Normal lymphocytes were found to adhere strongly to monolayer cultures of fibroblasts deficient in the lysosomal enzyme, j3-glucuronidase. During this co-culture, the fibroblasts acquired from the lymphocytes substantial amounts of this enzyme, which often accumulated at sites of contact between the two types of cell. Enzyme transfer was prevented by addition to the co-cultures either of purified lymphocyte plasma membranes or of antibody raised against such plasma membranes, but it was not inhibited by the addition of antibody raised against lymphocyte-derived β-glucuronidase. An active role for the lymphocyte in this contact-dependent process was suggested by interference contrast, immunofluorescence and scanning electron-microscopic studies. These revealed extensive arrays of projections of the lymphocyte that ramified over the fibroblast cell surface. By transmission electron microscopy, conspicuous clusters of micropinocytotic vesicles were evident in the cytoplasm of the ‘recipient’ fibroblasts, subjacent to the surface in regions closely apposed to adherent lymphocytes. Such high frequencies of these vesicles were restricted to sites of lymphocyte-fibroblast contact, suggesting that they may play an important part in the transfer of enzyme between these two types of cell.

A major group of inherited metabolic disorders has been identified as being associated with deficiencies of specific lysosomal enzymes (Stanbury et al. 1983). Therapy by replacement of the missing enzyme has been attempted in a number of ways, and transplantation of bone marrow from normal to enzyme-deficient individuals has been reported to have sometimes produced clinical improvements (Hugh-Jones et al. 1984).

Two mechanisms have been described whereby, at least in vitro, cells derived from individuals with inborn lysosomal storage diseases can acquire the missing enzymes. One, which is now well characterized (Creek & Sly, 1984), involves endocytosis, via mannose 6-phosphate receptors located on the cell surface, of ‘high uptake’ enzyme forms, which bear mannose 6-phosphate groups and are secreted into the medium by many types of cell. A second, quite different, mechanism has recently been described by which certain lysosomal enzymes are transferred to genetically deficient fibroblasts from normal lymphocytes (Olsen et al. 1983). This transfer is not inhibited by mannose 6-phosphate and requires cell-to-cell contact (Olsen et al. 1981, 1982). The present study examines the structural basis of this new type of contact-dependent enzyme transfer and investigates the importance of the lymphocyte plasma membrane in this process.

Cell culture

Lymphocytes were obtained from strain CBA mouse spleen and cultured for 72 h in the presence of 4μgml-1 concanavalin A (ConA) as described (Olsen et al. 1982). They were centrifuged (400g for 10min) and resuspended in complete medium (Eagle’s MEM containing 100μgml-1 streptomycin, 100 i.u. ml-1 penicillin and 10% foetal calf serum). Human fibroblasts deficient in β-glucuronidase (GM 151 cells) were obtained from the Human Genetic Mutant Repository and were grown in 35 mm plastic culture dishes in complete medium for at least 3 days by which time they had reached late logarithmic phase (approx. 3×104 to 4×104 cells per dish). For enzymeuptake and -transfer experiments only, they were grown for several more days, until they had reached confluence (105 cells per dish). In co-culture experiments the medium was replaced by 1 ml of fresh medium containing 107 lymphocytes and the culture was incubated again at 37°C in an atmosphere of 5 % CO2 in air for between 30 min and 24 h, as noted. At the end of this period, the non-adherent lymphocytes were carefully removed by washing the monolayer three times with warm PBS.

Cytochemical demonstration of β-glucuronidase

Monolayers of GM 151 cells were grown to logarithmic phase in culture dishes containing heat-sterilized coverslips (no. 1, 22mm2). After co-culture with 107 lymphocytes for 24h, the intracellular localization of β-glucuronidase activity was demonstrated by a modification of the procedure of Fishman & Goldman (1965) as previously described (Olsen et al. 1982). Incubation was carried out for 3h at 37°C, using the substrate, naphthol AS-BI-β-D-glucuronide, dissolved (0-lmgml-1) in O’lM-acetate buffer (pH4·0), which also served as a fixative. The reaction product was visualized by post-coupling with freshly prepared Fast Blue RR (1 mg ml-1) in 0·01 M-phosphate buffer (pH 7·4). After successive washes with PBS, methanol and distilled water, the coverslips were air-dried, inverted and mounted on slides with Styrolite for examination under the light microscope.

