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Microcirculation via IL-10Mac-1 in Endotoxemic Hepatic Selective Down-Regulation of Neutrophil

Cara and Paul KubesChristopher Curtis Matchett Waterhouse, Denise Carmona Gustavo Batista Menezes, Woo-Yong Lee, Hong Zhou,

http://www.jimmunol.org/content/183/11/7557doi: 10.4049/jimmunol.0901786November 2009;

2009; 183:7557-7568; Prepublished online 16J Immunol 

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6.DC1http://www.jimmunol.org/content/suppl/2009/11/16/jimmunol.090178

Referenceshttp://www.jimmunol.org/content/183/11/7557.full#ref-list-1

, 13 of which you can access for free at: cites 43 articlesThis article

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Print ISSN: 0022-1767 Online ISSN: 1550-6606. Immunologists, Inc. All rights reserved.Copyright © 2009 by The American Association of1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

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Selective Down-Regulation of Neutrophil Mac-1 inEndotoxemic Hepatic Microcirculation via IL-101

Gustavo Batista Menezes,* Woo-Yong Lee,* Hong Zhou,*Christopher Curtis Matchett Waterhouse,* Denise Carmona Cara,† and Paul Kubes2*

Hepatic neutrophil adhesion during endotoxemia is an integrin-independent, CD44-dependent process. Because integrins functionin other endotoxemic vasculatures, we used spinning disk confocal intravital microscopy to assess whether LPS down-modulatedintegrin functions in sinusoids. First, we applied fMLP onto the liver surface, and compared it with systemic LPS administration.Local fMLP caused neutrophil adhesion, crawling, and emigration for at least 2 h. Surprisingly, the number of adherent andcrawling neutrophils was markedly reduced in Mac-1�/� and ICAM-1�/� mice, but not in mice treated with anti-CD44 mAb. Bycontrast, systemic LPS injection induced a robust accumulation of neutrophils in sinusoids, which was dependent on CD44, butnot on integrins. Strikingly, local fMLP could not induce any integrin-dependent adhesion in endotoxemic mice treated withanti-CD44 mAb, indicating that Mac-1-dependent neutrophil adhesion was inhibited by LPS. This response was localized to thehepatic microvasculature because neutrophils still adhered via integrins in brain microvasculature. ICAM-1/ICAM-2 levels werenot decreased, but following LPS treatment, Mac-1 was down-regulated in neutrophils localized to liver, but not in the circulation.Mac-1 down-regulation in neutrophils was not observed in IL-10�/� mice. In vitro neutrophil incubation with IL-10 induced directdecrease of Mac-1 expression and adhesivity in LPS-stimulated neutrophils. Therefore, our data suggest that Mac-1 is necessaryfor neutrophil adhesion and crawling during local inflammatory stimuli in sinusoids, but during systemic inflammation, neutro-phils are exposed to high concentrations of IL-10, leading to a CD44-dependent, integrin-independent adhesion. This may be amechanism to keep neutrophils in sinusoids for intravascular trapping. The Journal of Immunology, 2009, 183: 7557–7568.

L iver dysfunction during systemic, uncontrolled inflamma-tion is a common clinical finding, which has been welldefined as a direct consequence of neutrophil accumula-

tion in liver parenchyma (1). Neutrophil-mediated liver injury hasbeen widely studied using a number of experimental models, in-cluding cecal ligation and puncture (2), ischemia-reperfusion (3),alcohol (4), endotoxin (5), and other insults (6). In these situations,neutrophils can be attracted to the liver parenchyma by a variety ofinflammatory mediators, including TNF-� and IL-1, which pro-duce increased levels of chemokines (7). The emigrated neutro-phils release reactive oxygen species (8), peroxidases (9), and pro-teases (10), which are molecules that cause liver parenchymaldamage.

In contrast to the neutrophil recruitment cascade derived frommesentery, muscle, brain, and skin in vivo and flow chambers in

vitro, the liver displays a distinct neutrophil recruitment paradigm(11). It is well accepted that in tissues like brain, selectins play animportant role in initial neutrophil tethering and rolling, and inte-grins (Mac-1 and LFA-1, mainly) are crucial to promote firm ad-hesion of neutrophils to the vessel wall (12). In contrast, neutrophilaccumulation within liver sinusoids during systemic inflammationis independent of selectins (13) and �2 integrins (7). In fact, theparticipation of these adhesion molecules in neutrophil recruitmentinto liver has only been described for postsinusoidal venules, butnot sinusoids (14). Although originally it was hypothesized thatneutrophil accumulation within inflamed sinusoids was a conse-quence of mechanical trapping of these cells in these narrow ves-sels (15), blockade of adhesion molecules such as CD44 and itsligand hyaluronan (HA)3 has recently been reported to preventboth neutrophil recruitment and the progression of neutrophil-de-rived liver injury (5, 16). CD44 avidity for HA did not increase inendotoxemia, but rather serum-derived HA-associated protein(SHAP), a molecule known to increase HA adhesivity, was notedto bind to HA in sinusoids in response to stimuli-like endotoxin(5). Clearly, there is a dominant role for neutrophil adhesion in theliver, albeit not via integrins. This is surprising in light of datasuggesting that basal recruitment of lymphocytes into liver oc-curred via integrins (17). This raises the following question: whydo the neutrophils not use integrins to adhere in liver sinusoids?

In addition to releasing many proinflammatory acute-phaseproteins, the liver has the capacity to release IL-10 and TGF-�as tolerogenic cytokines (18, 19). These regulatory cytokinescan protect the liver against severe injury and failure duringacute inflammatory processes, displaying an anti-inflammatory

*Calvin, Phoebe, and Joan Snyder Institute for Infection, Immunity, and Inflamma-tion, University of Calgary, Alberta, Canada; and †General Pathology Department,Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

Received for publication June 5, 2009. Accepted for publication October 4, 2009.

