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A comparison of the direct and indirect leukocyte migration tests in man
Journal of Immunological Methods, 10 (1976) 133--141 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
A COMPARISON OF THE DIRECT AND INDIRECT LEUKOCYTE MIGRATION TESTS IN MAN
R.J. W A R R I N G T O N *, S.K. B U E H L E R and K.B. ROBERTS
Division of Cell Studies, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada (Received 23 June 1975, accepted 14 July 1975)
In the Direct Leukocyte Migration Test, as few as 10% mononuclear cells are sufficient to produce antigen-specific inhibition of polymorphonuclear leukocyte migration in the presence of 100 pg of excipient-free PPD per ml. A clear distinction between tuberculinpositive and tuberculin-negative donors can be made. The inhibition of migration is blocked by the presence of actinomycin D. In the Indirect Test, significant inhibition of migration of pure polymorphs is produced by supernatants from stimulated tuberculinsensitive lymphocyte cultures. Inhibition is maximal at 6 h and is of a similar degree to that obtained in the direct test.
INTRODUCTION
The Leukocyte Migration Test (LMT), originally introduced by SCborg and Bendixen in 1967 and modelled on the Migration Inhibition Factor (MIF) assay of David et al. (1964) has been used increasingly in clinical medicine as an in vitro test of immunity. In the test, the migration of peripheral blood leukocytes from sensitised donors is inhibited in the presence of specific antigen. It has been more recently shown that the polymorphonuclear leukocytes respond to a factor produced by sensitised mononuclear cells stimulated by specific antigen (Clausen, 1973; Warrington, 1973; Rocklin, 1974; Weisbart et al., 1974) and detectable in the lymphocyte culture supernatants. The leukocyte inhibitory factor (LIF) elutes on column chromatography with albumin and is different from MIF (Rocklin, 1974). In addition, a migration enhancing factor has been described by Weisbart et al. (1974), also produced by stimulated sensitised mononuclear cells. Variability in results using the Leukocyte Migration Test and problems with its interpretation may be due to a number of factors e.g. variations in the proportions of mononuclear and polymorpholeukocytes used in the test, the use of toxic antigen preparations, the interaction of migration inhibition * Present address: Department of Clinical Immunology, Health Sciences Centre, 700, William Avenue, Winnipeg, Manitoba, R3E OZ3.
134 and enhancing factors and the possible involvement of a n t i g e n - a n t i b o d y complexes. In the following paper, a standardised direct leukocyte migration test using constant proportions of mononuclear to polymorphonuclear cells is compared with the indirect test, using purified polymorphonuclear leukocytes exposed to supernatants from stimulated l y m p h o c y t e cultures. MATERIALS AND METHODS
Donors Venous blood samples were obtained from normal healthy human donors, who were subsequently tested for tuberculin sensitivity with intradermal PPD (Connaught Laboratories, Toronto, Canada). Tuberculin-negative donors failed to show e r y t h e m a and induration at 48 h with the m a x i m u m dose of 250 T.U.
Preparation of cells Peripheral venous blood, anticoagulated with 2.7% EDTA (pH 7.4, 1 to 20 ml of blood) was diluted with an equal volume of isotonic saline. Mononuclear cells were separated on a cushion of 10 parts 37.5% Hypaque (sodium diatrizoate, Winthrop Laboratories, Aurora, Canada) and 24 parts 9% Ficoll (Pharmacia, Uppsala, Sweden) according to the m e t h o d of BCyum (1967). After washing with Hank's solution, lymphocytes comprised 92.6% (S.D. _+ 4.0) of the mononuclear cells (viability by trypan blue exclusion >99%), with phagocytic m o n o c y t e s making up the remainder. Polymorphonuclear leukocytes were prepared by diluting the red cell/ polymorph fraction with an equal volume of diluted plasma (retained from the original separation) and 6% dextran 110 (Pharmacia), 1 ml to 5 ml of the original fraction. This was left to sediment at a 45 ° angle at 4°C for 1--2 h. The leukocyte-rich supernatant was then removed and the cells were washed with Hank's solution. Red cell contamination was removed by osmotic lysis with 1 ml of distilled water for 15 sec and then an excess of Eagle's MEM containing 20% fetal calf serum, heat inactivated at 56°C for 30 min (HFCS) was added. Polymorphs comprised 98.7% (S.D. + 1.0) of the final cells (viability >90%) with lymphocytes making up the remainder.
