ANTIBODIES TO MEMBRANE ANTIGEN(S) COMMON TO THYMOCYTES AND A SUBPOPULATION OF LYMPHOCYTES IN INFECTIOUS-MONONUCLEOSIS SERA

399 TABLE VII-INDEX PATIENTS GROUPED BY NUMBER OF FIRST-DEGREE RELATIVES EXAMINED, SHOWING OBSERVED AND EXPECTED NUMBERS OF PATIENTS WITH AT LEAST ONE AFFECTED RELATIVE

ANTIBODIES TO MEMBRANE ANTIGEN(S) COMMON TO THYMOCYTES AND A SUBPOPULATION OF LYMPHOCYTES IN INFECTIOUS-MONONUCLEOSIS SERA D. B. THOMAS Middlesex

Department of Immunology, Hospital Medical School, London W.1

IgM antibodies against surface Summarybrane antigen(s), present

mem-

both human thymocytes and lymphocytes, have been identified in the sera of twelve out of forty-seven (25%) infectious-mononucleosis patients by indirect immunofluorescent staining of viable cells. Maximum staining was obtained at 3°C. There was an inverse relation between the number of immunoglobulin-bearing lymphocytes and those which stained with the IgM antibodies. It is suggested that such cold antibodies react with a fraction of thymus-derived (T) lymphocytes. on

recognised dominantly inherited condition, then, with a 50% chance of an individual first-degree relative being affected, the number of families expected to have at least one affected member can be calculated. In our series (see table VII) 19 families would be expected to have at least 1 affected member, but only 5 were found. This suggests that about a quarter of the survivors have the type-II disorder inherited as an autosomal dominant characteristic and so are what is called familial hyperbetalipoproteinaemia. Examination of the distribution of the cholesterol scores of the first-degree relatives of the patients with the type-ll abnormality shows that 9 first-degree relatives from 5 families have cholesterol scores more than 2 S.D. above the expected level. The distribution of cholesterol scores within these 5 families is compatible with the inheritance of hypercholesterolæmia in these families as a single-gene effect, when allowance is made for the fact that some affected firstdegree relatives will be dead. Examination of cholesterol scores in the whole group of first-degree relatives shows a significant shift of the whole distribution to the right. This suggests that in most index patients with type-II hyperlipidaemia, cholesterol levels are

determined by polygenic inheritance, with

minority patients exhibiting hypercholesterolaemia determined by a single-gene effect. There was an increase in triglyceride scores among some relatives of patients with type-II hyperlipidaemia, a

of

but no shift of the main distribution. The frequency of type-iv abnormality is greater in patients than controls, but comparatively less than type II, since the frequency of type IV is greater in the controls. There is a striking increase in the frequency of type IV in younger male and female survivors compared with the older age-groups. If type-iv hyperlipidaemia was always inherited as a dominant,19 13 families would be expected to have at least one affected member with the type-iv disorder. Only 5 were observed. Among the relatives of patients with type-iv hyperlipidasmia, there was a significant increase in mean triglyceride scores, but not of cholesterol scores. The numbers are too small, however, for us to draw any definite conclusions as to the mode of inheritance of the type-iv hyperlipidaemia encountered in survivors of myocardial infarction. We thank Dr. C. 0. Carter for his help and advice, Mrs. Patricia Hallifax and Mrs. Nancy Noble for their help, consultant physicians who allowed us to examine their patients, and the British Heart Foundation for a grant for secretarial help. Requests for reprints should be addressed to J. S.

Introduction

INFECTIOUS mononucleosis

(l.M.) presents

complex 2.3 are serological profile. and a of unusual increased, antibody specifivariety cities appear during the course of the disease. The presence of the heterophile agglutinin is a diagnostic featureand patients may produce anti-i (cold agglutinins),5, 6 rheumatoid factor,7, 8antinuclear factor,9 antibodies against Epstein-Barr (E.B.) virus,lo,11 and the membrane antigens of E.B.-virus-positive lympho3 1

a

Levels of IgG and IgM

blastoid cells.12, 1 I have found, in a proportion of l.M. sera, antibodies which are directed against membrane antigen(s) present on both human thymocytes and a subpopulation of

