Liver Elimination of Bacteria Taken up by the Neutrophil

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Complementary Adhesion Molecules Promote Neutrophil- Kupffer Cell Interaction and the Elimination of Bacteria Taken Up by the Liver This information is current as of September 24, 2021. Stephen H. Gregory, Leslie P. Cousens, Nico van Rooijen, Ed A. Döpp, Timothy M. Carlos and Edward J. Wing J Immunol 2002; 168:308-315; ; doi: 10.4049/jimmunol.168.1.308

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2002 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Complementary Adhesion Molecules Promote Neutrophil- Kupffer Cell Interaction and the Elimination of Bacteria Taken Up by the Liver1

Stephen H. Gregory,*† Leslie P. Cousens,† Nico van Rooijen,‡ Ed A. Do¨pp,‡ Timothy M. Carlos,* and Edward J. Wing*†

Most bacteria that enter the bloodstream are taken up by the liver. Previously, we reported that such organisms are initially bound extracellularly and subsequently killed by immigrating neutrophils, not Kupffer cells as widely presumed in the literature. Rather, the principal functions of Kupffer cells demonstrated herein are to clear bacteria from the peripheral blood and to promote accumulation of bactericidal neutrophils at the principal site of microbial deposition in the liver, i.e., the Kupffer cell surface. In a mouse model of listeriosis, uptake of bacteria by the liver at 10 min postinfection i.v. was reduced from approximately 60% of Downloaded from the inoculum in normal mice to ϳ15% in mice rendered Kupffer cell deficient. Immunocytochemical analysis of liver sections derived from normal animals at 2 h postinfection revealed the massive immigration of neutrophils and their colocalization with Kupffer cells. Photomicrographs of the purified nonparenchymal liver cell population derived from these infected mice demonstrated listeriae inside neutrophils and neutrophils within Kupffer cells. Complementary adhesion molecules promoted the interaction between these two cell populations. Pretreatment of mice with mAbs specific for CD11b/CD18 (type 3 complement receptor) or its counter-receptor, CD54, inhibited the accumulation of neutrophils in the liver and the elimination of listeriae. Complement http://www.jimmunol.org/ was not a factor; complement depletion affected neither the clearance of listeriae by Kupffer cells nor the antimicrobial activity expressed by infiltrating neutrophils. The Journal of Immunology, 2002, 168: 308–315.

3 he liver plays a major role in the clearance of bacterial nate (Cl2MDP-L) to eliminate Kupffer cells (9, 10). While their pathogens from the bloodstream (1–3). Blood clearance specific function(s) remains to be delineated, it is often assumed that Kupffer cells account for the marked (0.5–1 log ) decrease in T and the eventual elimination of bacteria taken up in the 10 liver are widely attributed to phagocytosis by Kupffer cells (1, 4). liver listeriae that typically occurs early during the course of in- Kupffer cells constitute the largest population of fixed tissue mac- fection (8). Previously we reported, however, that the bulk of lis- by guest on September 24, 2021 rophages found in the body (5). They generally reside within the teriae inoculated i.v. was cleared rapidly by the liver, initially lumen of the liver sinusoids, attached to fenestrated endothelial bound extracellularly, and subsequently killed by immigrating cells that compose the vessel walls, partially obscuring the vascu- neutrophils (11). lar channel. Neutrophils represent 1–2% of the total nonparenchymal cells Listeria monocytogenes is a Gram-positive, facultative, intracel- (NPCs) found in the liver of normal, noninfected mice (11). The lular, bacterial pathogen capable of causing severe infections in percentage of neutrophils increases rapidly following i.v. inocula- humans characterized by high mortality (6, 7). Listeriosis in mice tion of Listeria, reaching a maximum at 2 h postinfection and then is an experimental model used widely to explore the factors that declining thereafter with the elimination of extracellular bacteria effect host defenses to pathogens that replicate intracellularly (2, (11) (our unpublished observation). At 6 h postinfection, essen- 8). Kupffer cells play a prominent role in host resistance to both tially all the viable listeriae remaining in the liver were located primary and secondary listerial infections. The proliferation of inside hepatocytes, as judged by cell separation experiments and liver listeriae and mortality are increased markedly in mice pre- resistance of the organism to antibiotic treatment (11). Mice ren- treated with liposome-encapsulated dichloromethylene diphospho- dered neutrophil deficient by pretreatment with anti-granulocyte (RB6/8C5) mAb exhibited a sharp decrease in antimicrobial activity and, as a consequence, a marked increase in extracellular listeriae recovered in the liver at 6 h postinfection. Similarly, mice *Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, pretreated with RB6–8C5 mAb exhibited a reduced capacity to PA 15261; †Department of Medicine, Rhode Island Hospital and Brown University eliminate Escherichia coli, Staphylococcus aureus, and Klebsiella ‡ School of Medicine, Providence, RI 02903; and Department of Cell Biology, Vrije pneumoniae inoculated i.v. and taken up in the liver. These find- Universiteit, Amsterdam, The Netherlands ings were supported by more recent studies demonstrating in- Received for publication December 14, 2000. Accepted for publication October 22, 2001. creased numbers of S. typhimurium, Yersinia enterocolitica, and The costs of publication of this article were defrayed in part by the payment of page Clostridium piliforme in the livers of neutrophil-depleted mice in- charges. This article must therefore be hereby marked advertisement in accordance oculated i.v. (12–14). with 18 U.S.C. Section 1734 solely to indicate this fact. In view of the obligate requirement for neutrophils in killing, we 1 This work was supported in part by Public Health Service Grant DK44367 obtained undertook the series of experiments reported here to delineate the from the National Institutes of Health. 2 Address correspondence and reprint requests to Dr. Stephen H. Gregory, Depart- 3 ment of Medicine, Rhode Island Hospital/Brown University School of Medicine, 432 Abbreviations used in this paper: Cl2MDP-L, liposome-encapsulated dichlorometh- Pierre M. Galletti Building, 55 Claverick Street, Providence, RI 02903. E-mail ad- ylene diphosphonate; CD62L, CD62 ligand; CVF; cobra venom factor; NPC, non- dress: [email protected] parenchymal liver cells; PBS-L, liposome-encapsulated PBS.

