, MCP-1–induced calcium influx

was inhibited by pertussis toxin treatment. C,

Representative FACS plot gated on liver Ly6Chigh

monocytes showing the percentage of transferred

pertussis toxin-treated and control cells. Immuno-

fluorescence analysis of liver sections of recipients

is shown. Pertussis toxin-treated monocytes were

labeled with CFSE (green), control monocytes

were labeled with CMTMR (red), and hepatocyte

nuclei are stained with DAPI (original magnifica-

tion 320; scale bar, 70mm.).

FIGURE 5. CCR2 is dispensable for the bactericidal function of Ly6-

Chigh monocytes in the liver. CCR22/2 mice were infected with L. mon-

ocytogenes; 24 h later, mice were infused with Ly6Chigh monocytes sorted

from the bone marrow of infected CCR2+/+ Tg(CCR2-EGFP) or CCR22/2

Tg(CCR2-EGFP) donors. The numbers of viable L. monocytogenes bac-

teria in the livers of untreated CCR22/2 mice and CCR22/2 mice that

received CCR2+/+ Ly6Chigh monocytes or CCR22/2 Ly6Chigh monocytes

are plotted. pp , 0.05.

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performed immunofluorescence staining of infected livers andobserved induction of ICAM-1 in regions immediately adjacent tofoci of L. monocytogenes infection (Fig. 7A). ICAM-1 expressionpartially colocalizes with the endothelial marker CD31 (Fig. 7B),suggesting that sinusoidal endothelial cells upregulate ICAM-1expression in response to bacterial molecules. Ab-mediatedblockade of CD11b or ICAM-1 reduced retention of Ly6Chigh

monocytes in liver sinusoids and their accumulation at infectedfoci. In contrast, Ab-mediated blockade of CD11a, a subunit ofLFA-1 that also binds ICAM-1, did not reduce monocyte re-cruitment to the liver (Fig. 7C, 7D). Focal staining for ICAM-1persisted when monocyte recruitment was inhibited by CD11b

blockade or in CCR2-deficient mice (Fig. 7C), indicating thatinduced ICAM-1 is expressed by nonmonocytes. Nevertheless,because monocytes also express ICAM-1, it is possible that re-cruited monocytes enhance trafficking of circulating monocytes tofoci of infection in a positive feedback loop. CD44 was alsodemonstrated to function as a surface molecule that sequestersneutrophils in inflamed liver sinusoids (40). By blocking CD44,we observed 50–60% reduction of monocyte recruitment to theliver (Fig. 7D). Ab-mediated blockade of CD11b, ICAM-1, orCD44 did not result in the accumulation of monocytes in thebone marrow, indicating the trafficking was not blocked at theinterface between bone marrow and circulation. Thus, our resultssuggest that monocyte recruitment within the liver is mediated bychemokine-independent adhesion mechanisms involving CD11b,CD44, and ICAM-1.

DiscussionRecruitment and targeting of inflammatory cells during infection isa complex process involving multiple steps that are enabled, in part,by chemokine-dependent signals. In this study, we demonstratedthat Ly6Chigh monocytes are rapidly recruited from bone marrowto the liver during L. monocytogenes infection. CCR2 deficiencyimpairs recruitment and leads to a greater bacterial burden in theliver. We provided evidence that CCR2-mediated signals princi-pally enable monocyte egress from bone marrow and that onceLy6Chigh monocytes migrate into the circulation, they are targetedto hepatic foci of infection and provide immune defense, withouta requirement for additional CCR2-mediated signals. Our studyindicated that monocyte recruitment to hepatic foci involves thefocal induction of adhesion molecules.Our histologic data and previous studies (8) showed that the

distribution of bacterial infection in the liver is localized and re-stricted to inflammatory foci that consist of recruited leukocytesand infected hepatocytes. Intravital imaging demonstrated thatblood flow to the central region of infected hepatic foci wasmarkedly diminished. Bacterial infection seems to be restricted bythe recruitment of inflammatory monocytes to the region sur-rounding bacterially infected foci. In contrast to previous studies,in which leukocyte recruitment to the liver was studied in thesetting of widespread inflammation (29, 40), our study focused onmonocyte recruitment to focal sites of infection and inflammation.We found that bacterial infection selectively increased expressionof the adhesion molecule ICAM-1 in regions immediately adja-cent to infected foci. Focal induction of ICAM-1 during L. mon-ocytogenes infection may result from localized production anddiffusion of TLR ligands from areas of bacterial infection andhepatocyte necrosis.Leukocyte recruitment in liver sinusoids occurs by distinct

mechanisms that differ from the tether-and-roll paradigm gov-erning the adhesion and emigration in other vascular compartments(41). Hepatic sinusoidal endothelium does not express selectins,and selectins are not essential for recruitment of cells to sinusoids(42). In fact, visualization of leukocyte movement in the liverrevealed that adhesion in sinusoids often occurred independent ofrolling (41). Hepatic sinusoids are fenestrated and lack a basallamina and tight junctions. Thus, the recruitment of monocytesfrom sinusoids to foci of infection likely follows a scheme thatdiffers from chemokine-dependent endothelial transmigration.Although most previous studies showed that leukocyte chemotaxisinduced by chemokines is inhibited by pertussis toxin, certainchemokine receptors were shown to couple to pertussis toxin-sensitive and -resistant G proteins (43). Therefore, it is possiblethat pertussis toxin-insensitive signals mediated by chemokinereceptors contribute to monocyte recruitment.