peripheral lymphocytes. Materials and Methods and Lymphocyte Thymocyte Separation 25-30 ml. of venous blood from healthy adult volunteers DR. PATTERSON, DR. SLACK: REFERENCES Kannel, W. B., Dawber, T. R., Friedman, G. D., Glennon, W. E., McNamara, P. M. Ann. intern. Med. 1964, 61, 888. 2. Morris, J. N., Kagan, A., Pattison, D. C., Gardner, M. J., Raffle, P. A. B. Lancet, 1966, ii, 553. 3. Albrink, M. J., Man, E. B. Archs intern. Med. 1959, 103, 4. 4. Hatch, F. T., Russell, P. K., Poon-King, T. M. W., Canellos, G. P., Lees, R. S., Hagopian, L. M. Circulation, 1966, 33, 679. 1.

5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

15. 16. 17. 18.

19.

Slack, J. Lancet, 1969, ii, 1380. Heinle, R. A., Levy, R. I., Fredrickson, D. S., Gorlin, R. Am. J. Cardiol. 1969, 24, 178. Noble, R. P. J. Lipid Res. 1968, 9, 693. Nevin, N. C., Slack, J. J. med. Genet. 1968, 5, 9. Sperry, W. M., Webb, M. J. biol. Chcm. 1950, 187, 97. Van Handel, E., Zilversmidt, D. B. J. Lab. clin. Med. 1957, 50, 152. Chin, H. P., Blankenhorn, D. H. Clinica chim. Acta, 1968, 20, 305. Hamilton, M., Pickering, G. W., Fraser Roberts, J. A., Sowry, G. S. C. Clin. Sci. 1954, 13, 37. Fredrickson, D. S., Levy, R. I., Lees, R. S. New Engl. J. Med. 1967, 276, 34. Quarfordt, S., Levy, R. I., Fredrickson, D. S. J. clin. Invest. 1971, 50, 754. Bull. Wld Hlth Org. 1970, 43, 891. Fredrickson, D. S., Levy, R. I., Lees, R. S. New Engl. J. Med. 1967, 276, 215. Mills, G. L., Wilkinson, P. A. Br. Heart J. 1966, 28, 638. Rifkind, B. M., Lawson, D., Gale, M. J. Atheroscler. Res. 1968, 8, 167. Schreibman, P. H., Wilson, D. E., Arky, R. A. New Engl. J. Med. 1969, 281, 981.

400 added to heparinised containers and the erythrocytes sedimented with 0-8 volumes of 3-5% dextran (Pharmacia) in 0-9% saline solution for 30 minutes at 37°C. The supernatant was removed and centrifuged at 150 g for 10 minutes. The cell pellet was washed twice with Eagle’s minimum essential medium (M.E.M.) and resuspended in 2 ml. of 10% fetal calf serum in M.E.M. The cell suspension was applied to a glass bead column (1-5 X 10 cm.), previously equilibrated with the same medium, and incubated for 30 minutes at 37°C to remove adherent cells. The column eluate was washed twice before immunofluorescent staining. Tonsils, thymus, fetal spleen, or liver were teased in cold M.E.M. and the debris removed by standing at 30C for 10 minutes. Cells were washed three times before was

staining. Cord Blood Fresh cord blood was incubated with 0-8 volumes of 3-5% dextran in 0-9% saline solution for 30 minutes at 37°C. The erythrocyte sediment was washed three times with M.E.M. and used for absorption of sera.

Cell Culture

Peripheral blood lymphocytes were cultured in M.E.M. supplemented with 10% fetal calf serum in an atmosphere of 5 % carbon dioxide and 95 % air at a cell density of 106 per ml. Burkitt lymphoma cell lines EB1, EB2, and EB3 were maintained in M.E.M. supplemented with 10% human serum at a cell density of 8 x 105 to 2 x 106 per ml.

procedure

was

used, the temperature being maintained

at 3 DC: serum or normal rabbit serum for 30 minutes, washes. (b) Infectious-mononucleosis serum or normal human serum for 30 minutes, two washes. (c) Fluorescein-conjugated goat anti-rabbit immunoglobulin for 30 minutes, two washes. (d) Fluorescein-conjugated sheep anti-human IgM for 30 minutes, three washes and examination of cells under ultraviolet