Interference-contrast microscopy

Monolayers of GM151 cells were grown on coverslips to logarithmic phase (3×105 cells) and cocultured for 6h with 107 lymphocytes. They were washed and mounted in HEPES-buffered complete medium, then examined at room temperature under the ×100 oil-immersion objective on a Leitz Orthoplan microscope fitted with interference-contrast optics.

Immunofluorescence

Primary antibody was raised in rabbits against plasma membranes prepared from ConA-activated mouse lymphocytes, as described below, and was used at a dilution of 1:250. It was visualized by the subsequent application of a 1:50 dilution of fluorescein-isothiocyanate (FITC)- conjugated goat anti-rabbit immunoglobulin G (IgG). Working dilutions of all antibodies were stored at 4°C in PBS containing 0·1 % sodium azide, and were centrifuged for 5 min at 10000g before application to the cell monolayers.

Washed logarithmic phase GM151 monolayers, which had been co-cultured for 6h with 107 lymphocytes, were fixed at room temperature for 30 min with 1 % formal saline buffered to pH 7·3 and stored in the same solution at 4°C. Prior to the application of the primary antiserum, the coverslips were washed with PBS for 30 min at room temperature. All subsequent applications of antisera and the intervening 30-min washes with PBS were also carried out at this temperature. After the application of the secondary, FITC-conjugated, antiserum and a final wash the coverslips were inverted and mounted in freshly prepared phosphate-buffered 95% glycerol (pH 7·2), containing 0·1 % diphenylenediamine (to inhibit bleaching of the fluor). Slides were examined within 1 h under a Leitz Orthoplan microscope fitted with an epifluorescence illumination system under ×50 and ×100 water-immersion objectives. GM151 cells, cultured alone or co-cultured with mouse lymphocytes, were found to exhibit a very low level of general ‘background’ fluorescence, which was not modified when the primary antibody was replaced by pre-immune rabbit serum or when normal non-immune goat serum was intercalated between the rabbit anti-mouse lymphocyte primary antiserum and the goat anti-rabbit IgG-FITC conjugate.

Electron microscopy

Monolayers for examination by scanning electron microscopy (SEM) were grown on glass coverslips and co-cultured for 6h as described. They were fixed overnight at 4°C in modified Karnovsky’s fixative (Hayat, 1981), washed in 0·4 M-cacodylate buffer (pH7·3) containing 30% sucrose, and dehydrated through ascending concentrations of ethanol. They were then dried from carbon dioxide in a Polaron E5100 critical-point drier. The coverslips were mounted on aluminium stubs, sputter coated with gold/palladium, and examined under a Cambridge 5600 scanning electron microscope.

For examination under the transmission electron microscope (TEM), confluent monolayers of GM151 cells were grown on squares (lcm2) of sterile Melinex and co-cultured with 107 lymphocytes for 6 h. The monolayers were washed to remove non-adherent lymphocytes and fixed with 1 % glutaraldehyde in 0·05 M-cacodylate buffer containing 0·3 %’Ruthenium Red for l.h at room temperature. After rinsing in 0·05 M-cacodylate buffer containing 0T % Ruthenium Red, the cultures were post-fixed in 1 % osmium tetroxide in cacodylate-Ruthenium Red for 3 h at room temperature. Samples were dehydrated rapidly through ascending grades of ethanol and embedded in Spurr’s resin. Ruthenium Red was used in order to enhance the ‘cell coat’. After the Melinex had been removed from the block, ultrathin sections (60–80 nm) were cut, stained with aqueous uranyl acetate and Reynold’s lead citrate, and examined using a Philips 201 electron microscope.

