Plasticity of the tubular lysosomal compartment in macrophages

PHILIP E. KNAPP and JOEL A. SWANSON*

Department of Anatomy and Cellular Biology, Harvard Medical School, 220 Longzvood Avenue, Boston, MA 02115, USA

* Author for correspondence

Summary

Because bone marrow-derived macrophages differ-entiate in culture, their lysosomal compartment islargely devoid of the undigested particles that arecommon in macrophages removed from tissues.The morphology of this nearly vacant lysosomalcompartment was observed, after labeling withfluorescent endocytic tracers such as LuciferYellow, to be an extensive, tubuloreticular network,which underwent extensive rearrangements in ac-commodating endocytic loads. It was converted tospherical organelles when the lysosomal compart-ment was loaded with osmotically active solutessuch as sucrose or Acridine Orange. Enzymaticdegradation of intravacuolar sucrose by pinocy-tosed invertase resulted in the shrinkage of vacuolesand the re-formation of the tubular network. Afterphagocytosis of opsonized erythrocytes or latex

beads, tubular lysosomes wrapped around the pha-gosomes, then merged to form phagolysosomes.The disappearance of tubules was proportional tothe total surface area of particles ingested. Degra-dation of the phagocytosed contents permittedshrinkage of the phagolysosome and concomitantre-formation of the tubuloreticular network. Non-degradable contents such as latex beads preventedre-formation of the tubular network. These re-arrangements of the lysosomal compartment indi-cate that the organelle exhibits considerable plasti-city and interconnectedness, and that maturation oflysosomes after endocytosis does not necessarilyentail irreversible morphological changes.

Key words: macrophage, lysosome, phagocytosis.

Introduction

It has long been known that the lysosomal compartmentcan expand to accommodate content, and that materialentering the compartment via endocytosis can determineits shape. The most conspicuous example of this occursduring macrophage phagocytosis, in which small lyso-somes fuse with larger, particle-containing phagosomesand conform to the shape of the ingested particle (Cohnand Wiener, 1963; Cohn et al. 1966). In addition,membrane-impermeant solutes, which accumulate inlysosomes either through endocytosis or by pH-depen-dent trapping, can expand that compartment via osmoticeffects (Cohn and Ehrenreich, 1969; Ohkuma and Poole,1981).

The shape of the lysosomal compartment is alsoinfluenced by cytoplasmic factors. In some macrophages,lysosomes stained for acid phosphatase or labeled viapinocytosis of soluble tracers appear elongate and tubular(Nichols, 1982; Swanson et al. 1985). This shape isachieved in part through an association between lyso-somes and microtubules (Swanson et al. 1987). More-over, both the shape and the distribution of macrophagelysosomes can be altered by conditions that changecytoplasmic pH (Heuser, 1989).

The tubular morphology has been observed in some

Journal of Cell Science 95, 433-439 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

macrophages but not others. Lysosomes labeled bypinocytosis of the fluorescent dye Lucifer Yellow aretubular in cultured human monocytes, phorbol ester-treated peritoneal macrophages, J774 macrophages andbone marrow-derived macrophages, but those of un-stimulated peritoneal or alveolar macrophages are nottubular (Swanson et al. 1987). Lysosomes labeled inliving macrophages using the fluorescent probe AcridineOrange have not been described as tubular.

Acridine Orange is membrane permeant at neutral pH,but less so at lower pH; and therefore it accumulates inacidic intracellular compartments such as lysosomes.Cells incubated in low concentrations of Acridine Orangequickly accumulate the dye in lysosomes to high enoughconcentrations for the fluorescence to appear more red.Although lysosomes labeled with Acridine Orange for 10(Hart and Young, 1975) or 20 (Kielian et al. 1982) minappear vesicular, we wondered if this might be becausethe concentrations of Acridine Orange necessary for thered shift in its fluorescence emission spectrum might alsobe high enough to expand the compartment osmotically.

The purpose of this investigation was to determinehow endocytosed particles, endocytosed solutes or ac-cumulated Acridine Orange affect lysosome morphologyin bone marrow-derived macrophages. Labeling with lowconcentrations of highly fluorescent tracer molecules

433

permitted light microscopic observation of lysosomesduring and after phagocytosis or osmotic expansion. Ourresults indicate that macrophage lysosomes are tubularwhen they lack any endocytic load, but lose their tubularmorphology as their luminal content increases. More-over, they exhibit considerable plasticity in their move-ments and morphological rearrangements following en-docytosis.