Copyright © 2002 by The American Association of Immunologists 0022-1767/02/$02.00 The Journal of Immunology 309 precise role of Kupffer cells in host resistance to systemic bacterial on hepatocytes or circulating white blood cells, i.e., lymphocytes, granu- infections using listeriosis in mice as an experimental model. The locytes (including neutrophils), or mononuclear phagocytes (9, 22–25). results of these experiments indicate that Kupffer cells are required for the efficient clearance of listeriae from the bloodstream. The Monoclonal Abs subsequent eradication of those organisms is facilitated by the CD11b/CD18 (Mac-1)- and CD54 (ICAM-1)-dependent accumu- mAbs produced by the following hybridomas were obtained in the ascites fluid of pristane-primed, homozygous nude BALB/c mice injected with 107 lation of bactericidal neutrophils at the site of listeriae deposition, cells: rat IgG2b anti-mouse granulocyte (RB6-8C5) (11), rat IgG2b anti- i.e., on the Kupffer cell surface. Photomicrographs demonstrating mouse CD54 (YN1/1.7.4; American Type Culture Collection, Manassas, neutrophils inside macrophages at 2 h postinfection suggest that VA), and rat IgG2b anti-mouse CD11b (5C6; American Type Culture Col- Kupffer cells may play a crucial role in eliminating neutrophils lection). The Abs were purified by chromatography on a protein G column (Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s in- from the liver as extracellular organisms in the sinusoids are structions. Monoclonal rat IgG2b anti-mouse CD11a (FD441.8; American eradicated. Type Culture Collection), rat IgG1 anti-mouse CD106 (MK1.9; American Type Culture Collection), and rat IgG2a anti-mouse CD62 ligand (CD62L; Materials and Methods Mel-14; American Type Culture Collection) were derived from the super- Animals natants of cultured hybridomas. The Ig fraction was precipitated with ammonium sulfate, dialyzed against PBS, and concentrated by centrifugation. Female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were For experiments, test animals were inoculated i.p. with 0.5 mg purified Ab housed and cared for in accordance with the guidelines set forth by the 18 h before infection. Control mice received an equivalent concentration of institutional animal care and use committees, University of Pittsburgh and chromatographically purified normal rat IgG (Organon Technika-Cappel,

Rhode Island Hospital. Animals between 8 and 16 wk of age were used in Durham, NC). All mAbs used in these in vivo experiments possessed Downloaded from the experiments described. blocking or neutralizing activity (26–31). Conjugated mAbs used in ﬂow cytometric analyses to characterize the NPC populations derived from con-