Culture conditions Mononuclear cells were cultured at a cell concentration of 2.5 × 106 cells/ml in 2 ml of RPMI 1640 containing 20% HFCS, penicillin 100 units/ml and streptomycin 100 pg/ml. Cultures were stimulated with excipient-free PPD (Parke, Davis & Co., Montreal, Canada) in Hepes-buffered Medium 199 at concentrations of 2.5 pg and 10 pg/ml. Control cultures received an equal
135 volume (0.1 ml) of Medium 199. Cells were incubated in sterile plastic tubes (Falcon Plastics) in duplicate cultures at 37°C in 5% COs and air. At 24 h 1 ml of supernatant was removed from each culture and duplicate samples pooled and stored at - 20°C. Fresh warm RPMI 1640 with the appropriate a m o u n t of antigen and 20% HFCS was added back to the tubes, which were returned to the incubator. At 72 h, a further 1 ml of supernatant was removed from each culture, duplicates pooled and stored at --20 ° C. To assess the effects of inhibition of RNA synthesis, 1 pM actinomycin D (final concentration) (Sigma Chemical Company, St. Louis, Missouri) was added to stimulated and control cultures. Stimulation was assessed with uridine-6-3H (New England Nuclear, 25.3 c/mM) 1 pCi/ml of culture was added to each tube in 0.05 ml after three days culture. Cells were exposed to isotope for 2 h at 37°C and then harvested, using three washes of ice-cold isotonic saline, precipitation of nuclear protein with cold 5% TCA and two washes with 95% methanol. After drying~ precipitates were solubilized with NCS Tissue Solubilizer (Amersham/Searle Corporation). and radioactivity was assessed in a Nuclear Chicago Mark I counter, using Spectrofluor PPO (Amersham/Searle).
The Leukocyte Migration Test Initially, preseparated mononuclear cells were mixed with polymorphs in various proportions. However subsequently the test was standardized using 10% mononuclear cells and 90% polymorphs in the direct LMT and 'pure' polymorphs in the indirect LMT. Cell suspensions were prepared at a concentration of 30 X 106 cells/ml in Hepes-buffered Eagle's MEM containing 20% HFCS and were kept cold in ice. Capillary tubes (Non-heparinised Microhematocrit, L.D. 1 . 1 - 1 . 2 MM, Fisherbrand) were filled with the cell suspension and sealed at one end with vinyl plastic p u t t y . After spinning at 130 g for 2 min, tubes were cut at the interface and placed in Mackaness-type chambers (Disposable ' L e x y ' Culture Chambers, Mini-Lab, Duvernay, Laval, Quebec, Canada), two capillaries in each chamber, held in place with silicone grease. After sealing on a sterile coverslip with paraffin wax, the chambers were filled. In the direct LMT, a 1 : 1 mixture of Hepes-buffered MEM and 199 with penicillin 100 units/ml and streptomycin 100 pg/ml, 20% HFCS and excipient-free PPD, 10 pg/ml or 100 pg/ml was used. Control chambers were filled with medium and serum alone. Where the effects of a metabolic inhibitor on the antigen-specific migration inhibition was assessed, the medium contained, in addition, 1 pM actinomycin D. In the indirect LMT, stored supernatants from stimulated and control mononuclear cell cultures were taken and PPD was added to give equal concentrations in all samples i.e. a m a x i m u m of 10 pg PPD/ml. These were added to the chambers containing pure polymorphs in the capillary tubes i.e. w i t h o u t added mononuclear cells. These cells were obtained from donors
136
irrespective of tuberculin sensitivity, since the migration of 'pure' polymorphs from tuberculin-positive donors was not inhibited by up to 100 pg PPD/ml (see Results). Chambers were sealed with paraffin wax and incubated at 37°C for a total of 18 h. The area of migration was assessed at 6 h and at 18 h, by projection using a Projection microscope onto a sheet of stiff paper, 0.4 mm thick, and the whole fan was outlined. The area of migration was subsequently cut out and weighed. The results were expressed as: X % migration = ~ X 100 Where X = the weight of the area of migration in the presence of antigen or in supernatants from antigen-stimulated cultures; Y = the weight of the area of migration in the absence of antigen or in supernatants from control (unstimulated) cultures. RESULTS
Effects of variation-in-ratio of mononuclear cells to polymorphs in the direct LMT Experiments in which the proportion of mononuclear to polymorphonuclear leukocytes used in the test were varied are shown in Fig. 1. It was f o u n d that whereas 'pure' mononuclear cells and 'pure' polymorphonuclear cells from tuberculin-positive donors were n o t affected in their migration by specific antigen, mixtures of the two cell types were inhibited. Since it appeared that approximately 10% of mononuclear cells produced a maximal I00
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Fig. 1. T h e % m i g r a t i o n in t h e p r e s e n c e of 100 pg P P D / m l for r e c o n s t i t u t e d m i x t u r e s o f p o l y m o r p h s a n d m o n o n u c l e a r cells in various p r o p o r t i o n s f r o m t u b e r c u l i n - s e n s i t i v e don o r s ( s y m b o l s i n d i c a t e t h r e e d i f f e r e n t e x p e r i m e n t s ) . Results are t h e m e a n o f d u p l i c a t e assays.
