of Gus isolated from 3T3 cells (Dean et al. 1985) was added to the .... increased incubation time (Dean and Martin, 1988). Intracellular localization of Gus by ...
Contact-dependent transfer of a lysosomal enzyme from lymphocytes to fibroblasts
GEORGE BOU-GHARIOS1, JILL MOSS2, TERRY PARTRIDGE2, DAVID ABRAHAM1 and IRWIN OLSEN1 l
Cell Enzymology Unit, Kennedy Institute of Rheumatology, 6 Bute Gardens, London W6 7DW, UK Department of Histopathology, Charing Cross and Westminster Medical School, Fulham, Palace Road, London W6 8RF, UK
2
Summary In this study we have examined the mechanism underlying the contact-mediated transfer of a lysosomal enzyme from lymphocytes to fibroblasts in culture. We found that although antibody against the mannose 6-phosphate lysosomal targetting receptor (MPR) completely inhibited flbroblast uptake of the lysosomal enzyme /J-glucuronidase (Gus) from the culture medium, it had no effect on the transfer of the enzyme from normal lymphocytes. In contrast, the presence of antibody that prevented the adhesion of the lymphocytes to the fibroblasts inhibited Gus
acquisition but had no effect on endocytosis. Immunogold electron microscopy of the contact site between the two types of cell showed that the transfer of Gus involved uncoated vesicles localized near the cell surface of the fibroblast at sites of contact with the lymphocytes. The acquired lymphocyte enzyme was shown to be transported to the fibroblast lysosomes.
Introduction
bolic defect of the recipient cells (Abraham et al. 1985). Thus acquisition of a lysosomal enzyme from lymphocytes, a major cellular component arising in the bone marrow, could play an important part in enzyme replacement therapy in vivo. The present study examines the localization and transport of a representative lysosomal enzyme, /3-glucuronidase (Gus), during its transfer directly from lymphocytes to fibroblasts.
The lysosomal storage diseases are a major group of metabolic disorders resulting from the inherited deficiency of specific lysosomal enzymes (Stanbury et al. 1983). Therapeutic treatment by replacement of the missing enzyme has been attempted in a number of different ways with only limited success (Desnick, 1980). Transplantation with normal bone marrow, which is able to repopulate the haemopoietic compartment and could permanently provide the missing enzyme to affected cells of the recipient, has been shown to produce some clinical improvement (Hugh-Jones et al. 1984; Krivit and Paul, 1986). Although little is known about the mechanism(s) by which the correction of lysosomal deficiency disorders is achieved in vivo, two processes have been identified in vitro by which cells can acquire lysosomal enzymes. The better-characterized process involves the active endocytosis of'high-uptake' forms of the enzyme, which contain phosphorylated mannose residues, by fibroblasts and other types of cell (Wileman et al. 1985). This process is mediated by surface receptors specific for mannose 6-phosphate (Willingham et al. 1981; Kornfeld and Mellman, 1989), although other receptors have also been identified that recognize different residues on some lysosomal enzymes and other proteins (von Figura and Hasilik, 1986). A second process has been described whereby certain lysosomal enzymes are transferred directly from normal lymphocytes to deficient cells via a mechanism that requires cell-to-cell contact (Olsen et al. 1981, 1983). The transferred lymphocyte enzyme has been shown to be biologically functional, effectively correcting the metaJournal of Cell Science 100, 443-449 (1991) Printed in Great Britain © The Company of Biologists Limited 1991
Key words: lysosomal enzyme, lymphocytes, fibroblasts.
Materials and methods Preparation of murine lymphocytes Single-cell suspensions of lymphocytes from murine spleen were obtained as described previously (Olsen et al. 1982). They were cultured at an initial density of 106 cells ml" 1 , in RPMI 1640 medium supplemented with 5% foetal calf serum (FCS), 2mgml~ 1 of concanavalin A (Con A) and 5 X 1 0 ~ 5 M 2-mercaptoethanol. After 72 h the culture contained approximately 2.5xlO 6 cells ml" 1 , 80% of which were activated T-cells characterized by their morphological appearance as lymphoblasts.