Bacteria trol and Cl2MDP-L-treated mice were purchased from PharMingen (San Diego, CA). L. monocytogenes (EGD strain) was cultured and maintained as described previously (15). The organism was passed routinely in mice to sustain its virulence. Listeriae derived from cultures growing exponentially were used Complement depletion in the experiments described. The number of organisms recovered in the http://www.jimmunol.org/ livers and peripheral blood of infected animals was estimated from the Mice were rendered complement deficient by treatment i.p. with 30 ␮g number of colonies that grew on trypticase soy agar plates inoculated with cobra venom factor (CVF; Calbiochem-Novabiochem International, La an aliquot of organ homogenate (16). Total bacteria in the bloodstream Jolla, CA) 18 h before infection. Serum samples derived from mice at the were calculated assuming the blood volume to be 8% (v/w) of the weight time of infection and analyzed by zonal electrophoresis exhibited a Ͼ95% of each animal (17). reduction in the C3 component of complement. Cell preparation Total and purified nonparenchymal liver cell populations composed pri- Immunocytochemistry marily of leukocytes and endothelial cells were obtained following perfu- Cryostat sections (6 ␮m) derived from the livers of mice at 2 h postinfec- 7 sion of the liver with collagenase using the two-step method we reported tion i.v. with 2 ϫ 10 listeriae were fixed in acetone and air-dried. Mono- by guest on September 24, 2021 previously (11, 18). Transient blood cells circulating through the liver and nuclear phagocytes (macrophages) were detected by a modification of not firmly bound to the tissues were eliminated during the exhaustive per- methods previously described (32); sections were incubated with rat anti- fusion process. Isolated cells were suspended in HEPES-buffered RPMI mouse F4/80 Ag, washed, and reincubated with Alexa Fluor 488-conju- 1640 medium (BioWhittaker, Walkersville, MD) supplemented with 10% gated goat anti-rat IgG (Molecular Probes, Eugene, OR). Infiltrating (Ly- heat-inactivated FBS (Sterile Systems, Logan, UT). The purified NPC pop- 6Gϩ) neutrophils were detected by incubating the sections with rat IgG2b ulation was Ն99% viable and essentially free of parenchymal cell (hepa- anti-mouse granulocyte mAb (RB6-8C5). The slides were then washed and tocyte) contamination. Purified NPCs were sedimented onto glass slides incubated with Texas Red-conjugated goat anti-rat IgG (Jackson Immuno- using a cytocentrifuge (Shandon Elliott, Pittsburgh, PA) and stained with Research Laboratories, West Grove, PA). The slides were mounted with Wright-Giemsa stain. Neutrophils within a population of 200 cells were Vectashield (Vector, Burlingame, CA) and examined under a Nikon enumerated based upon morphology. Peripheral blood samples were ob- Eclipse E800 microscope (Melville, NY) equipped with dual filters for tained by cardiac puncture using EDTA-coated tuberculin syringes. Total FITC and Texas Red. Multiple tissue sections were surveyed and the num- and differential leukocyte counts were determined after sedimentation of ber of red fluorescent Ly-6Gϩ neutrophils in juxtaposition with yellow- the bulk of erythrocytes with 5% dextran T500, followed by treatment with green fluorescent F4/80ϩ Kupffer cells was determined and expressed as a 0.83% ammonium chloride to lyse the RBCs that remained. percentage of the total neutrophils counted. Alternatively, neutrophils in the liver were detected by immunohisto- Kupffer cell depletion chemistry. Cryostat sections were pretreated with avidin/biotin (Vector) to block nonspecific binding of peroxidase-conjugated avidin. The sections Multilamellar liposomes containing Cl2MDP (a gift obtained from Roche, Mannheim, Germany) were prepared as previously described (19). Mice were then incubated with biotin-conjugated rat IgG2b anti-mouse Ly-6G were rendered Ͼ95% Kupffer cell-depleted by the inoculation i.v. of 200 (PharMingen), washed, incubated with avidin-peroxidase, washed, and ␮ developed with 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO) and lCl2MDP-L (containing 1 mg/ml Cl2MDP) suspended in saline 3 days before experimental use (20, 21). Mice administered 200 ␮l normal saline hydrogen peroxide. or liposome-encapsulated PBS (PBS-L) served as controls. The effect of Cl MDP-L on resident cells in the liver was specific for Kupffer cells. 2 Myeloperoxidase Cl2MDP-L had no detectable effect on hepatocytes in vivo; aspartate aminotransferase levels were comparable in the sera obtained from control and The myeloperoxidase activity in homogenates prepared from the liver, Cl2MDP-L-treated mice on day 3 (21). Moreover, the addition of spleen, and peripheral blood was quantified by analyzing the H2O2-depen- Cl2MDP-L had no effect on the viability of hepatocytes in vitro (assessed dent oxidation of 3,3Ј,5,5Ј-tetramethylbenzidine (Sigma) spectrophoto- in terms of aspartate aminotransferase activity released into the culture metrically as described previously by Schierwagen et al. (33). medium over a 48-h incubation period) or the capacity of cultured hepatocytes to support the intracellular growth of Listeria (21). The NPC populations obtained from control (saline-treated) and Cl2MDP-L-treated mice Statistical analysis on day 3 following inoculation i.v. contained comparable percentages of Ly-6Gϩ neutrophils (1.8 Ϯ 1.0 vs 1.3 Ϯ 0.1), NK1.1ϪCD3ϩ T cells (7.1 Ϯ The results were analyzed using the SigmaStat statistics program (Jandel 3.2 vs 5.0 Ϯ 1.3), NK1.1ϩCD3Ϫ NK cells (14.6 Ϯ 2.4 vs 15.3 Ϯ 1.7), and Scientific, San Rafael, CA). Individual means were compared by nonpaired NK1.1ϩCD3ϩ natural T cells (16.4 Ϯ 3.6 vs 16.8 Ϯ 2.5; flow cytometric Student’s t test. Data derived from three or more groups were compared by analysis, control listed first). These data are consistent with the results of ANOVA, followed by Student-Newman-Keuls test. Differences at p Ͻ numerous other studies that failed to show a significant effect of Cl2MDP-L 0.05 were considered significant. 310 NEUTROPHIL-KUPFFER CELL INTERACTION

Results Kupffer cell depletion delays clearance of Listeria from the bloodstream L. monocytogenes injected i.v. into control groups of mice pretreated with saline or PBS-L was cleared rapidly from the bloodstream. Less than 0.1% of the inoculum remained in the blood (Fig. 1), while 40–60% was recovered in the liver (Fig. 2) at 10 min postinfection. In contrast, about 10% of the inoculum injected into Cl2MDP-L-pretreated, Kupffer cell-depleted mice persisted in the blood, and only 15% was recovered in the liver at 10 min following infection. Three to 5% of the inoculum was recovered in the spleens of both control and Kupffer cell-depleted mice at this time. It is relevant to note in this regard that Cl2MDP-L administered i.v. also renders animals deﬁcient in splenic macrophages (9, 10). The organ distribution of residual listeriae not recovered in the livers or spleens of such mice remains to be determined. None- theless, these results demonstrate the crucial role of Kupffer cells

