Phagocytosis of Apoptotic Cells by Liver

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Phagocytosis of Apoptotic Cells by Liver: A MorphologicalStudyLUCIANA DINI,* PATRIZIA PAGLIARA, AND EMANUELA C. CARLA

Department of Biological and Environmental Science and Technologies, University of Lecce, Lecce, Italy

KEY WORDS apoptosis; phagocytosis of apoptotic cells; hepatocytes; Kupffer cells; endothelialcells; lymphocytes

ABSTRACT The present review deals with the morphological features of the removal of apoptoticcells by liver. The engulfment of cells undergoing apoptosis can be considered a specialized form ofphagocytosis, playing a major role in the general tissue homeostasis in physiological and pathologicalconditions. In fact, defects of phagocytosis of apoptotic cells might have deleterious consequences forneighboring healthy cells, i.e., pathogenesis of inflammatory disease or dysregulation of the immunesystem. Phagocytosis of apoptotic cells by liver is a complex phenomenon, involving multiple molecularmechanisms of recognition (i.e., lectin-like receptors and receptors for externalized phosphatydilserine)

of both parenchymal (hepatocytes) and nonparenchymal (Kupffer and endothelial cells) liver cells, oftenoperating in cooperation. The data discussed in the present review are drawn from studies of phago-cytosis of apoptotic cells in the liver, carried out with in vivo and in situ adhesion experiments as wellas in vitro assays. Our results indicate that the three main liver cell types (hepatocytes, Kupffer, andendothelial cells) are able to recognize and internalize apoptotic cells by means of specific receptors(galactose and mannose-specific receptor; receptor for phosphatydilserine) and by cytoskeletal reorga-nization that favors the engulfment of the apoptotic cells. The flags for the identification of apoptoticcells by the liver are modifications of the surface of dead cells, i.e., sugar residues and phosphatydil-serine exposition. Vitronectin receptor is not involved in such a recognition. The adhesions betweenmodified cell surfaces of apoptotic cells and phagocytes generate cytoplasmatic signaling pathways thatdrive apoptotic cells to their final fate within the phagocytes (i.e., lysosomal digestion). Microsc. Res.Tech. 57:530540, 2002. 2002 Wiley-Liss, Inc.

LIVER CELLS PHAGOCYTOSIS OF

APOPTOTIC CELLS Although apoptosis occurs at a negligible rate in

normal liver, a variety of physiological conditions, dis-eases, and xenobiotic treatments can cause this form ofcell death (Columbano et al., 1985; Bursch et al., 1986,1992; Tessitore et al., 1989; Grasl Kraupp et al., 1994;Ledda-Columbano et al., 1996). In liver, as in otherorgans, the execution of apoptosis may be initiated bymany different signals, either from within or outsidethe cell, involving ligand-receptor or TGF-beta/TGF-receptor, or potentially by more unspecific signals suchas ceramide or DNA damage. During the modulation/induction phase of liver apoptosis many differentgenes, such as p53, c-myc, or Bcl-2/Bax have been

shown to be able to shift the balance either to cellsurvival or cell death (Kanzel and Galle, 2000; Valenteand Calabrese, 1999). Therefore, liver apoptotic pro-cess can be divided into four phases: an inductionphase, the nature of which depends on the specificdeath-inducing signals; an effector phase, duringwhich the central executioner is activated and the cellbecomes committed to die; a degradation phase, duringwhich the cell acquires the biochemical and morpho-logical features of endstage apoptosis. In this cascadeof events, the point of no return would be the step atwhich the cell becomes irreversibly committed to theloss of essential cellular functions. The fourth, and lastphase, is the engulfment of the dead corps by macro-

phages and other occasional phagocytes. The fact

that free or nonphagocytosed dying cells are rarelyobserved or identified as a frequent physiological eventin the liver is the result of the swift in vivo removal intoadjacent phagocytic cells (Fig. 1).

The rapid ingestion of apoptotic cells is beautifullyperformed by liver sinusoidal cells (Dini, 2000), thuspreventing secondary necrosis and subsequent leakageof potentially harmful materials; this, in turn, limitsthe potential for inflammatory reactions and autoim-mune responses. In fact, in spite of the nature of thereceptor involved, the molecular mechanism by whichapoptotic cells are removed is important in impedingthe subsequent proinflammatory response (Meagher etal., 1992; Savill et al., 1997; Fadok et al., 1998b; Savilland Fadok, 2000). Specific receptors mediate the par-ticular phagocytic activities of the sinusoidal cells.

