Ellegaard and Dimitrov [1973] have demonstrated the existence of two different

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1 Quarterly Journal of Experimental Physiology (1977) 62, ADENOSINE TRIPHOSPHATASE LOCATED ON UNSTIMULATED HUMAN SMALL LYMPHOCYTE CELL MEMBRANES. By A. S. COULSON, V. H. ZEITMAN, R. B. COHN, E. DONG, R. B. GRIEPP, E. B. STINSON and N. E. SHUMWAY. From the Departments of Surgery and Cardiovascular Surgery, Stanford University School of Medicine, Stanford, Calif., U.S.A. (Received for publication 27th February 1976) (Revised version 5th July 1976) This study was undertaken to localize the enzyme sodium-potassium dependent adenosine triphosphatase in unstimulated human small lymphocytes using the histochemical technique of McClurkin [1964]. The substrate adenosine 5' triphosphate is hydrolyzed by the ATPase resulting in a lead phosphate precipitate at the site of enzyme action, subsequently visualized as lead sulphide. The enzyme was demonstrated in three different patterns, and for each donor the pattern was constant both on all four of the test slides, and on different occasions. The patterns observed were: clusters of granules related to the cell membrane; positive staining localized to portions of the cell membrane, and, less commonly, the whole cell circumference. The significance of this distribution may relate to areas with large numbers of antigen recognition sites on the lymphocyte membrane. Coulson [1969] suggested that the antigen recognition site on sensitized small lymphocytes was a unique three dimensional structure incorporated in the cell membrane, that micro-deformation of the membrane occurred following sterochemical recognition of a specific antigen at the site, and that the resulting membrane distortion was transduced by non-specific cholinesterase and ATPase [Duncan, 1967] to initiate an intracellular stimulation pathway. Since the hypothesis was first proposed, cholinesterase has been demonstrated on small lymphocyte cell membranes [Coulson, 1970], and more recently Ellegaard and Dimitrov [1973] have demonstrated the existence of two different ATPase activities in homogenates of human lymphocytes, one of which was ouabain sensitive and possibly associated with the cell membranes. These findings are consistent with the probable existence of at least two ATPases in the cell, one associated with mitochondria and oxidative phosphorylation, and another associated with cation transport [Roodyn, 1967]. The study described in this paper was undertaken with small lymphocytes prior to any stimulation as part of an evaluation of the enzyme systems in the small lymphocyte which would be available in the event of antigenic stimulation. In particular, a search was undertaken for any unusual arrangement of the membrane ATPase in view of its postulated role as an antigen recognition site marker. By using the cytochemical technique developed by McClurkin [1964] it was hoped not only to confirm the existence of ATPase in the small lymphocyte membrane but also to investigate its distribution, in keeping with Lison's 181

2 182 Coulson, Zeitman, Cohn, Dong, Griepp, Stinson and Shumway principle [Dixon, 1970], 'savoir non pas seulement que telle substance se trouve dans tel organe, mais ou elle s'y trouve.' METHODS Lymphocyte separation Blood from ten normal healthy adult donors was used. The lymphocytes were separated and smears of the cells made in the manner described by Coulson and Kennedy [1971]. In some instances, slow defibrination [Pickup, 1949] was employed and the concentrated lymphocyte suspension was used in the same way to make smears. For each donor four slides were made for the test incubation medium and four for each of the control media. Three of the donors were bled a second time some weeks after the first occasion and the whole experiment repeated in toto. With four donors every control was included; four slides were incubated in a medium lacking magnesium, four in a separate medium with added calcium, four in a medium with added ouabain, and yet another four in a medium lacking sodium and potassium (total of 20 slides per donor counting the four test slides). Two further donors just had the sodium and potassium omission control, and another donor just had the ouabain control. Fixation and histochemistry Lymphocyte smears were air-dried, then fixed in buffered 4 % formalin at room temperature for 120 ± 10 secs. The slides were then washed in three changes of glass distilled water for a total of 6 min. Test slides were incubated in a medium containing the following constituents: adenosine 5' triphosphate (3 0 mmol.y1, Sigma, lot 12C-7660), Trismaleate buffer (ph 7T8, 24 mmol.1-1), lead nitrate (3 mmol.y1), and magnesium sulphate (3 mmol.1-1). The techniques described by McClurkin [1964] were used for dissolving the dibarium salt of ATP in very dilute hydrochloric acid, making up the medium and for removing the precipitate by centrifugation. The ph of the test medium had to be adjusted in most instances with one or two drops of Tris stock solution A to bring it to the required value of 7-2. In addition, sodium chloride and potassium chloride solutions were added to the test medium to a final concentration of 100 mmol.1-i and 30 mmol.l1 respectively. Incubation was continued for 60 ± 5 min at 37 C in disposable Petri dishes; in some instances, incubation times of 30 min were used. Slides were supported face down in the incubation medium to avoid non-specific deposits of any residual precipitate. After incubation, the slides were washed carefully by rinsing and agitation in three changes of glass distilled water for a total of 6 min. They were then immersed in freshly prepared ammonium sulphide solution (1 % weight/volume in distilled water), followed by three washes, in distilled water, totaling 6 min. At this point, the slides were stained with methylene blue for 1 min and then washed in distilled water to 'blue' the nuclei. Finally, the slides were mounted in glycerol jelly. Controls The following controls were employed: (1) sodium and potassium chloride solutions were omitted; (2) or ouabain (3 x 10-5 mol.1-1, Eli Lilly 7 GC 13A) was added to the incubation medium. Further control studies carried out included the omission of magnesium from the medium (3); and separately the addition of calcium chloride to a final concentration of 8 mmol.l- 1 (4). The purpose of the controls was to demonstrate that the lymphocyte membrane sodiumpotassium dependent ATPase had the same specificity as the sodium-potassium ATPase was known to have in vitro. The latter enzyme is known to require the presence of magnesium ions for its activity, and similarly to require the presence of sodium and potassium, on the other hand it is inhibited by ouabain and calcium ions.