Gus-deficient fibroblasts Fibroblasts from patients with mucopolysaccharidosis type VII (/Sglucuronidase-deficient; GM 151 cells) were supplied by the Human Genetic Mutant Repository (Camden, New Jersey). They were grown as monolayers in tissue culture flasks (10 cm2) or in Lab-Tek Flaskettes (ICN Biomedicals Ltd, UK) in Eagle's minimal essential medium (MEM) containing 10% FCS as described (Bou-Gharios et al. 1988a). Cells were used between passages 5 and 12 for biochemical and immunocytochemical analysis. 443
Co-culture of lymphocytes and fibroblasts Con-A-activated T-lymphocytes were centrifuged at 200 # for 10 min at 20°C, resuspended in full MEM and re-centrifuged. They were resuspended to a density of 107 cells ml" 1 in the full medium and placed directly onto the confluent monolayers of the Gus-deflcient fibroblasts (GM 151 cells) for 1,6 and 24 h.
Effect of antibodies on direct transfer and endocytosis The antibodies used were raised in rabbits against purified lymphocyte Gus (anti-Gus) (Bou-Gharios et al. 19886), the cationindependent mannose 6-phosphate receptor (anti-MPR) (gift from G. Griffiths, EMBL, Heidlberg, Germany) and purified lymphocyte plasma membranes (anti-PM) that did not detect MPR (Abraham et al. 1989). Each one was added separately, at concentrations of 0.04, 0.2 and 1.0 mg ml" 1 , to confluent fibroblast monolayers for l h prior to adding lymphocytes, and remained present during the subsequent culture period of 24 h. The nonadherent lymphocytes were then decanted and the fibroblast monolayer was washed three times with fresh medium and then with trypsin for 1-2 min at 37 °C, which removed most of the lymphocytes still attached to the monolayer. Extracts of the fibroblasts and remaining contaminating lymphocytes (only fewer than 1 % of the total number of lymphocytes added initially still remained adherent after this procedure, as determined by direct counting in a haemocytometer) were prepared using 0.1 % Triton X-100 and were assayed for Gus activity, as described previously (Olsen et al. 1982). Similar experiments were carried out to test the effect of these antibodies on enzyme uptake by receptor-mediated endocytosis, where in place of the lymphocytes, a 'high-uptake' molecular form of Gus isolated from 3T3 cells (Dean et al. 1985) was added to the fibroblast cultures. Control cultures consisted of fibroblast monolayers co-cultured with lymphocytes or 3T3 enzyme for 24 h, in the absence of any added antibody. The adhesive interaction of the lymphocytes with the fibroblasts was measured quantitatively as previously described (Abraham et al. 1989). In brief, lymphocytes labelled with [me£fryZ-3H]thymidine were added to confluent fibroblast monolayers and incubated for 1 h at 37 °C. The non-adherent cells were carefully removed by aspiration, and, after washing, the fibroblast monolayers and attached lymphocytes were solubilized and the radioactivity that remained associated with the fibroblast monolayer was measured by scintillation counting.
Biochemical analysis Subcellular fractionation of co-cultured fibroblasts. GM 151 fibroblasts were co-cultured with lymphocytes for 1, 6 and 24 h, the non-adherent lymphocytes decanted and the fibroblasts detached by trypsin treatment. The cells were disrupted by cavitation under 501bfin~2 (llbfin~ 2 =6.9Pa) nitrogen (Rome et al. 1979). Unbroken cells and debris were removed by centrifugation at 800 g for 10 min, and a particulate fraction was obtained by centrifuging the supernatant at 30 000 g for 10 min. The pellet, which contained more than 80 % of the total Gus activity, was centrifuged in a self-generating gradient of 50 % Percoll in isotonic sucrose, and fractions were collected and assayed for enzyme activity as described previously (Abraham et al. 1986). Enzyme assays. Gus and cv-D-mannosidase activities were measured in cell extracts and subcellular fractions using their respective fluorogenic 4-methylumbelliferyl substrates (Olsen et al. 1982,1988). One unit of activity (U) is defined as the amount of activity that liberates 1 nmol of 4-methylumbelliferone per hour. 5'-Nucleotidase, a marker enzyme for the plasma membrane, was assayed by the release of radiolabelled adenosine, as previously described (Abraham et al. 1986).