in efficient clearance of bacterial pathogens from the bloodstream. Downloaded from FIGURE 2. Kupffer cell depletion diminishes the uptake of Listeria by Regardless of the initial capacity to clear listeriae from the the liver. Control (saline or PBS-L) and Kupffer cell-depleted (Cl2MDP-L) blood, the livers of control and Kupffer cell-depleted mice con- mice were inoculated i.v. with 7.2 log10 listeriae. The number of organisms tained essentially the same number of organisms at 6 h postinfec- present in the liver was determined at periodic intervals postinfection. Val- tion. This latter finding contrasts a marked increase in the listerial ues are the mean Ϯ SD percentage of the inoculum obtained from groups burden found previously in the livers of neutrophil-depleted, rel- of four mice at the times indicated. Cl2MDP-L pretreatment impaired the -Sig ,ء .(ative to control, animals at 6 h postinfection (11). As such, Kupffer uptake of listeriae by the liver (p Ͻ 0.001, by two-way ANOVA http://www.jimmunol.org/ cells appeared to contribute little to the antimicrobial activity ex- nificantly fewer organisms were recovered in the livers of Cl2MDP-L- Ͻ pressed in the liver early during the course of infection. In agree- pretreated, relative to saline- or PBS-L-pretreated, mice (p 0.05, by ment with the published reports of other investigators (9, 10), how- Student-Newman-Keuls test). Comparable results were obtained in a second experiment. ever, Kupffer cell depletion exerted a significant effect on the listerial burden of the liver assessed later during infection. At 48 h postinfection, Ͼ10-fold more listeriae were recovered in the livers Neutrophil-Kupffer cell interaction in Listeria-infected mice of Cl2MDP-L-treated mice relative to control mice pretreated with saline or PBS-L (Fig. 3). Previously, we reported that most bacteria cleared from the blood-

stream and taken up by the liver were initially bound extracellu- by guest on September 24, 2021 larly and subsequently killed by inﬁltrating, bactericidal neutrophils; essentially all the organisms remaining in the liver at 6 h

FIGURE 1. Kupffer cell depletion delays the clearance of Listeria from the bloodstream. Control (saline or PBS-L) and Kupffer cell-depleted

(Cl2MDP-L) mice were inoculated i.v. with 7.2 log10 listeriae. At periodic FIGURE 3. Kupffer cell depletion promotes replication of liver listeriae. intervals postinfection the number of organisms remaining in the blood- Mice were treated with saline, PBS-L, or Cl2MDP-L at 72 h before infec- stream was determined. Values are the mean Ϯ SD percentage of the in- tion (solid line) or at 6 h postinfection (broken line) with 5 ϫ 103 listeriae. Ϯ oculum obtained from groups of four mice at the times indicated. Values are the mean SD log10 organisms present in the livers dissected Cl2MDP-L pretreatment impaired the clearance of listeriae from the blood- from groups of three mice at the times indicated. Cl2MDP-L treatment Significantly more organisms promoted the proliferation of listeriae in the liver (p Ͻ 0.001, by two-way ,ء .(stream (p Ͻ 0.001, by two-way ANOVA ء were present in blood obtained from Cl2MDP-L-pretreated mice relative to ANOVA). , Significantly more organisms were recovered in the livers of Ͻ saline- or PBS-L-pretreated mice at all comparable time points (p 0.05, both groups of mice treated with Cl2MDP-L relative to control mice pre- by Student-Newman-Keuls test). A second experiment yielded similar treated with saline or PBS-L (p Ͻ 0.05, by Student-Newman-Keuls test). results. A second experiment yielded similar results. The Journal of Immunology 311 postinfection were located intracellularly based upon their resistance to gentamicin treatment (11). The data reported here demonstrating the critical role of Kupffer cells in blood clearance indicate that the majority of those organisms recovered in the liver at 10 min postinfection are bound to the surface of Kupffer cells. Taken together with the results of our previous studies cited above (11), these data imply that the subsequent elimination of those bound organisms requires direct neutrophil-Kupffer cell interaction. Indeed, the number of neutrophils in the liver increased rapidly following i.v. inoculation of L. monocytogenes, peaking at 2 h postinfection and declining thereafter (Fig. 4). The rapid demar- gination and/or desequestration of cells may facilitate the accumulation of neutrophils in the liver subsequent to infection. The percentage of the circulating neutrophils increased from 5–15% of the PBL in uninfected mice to 40–50% of PBL assessed at 30 min postinfection. Immunocytochemical analysis of liver sections prepared from mice at 2 h postinfection corroborates the massive influx of Ly- 6Gϩ neutrophils (Fig. 5, A and B). These infiltrating neutrophils Downloaded from were restricted to the vascular sinusoids; the vast majority (79 Ϯ 3.5%) colocalized with F4/80ϩ mononuclear phagocytes (Kupffer cells; Fig. 5C). In accordance with the latter result demonstrating direct contact between neutrophils and Kupffer cells, substantially fewer neutrophils accumulated in the livers of Kupffer cell-depleted, relative to control, mice subsequent to Listeria infection. http://www.jimmunol.org/ Kupffer cell depletion resulted in an approximately 3-fold reduction in the number of neutrophils sequestered in the livers of animals at 2 h postinfection (Table I). This reduction did not reflect a generalized decrease in neutrophil count; blood derived from infected control and Cl2DMP-L-treated mice contained comparable neutrophil numbers. Assessment of the myeloperoxidase activity (an enzyme marker for neutrophils) in homogenates of livers obtained at 6 h postinfection yielded a similar conclusion, i.e., Kupffer cell depletion diminished the accumulation of liver neu- by guest on September 24, 2021 trophils (Table II). Conversely, myeloperoxidase activity was elevated slightly in the peripheral blood at this time, indicating the presence of an increased number of neutrophils under conditions in which sequestration in the liver was inhibited. Indeed, neutrophils represented 66.4 Ϯ 5.2% of the peripheral blood leukocytes ob-