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 by Canadian Institutes for Health Research operating grantand grant group. G.B.M. received a fellowship from Fundacao de Amparo a Pesquisade Minas Gerais, Brazil. C.M.W. is founded by a RE/MAX Fellowship through theAlberta Children’s Hospital Foundation, and is a Canadian Institutes for Health Re-search Training Fellow in the Canadian Child Health Clinician Scientist Program, inpartnership with SickKids Foundation and the Child and Family Research Institute ofBritish Columbia. D.C.C. is a Conselho Nacional do Desenvolvimento Científico eTecnologico (Conselho Nacional de Pesquisas) Scientist. P.K. is an Alberta HeritageFoundation for Medical Research Scientist and the Snyder Chair in Critical CareMedicine.2 Address correspondence and reprint requests to Dr. Paul Kubes, Calvin, Phoebe andJoan Snyder Institute for Infection, Immunity and Inflammation, HRIC 4A26A, Uni-versity of Calgary 3280 Hospital Drive N.W., Calgary, Alberta, T2N 4N1 Canada.E-mail address: pkubes@ucalgary.ca

3 Abbreviations used in this paper: HA, hyaluronan; SHAP, serum-derived HA-as-sociated protein.

Copyright © 2009 by The American Association of Immunologists, Inc. 0022-1767/09/$2.00

The Journal of Immunology

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response under these conditions (20, 21). The constant expres-sion of regulatory cytokines, such as IL-10 in the liver, mayaccount for the proposed tolerogenic role of the liver. Indeed,endotoxin and other bacterial products constantly arrive fromthe intestine into the liver, where they are cleared from thecirculation without promoting overt inflammatory responses(22). This raises the possibility that even during endotoxemia orsepsis, the liver releases factors that continuously reduce inte-grin adhesivity on neutrophils.

Using spinning disk confocal microscopy, we investigated dif-ferences in adhesion following exposure of neutrophils to stimuliwith different activating properties. Interestingly, direct local stim-ulation of neutrophils with formyl peptide agonist revealed thatintegrins and, more specifically, Mac-1 were essential for the earlyneutrophil recruitment into the liver. In sharp contrast, endotoxininduced a CD44-dependent, integrin-independent mechanism ofneutrophil recruitment into the liver. Our data also demonstratethat integrin function was abolished specifically in the liver duringendotoxemia via an IL-10-dependent mechanism, allowing CD44to become the dominant adhesive mechanism.

Materials and MethodsMice

C57BL/6 and IL-10�/� mice were purchased from The Jackson Labora-tory. ICAM-1�/� mice were a gift of D. Bullard (University of Alabama,Birmingham, AL). Lysm-enhanced GFP mice, in which the enhanced GFPgene was knocked into the murine lysozyme M locus (greater than 80% areneutrophils), were provided by T. Graf (Albert Einstein College of Med-icine, Bronx, NY) and generated, as previously described (23). Mac-1�/�

mice (24) were provided by Dr. C. M. Ballantyne (Methodist DeBakeyHeart Center and Baylor College of Medicine, Houston, TX). All micewere maintained in a specific pathogen-free, double-barrier unit at the Uni-versity of Calgary. The protocols used were in accordance with the guide-lines drafted by the University of Calgary Animal Care Committee and theCanadian Council on the Use of Laboratory Animals. IL-10�/� mice wereage matched (7 wk) to avoid time-related spontaneous inflammatory boweldisease complications, and other strains were used between 6 and 12 wkof age.

Blocking Abs to study involvement of adhesion molecules

mAbs (eBiosciences) against ICAM-1 (100 �g/mouse), Mac-1 (CD11b; 50�g/mouse), LFA-1 (CD11a; 50 �g/mouse), and CD44 (20 �g/mouse) wereinjected i.v. 30 min before the experiments (5, 25).

FIGURE 1. A local fMLP stimulus induceshepatic neutrophil adhesion. A and B, Three-di-mensional reconstruction of liver Z-stacks show-ing GFP-expressing neutrophils (in green) accu-mulated in sinusoids (red) due to a local stimulus(A, saline; B, fMLP; �10 objective). Neutrophilaccumulation due to fMLP stimulus is observedwithin 15 min (C) and progressively increasedover 2 h. The majority of neutrophils accumu-lated adjacent to the filter (D). Neutrophil accu-mulation was assessed by counting the numberof cells adhered in liver sinusoids in each half ofthe field of view, being one-half immediately ad-jacent to the filter and the other the distant fieldof view, labeled as distal (E and F; �10 objec-tive). �, Indicates statistically significant differ-ence compared with controls (saline). p � 0.05.

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Induction of local and systemic inflammatoryresponses

We developed a novel model of local liver inflammation using a 1-mm2

filter (paper filter grade 410; VWR Scientific) gently placed onto the liversurface impregnated with the synthetic peptide (WKYMV(D-Met)-NH2;Phoenix Pharmaceutical), which functions as a formyl peptide receptoragonist (called fMLP in this study). Liver samples were collected for his-tological analysis using Leder esterase stain.

LPS (Escherichia coli LPS; 0111:B4; Calbiochem) was used to induceendotoxemia (systemic inflammation; 0.5 mg/Kg; i.p.). In one set of ex-periments, E. coli (Xen14; Bioware; 107 CFU) were injected i.p., and the

liver was prepared for intravital microscopy to determine which adhesionpathway dominates.

