Cell-to-cell contact between normal fibroblasts and lymphoblasts deficient in lysosomal enzymes

Biochimica et Biophysica Acta, 1138 (1992) 143-148

143

© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00

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Cell-to-cell contact between normal fibroblasts and lymphoblasts deficient in lysosomal enzymes Paola Di Natale 1, Tiziana Annella 1, Aurora Daniele ~, Rossella Negri 2 and Lucio Nitsch 2 t Dipartimento di Biochimica e Biotecnologie Mediche, H Facolth di Medicina e Chirurgia, Universith degli Studi di Napoli "Federico H", Napoli (Italy) and 2 Dipartimento di Biologia e Patologia Cellulare e Molecolare, H Facolth di Medicina e Chirurgia, Universith degli Studi di Napoli "Federico H", Napoli (Italy)

(Received 6 August 1991)

Key words: Fibroblast; Lymphoblast;Cell-cell interaction; Lysosomalenzyme H u m a n lymphoblasts deficient in iduronate sulfatase or in a-N-acetylglucosaminidase acquire discrete levels of enzyme activity after co-culture with human normal skin fibroblasts. This occurs by direct cell-to-cell contact and not by uptake of secreted fibroblast enzyme. The process is dependent on time and on the number of fibroblasts used. Electron-microscopic examination of the co-culture of the two cell types reveals extensive region of intimate contact. Fibroblastic projections appear frequently in close apposition with lymphoblast invaginations; a diffuse micropinocytotic activity is evident only in fibroblastic cells.

Introduction Deficiencies of specific lysosomal enzymes have been associated with a major group of inherited metabolic diseases [1]. In the past few years, correction of the enzyme deficiency has been extensively studied in vitro by the addition of normal enzyme to deficient cells, derived from affected patients. The most common mechanism whereby cells, derived from patients with lysosomal storage disorders, can acquire the missing enzymes, involves endocytosis via mannose 6-phosphate receptors located on the cell surface [2]. These enzymes, known as 'high uptake' forms, bear mannose 6-phosphate groups and are secreted into the culture medium sourrounding the cells. This mechanism has been mainly shown in cultured skin fibroblasts. Different types of cell, however, express different surface receptors. Hepatocytes, for example, have receptors for galactose [3] as well as fucose [4], alveolar macrophages selectively bind mannose and N-acetylglucosamine [5]. A completely different mechanism has been described for concanavalin A-transformed lymphocytes.

Correspondence: P. Di Natale, Dipartimento di Biochimica e Biotecnologie Mediche, II Facolt?~ di Medicina e Chirurgia, Universit~ degli Studi di Napoli "Federico II", Via Sergio Pansini n. 5, 1-80131 Napoli, Italy.

These cells have been shown to transfer certain lysosomal enzymes to genetically deficient fibroblasts by a process that requires cell-to-cell contact, is independent from mannose 6-phosphate and is abolished by inhibitors of protein synthesis [6-8]. In more recent work, it has been shown that cell contact induces the de novo synthesis of a lysosomal enzyme precursor in normal lymphocytes and its direct transfer to deficient fibroblasts [9] and that the process is valid only for the lysosomal form of the enzymes [10]. In the present paper we report our studies on the effect of co-culture of normal fibroblasts with lymphoblasts from mucopolysaccharidosis patients, obtained by Epstein-Barr virus transformation of peripheral lymphocytes.

Materials and Methods

Materials

4-methylumbelliferyl-2-acetamido-2-deoxy-a-Dglucopyranoside was from Calbiochem corporation. Trypan blue solution, 0.5% in physiological saline was supplied from Serva Feinbiochemica (NY, U.S.A.). R P M I 1640 medium, fetal calf serum and trypsin were purchased from Flow Laboratories (Irvine, U.K.). Transwll TM Chambers (3412) were from Costar (MA, U.S.A.).

