JOURNAL OF CELLULAR PHYSIOLOGY 144:166-174 ( I 990)

Acquisition of a Lysosomal Enzyme by Myoblasts in Tissue Culture J O N A T H A N R. BEAUCHAMP, TERENCE A. PARTRIDGE, AND I R W I N OLSEN* Cell Enzymology Unit O.R.B., /.O.),Kennedy institute of Kheumatology, London, WG 7DW, and Department of Histopdtho/ogy (T.A.P.), Charing Crossl!Nestminster Medical School, London, Wh 8RF, United Kingdom Skeletal muscle myoblasts from different sources acquired high levels of the lysosomal enzyme p-glucuronidase, when they were cultured together with mitogen-activated lymphocytes. Immunofluorescent staining, thermal stability, and electrophoretic mobility showed that the increase in enzyme activity in the myobiasts was due to the presence of the lymphocyte form of (he enzyme. Although myoblasts were able to take up exogenous P-glucuronidase from the culture medium by mannose 6-phosphate receptor-mediated endocytosis, enzyme acquisition during co-culture with lymphocytes was independent of this pathway. Enzyme transfer from the lymphocytes was found to require direct cell-cell contact with the muscle cells, and was accompanied by an increase in P-glucuronidase activity in the lymphocytes themselves. Since this additional activity was also due to the presence of the lymphocyte form of the enzyme, these results indicate that interaction with the muscle cells induced the de novo synthesis of P-glucuronidase in the lymphocytes.

The lysosomal storage diseases are a related group of metabolic disorders caused by inherited deficiencies of specific lysosomal enzymes (Stanbury et al., 1983). The resulting accumulation of partially degraded substrates of these enzymes in various tissues usually leads to physical deterioration and progressive mental retardation and often to death at an early age (Watts and Gibbs, 1986). A number of attempts have been made to replace the deficient enzymes, and bone marrow transplantation has sometimes produced clinical improvement in affected children (Hugh-Jones et al., 1984) and apparent pathological remission in experimental animals (Gasper et al., 1984; Pate1 and Pentachev, 1989). The mechanisms which underlie the correction of these deficiencies in vivo are not yet known, but in tissue culture at least two distinct processes have been shown to be involved in the acquisition of missing lysosomal enzymes by cells from affected patients. The first is endocytosis from the culture medium of lysosoma1 enzymes containing phosphorylatcd mannose residues (Kornfeld, 1987), or sometimes other recognition signals for which recipient cells have corresponding surface receptors (Lennartz et al., 1989). The second mechanism involves the direct transfer of certain lysosomal enzymes from normal bone marrow-derived cells, particularly lymphocytes (Olsen et al., 1981, 1983). This latter mode of enzyme acquisition is not mediated via the mannose &phosphate receptor (MPR) but does require cell-to- cell contact (Dean et al., 1982). Enzyme transferred directly from lymphocytes has been shown to effectively correct the metabolic defect of enzyme-deficient dermal fibroblasts in vitro (Abraham et al., 1985). Some lysosomal disorders in man (Carpenter and 0 1990 WILEY-LISS, INC.

Karparti, 1986) and other species (Dorling, 1984) are associated with skeletal muscle pathology. Although affected muscle cells have been shown to be capable of acquiring exogenous 1ysosomal enzymes in tissue culture (Di Marco et al., 1985; van der Ploeg et al., 1988a), it is not yet known whether lysosomal enzymes can also be transferred to them by contact with normal lymphocytes. Because direct transfer from lymphocytes may play a central role in the success of enzyme replacement by bone marrow transplantation, this process is of particular interest in view of theraputic attempts already undertaken to correct the glycogen storage disease type IIa, a lysosomal deficiency disorder which involves muscle pathology (Harris et al., 1986). In the present study we have examined the acquisition of a representative lysosomal enzyme, p-glucuronidase, by skeletal muscle myoblasts in vitro, by contact-mediated enzyme transfer from lymphocytes, and also by receptor-mediated endocytosis.

MATERIALS AND METHODS Cell culture Skeletal muscle cell lines from the mouse (C, (Yaffe and Saxel, 1977) and Gs (Christian et al., 1977)) and the rat (L6 (Yaffe, 1968)) were cultured as monolayers in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% foetal calf serum (FCS), 100 Uiml penicillin, and 100 pgiml streptomycin. Human myoblasts, prepared by enzymatic disaggregation of foetal

Received January 18, 1990; accepted April 5 , 1990.

