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*
Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence, RI 02903; and
Department of Cell Biology, Vrije Universiteit, Amsterdam, The Netherlands
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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15% 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 was not a
factor; complement depletion affected neither the clearance of
listeriae by Kupffer cells nor the antimicrobial activity expressed by
infiltrating neutrophils. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Listeria monocytogenes is a Gram-positive, facultative, intracellular, bacterial pathogen capable of causing severe infections in humans characterized by high mortality (6, 7). Listeriosis in mice is an experimental model used widely to explore the factors that effect host defenses to pathogens that replicate intracellularly (2, 8). Kupffer cells play a prominent role in host resistance to both primary and secondary listerial infections. The proliferation of liver listeriae and mortality are increased markedly in mice pretreated with liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP-L)3 to eliminate Kupffer cells (9, 10). While their specific function(s) remains to be delineated, it is often assumed that Kupffer cells account for the marked (0.5–1 log10) decrease in liver listeriae that typically occurs early during the course of infection (8). Previously we reported, however, that the bulk of listeriae inoculated i.v. was cleared rapidly by the liver, initially bound extracellularly, and subsequently killed by immigrating neutrophils (11).
Neutrophils represent 1–2% of the total nonparenchymal cells (NPCs) found in the liver of normal, noninfected mice (11). The percentage of neutrophils increases rapidly following i.v. inoculation of Listeria, reaching a maximum at 2 h postinfection and then declining thereafter with the elimination of extracellular bacteria (11) (our unpublished observation). At 6 h postinfection, essentially all the viable listeriae remaining in the liver were located inside hepatocytes, as judged by cell separation experiments and resistance of the organism to antibiotic treatment (11). Mice rendered 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 pretreated with RB6–8C5 mAb exhibited a reduced capacity to eliminate Escherichia coli, Staphylococcus aureus, and Klebsiella pneumoniae inoculated i.v. and taken up in the liver. These findings were supported by more recent studies demonstrating increased numbers of S. typhimurium, Yersinia enterocolitica, and Clostridium piliforme in the livers of neutrophil-depleted mice inoculated i.v. (12, 13, 14).
In view of the obligate requirement for neutrophils in killing, we undertook the series of experiments reported here to delineate the precise role of Kupffer cells in host resistance to systemic bacterial infections using listeriosis in mice as an experimental model. The results of these experiments indicate that Kupffer cells are required for the efficient clearance of listeriae from the bloodstream. The subsequent eradication of those organisms is facilitated by the CD11b/CD18 (Mac-1)- and CD54 (ICAM-1)-dependent accumulation of bactericidal neutrophils at the site of listeriae deposition, i.e., on the Kupffer cell surface. Photomicrographs demonstrating neutrophils inside macrophages at 2 h postinfection suggest that Kupffer cells may play a crucial role in eliminating neutrophils from the liver as extracellular organisms in the sinusoids are eradicated.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME) were housed and cared for in accordance with the guidelines set forth by the institutional animal care and use committees, University of Pittsburgh and Rhode Island Hospital. Animals between 8 and 16 wk of age were used in the experiments described.
Bacteria
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 in the experiments described. The number of organisms recovered in the livers and peripheral blood of infected animals was estimated from the number of colonies that grew on trypticase soy agar plates inoculated with an aliquot of organ homogenate (16). Total bacteria in the bloodstream were calculated assuming the blood volume to be 8% (v/w) of the weight of each animal (17).
Cell preparation
Total and purified nonparenchymal liver cell populations
composed primarily of leukocytes and endothelial cells were obtained
following perfusion of the liver with collagenase using the two-step
method we reported previously (11, 18). Transient blood
cells circulating through the liver and not firmly bound to the tissues
were eliminated during the exhaustive perfusion process. Isolated cells
were suspended in HEPES-buffered RPMI 1640 medium (BioWhittaker,
Walkersville, MD) supplemented with 10% heat-inactivated FBS (Sterile
Systems, Logan, UT). The purified NPC population was 
99% viable and
essentially free of parenchymal cell (hepatocyte) contamination.
