Clin. exp. Immunol. (1976) 24, 26-32.
A direct macrophage migration inhibition test applied in man A. W. TEELKEN & J. M. MINDERHOUD Department of Neurology, University Hospital, Oostersingel, Groningen, The Netherlands
(Received 4 July 1975) SUMMARY
A direct macrophage migration test is described, which proved to be useful for the detection of cellular hypersensitivity in man. We used a modification of the method described by Hughes & Paty (1972). In this method the MLR is abolished by 100 rad y-irradiation of the peritoneal exudate cells prior to pooling with human lymphocytes. Experiments with various intensity of irradiation, PPD, muscle antigen and encephalitogenic factor were performed to check this method. In a pilot study lymphocytes of patients with diseases of the central or peripheral nervous system or of muscle were tested. This proved that a hypersensitivity to Ef was present in various diseases of the CNS, while in muscle diseases positive tests were found using muscle antigen. INTRODUCTION The macrophage migration inhibition test (David et al., 1964) which can easily be used to examine hypersensitivity reactions in test animals, was not found to be suitable in man. It was supposed that the production of macrophage inhibition factor (MIF) by the mixed lymphocyte reaction (MLR) between guinea-pig lymphocytes which are present in the peritoneal exudate cell suspension and human lymphocytes, obscured the inhibitory effect of added antigen with the human lymphocytes. On the other hand, Rajapakse & Glynn (1970) and Bartfield & Atoynatan (1971) did not find the MLR strong enough to disturb the observation of the result of lymphocyte-antigen interaction. Thor et al. (1968) and later Rocklin, Meyers & David (1970) avoided these problems in their methods. They used the supernatant of a 72 hr culture of human lymphocytes and antigen to influence the migration of the guinea-pig peritoneal exudate cells. For the same reason, S0borg & Bendixen (1967) introduced a direct leucocyte migration test (LMT) in which autologous peripheral blood leucocytes were used instead of guinea-pig peritoneal exudate cells. However, the LMT proved to be controversial in performance and in interpretation while the method of Rocklin et al. (1970) is rather complicated and allows dilution of MIF in the supernatant. Therefore, we tried to find a more practical and reliable test. Another way to avoid the supposed MLR was shown by Hughes & Paty (1972), who described a very sensitive micro-method in which the MLR was suppressed by y-irradiation of the peritoneal exudate cells prior to adding human lymphocytes and antigen. We used this method to evaluate a direct macrophage migration inhibition test which can be used to detect cellular hypersensitivity in man. Correspondence: Dr A. W. Teelken, Department of Neurology, University Hospital, Oostersingel 59, Groningen, The Netherlands.
26
A direct macrophage migration inhibition test
27
MATERIALS AND METHODS Human lymphocytes were separated from defibrinated peripheral blood using a gradient of sodium metrizoate (Nijegaard and Co., Oslo) and Ficoll (Pharmacia, Uppsala, Sweden) described by Boyum (1968). Human tuberculin purified protein derivative (PPD) without conservative was a generous gift of Dr Sirks, State Department of Public Health, Bilthoven, The Netherlands. Encephalitogenic factor (Ef) was prepared from fresh bovine brain by a method described by Deibler, Martenson & Kies (1972). Muscle antigen was prepared from guinea-pig gluteus muscle. The muscle tissue was homogenized in 099% saline (20%Y w/v) and centrifuged at 250 g for 10 min. The supernatant was dialysed overnight against demineralized water and freeze-dried.
PPD to be used in the test was dissolved in TC 199 at a concentration of 33 #g/ml, and myelin antigen (Ef) or muscle antigen at a concentration of 280 ,ug/mi. Macrophage migration inhibition was tested by the micro-method described by Hughes (1972) and Hughes & Paty (1972). In this method 10%Y human lymphocytes was added to guinea-pig peritoneal exudate cells (PC). After thorough mixing, the cells were centrifuged and resuspended in nutrient medium (20%/ foetal calf serum in TC 199) up to 0 5 x 108 PC and 0-5 x 107 human lymphocytes/ml. In most of the experiments the PC were treated with 100 rad y-irradiation prior to pooling with the human lymphocytes, while in others the PC and human lymphocytes were irradiated together. Gamma-irradiation was performed by means of a 6'Co source (Department of Radiotherapy). To obtain a homogenized dose the tubes with the cell suspension were placed vertically in a perspex water-bath and irradiated perpendicularly to one side of the bath. The absorbed dose was fixed at exactly 100 rad. Surgical quality polythene tubing (bore 0 58 mm) was
filled with a 3-4 cm column of the cell mixture by means of a hypodermic needle. After melting of the air
space end the capillaries were centrifuged at 600 g for 5 min. The capillaries were cut off with a razor blade at the cell interface on both ends and 4 mm pieces of these capillaries were rapidly mounted in a culture chamber with the aid of silicone grease. The chambers were filled with nutrient medium (20%0 foetal calf serum in TC 199, with or without antigen). One or two chambers (each containing three capillaries) were set up for each experiment. Chambers were incubated for 18 hr at 370C. Subsequently the outermost fringe of migrating cells on both sides of the capillaries was traced on standard filter paper using a microscope with a x 2 5 objective. The area of migration was obtained by cutting out and weighing the paper tracings (in grams). In each experiment the mean of six or twelve areas of migration was used. Three series of experiments were performed. At first the lymphocytes of normal persons were used in experiments to investigate the migration of irradiated and non-irradiated macrophages and the influence of human lymphocytes and PPD. Eight of these persons were known to have negative skin tests to tuberculin;
thirty-seven were positive. In a separate series eight normal persons were used in order to compare the MLR and the inhibition by
lymphocyte-antigen interaction in individuals.