“To whom reprint requestsicorrespondence should be addressed.

LYSOSOMAL ENZYME ACQUISITION BY MYOBLASTS

muscle using 0.1% pangestin, 0.6% trypsin in Ca"i Mg+'-free Hanks saline, buffered to pH 7.4 with HEPES buffer (Watt et al., 1984) and grown in the above medium containing a n additional supplement of 2% chick embryo extract, were provided by Dr. J. Morgan (Department of Histopathology, Charing Cross and Westminster Medical School, London). All muscle culture vessels were pre-coated with 0.1% gelatin in phosphate-buffered saline (PBS) and allowed to air-dry before use. Monolayer cultures of human fibroblasts deficient in p-glucuronidase (Mucopolysaccharidosis type VII, GM 151) were incubated in Eagle's Minimum Essential Medium (MEM) supplemented with 10% FCS, 100 U i ml penicillin, and 100 pg/ml streptomycin. Suspensions of lymphocytes were obtained from the spleens of CBA mice (or strain Balbic as noted) and cultured at lo6 cellsiml in RPMI 1640 supplemented with 5% FCS{ 100 U/ml penicillin, 100 pgiml streptomycin, 5 x 10M 2-mercaptoethanol, and 2 pgiml concanavalin A (Con A) for 72 h. This produced cultures containing 70-80% of activated T-lymphocytes, as determined by immunofluorescent staining with antibody specific for the Thy-1 antigen (Sera Lab, Berks, UK).

Co-culture of myoblasts with lymphocytes Myoblasts were cultured as monolayers in 10 ml of medium in 75 cm2 gelatin-coated tissue culture flasks. When the cultures were approximately 50% confluent (2 x lo6 cells), the medium was removed and replaced with 10 ml of a suspension containing lo8 Con A-activated CBA lymphocytes. unless indicated otherwise. These co-cultures were incubated for 16 h at 37°C in a n atmosphere of 10% CO, in air prior to removing and retaining the medium containing the non-adherent lymphocytes. The monolayers were then treated with 0.25% trypsin for 3 min a t 37°C to remove the majority of the bound lymphocytes without detaching the myoblast monolayer. The monolayers were again treated with trypsin for 10 min in order to obtain a suspension of myoblasts containing few contaminating lymphocytes, which were then removed as described below. Myoblasts and lymphocytes were also cultured alone, as controls. The human myoblasts and the enzyme-deficient human fibroblasts (GM 151 cells) were co-cultured with lymphocytes, in the same way as the mouse myoblasts.

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dium and divided equally between twenty 35 mm plastic petri dishes coated with 0.1% gelatin. These were incubated a t 37"C, and at 10 min intervals duplicate dishes were removed and the non-adherent cells discarded. The monolayers were washed and the adherent cells lysed in 500 pl 0.5% Triton X-100 and 0.1% SDS in 20 mM Tris-HC1 buffer, pH 7.4. The ratio of 14C:3H label in the lysates was measured in a scintillation counter, and used to determine the relative rates of adhesion of the myoblasts compared with the lymphocytes.

Co-culture of myoblasts and lymphocytes without direct cell-to-cell contact Myoblast monolayers were cultured in 35 mm dishes as described. These were then fitted with a 12 mm Millicell-CM Culture Plate Insert Filter (Millipore UK, Middlesex, UK) into which was placed lo7 lymphocytes in 1ml of fresh medium. The myoblasts were thus separated from the lymphocytes by a 0.4 pm pore size filter, allowing the two cell types to share a common medium whilst preventing cell-to-cell contact. After incubation at 37°C for 16 h, the insert was removed and lysates were prepared from the monolayers and assayed for p-glucuronidase activity. I n some experiments, 200 U of purified human platelet enzyme (see below) were added into the insert instead of the lymphocytes. Assay of P-glucuronidase activity in cell extracts Cultures of myoblasts and fibroblasts, and co-cultures, were detached by incubating the monolayers with trypsin, as described; the cells were pelleted by centrifugation at 350g for 10 min and resuspended in PBS. An aliquot was removed for direct haemocytometer counting following fixation and staining with 0.1% acetic acid containing 0.5% (wiv) crystal violet. The cells were again centrifuged and then lysed by the addition of 0.25% Nonidet P-40 in 0.25M sucrose containing 3.3 mM CaC1, for 30 min at P C , a treatment which disrupts the plasma membrane and leaves nuclei intact (Olsen and Harris, 1975). These were removed by centrifugation at 8OOg for 10 min and the supernatants, which contained more than 95% of the enzyme activity, were retained. p-Glucuronidase activity in these cytoplasmic fractions, and in extracts prepared in the same way from washed suspensions of lymphocytes and fibroblasts, was measured by a fluorometric procedure as subusing 4-methyl-umbelliferyl-~-D-glucuronide strate (Olsen et al., 1982). Depending upon the level of activity in the sample. incubation times varied between 1 and 20 h, during which the rate of reaction remained linear. One unit (U) of P-glucuronidase activity is equivalent to the liberation of 1 nmol of 4methylumbelliferone per h.

