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Department of Medicine, Rhode Island Hospital and Brown University School of Medicine, Providence
Correspondence: 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: sgregory{at}lifespan.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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Key Words: septicemia • liver • macrophage • granulocyte • Listeria monocytogenes • mouse
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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KUPFFER CELLS |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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35% of the nonparenchymal liver cells in normal adult mice [10
, 11
]. They reside principally within the lumen of the liver sinusoids, adherent to the endothelial cells that compose the blood vessel walls. Kupffer cells, found in greatest number in the periportal area, constitute the first macrophage population to come in contact with bacteria, bacterial endotoxin, and microbial debris derived from the gastrointestinal tract and transported to the liver via the portal vein [12
]. In fact, endotoxin derived from the gut is a normal component of the portal-venous blood estimated to range from 10 to 1000 pg/ml in concentration [13
]. Consequently, Kupffer cells are constantly exposed to proinflammatory factors, e.g., bacterial endotoxin, known to activate macrophages. Like endotoxin derived from the gastrointestinal tract, bacterial endotoxin injected intravenously (i.v.) in animal models is cleared principally by the liver and taken up almost exclusively by Kupffer cells [14 ]. Essentially no endotoxin is found associated with hepatocytes or endothelial cells until 2–3 days after clearance. Similarly, colloids (e.g., carbon particles in suspension) injected i.v. into mice are cleared quickly from the blood by phagocytes residing in the liver and spleen [15 ]. When the amount of material injected is small, Kupffer cells remove 80–90% of the particles. The majority of bacteria injected i.v. is also taken up by the liver [15 ]. The efficiency with which bacteria are taken up, however, is decreased significantly in animals pretreated with colloidal suspensions, e.g., thorotrast [16 ] or carbon particles [15 ], suggesting that the clearance of colloids and microbes involves the same cell population, Kupffer cell. This has led to the conclusion that circulating leukocytes play an important role only in eliminating bacteria that enter the bloodstream when the bacteria are trapped in fibrin clots or adherent to endothelial cells lining the capillaries; then, the organisms are eliminated in a process called "surface phagocytosis" [15 ].
In contrast to these early reports, a number of more recent studies indicate that Kupffer cells may play a less significant role in resolving systemic infections. The recovery of Escherichia coli and Klebsiella pneumoniae inoculated i.v., for example, was comparable in the livers of untreated mice and mice pretreated with carrageenan to deplete Kupffer cells [17 ]. Similarly, pretreatment with crystalline silica to destroy Kupffer cells had no effect on the capacity of rat livers to take-up Salmonella typhimurium in an in vitro perfusion model [18 ], and clearance of Salmonella by mouse livers was reduced only twofold [19 ]. Likewise, pretreatment with silica had only a marginal effect on the clearance of Trypanosoma musculi [20 ]. The results of these studies should be interpreted with caution, however, in light of experiments demonstrating: elevated cytokine production by macrophages treated with silica or carrageenan, the inhibitory effects of these agents on complement activation and/or neutrophil function, and the failure of these compounds to deplete Kupffer cells completely [17 , 21 22 23 ].
