Cytochemical and ultrastructural studies concerning the cell coat glycoproteins in normal and transformed human blood lymphocytes

Printrd in Sweden Copyright Q 1974 by Academic Press, Inc. All rights of reproduction in any/mm reserved

Experimental Cell Research84 (1974) 31-39

CYTOCHEMICAL AND ULTRASTRUCTURAL STUDIES CONCERNING THE CELL COAT GLYCOPROTEINS IN NORMAL AND TRANSFORMED HUMAN BLOOD LYMPHOCYTES I. Variations of Sialic Acid Containing Glycoproteins Subsequent to Transformation of T and B Lymphocytes by Various Kinds of Stimulating Agents ANNY ANTEUNIS Centre de Physiologie

et d’lmmunologie Cellulaires, INSERM U. 104, CNRS, et Association H6pital Saint-Antoine, Paris 12, France

Claude Bernard,

SUMMARY This is an ultrastructural study on variations occurring subsequent to blast transformation in the amount and/or topographical distribution of sialic acid end groups carried by the cell coat glycoproteins of human blood T and B lymphocytes as revealed by PTA staining according to the Rambourg technique. The transformation was immediately followed by a transient decrease of the surface labelling in both T- and B-derived lymph-cell lines, irrespective of the nature of the stimulating agents used for induction of transformation. Nevertheless, upon reaching their final cyto-differentiation step, cells of both lines seemed to recover their initial PTA staining patterns observed in the controls. This transient decrease in the relative number of sialic acid end groups during a period of dramatically rapid cell growth and multiplication may most plausibly be interpreted as reflecting an ‘incomplete maturation’ of the newly synthesized cell coat glycoproteins.

In a previous work [3] we succeeded in showing that a significant number of acidic amino sugar end groups are carried by the glycoprotein molecules which occur within the cell coat structures of mammalian and avian lymphocytes. The presenceof these end groups has been demonstrated by means of staining with the hydrochloric-phosphotungstic acid mixture (PTA) ad modam Rambourg [19]. Moreover, by screening with a series of glycolytic enzyme pretreatments, we demonstrated that only sialidase (of source CZ. perfringens) was able to completely prevent 3-741811

the ultrastructural staining of the lymphocyte cell coat by PTA at low pH. Hence, it became clear that, in intact lymphocytes, sialic acid seemed to be the sole type of acidic amino sugar accounting for PTA staining. The above-mentioned sialic acid residues may be involved in the expression of various lymphocyte functions as revealed by these cells’ relevancy for electrophoretic mobility [ 141as well as for their behaviour versus ALS [2, 51 and homing (i.e. ecostatic) properties [4, 11, 261. In this paper, we report our findings concerning the ultrastructural aspects which Exptl Cell Res 84 (1974)

32 Army Anteunis

seem to be correlated with the presence of sialic acid-containing glycoproteins in human blood lymphocytes subsequent to their transformation upon treatment with various agents known to be active against either B or T cells in other mammalian species[12]. MATERIAL

AND METHODS

Abbreviations used PTA-HCl, Phosphotungstic hydrochloric acid; ALS, anti lymphocyte serum; GERL, associated Golgi apparatus and smooth Endoplasmic Reticulum from which Lysosomes appear to form. of the lymphocytes: The lymphocytes were isolated from human blood. The technique used for their separation was essentially that of Boyum [6] with minor modifications. (2) Culture: lo6 lvmnhocvtes were cultured during 5 &ys in the presence of
cells exhibited clearcut positive reactions, occurring as discontinuous electron-dense layers, external to the cell membranes (fig. 1). Stimulated cultures

As previously described [l] at least two basic types of transformed lymphocytes were observed in the various stimulated culture samples as a function of the nature of the mitogen

employed:

the stimulation

by means

of either one of the anti intact heavy Ig chain sera resulted in genuine classical plasmoblastic aspects, while conversely, stimulation by means of either phytomitogens or ALS or tuberculin or even, surprisingly, by anti F(ab’)z sera resulted in immunoblasts which exhibited obvious morphological differences as compared with the plasmoblasts. In addition to these two main basic cell types, several intermediate forms have also been observed

VI.