Separation of myoblasts from contaminating lymphocytes A selective replating procedure was used to eliminate the contaminating lymphocytes from the suspensions of trypsin-detacted co-cultures of myoblasts and lymphocytes. This was assessed by radiolabelling the myoblasts for 48 h with [14C-methyl]-thymidine (0.2 pCiiml) (Amersham, Bucks, UK) during logarithmic Identification of lymphocyte enzyme in growth. The monolayer was then washed five times co-cultured myoblasts with medium and incubated for 1 h a t 37°C with ten times the number of Con A-activated lymphocytes, Immunofluorescent staining of lymphocyte pwhich had also been previously labelled for 48 h using glucuronidase. Human myoblasts were grown on a different isotope, [3H-methyl]-thymidine (0.2 pCiiml) glass coverslips in 35 mm culture dishes until logarith(Amersham, Bucks, UK). After co-culture, the non-at- mic phase. The medium was replaced and the myotached lymphocytes were discarded and the monolayer blasts were either cultured alone or co-cultured with and bound lymphocytes detached by trypsin treatment. lo7 murine lymphocytes at 37°C for 4 h. Other co-culThe suspension of mixed cells was washed with me- tures were incubated for 24 h and then washed to re-

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move non-adherent lymphocytes, and the myoblasts gradient to 500 mM NaC1; 0.5 ml fractions were colwere re-cultured alone for a further period of 24 h. The lected and assayed for P-glucuronidase activity. monolayers were then washed with PBS and the lymphocyte p-glucuronidase was detected by indirect im- Uptake of purified myoblast P-glucuronidase by human fibroblasts munofluorescence by using a rabbit antibody previously shown to be specific for the murine enzyme Myoblast P-glucuronidase was purified from cell ex(Olsen et al., 19861, as follows. Monolayers were fixed tracts by ion-exchange chromatography on DEAE-52 in 3% formaldehyde in PBS for 20 min a t 20°C and cellulose (Dean et al., 1982). Confluent monolayer culpermeabilized by incubation with PBS containing 0.1% tures containing 2 x lo5 human fibroblasts deficient Tween-20 and 4 mg/ml normal goat serum (PTG) in p-glucuronidase (Mucopolysaccharidosis type VII, (Olsen et al., 1988). The rabbit antibody was applied to GM 151)were incubated for 16 h in 1ml fresh medium the washed monolayers a t a concentration of 50 pgiml containing 50 U of myoblast enzyme in the absence and in PTG for 60 min a t 20°C. After washing, the cover- presence of 10 mM M6P. p-Glucuronidase activity was slips were incubated for 30 min with affinity-purified measured in extracts of the trypsin-detached cells, as FITC-conjugated goat anti-rabbit IgG (ICN Biomedi- described above. cals, Bucks, UK), diluted 1/20 in PTG. They were then RESULTS washed thoroughly with PBS, mounted in 90% glycerol in PBS, and visualized by using a Zeiss Axioplan FluSelective replating of orescence microscope. lymphocyte-myoblast co-cultures Selective heat-inactivationof myoblast enzyme. The time course of the adhesion of trypsin-treated C, Lysates prepared from L, myoblasts and CBA mouse lymphocytes were incubated a t 64°C in sealed plastic myoblasts and activated lymphocytes to a plastic surtubes. Triplicate aliquots of each were removed a t 15 face coated with 0.1% gelatin is shown in Figure 1. Of min intervals and cooled rapidly on ice before assaying the applied myoblasts more than 60% had readhered after 10 min a t 37"C, and a further 20% became atfor p-glucuronidase activity. Polyacrylamide gel electrophoresis (PAGE) un- tached during the subsequent 40 min. However, only der non- denaturing conditions. P-Glucuronidase in 1%of the lymphocytes adhered during the first 40 min extracts of L6 myoblasts and Balbic lymphocytes cul- period, after which the percentage attached steadily tured alone and co-cultured together were purified by increased. This increase probably reflects the binding ion-exchange chromatography by using DEAE-52 cel- of the lymphocytes to the myoblasts which had adhered lulose (Dean et al., 1982). An aliquot of each sample