Scand. J. ImmunoL, Vol. 5, 1976.
Measles Virus-Induced Migration Inhibition In Vitro of Leukocytes from Patients with Multiple Sclerosis H. J. NORDAL, S. S. FR0LAND, B. VANDVIK & E. NORRBY Institute of Immunology and Rheumatology and Department of Neurology, Rikshospitalet University Hospital, Oslo, Norway, and Department of Virology, Karolinska Institute, School of Medicine, Stockholm, Sweden
Nordal, H. J., Freland, S. S., Vandvik, B. & Norrby, E. Measles Virus-Induced Migration Inhibition In Vitro of Leukocytes from Patients with Multiple Sclerosis. Scand. J. Immunol. } , 587-591, 1976. The migration inhibition in vitro induced by measles virus material on leukocytes from 17 patients with multiple sclerosis (MS) was compared with that of 8 patients with various other central nervous system diseases and that of 12 healthy blood donors. Significant differences could not be demonstrated. The results do not indicate that MS patients' leukocytes show an altered behaviour to measles virus material in the migration inhibition test. H. J. Nordal, Institute of Immunology and Rheumatology, F. Qvamsgt. 1, Oslo 1, Norway
The possibility that multiple sclerosis (MS) may be associated with a persistent infection of paramyxovirus has been discussed by several authors (1, 4, 12, 17, 18, 20, 21, 28), and it has been hypothesized that a defective cellular immune response may exist in the disease. Lymphocyte transformation tests in -vitro in the presence of paramyxovirus have not provided evidence of such a defect (6). However, by means of migration inhibition tests with material from measles virus-infected cell cultures as antigen, a reduced inhibition of leukocytes from patients with MS as compared with leukocytes from healthy individuals has been found, and this finding has been regarded as an in vitro correlate of a deficient cell-mediated immunity to the virus (5, 11, 19, 25, 26). In the present study, crude measles virus material was used in the capillary modification of the migration inhibition test, and the results obtained with leukocytes from MS patients were compared with those obtained with leuko-
cytes from healthy blood donors and from patients with various other central nervous system (CNS) diseases. MATERIALS AND METHODS Leukocyte donors. Twelve men and five women with definite (22) MS were studied. The patients were all severely disabled, requiring institutional care, and in a chronic progressive state of the disease. Most of the patients had chronic urinary infections, but none of them showed signs of acute infectious disease when blood was drawn. The average age was 51 years (range, 36 to 61 years), and they had all had the disease for more than 10 years. Blood from 12 randomly selected healthy adults was collected at The Red Cross Blood Centre in Oslo and served as healthy controls. This donor group comprised seven men and five women, with an average age of 40 years (range, 20 to 60 years). Eight pa-
588
H. J. Nordal, S. S. Proland, B. Vandvik & E. Norrby
Ml 1.10
r-
1.00
-
.90
-
.80
•
o
.70 .60 .50 .40
Fig. 1. The inhibitory effect of measles cellassociated virus material on the migration (MI) of leukocytes from patients with multiple sclerosis (MS), from healthy blood donors (healthy controls), and from patients with various other central nervous system diseases (patient controls).
0
*
o oo
• *
Oo 1
1:10'
1
i:iO'
1:10°
Final dilutions of the measles virus preparation o MS
o Healthy c o n t r o l s
» Patient controls
Median
tients with non-infectious CNS diseases (four with parkinsonism, two with cerebrovascular diseases, one with traumatic encephalopathy, and one with a cerebral astrocytoma) were included as patient controls. The average age was 58 years (range, 45 to 71 years). .None of the persons included in the study received corticosteroid or cytostatic treatment. Leukocyte preparation. Fifty ml of blood (Heparin NOVO, Copenhagen, 10 IU/ml) was collected and layered on top of an Isopaquedextran solution (Nyegaard & Co., Oslo, Norway, and Pharmacia Fine Chemicals, Uppsala, Sweden) and incubated at 37 °C for 1 to 2 h (3). The leukocyte-rich plasma was pipetted off, special care being taken to avoid contamination by the Isopaque-dextran phase, and centrifuged for 10 min at 200 g. The cell pellet was washed, three times in Hanks's balanced salt solutioH (Grand Island Biological Co., New York, N.Y.), and the cells > were centrifuged for 5 min at 200 g after each washing. Differential counts of leukocyte suspensions prepared by this technique, and by sedimentation of whole blood at 37°C for 1-2 h, did not disclose any selective loss of any of the major leukocyte populations. Control experiments did not show any significant difference between the two separation procedures with respect to the effect on the migration inhibition results.