Purified NPCs were sedimented onto glass slides using a cytocentrifuge
(Shandon Elliott, Pittsburgh, PA) and stained with Wright-Giemsa stain.
Neutrophils within a population of 200 cells were enumerated based upon
morphology. Peripheral blood samples were obtained by cardiac puncture
using EDTA-coated tuberculin syringes. Total and differential leukocyte
counts were determined after sedimentation of the bulk of erythrocytes
with 5% dextran T500, followed by treatment with 0.83% ammonium
chloride to lyse the RBCs that remained.
Kupffer cell depletion
Multilamellar liposomes containing Cl2MDP (a gift obtained from Roche, Mannheim, Germany) were prepared as previously described (19). Mice were rendered >95% Kupffer cell-depleted by the inoculation i.v. of 200 µl Cl2MDP-L (containing 1 mg/ml Cl2MDP) suspended in saline 3 days before experimental use (20, 21). Mice administered 200 µl normal saline or liposome-encapsulated PBS (PBS-L) served as controls. The effect of Cl2MDP-L on resident cells in the liver was specific for Kupffer cells. Cl2MDP-L had no detectable effect on hepatocytes in vivo; aspartate aminotransferase levels were comparable in the sera obtained from control and Cl2MDP-L-treated mice on day 3 (21). Moreover, the addition of Cl2MDP-L had no effect on the viability of hepatocytes in vitro (assessed in terms of aspartate aminotransferase activity released into the culture 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 on day 3 following inoculation i.v. contained comparable percentages of Ly-6G+ neutrophils (1.8 ± 1.0 vs 1.3 ± 0.1), NK1.1-CD3+ T cells (7.1 ± 3.2 vs 5.0 ± 1.3), NK1.1+CD3- NK cells (14.6 ± 2.4 vs 15.3 ± 1.7), and NK1.1+CD3+ natural T cells (16.4 ± 3.6 vs 16.8 ± 2.5; flow cytometric analysis, control listed first). These data are consistent with the results of numerous other studies that failed to show a significant effect of Cl2MDP-L on hepatocytes or circulating white blood cells, i.e., lymphocytes, granulocytes (including neutrophils), or mononuclear phagocytes (9, 22, 23, 24, 25).
Monoclonal Abs
mAbs produced by the following hybridomas were obtained in the ascites fluid of pristane-primed, homozygous nude BALB/c mice injected with 107 cells: rat IgG2b anti-mouse granulocyte (RB6-8C5) (11), rat IgG2b anti-mouse CD54 (YN1/1.7.4; American Type Culture Collection, Manassas, VA), and rat IgG2b anti-mouse CD11b (5C6; American Type Culture Collection). The Abs were purified by chromatography on a protein G column (Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s instructions. Monoclonal rat IgG2b anti-mouse CD11a (FD441.8; American Type Culture Collection), rat IgG1 anti-mouse CD106 (MK1.9; American Type Culture Collection), and rat IgG2a anti-mouse CD62 ligand (CD62L; Mel-14; American Type Culture Collection) were derived from the supernatants of cultured hybridomas. The Ig fraction was precipitated with ammonium sulfate, dialyzed against PBS, and concentrated by centrifugation. For experiments, test animals were inoculated i.p. with 0.5 mg purified Ab 18 h before infection. Control mice received an equivalent concentration of chromatographically purified normal rat IgG (Organon Technika-Cappel, Durham, NC). All mAbs used in these in vivo experiments possessed blocking or neutralizing activity (26, 27, 28, 29, 30, 31). Conjugated mAbs used in flow cytometric analyses to characterize the NPC populations derived from control and Cl2MDP-L-treated mice were purchased from PharMingen (San Diego, CA).
Complement depletion
Mice were rendered complement deficient by treatment i.p. with 30 µg cobra venom factor (CVF; Calbiochem-Novabiochem International, La Jolla, CA) 18 h before infection. Serum samples derived from mice at the time of infection and analyzed by zonal electrophoresis exhibited a >95% reduction in the C3 component of complement.