(a) Rabbit A.L.C.

two

light. Cell I.M. sera were tested also for activity at 37 °C. suspensions were incubated with sera at 37°C for 30 minutes, washed three times with M.E.M., and treated with fluorescein-conjugated sheep anti-human IgM, as described above. At least 300 cells were counted for each sample. Dead cells were identified (and ignored) by their cytoplasmic staining. Human Sera l.M. sera had been stored at -20°C for periods between 3 weeks and 4 years before testing. In absorption studies, l.M. sera (1/5 dilution) were incubated with an equal volume of cells, or cell extract, at room temperature for one hour and then three hours at 3°C, and tested for residual activity at this dilution.

Results

Lymphocytes Twelve of forty-seven

Immunofluorescent Staining l.M. sera were tested for antibodies to lymphoid cells by indirect immunofluorescent staining of viable cells. Lymphocyte or thymocyte suspensions (3 x 106; 100 µl.) were incubated with an equal volume of the test serum (1/5 dilution) for 30 minutes at 3°C, washed three times

with cold M.E.M., and incubated for 30 minutes at 3°C with 50 µl. of fluorescein-conjugated sheep anti-human IgG or IgM (Wellcome Research Laboratories, Beckenham; 1/5 dilution). Cells were washed a further four times with cold M.E.M. and examined with a LeitzOrtholux ’ microscope under ultraviolet light. Equal numbers of normal control sera were included in each experiment together with an I.M. serum (HS-1) which served as a positive control. A similar procedure was used to demonstrate surface immunoglobulin on lymphocytes. Cell suspensions were incubated with an equal volume of rabbit anti-human

light-chain (A.L.C.)

serum

(1/8 dilution;

prepared

as

described by Greaves et al.14) followed, after washing, by incubation with fluorescein-conjugated goat anti-rabbit immunoglobulin (1/8 dilution). When the above two sera were applied to the same cell suspension, the following

patients with l.M. (Paul-Bunnell titres, 1/350-1/2000) contained antibodies binding at 3°C to surface-membrane antigen(s) of peripheral lymphocytes. Similar percentages of stained cells were obtained with individual lymphocyte donors and different I.M. sera (4 to 12) at the test dilution (1/10), titres of sera for maximum staining being between 1/15 and 1/40. The percentage of lymphocytes from venous blood of healthy donors, chronic lymphocytic leukaemia patients, and tonsils sera

from

which stained with either l.M. sera or rabbit A.L.c. serum at 3°C is shown in the table. The percentage of stained cells in tonsils from different donors varied considerably, but in each case an inverse relation held for the two sera. Thus, when the number of cells staining for surface immunoglobulin was high, only few cells were positive with l.M. sera, and vice versa. Also, if the two sera were used together, the percentage staining was always higher than that for the individual sera.

The antibodies

were

of the

IgM class in each of

INDIRECT IMMUNOFLUORESCENT STAINING OF PERIPHERAL BLOOD AND TONSILLAR LYMPHOCYTES WITH I.M. SERA HUMAN LIGHT CHAIN (A.L.C.) SERUM AT 30c

*

At least three positive sera were included in each determination; values for individual sera were within 14 % of the mean.

OR/AND

N.t.

RABBIT ANTI-

= Not determined.

401

changes during the differentiation of thymocytes. Common antigenic determinants were present on adult lymphocytes and fetal and adult thymocytes, since each cell type absorbed the activity for the others. Other Fetal Tissues Both fetal bone-marrow and spleen (20-23 weeks old) contained 10-15% cells which stained with l.M. sera. It was difficult to assess the type of stained cells by morphological criteria under visible light. Several LM. sera, including samples negative for the above specificity, contained antibodies against a membrane antigen of fetal liver cells (18-23 weeks old); the antibodies were of both IgG and IgM class. Antibodies against fetal liver cells could be distinguished from those which stained lymphoid cells. There was no reduction in staining of fetal thymocytes or adult lymphocytes after absorption of sera with fetal liver. Most infectious-mononucleosis sera contain smooth-muscle antibodies which cross-react with membrane antigens of liver cells." The activity for lymphoid cells was not removed also by absorption of sera with a smooth-muscle extract of uterus (prepared by Dr. U. Groeschel-Stewart, according to the Fig. 1—Change in percentage staining of peripheral blood lymphocytes with time in cell culture. Values represent mean and range for three determinations.