Spinning disk confocal intravital microscopy: visualization ofliver microvasculature

Murine liver intravital microscopy was performed, as previously described(5). Briefly, mice were anesthetized by i.p. injection of a mixture of 10mg/kg xylazine hydrochloride (MTC Pharmaceuticals) and 200 mg/kg ket-amine hydrochloride (Rogar/STB). The right jugular vein was cannulatedto provide additional anesthetic and for i.v. administration of Abs. Bodytemperature was maintained at 37°C using an infrared heat lamp. Mice

FIGURE 2. fMLP induces neutrophiladhesion, polarization, and emigration. Aand B, Lower magnification (�4 objec-tive) showing filter positioning on liversurface. Neutrophil (in green) accumu-lation in sinusoids due to local stimulus(A, saline; B, fMLP) was mainly ob-served adjacent to the filter in fMLP-treated mice. fMLP-stimulated neutro-phils exhibited polarized cell shape incomparison with controls (C, D, andG), with elongated axis (D, white ar-row). E and F, Histological confirma-tion of neutrophil accumulation by es-terase staining (Leder). Neutrophils (F,black arrows) were significantly in-creased in liver parenchyma following2 h of fMLP local stimulus (H). �, In-dicates statistically significant differ-ence compared with controls (saline).p � 0.05.

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were placed in a right lateral position on an adjustable microscope stage. Alateral abdominal incision along the costal margin to the midaxillary linewas made to exteriorize the liver, and all exposed tissues were moistenedwith saline-soaked gauze to prevent dehydration.

The liver was prepared for in vivo microscopic observation. Briefly, theliver was placed on the pedestal of an upright microscope and continuouslysuperfused with warmed buffer. The liver surface was then covered with acoverslip to hold the organ in position. The liver microvasculature wasvisualized using a spinning disk confocal head, and images were acquiredwith an Olympus BX51 upright microscope (Olympus) using a �4/0.16UplanSApo objective, �10/0.30 UplanFL N objective, and �20/0.45LUCplanFL N objective, as previously described (5). The microscope wasequipped with a confocal light path (WaveFx; Quorum) based on a mod-ified Yokogawa CSU-10 head (Yokogawa Electric). Lysm-enhanced GFPmice were used to visualize neutrophils in the hepatic vasculature andparenchyma. FITC anti-GR-1 (eBiosciences; 10 �g/mouse) was injectedi.v. to visualize neutrophils (in knockout mouse strains). PE-coupled anti-PECAM-1 (CD31) was used to stain liver sinusoidal endothelial cells. PE-coupled anti-ICAM-1 (2 �g/mouse) and Alexa Fluor 488-coupled anti-ICAM-2 (15 �g/mouse; Invitrogen) were used to quantify the expressionof these adhesion molecules during endotoxemia. Two laser excitationwavelengths (Cobalt) were used in rapid succession and visualized withthe appropriate long-pass filters (Semrock). A 512 � 512 pixel back-thinned electron-multiplying charge-coupled device camera (C9100-13;Hamamatsu) was used for fluorescence detection. Volocity software(Improvision) was used to drive the confocal microscope and to renderthree-dimensional reconstructions. Sensitivity settings were 232–240,and auto contrast was used. Images were captured at 16 bits/channel inred, green, and blue. Red, green, and blue channels were overlaid, whennecessary, using brightest point settings before export in .tiff or .aviformat (for movies).

Visualization of brain microvasculature

Intravital microscopy of the neutrophil-endothelium interactions in brainmicrocirculation was performed and analyzed, as previously described(26). Briefly, a craniotomy was performed using a high-speed drill (FineScience Tools), and the dura mater was removed to expose the underlyingpial microvasculature. To observe leukocyte endothelial interactions, leu-kocytes were fluorescently labeled by i.v. injection of PE anti-GR1 Abs (10�g/mouse). Neutrophil-endothelium interactions in the brain microcircu-lation were observed using a BX51W1 spinning disk confocal microscope,and movies were recorded for further analysis following similar parametersto liver microscopy analysis.

Analysis of liver intravital imaging videos

Cells were tracked and counted using ImageJ software version 1.41(National Institutes of Health). Time-lapse video exported from Voloc-ity was imported in .avi files, and then converted to 8-bit greyscale.Fluorescent cells were adjusted using threshold control, and noise par-ticles less than 2.0 pixels were removed by despeckle filter or medianfilter. Movement of cells was measured using manual tracking, and wascalculated for each track by manipulation of ImageJ output spread-sheets. The cell velocity was expressed in �m/min. Tracks of 15 min,covering all 2-h videos, were analyzed in each mouse. To quantifyneutrophil accumulation, fluorescent cells that adhered in liver sinu-soids were counted. In experiments using local stimulation, the wholefield was divided in two halves (distal and adjacent to the filter; Fig. 1,E and F), and cells were counted in each half to measure directionalneutrophil accumulation toward the chemotatic gradient. Neutrophilsthat stayed immobilized in the vasculature for more than 30 s wereconsidered as adherent cells. The expression of ICAM-1 and ICAM-2during endotoxemia was assessed by measuring the integrated densityof the fluorescence in liver three-dimensional reconstructions using Im-ageJ software.

ELISA

TNF-�, IL-6, and IL-10 were measured in liver homogenates and serum byELISA. Liver tissue was homogenized in PBS (pH 7.4) containing proteaseinhibitors (Roche Diagnostics), and protein concentration was quantifiedusing the dendritic cell protein assay (Bio-Rad), according to the manu-facturer’s protocol. Samples of liver homogenate and serum were measuredin duplicate for each of the above cytokines using OptEIA ELISA kits foreach (BD Biosciences), according to the manufacturer’s instructions. Livercytokine expression was expressed in pg/mg total protein in each sample.Serum cytokines were expressed in pg/ml.

