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The intracellular localisation of immunoglobulin in human lymphoid cells and haematopoietic cell lines by immunoperoxidase electron microscopy
Journal o f Immunological Methods, 37 (1980) 275--286
275
© Elsevier/North-Holland Biomedical Press
THE INTRACELLULAR LOCALISATION OF IMMUNOGLOBULIN IN HUMAN LYMPHOID CELLS AND HAEMATOPOIETIC CELL LINES BY I M M U N O P E R O X I D A S E E L E C T R O N M I C R O S C O P Y
D.G. NEWELL 1, C. BOHANE 1, S. PAYNE I and J.L. SMITH 2 Departments o f 1 Haematology and Experimental Pathology and the 2 Regional Immunology Service, Southampton General Hospital, Southampton, U.K.
(Received 29 February 1980, accepted 19 May 1980)
A technique is described for the ultrastructural localisation of intracellular immunoglobulin (Ig) in human lymphoid cell suspensions by the immunoperoxidase method. The technique involves restricted saponin digestion of glutaraldehyde prefixed cells to enhance conjugate penetration. With this method of staining Ig was located in the rough endoplasmic reticulum, perinuclear space and Golgi apparatus in human lymphoid cells from a variety of sources, which is consistent with previously published observations using other ultrastructural techniques. In contrast, diffuse intracytoplasmic staining was predominant in cells prefixed with glutaraldehyde but not treated with saponin. These differences in patterns are discussed in terms of membrane permeability. Although saponin treatment was necessary for consistent localisation of intracellular Ig it resulted in unavoidable loss of Ig from the surface of the cells.
INTRODUCTION Studies o f i m m u n o g l o b u l i n (Ig) s y n t h e s i s a n d surface e x p r e s s i o n b y neoplastic B cells in l y m p h o c y t i c disease are i m p o r t a n t f o r diagnosis, f o r classification a n d f o r d e f i n i n g r e l a t i o n s h i p s to t h e n o r m a l cell o f origin ( G o r d o n and S m i t h , 1 9 7 8 ; G o r d o n et al., 1978). H o w e v e r t h e Ig s y n t h e s i s p a t t e r n s r e p o r t e d are r e p r e s e n t a t i v e o f t h e t o t a l p o p u l a t i o n and d o n o t t a k e i n t o a c c o u n t individual cell v a r i a t i o n . This v a r i a t i o n m a y be r e l e v a n t to t h e Ig h e a v y and light c h a i n s y n t h e t i c i m b a l a n c e c o m m o n in c h r o n i c l y m p h o c y t i c l e u k a e m i a (Maino et al., 1 9 7 7 ; G o r d o n et al., 1978). A t t h e cellular level h o r s e r a d i s h p e r o x i d a s e ( H R P ) c o u p l e d to anti-Ig p r o v i d e s a sensitive and p e r m a n e n t label f o r l i g h t - m i c r o s c o p i c l o c a t i o n . T e c h n i c a l l i m i t a t i o n s , h o w ever, have r e s t r i c t e d its use at t h e u l t r a s t r u c t u r a l level. T h e s e l i m i t a t i o n s reflect conflict between adequate fixation, preservation of good ultrastruct u r a l detail, and c o n j u g a t e p e n e t r a t i o n o f t h e fixed m e m b r a n e . Labelling o f intracellular Ig a f t e r r e l a t i v e l y s t r o n g g l u t a r a l d e h y d e f i x a t i o n has b e e n r e p o r t e d ( R e y e s et al., 1 9 7 8 ) b u t is i n c o n s i s t e n t in o u r hands, probably because of restricted membrane permeability. Kraehenbuhl and J a m i e s o n ( 1 9 7 4 ) have u s e d F a b f r a g m e n t s o f Ig or m i c r o p e r o x i d a s e to im-
276 prove conjugate penetration, while others have used limited detergent solubilisation to increase membrane permeability (Hall et al., 1978). In this study we have attempted to establish a uniform technique for the ultrastructural localisation of Ig by the immunoperoxidase method using cell suspensions of normal and neoplastic lymphoid cells. MATERIALS AND METHODS
A n tibod y-peroxidase conjugate Sheep antibodies to human heavy and light chains, prepared by affinity purification, were purchased from Tenovus Laboratories, Southampton. These antibodies were conjugated to horseradish peroxidase (Grade V1, Sigma Chemicals Ltd.) using either the 2-step glutaraldehyde method (Avrameas and Ternynck, 1971) or the improved periodate method (Wilson and Nakane, 1978). The conjugated immunoglobulin was precipitated by ammonium sulphate and prepared on a Sephacryl 200 column (Pharmacia Ltd.). The conjugates were stored at 4°C at a concentration of 1 mg protein/ml in phosphate buffered saline (PBS), with 1 : 10,000 w/v methiolate. Control conjugates were prepared by the same methods using normal sheep IgG.
Isolation and preparation of cells Lymphoid cells were isolated from blood, bone marrow, tonsil, lymph nodes and spleen from normal donors and patients with lymphoproliferative disease. Cell suspensions were prepared from lymphoid tissue by teasing and filtration through a metal gauze. Haematopoietic cell lines were provided by Dr. A. Karpas, Department of Haematological Medicine, Cambridge and have been previously investigated for immunoglobulin expression and synthesis (Gordon et al., 1977). Jijoye and E B2 cell lines were purchased from Flow Laboratories. All cell populations were prepared on Ficoll-triosil gradients (B5yum, 1968). Harvested cells were washed twice in Hepes buffered minimal essential medium (MEM, Gibco-Biocult, Glasgow) and resuspended in 0.1, 0.5, 1.0 or 1.25% glutaraldehyde (Grade 1, Sigma Chemicals Ltd.) in 0.1 M Sorensen's phosphate buffer, pH 7.2, at room temperature for 15--30 min. Fixed cells were washed three times with phosphate buffer and stored in buffer containing 0.01% azide at 4°C until required. For all washing procedures after fixation an Eppendorf microcentrifuge was used and the microcentrifuge tubes were siliconised to reduce cell loss.