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Cells L y m p h o b l a s t s were o b t a i n e d by E p s t e i n - B a r r (EB) virus t r a n s f o r m a t i o n of p e r i p h e r a l lymphocytes. Cells were m a i n t a i n e d in R P M I - 1 6 4 0 m e d i u m s u p p l e m e n t e d with 15% fetal calf serum. Viable cells w e r e c o u n t e d using a h e m o c y t o m e t e r after t r y p a n b l u e (0.5%) stain. F i b r o b l a s t s w e r e grown in m i n i m a l essential m e d i u m ( M E M ) s u p p l e m e n t e d with 10% fetal calf serum. Co-culture experiments In co-culture e x p e r i m e n t s R P M I - 1 6 4 0 m e d i u m was used. M o n o l a y e r s of n o r m a l fibroblasts were grown up to 70% confluency, t h e n m e d i u m was r e m o v e d and a suspension of deficient l y m p h o b l a s t s w e r e a d d e d in fresh R P M I - 1 6 4 0 m e d i u m . A f t e r a given time of coculture, the n o n - a d h e r e n t l y m p h o b l a s t s were r e m o v e d , w a s h e d twice with 2 ml o f 0.9% NaC1, f r e e z e - t h a w e d 6 times and t e s t e d for enzyme activities. In o r d e r to remove l y m p h o b l a s t s as gently as possible a n d to avoid r e m o t i o n of a t t a c h e d fibroblasts, only a fraction of the total lymphoblasts, c o r r e s p o n d i n g to less t h a n 50%, was r e c o v e r e d . Enzyme assays l d u r o n a t e sulfatase was assayed as d e s c r i b e d b e f o r e [11] using 3H-disulfate d i s a c c h a r i d e as substrate. 10015(1 /xg of cell h o m o g e n a t e were i n c u b a t e d with substrate for 17 h. A unit of i d u r o n a t e sulfatase activity is the a m o u n t of enzyme catalyzing the hydrolysis of 1% of the s u b s t r a t e p e r hour. a - N - A c e t y l g l u c o s a m i n i d a s e was assayed by the m e t h o d of M a r s h a n d F e n s o m [12] using 4 - m e t h y l u m b e l l i f e r y l - 2 - a c e t a m i d o - 2 - d e o x y - a - D g l u c o p y r a n o s i d e as substrate. 5 0 - 1 0 0 /xg of cell hom o g e n a t e were i n c u b a t e d with s u b s t r a t e for 18 h. Protein was d e t e r m i n e d by the m e t h o d of Lowry et al. [13].

Fibroblasts secretions F i b r o b l a s t s w e r e grown to c o n f l u e n c e in 150 m m c u l t u r e dishes in m i n i m a l essential m e d i u m ( M E M ) s u p p l e m e n t e d with 10% fetal calf s e r u m (FCS). F o r p r e p a r a t i o n s of secretions, fresh m e d i u m free from FCS, s u p p l e m e n t e d with 15 m M a m m o n i u m c h l o r i d e was a d d e d . S e c r e t i o n s were h a r v e s t e d after 24 h and p r o t e i n s p r e c i p i t a t e d with 80% a m m o n i u m sulfate. Following c e n t r i f u g a t i o n of the s a m p l e at 11 325 × g for 30 min, the p r e c i p i t a t e was r e c o n s t i t u e d to approx. 1 / 1 0 0 of the initial volume with p h o s p h a t e - b u f f e r e d saline (PBS) a n d d i a l y z e d in that b u f f e r ( t h r e e c h a n g e s of a 2-liter bath). P r e p a r a t i o n s w e r e s t o r e d at - 2 0 ° C until used. Electron microscopy Cell c o - c u l t u r e s were fixed in situ in 2.5% g l u t a r a l d h e y d e in 0.1 M c a c o d y l a t e buffer ( p H 7.4), at r o o m t e m p e r a t u r e , postfixed in 1% o s m i u m tetroxide in cac o d y l a t e buffer, s t a i n e d en bloc with 1% a q u e o u s u r a n y l a c e t a t e , d e h y d r a t e d in e t h a n o l and e m b e b b e d in P o l y / B e d 812. T h i n sections w e r e s t a i n e d with uranyl a c e t a t e a n d l e a d c i t r a t e a n d e x a m i n e d u n d e r a Philips 400 e l e c t r o n m i c r o s c o p e . Results