Immunocytochemistry
Cryostat sections (6 µm) derived from the livers of mice at 2 h postinfection i.v. with 2 x 107 listeriae were fixed in acetone and air-dried. Mononuclear phagocytes (macrophages) were detected by a modification of methods previously described (32); sections were incubated with rat anti-mouse F4/80 Ag, washed, and reincubated with Alexa Fluor 488-conjugated goat anti-rat IgG (Molecular Probes, Eugene, OR). Infiltrating (Ly-6G+) neutrophils were detected by incubating the sections with rat IgG2b anti-mouse granulocyte mAb (RB6-8C5). The slides were then washed and incubated with Texas Red-conjugated goat anti-rat IgG (Jackson ImmunoResearch Laboratories, West Grove, PA). The slides were mounted with Vectashield (Vector, Burlingame, CA) and examined under a Nikon Eclipse E800 microscope (Melville, NY) equipped with dual filters for FITC and Texas Red. Multiple tissue sections were surveyed and the number of red fluorescent Ly-6G+ neutrophils in juxtaposition with yellow-green fluorescent F4/80+ Kupffer cells was determined and expressed as a percentage of the total neutrophils counted.
Alternatively, neutrophils in the liver were detected by immunohistochemistry. Cryostat sections were pretreated with avidin/biotin (Vector) to block nonspecific binding of peroxidase-conjugated avidin. The sections were then incubated with biotin-conjugated rat IgG2b anti-mouse Ly-6G (PharMingen), washed, incubated with avidin-peroxidase, washed, anddeveloped with 3-amino-9-ethylcarbazole (Sigma, St. Louis, MO) andhydrogen peroxide.
Myeloperoxidase
The myeloperoxidase activity in homogenates prepared from the liver, spleen, and peripheral blood was quantified by analyzing the H2O2-dependent oxidation of 3,3',5,5'-tetramethylbenzidine (Sigma) spectrophotometrically as described previously by Schierwagen et al. (33).
Statistical analysis
The results were analyzed using the SigmaStat statistics program (Jandel Scientific, San Rafael, CA). Individual means were compared by nonpaired Student’s t test. Data derived from three or more groups were compared by ANOVA, followed by Student-Newman-Keuls test. Differences at p < 0.05 were considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. Nonetheless, these
results demonstrate the crucial role of Kupffer cells in efficient
clearance of bacterial pathogens from the bloodstream.
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Previously, we reported that most bacteria cleared from the
bloodstream and taken up by the liver were initially bound
extracellularly and subsequently killed by infiltrating, bactericidal
neutrophils; essentially all the organisms remaining in the liver at
6 h 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
demargination 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.
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, A and B). These
infiltrating neutrophils were restricted to the vascular sinusoids; the
vast majority (79 ± 3.5%) colocalized with
F4/80+ 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.
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 neutrophils (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 ± 5.2% of the peripheral blood
leukocytes obtained 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
Listeria-infected mice.
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). This
finding supports our contention that the innate response to bacteria
cleared from the bloodstream entails the direct interaction of
neutrophils with resident tissue macrophages that line the liver
sinusoids. Moreover, it suggests that the potentially important role of
Kupffer cells in eliminating neutrophils from the liver (e.g., between
2 and 6 h postinfection i.v.; shown in Fig. 4
) as extracellular
listeriae in the sinusoids are killed.
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The cell surface adhesion molecule, CD11b/CD18 (Mac-1; type 3
complement receptor, CR3), expressed by neutrophils, but not Kupffer
cells (34, 35, 36), participates in a broad spectrum of
activities, including adherence to vascular endothelial cells,
extravasation, and phagocytosis (37). CD11b/CD18 is a
critical factor in host defenses to Listeria expressed in
the liver (38, 39). A series of experiments was undertaken
to explore the role of CD11b/CD18 in neutrophil-Kupffer cell
interaction and the elimination of listeriae taken up in the liver
early during the course of infection. Mice pretreated with mAb specific
for CD11b (clone 5C6, a blocking (noncytolytic) Ab demonstrated to
suppress recruitment of inflammatory cells in vivo (27, 31)) exhibited a normal capacity to clear listeriae from the
bloodstream; the numbers of organisms present 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.