Materials and methods

CellsBone marrow-derived macrophages were obtained from thefemurs of adult, female, ICR mice (Trudeau Institute, SaranacLake, NY) or C3H/HeJ mice (Jackson Labs, Bar Harbor, ME)as described previously (Swanson, 198%). Bone marrow exu-date was cultured for 6 or 7 days in growth medium consistingof Dulbecco's minimal essential medium (DME) with 30% Lcell-conditioned medium, 20% heat-inactivated fetal bovineserum and 2p.p.m. (parts per million) of /J-mercaptoethanol at37°C, 5 % CO2, with 10 ml additional growth medium added onday 3. After this growth period dishes were washed and cellsremoved in cold divalent cation-free phosphate-buffered saline(PD: 137 mM NaCl, 3 min KC1, 7mM phosphate buffer,pH7.4), and were plated onto 12mm, no. 1 circular coverslipsat 105 cells per coverslip or onto 22 mm square coverslips at3X 10s cells per coverslip. After 45 min at 37°C PD was replacedwith DME containing 10 % heat-inactivated fetal bovine serum(DME-10F), and returned to 37°C, S % CO2 for 24h.

Fluorescent labeling of lysosomesMacrophages were incubated in Lucifer Yellow CH, potassiumsalt (LY; Molecular Probes, Eugene, OR) at 0.5mgml~' inDME-10F at 37°C, 5% CO2 for l - 2 h and then washed andincubated in phosphate-buffered saline with 10 % heat-inacti-vated fetal bovine serum for 30 min. This protocol was alsoemployed to label lysosomes with fluorescein-dextran(l .OmgmP ; Sigma Chem. Co., average MT10000) TexasRed—dextran (1.0mgml~ , Molecular Probes, Inc., averageMr 10000) and Texas Red-labeled ovalbumin (40 jig ml"1; Mol-ecular Probes). Lysosomes were labeled with Acridine Orangeby incubating cells in 5 jigm\~l Acridine Orange in DME-10Ffor 5-20 min before rinsing and observing. To monitor lyso-some expansion and collapse, cells on coverslips were incubatedovernight in DME-10F±20mgml""1 sucrose. This medium wasreplaced with DME-10F containing 0.5mgml~ LY+sucrose,and incubated for another 3 h at 37 °C. Cells were then eitherwashed in PBS and observed, or were incubated in DME-10Fwith O.SmgmP1 invertase (Sigma Chem. Co., St Louis, MO)for an additional 3 h before microscopic observation.

PhagocytosisSheep red blood cells (SRBC; Cappell) were washed twice bycentrifugation (1200 revs min"1, lOmin) and resuspension inPBS (137 mM NaCl, 3 mM KC1, 0.5 mM MgCl2, 1 mM CaCl2,7mM phosphate buffer, pH7.4) at 4°C. They were opsonizedby resuspending 5xlO8 cells in 1 ml PBS containing 2j«l anti-SRBC IgG (Cappell), 30 min at 37°C, and washing three timesin cold PBS. SRBC ghosts were prepared by osmotic rupture. Atotal of 3xlO8 cells were suspended and lysed in lml of 20mosM sodium phosphate, pH7.0, 5 mM MgCl2 and lOmgmP1

Texas Red-dextran, average Mr10000 (TrDx) for 10 min at4°C. Sodium chloride (0.25 ml, 4.5%) was then added torestore physiological osmolarity and cells were allowed to reseal

for 45 min at 37°C. They were then washed twice in cold PBSby centrifugation (HOOrevsmin"1, 3 min) and opsonized asabove. Polystyrene microspheres (latex beads), 7.0 fim diameter(±0.04j/m) and 3.98(im diameter (±0.03;U.m) (Duke Scien-tific, Palo Alto, CA), were suspended in lOmgml"1 bovineserum albumin (BSA) in PBS for 1 h, washed in 1 mgml~' BSAin PBS, and finally resuspended in warm PBS.