L y m p h o b l a s t s from a H u n t e r patient, deficient in i d u r o n a t e sulfatase activity or l y m p h o b l a s t s from a S a n f i l i p p o B p a t i e n t , deficient in a - N - a c e t y l g l u c o s a m i n i d a s e activity were c u l t u r e d t o g e t h e r with n o r m a l fibroblasts. D u r i n g a 24 h co-culture, the m u t a n t lymp h o b l a s t s a c q u i r e d substantial a m o u n t s of the deficient e n z y m e s from the n o r m a l fibroblasts. In H u n t e r lymp h o b l a s t s the activity of i d u r o n a t e sulfatase i n c r e a s e d l l . 6 - f o l d , r e a c h i n g 30% of the activity p r e s e n t in ex-

TABLE l

Changes in lysosomal enzyme acticities in mucopolysaccharidosis lymphoblasts after co-culture with normal fibroblasts Enzyme activities of the donor cells (normal fibroblasts) were the following: iduronate sulfatase 12.5 U/mg protein; a-N-acetylglucosaminidase 11.8 nmol/h per mg protein. If fibroblasts were cultured without lymphoblasts no enzyme activity was found in the supernatant. Cells

Iduronate sulfatase activity (U/rag protein) ~

Hunter lymphoblasts Hunter lymphoblasts co-cultured with normal fibroblasts ~ Normal lymphoblasts Sanfilippo B lymphoblasts Sanfilippo B lymphoblasts co-cultured with normal fibroblasts c Normal lymphoblasts

0.094

a-N-Acetylglucosaminidase activity (nmol/h per mg protein) b

Increase (fold)

L1.6

1.09

3.6 0.039 + 0.014 0.26 _+0.066 3.41 +0.35

6.7

Data are mean value of three experiments, with a range less than 15%. b Data are mean_+ S.D. of nine experiments. c Normal fibroblasts were grown to log phase (5' 105 cells) in 60-ram culture dishes and the co-culture initiated by the addition of 7- 106 Hunter lymphoblasts or 2.5.106 Sanfilippo B lymphoblasts in 2 ml RPMI medium supplemented with 15% fetal calf serum (FCS). After 20 h of co-culture, the non-adherent lymphoblasts were removed, washed twice with 2 ml of 0.9% NaC1, freeze-thawed six times and tested for enzyme activities as described under Methods.

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Fig. 1. Time-course of cell-to-cell contact. Monolayers of approx. 5.105 normal fibroblasts were grown in 60 mm culture dishes and the co-culture initiated by the addition of 2.5.106 Sanfilippo B lymphoblasts. At the indicated times of the co-culture periods (1-48 h) non-adherent lymphoblasts were removed from each dish and frozen. At the end of the experiment a-N-acetylglucosaminidase activity was assayed and expressed as total nmol of methylumbelliferone released per each dish. Data represent mean values of three independent experiments, with a range less than 15%.

tracts of normal lymphoblasts. In Sanfilippo B lymphoblasts a-N-acetylglucosaminidase activity increased 6.7-fold, reaching 7.8% of the activity present in normal lymphoblasts (Table I). No increase in enzyme activity was observed when mutant lymphoblasts, for instance Hunter, were co-cultured with the same mutant fibroblasts, i.e. Hunter (data not shown). ce-N-acetylglucosaminidase activity, in Sanfilippo B lymphoblasts increased with time for the first 6 h, reaching a plateau that was maintained up to 48 h (Fig.

1). The plateau was probably due to the saturation of contact sites. The enzyme activity acquired by deficient lymphoblasts increased when the number of normal fibroblasts, used during 20 h of co-culture, was increased (Fig. 2). We have no explanation for the difference in the rate of increase for a-N-acetylglucosaminidase (Fig. 2A) and for iduronate sulfatase (Fig. 2B). When both types of cells were cultured together in transwell chambers (i.e. physically separated by filters), deficient lymphoblasts did not acquire any measurable a-N-acetylglucosaminidase activity. When secretions from normal fibroblasts, obtained by NH4C1 treatment of the cells, were used in transwell chambers, acquisition of enzyme activity in deficient lymphoblasts was barely increased (Table II). Co-culture of Sanfilippo B fibroblasts with normal fibroblasts in transwell chambers resulted in a 1.8-fold increase in specific activity of deficient cells (Table II); the addition of secretions from normal fibroblasts to deficient fibroblasts in transwell chambers, resulted in a 2.7-fold increase (Table II). Addition of mannose-6-P, a competitive inhibitor of receptor-mediated uptake of several hydrolases, at 1 mM caused inhibition of enzyme uptake in deficient fibroblasts, while no effect was demonstrated in co-culture experiments with Hunter or Sanfilippo B lymphoblasts (data not shown). Since the relationship existing between co-cultured lymphoblasts and fibroblasts is possibly relevant to enzyme transfer, we performed a study at the ultrastructural level. Our aim was to demonstrate whether a true, intimate contact between the two cell types was really established and whether special contact-sites or