Enzyme uptake and transfer

Confluent monolayers of 105 GM151 cells were cultured for 24h in 1 ml of complete medium either with 107 lymphocytes containing approximately 40 units of β-glucuronidase or with 40 units of a high-uptake form of β-glucuronidase purified by DEAE-cellulose chromatography (Dean et al. 1982) from extracts of NS-1 cells (a murine lymphoblastoid cell line). Intracellular β -glucuronidase activity was measured in the fibroblasts after co-culture with lymphocytes by a quantitative singlecell cytochemical procedure (Olsen et al. 1981, 1982). After culture with NS-1 β-glucuronidase, enzyme activity was measured directly in intact cells by the following biochemical assay. The culture dishes were washed three times with warm PBS, and 0·5 ml of 2mM-4-methylumbelliferyl- β-D-glucuronide in 0·4 M-acetate buffer (pH 4’5) was added to each. After incubation with the cells for 1 h at 37°C, this solution was removed and mixed with 0’5 ml of 0·4M-glycine buffer (pH 10’4). The fluorescence produced by the liberated 4-methylumbelliferone was used to determine β - glucuronidase activity as previously described (Olsen et al. 1982). /1-Glucuronidase activity in extracts of cells and in preparations of purified enzyme was measured in the same way.

Preparation of lymphocyte plasma membranes

Plasma membranes of ConA-stimulated lymphocytes were prepared under sterile conditions by sedimentation in a discontinuous sucrose gradient (Koizumi et al. 1981) after nitrogen cavitation at 50Ibf in-2 (1 lbfin-2 ≈ 6·9kPa) for 10min at 4°C (Abraham et al. 1986). From 109 cells, approximately 1 mg of protein was obtained from the interface between the 20 % and 42 % sucrose layers. The specific activity of 5’-nucleotidase, an integral plasma membrane protein, was between 20 and 50 times higher in this fraction than in homogenates of whole lymphocytes. The plasma membranes were washed by centrifugation at 100000g for 60 min at 4°C, and resuspended at a concentration of 1 mg ml-1 in sterile PBS.

Preparation of antibodies

Antibody was raised in rabbits by subcutaneous injection of 0-5 mg of the plasma membrane protein preparation dispersed by sonic disruption in an equal volume of Freund’s complete adjuvant, followed 12 days later by a ‘booster’ injection of 0·5 mg in an equal quantity of the incomplete adjuvant. Serum was taken 12–20 days after the second injection and the IgG fraction was isolated by protein A-Sepharose chromatography. The antibody titre was determined by immunoprecipitation, with Staphylococcus protein A, of the lymphocyte plasma membrane preparation previously labelled with 125I, using the lodogen reagent (Pierce Chem. Co.). The antibody was 100-fold more reactive against mouse lymphocyte plasma membrane than against plasma membrane of GM151 cells, prepared and radiolabelled in an identical manner.

Antibody was prepared in the same way to murine lymphocyte β-glucuronidase that had been purified to electrophoretic homogeneity by polyacrylamide gel electrophoresis (Olsen et al. 1983), except that approximately 100μg of protein was given in each immunizing injection. The purified IgG fraction did not immunoprecipitate any of five other murine lysosomal enzymes tested β- hexosaminidase, a-iduronidase, β-glucosidase, α-mannosidase and aryl sulphatase), and its titre against β-glucuronidase from both murine lymphocytes and NS-1 cells was nearly 500-fold greater than against β-glucuronidase purified from normal human fibroblasts.

Splenic lymphocytes from the mouse rapidly adhered to β-glucuronidase-deficient human fibroblasts growing in monolayer culture. Within 30 min at 37°C, at least one, and an average of 8–10 lymphocytes remained attached to each GM 151 cell after the monolayer had been washed three times with PBS. By 6 h of co-culture, the average number of lymphocytes adherent to each fibroblast had decreased to approximately 3, and this ratio remained unchanged during the subsequent 18 h. As we have reported (Olsen et al. 1981, 1982), the interaction between these two types of cell was accompanied by direct transfer of the lymphocyte β-glucuronidase to recipient GM 151 fibroblasts, in which it reached a level of activity comparable with that in fibroblasts of individuals heterozygous for β-glucuronidase deficiency, who are clinically normal. The cytochemical procedure described in Materials and Methods showed that the enzyme accumulated conspicuously in the recipient fibroblasts in dense granular perinuclear deposits, presumably in lysosomes (Fig. 1). High concentrations of activity were also evident in apparent extensions of the lymphocyte onto the fibroblast surface. The lower level of β-glucuronidase activity seen more diffusely distributed throughout the cytoplasm of both types of cell is consistent with reports that this enzyme is associated in part with the network of microsomal membranes (Lorbacher et al. 1967; Ide & Fishman, 1969; Swank & Paigen, 1973).