FIGURE 5. Colocalization of infiltrating neutrophils and Kupffer cells in the livers of mice early during the course of systemic listerial infection. Six-micrometer sections were prepared from the livers of uninfected mice (top) and mice at 2 h postinfection i.v. with 2 ϫ 107 listeriae (middle and bottom). Infiltrating neutrophils detected by immunohistochemistry in the top and middle panels are red/brown. F4/80ϩ macrophages and Ly-6Gϩ neutrophils in sections assessed by immunofluorescence microscopy ap- pear yellow-green and red, respectively (bottom). Original magnification, ϫ400.

Ϯ tained from Cl2MDP-L-treated mice vs 44.3 3.7% obtained from control mice at 6 h postinfection. Cl2MDP-L pretreatment did not affect myeloperoxidase activity expressed in the spleens of FIGURE 4. Kinetics of neutrophil infiltration of the liver subsequent to Listeria-infected mice. Listeria infection. The neutrophils comprising the nonparenchymal liver cell population derived from mice inoculated i.v. with 2 ϫ 107 listeriae Neutrophils containing intracellular listeriae were detected were enumerated at the times indicated. Data are the mean Ϯ SD obtained among the purified NPCs obtained at 2 h postinfection; neutrophils from six mice and three independent experiments (two mice per time point inside mononuclear phagocytes were also observed periodically, -Greater than time zero (p Ͻ 0.05, by one-way i.e., in about 2% of total macrophages (Fig. 6). This finding sup ,ء .(per experiment ANOVA). ports our contention that the innate response to bacteria cleared 312 NEUTROPHIL-KUPFFER CELL INTERACTION

Table I. Neutrophils are diminished in the livers, but not the blood, of Kupffer cell-depleted mice infected with Listeriaa

Nonparenchymal Liver Cells Peripheral Blood Leukocytes

Pretreatment Total (ϫ107) % neutrophils Neutrophils/liver (ϫ107) Total/␮l(ϫ103) % neutrophils Neutrophils/␮l(ϫ103)

Control 6.7 Ϯ 0.7 37.8 Ϯ 2.6 2.50 Ϯ 0.14 2.8 Ϯ 0.9 32.0 Ϯ 5.2 0.90 Ϯ 0.34 Ϯ Ϯ Ϯ Ϯ Ϯ Ϯ Cl2MDP-L 4.4 0.7* 17.0 4.5** 0.75 0.17** 2.5 0.3 38.2 3.2 0.89 0.10 a ϫ 7 Control (saline-pretreated) and Cl2MDP-L-treated (Kupffer cell-depleted) mice were inoculated i.v. with 2 10 listeriae. The total numbers of nonparenchymal liver cells, peripheral blood leukocytes, and neutrophils present within each population were quantified at 2 h postinfection. Values are the means Ϯ SD of cells obtained from four mice .p Ͻ 0.001; Student’s t test ,ءء ;p ϭ 0.003 ,ء :in a representative experiment. Significantly less than control from the bloodstream entails the direct interaction of neutrophils Anti-CD11b abrogates the accumulation of neutrophils in the with resident tissue macrophages that line the liver sinusoids. livers of Listeria-infected animals Moreover, it suggests that the potentially important role of Kupffer The increased recovery of listeriae in the livers of mice pretreated cells in eliminating neutrophils from the liver (e.g., between 2 and with monoclonal anti-CD11b correlated inversely with the accu- 6 h postinfection i.v.; shown in Fig. 4) as extracellular listeriae in mulation of neutrophils. The NPC population derived from mAb- the sinusoids are killed. treated mice at 2 h postinfection (i.e., at the height of neutrophil Downloaded from Anti-CD11b abrogates the initial killing of Listeria cleared by infiltration) contained 5-fold fewer neutrophils than did the NPCs the liver obtained from mice administered normal rat IgG before infection The cell surface adhesion molecule, CD11b/CD18 (Mac-1; type 3 (Table IV). CD11b/CD18 expression, therefore, exerted a signifi- complement receptor, CR3), expressed by neutrophils, but not cant influence on the sequestration of neutrophils in the livers of Kupffer cells (34–36), participates in a broad spectrum of activi- animals infected systemically. Anti-CD11b mAb treatment, on the other hand, failed to affect either the total leukocyte count (1.8 Ϯ