within the first 40 min of incubation, since the lymwas heated a t 64°C for 45 min. After concentration to phocytes did not themselves attach to surfaces coated 50 p1, the samples were centrifuged at 50,OOOg for 10 with gelatin alone (data not shown). The more rapid min, mixed with 10 p l of buffer (75 mM Tris-HC1, 67 attachment of the myoblasts resulted in a n increase in mM boric acid, 2 mM EDTA, 17% glycerol), and run on the initial ratio of one myoblast to three lymphocytes to a vertical 8% (wiv) polyacrylamide gel for 36 h a t a nearly 25:l in the adherent cell population after 40 rnin constant current of 30 mA. The gel was then washed of replating. This provided the means of removing conextensively in 0.2M sodium acetate buffer, pH 5.5, and taminating adherent lymphocytes from the co-cultured the P-glucuronidase activity was visualized by incuba- myoblasts. Thus, in subsequent experiments, myoblast tion of the gel a t 37°C by a simultaneous coupling pro- monolayers with adherent lymphocytes were detached cedure using 0.25 mM napthol-AS-BI-p-D-glucuronideby trypsin treatment and washed, and the mixed cell as substrate, in the acetate buffer containing 1.8 mM suspension was allowed to rcplate for 40 min. The nonpararosaniline and 1.8 mM sodium nitrite (Hayashi, adherent cells, mainly lymphocytes, were removed and the adherent cells, mainly myoblasts, were again de1965). tached with trypsin. This procedure produced populaUptake of purified human p-glucuronidaseby tions of cells containing more than 95% myoblasts. myoblasts and HPLC analysis The effects of co-culture on myoblast and 6-Glucuronidase was obtained from human platelets lymphocyte P-glucuronidase and purified by chromatography on DEAE-52 cellulose Table 1 shows p-glucuronidase activity in L, and C, (Dean e t al., 1982); 200 Uiml of the enzyme in 1 ml of medium was added to washed myoblast monolayers myoblasts that were co-cultured with activated mouse containing lo6 cells in the presence and absence of 10 lymphocytes. Enzyme activity in these myoblasts mM mannose 6-phosphate (M6P). After incubation for changed from 154 to 190, and from 40 to 58 U/107 L, 16 h at 37°C the monolayers were washed three times and C, cells: respectively, representing increases of 34 with PBS and P-glucuronidase activity was measured and 45%.It is notable t h a t co-culture also resulted in a in extracts of the cells as described. The extracts were increase of approximately 50% in the p-glucuronidase dialyzed against 5 mM Tris-HCI buffer, pH 8.0, concen- activity of the lymphocytes themselves compared with trated to a final volume of 500 pl, clarified by centrif- lymphocytes cultured alone. Moreover, the increases in ugation a t 50,OOOg for 10 min, and analyzed by using a lymphocyte and myoblast p-glucuronidase activities Gilson HPLC system fitted with a n anion-exchange were not inhibited by the addition to the co-culture column (TSK gel DEAE-5PW; 7.5 mm x 7.5 cm) equil- medium of M6P, a potent inhibitor of receptor-mediibrated in the mobile phase with the Tris buffer. Un- ated endocytosis of lysosomal enzymes via phosphorybound material was eluted a t 0.5 ml/min for 20 min, lated mannose residues (Sly and Fischer, 1982). The increases in P-glucuronidase in the myoblasts after which a linear NaCl gradient (0-250 mM) was established over 25 min, followed by a second 10 min and the lymphocytes were abolished, however, when

169 . i

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0

10

20

30

40

50

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70

80

90

Time (min) Fig. 1. Time course of adhesion of lymphocytes and C, myoblasts. A mixed suspension of 1i4C-methyll-thymidine-labelled myoblasts and ['H-methyll-thymidine-labelled lymphocytes was prepared and plated onto 0.1% gelatin-coated plastic as described in the Materials and

Methods. The percentages of myoblasts and lymphocytes which had adhered were determined from the amount of each label in the cells attached to the plastic. These values were used to calculate the ratio of adhered myoblasts to lymphocytes.