3 FIG. 1. Smears of lymphocytes were incubated in a medium containing ATP, lead ions and sodium and potassium, and lead phosphate deposited at the site of enzyme activity was converted to black lead sulphide. x Portions of the membrane (arrows) are stained indicating sites of ATPase activity. [To face page 183

4 ATP in lymphocyte cell membranes 183 RESULTS Localization of the ATPase Four slides were stained in each incubation medium from each donor. Three different patterns of positive staining were identified. The pattern for a given donor was seen in all four of his test slides on each occasion he was employed. The patterns were: either a cluster of granules or dots related to the cell membrane, the staining of a portion of the cell membrane, or of the whole of the cell membrane (Figs. 1 and 2). The cluster of granules appeared to be related to the cell membrane because when adjusting the fine focus under oil immersion the granules came into focus in the same plane as the membrane. A B FIG. 2. Diagrams showing three patterns of staining seen in the small lymphocytes: (a) clusters of granules, (b) portions of membrane (based on Fig. 1), and (c) the entire cell membrane. The last pattern was only seen with one donor. The experiment was done formally 13 times with 10 different donors (3 donors were used on a second occasion after an interval of several weeks); the granule pattern was seen nine times (2 donors on 2 occasions, 5 donors once only), the portion of membrane 3 times (one donor twice, one donor once only), and the whole membrane once. On any one test slide the positive staining of the lymphocytes was overwhelmingly one or the other of the three patterns. The percentage positivity of the small lymphocytes on the slides varied from 10 to 100%. The erythrocytes in the smear were consistently negative. Incubation for 60 min produced denser staining than 30 min. CONTROLS Controls With six donors (4 slides each), when sodium and potassium were omitted from the medium, three donors yielded trace positive results in the same staining

5 184 Coulson, Zeitman, Cohn, Dong, Griepp, Stinson and Shumway pattern as the test slides, and three others were completely negative. With five donors (4 slides each) where ouabain was added there was no staining. With the four donors where the magnesium omission controls and the calcium controls were carried out, staining of the lymphocytes was inhibited on all 32 of the slides. DISCUSSION Earlier studies on lymphocyte membrane enzymes possibly related to that cell's immunologic functions have included the localization of cholinesterase at the small lymphocyte cell membrane [Coulson, 1970], and nucleoside diphosphatase on the Golgi membranes [Coulson, 1965] of the transformed cell, the latter structure possibly being active in the secretion of lymphokines. A subsequent study [Coulson and Kennedy, 1971] showed that cyclic AMPase was localized on the nuclear membrane, although a definite role has not yet been established for this enzyme in the stimulation pathway. The experiments described in this paper have shown localization of ATPase in one of three patterns: clusters of dots, sections of the cell membrane, and the whole cell circumference. The sensitivity of the staining to ouabain inhibition and the effect of the various cations is good evidence that a sodium-potassium dependent ATPase is indeed responsible for the staining phenomena. Previous work by Fries, Bryon, Brunat, Bruchier, Revillard and Traeger [1967], indicated that ATPase could be demonstrated in electron microscope studies of transformed lymphocytes as grains 50 to 100 nm in diameter on the external face of the cell membrane. A maximum of one-third of the transformed cells were positively stained; small lymphocytes were reported to be negative. Gropp and Fischer [1964] similarly reported that small lymphocytes were unstained in cytochemical preparations undertaken to localize ATPase, but that PHA induced transformed cells were positive at the cell borders. Subsequently, Lichtman and Weed [1969], and Ellegaard and Dimitrov [1973] showed that small lymphocytes did possess a Na/K activated ATPase which was ouabain sensitive. The work of Quastel and Kaplan [1970] suggested that lymphocyte stimulation involved early and protracted activation of the Na/K ATPase associated with membrane transport and was critically dependent on a subsequent increase in intracellular potassium. The present demonstration of ATPase complements the recent work with whole cells in culture and with extracts of small lymphocytes; the reason for the successful demonstration of the enzyme in the present study, in contrast to earlier cytochemical studies, is probably related to the increased sensitivity of the McClurkin technique. The fact that the enzyme was localized to specific groups of dots and that the pattern was repeated from cell to cell is suggestive of some kind of subcellular organization. While we fully appreciate that the antigen recognition site is probably of the order of 4 nm diameter to match the size of the antigenic determinant, we suggest the possibility that there may be specialized areas of the lymphocyte membrane bearing antigen recognition sites, and that positive