In a second experiment lymphocytes of four normal persons and of two patients with a muscular dystrophy used to investigate the results of varying percentages of lymphocytes, concentrations of antigen or intensity of irradiation on the macrophage migration. At last fifteen normals and sixty patients were tested with muscle antigen and encephalitogenic factor. In each of these tests one or two controls were included. These controls consisted of the migration of macrophages of an irradiated PC-non-irradiated human lymphocyte mixture and the migration of macrophages after irradiation of PC and lymphocytes together in the presence of one of the antigens. In all the experiments the percentage of migration inhibition was calculated comparing the area of migration with a control: were
percentage inhibition= (|PC+ Lyl * +Ag)- (PC* + Ly+ Ag)
(|PC+Lyl*+Ag)
in which (IPC+LyI*+Ag) is the area of migration when PC and lymphocytes are irradiated together and incubated in nutrient medium with antigen, and (PC*+Ly+Ag) is the area of migration when PC are irradiated before pooling with lymphocytes (non-irradiated) and incubated in nutrient medium with antigen. All the examinations were done blind, i.e. they were done by a person who knew neither the patients nor their disease.
RESULTS MLR and PPD experiments as n
The mean of the migration of macrophages in nutrient medium during 18 hr at 370C expressed in the weight of the paper-tracings of the migrations was 0-242 g (s.d. 0032;
=24).
0B5_~ ~ * *3. A. W. Teelken & J. M. Minderhoud
28 0
0-30 _
s
i'.8.30@
..44
025
o_~~~~~~~~~~~~~~~~~~~~~ f~~~~~0-10 ~~
y 90 .0
CP~
_0
~
~
Iss:0
0~~~~~~
~ ~~
(i;) (
PC
0-242 x sd. 0-032 n 24
PC*
0-240
0-019 18
~
~
~
~
(iii) PC
Civ)
Ly
Ly PPD
N073 0-014 12
0073
PC
0012 8
~~~~~~C
P
(v;)
(vijO Cvii)
PC
PC
PC
Ly
Ly PPD Cpos
Ly PPD
Ly0 M/Ef
0260 0012 48
0073
( v) PC
0-030 37
0eg)
-248 0-027 8
(>269
0019 32
FIG. 1. Areas of macrophage migration (in grams) in different conditions. PC: peritoneal exudate cells; Ly: human lymphocytes; M: muscle antigen; Ef: myelin antigen (encephalitogenic factor).
Irradiation with 100 rad did not cause a significant influence on the migration (mean 0-240 g; s.d. 0-019; n= 18; Fig. 1(i) and (ii)). The influence of the probable MLR was found when 10% human lymphocytes was added. About 70YO inhibition of the macrophage migration was found (mean 0-073 g; s.d. 0-014; n= 12; Fig. l(iii)). This MLR could be avoided by y-irradiation of PC prior to pooling with human lymphocytes (mean 0-260; s.d. 0-012; n = 48; Fig. 1(v)). In forty-five experiments lymphocytes of normal persons and PPD were added to irradiated PC. In all persons with a positive skin test to tuberculin an inhibition of the migration was found varying between 41 % and 90%° (mean of the migration 0-0731 g; s.d. 0-030; n=37), while in eight tuberculin-negative persons only a slight or no inhibition at all was found (between 0Oy and 17%4; mean of migration 0-248 g; s.d. 0-027; n=8). Rajapakse & Glynn (1970) did not find the MLR sufficiently pronounced to disturb the observation of the migration inhibition caused by lymphocyte antigen interaction. However, we investigated the inhibition caused by the MLR without and with PPD in eight normal subjects (Table 1). It appeared that in all except one person with the most pronounced inhibition in our test, the inhibition due to PPD was obscured by the MLR. In the other persons and in the mean of the results (Fig. 1(iii) and (iv)) no difference could be found. Titrations In two experiments the number of human lymphocytes mixed with non-irradiated PC was changed. The maximum influence of the MLR on the migration of macrophages was
found already with 500 human lymphocytes pooled with PC (Fig. 2(i)).
29
A direct macrophage migration inhibition test TABLE 1. Comparison of the migration inhibition caused by MLR (PC+ Ly) and by MLR+ lymphocyte-antigen interaction in eight normal persons. MMI Inh. %Y: percentage of macrophage migration inhibition found with the test described here. Tub.: +: tuberculin positive persons;-: tuberculin negative persons Inhibition
Tube 1 2 3 4 5 6 7 8
+ + + + + + -
Inhibition MMI inhibition
PC (g)
PC + Ly (g)
(%)
PC + Ly + PPD (g)
(%)
(%)
0-236 (0-014) 0-241 (0-012) 0-233 (0 010) 0230 (0009) 0-241 (0007) 0-233 (0010) 0-266 (0-013) 0-240 (0-012)
0-072 (0 011) 0-084 (0-010) 0-076 (0-010) 0-063 (0008) 0-088 (0'009) 0-076 (0-010) 0 074 (0 009) 0-068 (0 010)
69 5 650 67-0 730 63-0 67-0 67-0 72-0
0 073 (0 007) 0086 (0009) 0 073 (0-007)
69-0 640 69-0 79.5* 63-0 67-0 66-0 70-0
65 0 62-0 61-0 750 605 63-0 0 0
0047 (0007) 0-089 (0008) 0077 (0009) 0-076 (0 009) 0-072 (0 009)
* Difference between PC+ Ly inhibition and PC+ Ly+ PPD inhibition significant (P< 0001). In each test n= 6 or n= 12.
