Acta Neurol Scand., 1985:71:303-308

Key words: Cell membranes; erythrocytes; granulocytes: lymphocytes; multiple sclerosis; Z ’ , 3’-cyclic neucleotide 3-phosphohydrolase

Membrane-bound 2’, 3’,-cyclic nucleotide 3’-phosphohydrolase activity of lymphocytes, granulocytes and erythrocytes in multiple sclerosis

S. C. Rastogi and J. Clausen The Neurochernical Institute, Copenhagen, Denmark

ABSTRACT - A sensitive fluorimetric method was used for the assay of 2’, 3‘-cyclic nucleotide 3’-phosphohydrolase (CNP) activity of lymphocyte, granulocyte and erythrocyte membranes from patients with multiple sclerosis (MS). The data obtained were compared with the corresponding data from normal individuals. CNP activities of granulocyte and erythrocyte membranes of the 2 groups did not differ sipficantly. However, a 40 % decreased membrane CNP activity of MS lymphocytes was found when the data were compared with the normal lymphocytes’ activity ( P < 0.0005) by both the non-parametric median test and Student’s t-test. A role of CNP in immu- noregulation is suggested.

Accepted for publication October 26, 1984

The enzyme 2’, 3’-cyclic nucleotide 3’-phosphohydrolase (CNP E.C.3.1.4.37) hydro- lyses ribonucleoside 2’, 3’-cyclic phosphates to the corresponding ribonucleoside 2’-phosphates. In 1962, Drummond et a1 (1) decribed the presence of CNP in the central nervous system (CNS). Since then, several reports have demonstrated that CNP was concentrated in CNS myelin, pe- ripheral myelin and in both oligodendrocytes and Schwann cells (2-9). The specific CNP activity of non-neural tissues has been found to be several- fold lower than that of the myelin-related mem- branes of central and peripheral nervous system

A decreased activity of CNP in white matter and in myelin from demyelinating diseases including multiple scleroses (MS) has been found (11, 12),

(1510).

especially in demyelinating areas (13). The release of myelin fragments to the cerebrospinal fluid (CSF) of patients with demyelinating diseases may be responsible for the increased activity of CNP in CSF from patients with MS and other demyelinat- ing diseases (14,15). However, in a recent report (16), normal levels of CNP in CSFs and sera from MS patients were found.

The presence of CNP activity in human erythrocyte membranes (17) and in rat erythrocytes (18) has been decribed. In a pre- liminary study, CNP activity of erythrocyte mem- branes from 5 MS patients was found to be lower than that from 5 normals (19). Thus, it was tempt- ing to compare the membrane CNP activities of hematogenous cells from the normal Danish pop- ulation with those of MS patients.

304 S. RASTOGI ET AL

Materials and methods Chemicals 2-(N-morpholino)ethane sulfonic acid (MES), 2', 3'-cyclic NADP, RNADPH (preweighed vials) and bovine albumin (fraction V) were purchased from Sigma Chemical Co., USA. Glucose- 6-phosphate (G6P) disodium salt and G6P-de- hydrogenase (G6P-DH) from yeast were the products of Boehringer Mannheim, West Ger- many. Ficoll-paque was obtained from Pharmacia Fine Chemicals, Sweden, and all other chemicals were of analytical grade from E. Merck, West Germany.

MS patients and normal individuals Peripheral blood from 29 definite MS patients (median age 46 years, range 23-78 years, 14 males and 15 females) was used in the present study. All the patients were diagnosed at various hospitals in the Copenhagen area but not hospitalized at the time of our study. Information about the diagnosis was obtained through the Danish Multiple Scle- rosis Register with the courtesy of Chief Neurolo- gist Dr. K. Hyllested to whom we express our gratitude. The diagnostic criteria of MS were those of Schumacher et a1 (20). None of the pa- tients were receiving immunosuppressive drugs. Twenty-four laboratory personnel and their rela- tives (median age 39 years, range 23-67 years, 14 males and 10 females) were used as normal con- trols in the present study.