Saponin treatment of cells for intracellular Ig staining Cells prefixed in 0.1 or 0.5% glutaraldehyde were treated with 1.0% saponin (Sigma Chemicals Ltd.) at 55°C for periods of up to 100 min.
277 The treated cells were washed 3 times by centrifugation with phosphatebuffered saline.
Labelling and staining of cells for Ig localisation Cells were incubated with conjugate either directly after prefixation or saponin treatment. The cells were pelleted and resuspended in at least twice their volume of conjugate (1 mg/ml) and incubated for 60 rain at room temperature before washing three times with PBS. Peroxidase activity was detected by the m e t h o d of Graham and Karnovsky (1966) as follows. Ten mg of diamino benzidine (DAB) were dissolved in 20 ml of 0.05 M Tris HC1 buffer (pH 7.6) and the pH readjusted to pH 7.6; 0.2 ml of 1% hydrogen peroxide solution was added to the DAB solution and the cells incubated in the reaction mixture for 30 min at room temperature in the dark. Post fixation with 1% glutaraldehyde in 0.1 M Sorensen's buffer, pH 7.2, for 15 min at room temperature was usually performed after the DAB incubation. After washing in PBS the cells were post fixed in 1% osmic acid in Palade's acetate buffer, pH 7.2, washed in distilled water and embedded in 2% agar, prior to embedding in Spurr resin. Thin sections were cut on glass knives and viewed w i t h o u t further staining on a Phillips 201 or 300 transmission electron microscope at 40 kV. RESULTS Significant immunoperoxidase staining was rarely observed in prefixed (1--1.25% glutaraldehyde, 15--30 min) leukaemic cells, tonsil cells or cell lines, previously shown by immunofluorescence to have intracellular Ig (Fig. 1, Table 1). Obvious membrane damage was associated with m a n y of these stained cells but this was insufficiently consistent to totally account for the conjugate penetration. The non-saponin treated cells occasionally had the same intracisternal staining pattern as the treated cells but more frequently the staining was extracisternal and diffuse (Table 1). This difference in localisation was particularly well illustrated by the cell line Raji. When fixed with 1.25% glutaraldehyde for 30 min, Raji cells demonstrated a diffuse and extracisternal staining pattern (Fig. 2), whilst the same cells, treated with saponin, showed a monoclonal staining of the RER and PNS (Fig. 3). This extracisternal staining did not appear to be non specific and was n o t observed to the same extent with the control conjugate. The treatment of mildly prefixed (0.1% glutaraldehyde for 10 min) cells with 1% saponin allowed consistent penetration of the cell membranes by the conjugate and staining of intracellular Ig was observed in the rough endoplasmic reticulum (RER), perinuclear space (PNS) and vesicles of the Golgi apparatus (Fig. 4). The proportion of cells stained for intracellular Ig was significantly higher (Table 1) and the staining stronger in the saponin
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Fig. 1. Intracellular Ig locaJised in the perinuclear space (arrowed) i~ a CLL cel] fixed with 1% glutaraldehyde for 30 min. x13,500.
treated samples than in similar preparations fixed but n o t treated with saponin. This technique allowed the visualisation of intracellular Ig in human lymphoid cells from several sources including m y e l o m a (Fig. 4) and leukaemic cells (Fig. 5) from peripheral blood, tonsil cells (Fig. 6) and lymphoblastoid cell lines (Fig. 7). The anti Fab-7-peroxidase conjugate produced by the 2-step glutaraldehyde m e t h o d was significantly more efficient at penetrating the membranes of saponin treated cells than the conjugate obtained by the periodate technique, as judged by both the number of cells stained and the intensity of staining. No stained cells were observed in non-saponin treated cells when incubated with the periodate conjugated material. The treatment of lightly fixed (0.1% glutaraldehyde for 15 min at room temperature) cells with 1% saponin at 55°C resulted in a variable e x t e n t of cell damage. This damage was manifest as discontinuous intracellular and plasma membranes, dispersed cytoplasm and swollen mitochondria and endoplasmic reticulum. The extent of cell damage was dependent on the times of exposure, the cell source and the concentration of the prefixative. Reduction of the time of exposure to saponin from the 100 min recommended for sheep immunoblasts (Hall et al., 1978) to less than 10 min did
Fig. 2. Raji cell fixed with 1.25% glutaraldehyde showing granular and diffuse extracisternal cytoplasmic staining, x10,500.
Fig. 3. Raji cell prefixed with 0.1% glutaraldehyde and treated with saponin showing weak staining in the rough endoplasmic reticulum and perinuclear space, x10,500.
281
Fig. 4. Intracellular Ig localised in the perinuclear space, rough endoplasmic reticulum and Golgi in myeloma cells fixed with 0.1% glutaraldehyde for 10 min followed by 1% saponin treatment, x l 2,000. D
Fig. 5. Intracellular Ig visualised in the perinuelear space and rough endoplasmic retieulum of the same CLL population as Fig. 1 using saponin treatment to enhance penetration. ×12,900.
Fig. 6. Lymphoplasmaeytoid cell from a tonsil cell preparation, x14,200.