Mean
Leukocyte migration test. The test system was set up as described by Soborg & Bendixen (24) and modified by Froland & Gaarder (8). Unheparinized capillary tubes with an internal diameter of 0.6 mm were filled with a suspension of leukocytes in culture medium (Me Coy's medium with 20% foetal calf serum. Flow Laboratories) and centrifuged at 2,000 g for 10 min, producing an approximately 3mm-long cell column in the bottom of the tube. The migration areas were measured after 18 h, using a projection microscope. The circumference of the migration areas was drawn on paper, which was then cut and weighed. The migration index (MI) (24) was defined as the ratio between the migration area in culture medium containing measles virus and that in culture medium alone. All experiments were done in triplicate. The average standard deviation within triplicates was 7% of the mean. Differences in migration inhibition were evaluated by the Wilcoxon rank sum test (23). Measles virus preparation. The Lee strain of measles virus (originally isolated from a patient with subacute sclerosing panencephalitis and kindly provided by Dr. H. Koprowski, The Wistar Institute, Philadelphia, Pa., USA) was grown in Vero cell cultures, and a pool of measles cell-associated virus material was prepared from a 30% (v/v) suspension of virus-infected cells in phosphate-buffered saline
Migration Inhibition of MS Leukocytes
(PBS), pH 7.4 and 0.05M, as described elsewhere (15). Control material was prepared in the same way from non-infected Vero cell cultures. Careful examination of the cell cultures and virus preparations failed to reveal the presence of any contaminating infectious agent. The virus and control preparations were stored in small aliquots at -20°C. Final dilution in the culture medium, followed by mild sonication, was done shortly before use. Titration of measles virus antibodies. Measles virus nucleocapsid antibodies in the sera of patient and control donors were assayed by means of a counterimmunoelectrophoretic technique, as described previously (14). RESULTS Preliminary experiments showed that control material from non-infected cell cultures had negligible inhibitory effect on leukocyte migration in dilutions of lilO^ or higher, whereas dilutions of the measles virus preparation of 1:102, 1:104, and 1:10® induced marked, moderate, and minimal inhibition, respectively. These dilutions of the virus preparation were therefore found most suitable for the comparative study. The migration indices of the individual MS patients, the healthy control donors, and the patients with the other neurological diseases are shown in Fig. 1. Only small differences, of marginal statistical significance, were found when the migration inhibition of leukocytes from MS patients was compared with that of leukocytes from the healthy blood donors and the patient controls. The largest difference in median migration inhibition between the MS and patient control groups was 0.04, which was found at a dilution of the virus preparation of 1:10*. This difference and the small differences seen at the two other dilutions were statistically insignificant (P > 0.05). The difference between the results of the MS group and the healthy control donor group was also small. The largest differences in median migration inhibition were found at dilutions of the virus preparation of 1:10^
589
Ml
.90 .80 .70 .60 .50 I I I I J
Measles Virus-Induced Migration Inhibition In Vitro of Leukocytes from Patients with Multiple Sclerosis H. J. NORDAL, S. S. FR0LAND, B. VANDVIK & E. NORRBY Institute of Immunology and Rheumatology and Department of Neurology, Rikshospitalet University Hospital, Oslo, Norway, and Department of Virology, Karolinska Institute, School of Medicine, Stockholm, Sweden
Nordal, H. J., Freland, S. S., Vandvik, B. & Norrby, E. Measles Virus-Induced Migration Inhibition In Vitro of Leukocytes from Patients with Multiple Sclerosis. Scand. J. Immunol. } , 587-591, 1976. The migration inhibition in vitro induced by measles virus material on leukocytes from 17 patients with multiple sclerosis (MS) was compared with that of 8 patients with various other central nervous system diseases and that of 12 healthy blood donors. Significant differences could not be demonstrated. The results do not indicate that MS patients' leukocytes show an altered behaviour to measles virus material in the migration inhibition test. H. J. Nordal, Institute of Immunology and Rheumatology, F. Qvamsgt. 1, Oslo 1, Norway
The possibility that multiple sclerosis (MS) may be associated with a persistent infection of paramyxovirus has been discussed by several authors (1, 4, 12, 17, 18, 20, 21, 28), and it has been hypothesized that a defective cellular immune response may exist in the disease. Lymphocyte transformation tests in -vitro in the presence of paramyxovirus have not provided evidence of such a defect (6). However, by means of migration inhibition tests with material from measles virus-infected cell cultures as antigen, a reduced inhibition of leukocytes from patients with MS as compared with leukocytes from healthy individuals has been found, and this finding has been regarded as an in vitro correlate of a deficient cell-mediated immunity to the virus (5, 11, 19, 25, 26). In the present study, crude measles virus material was used in the capillary modification of the migration inhibition test, and the results obtained with leukocytes from MS patients were compared with those obtained with leuko-
cytes from healthy blood donors and from patients with various other central nervous system (CNS) diseases. MATERIALS AND METHODS Leukocyte donors. Twelve men and five women with definite (22) MS were studied. The patients were all severely disabled, requiring institutional care, and in a chronic progressive state of the disease. Most of the patients had chronic urinary infections, but none of them showed signs of acute infectious disease when blood was drawn. The average age was 51 years (range, 36 to 61 years), and they had all had the disease for more than 10 years. Blood from 12 randomly selected healthy adults was collected at The Red Cross Blood Centre in Oslo and served as healthy controls. This donor group comprised seven men and five women, with an average age of 40 years (range, 20 to 60 years). Eight pa-
588
H. J. Nordal, S. S. Proland, B. Vandvik & E. Norrby
Ml 1.10
r-
1.00
-
.90
-
.80
•
o
.70 .60 .50 .40
Fig. 1. The inhibitory effect of measles cellassociated virus material on the migration (MI) of leukocytes from patients with multiple sclerosis (MS), from healthy blood donors (healthy controls), and from patients with various other central nervous system diseases (patient controls).