Immunofluorescence staining The washed monolayers were fixed in situ with 3 % formaldehyde in phosphate-buffered saline (PBS) for 15 min at 20 °C and washed 3 times in PBS. They were then treated for 5 min with freshly prepared O.Smgml"1 sodium borohydride in PBS, washed again with PBS, and incubated for 20 min in PBS containing 0.1% saponin, 20 % normal goat serum and 1 mM EGTA. After washing
444
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3 times with 0.1% saponin in PBS, anti-Gus was added (0.1 mg protein ml" 1 in saponin-goat serum) for 60 min at 20 °C. The monolayers were washed with 0.1% saponin and incubated for 30 min with the secondary antibody, FITC-conjugated goat antirabbit IgG (ICN Biomedicals Ltd., UK). After washing three times with 0.1 % saponin in PBS and then in PBS alone, the cells were mounted in 95 % (v/v) glycerol in PBS and examined under a Zeiss photomicroscope III fitted with an epi-fluorescence condenser III RS and a x25 objective. Electron microscopy Morphological examination of co-cultures. Confluent monolayers of GM 151 cells were grown in Lab-Tek Flaskettes (ICN Biomedicals Ltd., UK) and co-cultured with 107 lymphocytes ml" 1 for 1, 6 and 24 h. After removal of non-adherent lymphocytes, the monolayers were fixed in glutaraldehyde, postfixed in osmium tetroxide, processed and embedded in situ in Spurr's resin as previously described (Bou-Gharios et al. 1988a). Immunogold electron microscopy. Co-cultures were carried out as described above, and the monolayers were fixed in 0.5% glutaraldehyde and embedded in Lowicryl K4M resin (BouGharios et al. 1988a). Immunogold labelling was carried out as previously described (Bou-Gharios et al. 19886). Briefly, ultrathin sections of Lowicryl K4M-embedded co-cultures were incubated first with 50 ;d of 20 % normal goat serum in PBS containing 1 % bovine serum albumin and 1% Tween-20, for 20 min at 20 °C, followed by 2h incubation with the rabbit anti-Gus. After washing with buffer, the sections were incubated for 60 min with goat anti-rabbit IgG conjugated to 10 nm colloidal gold (Janssen, UK). After washing, the grids were stained with aqueous uranyl acetate and Reynold's lead citrate. Morphometric analysis. This was carried out to determine the statistical validity of the observed accumulation of gold staining (Gus antigen) within certain intracellular regions. The total area of the photomicrograph, and the areas of individual vesicles, were measured using a semi-automated image analysis system (Cambridge Electronics Design, Ltd., UK). The total number of gold particles in the whole area of the untrimmed photograph, and the number localized within particular organelles, were counted manually. We calculated the expected frequency of gold in each vesicle, which is the number of gold particles per unit area of the whole micrograph multiplied by the area of the vesicle. The probability (P) of finding the actual number of gold particles in each individual organelle was calculated from the Poisson distribution of the expected frequency.
Results Effect of antibodies on cell interaction and Gus activity in fibroblasts Addition of anti-PM, anti-Gus and anti-MPR had markedly different effects on lymphocyte—fibroblast interaction and the level of enzyme activity in the fibroblasts, as shown in Fig. 1. Whereas anti-PM strongly inhibited both the adhesion of the lymphocytes to the fibroblast monolayer and the acquisition of Gus activity from the lymphocytes, it had no effect on endocytic uptake of a purified Gus enzyme (from 3T3 cells) added to the fibroblast culture medium. In contrast, anti-Gus and antiMPR had no effect on either lymphocyte attachment or enzyme transfer from lymphocytes, but both antibodies prevented endocytosis by the fibroblasts of the highuptake form of Gus. Subcellular localization of Gus in recipient fibroblasts The contact-dependent acquisition of Gus activity by the GM 151 cells was further explored by fractionation on a Percoll density gradient. When the Gus-deficient fibroblasts were co-cultured with normal murine lymphocytes for l h and extracts centrifuged through the gradient,
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Fig. 1. The effect of antibodies on lymphocyte adhesion and the acquisition of Gus activity by GM 151 cells. Increasing concentrations of antibodies against: (A) lymphocyte plasma membranes (anti-PM); (B) Gus; and (C) MPR were added to GM 151 monolayers. In A, the binding of radiolabelled lymphocytes was measured as described in Materials and methods. (B) and (C) show the effects on the aquisition of Gus activity during co-culture with lymphocytes and incubation with 3T3 enzyme, respectively. The results are presented as the percentage of binding (A) and enzyme activity (B and C) of control cultures that did not contain any antibody.