137
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Fig. 2. The % migration for leukocytes from tuberculin-positive (closed circles) and tuberculin-negative (open squares) donors in the presence of PPD in the direct LMT. Results are the mean of duplicate assays. The position of t = -+3 standard deviations from the mean for tuberculin-negative donors in the presence of 100 pg PPD/ml is indicated. degree o f inhibition, this p r o p o r t i o n was used in the direct LMT in subseq u e n t experiments. In addition, in the indirect LMT, pure p o l y m o r p h s could be used irrespective of the tuberculin-sensitivity of the donor.
The direct L M T in tuberculin-positive and tuberculin-negative donors Th e migration of 10% m o n o n u c l e a r / 9 0 % p o l y m o r p h cell mixtures prepared f r o m d o n or s o f u n k n o w n cutaneous tuberculin reactivity were examined in the direct LMT in the presence of PPD 10 pg/ml and 100 pg/ml and in its absence. The results are indicated in fig. 2 and are here expressed with regard to subsequently determined cutaneous sensitivity. When the cells from tuberculin-positive donor s were exposed to 100 pg PPD/ml, the mean migration as a percentage of c ont r ol was 68% (S.D. -+ 7) whereas for tuberculin-negative donors the migration was 98% (S.D. -+ 4). These results differ significantly (P < 0.001) by Student's t test. At the lower antigen dose, 10 pg PPD/ml, there was no difference between the migration of cells from
138
tuberculin-positive and negative donors (94 + 14% and 98 + 13% respectively). The effect of actinomycin D on migration-inhibition It was found that 1 pM actinomycin D blocked PPD-specific inhibition of migration whilst reducing control migration only slightly (fig. 3). In the presence of 100 pg PPD/ml, mean migration was 56% (S.D. + 15) whilst in the presence of actinomycin D and PPD, migration was 106% (S.D. +_ 22). These results differ significantly (P < 0.001). The area of migration of control cells was reduced slightly by the actinomycin D, to 90% (S.D. +_ 12) but was not significantly different from control. In l y m p h o c y t e cultures with PPD, 1 pM actinomycin D reduced uridine-6 -3 H incorporation from 10 × 103 dpm/106 cells/h to 19 dpm/106 cells/h. A comparison of the direct and indirect LMT using cells from tuberculinpositive and tuberculin-negative donors In the direct LMT, significant inhibition of migration of the mononuclear/
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Actinomycin 1,tim
Fig. 3. T h e % m i g r a t i o n o f l e u k o c y t e s for t u b e r c u l i n - p o s i t i v e d o n o r s in t h e d i r e c t L M T in t h e p r e s e n c e o r a b s e n c e o f 1 0 0 p g P P D / m l a n d 1 p M a c t i n o m y c i n D. R e s u l t s are t h e m e a n o f d u p l i c a t e assays.
139
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INOIRECT LEUKOCYTE MIGRATION TEST
Fig. 4. T h e % migration o f l e u k o c y t e s from tuberculin-positive and tuberculin-negative d o n o r s in the direct and indirect LMT. T h e direct test was carried o u t w i t h 1 0 0 pg PPD/ ml. T h e indirect test w a s u n d e r t a k e n w i t h supernatants from PPD s t i m u l a t e d ( A = 2.5 p g / m l ; B = 1 0 p g / m l ) and c o n t r o l cultures. H o r i z o n t a l lines indicate t = + 3 S.D. for tuberculin-negative donors. Results are the m e a n o f duplicate assays. Short bars indicate mean for tuberculin-positive and tuberculin-negative donors.