Particles for phagocytosis were introduced to macrophages byadding 1 ml PBS with lXlO6 to 3X106 particles into a 16 mmplastic well containing macrophages on circular coverslips, thencentrifuging at 1000 revs min~ for 5 min. Cells were thenincubated for various times in DME-10F (37°C).

MicroscopyAll quantitative scoring experiments were performed usingmacrophages on 12 mm diameter, circular no. 1 coverslipsmounted on glass slides and sealed with Valap as describedpreviously (Swanson, 1989a). Micrographs were taken ofmacrophages either on 12 mm circular coverslips or on 22 mmsquare coverslips mounted on slides in the same manner.Observations were made using a Zeiss Plan-Neofluar 63 X lens(NA 1.25) in a Zeiss photomicroscope III equipped forepifluorescence with LY and rhodaniine filter sets. Use of theLY filter set allowed simultaneous observation of LY andTRDx. Color photographs were taken with Kodak Ectachromefilm, ASA 400, which was push processed. Black and whitephotos were taken with Kodak T-Max 400, which was also pushprocessed.

Results

Osmotic expansion of the lysosomal compartmentLysosomes observed in living cells after labeling viapinocytosis of LY appeared as a branched tubularnetwork extending throughout the cytoplasm (Fig. 1A).The network could also be labeled with a variety of otherfluorescent probes including Texas Red-conjugated ov-albumin and fluorescein- or Texas Red-conjugated dex-trans of various sizes (data not shown). Under theseconditions tubules labeled by pinocytosis were observedin more than 95 % of the cells. Some cells displayed web-like interconnections between tubules (Fig. ID).

Acridine orange altered the compartment morphology.After a 5-min incubation in 5 jtgml"1 Acridine Orange,red fluorescent macrophage lysosomes appeared tubular(Fig. 2A). However, after 10 min, the lysosomes began toappear discontinuous and vesicular (Fig. 2B). After20 min in Acridine Orange the lysosomes were enlargedand distended (Fig. 2C). The observed change in mor-phology indicates that the accumulation of AcridineOrange in lysosomes for as little as 10 min caused theosmotic expansion from their initial elongated mor-phology.

Osmotic expansion of the lysosomal compartment canalso be achieved by endocytosis of the membrane-impermeant solute sucrose (Cohn and Ehrenreich, 1969;Swanson et al. 1986). Incubation of bone marrow-derived macrophages in 20 mg ml" ' sucrose (60 mM) for 3(Fig. IB) or 15 h (Fig. 1C) resulted in a shape change inthe LY-labeled tubular lysosomal network. Tubules ap-peared first to blister (Fig. IB), and then those blistersexpanded into spherical organelles. Incubation of su-

434 P. E. Knapp andj. A. Swanson

Fig. 1. Reversible expansion of lysosomes by sucrose. Macrophages were first incubated overnight in DME-IOF (A,B,D,E)or in DME-L0Fplus20mgmr' sucrose (C,F). They were then incubated 3 h in DME-IOF plus 0.5 mgml"1 LY (A,D) or inDME-IOF, LYand 20mgml~' sucrose (B,C,E,F). Cells were then either rinsed in PBS and observed by fluorescencemicroscopy (A-C) or incubated 3h in DME-IOF containing 0.5mgml~' invertase (D-F) before being rinsed in PBS andobserved by fluorescence microscopy. Arrows indicate thin, sheet-like connections between several tubules. Bar,

crose-vacuolated macrophages in 0.5mgml ' invertaseallowed delivery of that enzyme into sucrose vacuoles, theconversion of sucrose to component monosaccharides,and the collapse of the spherical organelles (Cohn andEhrenreich, 1969). After incubation in invertase-contain-ing medium for 3 h, vacuoles containing sucrose regaineda tubular morphology (Fig. 1E,F). Thus, the distentionof tubular lysosomes by osmotic expansion was revers-ible.