In vitro incubation of neutrophils for assessment of adhesionmolecule expression and Mac-1/fibrinogen binding

To investigate the effect of IL-10 directly on LPS- and/or fMLP-stimulatedneutrophils, wild-type mice were anesthetized and blood was collected bycardiac puncture with heparized syringes (27). Blood samples were imme-diately placed in sterile tubes kept on ice, and LPS (100 ng/ml), fMLP (0.1�M), or IL-10 (100 ng/ml; Cedarlane Laboratories) was added to the sam-ples, as indicated. Samples were incubated for up to 4 h in a humidifiedincubator (37.5°C; 5% CO2), and then prepared for flow cytometry anal-ysis, as described below. Alexa Fluor 488-coupled fibrinogen (15 �g/ml;Invitrogen) was added to the samples at the end of the incubation processfor measurement of Mac-1 adhesivity (28). In one set of experiments, bonemarrow-derived neutrophils were purified (5) and incubated in HBSS(added to 10% of plasma) with LPS and/or IL-10 under the sameconditions.

Leukocyte harvest for flow cytometry analysis

Four hours after i.p. LPS or saline injection, mice were anesthetized and theperitoneal and thoracic cavities were opened for liver excision and blood

FIGURE 3. Local fMLP stimulus induces Mac-1/ICAM-1-dependent,CD44-independent neutrophil adhesion and crawling in liver sinusoids.Neutrophil adhesion (A), percentage of crawling cells (B), and crawlingvelocity (C) within sinusoids were assessed following 2 h of fMLP localstimulus. �, Indicates statistically significant difference compared with con-trols (saline filter), and †, in comparison with vehicle-injected fMLP-treated mice (C). p � 0.05.

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collection by cardiac puncture. LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), CD44 (Ly-24), and GR-1 (Ly-6G) expression were measured incirculating and in liver-infiltrating neutrophils in response to LPS usingflow cytometry, as previously described (29). Extraction of neutrophilsfrom the liver was performed, as described previously (30), adapted toneutrophil purification using Percoll gradient. Samples of 100 �l of wholeblood or 1 million liver-derived leukocytes were incubated with 1 �g ofFITC-conjugated mAb anti-GR-1, 3 �g of PE-conjugated mAb againstLFA-1 or CD44, and 3 �g of PE-Cy5-conjugated against Mac-1 mAb or

nonspecific isotype controls (all purchased from BD Biosciences) for 30min at room temperature. Blood samples were added to 100 �l of OptilyseB (Immunotech) to lyse RBC. Cells were washed and resuspended in aPBS/0.5% BSA/20 mM glucose solution, read by a BD FACScan flowcytometer (BD Biosciences) using CellQuest Pro software (BD Bio-sciences), and analyzed using FlowJo software (Tree Star). In all blood andliver neutrophil studies, flow cytometry was performed with parameterssetting to gate only granulocytes. Sequentially, 40,000 events were ana-lyzed for each sample and gated using side/forward scattergram to select

FIGURE 4. Effects of LPS sys-temic injection and neutrophil accu-mulation in the liver and brainmicrovasculature. A–D, Systemicadministration of LPS (0.5 mg/Kg)caused integrin-independent, CD44-dependent neutrophil accumulationin liver sinusoids. Neutrophils (ingreen) accumulated in liver sinu-soids (red) following 4 h of i.p. LPSinjection, displaying a very lowcrawling velocity (�10 objective).E–G, Integrin-dependent neutrophil(red) accumulation in brain vesselsfollowing i.p. LPS injection. �, In-dicates statistically significant dif-ference compared with controls (sa-line injection), and †, in comparisonwith vehicle-injected, LPS-treatedmice (C). p � 0.05.

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only polymorphonuclear cells and FLH-1 histogram to select only GR-1high-expressing cells (predominantly neutrophils). In a set of experiments,intracellular levels of integrins were also assessed by flow cytometry. Thecells were incubated with saturating concentrations of nonfluorescent(blocking) Abs for 30 min (10 times the regular dose; 30 �g of anti-LFA-1and anti-MAC-1) to block the majority (99%) of surface Ags. These cellswere then fixed and permeabilized (Cytofix/Cytoperm; BD Biosciences) tostain intracellular integrins with the fluorescent Abs. Mean of fluorescence

intensity of these populations was obtained and compared with control andexperimental groups.

Statistical analysis

All data are displayed as mean � SEM or percentage of control. Data wereanalyzed using standard statistical analysis (ANOVA and Student’s t test

FIGURE 5. A, Integrin-independent, CD44-dependent E. coli induced neutrophil accumula-tion in liver sinusoids. E. coli (107 CFU) inducesneutrophil (green) accumulation 4 h after i.p. ad-ministration. This is blocked with anti-CD44mAb (B and C), but not with anti-Mac-1 mAb(C). �, Indicates statistically significant differ-ence compared with saline (UT; E. coli injectionalone), and †, in comparison with vehicle-in-jected mice (UT). p � 0.05.

FIGURE 6. LPS decreases Mac-1-dependent neutrophil adhesion inliver. A, Neutrophil adhesion follow-ing 2 h of local fMLP or 4 h of i.p.LPS. Treatments were indicated bythe � sign. �, Indicates statisticallysignificant difference compared withcontrols (saline injection), and †, incomparison with LPS injection alone.B and C, Flow cytometry analysis ofblood and liver neutrophils showingselective down-regulation of Mac-1in neutrophils that home to the liver.Intracellular staining (D) for Mac-1and LFA-1 in liver neutrophils re-vealed that these molecules are notinternalized during systemic (LPS)inflammation. �, Indicates statisti-cally significant difference comparedwith controls (saline injection). p �0.05.

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with Bonferroni’s correction for multiple comparison, where appropriate).Statistical significance was set at p � 0.05.