polymorph mixtures occurred in the presence of 100 pg PPD/ml (fig. 4) whereas inhibition of cells from tuberculin-negative donors was not seen. When 24 h supernatants from stimulated lymphocyte cultures were assessed, using the indirect LMT and pure polymorphs as indicator cells, it was found that significant inhibition of migration occurred only with supernatants from cultures of tuberculin-positive donor lymphocytes stimulated with PPD. The degree of inhibition as measured at 6 h was similar to that seen in the direct test, but by 24 h, inhibition of migration was less marked. There thus appeared to be some degree of reversal of the migration inhibition. With 72 h culture supernatants, there was no longer a clear cut difference between tuberculin-positive and tuberculin-negative donors, although in some supernatants, inhibitory activity was still present. Other supernatants appeared to enhance migration. DISCUSSION In the present series of experiments, it was found that purified polymorphs, as well as mononuclear cells, were not inhibited in their migration by the presence of up to 100 pg of excipient-free PPD/ml. This lack of sen-
140 sitivity of purified cell preparations has been c o m m e n t e d on previously (S~borg, 1969; Clausen, 1970; Lockshin et al., 1973). However, as little as 10% of mononuclear cells added to the pure polymorphs resulted in migration inhibition provided that the mononuclear cells were obtained from a tuberculin-positive donor. Since migration inhibition appears to decrease with increasing proportions of mononuclear cells, and in b u f f y coat preparations the proportion of polymorphs to mononuclear cells varies considerably, it would appear advisable to control the cell proportions used in the direct LMT. Using 10% mononuclear and 90% polymorphonuclear cells it was possible to clearly discriminate between tuberculin-positive and tuberculin-negative individuals using an antigen concentration of PPD 100 pg/ml. The abolition of antigen-specific migration inhibition with 1 pM actinomycin D indicates that the inhibition was due to active synthesis of some inhibitory factor rather than the result of a direct toxic effect of the PPD. The advantage of actinomycin D over puromycin as used by Mitchell et al. (1972) and Maini et al. (1973) is that the normal movement of leukocytes in the controls is not significantly affected. A comparison of the direct and indirect LMT in a group of tuberculinpositive and tuberculin-negative donors indicated that there was a good correlation between the migration inhibition in the direct test and that occurring with 24 h l y m p h o c y t e culture supernatants, provided that migration was assessed at 6 h. However by 24 h, the degree of inhibition was in some instances reduced significantly. A similar finding has been reported in the direct LMT b y Brostoff (1973). This would tend to suggest that the migration inhibition is reversible, perhaps due to inactivation of the inhibitory factor. By 72 h, the culture supernatants from stimulated tuberculin-positive donor l y m p h o c y t e s no longer consistently contain inhibitory activity. Indeed, some supernatants now appear to enhance migration of the polymorphs. Thus, as Weisbart et al. (1974) have indicated, both migration inhibitory and enhancing factors may be found in l y m p h o c y t e culture supernatants, the result of the LMT depending u p o n the predominant factor formed. However, in the present series of experiments, it would appear that the inhibitory factor predominates in 24 h cultures, the period over which the direct LMT is carried out. Both these factors would appear to be low molecular weight lymphokines as indicated by Rocklin (1974) and Weisbart et al. (1974). In their studies inhibitory activity was associated with the albumin fraction and n o t with 7-globulins. Since the active supernatants may be assayed for inhibitory activity on purified homologous polymorphs, and may be stored for some weeks, the indirect assay may be useful in situations where insufficient polymorphs prevent the use of the direct test, because of excessive proportions of mononuclear cells e.g. in drug-induced or other forms of neutropenia, or where it is important to exclude the presence of a n t i g e n - a n t i b o d y complexes by fractionation studies.
141 ACKNOWLEDGEMENTS T h e a u t h o r s gratefully a c k n o w l e d g e the assistance o f Ms. P e n n y H a n s o n a n d Ms. D e b b i e H y a m . T h e w o r k was s u p p o r t e d in part b y a g r a n t f r o m T h e C a n a d i a n T u b e r c u l o s i s and R e s p i r a t o r y Diseases F o u n d a t i o n .
REFERENCES BSyum, A., 1968, Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 1. Brostoff, J., 1974, J. Immunol. Methods 4, 27. Clausen, J.E., 1970, Acta. Med. Scand. 188, 59. Clausen, J.E., 1973, J. Immunol. 110,546. David, J., S. A1-Askari, H.S. Lawrence and L. Thomas, 1964, J. Immunol. 93, 264. Lockshin, M.D., J. Waxman and M.W. Jenkins, 1973. Experientia 29, 340. Maini, R.N., L.M. Roffe, I.T. Magrath and D.C. Dumonde, 1973, Int. Arch. Allergy 45, 308. Mitchell, C.G., M.G.M. Smith, P.L. Golding, A.L.W.F. Eddleston and R. Williams, 1972, Clin. Exp. Immunol. 11,535. Rocklin, R.E., 1974, J. Immunol. 112, 1461. S~bborg, M., 1969, Acta Med. Scand. 185,221. S~bborg, M. and G. Bendixen, 1967, Acta. Med. Scand. 181,247. Warrington, R.J., 1973, Ph.D. Thesis Memorial University of Newfoundland. Weisbart, R.H., R. Bluestone, L.S. Goldberg and C.M., Pearson, 1974, Proc. Nat. Acad. Sci. U.S. 71,875.