the twelve

l.M. sera as shown by staining with specific fluorescein-conjugated antisera. Experience with a panel of thirty lymphocyte donors (and nineteen thymocyte donors, see below) makes it unlikely that staining is due to isoantibodies. All sera were tested

at

least three times on different donors and

were

consistently positive or consistently negative. The control serum (HS-1) was positive on every sample. Activity was not related to the titre for heterophile antibodies (positive sera, geometric mean titre i/1200, range 1/350 to 1/2300; negative sera, geometric mean

titre 1/1500, range 1/640 to 1/2500) and could not be

removed by absorption bf sera with an ox-blood preparation (" ox-cell antigen ", Oxoid). Thus the IgM antibodies are distinct from the heterophile agglutinin. Fig. 1 indicates the change in % staining of pe’ripheral blood lymphocytes with time in culture. The decrease in cell number was associated with a loss of immunoglobulin-bearing cells and a proportional increase of cells which stained with l.M. sera. Therefore, it would seem that l.M. sera react with a subpopulation of lymphocytes which do not express surface immunoglobulin and show preferential viability in cell culture. The immunofluorescent staining of peripheral blood lymphocytes is shown in fig. 2. There was considerable heterogeneity in staining pattern and cells showed either a ring or speckled staining of their surface.

Thymocytes A similar

staining pattern was observed on treating with thymocytes any of the twelve positive I.M. sera There a striking variation in the percentage was (fig. 3). of thymocytes staining at different ages, especially before parturition (fig. 4). This may reflect surface

Fig. 2-Immunofluorescent staining of peripheral blood lymphocytes with I.M.

serum.

(a) Visible. (b) Ultraviolet light.

402

Fig. 4-Variation in percentage of thymocytes staining with I.M. sera

at

different ages.

Values represent the range for

at

least three

l.M. sera.

could be eluted on incubation at 37 °C for 30 minutes. Thus the activity in l.M. sera is due to cold antibodies. However, the antibody specificity is not related to the cold agglutinins, anti-i or anti-I; activity of sera was not affected by absorption with adult or cord blood

erythrocytes. Discussion

Fig. 3-Immunofluorescent staining of fetal thymocytes (23 weeks) with I.M. serum. (a) Visible. (b) Ultraviolet light.

method of Namba and Grob 16). Thus the two specificities are distinct. Fetal brain (18-20 weeks old) was the only other tissue to be examined; absorption with brain did not remove the activity of I.M. sera for peripheral lymphocytes.

Burkitt Lymphoma Cell Lines I.M. sera contain antibodies against membrane antigens of E.B.-virus-positive lymphoblastoid cells which are absent from normal lymphocytes. 12,13 However, no significant staining of the Burkitt lymphoma cell lines EB1, EB2, EB3 has been found with four positive infectious-mononucleosis sera (normal human serum 8-10% stained cells; I.M. sera 8-12% stained cells). Also, absorption of i.M. sera with the three cell lines did

reduce subsequent staining of thymocytes or lymphocytes. The specificity for normal lymphoid cells is not related, therefore, to the membrane antigen(s) of E.B.-virus-positive lymphoblastoid cell lines. not

Effect of Temperature When tonsillar

Staining peripheral

on

blood

lymphocytes 37°C, there was no appreciable staining (15% of the value at 3°C). Also, the IgM antibodies absorbed by lymphocytes at 3°C

were

treated with

or

i.M.