TABLE 1. Changes in P-glucuronidase activity in myoblasts and lymphocytes

Cells L, rat myoblasts Mouse lymphocytes C, mouse myoblasts Mouse 1vmDhocvtes

Cultured alone 154 fi 5 40 -c 5 40 ? 1

34

?

3

P-glucuronidase activity (U/107cells) Co-cultured' Co-cultured' + M6P2 207 -c 10 190 i 12 64 ? 6 64 +- 6 58 i 2 59 c 4 45 2 3 46 ? 3

% increase after co-culture

-M6P 23 60 45 35 ~~

~

+ M6P ~

34 60 48 32

'Co-cultures were incubated for 16 h a t 37°C a t a 1ymphocyte:myoblast ratio of 50:1,as described in the Materials and Methods. 'M6P was added a t 10 mM. ( ? SEM (n=6)).

direct contact between the two types of cell was prevented. As shown in Figure 2, incubating the cells together in the same dish but physically separated from each other by a 0.4 pm pore size filter insert produced no increase in myoblast p-glucuronidase activity, compared with control co-cultures in which the lymphocytes were in direct contact with the myoblasts. Under the same conditions of cell separation, p-glucuronidase in the lymphocytes also did not increase, remaining the same as in control cells cultured alone (data not shown). The presence of the filter was found not to prevent the uptake of exogenous enzyme, since when human platelet p-glucuronidase was placed into the insert, its endocytosis by the myoblasts was the same as that of enzyme added directly to the monolayers in the absence of the filter (Fig. 2). The ratio of lymphocytes to L, myoblasts was also found to have a marked effect on the changes in activity in both types of cell. The results in Figure 3 show t h a t a relatively low proportion of lymphocytes in the co-culture (10,to one myoblast) produced a n increase of only 10% in myoblast activit and of approximately 20% in p-glucuronidase per 10 lymphocytes. However, as the proportion of added lymphocytes was increased, P-glucuronidase activity acquired by the myoblasts also became progressively higher, reaching a maximum of 40% a t a ratio of 80 lymphocytes per myoblast. In marked contrast, there was no parallel increase in lymphocyte P-glucuronidase activity, which actually declined progressively a s the proportion of added lymphocytes increased.

Y

Identification of acquired p- glucuronidase activity in co-cultured myoblasts Immunochemical detection of lymphocyte Pglucuronidase in myoblasts. I n these experiments normal murine lymphocytes were co-cultured with primary myoblasts of human origin, as described in Materials and Methods. An antibody specific for the murine form of p-glucuronidase (Olsen et al., 1986) was then used to detect the presence of the lymphocyte enzyme in the co-cultured human myoblasts. The results in Figure 4 show that only background fluorescence was obtained in the myoblasts cultured alone (Fig. 4a). After 4 h co-culture, low levels of lymphocyte p-glucuronidase were detected in the myoblasts, usually in close proximity to adherent lymphocytes, as shown in Figure 4b. Following 24 h co-culture and a further 24 h culture in the absence of lymphocytes, much greater amounts of the murine enzyme were readily visible within numerous, small cytoplasmic structures, often accumulated in the perinuclear region (Fig. 4c). Selective heat inactivation of endogenous myoblast activity. The thermal lability of p-glucuronidase is known to differ among various animal species (personal observations), as well as between certain strains of mice (Herrup and Mullen, 1979). When extracts of L, rat myoblasts were placed a t 64"C, their P-glucuronidase activity rapidly decreased, as shown in Figure 5. In contrast to this highly labile rat enzyme, which was almost completely inactivated after 45 min, identical

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BEAUCHAMP ET AL.

r---

0Control insert + filter insert - filter

Cultured alone

Fig. 2. Effects of cell-cell contact and separation on 6-glucuronidase acquisition by L, myoblasts. Myoblasts were cultured with lymphocytes or purified p-glucuronidase as described in the Materials and Methods. The lymphocytes or enzyme was added either directly to the

2

10

Cultured with P-glucuronidase

Co-cultured with lymphocytes

20

30

40

culture medium (- filter insert) or into the same medium but separated from the myoblasts by a 0.4 p,m-pore-diameter filter + filter insert). Control myoblasts were cultured alone.

50

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100

c (

2?