A COMPARISON OF THE DIRECT AND INDIRECT LEUKOCYTE MIGRATION TESTS IN MAN
R.J. W A R R I N G T O N *, S.K. B U E H L E R and K.B. ROBERTS
Division of Cell Studies, Faculty of Medicine, Memorial University of Newfoundland, St. John's, Newfoundland, Canada (Received 23 June 1975, accepted 14 July 1975)
In the Direct Leukocyte Migration Test, as few as 10% mononuclear cells are sufficient to produce antigen-specific inhibition of polymorphonuclear leukocyte migration in the presence of 100 pg of excipient-free PPD per ml. A clear distinction between tuberculinpositive and tuberculin-negative donors can be made. The inhibition of migration is blocked by the presence of actinomycin D. In the Indirect Test, significant inhibition of migration of pure polymorphs is produced by supernatants from stimulated tuberculinsensitive lymphocyte cultures. Inhibition is maximal at 6 h and is of a similar degree to that obtained in the direct test.
INTRODUCTION
The Leukocyte Migration Test (LMT), originally introduced by SCborg and Bendixen in 1967 and modelled on the Migration Inhibition Factor (MIF) assay of David et al. (1964) has been used increasingly in clinical medicine as an in vitro test of immunity. In the test, the migration of peripheral blood leukocytes from sensitised donors is inhibited in the presence of specific antigen. It has been more recently shown that the polymorphonuclear leukocytes respond to a factor produced by sensitised mononuclear cells stimulated by specific antigen (Clausen, 1973; Warrington, 1973; Rocklin, 1974; Weisbart et al., 1974) and detectable in the lymphocyte culture supernatants. The leukocyte inhibitory factor (LIF) elutes on column chromatography with albumin and is different from MIF (Rocklin, 1974). In addition, a migration enhancing factor has been described by Weisbart et al. (1974), also produced by stimulated sensitised mononuclear cells. Variability in results using the Leukocyte Migration Test and problems with its interpretation may be due to a number of factors e.g. variations in the proportions of mononuclear and polymorpholeukocytes used in the test, the use of toxic antigen preparations, the interaction of migration inhibition * Present address: Department of Clinical Immunology, Health Sciences Centre, 700, William Avenue, Winnipeg, Manitoba, R3E OZ3.
134 and enhancing factors and the possible involvement of a n t i g e n - a n t i b o d y complexes. In the following paper, a standardised direct leukocyte migration test using constant proportions of mononuclear to polymorphonuclear cells is compared with the indirect test, using purified polymorphonuclear leukocytes exposed to supernatants from stimulated l y m p h o c y t e cultures. MATERIALS AND METHODS
Donors Venous blood samples were obtained from normal healthy human donors, who were subsequently tested for tuberculin sensitivity with intradermal PPD (Connaught Laboratories, Toronto, Canada). Tuberculin-negative donors failed to show e r y t h e m a and induration at 48 h with the m a x i m u m dose of 250 T.U.
Preparation of cells Peripheral venous blood, anticoagulated with 2.7% EDTA (pH 7.4, 1 to 20 ml of blood) was diluted with an equal volume of isotonic saline. Mononuclear cells were separated on a cushion of 10 parts 37.5% Hypaque (sodium diatrizoate, Winthrop Laboratories, Aurora, Canada) and 24 parts 9% Ficoll (Pharmacia, Uppsala, Sweden) according to the m e t h o d of BCyum (1967). After washing with Hank's solution, lymphocytes comprised 92.6% (S.D. _+ 4.0) of the mononuclear cells (viability by trypan blue exclusion >99%), with phagocytic m o n o c y t e s making up the remainder. Polymorphonuclear leukocytes were prepared by diluting the red cell/ polymorph fraction with an equal volume of diluted plasma (retained from the original separation) and 6% dextran 110 (Pharmacia), 1 ml to 5 ml of the original fraction. This was left to sediment at a 45 ° angle at 4°C for 1--2 h. The leukocyte-rich supernatant was then removed and the cells were washed with Hank's solution. Red cell contamination was removed by osmotic lysis with 1 ml of distilled water for 15 sec and then an excess of Eagle's MEM containing 20% fetal calf serum, heat inactivated at 56°C for 30 min (HFCS) was added. Polymorphs comprised 98.7% (S.D. + 1.0) of the final cells (viability >90%) with lymphocytes making up the remainder.