0 25 0.20
PC + Ly Mixed lymphocyte reaction
0-15 _-\
0-10 -
a
0-05 _
I 2
0
3
4
5
6
Human lymphocytes 025L
8
lo
9
(%)
PC
0.20 C)
7
-1
05__ PC + Ly 10% 010O F
E
005
o0
(ii )
U)
e
0
10
100 (rod)
1000
y -Irradiation
0-25L
PC* +
0-20
0.15
Im W
0-10 _ 0-05 0
Ly 10% +M
sl IO 20 m -Antigen
50 (280 ug/ml ) (%)
100
FIG. 2. Titrations of respectively the mixed lymphocyte reaction with varying percentages of human lymphocytes (i) and varying intensity of irradiation (ii). In (iii) the migration in the presence of various antigen concentrations is shown. w: Weak lymphocyte sensitization to muscle antigen in a carrier of a muscular dystrophy; s: strong sensitization to muscle antigen in a muscular dystrophy patient.
30
A. W. Teelken & J. M. Minderhoud
In two other experiments the degree of suppression of the MLR by y-irradiation was tested. PC were just given a sham irradiation or they were irradiated with respectively 10, 100 and 1000 rad. The migration of those PC decreased a little by irradiation (in contrast with the results in the larger series; Fig. 1(i) and (ii)). When 10% human lymphocytes was added an inhibition of migration of the non-irradiated PC and of the PC irradiated with 10 rad was found. By irradiation with 100 rad the MLR was totally suppressed (Fig. 2(ii)). Experiments with muscle antigen (M) and encephalitogenic factor (Ef) In order to avoid artefacts caused by different numbers of cells or non-specific influence of antigen, the migration of irradiated macrophages in the presence of human lymphocytes and antigen was compared with the migration of macrophages also in the presence of human lymphocytes and the same antigen after y-irradiating the PC and the lymphocytes together. The mean area of migration in the presence of irradiated human lymphocytes (of patients with a disease of the central or peripheral nervous system) and muscle antigen or Ef did not differ from the mean area of migration of irradiated PC which were pooled with nonirradiated human lymphocytes without antigen (mean 0-269 g; n=32; s.d. 0 010 g; Fig. 1(viii)). There was only a slight increase of migration compared with the migration of irradiated or not irradiated PC alone. This increase was not significant. So y-irradiation seemed to abolish the interaction between heterogeneic lymphocytes or between antigen and human lymphocytes without influencing the migrating activity of macrophages. Calculating the percentage inhibition of migration in experiments with non-irradiated lymphocytes and as a control, with irradiated lymphocytes, gave a good impression of the cellular hypersensitivity to antigens. The amount of cellular hypersensitivity differs from one person to the other. In Fig. 2(iii) a titration of the test is shown in two patients with muscle disease, one with an intensive and one with a weaker cellular hypersensitivity to muscle antigen, in which the antigen concentration as parts of the concentration normally used (280 pg/ml) is changed. With the described method fifty-five persons, that is twenty-one patients with a disease or lesion of the central nervous system, fifteen patients with a peripheral nerve disease and nineteen patients with a muscle disease, and fifteen controls were tested. The results are shown in Table 2. TABLE 2. Summary of the results obtained in a pilot study in which normals and patients with a disease of the central nervous system (CNS), the peripheral nervous system (PNS) or a muscle disease were examined on cellular hypersensitivity to muscle antigen (M) and myelin antigen (Ef). Results expressed as the percentage of macrophage migration inhibition
Ef
Normals CNS PNS Muscle disease
M
(n= 15) 1-1% (-l10%/+ 11y) I-0% (-10%/+ 10%) 7-1% (-4%/32%) (n=21) 16 7% (1%/50%) (n= 15) 36-5% (0%y/68%Y) 290%(4%/60%) (n= 19) 5-3% (-10%/29%) 43W1% (12%/88%)
The majority of the patients with diseases of the central nervous system were found to be sensitized against Ef; the majority of the patients with a disease of the peripheral nervous system were sensitized to Ef and to muscle antigen and all the patients with a muscle disease showed a cellular hypersensitivity to muscle antigen. In later studies we also used peripheral Ef-antigen, which gave better results than muscle antigen or Ef in patients with a lesion of the peripheral nervous system.
A direct macrophage migration inhibition test
31
The results of this direct macrophage migration inhibition test in various neurological diseases are described elsewhere (Minderhoud & Teelken, 1974; Minderhoud et al., 1974).