Morphological rearrangements accompanyingphagocytosisMacrophages whose lysosomes had been labeled viapinocytosis of LY were allowed to phagocytose sheeperythrocyte ghosts that had been opsonized with goatanti-sheep erythrocyte antibody and loaded via osmoticrupture with Texas Red-labeled dextran (TRDx). Suf-ficient TRDx was loaded into the erythrocyte ghosts to

allow its detection with the LY filter set. This permittedsimultaneous morphological observation of both lyso-somes (LY) and phagosomes (TR) during phagosome-lysosome fusion (Fig. 3). Shortly after phagocytosis, LY-labeled tubules surrounded phagosomes containingTRDx-labeled ghosts. After membrane fusion the twofluorophores mixed, creating orange phagolysosomes.Thirty minutes to one hour after phagocytosis the tubularlysosomal network disappeared (Fig. 3C), as the mem-brane previously associated with the network was incor-porated into the combined phagolysosomal compart-ment. This compartment appeared as one or a few large,orange vesicles. Three hours after phagocytosis, thetubular network reappeared, labeled with a mixture ofLY and TRDx fluorescence, thus indicating that the newnetwork arose from the phagolysosome.

After phagocytosis of latex beads, the lysosomalnetwork disappeared, as it did after phagocytosis of

Macrophage lysosome plasticity 435

Fig. 2. Acridine Orange alters macrophage lysosomemorphology. Macrophages were incubated in 5^gml~'Acridine Orange in DME-10F for 5(A), 10(B) or 20(C) minat 37 °C, then were quickly washed and observed byfluorescence microscopy (rhodamine filter set). Theserepresentative micrographs show the rapid conversion oftubular lysosomes into a more distended vesicularcompartment. Bar, 10/un.

erythrocyte ghosts. But in this case LY fluorescenceremained surrounding the indigestible beads for manyhours after disappearance of the tubules. A network didnot reappear. Fig. 4 shows macrophages that werelabeled by pinocytosis of LY before phagocytosis, but

that also remained in the LY solution throughout phago-cytosis of latex beads and the subsequent 5 h chaseperiod. Continued pinocytosis would have labeled anynewly formed lysosomes. Although pinosomes werelabeled by this procedure, no new labeled tubular organ-elles appeared, indicating that the fluorescence surround-ing the ingested beads represents the entire lysosomalcompartment. The re-formation of a tubular networkafter phagocytosis of ghosts, but not after phagocytosis oflatex, indicates that the re-formation of tubular mor-phology requires the phagolysosome shrinkage that ac-companies digestion.

To quantify the depletion and reappearance of tubulesfollowing phagocytosis, macrophage lysosomes werelabeled by pinocytosis of LY, and then cells were incu-bated with latex beads or opsonized erythrocytes forphagocytosis. The percentage of cells containing tubuleswas determined for each condition at various times afterphagocytosis. As shown in Fig. 5, phagocytosis of opso-nized erythrocytes depleted the tubular network tran-siently, whereas phagocytosis of latex beads led to irre-versible depletion.

Using two sizes of beads we determined the extent oftubule disappearance with increasing phagocytic loads.For each number of beads ingested 10-20 macrophageswere scored for the presence or absence of tubules. Thepercentage of cells with tubules was plotted in respect ofthe number of beads per cell (Fig. 6A), the total amountof bead surface area internalized (Fig. 6B) or the totalamount of bead volume internalized (Fig. 6C). Phago-cytosis of 4/im diameter beads depleted the tubularnetwork to the same extent as phagocytosis of 7 fimdiameter beads when the quantity ingested was expressedas the total surface area of latex ingested (Fig. 6B).Complete distributions for beads of both sizes weredetermined on three separate occasions. There wasvariation between experiments in the number of beadsrequired to deplete the cell of tubules. For each of thethree trials, however, the total ingested surface areaprovided the closest correspondence between phagocyticload and tubule depletion.

Discussion

Because bone marrow-derived macrophages grow anddifferentiate in culture, their lysosomal compartmentcontains very little phagocytosed indigestible or undi-gested matter. The experiments described here demon-strate that in this relatively vacant condition lysosomesexist as an extensive tubular network. This networkdisappears as the lysosomal volume expands, then re-appears as volume decreases.