Culture conditions Mononuclear cells were cultured at a cell concentration of 2.5 × 106 cells/ml in 2 ml of RPMI 1640 containing 20% HFCS, penicillin 100 units/ml and streptomycin 100 pg/ml. Cultures were stimulated with excipient-free PPD (Parke, Davis & Co., Montreal, Canada) in Hepes-buffered Medium 199 at concentrations of 2.5 pg and 10 pg/ml. Control cultures received an equal
135 volume (0.1 ml) of Medium 199. Cells were incubated in sterile plastic tubes (Falcon Plastics) in duplicate cultures at 37°C in 5% COs and air. At 24 h 1 ml of supernatant was removed from each culture and duplicate samples pooled and stored at - 20°C. Fresh warm RPMI 1640 with the appropriate a m o u n t of antigen and 20% HFCS was added back to the tubes, which were returned to the incubator. At 72 h, a further 1 ml of supernatant was removed from each culture, duplicates pooled and stored at --20 ° C. To assess the effects of inhibition of RNA synthesis, 1 pM actinomycin D (final concentration) (Sigma Chemical Company, St. Louis, Missouri) was added to stimulated and control cultures. Stimulation was assessed with uridine-6-3H (New England Nuclear, 25.3 c/mM) 1 pCi/ml of culture was added to each tube in 0.05 ml after three days culture. Cells were exposed to isotope for 2 h at 37°C and then harvested, using three washes of ice-cold isotonic saline, precipitation of nuclear protein with cold 5% TCA and two washes with 95% methanol. After drying~ precipitates were solubilized with NCS Tissue Solubilizer (Amersham/Searle Corporation). and radioactivity was assessed in a Nuclear Chicago Mark I counter, using Spectrofluor PPO (Amersham/Searle).
The Leukocyte Migration Test Initially, preseparated mononuclear cells were mixed with polymorphs in various proportions. However subsequently the test was standardized using 10% mononuclear cells and 90% polymorphs in the direct LMT and 'pure' polymorphs in the indirect LMT. Cell suspensions were prepared at a concentration of 30 X 106 cells/ml in Hepes-buffered Eagle's MEM containing 20% HFCS and were kept cold in ice. Capillary tubes (Non-heparinised Microhematocrit, L.D. 1 . 1 - 1 . 2 MM, Fisherbrand) were filled with the cell suspension and sealed at one end with vinyl plastic p u t t y . After spinning at 130 g for 2 min, tubes were cut at the interface and placed in Mackaness-type chambers (Disposable ' L e x y ' Culture Chambers, Mini-Lab, Duvernay, Laval, Quebec, Canada), two capillaries in each chamber, held in place with silicone grease. After sealing on a sterile coverslip with paraffin wax, the chambers were filled. In the direct LMT, a 1 : 1 mixture of Hepes-buffered MEM and 199 with penicillin 100 units/ml and streptomycin 100 pg/ml, 20% HFCS and excipient-free PPD, 10 pg/ml or 100 pg/ml was used. Control chambers were filled with medium and serum alone. Where the effects of a metabolic inhibitor on the antigen-specific migration inhibition was assessed, the medium contained, in addition, 1 pM actinomycin D. In the indirect LMT, stored supernatants from stimulated and control mononuclear cell cultures were taken and PPD was added to give equal concentrations in all samples i.e. a m a x i m u m of 10 pg PPD/ml. These were added to the chambers containing pure polymorphs in the capillary tubes i.e. w i t h o u t added mononuclear cells. These cells were obtained from donors
136
irrespective of tuberculin sensitivity, since the migration of 'pure' polymorphs from tuberculin-positive donors was not inhibited by up to 100 pg PPD/ml (see Results). Chambers were sealed with paraffin wax and incubated at 37°C for a total of 18 h. The area of migration was assessed at 6 h and at 18 h, by projection using a Projection microscope onto a sheet of stiff paper, 0.4 mm thick, and the whole fan was outlined. The area of migration was subsequently cut out and weighed. The results were expressed as: X % migration = ~ X 100 Where X = the weight of the area of migration in the presence of antigen or in supernatants from antigen-stimulated cultures; Y = the weight of the area of migration in the absence of antigen or in supernatants from control (unstimulated) cultures. RESULTS
Effects of variation-in-ratio of mononuclear cells to polymorphs in the direct LMT Experiments in which the proportion of mononuclear to polymorphonuclear leukocytes used in the test were varied are shown in Fig. 1. It was f o u n d that whereas 'pure' mononuclear cells and 'pure' polymorphonuclear cells from tuberculin-positive donors were n o t affected in their migration by specific antigen, mixtures of the two cell types were inhibited. Since it appeared that approximately 10% of mononuclear cells produced a maximal I00
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Fig. 1. T h e % m i g r a t i o n in t h e p r e s e n c e of 100 pg P P D / m l for r e c o n s t i t u t e d m i x t u r e s o f p o l y m o r p h s a n d m o n o n u c l e a r cells in various p r o p o r t i o n s f r o m t u b e r c u l i n - s e n s i t i v e don o r s ( s y m b o l s i n d i c a t e t h r e e d i f f e r e n t e x p e r i m e n t s ) . Results are t h e m e a n o f d u p l i c a t e assays.