DISCUSSION Bartfield & Atoynaten (1971) and Rajapakse & Glynn (1970) were not able to show a clear MLR between two histoincompatible lymphocyte suspensions. Using their more sensitive micro-modification of the macrophage migration inhibition test Hughes & Paty (1972) were able to find a MLR after incubation during 16 hr at 350C. This MLR could be suppressed by y-irradiation. If in our experiments non-irradiated peritoneal exudate cells mixed with 1000 human lymphocytes were cultured in the presence of PPD no additional inhibition could be detected in five of the six patients with a positive skin test to tuberculin. In only one could additional migration inhibition be detected in the presence of antigen. Only this person could be scored positive in a method using non-irradiated macrophages instead of six who actually were positive to tuberculin. The advantage of the micro-method which has an increased sensitivity compared with the method of Thor et al. (1968) and Rocklin et al. (1970) is in particular the short period of incubation necessary to detect lymphocytelymphocyte interaction or lymphocyte-antigen interaction. This diminishes the necessity of absolute sterility and probably induces the possibility of sufficient y-irradiation without influencing the migrating abilities of macrophages. Visakorpi (1974) and others found that antigen-induced migration inhibition in guinea-pigs sensitized with albumin in Freund's adjuvant was still detectable after an in vitro irradiation of 5000 or 10,0C0 rad; in the investigations of Hughes & Paty (1972) and in our investigations using the micro-method, human lymphocytes and PPD, Ef or M-antigen, y-irradiation of 100 rad proved to be sufficient to suppress both the MLR and the antigen-human lymphocyte interaction, during the 18 hr of culture. Performed in this way the macrophage migration inhibition test is found to be sensitive and useful in detection of cellular hypersensitivity in man. Hughes & Paty (1972) found some deleterious effect on the migration of macrophages caused by the 100 rad y-irradiation. In their study the migration was 9-700 less than the migration of non-irradiated PC. In our experiments no reduction of the migration caused by the y-irradiation could be found (0-242 g; s.d. 0-032, versus 0-240 g; s.d. 0-019). A slight increase of migration was found in experiments in which human lymphocytes were pooled with irradiated PC (mean 0-260 g; s.d. 0-012 g), which was probably caused by a minimal one-way lymphocyte reaction. But this slight increase of migration was also found when irradiated PC and irradiated human lymphocytes of patients were incubated in nutrient medium containing antigen (mean 0-269 g; s.d. 0-010g). In order to avoid as many artefacts as possible we included in each test at least one or two controls: the migration of irradiated macrophages pooled with the human lymphocytes (Fig. l(v)), or mostly the migration of macrophages irradiated after pooling with the human lymphocytes and incubated in the presence of one of the antigens (Fig. l(viii)). The antigen-human lymphocyte interaction was measured as the percentage of inhibition of migration. This method proved to be successful in a pilot study in which lymphocytes of patients with different neurological diseases were examined. The results indicated that in diseases of muscle or the nervous system a cellular hypersensitivity against probable break-down products of these tissues is present and that this reaction can be measured by the described MMI test. We should like to thank Dr S. Kiestra for his technical advice about the y-irradiation and the technical staff of the Department of Radiotherapy (Head: Professor Dr H. C. Stam) for their assistance in the irradiation. We are also very grateful for the assistance of Miss M. J. F. van den Ouweland and Miss D. A. Woldendorp in taking blood samples and for their help in separating cells. And last but not least our gratitude goes to Mr G. Loof who performed the MMI test without knowing anything about the patients who were tested and about the results that could be expected.
32
A. W. Teelken & J. M. Minderhoud REFERENCES
BARTFIELD, H. & ATOYNATAN, T. (1971) Activities and properties of macrophage migration inhibitory factor produced by mixed lymphocyte cultures. Nature: New Biology, 230, 246. BOYUM, A. (1968) Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, supplement 97. DAVID, J.R., AL-ASKARI, S., LAWRENCE, H.S. & THOMAS, L. (1964) Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigens. J. Immunol. 93, 264. DEIBLER, G.E., MARTENSON, R.E. & KIES, M.W. (1972) Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Preparative Biochemistry, 2, 139. HUGHES, D. (1972) Macrophage migration inhibition test: a critical examination of the technique using a polythene capillary tubing micromethod. J. immunol. Methods, 1, 403. HUGHES, D. & PATY, D.W. (1972) Macrophage migration inhibition test applied to delayed hypersensitivity in man. Z. ImmunForsch. 142, 478. MINDERHOUD, J.M. & TEELKEN, A.W. (1974) Cellular hypersensitivity to myelin and muscle tissue in man examined with a direct macrophage
migration inhibition test. I. Diseases of the central and peripheral nervous system. Clin. Neurol. Neurosurg. 77, 225. MINDERHOUD, J.M., TEELKEN, A.W., LEENSTRABORSJE, H. & OOSTEN, J.K. (1974) Cellular hypersensitivity to myelin and muscle tissue in man examined with a direct macrophage migration inhibition test. II. Muscle diseases. Clin. Neurol. Neurosurg. 77, 234. RAJAPAKSE, D.A. & GLYNN, L.E. (1970) Macrophage migration inhibition test using guinea-pig macrophages and human lymphocytes. Nature (Lond.), 226, 857. ROCKLIN, R.E., MEYERS, O.L. & DAVID, J.R. (1970) An in vitro assay for cellular hypersensitivity in man. J. Immunol. 104, 95. S0BORG, M. & BENDIXEN, G. (1967) Human lymphocyte migration as a parameter of hypersensitivity. Acta med. scand. 181, 247. THOR, D.E., JURERIZ, R.E., VEACH, S.R., MILLER, E. & DRAY, S. (1968) Cell migration inhibition factor released by antigen from human peripheral lymphocytes. Nature (Lond.), 219, 755. VISAKORPI, R. (1974) The effect of irradiation on macrophage migration inhibition. Immunology, 27, 145.