T A B L E I1

a-N-Acetylglucosaminidase activity in Sanfilippo B cells in different culture conditions Fibroblasts were grown to log phase (1.105 cells) in 35-mm bottom dishes of transwell chambers (3412 Costar) in 2 ml RPMI-1640 medium containing 15% fetal calf serum. The co-culture was initiated by adding, in a total vol. of 1 ml: (a) 2.5.106 Sanfilippo B lymphoblasts on the fibroblast monolayer, after removal of the filter, i.e. cell-to-cell contact; (b) 2.5.106 Sanfilippo B lymphoblasts on filters placed on top of growing fibroblasts, i.e. physically separated; (c) 0.1.106 Sanfilippo B fibroblasts placed on filters; (d) secretions from normal fibroblasts placed on filters. In (e) Sanfilippo B lymphoblasts were placed in 35-mm bottom dishes of transwell chambers and 1 ml of secretions from normal fibroblasts was placed on filters. T h e culture continued for 20 h, after which cells were removed and tested for o~-N-acetylglucosaminidase activity. Normal fibroblast secretions were prepared as described under Methods. 1 ml of such preparation contained 16.2 n m o l / h (total enzyme activity). Results shown are m e a n s of three independent experiments, with a range less than 10%. Cells

a-N-Ace tylglucosaminidase activity ( n m o l / h per mg protein)

Sanfilippo B lymphoblasts cultured alone co-cultured with normal fibroblasts a co-cultured with normal fibroblasts in transwell chambers h grown in transwell chambers + secretions from normal fibroblasts e

0.038 0.122 0.037 0.052

Sanfilippo B fibroblasts cultured alone co-cultured with normal fibroblasts in transwell chambers c grown in transwell chambers + secretions from normal fibroblasts d

0.44 0.82 1.2

146 specialized junctions were present. We also investigated the presence of endocytic structures at, or near, the contact-sites. Electron microscopic examination of monolayers after 1 h (when enzyme transfer is already in place) and 24 h (when lymphocytes have acquired high levels of a-N-acetylglucosaminidase activity) revealed extensive regions of intimate contact between the two types of cells (Fig. 3a). In the zones of closest apposition the lymphoblast and fibroblast membranes were separated by only a few rim. Contacts were often performed by cytoplasmic processes of both cellular types, in particular fibroblastic projections appeared frequently in close apposition with lymphoblast invaginations (Fig. 3b). No specialized junctions were detected. A diffuse micropynocytotic activity is evident in fibroblastic cells, both at sites where cells are in contact with each other as in regions that are not adherent to lymphoblasts. Such activity is generally neglectable in the observed lymphoblasts.

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Fig. 2. Cell-to-cell contact as function of fibroblast number. Monolayers ranging from approx. 1 to 16" 105 normal fibroblasts were grown in 35, 60, 100 and 150-mm culture dishes, respectively. The co-culture initiated by the addition of 2.5- 106 Sanfilippo B lymphoblasts (panel A), or 7.0.106 Hunter lymphoblasts (panel B). Volume of medium above the co-cultures was 1, 2, 4, 8 ml, respectively. At the end of the co-culture period (20 h) non adherent lymphoblasts were removed and tested for acquired enzyme activities. Data represent mean values of three independent experiments, with a range less than 15%.