Fig. 7. Intracellular Ig localisation in the perinuclear space, rough endoplasmie reticulum and Golgi of the lymphoblastoid cell line 160. x6800.
283
Fig. 8. Jijoye cell prefixed with 0.1% glutaraldehyde and treated with saponin showing strong Ig staining in the perinuclear space, rough endoplasmic reticulum and Goigi apparatus as well as staining around the cell periphery which was also seen with the control conjugate. ×6500.
n o t cause any obvious change in either the location or the intensity of staining but significantly reduced cell damage. Cells from different tissue sources varied considerably in their sensitivity to saponin treatment. Circulating m y e l o m a cells and CLL cells when treated with saponin for 100 min, after 0.1% glutaraldehyde prefixation for 10 min, showed significantly less cell damage than cells isolated from tonsil, lymph node, spleen or peripheral blood. The cell lines were the most susceptible to damage by saponin but a comparison of cells fixed and processed for immunoperoxidase staining with cells fixed for conventional electron microscopy did not indicate any obvious subpopulation selectivity. An increase in concentration of glutaraldehyde to 0.5 or 1.0% significantly reduced the saponin-induced morphological damage but also reduced the staining intensity, frequently preventing Ig detection. Prefixation with 0.1% glutaraldeh y d e for 15 min at room temperature provided the most consistent and highest intensity staining of intracellularly localised Ig when combined with saponin treatment. Little surface Ig could be detected on cells shown to have intracellular Ig
284 by immunoperoxidase staining even though the presence of surface Ig was expected from immunofluorescence (Figs. 6 and 7, Table 1). It was apparent from a comparison of saponin treated and untreated cells that the surface Ig was stripped during exposure to saponin, even for short periods of less than 10 min after 0.1% or 0.5% glutaraldehyde fixation. Surface Ig was detected on cells fixed with 0.1% glutaraldehyde b u t was better preserved after fixation with 0.5% or 1.0% glutaraldehyde. No significant differences in the proportion of cells staining for surface Ig were detected between conjugates produced b y the periodate and t h e 2-step glutaraldehyde methods. Several types of non-specific intracellular staining were detected using the normal sheep IgG-peroxidase conjugate. When present, non-specific staining was observed as a diffuse, granular staining of the cytoplasm. In cells isolated from tissues or l y m p h o c y t e preparations with poor viability non-specific staining was usually associated with obviously necrotic cells. This type of non-specific staining could be eliminated b y removal of non-viable cells on a Ficoll-triosil gradient prior to fixation. In some cell lines a second type of non-specific staining was observed in both saponin treated and untreated cells. This staining had a similar diffuse cytoplasmic presentation b u t was associated with the cell surface (Fig. 8), and was detected with the control conjugate as well as b o t h anti-light chain conjugates. This stain was related to neither surface Ig expression nor to cell viability, and only occurred in 2 of the 6 cell lines so far tested. DISCUSSION The immunoperoxidase technique is extensively used in the diagnosis and classification of lymphomas b y light microscopy, b u t because of the impermeability of the glutaraldehyde fixed cell membrane to antibody-peroxidase conjugate (Kuhlmann et al., 1974) has not been used for localisation of intracellular Ig except in association with methods which involve removal of the cell membrane barrier such as those employing cryostat sections (Kraehenbuhl and Jamieson, 1974). The immunoperoxidase technique has, however, been extremely useful for ultrastructural demonstration of surface Ig on B l y m p h o c y t e s in suspension (Reyes et al., 1975) and during such studies Reyes et al. {1978) reported that antibody-peroxidase conjugate penetxates glutaraldehyde-fixed cell membranes. We suggest that the criteria for an acceptable technique for the ultrastructural localisation of Ig in human B l y m p h o c y t e s should include: (1) good morphological preservation comparable with routine ultrastructural pathological preparations, (ii) suitability of lymphoid cells obtained from a variety of tissues, including blood, and (iii} a direct labelling technique minimising the number of manipulations and reducing cell loss and contamination. Our studies confirm that intracellular localisation of Ig is possible after prefixation with glutaraldehyde alone b u t that not every monoclonal population of cells k n o w n to contain intracellular Ig is stained b y this method
285 and furthermore a significantly smaller population of cells was stained compared with other ultrastructural techniques. Cells stained in this way exhibit a diffusely cytoplasmic rather than intracisternal pattern. This pattern m a y be associated with restricted diffusion of conjugates through intracytoplasmic membranes. Hall et al. (1978) have demonstrated that the permeability of lightly glutaraldehyde-fixed sheep immunoblasts to peroxidase labelled antibody conjugates is enhanced b y treatment with 1% saponin for 100 min at 55°C. Our results indicate that a similar saponin treatment of human B lymphoid cells allows adequate penetration of the conjugate through the plasma and intracytoplasmic membranes to give reliable staining of Ig in the endoplasmic reticulum, t h e perinuclear cisternae and the Golgi apparatus. Staining patterns agree in both