A direct macrophage migration inhibition test applied in man A. W. TEELKEN & J. M. MINDERHOUD Department of Neurology, University Hospital, Oostersingel, Groningen, The Netherlands
(Received 4 July 1975) SUMMARY
A direct macrophage migration test is described, which proved to be useful for the detection of cellular hypersensitivity in man. We used a modification of the method described by Hughes & Paty (1972). In this method the MLR is abolished by 100 rad y-irradiation of the peritoneal exudate cells prior to pooling with human lymphocytes. Experiments with various intensity of irradiation, PPD, muscle antigen and encephalitogenic factor were performed to check this method. In a pilot study lymphocytes of patients with diseases of the central or peripheral nervous system or of muscle were tested. This proved that a hypersensitivity to Ef was present in various diseases of the CNS, while in muscle diseases positive tests were found using muscle antigen. INTRODUCTION The macrophage migration inhibition test (David et al., 1964) which can easily be used to examine hypersensitivity reactions in test animals, was not found to be suitable in man. It was supposed that the production of macrophage inhibition factor (MIF) by the mixed lymphocyte reaction (MLR) between guinea-pig lymphocytes which are present in the peritoneal exudate cell suspension and human lymphocytes, obscured the inhibitory effect of added antigen with the human lymphocytes. On the other hand, Rajapakse & Glynn (1970) and Bartfield & Atoynatan (1971) did not find the MLR strong enough to disturb the observation of the result of lymphocyte-antigen interaction. Thor et al. (1968) and later Rocklin, Meyers & David (1970) avoided these problems in their methods. They used the supernatant of a 72 hr culture of human lymphocytes and antigen to influence the migration of the guinea-pig peritoneal exudate cells. For the same reason, S0borg & Bendixen (1967) introduced a direct leucocyte migration test (LMT) in which autologous peripheral blood leucocytes were used instead of guinea-pig peritoneal exudate cells. However, the LMT proved to be controversial in performance and in interpretation while the method of Rocklin et al. (1970) is rather complicated and allows dilution of MIF in the supernatant. Therefore, we tried to find a more practical and reliable test. Another way to avoid the supposed MLR was shown by Hughes & Paty (1972), who described a very sensitive micro-method in which the MLR was suppressed by y-irradiation of the peritoneal exudate cells prior to adding human lymphocytes and antigen. We used this method to evaluate a direct macrophage migration inhibition test which can be used to detect cellular hypersensitivity in man. Correspondence: Dr A. W. Teelken, Department of Neurology, University Hospital, Oostersingel 59, Groningen, The Netherlands.
26
A direct macrophage migration inhibition test
27
MATERIALS AND METHODS Human lymphocytes were separated from defibrinated peripheral blood using a gradient of sodium metrizoate (Nijegaard and Co., Oslo) and Ficoll (Pharmacia, Uppsala, Sweden) described by Boyum (1968). Human tuberculin purified protein derivative (PPD) without conservative was a generous gift of Dr Sirks, State Department of Public Health, Bilthoven, The Netherlands. Encephalitogenic factor (Ef) was prepared from fresh bovine brain by a method described by Deibler, Martenson & Kies (1972). Muscle antigen was prepared from guinea-pig gluteus muscle. The muscle tissue was homogenized in 099% saline (20%Y w/v) and centrifuged at 250 g for 10 min. The supernatant was dialysed overnight against demineralized water and freeze-dried.
PPD to be used in the test was dissolved in TC 199 at a concentration of 33 #g/ml, and myelin antigen (Ef) or muscle antigen at a concentration of 280 ,ug/mi. Macrophage migration inhibition was tested by the micro-method described by Hughes (1972) and Hughes & Paty (1972). In this method 10%Y human lymphocytes was added to guinea-pig peritoneal exudate cells (PC). After thorough mixing, the cells were centrifuged and resuspended in nutrient medium (20%/ foetal calf serum in TC 199) up to 0 5 x 108 PC and 0-5 x 107 human lymphocytes/ml. In most of the experiments the PC were treated with 100 rad y-irradiation prior to pooling with the human lymphocytes, while in others the PC and human lymphocytes were irradiated together. Gamma-irradiation was performed by means of a 6'Co source (Department of Radiotherapy). To obtain a homogenized dose the tubes with the cell suspension were placed vertically in a perspex water-bath and irradiated perpendicularly to one side of the bath. The absorbed dose was fixed at exactly 100 rad. Surgical quality polythene tubing (bore 0 58 mm) was
filled with a 3-4 cm column of the cell mixture by means of a hypodermic needle. After melting of the air
space end the capillaries were centrifuged at 600 g for 5 min. The capillaries were cut off with a razor blade at the cell interface on both ends and 4 mm pieces of these capillaries were rapidly mounted in a culture chamber with the aid of silicone grease. The chambers were filled with nutrient medium (20%0 foetal calf serum in TC 199, with or without antigen). One or two chambers (each containing three capillaries) were set up for each experiment. Chambers were incubated for 18 hr at 370C. Subsequently the outermost fringe of migrating cells on both sides of the capillaries was traced on standard filter paper using a microscope with a x 2 5 objective. The area of migration was obtained by cutting out and weighing the paper tracings (in grams). In each experiment the mean of six or twelve areas of migration was used. Three series of experiments were performed. At first the lymphocytes of normal persons were used in experiments to investigate the migration of irradiated and non-irradiated macrophages and the influence of human lymphocytes and PPD. Eight of these persons were known to have negative skin tests to tuberculin;
thirty-seven were positive. In a separate series eight normal persons were used in order to compare the MLR and the inhibition by
lymphocyte-antigen interaction in individuals.