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NEUTROPHILS |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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70% of peripheral blood leukocytes in adult humans and 5–20% of the circulating leukocytes in mice depending on age and strain. Neutrophils constitute the first line of defense against most classes of pathogenic microorganisms. Their effector functions at sites of infection include phagocytosis, production of toxic metabolites, and the release of proteolytic enzymes [24
]. Although these functions facilitate the elimination of invading organisms, they can also cause severe tissue damage [24
, 25
]. Indeed, the dysregulated activation of neutrophils is thought to contribute to the exaggerated inflammatory response associated with acute respiratory distress syndrome and multiple organ dysfunction syndrome (MODS) during sepsis [26
]. The accumulation of neutrophils in the liver sinusoids is a distinguishing feature of endotoxemia and sepsis; reactive oxygen intermediates (ROI) and proteolytic enzymes released by neutrophils are a central cause of severe hepatic injury [27
]. Treatments that include the administration of neutrophil-depleting antibody, antioxidants, and protease inhibitors attenuate the liver dysfunction that otherwise occurs in animal models of sepsis demonstrating the role of these factors in liver injury [28
29
30
]. The timely elimination of inflammatory neutrophils sequestered in the liver during transient periods of bacteremia or endotoxemia, therefore, prevents the exaggerated response observed during sepsis and the development of MODS. |
LISTERIOSIS MODEL OF SYSTEMIC INFECTION |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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) production in host defenses to intracellular bacterial pathogens was first demonstrated in studies involving Listeria monocytogenes [32
33
34
]. Like many other pathogens that enter the bloodstream, the bulk (60%) of L. monocytogenes inoculated i.v. into mice is cleared rapidly and recovered in the liver [8
, 9
]. Most (>90%) of the organisms associated with the nonparenchymal liver cell population, including Kupffer cells isolated at 10 min postinfection, are bound extracellularly as judged by their sensitivity to antibiotic (gentamicin) treatment [8
]. Those organisms that escape clearance by the liver are found widely distributed in other tissues including spleen, bone marrow, and lungs where proliferation may occur [35
]. |
INNATE HOST DEFENSES TO LISTERIA EXPRESSED IN THE LIVER |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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Neutrophils
Neutrophils are an essential component of innate host resistance and the adaptive responses to primary and secondary listerial infections [42
43
44
]. Mice unable to mobilize neutrophils to the site of bacterial deposition in the liver and other tissues exhibit marked increases in listerial replication and death from infection. Virtually every relevant textbook currently in print characterizes phagocytosis by fixed tissue macrophages that line the liver sinusoids (i.e., Kupffer cells), as the principal mechanism by which bacterial pathogens are cleared from the bloodstream and eliminated. Recent experiments indicate, however, that immigrating neutrophils (not Kupffer cells) account for the bactericidal activity expressed in the liver early during the course of infection [8
]. Although neutrophils constitute a relatively small percentage (1–2%) of the nonparenchymal liver cells in a normal (uninfected) mouse, a dramatic 10- to 20-fold increase in neutrophils occurs within 2 h following infection. This influx correlates with a marked (0.5–1.0 log10) decrease in the listerial burden of the liver during the 4-h period that follows. Mice depleted of neutrophils by treatment with monoclonal (RB6-8C5) antigranulocyte antibody exhibit an increase in extracellular (gentamicin-sensitive) liver listeriae and hepatocyte damage monitored by aspartate aminotransferase (AST) activity in sera. Additional studies demonstrate a sharp decline in the capacity of neutrophil-depleted mice to kill other gram-positive and gram-negative organisms trapped in the liver [8
, 45
46
47
].
Kupffer cells
Although Kupffer cells are not major factors affecting phagocytosis and killing listeriae directly, adherence of microorganisms to the Kupffer cell surface plays a significant role in blood clearance. Mice rendered Kupffer cell-deficient by pretreatment with liposome-encapsulated dichloromethylene diphosphonate exhibit a marked increase in the number of organisms present in the blood and a significant (75%) decrease in the number recovered from the liver at 10 min postinfection [9
]. The specific mechanisms that underlie Kupffer cell-mediated attachment and clearance of bacteria from the bloodstream remain to be delineated. The reduced capacity of mice pretreated i.v. with sugars (e.g., methyl-
-D-mannoside) or neoglycoproteins (mannose-conjugated bovine serum albumin) to remove organisms from the blood suggests that clearance is mediated in part by the interaction of lectins expressed by Kupffer cells and carbohydrate residues expressed by the bacteria [48
, 49
]. Indeed, fixed tissue macrophages exhibit receptors specific for ligands present on the surface of microorganisms, 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
]. Not all microorganisms are efficiently cleared from the bloodstream by the liver. Pseudomonas aeruginosa and Morganella morganii inoculated i.v. into mice, for example, remained in the blood for an extended period of time and were bound less readily by purified Kupffer cells in culture [17
]. Reduced blood clearance and Kupffer cell binding correlated with decreased cell surface mannose and/or increased hydrophobicity exhibited by these microorganisms. Similarly, the rate with which Serratia marcescens was cleared from the bloodstream correlated with the presence of mannose on the microbe surface [52
].