Isolation of cell membranes Lymphocytes (21) and granulocytes (22) from heparinized blood were isolated as decribed. The cells were stored frozen (-20°C) until the enzyme assay was performed. The cell membranes were isolated on the same day as the enzyme activity was assayed. The cells were suspended in 0.6 ml of 0.1 4 M NaCl and disintegrated by ultrasonication at 50 watts for 10 sec (0°C). The sonicated cells were diluted to 5 ml with NaCl and centrifuged (4°C) at 16,OOOg for 30 min. The supernatant was discarded and the membranes were suspended in

0,5 ml MES buffer (200 mM MES + 30 mM MgClz + 1 mM EDTA, pH 6.0) by ultrasonication for 10 sec (0°C).

After the isolation of lymphocytes from the blood, the remaining erythrocytes were used for the isolation of erythrocyte membranes as de- scribed by Steck and Kant (23). The erythrocyte membranes were stored frozen (-20°C). They were diluted in MES buffer before use for the assay of CNP. Protein concentration of the mem- branes was determinbed by the method of Lowry et a1 (24).

CNP assay CNP was assayed fluorimetrically using cyclic NADP+ as substrate according to Weissbarth et a1 (25). Preliminary studies were performed utilizing lymphocyte, erythrocyte and granulocyte mem- branes from 2 MS patients and 2 normal individu- als. By varying the membrane protein con- centration, the substrate concentration and the incubation period at 30°C, the optimal conditions for enzyme assay were found to be as follows: 10 pg protein (erythorcyte membranes) or 20-30 pg protein (granulocyte and lymphocyte mem- branes) in 280 p1 MES buffer containing 6 mM G6P, 1.2 pg albumin/ml, 23 pg G6P-DWml, 4 mM cyclic NADP+/ml and 0.025 % Triton X-100, were incubated for 20 min at 30°C in a shaking water bath. The enzyme reaction was stopped by the addition of 2.52 ml 50 mM Na-carbonate buffer, pH 10.5, to the incubation mixture. The fluorescence of the reaction product (NADPH) was measured using 360 nm as excitation and 460 nm as emission filter. The enzyme activity was expressed as pmoles of NADPH produced/h/mg protein.

All samples were run in duplicate and duplicate reagent blanks, substrate blanks and blanks for the membrane protein were included in each ex- periment. Fluorescence of serial dilutions of R-NADPH in Na-carbonate buffer was used to plot a standard curve for each set of experiments. Preliminary studies revealed the enzyme activity to be resistant to freezing up to 3 months.

CNP OF MS BLOOD CELL MEMBRANES 305

> MS NORMAL Fig. 2. Membrane CNP activity of MS and normal lym- phoc.es. Ordinate: c,p activity (pmoles NADPW wmg protein).

I0 20 30 Fig. I . CNP activities of cell membranes from a normal subject as a function of protein concentration under optimal conditions. Abscissa: pg protein Ordinate: n moles NADPW20 min. A: Lymphocyte membranes, 0: Ganulocyte membranes, 0 : Erythrocyte membranes Results

The optimal conditions for the CNP assay re- vealed by the preliminary experiments were simi- lar to those described by Weissbarth et a1 (25) and Jones et a1 (19). Fig. 1 shows the membrane pro-

Statistics tein concentration depndent increase in CNP ac- The data of MS patients and normal individuals tivity under optimal conditions for the lympho- were compared employing the non-parametric cytes, granulocytes and erythrocytes of a normal median test (26) as well as using Student's t-test for subject. In all the cases, both MS patients and 2 independent variables. normal individuals, the CNP acitivity of

Table 1 Membrane CNP activity of lymphocytes, granulocytes and erythrocytes in MS patients and normal individuals

Cell type CNP activity ( p o l e N A D P W m g protein)

Mean f SD 2 SEM Median Mean ? SD f SEM Median MS(n = 29) Normals (n = 24)

Erythrocytes 15.13 2.67 0.25 15.01 14.77 2.42 0.18 15.64

Granulocytes 3.95 0.71 0.02 3.84 4.21 1.15 0.09 4.18

Lymphocytes 1.48. 0.55 0.08 1.41. 2.41' 0.93 0.04 2.30.