sera

at

In the mouse, cell-surface antigens have proved useful markers to distinguish two populations of lymphocytes. Thus surface immunoglobulin is considered a feature of bone-marrow-derived (B) lymphocytes and the &thgr; isoantigen 16 to be specific for thymocytes and thymus-derived (T) lymphocytes. Evidence in support of this view was the inverse relation between the number of peripheral lymphocytes expressing surface immunoglobulin or the a isoantigen after neonatal thymectomy or treatment with antilymphocytic serum.18-20 Recently, differences have been found in the viability of T and B murine lymphocytes in vitro.21 There was a striking reduction in the percentage of immunoglobulin-bearing lymphocytes after several days of cell culture and a corresponding increase in the number of a-positive cells. Also, there is a reported decrease of viability, in vitro, of peripheral blood lymphocytes from thymectomised mice. 22 It seems reasonable to suggest that the IgM antibodies of LM. sera are directed against a membrane antigen(s) of thymocytes and a subpopulation of T lymphocytes. Absorption experiments indicate a common membrane antigen on a proportion of thymocytes and peripheral lymphocytes. An inverse relation exists for the number of immunoglobulinbearing cells and lymphocytes which react with 10M. sera and an additive result is obtained on treating lymphocytes with both A.L.C. and l.M. serum (table). There is not complete summation with double staining, but this may be due to the technical limitations of the method. Thus the staining with l.M. sera was reduced in most instances by pretreatment of cells with normal rabbit serum. The differential viability of lymphocytes

403

in culture (fig. 3) is also consistent with findings in the mouse but contradict the reports by Hellstrom et al., 2and Biberfeld et al.24for man; no such decrease was found in the number of immunoglobulin-bearing cells in cultures of human peripheral blood. There are, however, several differences between the present membrane antigen(s) of human lymphoid cells and the murine isoantigen 6. The 6 determinant is present on most thymocytes (95-100%), a large

proportion of peripheral lymphocytes (65-85%, lymph node),19 and brain.1’ In contrast, no more than 60% of human thymocytes can be stained with i.M. sera, and this value varies with age of the tissue. Also, an appreciable number of lymphocytes (40-45% in peripheral blood, see table) do not stain with either A.L.C. serum or I.M. serum. The activity in i.M. sera was not affected by absorption with brain. A proportion of i.M. sera contain anti-i antibodies, and Shumak et al.25 have shown that the i antigen is adult lymphocytes. Absorppresent on most (>80%) tion studies indicate that the present IgM antibodies are distinct from the cold agglutinins. Although the circumstantial evidence is strong, it remains to be shown, unequivocally, that lymphocytes which react with i.M. sera are exclusively T cells. I thank Prof. I. M. Roitt, Dr. B. Phillips, and Dr. J. H. L. Playfair for helpful discussion; Dr. S. M. Worlledge, Dr. M. S. Pereira, and Dr. H. G. Smith for serum samples; the staff of the tissue bank, Royal Marsden Hospital, and Mr. I. M. Breckenridge for tissue samples; Prof. M. A. Epstein for Burkitt lymphoma cells; Miss G. Medley for able technical assistance; and to Mrs. G. Stead for preparation of the manuscript. This work was supported by the Medical Research Council.

EVALUATION OF CLONIDINE IN PROPHYLAXIS OF MIGRAINE Double-blind Trial and Follow-up

J. SHAFAR

Burnley and District Hospital Group P. A. KNOWLSON

Boehringer Ingelheim Ltd., Isleworth, Middlesex In a double-blind trial, clonidine 50 µg. twice a day was compared with placebo tablets in the prophylaxis of migraine. 65 patients entered the trial, but 15 defaulted. Of the other 50 patients, 8 exhibited very frequent symptoms and constituted a clearly defined subgroup: in these no significant evidence of drug effect was demonstrable. In the main group of 42 patients, clonidine was associated with a statistically significant reduction in the frequency of severe attacks of migraine: the placebo did not affect severe attacks. Side-effects were noted with both preparations, but no pattern of clinical importance emerged. After the trial the patients were invited to take clonidine in a follow-up investigation lasting twelve months. 37 patients accepted, including 5 from the subgroup with very frequent headaches; 7 patients subsequently withdrew. In comparison with the results in the double-blind trial, a statistically significant mean further improvement occurred in 27 patients with conventional migraine. Improvement was also seen in 3 patients with atypically frequent symptoms. In all cases the daily dose used was 0.15 mg. (six tablets), and side-effects occurred in only 2 patients.