ties, including adherence to vascular endothelial cells, extravasa- http://www.jimmunol.org/ 3 3 tion, and phagocytosis (37). CD11b/CD18 is a critical factor in 0.7 ϫ 10 /␮l (mAb-treated) vs 2.0 Ϯ 0.8 ϫ 10 /␮l (control)) or the host defenses to Listeria expressed in the liver (38, 39). A series of number of neutrophils (0.4 Ϯ 0.2 ϫ 103/␮l (mAb-treated) vs 0.6 Ϯ experiments was undertaken to explore the role of CD11b/CD18 in 0.4 ϫ 103/␮l (control)) present in the peripheral blood. Similarly, neutrophil-Kupffer cell interaction and the elimination of listeriae the administration of anti-ICAM-1 mAb affected neither the total taken up in the liver early during the course of infection. Mice PBL (2.2 Ϯ 0.8 ϫ 103/␮l) nor the peripheral blood neutrophil pretreated with mAb specific for CD11b (clone 5C6, a blocking (0.4 Ϯ 0.2 ϫ 103/␮l) count assessed in the same set of (noncytolytic) Ab demonstrated to suppress recruitment of inflam- experiments. matory cells in vivo (27, 31)) exhibited a normal capacity to clear listeriae from the bloodstream; the numbers of organisms present by guest on September 24, 2021 in the livers of control and Ab-treated mice at 10 min postinfection i.v. were comparable (Fig. 7). At 6 h postinfection, however, significantly more organisms were recovered in the livers of Ab- treated mice. Similarly, the listerial burden of the liver 6 h following inoculation was elevated in mice pretreated with mAb specific for CD54 (ICAM-1; CD11b/CD18 counter-receptor) expressed constitutively by Kupffer cells (40) (Table III). Thus, while neither CD11b/CD18 nor CD54 was a factor in clearance of listeriae from the bloodstream, expression of these complementary adhesion molecules was crucial for the elimination of liver listeriae between 10 min and 6 h postinfection. Pretreatment with blocking Abs specific for several other adhesion molecules known to modulate the trafficking and/or activity of neutrophils, i.e., CD62L (L-selectin), CD11a (LFA-1), and CD106 (VCAM-1), had no effect on the number of listeriae recovered in the liver at 6 h following infection.

Table II. Myeloperoxidase activity is diminished in the livers of Kupffer cell-depleted mice infected with Listeriaa

⌬ Myeloperoxidase Activity ( OD650/min/mg protein)

Population Control Cl2MDP-L Liver 0.033 Ϯ 0.009 0.019 Ϯ 0.005* Peripheral blood 6.160 Ϯ 0.460 7.563 Ϯ 0.598** Spleen 0.722 Ϯ 0.449 0.953 Ϯ 0.463

a Control (saline-pretreated) and Cl2MDP-L-treated (Kupffer cell-depleted) mice FIGURE 6. Photomicrographs of listeriae inside neutrophils (hollow ar- ϫ 7 Ϯ were inoculated i.v. with 2 10 listeriae. Values are the means SD myeloper- row, top) and neutrophils inside liver macrophages (solid arrow, bottom). oxidase activity in homogenates of the livers, peripheral blood, and spleens obtained p ϭ Cytocentrifuge smears of the NPCs obtained from mice at 2 h postinfection ,ء :from five animals at 6 h postinfection. Significantly different from control .p ϭ 0.01; Student’s t test. i.v. with 2 ϫ 107 listeriae. Original magnification, ϫ400 ,ءء ;0.03 The Journal of Immunology 313

Table IV. Anti-CD11b blocks the accumulation of neutrophils in the livers of Listeria-infected micea

Treatment NPCs (% neutrophils)

Normal rat IgG 36 Ϯ 4 Anti-CD11b 7 Ϯ 4

a Mice inoculated i.p. with 0.5 mg normal rat IgG or monoclonal rat anti-CD11b were challenged with Ϸ2 ϫ 107 Listeria 18 h later. The percentage of neutrophils constituting the NPC population was determined at 2 h postinfection. Values are the means Ϯ SD obtained from nine livers and three separate experiments. Values are statistically different ( p Ͻ 0.001; nonpaired Student’s t test).