ResultsLocal fMLP stimulus induces Mac-1/ICAM-1-dependent,CD44-independent neutrophil adhesion and crawlingin liver sinusoids

To study the features related to local liver inflammation, an fMLPimpregnated filter was placed on the liver surface, and neutrophilaccumulation was visualized using spinning disk confocal micros-copy. To better visualize neutrophil accumulation, we used three-dimensional computer reconstruction of liver Z-stacks (total depthof 80 �m) from liver microvasculature. Local administration offMLP (2 �g/filter) induced significant neutrophil adhesion and em-igration to liver parenchyma in comparison with controls (Fig. 1,A–D; supplementary video 1).4 Neutrophil adhesion was observedwithin 30 min of local exposure to fMLP with continued cell re-cruitment up to 2 h (Fig. 1C). The greatest quantity of cells waspresent immediately adjacent to the filter (Figs. 1, D–F, and 2, Aand B), and close to 100% of adherent neutrophils exhibited crawl-ing movement and polarized cell shape toward fMLP, as assessedby the longest cell axis (Fig. 2, C, D, and G). Many neutrophilswere seen to emigrate out of the sinusoids. The presence of neu-trophils in liver parenchyma was confirmed by histological stain-ing for neutrophil esterase (Fig. 2, E, F, and H). Using time lapsevideos, we observed that neutrophils were able to adhere and tocrawl inside sinusoids toward the fMLP gradient (supplementalvideo 2), a behavior not observed with saline-soaked filters (sup-plemental video 3). Observation of these cells for longer times (upto 4 h) did not reveal any changes in adhesion and crawling profile,indicating that 2 h of observation was adequate for subsequentstudies (data not shown). Some increase in neutrophil adhesioncould be noted even at 200 �m away from fMLP (labeled as distalin Fig. 1F). The majority of these cells migrated toward the che-moattractant (supplemental video 2). Macroscopically, no visuallesions were observed at the end of the experiment on the liversurface after filter placement, and sinusoidal morphology and per-fusion were not visually altered.

The number of adherent neutrophils was significantly decreasedin ICAM-1�/� and Mac-1�/� mice, but not in anti-CD44 or anti-LFA-1 mAb-treated mice (Fig. 3A). These data showing signifi-cant inhibition were confirmed using blocking Abs againstICAM-1 and Mac-1 in wild-type mice (data not shown). The per-centage of adherent cells that crawled was close to 100% and wasnot affected by either anti-LFA-1 or anti-CD44 mAb, but was abol-ished by ICAM-1 or Mac-1 deficiency (Fig. 3B). The few cells thatstill adhered and/or crawled in Mac-1�/� or ICAM-1�/� mice didso at extremely low velocity (Fig. 3C). Although anti-LFA-1 didnot alter the percentage of crawling cells, there was a partial re-duction in crawling velocity, indicating a contribution of LFA-1 tothis parameter (Fig. 3C). Treatment with anti-CD44 mAb did notalter the percentage of crawling neutrophils nor velocity of neu-trophil crawling, suggesting that this adhesion molecule has nodetectable role in this model of chemotactic-induced neutrophilrecruitment (Fig. 3, B and C).

Systemic LPS injection induced CD44-dependent neutrophilaccumulation in liver

Previous studies have reported no role for �2 integrins in neutro-phil adhesion in liver sinusoids following systemic LPS adminis-tration (14). LPS induced a marked increase in neutrophil adhesionin the sinusoids (Fig. 4A) compared with saline-injected mice (Fig.

4B). Several blanched areas were macroscopically observed on theliver surface (sign of malperfusion), and histological analysis con-firmed significant accumulation of neutrophils in liver vessels(data not shown). The adhesion in this model was entirely inde-pendent of Mac-1, LFA-1, or ICAM-1 (Fig. 4C) and CD18 (datanot shown). In striking opposition to fMLP, the neutrophil adhe-sion induced by LPS was blocked 70% with the anti-CD44 mAb.More than 90% of adhered neutrophils were static, and did notcrawl or crawled for a very short distance in response to LPS (Fig.4D) with erratic crawling movements (supplemental video 4). Themajority of neutrophils remained within the vasculature and didnot emigrate.4 The online version of this article contains supplemental material.

FIGURE 7. LPS does not affect ICAM-1 and ICAM-2 expression inliver sinusoids. PE-coupled anti-ICAM-1 (red; 2 �g/mouse) and AlexaFluor 488-coupled anti-ICAM-2 (green; 15 �g/mouse; Invitrogen) wereused to quantify the expression of these adhesion molecules during endo-toxemia. The expression of ICAM-1 and ICAM-2 during endotoxemia wasassessed by measuring the integrated density of the fluorescence in liverthree-dimensional reconstructions using ImageJ software. Iso � isotypecontrols of mAb; C � control 4 h; LPS � inflammatory protein LPSinjection 4 h.

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To evaluate the role of integrins in neutrophil adhesion duringendotoxemia in a nonhepatic tissue, the brain microvasculaturewas observed using spinning disk confocal microscopy under thesame conditions as those described for the liver. We observed thatsystemic LPS injection induced a significant increase in adherentneutrophils in brain venules in comparison with controls (Fig. 4,E–G). Treatment with anti-LFA-1 mAb reduced the number ofadherent neutrophils, but no statistically significant difference wasobserved in Mac-1�/� mice. However, the treatment of endotox-emic Mac-1�/� mice with anti-LFA-1 mAb further decreased thenumber of adherent neutrophils, suggesting that both integrins me-diated neutrophil adhesion in brain during systemic LPSinflammation.