In a second experiment lymphocytes of four normal persons and of two patients with a muscular dystrophy used to investigate the results of varying percentages of lymphocytes, concentrations of antigen or intensity of irradiation on the macrophage migration. At last fifteen normals and sixty patients were tested with muscle antigen and encephalitogenic factor. In each of these tests one or two controls were included. These controls consisted of the migration of macrophages of an irradiated PC-non-irradiated human lymphocyte mixture and the migration of macrophages after irradiation of PC and lymphocytes together in the presence of one of the antigens. In all the experiments the percentage of migration inhibition was calculated comparing the area of migration with a control: were
percentage inhibition= (|PC+ Lyl * +Ag)- (PC* + Ly+ Ag)
(|PC+Lyl*+Ag)
in which (IPC+LyI*+Ag) is the area of migration when PC and lymphocytes are irradiated together and incubated in nutrient medium with antigen, and (PC*+Ly+Ag) is the area of migration when PC are irradiated before pooling with lymphocytes (non-irradiated) and incubated in nutrient medium with antigen. All the examinations were done blind, i.e. they were done by a person who knew neither the patients nor their disease.
RESULTS MLR and PPD experiments as n
The mean of the migration of macrophages in nutrient medium during 18 hr at 370C expressed in the weight of the paper-tracings of the migrations was 0-242 g (s.d. 0032;
=24).
0B5_~ ~ * *3. A. W. Teelken & J. M. Minderhoud
28 0
0-30 _
s
i'.8.30@
..44
025
o_~~~~~~~~~~~~~~~~~~~~~ f~~~~~0-10 ~~
y 90 .0
CP~
_0
~
~
Iss:0
0~~~~~~
~ ~~
(i;) (
PC
0-242 x sd. 0-032 n 24
PC*
0-240
0-019 18
~
~
~
~
(iii) PC
Civ)
Ly
Ly PPD
N073 0-014 12
0073
PC
0012 8
~~~~~~C
P
(v;)
(vijO Cvii)
PC
PC
PC
Ly
Ly PPD Cpos
Ly PPD
Ly0 M/Ef
0260 0012 48
0073
( v) PC
0-030 37
0eg)
-248 0-027 8
(>269
0019 32
FIG. 1. Areas of macrophage migration (in grams) in different conditions. PC: peritoneal exudate cells; Ly: human lymphocytes; M: muscle antigen; Ef: myelin antigen (encephalitogenic factor).
Irradiation with 100 rad did not cause a significant influence on the migration (mean 0-240 g; s.d. 0-019; n= 18; Fig. 1(i) and (ii)). The influence of the probable MLR was found when 10% human lymphocytes was added. About 70YO inhibition of the macrophage migration was found (mean 0-073 g; s.d. 0-014; n= 12; Fig. l(iii)). This MLR could be avoided by y-irradiation of PC prior to pooling with human lymphocytes (mean 0-260; s.d. 0-012; n = 48; Fig. 1(v)). In forty-five experiments lymphocytes of normal persons and PPD were added to irradiated PC. In all persons with a positive skin test to tuberculin an inhibition of the migration was found varying between 41 % and 90%° (mean of the migration 0-0731 g; s.d. 0-030; n=37), while in eight tuberculin-negative persons only a slight or no inhibition at all was found (between 0Oy and 17%4; mean of migration 0-248 g; s.d. 0-027; n=8). Rajapakse & Glynn (1970) did not find the MLR sufficiently pronounced to disturb the observation of the migration inhibition caused by lymphocyte antigen interaction. However, we investigated the inhibition caused by the MLR without and with PPD in eight normal subjects (Table 1). It appeared that in all except one person with the most pronounced inhibition in our test, the inhibition due to PPD was obscured by the MLR. In the other persons and in the mean of the results (Fig. 1(iii) and (iv)) no difference could be found. Titrations In two experiments the number of human lymphocytes mixed with non-irradiated PC was changed. The maximum influence of the MLR on the migration of macrophages was
found already with 500 human lymphocytes pooled with PC (Fig. 2(i)).
29
A direct macrophage migration inhibition test TABLE 1. Comparison of the migration inhibition caused by MLR (PC+ Ly) and by MLR+ lymphocyte-antigen interaction in eight normal persons. MMI Inh. %Y: percentage of macrophage migration inhibition found with the test described here. Tub.: +: tuberculin positive persons;-: tuberculin negative persons Inhibition
Tube 1 2 3 4 5 6 7 8
+ + + + + + -
Inhibition MMI inhibition
PC (g)
PC + Ly (g)
(%)
PC + Ly + PPD (g)
(%)
(%)
0-236 (0-014) 0-241 (0-012) 0-233 (0 010) 0230 (0009) 0-241 (0007) 0-233 (0010) 0-266 (0-013) 0-240 (0-012)
0-072 (0 011) 0-084 (0-010) 0-076 (0-010) 0-063 (0008) 0-088 (0'009) 0-076 (0-010) 0 074 (0 009) 0-068 (0 010)
69 5 650 67-0 730 63-0 67-0 67-0 72-0
0 073 (0 007) 0086 (0009) 0 073 (0-007)
69-0 640 69-0 79.5* 63-0 67-0 66-0 70-0
65 0 62-0 61-0 750 605 63-0 0 0
0047 (0007) 0-089 (0008) 0077 (0009) 0-076 (0 009) 0-072 (0 009)
* Difference between PC+ Ly inhibition and PC+ Ly+ PPD inhibition significant (P< 0001). In each test n= 6 or n= 12.