Discussion The rapid clearance of bacterial pathogens from the bloodstream and the subsequent elimination of those pathogens from the liver are generally attributed to fixed tissue macrophages, i.e., Kupffer cells, that line the liver sinusoids (1, 42). Indeed, in the case of listeriosis in mice, it is often suggested that phagocytosis by Downloaded from FIGURE 7. Pretreatment with anti-CD11b/CD18 inhibits the elimina- Kupffer cells accounts for the decrease in number of liver listeriae tion of listeriae early during the course of infection. Data are the mean Ϯ that typically occurs between 10 min and 6 h postinfection i.v. (8, SD number of listeriae recovered in the livers derived from four mice 43). Previously we reported, however, that most listeriae taken up pretreated with normal rat IgG or monoclonal anti-CD11b 18 h before in the liver were initially bound extracellularly and subsequently infection i.v. with 2.7 ϫ 104 listeriae. Similar results were obtained in three killed by immigrating neutrophils (11). The failure of Kupffer cells Significantly greater than the comparable group to contribute substantially to the destruction of these organisms ,ء .additional experiments ϭϽ http://www.jimmunol.org/ of mice administered normal rat IgG 18 h before infection (p 0.001, by correlates with in vitro experiments that demonstrate the dimin- nonpaired Student’s t test). ished capacity of Kupffer cells to generate reactive oxygen intermediates and to kill intracellular pathogens, i.e., Toxoplasma gon- dii trophozoites and L. donovani amastigotes (36). Complement is not a factor in early host defenses to liver While Kupffer cells play a relatively minor role in killing bac- Listeria terial pathogens directly, the results reported herein indicate that While complement is an important factor in host resistance to Lis- they are a critical factor in blood clearance. Kupffer cell-depleted teria (41), it did not play a substantial role in the inhibitory effect mice exhibited a significant increase in the number of listeriae of anti-CD11b (i.e., anti-type 3 complement receptor) on the initial remaining in the bloodstream and an approximately 75% decrease by guest on September 24, 2021 killing of listeriae taken up in the liver. Groups of control mice and in the number recovered in the liver at 10 min postinfection i.v. mice rendered complement deficient by pretreatment with CVF This finding correlates with earlier reports demonstrating the cru- exhibited equivalent capacities to clear listeriae from the blood- cial role of Kupffer cells in clearance of bacteria from circulating stream (evident at 10 min postinfection) and to eliminate liver blood (1). It contradicts more recent studies that showed a negli- listeriae during the following 6 h (Fig. 8). Moreover, the percent- gible effect of two macrophage toxins, i.e., crystalline silica and age of neutrophils recovered within the NPC populations derived from these two groups of animals at 2 h postinfection was essentially the same (data not shown). Thus, complement did not affect the immigration or the antimicrobial activity of neutrophils expressed early during the course of listerial infection. Complement, however, did play a significant role in the elimination of liver listeriae later in infection. At 2 days postinfection, the livers of Ͼ complement-depleted (CVF-pretreated) mice contained 1 log10 more bacteria than did the livers derived from control animals.

Table III. mAbs speciﬁc for the complementary adhesion molecules CD11b/CD18 and CD54 block killing of Listeria cleared by the liver a

Antigenic Determinant Listeriae/Liver (ϫ103)

Control 4.5 Ϯ 0.5 CD11b 11.1 Ϯ 1.0b CD54 7.4 Ϯ 0.7b CD62L 5.8 Ϯ 0.9 Ϯ CD11a 4.4 1.3 FIGURE 8. Increased replication of Listeria in the livers of comple- CD106 4.8 Ϯ 0.7 ment-depleted mice. Control and complement-depleted (CVF-treated) a Mice inoculated i.p. with 0.5 mg normal IgG (control) or mAb specific for the mice were inoculated i.v. with 4 ϫ 104 listeriae. Data are the mean Ϯ SD antigenic determinants listed were challenged i.v. with 4.3 log10 Listeria 18 h later. number of organisms recovered in the livers of four mice at the times Ϯ Data are the means SD Listeria per liver dissected from groups of four mice at 6 h indicated in a single representative experiment. Two additional experi- ء .postinfection. Two additional experiments yielded comparable results b Significantly greater than in those animals treated with normal rat IgG before ments yielded comparable results. , CVF-treated is significantly different infection ( p Ͻ 0.05; one-way ANOVA). from control (p ϭ 0.008, by nonpaired Student’s t test). 314 NEUTROPHIL-KUPFFER CELL INTERACTION carrageenan, on the ability of the liver to clear pathogens from the ination of liver listeriae between 10 min and 6 h postinfection was bloodstream (44, 45). It has been suggested, however, that the inhibited. The diminished capacity of Ab-treated mice to eliminate failure of either silica or carrageenan to diminish blood clearance Listeria correlated with decreased sequestration of neutrophils in reflects the ability of these agents to compromise Kupffer cell func- the liver early during the course of infection (i.e., at 2 h postin- tion without actually eliminating Kupffer cells or affecting their fection when the accumulation of neutrophil was otherwise max- capacity to bind bacteria extracellularly (44, 46). Moreover, it is imal). Pretreatment with CVF protein failed to exert the same ef- relevant to note that in contrast to these other reagents, the admin- fect, indicating that complement was not an intermediary factor istration of Cl2MDP-L prohibits, rather than induces, the produc- affecting the infiltration and/or listericidal activity expressed by tion of proinflammatory cytokines by macrophages (23, 46). neutrophils. Rather, the results of experiments involving anti- In addition to playing a crucial role in blood clearance, Kupffer CD11b/CD18 pretreatment suggest that the interaction of CD11b/ cells exerted a significant, inhibitory effect on the subsequent CD18 with its counter-receptor, CD54, facilitates the accumulation growth of listeriae taken up by the liver. Thus, at 24 and 48 h of neutrophils at the principal site of listerial deposition in the liver postinfection Ͼ1 log more listeriae were recovered in the livers 10 (i.e., on the Kupffer cell surface). This conclusion is supported by of Cl MDP-L-treated mice than in control mice pretreated with 2 experiments demonstrating the negative effect of anti-CD54 mAb, saline or PBS-L. While the specific factors that contribute to this but not mAb specific for VCAM-1 (CD106), LFA-1 (CD11a/ increase are presently unclear, it is notable that Kupffer cells syn- CD18), or L-selectin (CD62L), on the early elimination of listeriae thesize a variety of cytokines (i.e., IL-1␤, IL-6, IL-12, and TNF-␣) cleared by the liver. Both the constitutive expression of CD54 by that promote the proinflammatory and/or antimicrobial activity of