To compare LPS and fMLP responses to a bacterial infection,we inoculated mice i.p. with E. coli (107 CFU; 4 h). It could beargued that in this model both LPS and fMLP pathway might beactivated. Surprisingly, E. coli infection caused marked neutrophiladhesion in liver sinusoids (Fig. 5A) that was inhibited with anti-CD44 mAb, but not with anti-Mac-1 mAb (Fig. 5, B and C). Thissuggests that E. coli infection causes a profile of hepatic neutrophiladherence more similar to that seen with LPS than with fMLPtreatment, or that the former pathway deactivated the latter.

Systemic LPS-induced liver inflammation modifies local hepaticinflammatory response

To determine whether �2 integrins could be activated in endotox-emic liver, we examined whether �2 integrin-dependent adhesionwith fMLP could still occur in endotoxemic mice. Interestingly,local fMLP (2 h) did not induce any further adhesion (Fig. 6A) andcould not induce crawling of neutrophils when LPS was present

(data not shown). Anti-CD44 mAb inhibited 70% of neutrophiladhesion during endotoxemia in liver sinusoids (Fig. 6A). Addingthe fMLP-laden filter under these conditions did not induce anyadditional neutrophil adhesion (Fig. 6A) nor crawling (data notshown), indicating that a Mac-1-dependent mechanism could notbe induced in the liver in the presence of a systemic LPS injection.

Up-regulation of Mac-1 expression on circulating neutrophilsinduced by systemic LPS injection is not maintained byneutrophils that home to the liver

To test our hypothesis that LPS dampens Mac-1-mediated adhe-sion in liver, we tracked the expression of Mac-1, LFA-1, andCD44 on both circulating and liver-harvested neutrophils afterLPS treatment. Systemic LPS induced a significant increase inMac-1 expression, but not in LFA-1 nor CD44, on circulating neu-trophils (Fig. 6B). In sharp contrast, neutrophils harvested from theliver of LPS-injected mice had reduced Mac-1 compared with neu-trophils from untreated mice (Fig. 6C); however, no significantchanges were observed in LFA-1 nor in CD44 expression (Fig. 6,B and C).

Next, we investigated whether the decreased expression ofMac-1 in liver neutrophils after LPS administration was due tointernalization of this integrin or an inability to mobilize Mac-1 tothe membrane surface. For this, we blocked surface-expressedMac-1 and then permeabilized cells and stained for intracellularintegrins. We found no difference in intracellular levels of Mac-1and LFA-1 between control and LPS-treated mice, suggesting thatthe decreased expression of Mac-1 is not due to internalization, butis instead probably due to shedding of this integrin from the neu-trophil surface (Fig. 6D).

FIGURE 8. IL-10 as a candidatefor Mac-1 down-regulation in liverneutrophils. Dosage of IL-10, TNF-�,and IL-6 levels in liver tissue (A) andblood (B). Disproportionate amountsof IL-10 are expressed in liver tissue,in contrast to the lower levels inblood. C, Flow cytometry analysis ofMac-1 expression on neutrophils fol-lowing LPS incubation (100 ng/ml) inthe presence and absence of IL-10 (10ng/ml). �, Indicates statistically sig-nificant difference compared with in-cubation with LPS alone. p � 0.05.D, Flow cytometry analysis of Mac-1adhesivity. Blood samples were incu-bated with LPS (100 ng/ml), fMLP(0.1 �M), or IL-10 (100 ng/ml) for4 h. Alexa Fluor 488-coupled fibrin-ogen was added to the samples at theend of the incubation process formeasurement of Mac-1 adhesivity. �,Indicates statistically significant dif-ference compared with incubationwith saline, and †, compared withfMLP plus LPS incubation. p � 0.05.

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For completeness, we assessed by confocal intravital micros-copy whether the endothelial ligands for Mac-1 (ICAM-1 andICAM-2) were down-regulated during endotoxemia. Interestingly,we found no differences in the expression of both adhesion mol-ecules during endotoxemia (Fig. 7).

IL-10 as a candidate for Mac-1 suppression in liver neutrophils

Our data suggested that the low levels of Mac-1 expression arerestricted to liver neutrophils, because circulating neutrophils ex-pressed elevated levels of Mac-1 after LPS stimulation. Thesefindings led us to hypothesize that a local mediator produced inhigh amounts selectively in the liver may be affecting adherent andemigrated neutrophils within the liver microenvironment, therebymaintaining low Mac-1 expression in these cells. Data using Lu-minex array revealed that serum levels of many proinflammatorycytokines and chemokines (such as IL-1�, IFN-�, TNF-�, MIP-1�, RANTES, KC, and IL-6) are up-regulated during endotoxemia(data not shown). In addition, we noted IL-10 as the one anti-inflammatory cytokine significantly up-regulated during this pro-cess (Fig. 8). To further assess the production of cytokines in liverduring LPS challenge, we collected liver samples. Interestingly,we found that disproportionately high amounts of IL-10 (�650pg/mg tissue) were constitutively expressed in liver when com-pared with IL-6 and TNF-� (�25 pg/mg; Fig. 8A), the two highestproinflammatory cytokines. Within the liver, high levels of IL-10were observed over the 4 h of LPS injection. In striking contrast,serum levels of IL-10 were much lower than IL-6 over the 4 h ofLPS injection (Fig. 8B). Clearly, whereas disproportionately highlevels of IL-10 were seen in the liver tissue, serum IL-10 levelsincreased notably, but less than IL-6 and TNF-� in blood (Fig. 8B).

Because IL-10 is described as an anti-inflammatory cytokine, itselevated expression in liver tissue led us to investigate the effect ofthis cytokine on Mac-1 expression by neutrophils. Blood samplesfrom wild-type mice were collected and incubated in vitro withLPS in the presence or absence of IL-10. Incubation of blood cellswith LPS (100 ng/ml) induced a significant increase in Mac-1 ex-pression on neutrophils by 1 h, which was further increased at 4 hafter incubation. Strikingly, IL-10 directly inhibited LPS-inducedMac-1 up-regulation by neutrophils (Fig. 8C). Interestingly, IL-10in the absence of LPS caused a decrease in Mac-1 expression. Asa negative control, incubation of blood cells with sterile PBS for4 h did not alter Mac-1 expression levels (data not shown).