0 25 0.20
PC + Ly Mixed lymphocyte reaction
0-15 _-\
0-10 -
a
0-05 _
I 2
0
3
4
5
6
Human lymphocytes 025L
8
lo
9
(%)
PC
0.20 C)
7
-1
05__ PC + Ly 10% 010O F
E
005
o0
(ii )
U)
e
0
10
100 (rod)
1000
y -Irradiation
0-25L
PC* +
0-20
0.15
Im W
0-10 _ 0-05 0
Ly 10% +M
sl IO 20 m -Antigen
50 (280 ug/ml ) (%)
100
FIG. 2. Titrations of respectively the mixed lymphocyte reaction with varying percentages of human lymphocytes (i) and varying intensity of irradiation (ii). In (iii) the migration in the presence of various antigen concentrations is shown. w: Weak lymphocyte sensitization to muscle antigen in a carrier of a muscular dystrophy; s: strong sensitization to muscle antigen in a muscular dystrophy patient.
30
A. W. Teelken & J. M. Minderhoud
In two other experiments the degree of suppression of the MLR by y-irradiation was tested. PC were just given a sham irradiation or they were irradiated with respectively 10, 100 and 1000 rad. The migration of those PC decreased a little by irradiation (in contrast with the results in the larger series; Fig. 1(i) and (ii)). When 10% human lymphocytes was added an inhibition of migration of the non-irradiated PC and of the PC irradiated with 10 rad was found. By irradiation with 100 rad the MLR was totally suppressed (Fig. 2(ii)). Experiments with muscle antigen (M) and encephalitogenic factor (Ef) In order to avoid artefacts caused by different numbers of cells or non-specific influence of antigen, the migration of irradiated macrophages in the presence of human lymphocytes and antigen was compared with the migration of macrophages also in the presence of human lymphocytes and the same antigen after y-irradiating the PC and the lymphocytes together. The mean area of migration in the presence of irradiated human lymphocytes (of patients with a disease of the central or peripheral nervous system) and muscle antigen or Ef did not differ from the mean area of migration of irradiated PC which were pooled with nonirradiated human lymphocytes without antigen (mean 0-269 g; n=32; s.d. 0 010 g; Fig. 1(viii)). There was only a slight increase of migration compared with the migration of irradiated or not irradiated PC alone. This increase was not significant. So y-irradiation seemed to abolish the interaction between heterogeneic lymphocytes or between antigen and human lymphocytes without influencing the migrating activity of macrophages. Calculating the percentage inhibition of migration in experiments with non-irradiated lymphocytes and as a control, with irradiated lymphocytes, gave a good impression of the cellular hypersensitivity to antigens. The amount of cellular hypersensitivity differs from one person to the other. In Fig. 2(iii) a titration of the test is shown in two patients with muscle disease, one with an intensive and one with a weaker cellular hypersensitivity to muscle antigen, in which the antigen concentration as parts of the concentration normally used (280 pg/ml) is changed. With the described method fifty-five persons, that is twenty-one patients with a disease or lesion of the central nervous system, fifteen patients with a peripheral nerve disease and nineteen patients with a muscle disease, and fifteen controls were tested. The results are shown in Table 2. TABLE 2. Summary of the results obtained in a pilot study in which normals and patients with a disease of the central nervous system (CNS), the peripheral nervous system (PNS) or a muscle disease were examined on cellular hypersensitivity to muscle antigen (M) and myelin antigen (Ef). Results expressed as the percentage of macrophage migration inhibition
Ef
Normals CNS PNS Muscle disease
M
(n= 15) 1-1% (-l10%/+ 11y) I-0% (-10%/+ 10%) 7-1% (-4%/32%) (n=21) 16 7% (1%/50%) (n= 15) 36-5% (0%y/68%Y) 290%(4%/60%) (n= 19) 5-3% (-10%/29%) 43W1% (12%/88%)
The majority of the patients with diseases of the central nervous system were found to be sensitized against Ef; the majority of the patients with a disease of the peripheral nervous system were sensitized to Ef and to muscle antigen and all the patients with a muscle disease showed a cellular hypersensitivity to muscle antigen. In later studies we also used peripheral Ef-antigen, which gave better results than muscle antigen or Ef in patients with a lesion of the peripheral nervous system.
A direct macrophage migration inhibition test
31
The results of this direct macrophage migration inhibition test in various neurological diseases are described elsewhere (Minderhoud & Teelken, 1974; Minderhoud et al., 1974).