monoclonality and distribution with those observed b y immunofluorescent methods (Gordon et al., 1977). Damage resulting from exposure of cells to saponin is considerable, in particular loss of membrane and cytosol integrity, swelling of mitochondria and endoplasmic reticulum and significant loss of surface immunoglobulin. Differences in the susceptibility of different cell types to saponin treatment were apparent; for example, cells from lymphoid tissues were considerably more susceptible than blood lymphocytes. Some of these differences may be due to damage caused during tissue dissociation. Leukaemic cells tended to be the least susceptible while the lymphoblastoid cell lines were the most sensitive to digestion with saponin. Saponin-induced damage did not result in significant non-specific staining but the presence of non-viable cells before glutaraldehyde fixation produced non-specific, diffuse cytoplasmic staining in samples incubated with control conjugates as well as with anti Fab~,-peroxidase conjugates. The removal of dead and dying cells on Ficoll triosil gradients was therefore considered essential. Much of the morphological damage resulting from cell exposure to saponin was prevented by reduction of the exposure time to 5 or 10 min. This neither inhibited conjugate penetration nor altered immunoglobulin location. Attempts to further reduce damage by increasing the initial glutaraldehyde fixation were unsuccessful presumably because of the resulting more extensive cross-linking of membrane proteins. Although a fixation regime of 0.1% glutaraldehyde is considered the most suitable for immunoelectron-microscopic detection of immunoglobulin (Van Ewijk et al., 1980), this is not optimal for morphological preservation and may be insufficient to prevent antigen movement. However the intracellular Ig localisation patterns in cells from human tonsil and spleen fixed in this way are consistent with those seen with other ultrastructural techniques, such as periodate-lysine-paraformaldehyde fixation and cryostat sections (Tsunoda et al., 1978). Similar localisation patterns for IgG were observed in rabbit plasmacytes with formaldehyde fixation (Kraehenbuhl and Jamieson, 1974), and for anti-peroxidase antibodies with peroxidase as both immunogen and marker (Leduc et al., 1968). This suggests that neither mild
286 g l u t a r a l d e h y d e f i x a t i o n n o r saponin t r e a t m e n t causes r e d i s t r i b u t i o n o f imm u n o g l o b u l i n w i t h i n the cell. T h e p e r i o d a t e m e t h o d o f c o n j u g a t i o n is highly e f f i c i e n t in conjugating H R P t o IgG (Wilson and Nakane, 1 9 7 8 ) , b u t this c o n j u g a t e was relatively p o o r at labelling intracellular Ig. This m a y be d u e to t h e larger size o f t h e c o n j u g a t e (up t o 5 or 6 m o l e s H R P t o I m o l e IgG) (Wilson and N a k a n e , 1 9 7 8 ) c o m p a r e d w i t h t h a t p r o d u c e d b y t h e 2-step g l u t a r a l d e h y d e m e t h o d (1 m o l e H R P to 1 m o l e IgG) ( K u h l m a n et al., 1 9 7 4 ) . Also such larger conjugates have d e c r e a s e d a n t i b o d y activity d u e t o the extensive H R P binding (Wilson and Nakane, 1 9 7 8 ) . T h e loss o f surface Ig d u r i n g the p r e p a r a t i o n p r o c e d u r e m e a n s t h a t surface and intracellular Ig c a n n o t b o t h be visualised at the same t i m e b y this t e c h n i q u e . This is a disadvantage for studies correlating the d i s t r i b u t i o n o f surface and intracellular Ig d u r i n g l y m p h o c y t e d i f f e r e n t i a t i o n , and w h e n h e t e r o g e n e o u s cell p o p u l a t i o n s f r o m p a t h o l o g i c a l specimens are being e x a m i n e d . H o w e v e r t h e t e c h n i q u e d e s c r i b e d appears to be a d e q u a t e for studies o f m o n o c l o n a l p o p u l a t i o n s and will be used to investigate the sites o f synthesis and secretion o f Ig in r e l a t i o n to b i o s y n t h e t i c p a t t e r n s and f o r c o m p a r i s o n with various light m i c r o s c o p i c m e t h o d s o f Ig d e t e c t i o n . ACKNOWLEDGEMENT Dr. D.G. Newell and Dr. J.L. S m i t h are in r e c e i p t o f a grant f r o m the Cancer Research Campaign for this p r o j e c t . REFERENCES Avrameas, S. and T. Ternynck, 1971, Immunochem. 8, 1175. BSyum, A., 1968: Scand. J. Clin. Lab. Invest. 21, Suppl. 97. Gordon, J. and J.L. Smith, 1978, Clin. Exp. Immunol. 31,244. Gordon, J., D. Hough, A. Karpas and J.L. Smith, 1977, Immunology 32, 559. Gordon, J., A.R. Howlett and J.L. Smith, 1978, Immunology 34,397. Graham, R.C. and M.J. Karnovsky, 1966, J. Histochem. Cytochem. 14,291. Hall, J.G., M.S.C. Birbeck, D. Robertson, J. Peppard and E. Orland, 1978, J. Immunol. Methods 19, 351. Kraehenbuhl, J.P. and J.D. Jamieson, 1974, Int. Rev. Exp. Pathol. 13, 1. Kuhlman, W.D., S. A~ameas and T. Ternynck, 1974, J. Immunol. Methods 5, 33. Leduc, E.H., S. Avrameas and M. Bouteille, 1968, J. Exp. Med. 127, 109. Maino, V.C., J.T. Kurnick, R.T. Kubo and H.M. Grey, 1977, J. Immunol. 118, 742. Reyes, F., J.L. Lejonc, M.F. Gourdin, P. Mannini and B. Dreyfus, 1975, J. Exp. Med. 141,392. Reyes, F., M.F. Gourdin, J.P. Farcet, J. Breton-Gorius and B. Dreyfus, 1978, Recent Results Cancer Res. 64,176. Tsunoda, R., K. Terashima, K. Takahashi and M. Kijima, 1978, Acta Path. Jap. 28, 53. Van Ewijk, W., R.C. Coffman and I.J. Weissman, 1980, Histochem. J. 12,349. Wilson, M.B. and P.K. Nakane, 1978, in: Immunofluorescent and Related Staining Techniques, eds. W. Knapp, K. Holubar and G. Wicks (Elsevier, Amsterdam) p. 215.