The re-formation of the tubular morphology aftershrinkage of sucrose vacuoles or phagolysosomes indi-cates that the compartment exhibits considerable plasti-city at late stages in its maturation. The tubular networkformed hours after phagocytosis of erythrocytes appearsvery similar to that labeled by a brief incubation with LY.This complicates morphological discrimination betweenearly and late lysosomes in the macrophage. It also

436 P. E. Knapp andj. A. Swanson

Fig. 3. Reorganization of lysosomes following phagocytosis.Macrophage lysosomes were first labeled by pinocytosis of LY(yellow fluorescence). They were then provided withopsonized erythrocvte ghosts that had been preloaded byosmotic rupture with TRDx (red fluorescence). A. Eight minafter phagocytosis, red phagosomes appear independent of theyellow tubular lysosomal network. B. Fifteen min afterphagocytosis, tubular lysosomes appear closely apposed tosome phagosomes. Some mixing of the fluorophores isevident, indicating phagosome-lysosome fusion. C. Sixty minafter phagocytosis, the two fluorophores are completely mixedinside phagolysosomes, and the tubular network hasdisappeared. D. Three hours after phagocytosis, a newtubular lysosomal network has re-formed from shrunkenphagolysosomes.

Fig. 4. Phagocytosis of latex beads causes irreversible redistribution of the tubular lysosomai compartment. Macrophagespreloaded with LY were incubated with latex beads and LYfor lOmin (A,B) or 5h (C,D). Phagosome-lysosome fusiondepleted the tubular network. Despite continued incubation in LYfor 5h, no tubular endocytic compartment reappeared.A and C, phase micrographs; B and D, corresponding fluorescence images. Bar, 10/im.

demonstrates that the tubular compartment is not simplyan intermediate shape in the morphogenesis of a sphericallysosome. Instead, the tubular morphology is that of astable, interconvertible organelle that can reversiblyaccommodate endocytic loads.

The traditional view of the lysosomai compartment as apopulation of discrete vesicles should be reconsiderednow in light of the continuity we observe here. Theanastomosing network, in its form and in its movements,indicates a considerable traffic of lysosomes in cytoplasm.Microtubules contribute to this traffic, possibly viamicrotubule-dependent motors such as kinesin (Swansonet al. 1987; Vale et al. 1985). However, microtubule-dependent lysosome movement is not rate-limiting foreither phagosome-lysosome fusion or hydrolytic degra-dation of phagocytosed content, as microtubule-depoly-merizing drugs have little or no effect on these rates(P.E.K., data not shown; Pesanti and Axline, 1975a,b).Microtubule association may be more important duringthe reorganization of the lysosomai structure followingdegradation of phagolysosomal contents. We have ob-served that the re-formation of tubular lysosomainetworks after phagocytosis is prevented by depolymeriz-ation of microtubules with nocodazole (P.E.K., data not

0 1 2 3 4Time after phagocytosis (h)

Fig. 5. The tubular lysosomai network reassembles afterphagocytosis of digestible particles. Macrophages preloadedwith LY were provided with latex beads or opsonizederythrocytes for phagocytosis, then were incubated inDME-10F for the times shown. Cells were scored for thepresence of tubular lysosomes. (O) No phagocytosis; (•)9.1±5.5 latex beads per cell; (•) 8.4±5.6 erythrocytes percell; (•) 15.9+4.7 erythrocytes per cell. Similar results wereobtained on three separate occasions. Variations in thenumber of particles phagocytosed precluded pooling of thedata.

Macrophage lysosome plasticity 437

100 200 300 400 500 600 700 800 900Surface area internalized 2

100 200 300 400 500 600Volume internalized (,«m3)

Fig. 6. Tubular lysosome disappearance correlates with thetotal surface area ingested by phagocytosis. Macrophagespreloaded with LY were allowed to phagocytose latexbeads, then the percentage of cells containing tubularlysosomes greater than 10 /m\ in length were scored for eachbead load. A. Fewer 7 urn diameter beads (A) wererequired to deplete the tubular lysosomal network than4;<m diameter beads ( • ) . B. The data of A expressed interms of total surface area ingested: (O) 7jum diameterbeads, (•) 4,um diameter beads. C. The data of Aexpressed in terms of total bead volume ingested: (D) 7,umdiameter beads, (•) 4;«m diameter beads.

shown). Under these conditions, the absence of micro-tubule tracks along which to extend the lysosome maypermit other morphological arrangements of that mem-brane (e.g. see Swanson et al. 1986).