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The increased recovery of listeriae in the livers of mice
pretreated with monoclonal anti-CD11b correlated inversely with the
accumulation of neutrophils. The NPC population derived from
mAb-treated mice at 2 h postinfection (i.e., at the height of
neutrophil infiltration) contained 5-fold fewer neutrophils than did
the NPCs obtained from mice administered normal rat IgG before
infection (Table IV). CD11b/CD18
expression, therefore, exerted a significant influence on the
sequestration of neutrophils in the livers of animals infected
systemically. Anti-CD11b mAb treatment, on the other hand, failed to
affect either the total leukocyte count (1.8 ± 0.7 x
103/µl (mAb-treated) vs 2.0 ± 0.8 x
103/µl (control)) or the number of neutrophils
(0.4 ± 0.2 x 103/µl (mAb-treated)
vs 0.6 ± 0.4 x 103/µl (control))
present in the peripheral blood. Similarly, the administration of
anti-ICAM-1 mAb affected neither the total PBL (2.2 ±
0.8 x 103/µl) nor the peripheral blood
neutrophil (0.4 ± 0.2 x 103/µl)
count assessed in the same set of experiments.
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While complement is an important factor in host resistance to
Listeria (41), it did not play a substantial
role in the inhibitory effect of anti-CD11b (i.e., anti-type 3
complement receptor) on the initial killing of listeriae taken up in
the liver. Groups of control mice and mice rendered complement
deficient by pretreatment with CVF exhibited equivalent capacities to
clear listeriae from the bloodstream (evident at 10 min postinfection)
and to eliminate liver listeriae during the following 6 h (Fig. 8). Moreover, the percentage 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 complement-depleted
(CVF-pretreated) mice contained >1 log10 more
bacteria than did the livers derived from control animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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While Kupffer cells play a relatively minor role in killing bacterial pathogens directly, the results reported herein indicate that they are a critical factor in blood clearance. Kupffer cell-depleted mice exhibited a significant increase in the number of listeriae remaining in the bloodstream and an approximately 75% decrease in the number recovered in the liver at 10 min postinfection i.v. This finding correlates with earlier reports demonstrating the crucial role of Kupffer cells in clearance of bacteria from circulating blood (1). It contradicts more recent studies that showed a negligible effect of two macrophage toxins, i.e., crystalline silica and carrageenan, on the ability of the liver to clear pathogens from the bloodstream (44, 45). It has been suggested, however, that the failure of either silica or carrageenan to diminish blood clearance reflects the ability of these agents to compromise Kupffer cell function without actually eliminating Kupffer cells or affecting their capacity to bind bacteria extracellularly (44, 46). Moreover, it is relevant to note that in contrast to these other reagents, the administration of Cl2MDP-L prohibits, rather than induces, the production of proinflammatory cytokines by macrophages (23, 46).
In addition to playing a crucial role in blood clearance, Kupffer cells
exerted a significant, inhibitory effect on the subsequent growth of
listeriae taken up by the liver. Thus, at 24 and 48 h
postinfection >1 log10 more listeriae were
recovered in the livers of Cl2MDP-L-treated mice
than in control mice pretreated with saline or PBS-L. While the
specific factors that contribute to this increase are presently
unclear, it is notable that Kupffer cells synthesize a variety of
cytokines (i.e., IL-1
, IL-6, IL-12, and TNF-
) that promote the
proinflammatory and/or antimicrobial activity of other cell types that
infiltrate or reside within the liver. These cytokines exert
significant effects on the metabolic activity and biological functions
of hepatocytes, for example, and play a crucial role in the
hepatocellular dysfunction associated with endotoxemia and septicemia
(47). During listerial infections, IL-6 produced by
Kupffer cells induces STAT3 activation and the synthesis of acute phase
reactants by hepatocytes that may promote host resistance (21, 48). Such findings suggest that a reduction in cytokine
production contributes to the increased proliferation of
Listeria in the livers of Kupffer cell-depleted
mice.