The interaction of L. monocytogenes with Toll-like receptors (TLRs) expressed by tissue macrophages may initiate the proinflammatory response induced in the liver subsequent to infection. TLRs play a significant role in innate immunity and the regulation of specific immune responses in mice and humans. TLR4 in rodents, for example, governs the response of cells including Kupffer cells to bacterial endotoxin [lipopolysaccharides (LPS)] [53
, 54
]. A mutation in the tlr4 gene accounts for the hyporeactivity of C3H/HeJ mice to LPS and renders these mice highly susceptible to infections by gram-negative organisms [55
]. Conversely, TLR2 mediates the response to peptidoglycan and lipoproteins and promotes monocyte activation by L. monocytogenes [56
]. The interaction of bacterial products with TLRs leads to the activation of transcription factors such as nuclear factor-
B, which promotes the production of proinflammatory cytokines [e.g., interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF-)] involved in innate host defenses [57
].
Depletion experiments demonstrate that Kupffer cells are the principal source of IL-1ß, IL-6, IL-12, and TNF- produced in the livers of mice immediately following i.v. inoculation of bacteria or bacterial endotoxin [58
, 59
]. Experiments using competitive quantitative-polymerase chain reaction to determine the kinetics of cytokine mRNA expression indicate that mRNA levels peak between 30 min and 2 h postinoculation and subsided sharply thereafter. Four- to fivefold less IL-6 message, for example, is found in the livers of animals at 6 h than at 30 min postinfection with Listeria [58
]. A variety of cells types including hepatocytes, T lymphocytes, immigrating monocytes, and neutrophils may contribute to the production of IL-1ß, IL-6, IL-12, and TNF- in the liver later during the course of infections [60
61
62
].
Complementary adhesion molecules (CD11b/CD18 and CD54) facilitate neutrophil-Kupffer cell interaction
Findings to date suggest that the majority of listeriae taken up in the liver is initially bound to the surface of Kupffer cells and subsequently killed by infiltrating bactericidal neutrophils [8
, 9
]. Indeed, the bulk (>75%) of infiltrating neutrophils is found in juxtaposition with Kupffer cells in liver sections prepared 2 h after i.v. inoculation of Listeria [9
]. Complementary adhesion molecules facilitate the sequestration of neutrophils in the liver and their interaction with the Kupffer cells of mice infected systemically.
CD11b/CD18 (Mac-1; type 3 complement receptor) expressed by neutrophils but not Kupffer cells participates in a broad spectrum of activities including adherence to vascular endothelial cells, extravasation, and phagocytosis [63 ]. CD11b/CD18 molecules play an elemental role in the sequestration of neutrophils and liver injury observed in animals inoculated with endotoxin. Mice pretreated with anti-CD11b monoclonal antibody (mAb) exhibit reductions in liver neutrophils, glutathione disulfide formation (an index of oxidant stress), and serum AST activity indicative of hepatocyte damage [64 ].
CD11b/CD18 is a critical factor in host defenses to Listeria expressed in the liver [65 ]. Although mice pretreated with anti-CD11b mAb (clone 5C6; a noncytotoxic-blocking antibody) exhibit a normal capacity to clear listeriae from the bloodstream, significantly more organisms are recovered in the liver at 6 h postinfection [9 ]. Similarly, the listerial burden of the liver is elevated in mice administered mAb-specific for CD54 [intercellular adhesion molecule-1 (ICAM-1); CD11b/CD18 counter-receptor] expressed constitutively by Kupffer cells [66 ]. Thus, although neither CD11b/CD18 nor CD54 played a role in clearance of listeriae from the bloodstream, expression of these complementary adhesion molecules is crucial for the subsequent elimination of those organisms from the liver. The administration of blocking antibodies specific for several other adhesion molecules known to modulate the trafficking and/or activity of neutrophils, i.e., CD62L (L-selectin), CD11a (lymphocyte function-associated antigen-1), and CD106 (vascular cell adhesion molecule-1), had no effect on the number of listeriae recovered in the livers of mice during the 6 h following infection. These findings correlate with results demonstrating the roles of ICAM-1 and CD11b, but not P-, E-, or L-selectin in the accumulation of neutrophils in the liver during inflammation [67 ]. Indeed, the increased recovery of listeriae in the livers of mice pretreated with anti-CD11b mAb correlates inversely with a fivefold reduction in the number of neutrophils sequestered in the liver at 2 h postinfection, the normal height of neutrophil infiltration [9 ].