PHA-blast transformed cells showed discontinuous PTA-positive figures of varying density. Nevertheless, the negative zones were more important than those observed in the controls. It should be noted here that in the large blastic reticular-like cells, the Golgi regions which are well developed in this type (4) Hydrochloric-phosphotungstic acid (PTA) stainof cells (probably Gerl [ 171)always underwent ing: The cell suspensions were centrifuged at 400 g strongly positive reactions (fig. 2). No change during 10 min. The pellets were subsequently fixed in 1.5 % phosphate-buffered glutaraldehyde during 1 h in the distribution of PTA positivity has been at 4°C and finally rinsed overnight in the same buffer. observed in the cell coat during mitosis. After glycolmethacrylate embedding according to Leduc & Bernhard [13] gold-brown sections were preThe immunoblast-like cell-type observed pared and floated during 30 min at room temperature in the cultures stimulated by Con A, MLR on a 3 % hydrochloric-phosphotungstic acid solution according to Rambourg [20]. and tuberculin, exhibited a marked decrease The sections were then picked up on naked grids in the PTA-positive staining. (The staining and examined in the Siemens Elmiskop I without counterstaining. occurred as a thin dotted line, which was clearly localized outside the external leaflet of RESULTS the cell membranes) (figs 3, 4). Control cultures Upon incubation with ALS, the cell coat The 5-day-old control cultures carried out of the transformed cells showed the same under the above-mentioned conditions con- characteristics as the PHA-stimulated lymsisted of predominantly small lymphocyte phocytes, except that the transformed cells populations. On staining with PTA these bore cone-shaped PTA-positive deposits Exptl Cell Res 84 (1974)

Surface sialic acid on transformed lymphoc ytes

Figs.l-8. U1trastnztura1.aspect.s of the cell coat of normal and transformed human blood lymphocy rtes stzlined by the PTA-HCl technique. Fig. 1. Normal human blood small lymphocyte. Cell coat intensly stained . x24 Fig. 2. PHA-stimulated lymphocyte. Note the patchy distribution of the PTA-HCI label. Golgi areta (GEEi~ strongly positive. x 16 000. Exptl Cell Res 84 (1974)

34 Anny Anteunis

Fig. 3. ConA-stimulated lymphocyte. x 14 000. Fig. 4. Tuberculin-stimulated lymphocyte. x 18 000. The surface staining of both ConA- and tuberculin-stimulated lymphocytes presents a dotted-line appearance clearly localized outside of the external leaflet of the cell membrane (see enlargement, fig. 3). Exptl

Cell Res 84 (1974)

Surface sialic acid on transformed lymphocytes

35

Fig. 5. ALS-stimulated lymphocyte. Distribution of the label is similar to that noted for PHA, except for shaped strong PTA positive deposits (-). x 20 000. Fig. 6. Anti-F(ab’), serum-stimulated lymphocyte. x 36 500. Exptl Ceil Res 84 (1974)

36 Amy Anteunis

Fig. 7. Anti 8 chain serum-stimulated lymphocyte. Note for these (figs 6, 7) transformed cell-ty1)es a weak punctilinious PTA staining. x 12 000. Fig. 8. Plasmoblast observed upon incubation with anti c( chain serum. This cell type shows a stroi ng PClsitive PTA staining similar to that noted for the control. x 12 000.

Exptl Cell Res 84 (1974)