Kupffer cells in vivo and the elevated expression of CD54 by Downloaded from other cell types that infiltrate or reside within the liver. These cytokines exert significant effects on the metabolic activity and bio- Kupffer cells following endotoxin stimulation are well documented logical functions of hepatocytes, for example, and play a crucial (40). It is attractive to speculate, therefore, that the direct interac- role in the hepatocellular dysfunction associated with endotoxemia tion of CD11b/CD18 and CD54 expressed by neutrophils and and septicemia (47). During listerial infections, IL-6 produced by Kupffer cells, respectively, facilitates neutrophil accumulation and Kupffer cells induces STAT3 activation and the synthesis of acute the elimination of listeriae taken up by the liver. It is important to phase reactants by hepatocytes that may promote host resistance note, however, that other cell types present in the liver may express http://www.jimmunol.org/ (21, 48). Such findings suggest that a reduction in cytokine pro- the same molecules (e.g., CD54 by hepatocytes and endothelial duction contributes to the increased proliferation of Listeria in the cells that line the blood vessels) and influence these events (38). livers of Kupffer cell-depleted mice. Kupffer cell depletion diminished but did not completely negate, The particular mechanisms that underlie Kupffer cell-mediated blood clearance of listeriae by the liver (Figs. 1 and 2). Thus, while clearance of listeriae from the bloodstream remain to be delin- Kupffer cells constitute the predominant cell factor in clearance, eated. The reduced capacity of mice pretreated i.v. with sugars or other resident liver cell types must also be involved and/or com- neoglycoproteins to remove organisms from the blood suggests pensate for Kupffer cells in their absence. The accumulation of that, in at least some cases, clearance is mediated by the interaction neutrophils in the livers of Listeria-infected, Kupffer cell-depleted of lectins expressed by Kupffer cells and carbohydrate residues mice was reduced in accordance with the number of micro-organ- by guest on September 24, 2021 expressed by the bacteria (49). Indeed, fixed tissue macrophages isms taken up. The importance of these neutrophils in host de- exhibit receptors specific for ligands present on the surface of mi- fenses is supported by a marked increase in the proliferation of cro-organisms, but not normally displayed by host cells. Macro- extracellular listeriae in the livers of mice depleted of both neu- phage scavenger receptors, for example, have a high affinity for an trophils and Kupffer cells relative to animals depleted of Kupffer unusually broad range of polyanionic ligands, including lipotei- cells alone (our unpublished observation). choic acid, a component of Gram-positive bacteria such as L. The appearance of neutrophils inside macrophages recovered monocytogenes (50, 51). As such, macrophage scavenger receptors from the livers of mice at 2 h postinfection (Fig. 5) suggests that may participate in host defenses by binding and clearing Gram- Kupffer cells may play a critical role in eliminating neutrophils that positive bacteria from the bloodstream and tissues (50, 51). accumulate in the liver subsequent to systemic infection. Similar Type 3 complement receptors (CR3) specific for the iC3b com- findings, i.e., the presence of neutrophils within Kupffer cells, were ponent of complement are a major factor effecting the internaliza- reported for rats inoculated i.v. with a lyophilized streptococcal tion and killing of bacteria by phagocytes in vitro (52). CR3, how- preparation (53). These results correlate with the widely held prop- ever, were not involved in the clearance of systemic listeriae by osition that neutrophils in inflammatory lesions eventually undergo Kupffer cells. Neither complement depletion nor pretreatment of apoptosis, followed by phagocytosis and removal by neighboring mice with mAb specific for CR3 (i.e., anti-CD11b/CD18) affected macrophages (54, 55). Considered in conjunction with the results the recovery of listeriae in the liver at 10 min postinfection. This of previous experiments (11), the findings reported herein suggest finding is consistent with results demonstrating the failure of Kupffer cells to express CR3 at a significant level (34–36). Al- an entirely new paradigm for the role of Kupffer cells in early host though complement was not a factor in Kupffer cell-mediated defenses to systemic bacterial infections. We propose that the ma- clearance of listeriae from the bloodstream or in neutrophil-medi- jority of organisms that enter the bloodstream are taken up in the ated killing of liver listeriae early in infection, complement was a liver and bound extracellularly by Kupffer cells. Subsequently, critical component of host defenses expressed later. Approxi- complementary adhesion molecules (i.e., CD11b/CD18 and CD54) up-regulated in response to infection promote the accumu- mately 1 log10 more listeriae was recovered in the livers of complement-depleted animals at 48 h postinfection. This finding is lation of neutrophils at the Kupffer cell surface and the elimination consistent with previous reports demonstrating the importance of of bound bacteria. Finally, Kupffer cells ingest and destroy adher- complement in host resistance to Listeria and the contribution of ent neutrophils as the extracellular organisms in the liver sinusoids complement to the inflammatory response of mononuclear phago- are eradicated. The role of Kupffer cells and the specific mecha- cytes occurring during the later stages of infection (41). nisms that underlie the elimination of neutrophils sequestered in While mice pretreated with anti-CD11b/CD18 mAb exhibited a hepatic sinusoids during systemic infections are matters of ongo- normal capacity to clear Listeria from the bloodstream, the elim- ing investigation in our laboratories. The Journal of Immunology 315

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