6 ATP in lymphocyte cell membranes 185 staining visible at the light microscopic level may represent large numbers of these clustered in such special areas. In the event ATPase is proven to have immunological significance its precise location may point to the site where it and cholinesterase are activated during membrane microdeformation immediately following binding and recognition of an antigen. It is too early to attribute a physiological significance to the arrangement of the enzyme pattern but the fact that it is located at the cell surface lends support to the original hypothesis [Coulson, 1969]. The study of antigen recognition sites is somewhat frustrating, as there is as yet no technique available which permits direct visualization of the sites, let alone study of the interaction of antigens and site. In the absence of otherwise suitable technology, resort has been made to indirect studies, including genetics [David, Frelinger and Schreffier, 1974], binding of labelled antigen [DeLuca, Decker, Miller and Sercarz, 1974], or of antibodies to the presumed site [Solheim and Thorsby, 1974]. The study of enzymes which may be related to the antigen recognition site is another line of circumstantial evidence. There is, however, one further interesting aspect to considering the enzymology of the recognition site, and that is that the deployment of specific enzyme inhibitors may permit a more specific pharmacological approach to immunosuppression. ACKNOWLEDGMENTS This study was supported by the University of London Laura de Saliceto Studentship and the American College of Surgeons Schering Scholarship. REFERENCES COULSON, A. S. (1965). The Golgi apparatus in small lymphocytes and in transformed blast cells. Quarterly Journal of Experimental Physiology, 50, COULSON, A. S. (1969). Recognition pathway in lymphocytes. Journal of Theoretical Biology, 25, COULSON, A. S. (1970). Open discussion following 'Biochemical and Morphological aspects of lymphocyte transformation' presentations. Proceedings of the Fifth Leukocyte Culture Conference, Ed. Harris, J. E. Academic Press, London and New York, pp COULSON, A. S. and KENNEDY, L. A. (1971). Lymphocyte membrane enzymes II. Cyclic 3',5'-adenosine monophosphatase located on unstimulated human small lymphocyte nuclear membranes. Blood, 38, DAVID, C. S., FRELINGER, J. A. and SCHREFFLER, D. C. (1974). New lymphocyte antigens controlled by the Ir-IgG region of the H-2 gene complex. Transplantation, 17, DELUCA, D., DECKER, J., MILLER, A. and SERCARZ, E. (1974). Antigen binding to lymphoid cells from unimmunized mice: high frequency of beta-galactosidose binding cells at optimal conditions. Journal of Cellular Immunology, 10, DIXON, K. C. (1970). Histochemical studies on the skin. An Introduction to the Biology of the Skin. Ed. Champion, R. H., Gillman, T., Rook, A. J. and Sims, R. T. Blackwell, Oxford and Edinburgh, pp DUNCAN, C. J. (1967). The Molecular Properties and Evolution of Excitable Cells. Pergamon Press, Oxford.

7 186 Coulson, Zeitman, Cohn, Dong, Griepp, Stinson and Shumway ELLEGAARD, J. and DIMrmoV, N. V. (1973). Ouabain-sensitive and oligomycin-sensitive adenosine-triphosphatase activities of normal human lymphocytes. British Journal of Haematology, 25, FRiEs, D., BRYON, P. A., BRUNAT, N., BROCHIER, J., REVILLARD, J-P., and TRAEGER, J. (1967). IAtude en microscopie electronique de l'activite adenosine-triphosphatase des membranes cytoplasmatiques des lymphocytes transformes 'in vitro'. Pathologie et Biologie (Paris) 15, GROPP, A. and FISCHER, R. (1964). Untersuchungen zur Phytohamagglutinin-stimulierten Umwandlung von menschlichen Blut lymphocytes zu blasten antigen Zellen. Virchows Archivs (Pathol. Anat.) 338, LICHTMAN, M. A. and WEED, R. I. (1969). The monovalent cation content and adenosine triphosphatase activity of human normal and leukemic granulocytes and lymphocytes: relationship to cell volume and morphologic age. Blood 34, MCCLURKIN, J. T. (1964). A method for the cytochemical demonstration of sodium-activated adenosive triphosphatase. Journal of Histochemistry and Cytochemistry, 12, PICKUP, D. M. (1949). Extraction of lymphocytes from rabbit's blood. Nature, 164, QUASTEL, M. R. and KAPLAN, J. G. (1970). Lymphocyte stimulation: the effect of ouabain on nucleic acid and protein synthesis. Experimental Cell Research, 62, ROODYN, D. B. (1967). Enzyme Cytology, Academic Press, London and New York. SOLHEIM, B. G. and THORSBY, E. (1974). B-2-microglobulin is part of the HL-A molecule in the lymphocyte membrane. Nature, 249,