(6.7 - 19.67) (7.5 - 19.74)

(2.9 - 5.52) (3.09 - 7.75)

(0.36 - 2.50) (1.06 - 4.18)

Numbers in parenthesis represent range. Compared to normal, P < 0.0005.

20

306 S. RASTOGI ET AL

erythrocyte membranes was found to be the high- est followed by that of granulocyte and lympho- cyte membranes. The CNP activities of lympho- cytes, granulocytes and erythrocytes did not correlate. Furthermore, the CNP activities of the cell membranes assayed were not found to be related with age or sex of the individuals studied:

The data on membrane CNP activities of lym- phocytes, erythrocytes and granulocytes are shown in Table 1. It is evident from Table 1 that both mean and median lymphocyte membrane CNP activity of normal individuals was 62 % higher than those of MS patients. Both the non- parametric median test and Student’s t-test re- vealed a sigmfxant difference between the MS and the normal lymphocyte membrane CNP ac- tivity at the level of P < 0.0005. Although overlap- ping of data for MS patients and normal individu- als was observed (Fig. 2), 34 % of the CNP activities of normal lymphocyte membranes were higher than the highest value for the MS lympho- cyte membrane CNP activity measured in the present study. Similarly, 17 % of the MS lympho- cyte membrane CNP activities were found to be lower than the lowest CNP activity traced in nor- mal lymphocyte membranes. The membrane CNP activities of granulocytes and erythrocytes of MS patients and normal individuals were found to be comparable (Table 1).

Discussion The present study reveals that CNP activity is also present in lymphocyte and granulocyte mem- branes besides that in erythrocyte membranes. The CNP activity of erythrocyte membranes was found to be higher than that of granulocytes fol- lowed by lymphocytes. The CNP activity of cell membranes was not found to depend on age or sex of the individuals studied. The mean erythrocyte membrane CNP activity of normal individuals found in the present investigation was 14.77 units (Table 1). In a preliminary study, Jones et al(19) found that the erythrocyte membrane CNP ac- tivity of 5 normal individuals (> 20 units) was higher than that of 5 MS patients (< 20 units). Our data on erythrocyte membrane CNP activity in MS patients and normal individuals revealed that the enzyme activity of the 2 groups was compara- ble (Table 1).

A 40 % decrease in the lymphocyte membrane CNP activity of MS patients compared to normal individuals was traced in the present study. Al- though the data of MS patients and normal indi- viduals were found to overlap, a significant dif- ference (P < 0.0005) between the lymphocyte membrane CNP activities of MS patients and nor- mal individuals was found. This may indicate that MS lymphocytes in general were deficient in CNP activity. The 3 possible causes for decreased CNP activity in MS lymphocyte membranes seem to be: 1) decreaseddefective synthesis of the enzyme in lymphocytes; 2) defective incorporation of CNP in the membranes; and 3) an altered distribution of various subpopulations of lymphocytes with low membrane CNP activity in MS. The assay of intra- cellular CNP activity in lymphocytes of 5 MS pa- tients and 5 normal individuals revealed (data not shown) that the intracellular CNP activities in both MS and normal lymphocytes were similar to those present in the membranes (89-98 % for MS patients and 85-104 % for normal individuals). This may indicate that the membrane CNP defi- ciency in lymphocytes from MS patients may be associated with a defective synthesis of this en- zyme in MS lymphocytes. However, further stud- ies will be necessary to elucidate the cause of lym- phocyte membrane CNP deficiency in MS patients.

The role of CNP in vivo has not yet been fully understood and the physiological substrate for this enzyme is still unknown. However, it has been shown that CNP hydrolyses 2’, 3’-cyclic mono- phosphates of adenosine, guanosine, cytidine and uridine as well as some oligonucleotides con- taining a 2’, 3’-cyclic nucleotide terminus (3). Moreover, it catalyses the hydrolysis of adenosine 2’, 3’-cyclic-phosphate 5’-phosphosulfate and compounds of similar structure (15). Thus, an in- creased cyclic nucleotide level associated with a decreased CNP activity may be present in the lymphocytes of MS patients. An increased level of cyclic nucleotide or the substances that augment the cyclic nucleotide levels in lymphocytes have been shown to inhibit natural killer (NK) activity of the lymphocytes (27, 28). NK-cell activity of MS lymphocytes has been shown to be reduced (29, 30, 31). The results of the present study thus warrant the tracing of the relationship between

CNP OF MS BLOOD CELL MEMBRANES 307

CNP activity and the NK-cell activity of peripheral blood lymphocytes which may lead to an abnor- mal immunoregulation in MS and other inflamma- tory diseases.