Among the several alternative mechanisms reportedfor removal of apoptotic cells, which are mainly relatedto the cell type and system used (i.e., lectins, throm-bospondin (TPS), CD14, scavenger receptors, v3,CD36, ABC1 (ATP binding Cassette transporter),

*Correspondence to: Prof.ssa Luciana Dini, Department of Biology, Universityof Lecce, strada prov.le per Monteroni 73100 Lecce Italy. [email protected]

Received 20 March 2001; accepted 13 July 2001

DOI 10.1002/jemt.10107

Published online in Wiley InterScience (www.interscience.wiley.com).

MICROSCOPY RESEARCH AND TECHNIQUE 57:530540 (2002)

2002 WILEY-LISS, INC.


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Ced-6, Ced-7, Ced-5, Ced2, Ced10, DOCK180), in theliver the recognition and phagocytosis of apoptotic cellsare mostly performed by means of hepatic lectin-likereceptors (Morris et al., 1984; Savill et al., 1990, 1992;Dini et al., 1992; Flora and Gregory, 1994; Ren et al.,1995; Luciani and Chimini, 1996; Devitt et al., 1998;Fadok et al., 1998a; Liu and Hengartner, 1998; Savill,1998; Wu and Horvitz, 1998; Schlegel et al., 1999; Dini,2000; Savill and Fadok, 2000).

The first demonstration that liver carbohydratereceptors are involved in the phagocytosis of apopto-tic liver cells by healthy ones was performed on new-born hepatocyte cultures induced to undergo apopto-

sis by hormonal treatments (Dini et al., 1992) andfurther confirmed on normal adult cells (Fig. 2). In-hibition experiments, in which competing saccha-rides or antibodies to the asialoglycoprotein recep-tors were added to the culture medium, demon-strated that the hepatocyte recognition andinternalization of apoptotic cells is due to the expo-sition of several glycans (in particular, galactose/N-acetyl-galactosamine) on the surface of apoptoticcells, rendering them available for interaction withlectin-like receptors on hepatocytes (Dini et al.,1992). The vitronectin receptor was not involved inthis recognition, since the tetrapeptides Arg-Gly-

Fig. 1. Light (a d) and elec-tron (e,f) micrographs of rat liver5 days after a single injection oflead nitrate (10 mmoles/100 gb.w.). The intoxication with theheavy metal generates apoptoticcells that are visible as single scat-tered apoptotic hepatocytes (a,d,e)inside parenchymal liver cells (ar-rows) and (b) inside the sinusoids(arrows). e: A dying cell embeddedin the organ that has lost contactwith its neighbors, its microvilli,and any other rufflings of the cellsurface (arrows). Cellular organ-elles are well preserved, as is thenuclear envelope. The chromatin iscondensed along the nuclear enve-

lope. The entire dying cell, at thelater stages of apoptosis, collapses(i.e., decreased volume) and isquickly engulfed by macrophagesor by living hepatocytes. c,f: Apo-ptotic cells phagocytosed byKupffer cells (arrows). The apopto-tic cell is entirely surrounded bytiny protrusions of the liver macro-phages, occupying most of the si-nusoid (f). Highly condensed chro-matin is observed with light aswell as electron microscopy. Mag-nifications: (a) 1,000; (b,c)1,200; (d) 800; (e) 13,000; (f)5,500.

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Fig. 2. Normansky light micrographs of cultured isolated hepato-cytes treated for 48 hours with 30 g/ml of lead nitrate. The incuba-tion with the heavy metal generates apoptosis in the hepatocytescultures. In both a and b, micrographs at different stages of theapoptotic process can be recognized: (a) cells are condensing cyto-plasm and losing their connections with the Petri dish (arrows).

Round hepatocytes showing condensed nuclei and detaching from theculture dish are observed (b, arrows); (b, double arrow) a floatingapoptotic body recognized by a hepatocyte is visible. The final stage ofapoptotic process is the engulfment of apoptotic hepatocyte by theliving ones (b arrowheads). Magnifications: (a,b) 1,200.