Surface sialic acid on transformed lymphocytes

corresponding to the hair-like 1.?. cytoplasmic extensions previously described for this celltype [l] (fig. 5). Upon incubation with anti-Ig sera directed against human F(ab’), and Fc fragments, as well as against intact human heavy Ig-chains, all the transformed cell-types we were able to observe exhibited weak, punctilinious PTA staining (figs 6, 7). Nevertheless in plasmocytes which apparently correspond to the final cytodifferentiation step of the Bderived antibody synthesizing cell lines (unpublished data) the PTA-positive ultrastructural aspects were closely similar to those observed in the controls (fig. 8). DISCUSSION The superficial structures which coat the external membranes of the lymphocytes as well as of most other eukaryotic cell-types, mainly consist of glycoproteins which contain sialic acid residues [18, 191.The observation that most sialic acid residues which are located on the lymphocyte’s surface are released upon sialidase pretreatment, thus leading to a marked decreasein the cellular electrophoretic mobility [2, 141,clearly suggests that these acidic amino sugar molecules must be attached in terminal positions on the oligosaccharide side chains. Moreover, the fact that theseneuraminidase-sensitive anionic groups are able to bind selectively some chemical labels as PTA provides the technical opportunity to use such labels as specific markers of the existence of sialic acid end group carrying macromolecules within the cell coat. As only two physicochemical parameters (the density and the stereochemical availability of those sialic acid end groups) seem to account completely for this type of histochemical reactivity at the cell coat level in small lymphocytes of human blood, it be-

31

comes obvio,us,that the Rambourg technique for PTA staining provides highly reliable data concerning cell coat morphochemistry. Roughly identical staining patterns have been observed on the surfaces of lymphocytes of various origins, irrespective of the fact that they had been harvested from rats [19] from mice or from chicken donors [3]. The blast transformation phenomenon, which is characterized by important physiological and biochemical modifications such as (a) dramatic increase of the cell volume, (b) development of the vacuolar system [22] and (c) of the protein synthesis apparatus, (d) cortical and (e) endoplasmic viscosity modifications [8], also involves (f)alterations of the cell surface configuration. Thus, a significant increase of the intramembranous granule population is observed in PHA-transformed lymphocytes [24]. Nevertheless, our results demonstrate that staining of the superficial sialoproteins by PTA is lessin all the observed transformed cells than in the controls. This decrease of the surface labelling does not seem to be related to the nature of the stimulating agent used for induction of transformation, since this phenomenon has been observed both after treatment with nonspecific agents known to act on T-derived lymphocytes (e.g. PHA, Con A, ALS) or on B-derived lymphocytes (anti F(ab’),, anti Fc and anti intact heavy Ig-chains sera) as well as subsequently to specific antigenic stimulation, e.g. with tuberculin. In the transformed cells, the topographical distribution patterns of the sialic acid residues over the entire cellcoat surface occurred as discontinuous small agglomerations, dispersedoutside the external leaflet of the unit plasma membrane, thus roughly resembling a dotted line. This transient decrease of the density of PTA-labeled structures on the cell surface may plausibly be accounted for by one or the other of the following mechanisms: Exptl Cell Res 84 (1974)

38 Anny Anteunis

0

oligora:ciarid~

chain

9. Various possible situations oligosaccharidic chain maturation.

Fig. glycopratein

bclckione

(a) either the occurrence of some alteration in the pK of the sialic acid residues, involving a subsequent decreaseof the entire sialoprotein histochemical reactivity, (b) or the occurrence of a relative decrease in the surface density of the sialic residues per pm2 due to the ‘topographical dilution’ of the superficial sialic acid-carrying glycoproteins entailed by the rapid cell growth which regularly follows a very recent triggering of blast transformation, (c) or, finally, the occurrence of some genuine (i.e. absolute) decrease in the number of the terminal sialic acid residues, which may be accounted for either by: (i) an enzymatic loss ‘post synthesis’; or (ii) an incomplete ‘maturation’ of the newly (and, thus, ‘too rapidly’) synthesized glycoproteins, which subsequently are devoid of their terminal sugar subunits, hence, in this given instance, the sialic acid ones (fig. 9). The last interpretation seems to be the most plausible, being consistent with both modern molecular cell genetics knowledge, as well as with similar observations recently published concerning the incomplete maturation of other kinds of glycoproteins [9, 15,211. These differences in the number of the sialic acid residues, available on the surface of Exptl Cell Res 84 (1974)