more than 90 % of the Gus activity was present in a lowdensity region (density 1.04-1.06 g ml ; Fig. 2A). However, after 6 h of co-culture approximately 20 % of the total enzyme activity was recovered from a region of higher density (1.08-1.lOgml" 1 ; Fig. 2B). When the lymphocyte-fibroblast co-culture was extended to 24 h, the proportion of activity in this latter region increased further, to 35 % of the total (Fig. 2C). A previous study has shown that exogenous, purified Gus internalized into GM 151 cells by receptor-mediated endocytosis was also transported from a low- to a high-density fraction with increased incubation time (Dean and Martin, 1988). Intracellular localization of Gus by immunofluorescence The pattern of murine Gus-specific immunofluorescence in the human fibroblasts was also found to depend on the period they had been co-cultured with the normal murine lymphocytes. After l h , the lymphocyte enzyme was
12 16 Fraction number
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Fig. 2. The distribution of lymphocyte Gus activity in subcellular fractions of GM 151 cells. Lymphocytes were cocultured with GM151 cells for l h (A), 6h (B), and 24h (C). Fibroblast extracts were prepared and analysed on a Percoll density gradient. The density profile of the gradient is shown by the continous line in A and peak regions indicated by arrows. Note the progressive increase in Gus activity in the high-density region of the gradient. The continuous line in Fig. 2B shows the bi-modal distribution of the endogenous O--Dmannosidase activity present at all times in the GM 151 cells. (C) shows the location in the gradient of the GM 151 plasma membrane enzyme 5'-nucleotidase, whose activity was found mainly at a very low density (approximately 1.02gml"1).
detected scattered throughout the cytoplasm of the recipient cells (Fig. 3A), whereas after 24 h of co-culture Gus staining was also observed in discrete granules that accumulated mainly in the perinuclear region of the fibroblasts (Fig. 3B). In control experiments, no fluorescent labelling was seen in enzyme-deficient human fibroblasts that had been cultured in the absence of the murine lymphocytes. Ultrastructural changes in fibroblasts co-cultured with lymphocytes Transmission electron microscopy of fibroblast monolayers co-cultured with lymphocytes for either 1, 6 or 24 h showed the presence of large numbers of small vesicles accumulated within the enzyme-deficient cells at sites of contact with the lymphocyte (Fig. 4A). When observed at higher magnification, these structures did not appear to be coated (Fig. 4B). Fibroblasts cultured alone contained very few vesicles of this type (not shown). Contact-dependent transfer of a lysosomal enzyme
445
Fig. 3. Immunofluorescence localization of Gus in GM 151 cells. After 1 h of co-culture (A), lymphocyte Gus is seen distributed throughout the cell body of the fibroblasts, while after 24 h of co-culture (B), the staining is also localized in punctate structures predominantly in the perinuclear region (x900).
Immunogold detection of lymphocyte Gus in recipient fibroblasts Immunogold labelling showed that after 1 h of co-culture with lymphocytes the murine Gus antigen could readily be detected in the GM 151 cells. At this time, gold particles localized predominantly in small, non-coated vesicles near the cell surface of the fibroblast (Fig. 5A,B), and were also sometimes found in larger organelles more distal to the fibroblast plasma membrane (Fig. 5A). Morphometric analysis, carried out as described in Materials and methods, showed that the P values for the frequencies of gold in the small, peripheral vesicles are =£6.5xlO~8 (Fig. 5A) and 5=5.1x10"* (Fig. 5B), while the probability for the actual gold found in the larger, distal vesicle is s=2.1xl(T 5 (Fig. 5A). After 6 h of co-culture some lymphocyte Gus antigen was found to have accumulated in electron-dense bodies within the cell body of the fibroblast (Fig. 5C), while after 24 h it was also present in large, multivesicular bodies (Fig. 5D). In control experiments gold labelling was not found in the enzyme-deficient human fibroblasts cultured alone, or when anti-Gus (primary antibody) was either omitted or substituted by non-immune rabbit immunoglobulin. Discussion It is now well known that many types of cells are capable of internalizing macromolecules by a process that involves specific receptors localized at the cell surface. In the case of lysosomal enzymes, this is mediated by mannose 6phosphate receptors (MPR), which are also responsible for 446