The particular mechanisms that underlie Kupffer cell-mediated clearance of listeriae from the bloodstream remain to be delineated. The reduced capacity of mice pretreated i.v. with sugars or neoglycoproteins to remove organisms from the blood suggests that, in at least some cases, clearance is mediated by the interaction of lectins expressed by Kupffer cells and carbohydrate residues expressed by the bacteria (49). Indeed, fixed tissue macrophages exhibit receptors specific for ligands present on the surface of micro-organisms, but not normally displayed by host cells. Macrophage scavenger receptors, for example, have a high affinity for an unusually broad range of polyanionic ligands, including lipoteichoic acid, a component of Gram-positive bacteria such as L. monocytogenes (50, 51). As such, macrophage scavenger receptors may participate in host defenses by binding and clearing Gram-positive bacteria from the bloodstream and tissues (50, 51).
Type 3 complement receptors (CR3) specific for the iC3b component of complement are a major factor effecting the internalization and killing of bacteria by phagocytes in vitro (52). CR3, however, were not involved in the clearance of systemic listeriae by Kupffer cells. Neither complement depletion nor pretreatment of mice with mAb specific for CR3 (i.e., anti-CD11b/CD18) affected the recovery of listeriae in the liver at 10 min postinfection. This finding is consistent with results demonstrating the failure of Kupffer cells to express CR3 at a significant level (34, 35, 36). Although complement was not a factor in Kupffer cell-mediated clearance of listeriae from the bloodstream or in neutrophil-mediated killing of liver listeriae early in infection, complement was a critical component of host defenses expressed later. Approximately 1 log10 more listeriae was recovered in the livers of complement-depleted animals at 48 h postinfection. This finding is consistent with previous reports demonstrating the importance of complement in host resistance to Listeria and the contribution of complement to the inflammatory response of mononuclear phagocytes occurring during the later stages of infection (41).
While mice pretreated with anti-CD11b/CD18 mAb exhibited a normal capacity to clear Listeria from the bloodstream, the elimination of liver listeriae between 10 min and 6 h postinfection was inhibited. The diminished capacity of Ab-treated mice to eliminate Listeria correlated with decreased sequestration of neutrophils in the liver early during the course of infection (i.e., at 2 h postinfection when the accumulation of neutrophil was otherwise maximal). Pretreatment with CVF protein failed to exert the same effect, indicating that complement was not an intermediary factor affecting the infiltration and/or listericidal activity expressed by neutrophils. Rather, the results of experiments involving anti-CD11b/CD18 pretreatment suggest that the interaction of CD11b/CD18 with its counter-receptor, CD54, facilitates the accumulation of neutrophils at the principal site of listerial deposition in the liver (i.e., on the Kupffer cell surface). This conclusion is supported by experiments demonstrating the negative effect of anti-CD54 mAb, but not mAb specific for VCAM-1 (CD106), LFA-1 (CD11a/CD18), or L-selectin (CD62L), on the early elimination of listeriae cleared by the liver. Both the constitutive expression of CD54 by Kupffer cells in vivo and the elevated expression of CD54 by Kupffer cells following endotoxin stimulation are well documented (40). It is attractive to speculate, therefore, that the direct interaction of CD11b/CD18 and CD54 expressed by neutrophils and Kupffer cells, respectively, facilitates neutrophil accumulation and the elimination of listeriae taken up by the liver. It is important to note, however, that other cell types present in the liver may express the same molecules (e.g., CD54 by hepatocytes and endothelial cells that line the blood vessels) and influence these events (38).