In an additional set of experiments, we examined the adhesivityof Mac-1 by measuring fibrinogen binding to neutrophils, a spe-cific Mac-1 ligand. IL-10 was able to block the increased adhe-sivity of Mac-1 induced by LPS and fMLP (Fig. 8D).

We also investigated the effect of IL-10 on purified bone mar-row-derived neutrophils. We confirmed that IL-10 was also able tosignificantly reduce LPS-induced Mac-1 up-regulation in bonemarrow-derived neutrophils. However, the magnitude of the re-duction was much smaller (20%). Moreover, the down-regulationof constitutively expressed Mac-1 by IL-10 was not observed (datanot shown).

Low Mac-1 expression in liver neutrophils during LPS challengeis not observed in the absence of IL-10

To investigate the role of IL-10 in the regulation of Mac-1 expres-sion on neutrophils in vivo, we harvested neutrophils from bloodand liver of IL-10�/� mice treated with LPS. We observed thatafter systemic LPS inflammation, blood neutrophils from IL-10�/� mice displayed a similar phenotype to neutrophils fromwild-type mice, in that Mac-1 expression was up-regulated (Fig.9A). However, Mac-1 levels in liver neutrophils from IL-10�/�

mice were not low after LPS administration, in contrast to liver

neutrophils from wild-type mice (Fig. 9B). CD44 levels were notsignificantly altered by the presence or absence of IL-10 (Fig. 9, Aand B).

Because Mac-1 levels did not decrease in neutrophils harvestedfrom livers in endotoxemic IL-10�/� mice, we examined whetherthe presence of Mac-1 had a physiologic function and now con-tributed to neutrophil adhesion in liver sinusoids during endotox-emia. Interestingly, anti-Mac-1 Ab blocked LPS-induced neutro-phil adhesion in IL-10�/� mice by more than 60%, indicating thatthe retained expression of Mac-1 in neutrophils in IL-10�/� liversin response to LPS injection can now contribute to neutrophil ad-hesion within sinusoids (Fig. 9C). Although some adhesion viaCD44 was retained, the 70% inhibition seen in wild-type miceusing anti-CD44 mAb (Fig. 6A) was of much smaller magnitude(Fig. 9C) in IL-10�/� mice. Anti-Mac-1 mAb could furtherreduce neutrophil adhesion in anti-CD44 mAb-treated IL-10�/�

FIGURE 9. Down-regulation of Mac-1 in liver neutrophils during LPSchallenge is not observed in the absence of IL-10. Blood (A) and liver (B)neutrophils from wild-type (WT) or IL-10�/� mice were harvested for flowcytometry analysis of Mac-1 and CD44 expression following LPS i.p. in-jection. �, Indicates statistically significant difference compared with con-trols (saline injected). C, Effect of Ab blockade of Mac-1 and CD44 duringLPS systemic inflammation in IL-10�/� mice. �, Indicates statistically sig-nificant difference compared with controls (saline injected), and †, in com-parison with vehicle (UT)-treated mice. p � 0.05.

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mice (data not shown). Addition of anti-Mac-1 and anti-CD44Abs to wild-type mice did not reduce adhesion any more thananti-CD44 Ab treatment alone (data not shown).

DiscussionNeutrophil accumulation in liver may be paradoxical in that thesecells play a vital role in promoting effective bacterial clearance(31), but neutrophil infiltration in the liver can also result in seriousand progressive parenchymal damage, leading to liver failure andsevere clinical outcomes. Acute and chronic diseases of the liverare not uncommon, and in worst case scenarios may even requireliver transplantation as a life-saving procedure (32). In this study,it was our goal to investigate the mechanisms involved in hepaticneutrophil accumulation during endotoxemia, compared with a lo-cal stimulus that directly activated neutrophils. Unexpectedly, themechanisms were drastically different, in that an acute stimuluslike fMLP that directly activates neutrophils made use of standardneutrophil-adhesive mechanisms, including Mac-1, an adhesionmolecule that played no role following LPS administration. In fact,when fMLP was superimposed onto LPS-induced neutrophil re-cruitment, Mac-1-mediated neutrophil adhesion could no longer beobserved. The underlying mechanism involved a novel inhibitoryrole for IL-10 down-regulating Mac-1 on neutrophils in endotox-emia. It has previously been reported that the liver is a tolerogenicorgan dampening T cell activation and releasing anti-inflammatorymolecules when challenged with activating stimuli (22). Decreasedlevels of Mac-1 could also function as a strategic mechanism toprevent unwarranted adhesion of neutrophils. However, when thestimulus is sufficiently severe, CD44 and its ligand HA (expressedprimarily in the liver) recruit neutrophils to the liver.