G. Bou-Gharios et al.
the intracellular trafficking to the lysosomes of endogenous lysosomal enzymes containing mannose 6-phosphate (reviewed by Kornfeld, 1987). A number of studies have shown that high levels of lysosomal enzymes can be acquired not only by receptor-mediated endocytosis, but also by direct transfer during cell-to-cell contact with, bone marrow-derived cells (Olsen et al. 1983; McNamara et al. 1985). This type of process is likely to play an important part in the successful therapy of lysosomal deficiency diseases by bone marrow transplantation (Hobbs, 1989), since lysosomal enzymes have been shown to be transferred from lymphocytes with high efficiency (Olsen et al. 1982) and to be biologically effective in correcting the metabolic defect in vitro (Abraham et al. 1985). In the present study we have found, by antibody blocking experiments, that the MPR is not involved in this process, since anti-MPR had no effect on the transfer of Gus from normal lymphocytes to enzyme-deficient fibroblasts. Our observation that anti-Gus, like anti-MPR, effectively inhibited endocytosis of a 'high-uptake' form of Gus from the culture medium but did not affect the acquisition of the lymphocyte enzyme, also suggests that the enzyme is not transferred by secretion and endocytosis via the MPR. This result confirmed our previous findings that mannose 6-phosphate itself, a potent inhibitor of MPR-mediated endocytosis via the MPR, had no effect on the contact-dependent transfer of Gus (Olsen et al. 1986). However, the finding that the MPR is not involved in this process does not necessarily preclude a role for a different transport mechanism that could enable the enzyme to migrate through 'protected clefts' that form along zones of contact between cells (Dean et al. 1991).
Fig. 4. Transmission electron microscopy of a contact site between a lymphocyte (L) and afibroblast(Fl). Monolayer co-cultures were embedded in Spurr's resin and cut at right angles. (A) shows the presence of small vesicles (arrows) within the nbroblast cytoplasm along the site of contact with a lymphocyte (x 15 500). (B) A higher magnification of this region (x 43 500). Very few vesicles were observed at the surface offibroblast(F2) that was not in contact with a lymphocyte.
The results of subcellular fractionation and immunofluorescence show that, like MPR-mediated internalization of lysosomal enzymes, enzyme transferred from lymphocytes is found first in a low-density fibroblast compartment and then, at later times, also in a higher-density region of the Percoll gradient. While the latter is known to contain lysosomal enzyme activity and lysosomal membrane proteins but no MPR (Olsen et al. 1990), the former fraction is far more heterogeneous and is likely to contain components originating in the endosomal and Golgi complex (Abraham et al. 1986). These changes in the subcellular distribution of the transferred lymphocyte lysosomal enzyme correspond to its detection by immunofiuorescence initially within the body and peripheral cytoplasmic regions of the recipient fibroblasts, and subsequently its accumulation in distinct vesicles in the perinuclear area. Similar intracellular transport has also been reported for Gus and other lysosomal enzymes internalized by endocytosis (Dean and Martin, 1988; Rome et al. 1979; Merion and Sly, 1983). The perinuclear vesicles have also previously been shown to contain endogenous lysosomal enzymes (Olsen etal. 1988; Lagunoffei al. 1973)