Fig. 3. In situ adhesion experi-ments of lymphocytes to sinusoidalliver cells. a: An apoptotic lympho-cyte showing an extensive bleb (as-terisks) is establishing contactwith an endothelial cell (arrow).Note the absolute lack of cellularorganelles inside the bleb. c: Anendothelial cell is surrounding anapoptotic lymphocyte (asterisks),which occupies the entire sinusoi-dal lumen. b,d: Micrographs show-

ing phagosomes containing apo-ptotic lymphocytes (asterisks) atdifferent degrees of digestion. Inb, bodies derived from fragmenta-tion of apoptotic cells are shown(arrows). In d, a large phagosomeis shown in which chromatin isstill recognizable, but not anyother organelles. Magnifications:(a) 4,500; (b) 9,500; (c) 8,000;(d) 10,500.

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Asp-Ser (RGDS) and Arg-Gly-Glu-Ser (RGES) fail toexert any inhibitory action (Dini et al., 1992).

Sinusoidal liver cells are able to clear from the bloodgalactose and mannose-terminated particles (eventhose of large size) and a wide range of molecules with

a net negative charge by means of scavenger receptorsand receptors specific for galactose and mannose resi-dues (Steer and Clarenburg, 1979; Kolb-Bachofen etal., 1982; Steffan et al., 1986; Praaning-van Dalen etal., 1987; Van Berker et al., 1992). Due to their locationin the sinusoids and combined with the fact that theyrepresent the majority of the bodys fixed macrophages,Kupffer cells are the first cells of the mononuclearphagocyte system to come into contact with particulateand immunoreactive materials coming from the blood.Therefore, together these properties enable them toexert an efficient and fast recognition and engulfmentof apoptosing cells. In fact, they are primary actors inclearing potentially noxious materials like apoptoticcells.

MORPHOLOGICAL ASPECTS OF APOTOTICCELL PHAGOCYTOSIS

Here the morphological aspects of recognition andengulfment of apoptotic cells by hepatocytes, endothe-lial, and Kupffer cells are highlighted. The data re-ported below have been obtained from observationswith light and electron microscopy of in vivo, in situ,and in vitro experiments. Briefly, the experiments werecarried out as follows.

In Vivo Induction of Apoptosis

Inbred 5-week-old male Swiss mice (20 30 g) or maleWistar rats (150 200 g) purchased from Morini (ReggioEmilia, Italy) were used. All the animals, maintainedon a 12-hour daynight rhythm, with free access towater and food (standard diet), received humane careand the study protocols complied with national laws.Before surgery, animals were anesthetized by an i.p.injection with Farmotal (Farmitalia, Italy) 10 mg/100 g body weight. Lead nitrate (Carlo Erba, Milano,

Italy), dissolved in distilled water, was injected i.v. at adose of 10 mmoles/100g body weight. The animals werekilled at various time intervals after treatment. Con-trol rats received an equivalent amount of NaCl 0.9%.

In Situ Adhesion Experiments

Livers were perfused in a nonrecirculating system ata flow rate of 1 ml/min. Apoptotic lymphocytes 1 106

labeled with Hoechst 33342 were injected through theportal vein into liver circulation. Peripheral bloodmononuclear cells were obtained after Ficoll gradientseparation of buffy coats from blood donations of non-smoking healthy males, age 25 45 years. Lympho-cytes, separated from monocytes by double adherenceto plastic and maintained at a cell density of 1 106

cells/ml in complete culture medium, were used on thefirst day of explant. Apoptosis, induced with cycloex-himide (CHX) 10-2 M for 18 hours, followed by 1-hourrecovery in fresh medium, 10 g/ml puromycin (PMC),or by keeping lymphocytes in water baths equilibratedto 43C for 1 hour followed by 8 hours of recovery at37C was evaluated by flow cytometry, by light micros-copy on cytospin, and by electron microscopy. Modifi-cations of the cell surface were evaluated by using abroad panel offluorescent lectin conjugates with differ-ent specificities: Concanavalin-A (Con-A) and succinylConcanavalin-A (sCon-A) (a-D-mannosyl); Phaseouluslimensis (PHAE) (N-acetyl-D-galactosamine); Ricinuscommunis (RCA) (D-Galactosyl); Ulex europaeus (UEA)