Lysosomes left distended for long periods of time maylose some plasticity. In J774 macrophages, sucrosevacuoles formed by prolonged incubation in sucrosecollapse upon invertase treatment into whorled residualbodies, instead of reverting to the tubular morphology ofnon-vacuolated cells (Swanson et al. 1986). A similarrearrangement was detectable in some of the bonemarrow-derived macrophages observed by fluorescenceafter prolonged sucrose vacuolation and subsequentinvertase-induced collapse (J.S., data not shown). Itindicates that loss of lysosome plasticity, and the forma-tion of residual bodies, is a maturation event that occursonly after prolonged occupancy of the compartment.

The usual morphology of lysosomesIt is possible that the fluorescent probes delivered byendocytosis or pH-dependent accumulation themselvesinduce the tubular morphology. However, tubular lyso-

somes can be recognized ultrastructurally using acidphosphatase histochemistry in macrophages fixed with-out any prior endocytosis of tracer molecules (Swanson etal. 1987).

Tubular lysosomes are difficult to preserve in fixedcells, and macrophage lysosomes often appear by light orelectron microscopy as vesicles or rows of vesicles(Phaire-Washington et al. 1980; Swanson et al. 1987).Such difficulties in preservation, together with the factthat Acridine Orange disrupts tubular lysosome mor-phology in living macrophages, may explain why theextensive networks were not described until recently (cf.Buckley, 1973; Nichols, 1982). This is not to say thattubular lysosomes are ubiquitous. Many cultured celllines, when labeled with fluorescent tracers, displayspherical lysosomes (Miller et al. 1983). Tubular lyso-somal networks in macrophages probably result from anincreased content of lysosomal membrane relative toother cells. This membrane remains in the high surface-to-volume tubular morphology until its volume increasesby accumulation of either solutes or phagocytosed par-ticles. Tubule depletion is proportional to the amount ofparticle surface area ingested.

That lysosomes appear tubular even after considerableendocytosis of LYor TRDx suggests that these moleculesdo not reach concentrations that expand the compart-ment osmotically. Although LY is highly water-soluble(Stewart, 1978) and therefore potentially capable ofosmotic expansion of lysosomes, it is added in theseexperiments at a concentration of only 1 raM, much lowerthan that of sucrose (60mM), which does cause osmoticvesiculation. Therefore, LY does not appear to accumu-late to disruptive concentrations during the incubationtimes used here. Rapid expansion of lysosomes byAcridine Orange is achieved at even lower extracellularconcentrations (3[iM). Its pH-dependent concentrationinside lysosomes, then, must occur much more rapidlythan the endocytosis-dependent concentration of sucrose.. Why are tubular lysosomes evident in some macro-

phages but not others? Lysosomes of unstimulated,thioglycolate-elicited, mouse peritoneal macrophages sel-dom appear tubular. These macrophages normally con-tain internalized agar from the thioglycolate broth, whichcreates a permanently enlarged phagolysosomal compart-ment. Treatment with the tumor promoter phorbolmyristate acetate rapidly leads to the appearance oftubular lysosomes (Swanson et al. 1987), suggesting thatdespite the partial expansion of the lysosomal compart-ment by agar, some membrane is available to formtubules. It also indicates that in the absence of stimu-lation these cells do not normally promote lysosomeextension along microtubules. Similarly, the lysosomes ofcultured cells often cluster in the vicinity of the nucleus,away from peripheral regions of cytoplasm (Zelenin,1966; Herman and Albertini, 1984). Centrifugal move-ment of lysosomes may require activation of cytoplasmicmotors on microtubules, and the activity of such motorsmay vary with the physiological state of the cell. Thedramatic redistributions of lysosomes elicited by chang-ing cytoplasmic pH are consistent with this hypothesis(Heuser, 1989).

438 P. E. Knapp andjf. A. Swanson

Thus, two factors contribute to the formation of thetubular lysosomal compartment: the contents of thelumen and the interactions of the organelle with cytoplas-mic microtubules. In bone marrow-derived macro-phages, the lysosomal compartment is large, relativelyvacant, and extended along microtubules. Conversely,lysosomes of thioglycollate-elicited macrophages arepartly distended by indigestible agar and interact poorlywith microtubules until the cell is stimulated withphorbol esters.

The authors thank Gregory Cannon for helping to developsome of the methods used here, Esther Racoosin and PeterHollenbeck for suggestions and Angela Riccardo for secretarialassistance. This work was supported by NIH grant CA 44328 toJ.S.

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(Received 22 September 1989 - Accepted 17 November 1989)

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