and are considered to be lysosomes (Parenti et al. 1987; Willingham, 1983). The results of transmission electron microscopy suggest that initial routing of the lymphocyte enzyme in the fibroblasts is likely to be via non-coated vesicles that are formed at the contact sites between the two types of cell. This is supported by immunogold staining, which shows that the lymphocyte Gus antigen is present in small structures localized near the fibroblast cell surface. The origin of these micropinocytic vesicles is unknown, but they are probably derived entirely from the fibroblast plasma membrane, since, in experiments not shown here, they were found not to contain murine plasma membrane antigens. Similar non-coated microinvaginations may be involved in endocytosis of certain exogenous ligands (Heut et al. 1980; Montesano et al. 1982), and may also be responsible for the contact-induced increase in pinocytotic rates in fibroblasts and epithelial cell cultures (Kaplan, 1976). Immunogold electron microscopy also showed that, in the initial stages of lymphocyte enzyme transfer, Gus is also present in larger organelles, more distal to the Contact-dependent transfer of a lysosomal enzyme
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Fig. 5. Immunogold labelling of lymphocyte Gus in GM 151 cells. Lymphocytes were co-cultured with the fibroblasts for: (A,B) 1 h, (C) 6h and (D) 24 h, and the monolayers fixed and embedded in Lowicryl K4M resin as described in Materials and methods. In A and B, gold particles are seen in small, peripheral non-coated vesicles (small arrows) near the cell surface (arrowheads). In A, gold is also seen in a larger vesicle (asterisk), while in B no gold is found in coated vesicles (large arrow) (x 54 000). In C the gold labelling is accumulated within an electron-dense body (x 42 000); and by 24 h (D) gold particles are also present in a large multivesicular body (x 42 000).
fibroblast cell surface. These appear to be associated with a vesicular complex that may have formed by the fusion of uncoated vesicles, possibly 'early endosomes' (Helenius et al. 1983). It is notable that other studies have shown that ligands internalized via coated pits also reach the same endosomal compartment as those associated with non448
G. Bou-Gharios et al.
coated vesicles (Tran et al. 1987). At later times of interaction, a substantial amount of Gus is seen to accumulate in other distinct organelles, which are probably lysosomes, as suggested by subcellular fractionation. Although the mechanism underlying the egress of lysosomal enzymes from lymphocytes is still unknown, it
has been shown that it is the high molecular weight precursor forms of these enzymes that are transferred from the low-density 'pre-lysosomal' compartment (Olsen et al. 1988). This finding is consistent with the observation that during cell-to-cell interaction there is a re-orientation of the biosynthetic/cytoskeletal network of lymphocytes towards sites of contact with other cells (Dennert et al. 1986; Henkart, 1985; Andr6 et al. 1990), suggesting that Gus is most likely to be transferred from either the rough endoplasmic reticulum or the Golgi complex of the lymphocytes. The experiments described here thus show that lysosomal enzymes transferred from lymphocytes by cell-tocell contact are transported intracellularly by a mechanism that may be very similar to that involved in the trafficking of ligands internalized by endocytosis. Although these two processes may not occur with precisely the same kinetics, their functional consequence is the delivery of the enzyme to the lysosome, wherein it is metabolically, and hence clinically, effective in the breakdown of storage products that accumulate in lysosomal deficiency diseases.
clustering and endocytosis of HLA antigens on cultured human fibroblasts. Cell 21, 429-438. HOBBS, J. (1989). Correction of Certain Genetic Diseases by Transplantation (ed. J. R. Hobbs), published by the Cogent Fund, c/o Westminster Medical School Research Trust, London.
The authors express their gratitude to the Wellcome Trust and Action Research for supporting this work. We are grateful to Dr M. Barrett for the image analysis, Mrs G. Adams for the preparation of Gus antibody, Mr J. Beauchamp for his technical assistance and Ms A. Winter for typing the manuscript.
MONTESANO, R., ROTH, J., ROBERT, A. AND ORCI, L. (1982). Non-coated
References ABRAHAM, D., BOU-GHARIOS, G., MUIR, H. AND OLSEN, I. (1989).
Adhesion of lymphoid cells to fibroblasts in tissue culture. Cell. Immun. 122, 33-47. ABRAHAM, D., BOU-GHARIOS, G., OLSEN, I., WINCHESTER, B., MUIR, H.