Complement is not a factor in early host defenses to liver Listeria
Although complement is an important factor in host resistance to Listeria [68
], it exerts negligible effects on the immigration and antimicrobial activity of neutrophils expressed in the liver early during the course of infection. Control mice and mice rendered complement-deficient by pretreatment with cobra venom factor (CVF) exhibit equivalent capacities to clear listeriae from the bloodstream (evident at 10 min postinfection) and to eliminate liver listeriae during the subsequent 6 h [9
]. Moreover, the percentages of neutrophils recovered in the nonparenchymal cell populations derived from these two groups of animals at 2 h postinfection are comparable. Later during the course of infection, however, complement plays a significant role in the elimination of liver listeriae. At 2 days, the livers of complement-depleted (CVF-pretreated) mice contain >1 log10 more bacteria than do the livers derived from control animals [9
]. This latter finding is consistent with reports demonstrating the importance of complement in host resistance to Listeria and its contribution to the inflammatory response of mononuclear phagocytes occurring later during infection [68
].
Kupffer cells phagocytose inflammatory neutrophils sequestered in the livers of Listeria-infected mice
Clearance of infiltrating neutrophils from inflamed tissues is required for the resolution of inflammation [69
, 70
]. There is little evidence to suggest that these neutrophils return to the circulation from inflammatory sites or that lymphatic channels provide a route of egression [71
]. Rather, it is generally accepted that neutrophils undergo programmed cell death (apoptosis) in situ [72
]. The onset of apoptosis by neutrophils in vitro is associated with down-regulation of key proinflammatory functions (e.g., chemotaxis, cell surface receptor expression, phagocytosis, degranulation, and respiratory burst) and cell surface changes that enable their swift recognition and ingestion by macrophages [69
, 70
, 72
73
74
75
]. Limited histologic evidence indicates that apoptosis of inflammatory neutrophils also occurs in vivo and that these intact neutrophils are subsequently removed by macrophages, preventing the tissue damage associated with the uncontrolled discharge of toxic metabolic products and proteolytic enzymes [76
, 77
]. Activated neutrophils infused experimentally into mice localize primarily in the liver suggesting that the liver is actively involved in the retention and disposal of neutrophils, thus maintaining homeostasis of the circulating population [78
]. Immunohistochemical detection of Kupffer cells positive for myeloperoxidase (an enzyme commonly associated with neutrophils) in sections of mouse and human livers supports the role of Kupffer cells in neutrophil elimination [79
]. Recently, Shi et al. [80
81
82
] showed neutrophils inside Kupffer cells in liver sections derived from rats inoculated with bacterial endotoxin or a lyophilized streptococcal preparation. Similarly, neutrophils were found in 2% of macrophages recovered from the livers of mice at 2 h postinfection with a lethal dose of listeriae [9
]. Taken together, these findings suggest that Kupffer cells play a critical role in eliminating activated neutrophils that accumulate in the liver sinusoids subsequent to clearance of bacteria, bacterial endotoxin, and microbial debris from the bloodstream.
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FACTORS EFFECTING APOPTOSIS OF NEUTROPHILS |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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ROI
Membrane-associated reduced nicotinamide adenine dinucleotide phosphate oxidase and the production of ROI (factors that enable neutrophils to kill ingested microorganisms) may play a significant role in the induction of apoptosis [83
]. This conclusion is supported by the reduced rates of spontaneous and induced apoptosis exhibited by neutrophils obtained from patients with chronic granulomatosis disease, characterized by the diminished capacity to generate ROI [84
]. Moreover, apoptosis by neutrophils derived from normal individuals correlates with a down-regulation in superoxide dismutase and glutathione, key elements in the antioxidant-defense system [83
]. A variety of factors including several gram-positive and gram-negative bacterial pathogens, e.g., E. coli, Streptococcus pneumoniae, and Mycobacterium tuberculosis, promote ROI production and/or apoptosis by neutrophils [85
86
87
].
Fas-Fas ligand (FasL) interaction
Both human and mouse neutrophils express cell surface Fas (CD95) constitutively [88
, 89
]. Ligation of Fas accelerates the oxygen-dependent apoptosis of resting neutrophils in vitro [84
]. Human peripheral blood mononuclear phagocytes activated in vitro express cell surface FasL (CD95L) and induce apoptosis by neutrophils [90
]. The majority of mouse Kupffer cells express FasL and account for 80% of the FasL+ cells in normal liver [91
]. In a rat model of endotoxemia, anti-Fas antibody treatment partially blocked the accumulation of apoptotic neutrophils in the liver demonstrating the role of Fas, but not necessarily the involvement of Kupffer cells [81
].