275
© Elsevier/North-Holland Biomedical Press
THE INTRACELLULAR LOCALISATION OF IMMUNOGLOBULIN IN HUMAN LYMPHOID CELLS AND HAEMATOPOIETIC CELL LINES BY I M M U N O P E R O X I D A S E E L E C T R O N M I C R O S C O P Y
D.G. NEWELL 1, C. BOHANE 1, S. PAYNE I and J.L. SMITH 2 Departments o f 1 Haematology and Experimental Pathology and the 2 Regional Immunology Service, Southampton General Hospital, Southampton, U.K.
(Received 29 February 1980, accepted 19 May 1980)
A technique is described for the ultrastructural localisation of intracellular immunoglobulin (Ig) in human lymphoid cell suspensions by the immunoperoxidase method. The technique involves restricted saponin digestion of glutaraldehyde prefixed cells to enhance conjugate penetration. With this method of staining Ig was located in the rough endoplasmic reticulum, perinuclear space and Golgi apparatus in human lymphoid cells from a variety of sources, which is consistent with previously published observations using other ultrastructural techniques. In contrast, diffuse intracytoplasmic staining was predominant in cells prefixed with glutaraldehyde but not treated with saponin. These differences in patterns are discussed in terms of membrane permeability. Although saponin treatment was necessary for consistent localisation of intracellular Ig it resulted in unavoidable loss of Ig from the surface of the cells.
INTRODUCTION Studies o f i m m u n o g l o b u l i n (Ig) s y n t h e s i s a n d surface e x p r e s s i o n b y neoplastic B cells in l y m p h o c y t i c disease are i m p o r t a n t f o r diagnosis, f o r classification a n d f o r d e f i n i n g r e l a t i o n s h i p s to t h e n o r m a l cell o f origin ( G o r d o n and S m i t h , 1 9 7 8 ; G o r d o n et al., 1978). H o w e v e r t h e Ig s y n t h e s i s p a t t e r n s r e p o r t e d are r e p r e s e n t a t i v e o f t h e t o t a l p o p u l a t i o n and d o n o t t a k e i n t o a c c o u n t individual cell v a r i a t i o n . This v a r i a t i o n m a y be r e l e v a n t to t h e Ig h e a v y and light c h a i n s y n t h e t i c i m b a l a n c e c o m m o n in c h r o n i c l y m p h o c y t i c l e u k a e m i a (Maino et al., 1 9 7 7 ; G o r d o n et al., 1978). A t t h e cellular level h o r s e r a d i s h p e r o x i d a s e ( H R P ) c o u p l e d to anti-Ig p r o v i d e s a sensitive and p e r m a n e n t label f o r l i g h t - m i c r o s c o p i c l o c a t i o n . T e c h n i c a l l i m i t a t i o n s , h o w ever, have r e s t r i c t e d its use at t h e u l t r a s t r u c t u r a l level. T h e s e l i m i t a t i o n s reflect conflict between adequate fixation, preservation of good ultrastruct u r a l detail, and c o n j u g a t e p e n e t r a t i o n o f t h e fixed m e m b r a n e . Labelling o f intracellular Ig a f t e r r e l a t i v e l y s t r o n g g l u t a r a l d e h y d e f i x a t i o n has b e e n r e p o r t e d ( R e y e s et al., 1 9 7 8 ) b u t is i n c o n s i s t e n t in o u r hands, probably because of restricted membrane permeability. Kraehenbuhl and J a m i e s o n ( 1 9 7 4 ) have u s e d F a b f r a g m e n t s o f Ig or m i c r o p e r o x i d a s e to im-
276 prove conjugate penetration, while others have used limited detergent solubilisation to increase membrane permeability (Hall et al., 1978). In this study we have attempted to establish a uniform technique for the ultrastructural localisation of Ig by the immunoperoxidase method using cell suspensions of normal and neoplastic lymphoid cells. MATERIALS AND METHODS
A n tibod y-peroxidase conjugate Sheep antibodies to human heavy and light chains, prepared by affinity purification, were purchased from Tenovus Laboratories, Southampton. These antibodies were conjugated to horseradish peroxidase (Grade V1, Sigma Chemicals Ltd.) using either the 2-step glutaraldehyde method (Avrameas and Ternynck, 1971) or the improved periodate method (Wilson and Nakane, 1978). The conjugated immunoglobulin was precipitated by ammonium sulphate and prepared on a Sephacryl 200 column (Pharmacia Ltd.). The conjugates were stored at 4°C at a concentration of 1 mg protein/ml in phosphate buffered saline (PBS), with 1 : 10,000 w/v methiolate. Control conjugates were prepared by the same methods using normal sheep IgG.
Isolation and preparation of cells Lymphoid cells were isolated from blood, bone marrow, tonsil, lymph nodes and spleen from normal donors and patients with lymphoproliferative disease. Cell suspensions were prepared from lymphoid tissue by teasing and filtration through a metal gauze. Haematopoietic cell lines were provided by Dr. A. Karpas, Department of Haematological Medicine, Cambridge and have been previously investigated for immunoglobulin expression and synthesis (Gordon et al., 1977). Jijoye and E B2 cell lines were purchased from Flow Laboratories. All cell populations were prepared on Ficoll-triosil gradients (B5yum, 1968). Harvested cells were washed twice in Hepes buffered minimal essential medium (MEM, Gibco-Biocult, Glasgow) and resuspended in 0.1, 0.5, 1.0 or 1.25% glutaraldehyde (Grade 1, Sigma Chemicals Ltd.) in 0.1 M Sorensen's phosphate buffer, pH 7.2, at room temperature for 15--30 min. Fixed cells were washed three times with phosphate buffer and stored in buffer containing 0.01% azide at 4°C until required. For all washing procedures after fixation an Eppendorf microcentrifuge was used and the microcentrifuge tubes were siliconised to reduce cell loss.