AND SMITH, R. (1986). Effects of mitogenic stimulation on lymphocytes o--D-mannosidases. Eur. J. Cell Biol 40, 167-175. ABRAHAM, D., MUIR, H., OLSEN, I. AND WINCHESTER, B. (1985). Direct
enzyme transfer from lymphocytes corrects a lysosomal storage disease. Biochem. biophys. Res. Commun. 129, 417-425. ANDRE, P., BENOLIEL, A., CAPO, C, FOA, C , BUFERNE, M., BOYER, C ,
SCHMITT-VERHULST, A. AND BONGRAND, P. (1990). Use of conjugates
made between a cytolytic T cell clone and target cells to study the redistribution of membrane molecules in cell contact areas. J. Cell Sci. 97, 335-347. BOU-GHARIOS, G., ADAMS, G., MOSS, J., SHORE, I. AND OLSEN, I. (1988a).
A simple technique for in situ embedding of monolayer cultures in Lowicryl K4M. J. Microsc. 150, 161-163. BOU-GHAROIS, G., MOSS, J., OLSEN, I. AND PARTRIDGE, T. (19886).
Ultrastructural localisation of a lysosomal enzyme in resin-embedded lymphocytes. Histochemistry 89, 69-74. DEAN, M. F., DIMENT, S. A., OSTLUND, C , JENNE, B. M. AND
CONTRECTOR, S. (1985). Iodinated fibroblast (3-glucuronidase is a ligand for receptor mediated endocytosis. Biochem. J. 229, 213-219. DEAN, M. F. AND MARTIN, C. (1988). Intracellular localization of /3glucuronidase in fibroblasts after direct transfer from macrophages. Biochem. J. 256, 335-341. DEAN, M. F., RODMAN, J., LEVY, M. AND STAHL, P. (1991). Contact
formation and transfer of mannose BSA gold from macrophages to cocultured fibroblasts. Expl Cell Res. 192, 536-542. DENNERT, G., KUPFER, A., ANDERSON, C. G. AND SINGER, S. J. (1986).
Reorientation of the Golgi apparatus and the microtubule organizing center: Is it a means to polarize cell-mediated cytotoxicity? In Mechanisms of Cell-mediated Cytotoxicity. II. Adv. exp. Med. Biol. vol. 184 (ed. Pierre Henkart and Eric Martz), pp. 83-117, Plenum Press, NY. DESNICK, R (ed) (1980). Enzyme Therapy in Genetic Diseases, vol. 2, Birth Defects Original Article Series, vol. XVI. Alan R. Liss, New York. HELENIUS, A., MELLMAN, I., WALL, D. AND HUBBARD, A. (1983).
Endosomes. Trends biochem. Sci. 8, 245-250. HENKART, P. A. (1985). Mechanism of lymphocyte-mediated cytotoxicity. A. Rev. Immun. 3, 31-58. HEUT, C, ASH., J. AND SINGER, S. (1980). The antibody-induced
HUGH-JONES, K., HOBBS, J., CHAMBERS, D., WHITE, S., BYROM, N., WILLIAMSON, S., BARRETT, J., HENRY, K. AND PATRICK, D. (1984). Bone
marrow transplantation in mucopolysaccharidoses. In Molecular Basis of Lysosomal Storage Diseases (Barranger J. A. and Brady R. O., eds), pp. 411-428, Academic Press, New York. KAPLAN, J. (1976). Cell contact induces an increase in pinocytotic rate incultured epithelial cells. Nature 263, 596-597. KORNFELD, S. (1987). Trafficking of lysosomal enzymes. FASEB J. 1, 462-468. KORNFELD, S. AND MELLMAN, I. (1989). The biogenesis of lysosomes. A. Rev. Cell Biol. 5, 483-525. KRIVIT, W. AND PAUL, N. W., eds (1986). Bone marrow transplantation for treatment of lysosomal storage diseases. March of Dime Birth Defect Foundation, Birth Defects: Original Article Series, vol. 22, no. 1. Alan R. Liss, Inc, New York. LAGUNOFF, D., NICOL, D. M. AND PRITZL, P. (1973). Uptake of /3-
glucuronidase by deficient human fibroblasts. Lab. Invest. 29, 449-453. MCNAMARA, A., JENNE, B. M. AND DEAN, M. F. (1985). Fibroblasts