To date, no role for integrins as contributors to neutrophil re-cruitment into liver sinusoids has been described. In particular, nointegrins are involved in endotoxin or sepsis-related neutrophil ac-cumulation into sinusoids (14). In this study, for the first time, wehave demonstrated in vivo that adherent neutrophils can adhereand crawl directionally toward a chemotactic molecule within si-nusoids using the integrin Mac-1. Up-regulation of Mac-1 expres-sion by fMLP-stimulated neutrophils has been described in vitro(33), and Mac-1-mediated neutrophil crawling is an important stepduring efficient leukocyte extravasation in vivo in nonhepatic tis-sues (25, 34) and in vitro across endothelial monolayers (35). Theparticipation of integrins in neutrophil adhesion in liver was de-scribed, but only in postsinusoidal venules, and not the more abun-dant sinusoids where CD44 is dominant (6). In the fMLP-inducedrecruitment, the neutrophils not only crawled via Mac-1, but avidlyemigrated. By contrast, in LPS-induced inflammation, neutrophilssimply adhered and did not crawl, and only a few emigrated out ofthe blood vessels. No role for Mac-1 was noted in this systemicinflammation. It is reasonable to speculate that when LPS is de-tected in the vasculature, the system attempts to retain neutrophilsin the vasculature, where they may trap bacteria perhaps using therecently described neutrophil extracellular traps (31, 36). There-fore, we propose a paradigm in which the CD44/HA pathway isinduced to allow for retention of neutrophils by adhering in sinu-soids, whereas down-modulation of Mac-1 may serve to retainneutrophils in the sinusoids, preventing them from emigrating outof the vasculature.

Recent reports have highlighted CD44-HA interaction as adominant mechanism for neutrophil adhesion in sinusoids duringendotoxemia (5) and ischemia-reperfusion (37). Interestingly,

FIGURE 10. Proposed mechanism for Mac-1 down-regulation in liver neutrophils during endotoxemia. Local liver stimulus (fMLP) displays a con-ventional integrin-dependent neutrophil adhesion mechanism, which is abolished by LPS systemic inflammation. Kupffer cells, liver sinusoidal endothelialcells, and other liver parenchymal cells may be large sources of IL-10, creating an environment where neutrophils would be exposed to high amounts ofthis anti-inflammatory cytokine, down-regulating Mac-1 expression on the neutrophil surface, while permitting CD44 to retain neutrophils in the liversinusoids, where they may enhance bacterial trapping.

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anti-CD44 Ab-treated mice, and also CD44�/� mice (data notshown), displayed normal adhesive responses due to fMLP stim-ulus as a result of Mac-1 engagement. By contrast, LPS-inducedsystemic inflammation required CD44/HA interactions to seques-ter neutrophils in liver sinusoids (5). We observed no change inCD44 levels with LPS, entirely consistent with previous work (5).The prevailing view is that the CD44 ligand HA is modified bySHAP to change its quaternary conformation to enhance binding toCD44 (5). Indeed, we previously reported increase in levels ofSHAP binding in sinusoids post-LPS treatment. Moreover, it hasbeen shown that SHAP binding to HA greatly increases cellularadhesivity in vitro using flow chambers (38). Although the CD44/SHAP/HA pathway is induced with LPS, a simultaneous loss ofMac-1 adhesivity is observed through an IL-10-dependentmechanism.

IL-10 is known to play an important role as an anti-inflammatory molecule, by down-regulating NF-�B-mediated in-flammatory processes and reducing production of cytokines,thereby inhibiting de novo synthesis of adhesion molecules (39). Inour study, we report that IL-10 can down-modulate Mac-1 expres-sion presumably via a protein synthesis-independent mechanism,most likely involving Mac-1 shedding, an inactivating process pre-viously reported for other adhesion molecules (40). A recent reporthas shown that during endotoxemia, monocyte subpopulations canundergo apoptosis and, concomitantly, these cells down-regulateCD18 expression (41). We provide evidence that IL-10 is highlyexpressed in liver, and in the absence of this cytokine in IL-10�/�

mice, Mac-1 expression in neutrophils is not low. Kupffer cellslocated in the vasculature release large amounts of IL-10 in re-sponse to LPS (22, 42), making them a likely candidate for thisfunction. In fact, in the absence of IL-10, it was most interesting tosee that LPS administration led to Mac-1-dependent neutrophiladhesion in sinusoids. Interestingly, IL-10 levels were high con-stitutively in liver, but fMLP was able to induce Mac-1-dependentadhesion. This may reflect a lack of release of IL-10 from cellularstores unless LPS is added.

The IL-10 effects were restricted to the liver as �2 integrin func-tion was retained in the brain during endotoxemia. In contrast tothe 10- to 20-fold higher levels of IL-10 relative to molecules likeIL-6 and TNF-� in liver tissue, we found that serum levels ofIL-10 were notably less (5-fold) than IL-6 and TNF-� levels. This50- to 100-fold difference in anti-inflammatory vs proinflammatorylevels in circulation can explain the absence of Mac-1 down-mod-ulation in circulating neutrophils. Thus, we propose that the dis-proportionate expression of IL-10 in the liver overcomes the proin-flammatory effects of circulating cytokines, driving a regulatoryenvironment in the hepatic sinusoids. Whether there is a proteinthat retains IL-10 on the sinusoidal surface, thereby increasinglocal levels of IL-10 in the liver, is not known. Using a teleologicalargument, it may be that down-regulation of Mac-1 in liver duringsystemic infection would allow neutrophils to stop inside sinu-soids, increasing the probability of bacterial encounter to executeintravascular bactericidal activity. Indeed, there is a growing bodyof evidence that the liver sinusoids are an important site of theimmune system to prevent bacterial dissemination (43).

In Fig. 10, we summarize our major findings and propose amechanism to explain the dynamics of neutrophil adhesion mole-cules in liver during local and systemic inflammation. Collectively,our data suggest that Mac-1 is necessary for neutrophil adhesionand crawling during local inflammatory stimuli in liver. DuringLPS-induced systemic inflammation, HA becomes adhesive forCD44, thereby mediating neutrophil adhesion in liver. Simulta-neously, neutrophils become exposed to high concentrations ofIL-10 in liver, which down-regulates Mac-1 in these cells, thereby

reducing their capacity to crawl and emigrate, but perhaps increas-ing the intravascular filtering capacity of the liver for pathogens.

AcknowledgmentsWe thank Lori Zbytnuik for her assistance in experiments.

DisclosuresThe authors have no financial conflict of interest.

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