FIGURE 7. Focal induction of ICAM-1 mediates targeting of Ly6Chigh

monocytes. A, Immunofluorescence staining of liver sections from uninfected

or L. monocytogenes-infected C57BL/6J mice with L. monocytogenes and

ICAM-1. B, Sinusoidal endothelial cells express ICAM-1. Costaining of

ICAM-1 and the endothelial marker CD31 is shown.C, Ab-mediated blockade

of CD11b diminished the targeting of Ly6Chigh monocytes to foci of infection.

Infection of CCR2+/+ Tg(CCR2-EGFP) mice resulted in the accumulation of

EGFP-expressing monocytes at sites of L. monocytogenes infection and

ICAM-1 expression (left panel). Treatment with anti-CD11b blocking Ab

prevented monocyte localization but not ICAM-1 expression (middle panel).

Similarly, in the absence of CCR2, ICAM-1 expression surrounded foci of

infection in the absence ofmonocytes (right panel) (originalmagnification320;

scale bar, 70mm.). D, Blocking CD11b, CD44, and ICAM-1 diminished

monocyte recruitment to the liver. The F(ab9)2 fragments of indicated blocking

Abs were injected into C57BL/6 mice 12 h following infection. Liver mono-

cytes were quantified by FACS 6 h after Ab inoculation. The relative frequency

of Ly6Chigh monocytes in the livers of Ab-treated mice relative to untreated

control mice is plotted for three mice per treatment group.

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Previous studies showed that neutrophil sequestration in inflamedliver sinusoids following systemic LPS administration is mediatedby the interaction of CD44 on neutrophils with extracellular hyalur-onan (HA) (40). Systemic administration of LPS, in addition to re-sulting in hepatic inflammation, induces the expression of serum-derived HA-associated protein, which enhances the association ofHA with CD44. In our experiments, we found that Ab-mediatedblockade of CD44 markedly reduced the number of Ly6Chigh mon-ocytes detected by FACS in L. monocytogenes-infected livers (Fig.7D). Thus, our studies demonstrated a role for CD44 in inflammatorymonocyte recruitment to the liver. However, it is unclear whetherL. monocytogenes infection induces serum-derived HA-associatedprotein expression and, thus, promotes monocyte recruitment byenhancing interactions between CD44 and HA or whether HA ex-pression is altered in areas adjacent to foci of L. monocytogenes in-fection. Further studies are necessary to determine how CD44contributes to the targeting of monocytes to liver during L. mono-cytogenes infection.Studies with CX3CR1-GFP mice using intravital microscopy

documented the patrolling behavior of resident monocytes innoninflamed dermis (44). In those studies, specific Ab-mediatedblockade of LFA-1 led to detachment of CX3CR1-expressingmonocytes from the endothelial cell surface, suggesting that LFA-1was essential for maintaining monocyte attachment to the endo-thelium under conditions of shear force. Although we observedsimilar patrolling of Ly6Chigh monocytes in L. monocytogenes-infected liver, LFA-1 blockade did not result in a reduction inmonocyte recruitment to the liver or detachment of monocytes fromthe sinusoid wall or from foci of infection. It is possible that the roleof LFA-1 in patrolling differs between the inflammatory CCR2+

monocyte subset and the CX3CR1+ subset. Alternatively, adhesionmechanisms during infection may differ from those in the absenceof inflammation. In addition, monocyte patrolling in liver sinusoidsmay differ from that in dermal vasculature.In summary, we demonstrated that Ly6Chigh monocytes are

rapidly recruited from bone marrow to foci of infection in the liverwhere they mediate clearance of L. monocytogenes. Althoughmonocyte egress from bone marrow is CCR2 dependent, entryinto the liver parenchyma and localization to foci of bacterialinfection are chemokine receptor independent. Focal induction ofICAM-1 facilitates the targeting of inflammatory monocytes toregions immediately adjacent to foci of bacterial infection. Ly6-Chigh monocytes are increasingly recognized as essential media-tors of defense against viral, bacterial, fungal, and protozoanpathogens (8, 10, 11, 13, 45–47). Deciphering the mechanismsthat enable monocyte trafficking to peripheral sites of bacterialinfection may provide new approaches to enhance immune de-fense against these pathogens.

AcknowledgmentsWe thank Dan Portnoy for providing L. monocytogenes strain expressing

red fluorescent protein and Natalya Serbina and Ting Jia for helpful com-

ments and suggestions.

DisclosuresThe authors have no financial conflicts of interest.

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