Fig. 4. Phagosomes containing apoptotic cells can present differ-ent morphologies that are related both to digestion as well as to thetype of particle ingested (i.e., apoptotic cell, apoptotic body with orwithout condensed chromatin). a: Two phagosomes are inside anendothelial cell: one still shows chromatin (arrows), while the other(asterisks) is at the very late stage of digestion. b: A large phagosomeinside a Kupffer cell containing remnants of one unfragmented apo-ptotic cell. Condensed chromatin is still present associated with anucleolus (arrows); cytoplasmatic organelles are not more morpholog-ically recognizable. Magnifications: (a) 12,500; (b) 21,000.



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(a-L-Fucosyl); Triticum vulgare (WGA) (N-acetyl-D-glucosamine); Dolichos biflorus (DBA) (N-acetyl-D-galactosamine); Pisum sativum (PSA) (fucosylresidues); Arachis hypogaea (PNA) (N-acetyl-D-galac-tosamine); Limulus polyphemus (LPA) (N-acetyl-D-

galactosamine, N-acetyl-D-glucosamine, N-acetyl-neuramicic acid).

Samples were processed for light and electron mi-croscopy to assess the adhesion of lymphocytes to thesinusoidal wall. Adhesion specificity was tested in par-allel inhibition experiments by adding 80 mM (finalconcentration) of N-acetyl-D glucosamine (GlcNAc) andN-acetyl-D-galactosamine (GalNAc) into the perfusiontube before lymphocyte administration.

In Vitro Adhesion Experiments

Mouse liver sinusoidal cells (endothelial and Kupffercells) were isolated by enzymatic (0.05% D-Collagenaseand 0.1% Pronase), perfusion of livers and separatedfrom parenchymal and blood cells through centrifuga-tion in a 30% metrizamide gradient. Cells were platedonto type I collagen-coated 24-well plates at a concen-tration of 1 106 cells/ml/well in DMEM medium.Normal or apoptotic 5 105 lymphocytes labeled withHoechst 33342 were added to 24-well-plate culturedsinusoidal liver cells and incubated for 20, 60, and120 minutes. Sinusoidal liver cells were incubated inthe presence of 1 ng/ml lypopolysaccharide (LPS) for6 hours or 300 ng/ml recombinant human interleukin1 (rhIL-1) for 4 hours before addition of lymphocytes.Inhibition experiments were performed, incubating thecells with a solution of galactose, N-acetyl-galac-tosamine, and mannose (Gal/GlcNAc/Man) (final con-centration of sugar cocktail 80 mM) for 20 minutes at37C before incubation with apoptotic lymphocytes.The number of adhering cells was determined by afluorescence measurement system.

In Vivo Studies

In spite of the complexity of the phenomenon, theinvestigation of liver apoptosis in vivo or as a whole in

situ led to study of different aspects of the process atthe same time. In particular, taking into account theadvantage of the simultaneous presence of dying andhealthy cells in the same sample, the distribution, themorphology of dying liver cells, and the recognitionactivities of the healthy ones were investigated.

By using an in vivo model of liver apoptosis (Colum-bano et al., 1985), the rapid removal from the tissue ofapoptotic liver cells was clearly shown. Dead cells werealso frequently phagocytosed by nonprofessionalphagocytes. In fact, dying hepatocytes, hampered inreaching the circulation, were engulfed by the neigh-boring ones (Fig. 1). These data are in line with othersin the literature, describing (even in invertebrates, i.e.,Caenorhabditis elegans) examples of nonprofessional

phagocytes phagocyting dying cells (Ellis et al., 1991).

Fig. 5. In situ adhesion experiments of apoptotic lymphocytes toendothelial (a,c) and Kupffer cells (b,c). The first step for phagocyto-sis of apoptotic cells is their recognition and blockade by the sinusoi-dal wall. Circulating apoptotic lymphocytes injected through the he-patic circulation are retained by both endothelial (a,c, arrows) as wellas Kupffer cells (b,c, arrowheads); however, the percentage of Kupffercells with phagosomes containing apoptotic cells and/or remnants ofapoptotic cells is always higher than endothelial cells, thus confirmingthe well-known phagocytic activity of liver macrophages. In c a largeKupffer cell surrounding an apoptotic cell with a recognizable frag-mented nucleus is shown (arrowhead). Magnifications: 1,500.