Saponin treatment of cells for intracellular Ig staining Cells prefixed in 0.1 or 0.5% glutaraldehyde were treated with 1.0% saponin (Sigma Chemicals Ltd.) at 55°C for periods of up to 100 min.
277 The treated cells were washed 3 times by centrifugation with phosphatebuffered saline.
Labelling and staining of cells for Ig localisation Cells were incubated with conjugate either directly after prefixation or saponin treatment. The cells were pelleted and resuspended in at least twice their volume of conjugate (1 mg/ml) and incubated for 60 rain at room temperature before washing three times with PBS. Peroxidase activity was detected by the m e t h o d of Graham and Karnovsky (1966) as follows. Ten mg of diamino benzidine (DAB) were dissolved in 20 ml of 0.05 M Tris HC1 buffer (pH 7.6) and the pH readjusted to pH 7.6; 0.2 ml of 1% hydrogen peroxide solution was added to the DAB solution and the cells incubated in the reaction mixture for 30 min at room temperature in the dark. Post fixation with 1% glutaraldehyde in 0.1 M Sorensen's buffer, pH 7.2, for 15 min at room temperature was usually performed after the DAB incubation. After washing in PBS the cells were post fixed in 1% osmic acid in Palade's acetate buffer, pH 7.2, washed in distilled water and embedded in 2% agar, prior to embedding in Spurr resin. Thin sections were cut on glass knives and viewed w i t h o u t further staining on a Phillips 201 or 300 transmission electron microscope at 40 kV. RESULTS Significant immunoperoxidase staining was rarely observed in prefixed (1--1.25% glutaraldehyde, 15--30 min) leukaemic cells, tonsil cells or cell lines, previously shown by immunofluorescence to have intracellular Ig (Fig. 1, Table 1). Obvious membrane damage was associated with m a n y of these stained cells but this was insufficiently consistent to totally account for the conjugate penetration. The non-saponin treated cells occasionally had the same intracisternal staining pattern as the treated cells but more frequently the staining was extracisternal and diffuse (Table 1). This difference in localisation was particularly well illustrated by the cell line Raji. When fixed with 1.25% glutaraldehyde for 30 min, Raji cells demonstrated a diffuse and extracisternal staining pattern (Fig. 2), whilst the same cells, treated with saponin, showed a monoclonal staining of the RER and PNS (Fig. 3). This extracisternal staining did not appear to be non specific and was n o t observed to the same extent with the control conjugate. The treatment of mildly prefixed (0.1% glutaraldehyde for 10 min) cells with 1% saponin allowed consistent penetration of the cell membranes by the conjugate and staining of intracellular Ig was observed in the rough endoplasmic reticulum (RER), perinuclear space (PNS) and vesicles of the Golgi apparatus (Fig. 4). The proportion of cells stained for intracellular Ig was significantly higher (Table 1) and the staining stronger in the saponin
278
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Fig. 1. Intracellular Ig locaJised in the perinuclear space (arrowed) i~ a CLL cel] fixed with 1% glutaraldehyde for 30 min. x13,500.
treated samples than in similar preparations fixed but n o t treated with saponin. This technique allowed the visualisation of intracellular Ig in human lymphoid cells from several sources including m y e l o m a (Fig. 4) and leukaemic cells (Fig. 5) from peripheral blood, tonsil cells (Fig. 6) and lymphoblastoid cell lines (Fig. 7). The anti Fab-7-peroxidase conjugate produced by the 2-step glutaraldehyde m e t h o d was significantly more efficient at penetrating the membranes of saponin treated cells than the conjugate obtained by the periodate technique, as judged by both the number of cells stained and the intensity of staining. No stained cells were observed in non-saponin treated cells when incubated with the periodate conjugated material. The treatment of lightly fixed (0.1% glutaraldehyde for 15 min at room temperature) cells with 1% saponin at 55°C resulted in a variable e x t e n t of cell damage. This damage was manifest as discontinuous intracellular and plasma membranes, dispersed cytoplasm and swollen mitochondria and endoplasmic reticulum. The extent of cell damage was dependent on the times of exposure, the cell source and the concentration of the prefixative. Reduction of the time of exposure to saponin from the 100 min recommended for sheep immunoblasts (Hall et al., 1978) to less than 10 min did
Fig. 2. Raji cell fixed with 1.25% glutaraldehyde showing granular and diffuse extracisternal cytoplasmic staining, x10,500.
Fig. 3. Raji cell prefixed with 0.1% glutaraldehyde and treated with saponin showing weak staining in the rough endoplasmic reticulum and perinuclear space, x10,500.
281
Fig. 4. Intracellular Ig localised in the perinuclear space, rough endoplasmic reticulum and Golgi in myeloma cells fixed with 0.1% glutaraldehyde for 10 min followed by 1% saponin treatment, x l 2,000. D
Fig. 5. Intracellular Ig visualised in the perinuelear space and rough endoplasmic retieulum of the same CLL population as Fig. 1 using saponin treatment to enhance penetration. ×12,900.
Fig. 6. Lymphoplasmaeytoid cell from a tonsil cell preparation, x14,200.
Fig. 7. Intracellular Ig localisation in the perinuclear space, rough endoplasmie reticulum and Golgi of the lymphoblastoid cell line 160. x6800.