Cytokines
Cytokines influence many aspects of neutrophil activity including apoptosis. TNF-, for example, stimulates CD11b/CD18-dependent adherence to extracellular matrix proteins, phagocytosis, and the production of ROI in vitro [92
]. TNF- exhibits anti-inflammatory activity that is ascribed, in part, to its ability to induce cell death in neutrophils. TNF- enhances Fas- and oxygen-independent apoptosis of neutrophils induced by a variety of agents including E. coli, immobilized immunoglobulin G, and complement-coated erythrocytes in vitro [93
]. In vivo, treatments that diminish TNF- activity promote the accumulation of neutrophils in the lungs and delay the resolution of neutrophilic alveolitis in mice exposed to aerosolized P. aeruginosa or Legionella pneumophila [94
, 95
]. Membrane-bound TNF- expressed by macrophages induces apoptosis by neutrophils in a wound-healing model [96
].
In contrast to TNF-, several proinflammatory cytokines, e.g., IL-1ß and IL-6, prolong the survival of neutrophils in culture and abrogate apoptosis induced by TNF- or anti-Fas treatment [88
, 97
, 98
]. Thus, although Kupffer cells may express factors (e.g., FasL and TNF-) that promote apoptosis, they may also elaborate cytokines that sustain the viability of neutrophils that accumulate in the liver sinusoids during systemic infections.
Adhesion molecules
CD11b/CD18 engagement stimulates phagocytosis, cytokine-induced respiratory burst, ROI production, and apoptosis by neutrophils in vitro [63
, 92
, 99
]. As expected, ROI production and apoptosis induced by phagocytosis are diminished in cultures of neutrophils derived from CD11b/CD18-deficient mice or human neutrophils incubated in the presence of anti-CD11b/CD18 mAb [99
]. In contrast to these findings, however, other investigators demonstrate that neutrophils activated by cross-linking cell surface ß2 integrin molecules with anti-CD11b mAb exhibit elevated, intracellular, antioxidant (glutathione) levels and resistance to Fas-mediated apoptosis [100
].
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RECOGNITION AND INGESTION OF NEUTROPHILS SEQUESTERED IN THE LIVER |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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vß3 integrin/CD36 complex mediate the uptake of neutrophils by macrophages in vitro [101
, 102
]. Notably, it is presently unclear whether inflammatory neutrophils that accumulate in the liver sinusoids must undergo apoptosis prior to phagocytosis and removal by Kupffer cells. The elevated expression of human bcl-2 in neutrophils derived from transgenic mice inhibited apoptosis but not phagocytosis of the neutrophils by macrophages in culture [103
]. Similarly, 30% of phagocytosed neutrophils found in the liver sinusoids of rats at 6 h postinoculation with endotoxin showed no signs of DNA fragmentation [81
]. These findings suggest that senescent neutrophils may exhibit cell surface changes independent of apoptosis that enable their recognition and subsequent ingestion.
PS-specific receptor
Apoptotic cells are characterized by a loss of normal membrane asymmetry that localizes PS to the inner leaflet of the plasma membrane lipid bilayer [104
]. Externalization of PS targets apoptotic neutrophils for phagocytosis by macrophages, preventing the release of cytotoxic factors/inflammatory mediators and thus minimizing tissue damage [105
106
107
]. PS exposure alone enables recognition and ingestion of apoptotic neutrophils by activated (e.g., ß-1–3-glucan-treated), but not unstimulated macrophages in vitro [101
, 102
]. Annexin V-biotin administered in vivo binds PS and blocks Kupffer cell-mediated phagocytosis of neutrophils that accumulate in the livers of endotoxin-treated rats [81
].