Discussion

In the present study we show that when lymphoblasts from a Hunter or from a Sanfilippo B patient are cultured together with normal fibroblasts they acquire discrete levels of the missing lysosoma[ enzyme. Acquisition of enzyme activity by deficient lymphoblasts varied from 2 - 8 % of the activity present in normal fibroblasts (Table I). The process was time-dependent, at least for the first 6 h (Fig. l) and the increase of enzyme activity was dependent on the number of fibroblasts used (Fig. 2). Enzyme transfer occurs by direct cell-to-cell contact and not by uptake of secreted fibroblast enzymes. Acquisition, in fact, was not reduced in the presence of mannose 6-phosphate, but was abolished when both types of cell were cultured together while separated by filters in transwell chambers. We do not known the mechanism by which these enzymes are transferred from fibroblasts to lymphoblasts. In the opposite system, i.e. transfer of enzyme from normal lymphocytes to deficient fibroblasts an hypotesis was formulated by others: the enzymes could be transported to the cell surface and released by the cells into 15-nm intercellular gaps that have been observed in co-cultures [8]. In our co-culture system we have also found extensive regions of donor cell acceptor cell contact (Fig. 3). The plasma membranes were in such close apposition one to the other, in some places, to suggest the possibility of forming junctions of the gap - a n d / o r adherens type. No such junctions could however be demonstrated. On the other side, no junction is known yet that would allow selective and direct transfer, from one cell to another, of molecules as large as proteins. It should also be noted that, at variance with fibroblasts, cultured lymphoblasts do not show, at contact sites, any significant micropinocytotic activity which would be indicative of an endocytotic pathway of internalization. Recent results obtained in Olsen's laboratory have shown that cell-to-cell contact provided the molecular signals necessary for the activation of the lymphocytes. This was accompanied by the biogenesis of the lysosom a l / e n d o c y t i c compartment in the activated cells; moreover only the precursor form of the enzyme was transferred and then correctly processed and transported to the lysosomes of the deficient fibroblasts [9-10]. We have not performed yet such kind of studies and thus we can not assess if the same mechanism holds true also in our system. Although the biosynthesis of [ysosomal enzymes has not been fully elucidated in concanavalin-A transformed lymphocytes or in Epstein-Barr derived lymphoblasts, these cells probably have a biosynthetic pathway different from that of fibroblasts, mannose 6-phosphate dependent. The existence of a mannose

147

Fig. 3. Electron microscopy of lymphoblast-fibroblast interaction after 1 h (3a) and 24 h (3b) of co-culture. (a) Shows an extensive region of intimate contact: in the sites of closest apposition the gap between the membranes is only of few nm (inset). An extracellular matrix basal lamina-like can be seen between the cells in some region of membrane apposition (arrow). Micropinocytotic vescicles are evident only in the fibroblastic cells (arrowheads). F, fibroblast; L, lymphoblast; Bar, 0.25 /xm inset, bar, 0.1 #m. (b) A close association between the cells is frequently established by fibroblastic projections (F). Bar, 0.25/zm.

6-phosphate-independent transport has been suggested by several lines of evidence [1,2,14]. It is well known that in I-cell patients many cell types (including hepatocytes, Kupffer cells and ieukocytes) and several organs (including liver, spleen, kidney and brain) have nearly normal levels of many lysosomal enzymes although these cells and tissues are deficient in Nacetylglucosaminyl 1-phospho-transferase activity. Whatever the biosynthetic mechanism of lysosomal enzymes in lymphocytes and in lymphocyte-derived cells (lymphoblasts), these cells can either donate their normal enzymes to deficient fibroblasts [6-10] or acquire the missing enzyme from normal fibroblasts (this paper), through a contact-mediated transfer.

Acknowledgements We thank the assistance of V. Brescia for preparing cell cultures. We also thank R. Baldoni for preparing

the manuscript. This investigation was supported by grants from CNR, Progetto Finalizzato lngegneria Genetica, Ministry of Education (MURST 40%) and Ministry of Health, Roma, Italy.

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11 Morabito, E., Giambarrasi, I., Rocchi, M. and Di Natale, P. (1989) Clin. Chim. Acta 181, 125-134. 12 Marsh, J. and Fensom, A.H. (1985) Clin. Genet. 27, 258-262. 13 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. 14 Gabel, C.A., Goldberg, D.E. and Kornfeld, S. (1984) in "Molecular Basis of Lysosomal Storage Disorders" (Barranger, J.A. and Brady, R.O., eds.), pp. 175-193, Academic Press, New York.