Lymphocyte : myoblast ratio

Fig. 3. Effect of lymphocyte : myoblast ratio during co-culture on changes in P-glucuronidase activity in myoblasts and lymphocytes.

treatment of CBA mouse lymphocyte extracts reduced their initial p-glucuronidase activity by only lo%, showing that this enzyme is heat-stable. Thus, the amount of lymphocyte p-glucuronidase activity present in extracts of co-cultured L, myoblasts can readily be determined by enzyme assay of the extract before and after heat treatment. The analysis of p-glucuronidase thermal stability in extracts of co-cultured cells is shown in Table 2. Myoblast activity increased by 35% during co-culture, and heat treatment inactivated only 73% of the total activity compared to 97% of the activity of control L, cells cultured alone. Moreover, the increase of heat-stable activity after co-culture (51 U/107 cells) corresponded to the observed total increase in activity acquired during the co-culture (54 U/107 myoblasts). Although the lymphocyte enzyme level also increased during co-culture, from 40 to 60 U/107 cells, less than 5% of the activity was inactivated by heat. This shows that the increased activity in these co-cultured lymphocytes

was endogenous lymphocyte enzyme, and did not result from the acquisition of myoblast enzyme by the lymphocytes . PAGE analysis under non-denaturing conditions. The form of P-glucuronidase in L, myoblasts was readily distinguishable from the enzyme of the Ralbic strain of mouse by PAGE under non-denaturing conditions, as shown in Figure 6. Myoblast pglucuronidase was identified as a single, diffuse band of enzyme activity which migrated slowly (lane 1)and which could not be visualized after heat treating the extracts (lane 2). In contrast, Balbic lymphocyte enzyme was found to have a faster electrophoretic mobility (lane 3) and was not heat-inactivated (lane 4). Extracts of co-cultured L, myoblasts produced both of these bands (lane 5 ) , and heat treatment of the extract confirmed that the slower band was the endogenous thermo-labile myoblast enzyme, whilst the faster band was of the heat-stable type derived from the lymphocytes (lane 6 ) .

LYSOSOMAL ENZYME ACQUISITION BY MYOBLASTS

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cells by approximately 100%,although the absolute increase in p-glucuronidase varied considerably between the cells (380, 42, and 102 U/107 cells, respectively). The uptake of human enzyme from the medium was, however, almost totally abolished in the presence of M6P. HPLC analysis of lysates of C, myoblasts showed the presence of two peaks of activity of equivalent size (Fig. 7c), one corresponding to the endogenous C, enzyme (Fig. 7b) and the other to human platelet p-glucuronidase (Fig. 7a).

Uptake of myoblast P-glucuronidase by deficient human fibroblasts The muscle cells were found t o produce a high-uptake form of P-glucuronidase. When the enzyme was purified from either L, or G, myoblasts and added t o cultures of GM 151 fibroblasts, the activity of these latter cells increased from a basal level of 9 U/107 cells to approximately 400 U/107 cells (Table 4). This uptake was completely inhibited by the addition of M6P to the culture medium.

Fig. 4. Identification by immunofluorescence staining of acquired murine lymphocyte P-glucuronidase activity in human myoblasts. Human myoblasts were co-cultured with mouse lymphocytes and then stained with anti-mouse P-glucuronidase antibody, as described in the Materials and Methods. a: Control human myoblasts cultured alone. b Human myoblasts co-cultured with mouse lymphocytes for 4 h. The arrowheads indicate intensely stained lymphocytes attached to the myoblasts. c: Human myoblasts co-cultured with mouse lymphocytes for 24 h. The lymphocytes were then removed and the myoblasts were cultured alone for a further 24 h. The rabbit antibody reacts with the murine form of p-glucuronidase only, and not with the enzyme present in human cells (Olsen et al., 1986).