The CNP activity in CSF of MS patients has been found to be normal (16) or higher (14, 15) than in CSF of non-demyelinating diseases. It is well known that the lymphocyte counts found in the CSF of patients with MS are much higher than those in non-demyelinating diseases. Thus, a con- tribution of CNP by the dead cells in the CSF of patients with MS cannot be ruled out. Therefore, it will be advisable to centrifuge the fresh CSF at high speed before the assay of CNP should be made.

Acknowledgement We thank Miss Lisbeth JBrgensen for her excellent technical assistance.

References 1. Drummond G I, Iyer N T, Keith J. Hydrolysis of

ribolucleoside 2’, 3’-cyclic phosphate by a diesterase from brain. J Biol Chem 1962:2373535-3539.

2. Kurihara T, Tsukada Y. The regional and subcellular dis- tribution of 2’, 3’-cyclie nucleotide 3’-phosphohydrolase in the central nervous system. J Neurochem 1967:14:1167- 1174.

3. Olafson R W, Drummond G I, Lee J F. Studies on 2’ 3’-cyclic nucleotide-3’-phosphohydrolase from brain. Can J Biochem 1969:47961-966.

4. Poduslo S E, Norton W T. Isolation and some chemical properties of oligodendroglia from calf brain. J Neurochem 1972:19:727-736.

5. Uyemura K, Tobari C, Hirano S. Tsukada Y. Comparative studies on the myelin proteins of bovine peripheral nerve and spinal cord. J Neurochem 1972:19:2607-2614.

6. Waehneldt T V. Ontogenic study of a myelin-derived frac- tion with 2’. 3’-cyclic nucleotide 3’-phosphohydrolase ac- tivity higher than that of myelin. Biochem J 1975:151:435- 437.

7. Mikoshiba K, Aoki E, Tsukada Y. 2’-3’-cyclic nucleotide 3’-phosphohydrolase activity in the central nervous system of a myelin deficient mutant (shiverer). Brain Res

8. Matthieu J-M, Costantino-Ceccarini E, Beny M, Reigner J. Evidence for the association of 2’, 3‘-cyclic nucleotide 3’-phosphodiesterase with myelin related membranes in pe- ripheral nervous system. J Neurochem 1980:35: 1345-1350.

9. Reddy N B, Askanas B, Engel W K. Demonstration of 2’. 3’-cyclic nucleotide 3’-phosphohydrolase in cultured human Schwann cells. J Neurochem 1982:39:887-889.

1980:192: 195-204.

10. Weissbarth S, Maker H S, Raes I, Brannan T S, Lapin E P, Lehrer G M. The activity of 2’. 3’-cyclic nucleotide 3’-phosphodiesterase in rat tissues. J Neurochem

11. Gdpfert E, Pytlik S, Debuch H. 2’ , 3’-cyclic nucleotide 3’-phosphohydrolase and lipids of myelin from multiple sclerosis and normal brains. 1980:34:732-739.

12. Lees M B, Sapirstein V S, Reiss D S, Kolodny E H. Car- bonic anhydrase and 2’, 3’ cyclic nucleotide 3’-phosphohydrolase activity in normal human brain and in demyelinating diseases. Neurology 1980:30:719-725.

13. Riekkinen P J, Rinne U K, Arstila A U, Kurihara T, Pel- lineimi T T. Studies on the pathogenesis of multiple xle- rosis. 2’, 3’-cyclic nucleotide 3-phosphohydrolase as a marker of demyelination and correlation of findings with lysosomal changes. J Neurol Sci 1972:15:113-120.