283
Fig. 8. Jijoye cell prefixed with 0.1% glutaraldehyde and treated with saponin showing strong Ig staining in the perinuclear space, rough endoplasmic reticulum and Goigi apparatus as well as staining around the cell periphery which was also seen with the control conjugate. ×6500.
n o t cause any obvious change in either the location or the intensity of staining but significantly reduced cell damage. Cells from different tissue sources varied considerably in their sensitivity to saponin treatment. Circulating m y e l o m a cells and CLL cells when treated with saponin for 100 min, after 0.1% glutaraldehyde prefixation for 10 min, showed significantly less cell damage than cells isolated from tonsil, lymph node, spleen or peripheral blood. The cell lines were the most susceptible to damage by saponin but a comparison of cells fixed and processed for immunoperoxidase staining with cells fixed for conventional electron microscopy did not indicate any obvious subpopulation selectivity. An increase in concentration of glutaraldehyde to 0.5 or 1.0% significantly reduced the saponin-induced morphological damage but also reduced the staining intensity, frequently preventing Ig detection. Prefixation with 0.1% glutaraldeh y d e for 15 min at room temperature provided the most consistent and highest intensity staining of intracellularly localised Ig when combined with saponin treatment. Little surface Ig could be detected on cells shown to have intracellular Ig
284 by immunoperoxidase staining even though the presence of surface Ig was expected from immunofluorescence (Figs. 6 and 7, Table 1). It was apparent from a comparison of saponin treated and untreated cells that the surface Ig was stripped during exposure to saponin, even for short periods of less than 10 min after 0.1% or 0.5% glutaraldehyde fixation. Surface Ig was detected on cells fixed with 0.1% glutaraldehyde b u t was better preserved after fixation with 0.5% or 1.0% glutaraldehyde. No significant differences in the proportion of cells staining for surface Ig were detected between conjugates produced b y the periodate and t h e 2-step glutaraldehyde methods. Several types of non-specific intracellular staining were detected using the normal sheep IgG-peroxidase conjugate. When present, non-specific staining was observed as a diffuse, granular staining of the cytoplasm. In cells isolated from tissues or l y m p h o c y t e preparations with poor viability non-specific staining was usually associated with obviously necrotic cells. This type of non-specific staining could be eliminated b y removal of non-viable cells on a Ficoll-triosil gradient prior to fixation. In some cell lines a second type of non-specific staining was observed in both saponin treated and untreated cells. This staining had a similar diffuse cytoplasmic presentation b u t was associated with the cell surface (Fig. 8), and was detected with the control conjugate as well as b o t h anti-light chain conjugates. This stain was related to neither surface Ig expression nor to cell viability, and only occurred in 2 of the 6 cell lines so far tested. DISCUSSION The immunoperoxidase technique is extensively used in the diagnosis and classification of lymphomas b y light microscopy, b u t because of the impermeability of the glutaraldehyde fixed cell membrane to antibody-peroxidase conjugate (Kuhlmann et al., 1974) has not been used for localisation of intracellular Ig except in association with methods which involve removal of the cell membrane barrier such as those employing cryostat sections (Kraehenbuhl and Jamieson, 1974). The immunoperoxidase technique has, however, been extremely useful for ultrastructural demonstration of surface Ig on B l y m p h o c y t e s in suspension (Reyes et al., 1975) and during such studies Reyes et al. {1978) reported that antibody-peroxidase conjugate penetxates glutaraldehyde-fixed cell membranes. We suggest that the criteria for an acceptable technique for the ultrastructural localisation of Ig in human B l y m p h o c y t e s should include: (1) good morphological preservation comparable with routine ultrastructural pathological preparations, (ii) suitability of lymphoid cells obtained from a variety of tissues, including blood, and (iii} a direct labelling technique minimising the number of manipulations and reducing cell loss and contamination. Our studies confirm that intracellular localisation of Ig is possible after prefixation with glutaraldehyde alone b u t that not every monoclonal population of cells k n o w n to contain intracellular Ig is stained b y this method
285 and furthermore a significantly smaller population of cells was stained compared with other ultrastructural techniques. Cells stained in this way exhibit a diffusely cytoplasmic rather than intracisternal pattern. This pattern m a y be associated with restricted diffusion of conjugates through intracytoplasmic membranes. Hall et al. (1978) have demonstrated that the permeability of lightly glutaraldehyde-fixed sheep immunoblasts to peroxidase labelled antibody conjugates is enhanced b y treatment with 1% saponin for 100 min at 55°C. Our results indicate that a similar saponin treatment of human B lymphoid cells allows adequate penetration of the conjugate through the plasma and intracytoplasmic membranes to give reliable staining of Ig in the endoplasmic reticulum, t h e perinuclear cisternae and the Golgi apparatus. Staining patterns agree in both monoclonality and distribution with those observed b y immunofluorescent