DISCUSSION Bartfield & Atoynaten (1971) and Rajapakse & Glynn (1970) were not able to show a clear MLR between two histoincompatible lymphocyte suspensions. Using their more sensitive micro-modification of the macrophage migration inhibition test Hughes & Paty (1972) were able to find a MLR after incubation during 16 hr at 350C. This MLR could be suppressed by y-irradiation. If in our experiments non-irradiated peritoneal exudate cells mixed with 1000 human lymphocytes were cultured in the presence of PPD no additional inhibition could be detected in five of the six patients with a positive skin test to tuberculin. In only one could additional migration inhibition be detected in the presence of antigen. Only this person could be scored positive in a method using non-irradiated macrophages instead of six who actually were positive to tuberculin. The advantage of the micro-method which has an increased sensitivity compared with the method of Thor et al. (1968) and Rocklin et al. (1970) is in particular the short period of incubation necessary to detect lymphocytelymphocyte interaction or lymphocyte-antigen interaction. This diminishes the necessity of absolute sterility and probably induces the possibility of sufficient y-irradiation without influencing the migrating abilities of macrophages. Visakorpi (1974) and others found that antigen-induced migration inhibition in guinea-pigs sensitized with albumin in Freund's adjuvant was still detectable after an in vitro irradiation of 5000 or 10,0C0 rad; in the investigations of Hughes & Paty (1972) and in our investigations using the micro-method, human lymphocytes and PPD, Ef or M-antigen, y-irradiation of 100 rad proved to be sufficient to suppress both the MLR and the antigen-human lymphocyte interaction, during the 18 hr of culture. Performed in this way the macrophage migration inhibition test is found to be sensitive and useful in detection of cellular hypersensitivity in man. Hughes & Paty (1972) found some deleterious effect on the migration of macrophages caused by the 100 rad y-irradiation. In their study the migration was 9-700 less than the migration of non-irradiated PC. In our experiments no reduction of the migration caused by the y-irradiation could be found (0-242 g; s.d. 0-032, versus 0-240 g; s.d. 0-019). A slight increase of migration was found in experiments in which human lymphocytes were pooled with irradiated PC (mean 0-260 g; s.d. 0-012 g), which was probably caused by a minimal one-way lymphocyte reaction. But this slight increase of migration was also found when irradiated PC and irradiated human lymphocytes of patients were incubated in nutrient medium containing antigen (mean 0-269 g; s.d. 0-010g). In order to avoid as many artefacts as possible we included in each test at least one or two controls: the migration of irradiated macrophages pooled with the human lymphocytes (Fig. l(v)), or mostly the migration of macrophages irradiated after pooling with the human lymphocytes and incubated in the presence of one of the antigens (Fig. l(viii)). The antigen-human lymphocyte interaction was measured as the percentage of inhibition of migration. This method proved to be successful in a pilot study in which lymphocytes of patients with different neurological diseases were examined. The results indicated that in diseases of muscle or the nervous system a cellular hypersensitivity against probable break-down products of these tissues is present and that this reaction can be measured by the described MMI test. We should like to thank Dr S. Kiestra for his technical advice about the y-irradiation and the technical staff of the Department of Radiotherapy (Head: Professor Dr H. C. Stam) for their assistance in the irradiation. We are also very grateful for the assistance of Miss M. J. F. van den Ouweland and Miss D. A. Woldendorp in taking blood samples and for their help in separating cells. And last but not least our gratitude goes to Mr G. Loof who performed the MMI test without knowing anything about the patients who were tested and about the results that could be expected.
32
A. W. Teelken & J. M. Minderhoud REFERENCES
BARTFIELD, H. & ATOYNATAN, T. (1971) Activities and properties of macrophage migration inhibitory factor produced by mixed lymphocyte cultures. Nature: New Biology, 230, 246. BOYUM, A. (1968) Separation of leucocytes from blood and bone marrow. Scand. J. clin. Lab. Invest. 21, supplement 97. DAVID, J.R., AL-ASKARI, S., LAWRENCE, H.S. & THOMAS, L. (1964) Delayed hypersensitivity in vitro. I. The specificity of inhibition of cell migration by antigens. J. Immunol. 93, 264. DEIBLER, G.E., MARTENSON, R.E. & KIES, M.W. (1972) Large scale preparation of myelin basic protein from central nervous tissue of several mammalian species. Preparative Biochemistry, 2, 139. HUGHES, D. (1972) Macrophage migration inhibition test: a critical examination of the technique using a polythene capillary tubing micromethod. J. immunol. Methods, 1, 403. HUGHES, D. & PATY, D.W. (1972) Macrophage migration inhibition test applied to delayed hypersensitivity in man. Z. ImmunForsch. 142, 478. MINDERHOUD, J.M. & TEELKEN, A.W. (1974) Cellular hypersensitivity to myelin and muscle tissue in man examined with a direct macrophage
migration inhibition test. I. Diseases of the central and peripheral nervous system. Clin. Neurol. Neurosurg. 77, 225. MINDERHOUD, J.M., TEELKEN, A.W., LEENSTRABORSJE, H. & OOSTEN, J.K. (1974) Cellular hypersensitivity to myelin and muscle tissue in man examined with a direct macrophage migration inhibition test. II. Muscle diseases. Clin. Neurol. Neurosurg. 77, 234. RAJAPAKSE, D.A. & GLYNN, L.E. (1970) Macrophage migration inhibition test using guinea-pig macrophages and human lymphocytes. Nature (Lond.), 226, 857. ROCKLIN, R.E., MEYERS, O.L. & DAVID, J.R. (1970) An in vitro assay for cellular hypersensitivity in man. J. Immunol. 104, 95. S0BORG, M. & BENDIXEN, G. (1967) Human lymphocyte migration as a parameter of hypersensitivity. Acta med. scand. 181, 247. THOR, D.E., JURERIZ, R.E., VEACH, S.R., MILLER, E. & DRAY, S. (1968) Cell migration inhibition factor released by antigen from human peripheral lymphocytes. Nature (Lond.), 219, 755. VISAKORPI, R. (1974) The effect of irradiation on macrophage migration inhibition. Immunology, 27, 145.