Recently, Fadok et al. [108
] identified the stereospecific PS receptor, a glycosylated transmembrane protein (molecular weight, 70 K) expressed by activated mouse and human macrophages. Additionally, macrophages express a number of receptors with broad, lipid-binding activity that are capable of binding PS, e.g., CD36 [109
], CD68 [110
], CD14 [111
], LOX-1 [112
], scavenger receptor-B1 (SR-B1) [113
], and SR-PSOX [114
]. Although macrophages exhibiting these other receptors may bind apoptotic cells, ingestion occurs only in the presence of PS-specific receptor expression [115
]. This latter finding supports the "tether and tickle" model proposed by Hoffmann and co-workers [115
, 116
]. That is, cell surface molecules such as CD14, CD36, and CD68 bind (tether) the dying cells to macrophages; engagement (tickle) of the PS-specific receptor induces macropinocytosis.
vß3 Integrin/CD36 complex
In contrast to activated macrophages, unstimulated (e.g., bone marrow-derived) macrophages internalize apoptotic neutrophils in culture via, primarily, a complex composed of vß3 integrin (vitronectin receptor) and CD36 primarily [117
]. The vß3 integrin/CD36 complex recognizes thrombospondin bound to the surface of apoptotic neutrophils by an unidentified, thrombospondin-binding ligand; thrombospondin therefore serves as a bridge mediating recognition and uptake [117
]. Increasing evidence indicates that the first component of complement (C1q) can also couple apoptotic cells to phagocytes in a manner analogous to thrombospondin [118
]. Macrophages derived from C1q-deficient mice exhibit a reduced capacity to phagocytose apoptotic T cells although phagocytosis of apoptotic neutrophils was not tested [118
]. CD31 (PECAM-1) expressed by neutrophils is an additional ligand recognized by the vitronectin receptor [119
]. Anti-CD36 mAb blocks ingestion of apoptotic neutrophils by unstimulated as well as stimulated macrophages, implying that CD36 is involved in vß3 integrin- and PS-specific receptor-mediated neutrophil uptake [102
]. This latter finding supports the suggestion that vß3 integrin, PS-specific receptor, and CD36 are all components of a single, large receptor complex [102
].
Kupffer cell receptor
In vitro, amino sugars and amino acids inhibit vß3 integrin/CD36 complex-mediated uptake of apoptotic neutrophils by macrophages in a charge-dependent manner by masking anionic groups expressed on the neutrophil surface [73
]. The elevated levels of amino sugars, basic amino acids, and hydrogen ions common at inflammatory sites suggest that the vß3 integrin/CD36 complex may not be a critical factor mediating uptake of apoptotic neutrophils by macrophages in vivo. Rather, in the case of Kupffer cells, carbohydrate-specific receptors may facilitate the ingestion of apoptotic cells [120
]. Kupffer cell receptors (unique to Kupffer cells as the name implies) bind galactose-, N-acetyl-galactose-, and fucose-terminating oligosaccharides in a Ca++-dependent manner [120
]. These receptors exhibit significantly greater affinity for particles than for soluble factors suggesting that their physiologic function may be to clear microorganisms, as discussed above, and/or apoptotic cells from the bloodstream [121
].
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INGESTED NEUTROPHILS MODULATE MACROPHAGE FUNCTION |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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, MIP-1ß, and MIP-2], and soluble factors (e.g., leukotriene B4) [27
, 60
, 122
123
124
]. Recent studies suggest that apoptosis and ingestion of these neutrophils by Kupffer cells abrogate hepatic injury that may otherwise result from the uncontrolled release of such factors [8
, 80
81
82
]. Furthermore, the ingestion of apoptotic neutrophils may enhance or abrogate the inflammatory response of macrophages depending on the identity of the receptors mediating uptake. Phagocytosis of apoptotic neutrophils via the vß3 integrin/CD36 complex suppresses the production of proinflammatory cytokines [i.e., IL-1ß, IL-8, IL-10, granulocyte macrophage-colony stimulating factor (GM-CSF), and TNF-] and lipid mediators [i.e., thromboxane B2 (TxB2) and leukotriene C4 (LTC4)] by unstimulated as well as endotoxin-stimulated macrophages [125
]. Uptake of neutrophils mediated by Fc receptors, on the other hand, induces IL-1ß, IL-8, IL-10, GM-CSF, and TNF- production by macrophages [125
]. Treatment with anti-transforming growth factor (TGF-ß1) antibody restores the production of proinflammatory cytokines by macrophages that ingest apoptotic neutrophils via the vß3 integrin/CD36 complex demonstrating the role of TGF-ß1 in down-regulating the proinflammatory response. Indomethacin, a prostaglandin E2 (PGE2) inhibitor, blocks TGF-ß1 synthesis and promotes the production of IL-1ß and TNF- indicating the important intermediary function of PGE2. Besides suppressing proinflammatory cytokine synthesis, TGF-ß1 stimulates thrombospondin-dependent recognition of apoptotic neutrophils [126
] and inhibits nitric oxide (NO•) production by macrophages [127
].