Sum ary

Introduction

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

9. 10.

11. 12. 13.

14. 15. 16. 17. 18.

19. 20. 21. 22. 23.

L. in Infectious Mononucleosis (edited by R. L. Carter and H. G. Penman); p. 177. Oxford, 1969. Wollheim, F. A., Williams, R. C., Jr. New Engl. J. Med. 1960, 275, 61. Wollheim, F. A. Scand. J. Hœmat. 1968, 5, 97. Paul, J. R., Bunnell, W. W. Am. J. med. Sci. 1932, 183, 90. Rosenfield, R. E., Schmidt, P. J., Calvo, R. C., McGinnis, M. H. Vox Sang. 1965, 10, 631. Costea, N., Yakulis, V., Heller, P. Science, 1966, 152, 1520. Dresner, E., Trombly, P. New Engl. J. Med. 1959, 261, 981. Holborow, E. J., Asherson, G. J., Johnson, G. D., Barnes, R. D. S., Carmichael, D. S. Br. med. J. 1963, i, 656. Johnson, G. D., Holborow, E. J. Nature, 1963, 198, 1316. Henle, G., Henle, W., Diehl, V. Proc. natn. Acad. Sci., U.S.A. 1968, 59, 94. Gerber, P., Hamre, D., Moy, R. A., Rosenblum, E. N. Science, 1968, 161, 173.

Davidsohn, I., Lee, C.

Klein, G., Pearson, G., Henle, G., Henle, W., Diehl, V., Niederman, J. C. J. exp. Med. 1968, 128, 1021. Klein, G., Pearson, G., Henle, G., Henle, W., Goldstein, G., Clifford, P. ibid. 1969, 129, 697. Greaves, M. F., Torrigiani, G., Roitt, I. M. Clin. exp. Immun. 1971, 9, 313. Holborow, E. J. Personal communication. Namba, T., Grob, D. Ann. N. Y. Acad. Sci. 1966, 135, 606. Reif, A. E., Allen, J. M. Nature, 1966, 209, 521. Raff, M. C. Immunology, 1970, 19, 637. Jones, G., Torrigiani, G., Roitt, I. M. J. Immun. 1971, 106, 1425. Schlesinger, M., Yron, I. Science, 1969, 164, 1412. Jones, G., Roitt, I. M. Cell. Immun. (in the press). Doenhoff, M. J., Davies, A. J. S., Leuchars, E., Wallis, V. Proc. R. Soc. B, 1970, 176, 69. Hellström, U., Zeromski, J., Perlmann, P. Immunology, 1971, 20, 1099.

24. 25.

Biberfeld, P., Biberfeld, G., Perlmann, P. Expl Cell Res. 1971, 66, 177. Shumak, K. H., Rachkewich, R. A., Crookston, M. C., Crookston, J. H. Nature, New Biol. 1971, 231, 148.

E. R. TALLETT

THE pharmacological properties of the imidazoline derivative clonidine have endowed this compound with two distinct activities. Acutely in animals clonidine reduces central sympathetic outflow, 1,2 while chronic administration results in a diminished responsiveness of peripheral vessels to both dilator and constrictor stimuli. 3,4 These actions have led to the use of the drug in the treatment of arterial hypertension.5-9 Zaimis and Hanington3 suggested that the peripheral vascular effect of chronic administration would be of value in the prophylactic management of migraine, a condition in which changes in the cerebral and extracranial blood-vessels are considered to be of fundamental significance. Early results have been published, and include not only those from open-label assessment in patients whose migraine had not responded to other therapy, 10, 11 but also those obtained in a double-blind comparison with placebo.122 The response has been encouraging; in Sjaastad and Stensrud’s trial, 26 patients completed three weeks on each trial preparation; 13 were substantially and 3 moderately improved while taking clonidine 25 µg. a day, the difference from placebo effect being statistically significant (P<0-025). Barrie et al.,13 utilising six-week treatment periods in a double-blind trial, demonstrated that 59% of 107 patients responded to clonidine at a dose of 25 or 50 µg. twice a day, while the placebo improved

three times