methods (Gordon et al., 1977). Damage resulting from exposure of cells to saponin is considerable, in particular loss of membrane and cytosol integrity, swelling of mitochondria and endoplasmic reticulum and significant loss of surface immunoglobulin. Differences in the susceptibility of different cell types to saponin treatment were apparent; for example, cells from lymphoid tissues were considerably more susceptible than blood lymphocytes. Some of these differences may be due to damage caused during tissue dissociation. Leukaemic cells tended to be the least susceptible while the lymphoblastoid cell lines were the most sensitive to digestion with saponin. Saponin-induced damage did not result in significant non-specific staining but the presence of non-viable cells before glutaraldehyde fixation produced non-specific, diffuse cytoplasmic staining in samples incubated with control conjugates as well as with anti Fab~,-peroxidase conjugates. The removal of dead and dying cells on Ficoll triosil gradients was therefore considered essential. Much of the morphological damage resulting from cell exposure to saponin was prevented by reduction of the exposure time to 5 or 10 min. This neither inhibited conjugate penetration nor altered immunoglobulin location. Attempts to further reduce damage by increasing the initial glutaraldehyde fixation were unsuccessful presumably because of the resulting more extensive cross-linking of membrane proteins. Although a fixation regime of 0.1% glutaraldehyde is considered the most suitable for immunoelectron-microscopic detection of immunoglobulin (Van Ewijk et al., 1980), this is not optimal for morphological preservation and may be insufficient to prevent antigen movement. However the intracellular Ig localisation patterns in cells from human tonsil and spleen fixed in this way are consistent with those seen with other ultrastructural techniques, such as periodate-lysine-paraformaldehyde fixation and cryostat sections (Tsunoda et al., 1978). Similar localisation patterns for IgG were observed in rabbit plasmacytes with formaldehyde fixation (Kraehenbuhl and Jamieson, 1974), and for anti-peroxidase antibodies with peroxidase as both immunogen and marker (Leduc et al., 1968). This suggests that neither mild
286 g l u t a r a l d e h y d e f i x a t i o n n o r saponin t r e a t m e n t causes r e d i s t r i b u t i o n o f imm u n o g l o b u l i n w i t h i n the cell. T h e p e r i o d a t e m e t h o d o f c o n j u g a t i o n is highly e f f i c i e n t in conjugating H R P t o IgG (Wilson and Nakane, 1 9 7 8 ) , b u t this c o n j u g a t e was relatively p o o r at labelling intracellular Ig. This m a y be d u e to t h e larger size o f t h e c o n j u g a t e (up t o 5 or 6 m o l e s H R P t o I m o l e IgG) (Wilson and N a k a n e , 1 9 7 8 ) c o m p a r e d w i t h t h a t p r o d u c e d b y t h e 2-step g l u t a r a l d e h y d e m e t h o d (1 m o l e H R P to 1 m o l e IgG) ( K u h l m a n et al., 1 9 7 4 ) . Also such larger conjugates have d e c r e a s e d a n t i b o d y activity d u e t o the extensive H R P binding (Wilson and Nakane, 1 9 7 8 ) . T h e loss o f surface Ig d u r i n g the p r e p a r a t i o n p r o c e d u r e m e a n s t h a t surface and intracellular Ig c a n n o t b o t h be visualised at the same t i m e b y this t e c h n i q u e . This is a disadvantage for studies correlating the d i s t r i b u t i o n o f surface and intracellular Ig d u r i n g l y m p h o c y t e d i f f e r e n t i a t i o n , and w h e n h e t e r o g e n e o u s cell p o p u l a t i o n s f r o m p a t h o l o g i c a l specimens are being e x a m i n e d . H o w e v e r t h e t e c h n i q u e d e s c r i b e d appears to be a d e q u a t e for studies o f m o n o c l o n a l p o p u l a t i o n s and will be used to investigate the sites o f synthesis and secretion o f Ig in r e l a t i o n to b i o s y n t h e t i c p a t t e r n s and f o r c o m p a r i s o n with various light m i c r o s c o p i c m e t h o d s o f Ig d e t e c t i o n . ACKNOWLEDGEMENT Dr. D.G. Newell and Dr. J.L. S m i t h are in r e c e i p t o f a grant f r o m the Cancer Research Campaign for this p r o j e c t . REFERENCES Avrameas, S. and T. Ternynck, 1971, Immunochem. 8, 1175. BSyum, A., 1968: Scand. J. Clin. Lab. Invest. 21, Suppl. 97. Gordon, J. and J.L. Smith, 1978, Clin. Exp. Immunol. 31,244. Gordon, J., D. Hough, A. Karpas and J.L. Smith, 1977, Immunology 32, 559. Gordon, J., A.R. Howlett and J.L. Smith, 1978, Immunology 34,397. Graham, R.C. and M.J. Karnovsky, 1966, J. Histochem. Cytochem. 14,291. Hall, J.G., M.S.C. Birbeck, D. Robertson, J. Peppard and E. Orland, 1978, J. Immunol. Methods 19, 351. Kraehenbuhl, J.P. and J.D. Jamieson, 1974, Int. Rev. Exp. Pathol. 13, 1. Kuhlman, W.D., S. A~ameas and T. Ternynck, 1974, J. Immunol. Methods 5, 33. Leduc, E.H., S. Avrameas and M. Bouteille, 1968, J. Exp. Med. 127, 109. Maino, V.C., J.T. Kurnick, R.T. Kubo and H.M. Grey, 1977, J. Immunol. 118, 742. Reyes, F., J.L. Lejonc, M.F. Gourdin, P. Mannini and B. Dreyfus, 1975, J. Exp. Med. 141,392. Reyes, F., M.F. Gourdin, J.P. Farcet, J. Breton-Gorius and B. Dreyfus, 1978, Recent Results Cancer Res. 64,176. Tsunoda, R., K. Terashima, K. Takahashi and M. Kijima, 1978, Acta Path. Jap. 28, 53. Van Ewijk, W., R.C. Coffman and I.J. Weissman, 1980, Histochem. J. 12,349. Wilson, M.B. and P.K. Nakane, 1978, in: Immunofluorescent and Related Staining Techniques, eds. W. Knapp, K. Holubar and G. Wicks (Elsevier, Amsterdam) p. 215.