Fig. 1.

Cytochemical localization of β-glucuronidase. GM151 cells, grown as monolayers on glass, were co-cultured with normal murine lymphocytes for 24 h. After washing and fixation, the coverslips were incubated with naphthol AS-BI-β-D-glucuronide and postcoupled with Fast Blue RR as described in Materials and Methods. Note the presence of enzyme reaction product in the fibroblast (fib) as darkly staining granules, whose distribution resembles that of lysosomes, and also in the adjacent region of the adherent lymphocyte (lym). The arrow indicates a high concentration of β-glucuronidase localized in what appears to be a cytoplasmic extension of the lymphocyte. The grey colour indicates the widespread, probably microsomal, distribution of the enzyme in the cell. GM151 cells do not stain at all by this procedure when they are cultured alone. ×800.

Fig. 1.

Cytochemical localization of β-glucuronidase. GM151 cells, grown as monolayers on glass, were co-cultured with normal murine lymphocytes for 24 h. After washing and fixation, the coverslips were incubated with naphthol AS-BI-β-D-glucuronide and postcoupled with Fast Blue RR as described in Materials and Methods. Note the presence of enzyme reaction product in the fibroblast (fib) as darkly staining granules, whose distribution resembles that of lysosomes, and also in the adjacent region of the adherent lymphocyte (lym). The arrow indicates a high concentration of β-glucuronidase localized in what appears to be a cytoplasmic extension of the lymphocyte. The grey colour indicates the widespread, probably microsomal, distribution of the enzyme in the cell. GM151 cells do not stain at all by this procedure when they are cultured alone. ×800.

This acquisition of β-glucuronidase was progressively inhibited by the addition to live co-cultures of increasing amounts of the antibody raised against lymphocyte plasma membrane (Table 1). The transfer of enzyme from 107 lymphocytes was 90% inhibited by 0·2 mg of antibody, approximately the amount required to immunoprecipitate the plasma membrane protein prepared from the equivalent number of lymphocytes. This antibody, even at Imgml-1, neither immunoprecipitated β-glucuronidase purified from the lymphocytes, nor inhibited receptor-mediated endocytosis by the GM 151 cells of a high-uptake form of β-glucuronidase prepared from NS-1 cells (89% of control).

Table 1.

Effects of antibodies and lymphocyte plasma membrane on the acquisition of P-glucuronidase activity by GM151 cells

Effects of antibodies and lymphocyte plasma membrane on the acquisition of P-glucuronidase activity by GM151 cells
Effects of antibodies and lymphocyte plasma membrane on the acquisition of P-glucuronidase activity by GM151 cells

When lymphocyte plasma membranes themselves were added during the coculture, they also markedly inhibited the transfer of β-glucuronidase, as shown in Table 1. As little as 0-02 mg of plasma membrane protein, derived from approximately 2×107 lymphocytes, reduced enzyme transfer to 46% of control, whereas 10 times this amount had no significant effect on uptake by these cells of NS-1 β-glucuronidase (87% of control).

Conversely, when antibody prepared against lymphocyte β-glucuronidase was added to co-cultures, even at a level (1 mg per culture) sufficient to precipitate the enzyme activity present in 109 lymphocytes (i.e. 100 times the number actually present in the co-culture), it had little effect on the direct transfer of the enzyme from the lymphocytes to the GM151 cells (87% of control). It did, however, inhibit endocytosis by the fibroblasts of high-uptake NS-1 β-glucuronidase (14% of control), an effect similar to that previously reported for antibody to some other lysosomal enzymes (Von Figura & Weber, 1978; Hasilik et al. 1981).

In control experiments, mannose 6-phosphate, as before (Olsen et al. 1981, 1982), had no effect on the contact-dependent transfer of β-glucuronidase from lymphocytes to GM 151 cells, but completely inhibited endocytosis of NS-1 β-glucuronidase (4% of control).