Uptake of exogenous P-glucuronidase Myoblasts also had the ability t o acquire additional lysosomal enzyme activity from the culture medium by endocytosis mediated by M6P receptors. This was shown in experiments in which purified human p-glucuronidase was added to cultures of L,, C, and a third muscle cell line, G8 (Table 3). Endocytosis of the human enzyme increased the activity of all the muscle

DISCUSSION A number of previous studies have shown that both skeletal muscle cells (Fischer et al., 1980; Salminen, 1984) and certain muscle cell lines (Reuser et al., 1984) express the MPR and are capable of internalizing phosphorylated forms of exogenous lysosomal enzymes (Reuser et al., 1984). In the case of muscle cells from Pompe’s disease patients deficient in the lysosomal enzyme acid a-glucosidase, the enzyme acquired by endocytosis via MPR reversed the accumulation of glycogen previously present in the affected cells (Di Marco et al., 1985; van der Ploeg et al., 1988b). In the present study we have demonstrated that non-deficient myoblasts can also utilise the MPR pathway for internalizing another lysosomal enzyme, p-glucuronidase, from the medium, significantly increasing the total enzyme activity in these myogenic cells. Our observation that these cells also produced a phosphorylated form of pglucuronidase, which could be endocytosed by MPRs present in other type of cell, confirms the presence of the MPR pathway in myogenic cells. A major finding of our investigation is that skeletal muscle myoblasts can also acquire additional lysosoma1 enzyme by an entirely different mechanism, which has previously been demonstrated only for dermal fibroblasts as recipient cells (Olsen et al., 1983). In this process, when myoblasts were cultured together with normal lymphocytes, we found a significant increase in p-glucuronidase activity in both types of cell. By coculturing lymphocytes and myoblasts which produced different forms of this enzyme, readily distinguished by thermal lability and electrophoretic mobility, the increase in myoblast activity was found to be due to the acquisition of enzyme derived directly from the lymphocytes, and not due to the activation of endogenous myoblast enzyme. Nor was this increased activity due to the presence of contaminating lymphocytes, since these accounted for less than 5% of the total cells and could have contributed less than 5% of the increase in enzyme activity measured after co-culture. Since the lymphocytes used in these studies do not secrete lysosoma1 enzymes, as has been reported previously (Olsen et al., 19821,enzyme acquisition by the myoblasts could

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BEAUCHAMP ET AL. T

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4

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2 4

CBA mouse lymphocyte P-glucuronidase

L6 rat myoblast P-glucuronidase

0

60

30

90

Time (min a t 64°C) Fig. 5. Thermal stability of P-glucuronidase activity in extracts of L, rnyoblasts and CBA mouse lymphocytes. (Error bars = SEM (n = 31.)

TABLE 2. Thermal stability of p-glucuronidase in L, myoblasts and CBA lymphocytes’ p-glucuronidase activity (U/107 cells) Before heating After heating % inactivation

L, rnyoblasts: Cultured alone After co-culture CBA lymphocytes: Cultured alone After co-culture

5

155 209

56

97 73

40 60

38 58

5 3

‘Lysates were prepared from myoblasts and lymphocytes that had been cultured alone and from cells that had been co-cultured for 24 h a t 37°C. P-glucuranidase activity was measured before and after heating the lysates a t 64°C for 45 min, as described in the Materials and Methods.

1

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6

Balb/c m o u s e 1YmPhOCYte P-glucuronidase Fig. 6. Identification by PAGE of acquired P-glucuronidase activity in co-cultured myoblasts. Cell extracts were prepared, run on a n 8% polyacrylamide gel, and stained for p-glucuronidase activity as described in the Materials and Methods Lanes 1 and 2 contain extracts from L, myoblasts cultured alone, before, and after heat treatment, respectively. Lanes 3 and 4 contain Balbic mouse lymphocyte extract before and after heating. Lanes 5 and 6 contain extracts from I,, myoblasts which had been co-cultured with Balbic lymphocytes before and after heat treatment.

not have occurred by MPR-mediated endocytosis of pglucuronidase secreted from the lymphocytes into the culture medium. This was conclusively demonstrated in experiments in which we found that the increase in myoblast activity after co-culture was not inhibited by