acquire /5-glucuronidase by direct and indirect transfer during coculture with macrophages. Expl Cell Res. 160, 150-157. MERION, M. AND SLY, W. (1983). The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts. J. Cell Biol. 96, 644-650. membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature 296, 651-653. OLSEN, I., ABRAHAM, D., SHELTON, I., BOU-GHARIOS, G., MUIR, H. AND
WINCHESTER, B. (1988). Cell contact induces the synthesis of a lysosomal enzyme precursor in lymphocytes and its direct transfer to fibroblasts. Biochim. biophys. Ada 968, 312-322. OLSEN, I., BOU-GHARIOS, G. AND ABRAHAM, D. (1990). The activation of
resting lymphocytes is accompanied by the biogenesis of lysosomal organelles. Eur. J. Immun. 20, 2161-2170. OLSEN, I., DEAN, M., HARRIS, G. AND MUIR, H. (1981). Direct transfer of
a lysosomal enzyme from lymphoid cells to deficient fibroblasts. Nature 291, 244-247. OLSEN, I., DEAN, M., MUIR, H. AND HARRIS, G. (1982). Acquisition of/?-
glucuronidase activity in deficient fibroblasts during direct contact with lymphoid cells. J. Cell Sci. 55, 211-231. OLSEN, I., MUIR, H., SMITH, R., FENSOM, A. AND WATT, D. (1983). Direct
enzyme transfer from lymphocytes is specific. Nature 306, 75-77. OLSEN, I., OLIVER, T., MUIR, H., SMITH, R. AND PARTRIDGE, T. (1986).
Role of cell adhesion in contact-dependent transfer of a lysosomal enzyme from lymphocytes to fibroblasts. J. Cell Sci. 85, 231-244. PARENTI, G., WILLEMSEN, R., HOOGEVEEN, A., VERLEUN-MOOYMAN, M.,
VAN DONGEN, J. AND GALJAARD, H. (1987). Immunocytochemical
localization of lysosomal acid phosphatase in normal and 'I-cell' fibroblasts. Eur. J. Cell Biol. 43, 121-127. ROME, L., GARVIN, A., ALLIETTA, M. AND NEUFELD, E. (1979). Two
species of lysosomal organelles in cultured human fibroblasts. Cell 17, 143-153. STAHL, P., RODMAN, J., MILLER, M. AND SCHLESINGER, P. (1978).
Evidence for receptor-mediated binding of glycosidases by alveolar macrophages. Proc. natn. Acad. Sci. U.S.A. 75, 1399-1403. STANBURY, J., WYNGAARDEN, J., FREDRICKSON, D., GOLDSTEIN, J. AND
BROWN, M., eds (1983). Metabolic Basis of Inherited Disease, 5th edition. McGraw-Hill, New York. TRAN, D., CARPENTIER, J., SAWANO, F., GORDEN, P. AND ORCI, L. (1987).
Ligands internalized through coated or noncoated invagination follow a common intracellular pathway. Proc. natn. Acad. Sci. U.S.A. 84, 7957-7691. VON FIGURA, K. AND HASILIK, A. (1986). Lysosomal enzymes and their receptors. A. Rev. Biochem. 55, 167-193. WILEMAN, T., HARDING, C. AND STAHL, P. (1985). Receptor-mediated
endocytosis. Biochem. J. 232, 1-4. WILLINGHAM, M. (1983). An alternative fixation-processing method for preembedding ultrastructural immunocytochemistry of cytoplasmic antigens: The GBS (Glutaraldehyde-borohydride-saponin) procedure. J. Histochem. Cytochem. 31, 791-798. WILLINGHAM, M., PANSTAN, H., SAHAGIAN, G., JOURDIAN, G. AND
NEUFELD, E. (1981). Morphologic study of the internalization of a lysosomal enzyme by the mannose 6-phosphate receptor in cultured Chinese hamster ovary cells. Proc. natn. Acad. Sci. U.S.A. 78, 6967-6971. (Received 25 June 1991 - Accepted 5 August 1991)
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