The morphological features of contacts between lymphocytes and GM 151 cells were examined in the living co-cultures by interference-contrast light microscopy. Fig. 2 demonstrates the extensive dendritic processes produced by some lymphocytes, apparently in intimate association with the fibroblast cell surface. This feature was demonstrated more vividly and more frequently when fixed co-cultures were stained with the rabbit antibody against mouse lymphocyte plasma membrane and then visualized with FITC-conjugated goat anti-rabbit IgG (Fig. 3). The human fibroblasts were not stained by this procedure but the lymphocytes showed prominent staining, which appeared to be localized on their surfaces and again revealed an extensive array of projections of these cells that ramified over the fibroblasts. No comparable structures were observed on those lymphocytes that were not in close association with fibroblasts. No fluorescent staining was observed in control experiments in which the FITC-conjugated secondary antibody alone was applied to the co-cultures or in which the primary antibody was replaced by pre-immune serum from the same animal.

Fig. 2.

Interference contrast microscopy of unfixed co-cultures of GM151 cells and normal murine lymphocytes. Note the extensive array of cytoplasmic processes from the lymphocyte (lym), indicated by arrows, ramifying over the surface of the fibroblasts (fib). These extensions appear to be on the undersurface of the fibroblast for they lie in the same focal plane as the glass substratum. ×600.

Fig. 2.

Interference contrast microscopy of unfixed co-cultures of GM151 cells and normal murine lymphocytes. Note the extensive array of cytoplasmic processes from the lymphocyte (lym), indicated by arrows, ramifying over the surface of the fibroblasts (fib). These extensions appear to be on the undersurface of the fibroblast for they lie in the same focal plane as the glass substratum. ×600.

Fig. 3.

Immunofluorescence staining of murine lymphocytes in co-culture with GM 151 cells by rabbit anti-mouse lymphocyte plasma membrane IgG followed by FITC-conjugated goat anti-rabbit IgG. Viewed by epi-illumination with 490nm light. The arrows indicate the extensive dendritic processes produced by the lymphocytes in close association with the barely visible fibroblasts. μ600.

Fig. 3.

Immunofluorescence staining of murine lymphocytes in co-culture with GM 151 cells by rabbit anti-mouse lymphocyte plasma membrane IgG followed by FITC-conjugated goat anti-rabbit IgG. Viewed by epi-illumination with 490nm light. The arrows indicate the extensive dendritic processes produced by the lymphocytes in close association with the barely visible fibroblasts. μ600.

The intimate relationship established in co-culture between lymphocytes and GM 151 fibroblasts was examined at the ultrastructural level. Scanning electron microscopy showed many projections from lymphocytes onto the surfaces of the fibroblasts (Fig. 4), although these were noticeably less extensive than those observed by light microscopy (Figs 2, 3). This may reflect disruption of such structures by the fixation and processing procedures, since in specimens prepared in this way the ratio of adherent lymphocytes to fibroblasts was substantially reduced. It is also possible that the very extensive projections of lymphocytes tend to form, as is the case in Fig. 2, between the fibroblasts and the glass substratum where they would not be seen by scanning electron microscopy.

Fig. 4.

SEM of GM151 cells co-cultured for 6 h with murine lymphocytes. Note the projections of the lymphocytes attached closely to the surfaces of the underlying fibroblasts, as indicated by the arrows. ×8500.

Fig. 4.

SEM of GM151 cells co-cultured for 6 h with murine lymphocytes. Note the projections of the lymphocytes attached closely to the surfaces of the underlying fibroblasts, as indicated by the arrows. ×8500.