the addition of M6P, in contrast to the effect of M6P on the endocytosis of human enzyme by the myoblasts. It is notable that co-culture with myoblasts also resulted in increased P-glucuronidase activity in the lymphocytes themselves. Since there was no evidence for the presence of a myoblast form of the enzyme in the lymphocytes, it is likely that contact with the cell surface of the myoblasts was a n accessory activation signal for the lymphocytes, stimulating the de novo synthesis of new enzyme in these cells. These findings are consistent with previous observations t h a t cycloheximide, a n inhibitor of protein synthesis, prevented both the increase in enzyme activity in the lymphocytes and the transfer of enzyme to fibroblasts (Olsen et al., 1982, 1988). The direct transfer of P-glucuronidase from lymphocytes to myoblasts was wholly dependent upon direct contact between these cells. This was shown by experiments in which the cells were cultured in a common medium, separated from each other by a filter which prevented physical contact but not the exchange of macromolecules. Under these conditions both the transfer of enzyme to the myoblasts and the increase in lymphocyte activity were abolished. Contact dependence may also explain the effects of changes in lymphocyte:myoblast ratios on the levels of P-glucuronidase activity during co-culture. As increasing numbers of lymphocytes were applied to the myoblasts, a maximum level of enzyme acquisition by the myoblasts was reached, presumably because there was only a limited number of myoblast sites to which the lymphocytes could adhere. This would also explain why the lymphocyte activity per cell decreased as the proportion of lymphocytes increased, since a fixed number of myoblasts would be able to stimulate only a certain number of lymphocytes during co-culture. The addition of increasing numbers of lymphocytes would consequently reduce the ratio of stimulated to unstimulated cells and thus lead to a smaller increase in enzyme activity calculated on the basis of p-glucuronidase per lymphocyte. It is of interest that while in previous experiments human dermal fibroblasts co-cultured with murine

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LYSOSOMAL ENZYME ACQUISITION BY MYOBLASTS TABLE 3. Uptake of purified human platelet p-glucuronidase by myoblasts' p-glucuronidase activity (U/107myoblasts) Myoblast line L6 C, Gs

Cultured alone 340 37 186

t

Human enzyme + M6P

+ M6P

+ Human enzyme

3 20

720

36 177

79

360 30

288

189

'200 U of human platelet Ij-glucuronidase and 10 mM M6P were added in 1 ml of medium to monolayer myoblast cultures containing lo6 myoblasts, and incubated for 16 h a t 37°C as described in the Materials and Methods.

TABLE 4.Uptake of purified myoblast 6-glucuronidase by deficient human fibroblasts' p-glucuronidase activity (U/107 fibroblasts) Additions to fibroblast culture None L, P-glucuronidase G. B-alucuronidase

-

M6P 9 439 390

+ M6P 19 12 11

'50 U of purified myoblast P-glucuronidase and 10 mM M6P were added in 1ml of medium to monolayer fibroblast cultures containing 2 x 10' cells, and incubated for 16 h at 3 7 T , ias described in the Materials and Methods.

3

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d

lo ]

5

0 0

10

20

30

40

Fraction number Fig. 7. HPLC analysis of p-glucuronidase activity in C, myoblasts following culture with human platelet enzyme. Extracts were prepared from (a)human platelets, (b)C, myoblasts, and ( c ) C, myoblasts cultured in medium containing purified human platelet p-glucuronidase and analysed on a n HPLC anion-exchange column, as described in the Materials and Methods.

lymphocytes acquired approximately 300 U of p-glucuronidase/107 cells (Olsen et al., 19821, in the present experiments only some 20% of this amount was transferred to the L, myoblasts and only 10%to the C, cells. Our observation that the L, cell line is more fibroblastic in appearance and less myogenic than the C, myoblasts suggests that the expression of components involved in lymphocyte binding, stimulation, and subsequent transfer is greater on fibroblastic than on myogenic cells. Moreover, within the myogenic lineage itself there may also be a down-regulation of these molecules during development, being far lower on the C, cells and, in experiments not described here, very greatly reduced on fully differentiated muscle cells. The transfer of lysosomal enzymes from lymphocytes

to myoblasts could be of importance in vivo, since these enzymes have been implicated in normal muscle development, particularly in fusion (Colella and Bird, 1982; Holland and Herscovics, 1986). Lysosomal activities have also been reported to be elevated in a number of myopathic conditions characterized both by leucocyte infiltration and by muscle degeneration and regeneration (Kar and Pearson, 1980; Salminen et al., 1988). Although the inflammatory cells undoubtedly directly contribute a significant proportion of this enhanced activity, the muscle cells have also been shown to have substantially increased enzyme activity, both in degenerating and in adjacent, unaffected areas (Salminen and Kihlstrom, 1985). The direct transfer of lysosomal enzymes from lymphocytes, involving specific surface interactions, could thus play a central role in targeting such enzymes to satellite cells and myoblasts. This may be involved in promoting myoblast migration (Hughes and Blau, 19891, muscle fusion, and repair. Enzyme transfer may also occur to highly catabolic muscle fibres which have been suggested to serve as a source of amino acids and carbohydrates for the adjacent, regenerating fibres (Salminen and Kihlstrom, 1985).

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