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Inflammatory injury in the liver parenchyma wasnever observed. Hepatocytes surrounding the apoptoticcells showed normal cytoplasm without any signs oforganelle swelling and/or degradation (Fig. 1). There-fore, the protective role of the phagocyte recognition ofapoptotic self by preventing the leakage of noxioussubstances and by limiting the development of autoim-mune responses was successfully confirmed in theliver.

The increased number of apoptotic cells, produced bylead nitrate treatment and mainly poured into theblood, induces sinusoidal liver cells (i.e., Kupffer andendothelial cells) to actively phagocyte both apoptotichepatocytes and circulating apoptotic cells, and alsothose derived from other body areas (Figs. 3, 4). Thepeak of phagocytic activity of Kupffer cells (3-fold thecontrol) was measured at 5 and 15 days from leadnitrate injection (Ruzittu et al., 1999; Dini and Carla,1998; Dini, 2000), thus confirming the capacity (in par-ticular for the interaction with particulate materials) ofthe hepatic sinusoidal wall to operate as a protectivebarrier for the systemic circulation (Steffan et al., 1986;Wardle, 1987; Dini and Kolb-Bachofen, 1989; Kolb-Bachofen, 1992; Toth and Thomas, 1992).

In Situ Adhesion Experiments

To further explore the liver phagocytosis of apoptoticcells, in situ adhesion experiments were carried out.With this type of experiment the overall recognitioncapacity of the sinusoidal wall can be assayed. In theexperimental protocol, liver blood is replaced by culturemedium containing apoptotic cells. In particular, apo-ptotic lymphocytes were used, due to the fact that theliver is the specialized site where T cells undergoingapoptosis in vivo are eliminated. However, the molec-ular mechanism(s) that control this accumulation isstill unknown (Huang et al., 1994). Once injected intothe mouse hepatic circulation, apoptotic lymphocytes,but not normal ones, are efficiently removed by sinu-soidal cells by means of carbohydrate receptors, asconfirmed by inhibition studies (Ruzzittu et al., 1999;Dini, 2000). The amount of retained lymphocytes isstrictly dependent on the number of exposed bindingsites on the cell surface of sinusoidal liver cells. Inagreement with this are data showing that apoptoticlymphocytes retained by sinusoidal cells of the peripor-tal tract are double those retained in the perivenousregion. In fact, the number of carbohydrate receptorsexpressed on cell surface of endothelial cells from the

Fig. 6. Lymphocytes stainedwith Con-A-FITC for sugar residuesexposure. a: Control (untreated)cells were unstained. b, c,d: cellstreated with PMC 10 g/ml for 2, 4,and 6 hours respectively displayedbrightly marked membranes thatincrease with time of treatment.

Magnifications: 1,200.



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periportal tract is double those quantified on endothe-lial cells of the perivenous tract (Dini and Carla, 1998).

The immediate fate of apoptotic lymphocytes afterinjection into liver circulation, independent of the mo-dality of induction of apoptosis (i.e., by oxidative, hy-perthermic stress, or drugs) is their absorption ontoendothelial and Kupffer cells (Fig. 5), thus indicatingthat liver recognition of apoptotic cells is a fundamen-tal step for their subsequent sequestration, internal-ization, and digestion. Even if the ability to block apo-ptotic cells is similar for both endothelial and Kupffercells, these latter cells show a higher rate of apoptoticcell engulfment, Kupffer cells being more active andfaster than endothelial cells. The very rapid ingestionof apoptotic cells, which occurs immediately after theirbinding to Kupffer cell surfaces, was repeatedly ob-served in in situ adhesion experiments, as well as in in