14. BanikNL,MauldinLB, HoganEL.Activityof2’,3’-cyclic nucleotide 3’-phosphohydrolase in human cerebrospinal fluid. Ann Neurol 1979:5:539-541.

15. Sprinkle T J, McKhann G M. Activity of 2’, 3’-cydic-nu- cleotide 3’-phosphodiesterase in cerebrospinal fluid of pa- tients with demyelinating disorders. Neurosci Lett 1978: 7D3-206.

16. Clapshaw P A, MUler H W, Wietholter H , Seifert W. Simultaneous measurement of 2’, 3’ cyclic-nucleotide 3’ phosphodiesterase and RNase activities in sera and spinal fluids of multiple sclerosis patients. J Neurochem 1983:42:12-15.

1981 ~37677-680.

17. Sudo T, Kikumo M, Kurihara T. 2’. 3’-cyclic nucleotide 3’-phosphohydrolase in human erythrocyte membranes. Biochim Biophys Acta 1973:255:640-646.

18. Dreilling C. Localization of 2’, 3’-cyclic nucleotide 3’-phosphohydrolase in human erythrocyte membranes. Biochim Biophys Acta 1981:649587-594.

19. Jones R L, Wont Jr, E K, Ibsen K H, Leopold I H. Erythrocyte membrane 2’, 3’-cyclic nucleotide 3’-phosphodiesterase activity in multiple sclerosis. Metab Ped System Opthalmol 1983:725-30.

20. Schumacher G A, Beebe G, Kilber R F, et al. Problems of experimental therapy in multiple sclerosis. Ann N Y Acad Sci 1965:122:552-565.

21. Rastogi S C, Clausen J . In vitro stimulations of multiple sclerosis (MS) T and B lymphocytes in the presence of MS-specific brain antigens. Clin Immunol Immunopathol

22. Rastogi S C, Clausen J, Melchior J C, Dyggve H V, Jensen G E. Lysosomal (leucocyte) proteinase and sulfatase levels in Dyggve-Melchior-Clauen (DMC) syndrome. Acta Neu- rol Scand 19775639-396.

23. Steck T L, Kant J A. Preparation of impermeable ghosts and inside-out vescicles from human erythrocyte membranes. Meth Enzym 1974:XXXi:172-180,

24. Lowry 0 H, Rosenbrough N J, Fan A L. Randall P. Protein measurement with F o b reagent. J Biol Chem 1951 :193:265-275.

25. Weissbarth S, Maker H S, Lehrer G M, Schneider S, Born- stein M B. A sensitive fluorometric assay for 2’, 3’-cyclic nucleotide 3’-phosphohydrolase. J Neurochem 1980:35:503- 505.

26. Siege1 S. Non-parametric statistics for the behavioral sci-

1982:22:68-74.

20’

308 S. RASTOGl ET AL

ences, New York: McGraw Hill, 1956: 111-116. 27. Katz P, Zaytoun A M, Faun A S. Mechanisms of human

cell-mediated cytotoxicity. 1. Modulation of natural killer cell activity by cyclic nucleotides. J Immunol1982:129:287- 296.

28. Henriksson T. Inhibition of natural killing by adenosine ribonucleotides. Immunol Lett 1983:7: 171-176.

29. Benczur M, Petranyi G G, Palffy G, et al. Dysfunction of natural killer cells in multiple sclerosis - a possible patho- genic factor. Clin Exp Immunol 1980:39:657-662.

30. Hauser S L, Ault K A, Levin M J, Garovoy M R, Weiner H L. Natural killer cell activity in multiple sclerosis. J Immunol 1981:1271114-1117.

31. Merrill J, Jondal M, Seeley J, Ullberg M, Siden A. De- creased NK killing in patients with multiple sclerosis: An analysis of the level of the single effector cell in peripheral blood and cerebrospinal fluid in relation to the activityof the disease. Clin Exp Immunol 198247419-430.

Address S. C. Rastogi, Ph.D. The Neurochemical Institute 58, Raadmandsgade DK-2200, Copenhagen N Denmark