Depletion experiments indicate that Kupffer cells constitute the principal site of inducible NO synthase (iNOS) expression and NO• production in the livers of mice inoculated i.v. with bacterial endotoxin [128
]. NO• is an important mediator implicated in a wide variety of physiologic events including (but not restricted to) vasodilatation, neutrophil chemotaxis, and the adhesion of neutrophils to the vascular endothelium [129
130
131
]. The elevated expression of iNOS and overproduction of NO•, however, can suppress host resistance and the antigen-specific response of T cells to some organisms including Listeria [132
]. iNOS expression by macrophages is regulated by cytokines; TNF- promotes, for example, and TGF-ß1 inhibits NO• synthesis [127
, 133
]. Although the ingestion of apoptotic neutrophils may suppress NO• synthesis, a recent study found alternatively that endotoxin-activated neutrophils can induce NO• production by Kupffer cells [134
]. Taken together, these findings suggest that neutrophils infiltrating the liver subsequent to infection exert a profound effect on host defenses by modulating cytokine production and the synthesis of NO• by Kupffer cells. Indeed, the consequences of ingesting apoptotic neutrophils could account for the sharp decline in cytokine message levels noted above following peak expression and occurring shortly after i.v. inoculation of bacteria or bacterial endotoxin [58
, 59
].
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PARADIGM: NEUTROPHIL-KUPFFER CELL INTERACTION DICTATES INNATE HOST DEFENSES TO SYSTEMIC BACTERIAL INFECTIONS |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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PROSPECTS AND CONCLUDING REMARKS |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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Results demonstrating the ingestion of neutrophils by Kupffer cells may also have profound implications with regard to the development and expression of adaptive immunity in the liver. Many observations, e.g., the prolonged survival of allogeneic transplants, indicate that the liver is predisposed toward immune tolerance [135 ]. It has been suggested, in fact, that the liver is actively tolerogenic and plays a key role in preventing the destructive consequences of generalized inflammation by eliminating circulating effector T cells specific for antigens disseminated systemically [135 , 136 ]. This suggestion is supported by experiments demonstrating the accumulation of apoptotic CD8+ T cells in the liver during the clearance phase of peripheral immune responses [135 , 136 ]. Kupffer cells have been implicated in the selective retention and apoptosis of CD8+ T cells; glycolipid-binding receptors (e.g., PS-specific receptor), integrin receptors (i.e., ICAM-1), and FasL expressed by Kupffer cells may play roles [91 , 136 137 138 ].
Recent studies suggest that antigens derived from bacteria and bacterial products taken up by the liver and subsequently degraded (processed) by neutrophils may be "cross-presented" by Kupffer cells. Macrophages in vitro cross-present epitopes derived from ingested apoptotic cells via the vacuolar alternate MHC class I pathway [139 ]. Cultured macrophages can also acquire epitopes from viable neutrophils following their release or regurgitation [140 ]. In the absence of appropriate costimulatory molecules, cross-presentation of antigens by macrophages is more likely to promote tolerance than induce adaptive immunity in naïve T cells [139 , 141 , 142 ]. As such, epitopes derived from neutrophils and cross-presented by Kupffer cells may be a key element in the deletion of specific CD4+ and CD8+ T cell populations, predisposing the liver toward tolerance. Thus, in addition to down-regulating the proinflammatory response of Kupffer cells, ingested apoptotic neutrophils may exert a significant influence on the development and expression of antigen-specific CD4+ and/or CD8+ T cell-mediated immunity in the liver, where sensitized T cells are activated and naïve T cells are tolerized.
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ACKNOWLEDGEMENTS |
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Received January 25, 2002; revised April 1, 2002; accepted April 3, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONKUPFFER CELLSNEUTROPHILSLISTERIOSIS MODEL OF SYSTEMIC...INNATE HOST DEFENSES TO...FACTORS EFFECTING APOPTOSIS OF...RECOGNITION AND INGESTION OF...INGESTED NEUTROPHILS MODULATE...PARADIGM: NEUTROPHIL-KUPFFER...PROSPECTS AND CONCLUDING REMARKSREFERENCES |
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