vitro adhesion experiments.The recognition process of apoptotic cells, as exten-

sively reported in literature, is triggered by modifica-tions of the surface of the dead cells (Platt et al., 1998;Ren and Savill, 1998). Modifications of the glycidicresidues of glycoproteins of the plasma membrane ofapoptotic lymphocyte cell surfaces are major candi-dates as the eat me signal. Substantial changes ofexposed sugar residues were observed on apoptoticlymphocyte surfaces when compared to normal cells(Falasca et al., 1996; Dini, 2000). It is still unknownhow these cell surface carbohydrate modificationscould occur, but they appear to be a common mecha-nism for recognition of unwanted cells by the liver(i.e., the removal of aged erythrocytes by the liver; Kolbet al., 1981). In addition, progressive glycan modifica-tions (Fig. 6) are achieved in parallel with the morpho-logical modifications that characterize apoptotic cells.Therefore, taking into account the progressive surface

modifications, the execution process of apoptosis is di-vided into three stages, each characterized by qualita-tive and quantitative modifications of the cell surface:early, mature, and late/necrotic. Even if apoptosis isnot a synchronized phenomenon, it is nevertheless pos-sible to obtain a cell population enriched for each stageof apoptosis by using time-course experiments. En-riched cell suspension of early, mature, or late/necroticapoptotic cells were used in in vitro and in situ adhe-sion experiments. The mature apoptotic cell popula-tion was quickly recognized by sinusoidal cells whencompared to the recognition time of early or late/necrotic apoptotic cells (manuscript in preparation).

In Vitro Phagocytosis Experiments

Liver cells (i.e., hepatocytes, Kupffer cells, endothe-lial cells, as well as pit and fat storing cells) can bedissociated, purified, and maintained in suspension orin adhesion cultures for some time (depending on thecell type). Therefore, isolated liver cells, and sinusoidalcells in particular, are useful tools for studies of phago-cytosis of apoptotic cells.

Hepatocytes maintained in adhesion cultures for ashort time do not show significant rates of proliferationand apoptosis unless they are treated with specificproliferative or apoptotic inducers, i.e., retinoic acidand estrogens. Considering that under the above con-ditions the apoptotic rate is about 30%, it is possible, bymeans of microscopy analysis, to discriminate in the

Fig. 7. Transmission electron micrographs of the interaction be-tween apoptotic lymphocytes and cultured human Kupffer cells atdifferent stage of interaction. Apoptotic lymphocytes when incubatedwith Kupffer cells at 37C are promptly bound (a) and then phagocy-tosed (b). An apoptotic lymphocyte (asterisk) whose chromatin beginsto aggregate into dense masses adhering closely to the plasma mem-brane of a human Kupffer cell (Kc) at 5 min of incubation. Within5 minutes of co-culture almost all the apoptotic lymphocytes arebound to the plasma membrane of Kupffer cells, while after10 minutes of incubation the majority of apoptotic cells are internal-ized by the Kupffer cells, thus suggesting a very rapid mechanism ofrecognition (b). Phagosomes, containing dark material, which repre-sent residual of the partially digested apoptotic lymphocytes, are

visible inside Kupffer cells (b, arrow). Magnifications: (a) 7,500; (b)4,500.

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same culture dish apoptotic and healthy hepatocytesand to verify the phagocytic ability of the latter forapoptotic cells. The micrographs in Figure 2 show he-patocyte cultures treated with hormones. Many scat-tered apoptotic cells are observed: some of them aredetaching from the culture dish, others are undergoingrecognition before engulfment, and others have been

internalized by the healthy hepatocytes and are visibleas membrane-enclosed phagosomes (Fig. 2). All thesedata support the idea that hepatocytes are able tointernalize apoptotic cells when necessary.

In vitro adhesion and uptake experiments were per-formed by using cultures of isolated and purified endo-thelial and Kupffer cells. Sinusoidal liver cells wereincubated with apoptotic lymphocytes at differenttimes (Figs. 79). As mentioned above, lymphocyteswere chosen because in vivo they are a physiologicalsource of apoptotic cells/bodies recognized and phago-cytosed by liver cells. In fact, in vivo apoptotic lympho-cytes are recognized and phagocytosed well before thefinal stages of DNA degradation and cell lysis (Pradhan