137
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100
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Fig. 2. The % migration for leukocytes from tuberculin-positive (closed circles) and tuberculin-negative (open squares) donors in the presence of PPD in the direct LMT. Results are the mean of duplicate assays. The position of t = -+3 standard deviations from the mean for tuberculin-negative donors in the presence of 100 pg PPD/ml is indicated. degree o f inhibition, this p r o p o r t i o n was used in the direct LMT in subseq u e n t experiments. In addition, in the indirect LMT, pure p o l y m o r p h s could be used irrespective of the tuberculin-sensitivity of the donor.
The direct L M T in tuberculin-positive and tuberculin-negative donors Th e migration of 10% m o n o n u c l e a r / 9 0 % p o l y m o r p h cell mixtures prepared f r o m d o n or s o f u n k n o w n cutaneous tuberculin reactivity were examined in the direct LMT in the presence of PPD 10 pg/ml and 100 pg/ml and in its absence. The results are indicated in fig. 2 and are here expressed with regard to subsequently determined cutaneous sensitivity. When the cells from tuberculin-positive donor s were exposed to 100 pg PPD/ml, the mean migration as a percentage of c ont r ol was 68% (S.D. -+ 7) whereas for tuberculin-negative donors the migration was 98% (S.D. -+ 4). These results differ significantly (P < 0.001) by Student's t test. At the lower antigen dose, 10 pg PPD/ml, there was no difference between the migration of cells from
138
tuberculin-positive and negative donors (94 + 14% and 98 + 13% respectively). The effect of actinomycin D on migration-inhibition It was found that 1 pM actinomycin D blocked PPD-specific inhibition of migration whilst reducing control migration only slightly (fig. 3). In the presence of 100 pg PPD/ml, mean migration was 56% (S.D. + 15) whilst in the presence of actinomycin D and PPD, migration was 106% (S.D. +_ 22). These results differ significantly (P < 0.001). The area of migration of control cells was reduced slightly by the actinomycin D, to 90% (S.D. +_ 12) but was not significantly different from control. In l y m p h o c y t e cultures with PPD, 1 pM actinomycin D reduced uridine-6 -3 H incorporation from 10 × 103 dpm/106 cells/h to 19 dpm/106 cells/h. A comparison of the direct and indirect LMT using cells from tuberculinpositive and tuberculin-negative donors In the direct LMT, significant inhibition of migration of the mononuclear/
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PPD 100,ug]ml ActinomycinI~uM
Actinomycin 1,tim
Fig. 3. T h e % m i g r a t i o n o f l e u k o c y t e s for t u b e r c u l i n - p o s i t i v e d o n o r s in t h e d i r e c t L M T in t h e p r e s e n c e o r a b s e n c e o f 1 0 0 p g P P D / m l a n d 1 p M a c t i n o m y c i n D. R e s u l t s are t h e m e a n o f d u p l i c a t e assays.
139
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INOIRECT LEUKOCYTE MIGRATION TEST
Fig. 4. T h e % migration o f l e u k o c y t e s from tuberculin-positive and tuberculin-negative d o n o r s in the direct and indirect LMT. T h e direct test was carried o u t w i t h 1 0 0 pg PPD/ ml. T h e indirect test w a s u n d e r t a k e n w i t h supernatants from PPD s t i m u l a t e d ( A = 2.5 p g / m l ; B = 1 0 p g / m l ) and c o n t r o l cultures. H o r i z o n t a l lines indicate t = + 3 S.D. for tuberculin-negative donors. Results are the m e a n o f duplicate assays. Short bars indicate mean for tuberculin-positive and tuberculin-negative donors.