By transmission electron microscopy, the complex interdigitation of cytoplasmic profiles was consistent with the appearance in the light microscope, but identification of individual profiles as belonging to either lymphocytes or fibroblasts was possible only rarely, where continuity with the cell bodies from which they originated was maintained in the plane of the section. In this study we have therefore limited our observations to adherent cell profiles that can be identified with certainty. In these, extensive regions of close apposition of lymphocytes and fibroblasts of the adhaerens type (Farquhar & Palade, 1963) were commonly seen; the two plasma membrane profiles being separated by a uniform gap of some 10–20 nm (Fig. 5). No specializations resembling tight junctions, gap junctions or desmosomes were ever observed. Nor was there any evidence of structures such as the cytoplasmic connections (Schoenberg et al. 1964) or ‘septate-like’ junctions (Siebert, 1979) reported between lymphocytes and macrophages. However, in the present experiments there was a marked difference between the regions of fibroblast surface that were in contact with lymphocytes and other parts of these cells. As shown in Fig. 5, pits and vesicles, with a mean diameter of 80µm (s.D. 19-3), a size corresponding to micropinocytotic vesicles (Ghadially, 1982), were very prominent on the fibroblastic side of the regions of contact. The frequency distributions of such vesicles underlying random 3 μm lengths of electron-microscopic profiles along the fibroblast surface are shown in Fig. 6. Thus, for regions of fibroblasts in these cultures identified unequivocally as lymphocyte-adherent, 48 % of such sample lengths had more than five vesicles and three had more than 30. In marked contrast, in fibroblasts cultured alone 56 % of such sample lengths contained no vesicles and only 10% had five or more; similarly, low numbers of vesicles were found in cocultures in regions of fibroblast surface that were not apposed to lymphocytes.

Fig. 5.

TEM of a region of contact between a typical lymphocyte (above) and a GM151 fibroblast (below) showing the characteristic vacuolation of its cytoplasm. Numerous micropinocytotic vesicles (arrowed) can be seen just below the fibroblast surface in the region of contact. ×28 000.

Fig. 5.

TEM of a region of contact between a typical lymphocyte (above) and a GM151 fibroblast (below) showing the characteristic vacuolation of its cytoplasm. Numerous micropinocytotic vesicles (arrowed) can be seen just below the fibroblast surface in the region of contact. ×28 000.

Fig. 6.

Histogram of numbers of juxta-surface micropinocytotic vesicles in GM151 cells encountered in random 3 μm lengths of : A, unselected regions of surface profiles of fibroblasts cultured alone; B, regions of surface profiles of fibroblasts apposed to murine lymphocytes, examined after 6h co-culture.

Fig. 6.

Histogram of numbers of juxta-surface micropinocytotic vesicles in GM151 cells encountered in random 3 μm lengths of : A, unselected regions of surface profiles of fibroblasts cultured alone; B, regions of surface profiles of fibroblasts apposed to murine lymphocytes, examined after 6h co-culture.

GM 151 cells acquire high levels of β-glucuronidase activity as a consequence of contact with normal mouse lymphocytes. The phenomenon of contact-dependent enzyme transfer seems to be widespread, since co-culture with normal lymphocytes enhanced β-glucuronidase activity not only in completely deficient GM 151 cells but also in fibroblasts from individuals heterozygous for β-glucuronidase deficiency or from the C3H strain mouse, both of which have intermediate levels of this enzyme (Olsen et al. 1982). Most notable of the present findings is that under conditions of efficient β-glucuronidase transfer, contacts were formed between lymphocytes and fibroblasts, which were both more extensive and more specialized than have previously been described for these two types of cell. The ramifying extensions of the lymphocytes over the surfaces of fibroblasts, revealed by the two light-microscopic techniques used, were particularly striking. Such structures appeared to be developed by lymphocytes in the regions of their contact with fibroblasts and, by virtue of the massive extent to which they would increase the area of such contacts, they seem likely to be relevant to the high efficiency of this mechanism of enzyme transfer (Olsen et al. 1981, 1982).