0
*
o oo
• *
Oo 1
1:10'
1
i:iO'
1:10°
Final dilutions of the measles virus preparation o MS
o Healthy c o n t r o l s
» Patient controls
Median
tients with non-infectious CNS diseases (four with parkinsonism, two with cerebrovascular diseases, one with traumatic encephalopathy, and one with a cerebral astrocytoma) were included as patient controls. The average age was 58 years (range, 45 to 71 years). .None of the persons included in the study received corticosteroid or cytostatic treatment. Leukocyte preparation. Fifty ml of blood (Heparin NOVO, Copenhagen, 10 IU/ml) was collected and layered on top of an Isopaquedextran solution (Nyegaard & Co., Oslo, Norway, and Pharmacia Fine Chemicals, Uppsala, Sweden) and incubated at 37 °C for 1 to 2 h (3). The leukocyte-rich plasma was pipetted off, special care being taken to avoid contamination by the Isopaque-dextran phase, and centrifuged for 10 min at 200 g. The cell pellet was washed, three times in Hanks's balanced salt solutioH (Grand Island Biological Co., New York, N.Y.), and the cells > were centrifuged for 5 min at 200 g after each washing. Differential counts of leukocyte suspensions prepared by this technique, and by sedimentation of whole blood at 37°C for 1-2 h, did not disclose any selective loss of any of the major leukocyte populations. Control experiments did not show any significant difference between the two separation procedures with respect to the effect on the migration inhibition results.
Mean
Leukocyte migration test. The test system was set up as described by Soborg & Bendixen (24) and modified by Froland & Gaarder (8). Unheparinized capillary tubes with an internal diameter of 0.6 mm were filled with a suspension of leukocytes in culture medium (Me Coy's medium with 20% foetal calf serum. Flow Laboratories) and centrifuged at 2,000 g for 10 min, producing an approximately 3mm-long cell column in the bottom of the tube. The migration areas were measured after 18 h, using a projection microscope. The circumference of the migration areas was drawn on paper, which was then cut and weighed. The migration index (MI) (24) was defined as the ratio between the migration area in culture medium containing measles virus and that in culture medium alone. All experiments were done in triplicate. The average standard deviation within triplicates was 7% of the mean. Differences in migration inhibition were evaluated by the Wilcoxon rank sum test (23). Measles virus preparation. The Lee strain of measles virus (originally isolated from a patient with subacute sclerosing panencephalitis and kindly provided by Dr. H. Koprowski, The Wistar Institute, Philadelphia, Pa., USA) was grown in Vero cell cultures, and a pool of measles cell-associated virus material was prepared from a 30% (v/v) suspension of virus-infected cells in phosphate-buffered saline
Migration Inhibition of MS Leukocytes
(PBS), pH 7.4 and 0.05M, as described elsewhere (15). Control material was prepared in the same way from non-infected Vero cell cultures. Careful examination of the cell cultures and virus preparations failed to reveal the presence of any contaminating infectious agent. The virus and control preparations were stored in small aliquots at -20°C. Final dilution in the culture medium, followed by mild sonication, was done shortly before use. Titration of measles virus antibodies. Measles virus nucleocapsid antibodies in the sera of patient and control donors were assayed by means of a counterimmunoelectrophoretic technique, as described previously (14). RESULTS Preliminary experiments showed that control material from non-infected cell cultures had negligible inhibitory effect on leukocyte migration in dilutions of lilO^ or higher, whereas dilutions of the measles virus preparation of 1:102, 1:104, and 1:10® induced marked, moderate, and minimal inhibition, respectively. These dilutions of the virus preparation were therefore found most suitable for the comparative study. The migration indices of the individual MS patients, the healthy control donors, and the patients with the other neurological diseases are shown in Fig. 1. Only small differences, of marginal statistical significance, were found when the migration inhibition of leukocytes from MS patients was compared with that of leukocytes from the healthy blood donors and the patient controls. The largest difference in median migration inhibition between the MS and patient control groups was 0.04, which was found at a dilution of the virus preparation of 1:10*. This difference and the small differences seen at the two other dilutions were statistically insignificant (P > 0.05). The difference between the results of the MS group and the healthy control donor group was also small. The largest differences in median migration inhibition were found at dilutions of the virus preparation of 1:10^
589
Ml
.90 .80 .70 .60 .50 I I I I J