of

the small and of the transformed lymphocytes may easily account for the occurrence of apparently ‘new’ antigens. Indeed, it is well known that following sialidase pretreatment different kinds of cell types apparently increase their antigenicity [7, 10, 16, 23, 251 or unmask ‘new’ antigens which are normally ‘covered’ (and, thus, sterically hidden) by the sialoproteins located within the cell coat. The modifications observed in the distribution of sialic acid residues on the lymphocyte surfaces are always the same, irrespective to the fact that the transformed cells showing a blasto’id- or immunoblast-like aspect (subsequent to stimulation by phytomitogens, ALS, tuberculin and anti-F(ab’), sera) or a plasmoblast-like aspect (subsequent to stimulation by means of anti-Fc and anti intact heavy Ig-chains sera). However, upon stimulation with antia-serum, the surface staining of some transformed cells which exhibited typical plasma cell ultrastructural characteristics became closely similar to those of the controls. In a previous work, we have reported that the in vitro lymphocyte blast transformation may be considered as a phenomenon of ‘Corward’ cell differentiation (unpublished data). Thus, the modifications occurring in the

Surface sialic acid on transformed lymphocytes

distribution of the sialic acid end groups observed on the cell surface during this stepwise differentiation process seemto be closely related to the ‘juvenile’ aspect which regularly occurs in cells undergoing dramatic biochemical and physiological changes. Nevertheless, upon reaching the end point of their differentiation process (subsequent to stimulation by means of anti intact heavy Ig-chain sera) the B-derived lymphocytes occur as antibody-secretory cells, whose cell coats are apparently regaining the original morphochemical patterns, characteristic of the initial small lymphocyte.

REFERENCES 1. Anteunis, A, Arch biol (Bruxelles) 84 (1973) 281. 2. Bona, C, Anteunis, A, Robineaux, R & Halpem, B, Clin expl immunol 12 (1972) 377. 3. Bona. C & Anteunis. A. Ann Pasteur 124~ (1973) 4. Bemey, S N & Gesner, B M, Immunology 18 (1970) 681. 5. Bert, G, Jasolodi Cossano, D, Pecco, P & Mazzei, D, Lance% I (1970) 365. 6. Boyum, A, Stand j clin lab invest 21 (1968) suppl. 97. 7. Currie, G A, Van Doomick, W & Bagshawe, K B, Nature 219 (1968) 191. 8. De Robertis, E D, Nowinski, W W & Saez, F A,

39

Cell biology, 5th edn, p. 255. Saunders Co., Philadelphia (1970). 9. Dumitrescu. M S. Moraru. I & Popescu. S. Excerpta medica international congress series 233 (1971) 60. 10. Flye, M W, Grothaus, E A & Amos, D B, Surg forum 22 (1971) 97. 11. Gesner, B M & Ginsburg, V, Proc natl acad sci US 52 (1964) 750. 12. Greaves, M F & Janossy, G, Tramp1 rev 11 (1972) 87. 13. Leduc. E & Bernhard. W. J roval micro sot 81 (1963)‘114. ’ ’ 14. Lichtman, M A A & Weed, R I, Blood 35 (1970) 11 15. Mbraru, I, Dumitrescu, S M & Sulica, A I, Eur j clin biol res XVII no. 5 (1972) 451. 16. Oxley, S & Griffen, W 0, Surg forum 22 (1971) 113. 17. Novikoff, P N, Novikoff, A B, Quintana, N & Hauw, J J, J cell biol 50 (1971) 859. 18. Rambourg, A & Leblond, C P, J cell biol 32 (1967) 27. 19. Rambourg, A, J micr 8 (1969) 325. 20. - Ibid 11 (1971) 163. 21. Rivat, L, Ropartz, C, Lebreton, J P, Minnoni, P, Delarue. F. Gerval. A. Salmon. C. Reviron. J & Rochant, H, Nouv’rev fr hematoll (1970)‘371. 22. Robineaux, R, Anteunis, A, Bona, C & Chauvet, G, Co11 intemat CNRS sur “Le serum antilymphocytaire” no. 190 (1971) 19. 23. Sanford, B H, Transplantation 5 (1967) 1273. 24. Scott, R E & Marchesi, V T, Cell immunol 3 (1972) 301. 25. Simmons, R L, Rios, A & Ray, P K, Nature new biol 23 (1971) 179. 26. Woodruff, J & Gesner, B M, J exptl med 129 (1969) 551. Received September 11, 1973

Exptl Cell Res 84 (1974)