et al., 1994; Huang et al., 1994). Kupffer and endothe-lial cells in culture phagocyte in a very efficient mannerlymphocytes undergoing apoptosis induced by differentstimuli (heat-shock 43C; cycloheximide), but not nor-mal living ones (Dini and Carla, 1998; Dini, 2000)(Figs. 79). Since endocytosis is a multistep processthat includes cellular movements, in particular the

extension of pseudopodia, cytoskeletal integrity mustbe important. In fact, a relationship between Kupffercell shape and phagocytic activity has been recentlyreported (Dini et al., 1998). To accomplish phagocytosisof apoptotic cells, the recognition process must be fol-lowed by internalization. This latter phenomenon re-quires cytoplasmic movements that generate fine fila-mentous processes immediately adjacent to the apopto-tic lymphocyte (Fig. 8). In the meantime, theinternalization of apoptotic cells requires the recruit-ment of cell-surface receptors on the extending pseu-dopodia into positions in which they can interact withthe appropriate ligands. In fact, we repeatedly foundthat phagocytosis is inhibited by the presence in the

Fig. 8. Scanning electron mi-crographs of cocultures of apo-ptotic lymphocytes and humanKupffer cells. a: Human Kupffercells are characterized by promi-nent membrane ruffling with mi-crovilli of variable length accompa-nied by numerous pseudopodiawhen cultured in normal condi-tions. Conversely, apoptotic cellsare recognized by their round andsmooth surface that is a conse-quence of the disappearance of mi-crovilli during the apoptotic pro-cess. a: Apoptotic lymphocytesadded to the culture medium ad-here to the surface of the Kupffercells (arrow). b: A Kupffer cell atthe beginning of the phagocyticprocess: the macrophage is tether-ing the dead corpse (arrows). c: Afew minutes, later Kupffer cells,which are very active in phagocy-tosis, have completely internalizedthe apoptotic lymphocytes. After15 minutes of co-culture roundprotrusions (representing the in-ternalized apoptotic lymphocytes)are often visible inside the cells(arrow). When Kupffer cells wereincubated with the carbohydrate-specific receptor inhibitors (i.e.,sugars or modified glycoproteins),before and during the incubationwith apoptotic lymphocytes, theirphagocytic activity is dramatically

reduced. The addition of healthylymphocytes to the Kupffer cellcultures does not result in the rec-ognition and internalization of theblood cells. Magnifications: (a)5,500; (b) 11,000; (c) 10,000.



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culture medium of inhibitors of galactose- and man-nose-specific receptors (i.e., sugar residues both as sin-gle moieties or as cocktail and desialylated glycopro-teins, but not by unmodified ones) and to a lower extentby desialylated glycoproteins, but not by unmodifiedglycoproteins (Dini, 2000).

A difference in phagocytic activity is easily observedbetween isolated endothelial and Kupffer cells, the lat-ter being much more active than endothelial cells. Therecognition of the apoptotic lymphocytes once added tohuman Kupffer cell cultures is almost entirely com-pleted within a few minutes of incubation and theapoptotic cells are detected as dark material insidelarge phagosomes (Fig. 7). On the other hand, endothe-lial cells need more time to complete engulfment ofapoptotic lymphocytes. One explanation, of course, is

related to the different functions in the liver of endo-thelial and Kupffer cells, that being that macrophagesare characterized by high phagocytic activity. How-ever, it is worth noting that apoptotic recognition maybe regulated by the state of the phagocyte and byexternal influences (Savill et al., 1993). The exposure ofphagocytes to cytokines known to be present at inflam-mation sites (i.e., granulocyte-macrophage colony stim-ulating factor; GM-CSF) or implicated in the repair ofinjured tissue (i.e., transforming growth factor, TGFplatelet-derived growth factor, PDGF) and those in-

volved in the initiation of inflammation (i.e., interferongamma, IFN interleukin-1, IL-1, and tumor necro-sis factor : TNF) increased the recognition of apopto-tic human neutrophils (Savill et al., 1993). LPS and

IL1 upregulate the mannose receptor expression ofliver cells and consequently the phagocytic activity ofsinusoidal cells (Dini et al., 1995).