polymorph mixtures occurred in the presence of 100 pg PPD/ml (fig. 4) whereas inhibition of cells from tuberculin-negative donors was not seen. When 24 h supernatants from stimulated lymphocyte cultures were assessed, using the indirect LMT and pure polymorphs as indicator cells, it was found that significant inhibition of migration occurred only with supernatants from cultures of tuberculin-positive donor lymphocytes stimulated with PPD. The degree of inhibition as measured at 6 h was similar to that seen in the direct test, but by 24 h, inhibition of migration was less marked. There thus appeared to be some degree of reversal of the migration inhibition. With 72 h culture supernatants, there was no longer a clear cut difference between tuberculin-positive and tuberculin-negative donors, although in some supernatants, inhibitory activity was still present. Other supernatants appeared to enhance migration. DISCUSSION In the present series of experiments, it was found that purified polymorphs, as well as mononuclear cells, were not inhibited in their migration by the presence of up to 100 pg of excipient-free PPD/ml. This lack of sen-
140 sitivity of purified cell preparations has been c o m m e n t e d on previously (S~borg, 1969; Clausen, 1970; Lockshin et al., 1973). However, as little as 10% of mononuclear cells added to the pure polymorphs resulted in migration inhibition provided that the mononuclear cells were obtained from a tuberculin-positive donor. Since migration inhibition appears to decrease with increasing proportions of mononuclear cells, and in b u f f y coat preparations the proportion of polymorphs to mononuclear cells varies considerably, it would appear advisable to control the cell proportions used in the direct LMT. Using 10% mononuclear and 90% polymorphonuclear cells it was possible to clearly discriminate between tuberculin-positive and tuberculin-negative individuals using an antigen concentration of PPD 100 pg/ml. The abolition of antigen-specific migration inhibition with 1 pM actinomycin D indicates that the inhibition was due to active synthesis of some inhibitory factor rather than the result of a direct toxic effect of the PPD. The advantage of actinomycin D over puromycin as used by Mitchell et al. (1972) and Maini et al. (1973) is that the normal movement of leukocytes in the controls is not significantly affected. A comparison of the direct and indirect LMT in a group of tuberculinpositive and tuberculin-negative donors indicated that there was a good correlation between the migration inhibition in the direct test and that occurring with 24 h l y m p h o c y t e culture supernatants, provided that migration was assessed at 6 h. However by 24 h, the degree of inhibition was in some instances reduced significantly. A similar finding has been reported in the direct LMT b y Brostoff (1973). This would tend to suggest that the migration inhibition is reversible, perhaps due to inactivation of the inhibitory factor. By 72 h, the culture supernatants from stimulated tuberculin-positive donor l y m p h o c y t e s no longer consistently contain inhibitory activity. Indeed, some supernatants now appear to enhance migration of the polymorphs. Thus, as Weisbart et al. (1974) have indicated, both migration inhibitory and enhancing factors may be found in l y m p h o c y t e culture supernatants, the result of the LMT depending u p o n the predominant factor formed. However, in the present series of experiments, it would appear that the inhibitory factor predominates in 24 h cultures, the period over which the direct LMT is carried out. Both these factors would appear to be low molecular weight lymphokines as indicated by Rocklin (1974) and Weisbart et al. (1974). In their studies inhibitory activity was associated with the albumin fraction and n o t with 7-globulins. Since the active supernatants may be assayed for inhibitory activity on purified homologous polymorphs, and may be stored for some weeks, the indirect assay may be useful in situations where insufficient polymorphs prevent the use of the direct test, because of excessive proportions of mononuclear cells e.g. in drug-induced or other forms of neutropenia, or where it is important to exclude the presence of a n t i g e n - a n t i b o d y complexes by fractionation studies.
141 ACKNOWLEDGEMENTS T h e a u t h o r s gratefully a c k n o w l e d g e the assistance o f Ms. P e n n y H a n s o n a n d Ms. D e b b i e H y a m . T h e w o r k was s u p p o r t e d in part b y a g r a n t f r o m T h e C a n a d i a n T u b e r c u l o s i s and R e s p i r a t o r y Diseases F o u n d a t i o n .
REFERENCES BSyum, A., 1968, Scand. J. Clin. Lab. Invest. 21, Suppl. 97, 1. Brostoff, J., 1974, J. Immunol. Methods 4, 27. Clausen, J.E., 1970, Acta. Med. Scand. 188, 59. Clausen, J.E., 1973, J. Immunol. 110,546. David, J., S. A1-Askari, H.S. Lawrence and L. Thomas, 1964, J. Immunol. 93, 264. Lockshin, M.D., J. Waxman and M.W. Jenkins, 1973. Experientia 29, 340. Maini, R.N., L.M. Roffe, I.T. Magrath and D.C. Dumonde, 1973, Int. Arch. Allergy 45, 308. Mitchell, C.G., M.G.M. Smith, P.L. Golding, A.L.W.F. Eddleston and R. Williams, 1972, Clin. Exp. Immunol. 11,535. Rocklin, R.E., 1974, J. Immunol. 112, 1461. S~bborg, M., 1969, Acta Med. Scand. 185,221. S~bborg, M. and G. Bendixen, 1967, Acta. Med. Scand. 181,247. Warrington, R.J., 1973, Ph.D. Thesis Memorial University of Newfoundland. Weisbart, R.H., R. Bluestone, L.S. Goldberg and C.M., Pearson, 1974, Proc. Nat. Acad. Sci. U.S. 71,875.