The importance of such interactions between the surfaces of the two types of cell during enzyme transfer is further supported by the finding that the acquisition of glucuronidase by the GM 151 cells is inhibited by the addition of an excess either of lymphocyte plasma membranes or of antibody prepared against them. Furthermore, the failure of even very high levels of anti-;β-glucuronidase antibody to inhibit transfer indicates that this enzyme is not exposed to the bulk phase of the extracellular medium during its passage between the donor and recipient cells. Such sequestration of the enzyme during transfer might be accounted for if, for example, small enzyme-containing fragments of vesicles derived from lymphocytes, perhaps from their fine processes, were taken up by the recipient fibroblasts. This type of mechanism would be consistent with the inhibition of enzyme transfer both by the lymphocyte plasma membrane preparation, which would competitively inhibit uptake of enzyme-containing fragments, and by antibody against lymphocyte plasma membrane, which would precipitate these fragments. However, we do not see any electron-microscopic evidence of such cell fragments, nor do the immunofluorescent studies reveal the presence in the recipient fibroblasts of any lymphocyte-derived plasma membrane antigens. Moreover, enzyme transfer by pinching off cytoplasmic fragments from lymphocytes and their subsequent ingestion by fibroblasts is difficult to reconcile with our previous observations that transfer is contact-dependent and is restricted to certain lysosomal enzymes; excluding not only a number of cytosoluble enzymes but also β-glucosidase, a lysosomal enzyme known to be firmly membranebound (Olsen et al. 1983). It seems more likely, therefore, that enzyme transfer occurs in the zones of closest approach between the lymphocyte and fibroblast surface membranes, where they appeared to be separated by gaps of 10–20 nm. A gap of this size, which would be even further diminished by the presence within it of any unstained glycocalyx, would permit, at most, very restricted entry of anti-β- glucuronidase antibody; IgG molecules having dimensions of 12— 14nm ×7nm (Green, 1969). At the same time, such a gap, being of similar size to the synaptic cleft (Threadgold, 1976), is clearly compatible with rapid macromolecular diffusion between cells. We have yet to establish the ultrastructural effect of the lymphocyte plasma membrane and of anti-membrane antibody on these areas of close approach between donor and recipient cells.

At the ultrastructural level the most conspicuous feature of these lymphocyte/ fibroblast contact regions was the clusters of micropinocytotic vesicles, which were confined to the fibroblastic cells. Although micropinocytotic vesicles are by no means an uncommon feature in these cells, their concentration into focal clusters at sites of contact between lymphocytes and fibroblasts suggests that they are produced in large numbers as a local response by the fibroblast to contact with the lymphocyte. This evidence implicates these vesicles in the process by which β-glucuronidase is taken up into fibroblasts during contact with lymphocytes, and it is hoped that, by using the antibody to lymphocyte β-glucuronidase in immunolabelling techniques, it will be possible to demonstrate the presence of enzyme within these vesicles.

In contrast with this proposed mechanism, the endocytosis of ‘high-uptake’ lysosomal enzymes from the fluid medium appears to involve ‘coated’ pits and vesicles of a much larger size (Ghadially, 1982; Mellman, 1984). It is notable that such differences in the size of vesicles involved in the initial phases of enzyme acquisition by these two different mechanisms may also be reflected in differences between the transport and fate of the acquired enzyme within the recipient cell (Olsen et al. unpublished data).

The diffuse micropinocytotic activity occasionally seen in regions of contact between two fibroblasts may represent a more general mechanism whereby cells exchange macromolecular components. This is consistent with the observation that lymphocytes, which only very rarely contained such vesicles (Fig. 5), do not themselves acquire additional β-glucuronidase activity when they are co-cultured with normal human fibroblasts (Olsen, Abraham & Marsh, unpublished data).

The therapeutic use of grafts of normal tissue in subjects suffering from inherited deficiencies of specific lysosomal enzymes depends on an efficient and permanent means of distributing the relevant enzyme to all affected tissues. Lymphocytes are particularly well suited to such a role because of their circulation through the extravascular compartments (Gowans, 1966), non-lymphoid as well as lymphoid (Smith et al. 1970). Indeed, the transfer of lysosomal enzymes directly from a lymphocyte to other cells has also been implicated in lymphocyte-mediated cytotoxicity (Zucker-Franklin et al. 1983) and may reflect only one aspect of the lymphocyte’s repertoire of contact-dependent activities, such as interaction with endothelia during migration from the blood vascular system (Butcher et al. 1979; Stevens et al. 1982; Wenk et al. 1974; de Bono, 1976), with macrophages during the immune response (Schoenberg et al. 1964; Holbrook et al. 1977; Brelinska & Pilgrim, 1983) and with, for example, hepatocytes during chronic active hepatitis (Paronetto, 1970; Kawanishi, 1977), with synovial cells in rheumatoid joints (Kobayashi & Ziff, 1973) and with muscle fibres in some myopathies (Arahata & Engel, 1984).

This work was funded in part by a grant from the Wellcome Trust. We thank Dr Jill Moss and Mr T. Bull for their help and advice on the electron-microscopic aspects of this study.

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