Kupffer cell depletion diminished but did not completely negate, blood
clearance of listeriae by the liver (Figs. 1 and 2). Thus, while
Kupffer cells constitute the predominant cell factor in clearance,
other resident liver cell types must also be involved and/or compensate
for Kupffer cells in their absence. The accumulation of neutrophils in
the livers of Listeria-infected, Kupffer cell-depleted mice
was reduced in accordance with the number of micro-organisms taken up.
The importance of these neutrophils in host defenses is supported by a
marked increase in the proliferation of extracellular listeriae in the
livers of mice depleted of both neutrophils and Kupffer cells relative
to animals depleted of Kupffer cells alone (our unpublished
observation).
The appearance of neutrophils inside macrophages recovered from the
livers of mice at 2 h postinfection (Fig. 5) suggests that Kupffer
cells may play a critical role in eliminating neutrophils that
accumulate in the liver subsequent to systemic infection. Similar
findings, i.e., the presence of neutrophils within Kupffer cells, were
reported for rats inoculated i.v. with a lyophilized streptococcal
preparation (53). These results correlate with the widely
held proposition that neutrophils in inflammatory lesions eventually
undergo apoptosis, followed by phagocytosis and removal by neighboring
macrophages (54, 55). Considered in conjunction with the
results of previous experiments (11), the findings
reported herein suggest an entirely new paradigm for the role of
Kupffer cells in early host defenses to systemic bacterial infections.
We propose that the majority of organisms that enter the bloodstream
are taken up in the liver and bound extracellularly by Kupffer cells.
Subsequently, complementary adhesion molecules (i.e., CD11b/CD18 and
CD54) up-regulated in response to infection promote the accumulation of
neutrophils at the Kupffer cell surface and the elimination of bound
bacteria. Finally, Kupffer cells ingest and destroy adherent
neutrophils as the extracellular organisms in the liver sinusoids are
eradicated. The role of Kupffer cells and the specific mechanisms that
underlie the elimination of neutrophils sequestered in hepatic
sinusoids during systemic infections are matters of ongoing
investigation in our laboratories.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Stephen H. Gregory, Department of Medicine, Rhode Island Hospital/Brown University School of Medicine, 432 Pierre M. Galletti Building, 55 Claverick Street, Providence, RI 02903. E-mail address: sgregory{at}lifespan.org
3 Abbreviations used in this paper: Cl2MDP-L, liposome-encapsulated dichloromethylene diphosphonate; CD62L, CD62 ligand; CVF; cobra venom factor; NPC, nonparenchymal liver cells; PBS-L, liposome-encapsulated PBS.
Received for publication December 14, 2000. Accepted for publication October 22, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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H. Wu, J. E. Prince, C. F. Brayton, C. Shah, D. Zeve, S. H. Gregory, C. W. Smith, and C. M. Ballantyne Host Resistance of CD18 Knockout Mice against Systemic Infection with Listeria monocytogenes Infect. Immun., October 1, 2003; 71(10): 5986 - 5993. [Abstract] [Full Text] [PDF]
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 C. A. Rivera, M. H. Tcharmtchi, L. Mendoza, and C. W. Smith Endotoxemia and hepatic injury in a rodent model of hindlimb unloading J Appl Physiol, October 1, 2003; 95(4): 1656 - 1663. [Abstract] [Full Text] [PDF]
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M. Miyamoto, M. Emoto, Y. Emoto, V. Brinkmann, I. Yoshizawa, P. Seiler, P. Aichele, E. Kita, and S. H. E. Kaufmann Neutrophilia in LFA-1-Deficient Mice Confers Resistance to Listeriosis: Possible Contribution of Granulocyte-Colony-Stimulating Factor and IL-17 J. Immunol., May 15, 2003; 170(10): 5228 - 5234. [Abstract] [Full Text] [PDF]
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S. H. Gregory and E. J. Wing Neutrophil-Kupffer cell interaction: a critical component of host defenses to systemic bacterial infections J. Leukoc. Biol., August 1, 2002; 72(2): 239 - 248. [Abstract] [Full Text] [PDF]
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