CONCLUDING REMARKS

This brief discussion of the recognition and ingestionof apoptotic cells by hepatocytes, Kupffer, and endothe-lial cells shows clearly that liver cells are active par-ticipants in the removal of apoptotic cells and that thisremoval is swift and efficient despite its complexity. Toachieve recognition of apoptotic cells, signals in theform of molecular modifications of the plasma mem-brane must occur on the dying cell surface (i.e., modi-fication of the membrane lipid asymmetry, external

exposition of phosphatidylserine, and normally hiddensugar residues). The morphological study of apoptoticcells is not sufficient to detect the very early stages ofthe process, those characterized by plasma membranemodifications without visible nuclear modifications.Conversely, it is very useful for the detection of apopto-tic cells containing phagosomes, especially when dyingcells have been labeled with fluorescent dyes or elec-tron-dense markers. On the other hand, due to thecharacteristic chromatin condensation and roundshape of the apoptotic cells, the phagocytosis of themature/late stages of apoptotic cells is easily studiedwith both light and electron microscopy.

The adhesion to the apoptotic cell is the first stepthat allows engulfment of dead cells and this in turnallows apoptotic cells to reach their final fate within

the phagocytes. The recognition of dead cells could be amultistep process complicated by the existence of re-gional specialization and by the display on the apopto-tic cells of multiple signals to increase the probabilityof their removal and consequently the safety of thewhole organism. To engulf the apoptotic cells, cytoskel-etal reorganization is also necessary, as shown by thedramatic modification of the phagocytic cellular shape.In addition, as reported for the liver, cooperationamong different cellular types sharing the same recep-tor system is shown for the removal of apoptotic cells.In fact, hepatocytes, Kupffer, and endothelial cells op-erate, at the same time, in the plasma clearance ofapoptotic cells generated during the involuting phaseof liver hyperplasia induced by a single injection of lead

nitrate by means of a sugar recognition mechanism(Dini et al., 1995; Ruzzittu et al., 1999). These data,together with the fact that the phagocytic activity inendothelial cells can be enhanced in macrophage-de-pleted rats (Bogers et al., 1991) and that IL-1 inducesin vitro overexpression of mannose-specific receptorson endothelial cells, further support the idea of coop-eration among liver cells during phagocytosis of apo-ptotic cells (Dini et al., 1995; Dini et al., 1998). How-ever, the process of phagocytosis of apoptotic cells,which is an ancient process present in invertebrates aswell as in vertebrates, has developed species-specificmechanisms whose biological significance is still ob-scure.

Fig. 9. Scanning electron mi-crographs of in vitro (a) and in situ(b) adhesion experiments. Co-cul-tures of apoptotic lymphocytes andendothelial cells is shown in a. Af-ter 15 minutes of incubation apo-ptotic lymphocytes were observedin the process of binding to the sur-face of the endothelial cells (thefenestrae are clearly visible) (ar-rows). b: Apoptotic lymphocytes(arrows) engulfed by the sinusoi-dal wall of mouse liver after intra-portal injection. Magnifications:(a) 16,000; (b) 9,000.

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It is worth noting that the study of phagocytosisduring the process of apoptosis is not merely a specu-lative exercise, since defects of phagocytosis of apopto-tic cells might have deleterious consequence for neigh-boring healthy cells. The logical consideration of theimportance of phagocytosis leads to thoughts on thecontribution of defective clearance as a factor in thepathogenesis of inflammatory diseases. The relevanceof phagocytosis to the dysregulation of the immunesystem that underlies specific pathological conditionsrequires examination: for example, whether compro-mising the capability to ingest apoptosing cells contrib-utes to autoantibody production (Bellone et al., 1997;Botto et al., 1998; Hermann et al., 1998).

The studies of mutations affecting the clearance ofdying cells by professional phagocytes in Drosophilawill help to unravel the complexity inferred from inhib-itor studies in mammalian systems. However, furtherinvestigations of the mechanisms of recognition andingestion of apoptotic cells are urgently required toaddress regulatory roles in inflammation, immune re-sponses, and tissue remodeling. This in turn may allowmanipulation of phagocyte responses to apoptotic cellstimuli and the development of novel therapeutic strat-egies (for example, during tissue repair) as an effectiveantiinflammatory and immunosuppressive strategy(Voll et al., 1997; Fadok et al., 1998; Botto et al., 1998;Herrmann et al., 1998).

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Phagocytosis of Apoptotic Cells by Liver