Journal of lmmunological Methods 7 (1975) 123-142 © North-Holland Publishing Company
A MODIFIED, QUANTITATIVE ASSAY FOR SUBSTANCES TI-IAT INHIBIT THE MIGRATION OF MACROPHAGES AND OTHER LEUCOCYTES: THE USE OF CONCANAVALIN A AS A REFERENCE REAGENT E.L. FENTON and Morag M. TAYLOR Department of Biological Chemistry, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent, U.K.
Received 27 August 1974,
accepted 15 October 1974
Supernatants from long term cultures of HeLa cells and from short term, Con A-stimulated human peripheral lymphocyte cultures, are shown to inhibit the migration of lymphoid cells from continuous lines (LCL), in the capillary tube assay. Con A is used to calibrate parallel line assays on the HeLa inhibitor; data are given on the precision and reproducibility of the method. LCL cells and guinea-pig macrophages are compared as capillary test cells for inhibition by supernatants from human lymphocyte cultures.
1. Introduction When sensitised lymphoid cells of animal or human origin are cultured with sensitising antigen, or when lymphoid cells are incubated with certain plant lectins in tissue culture medium under defined conditions, a number of soluble factors, the 'mediators of cellular i m m u n i t y ' , or lymphokines, are liberated into the medium (Bloom and Bennett, 1966; David, 1966; Bennett and Bloom, 1968; Ruddle and Waksman, 1968; Dumonde et al., 1969; Ward et al., 1969). Interest in these factors arises from their possible role in augmenting the immune response, and among them, the macrophage migration inhibition factor (abbr M1F) has been extensively studied. This factor is characterised by its ability to limit the extent to which macrophages migrate from capillary tubes in tissue culture medium, a property which formed the basis for several well-known and widely used test systems (George and Vaughan, 1962; Bloom and Bennett, 1966; David, 1966). Generally, these methods employ macrophages from guinea-pig peritoneal exudates as migration test cells in the capillaries; the alternative and often more convenient use of human lymphoid cells from continuously maintained lines was suggested by Glade and Hischhorn (1970) and Glade (1971). 123
124
E.L. Fenton, M.M. Taylor, Assay Jor m~,ration inhibition factors
In general, the test cells migrate from capillaries during incubation in a closed environment of tissue culture medium and form zones on fiat glass surfaces of specially constructed chambers. Many reproductions of such zones are to be found in the literature (Bloom and Bennett, 1966; David, 1966; Dumont et al., 1972). In the earlier methods, indices of inhibition for 'unknowns' were simply expressed as percentages of controls, and among the recent improvements are notably those of Williams et al. (1973) and of Morley et al. (1972). Both feature the application of accepted principles of biological assay in the field of lymphocyte mediators by the use of the 4 point parallel line design (i.e. 2 doses each of an 'unknown' and of a 'standard') in the assay of MIF. Morley et al. (1972) extended the 4 point assay to the measurement of increased vascular permeability and to lymphocyte mitogenic activity in addition to MIF. Both groups of workers used a partially purified guinea-pig lymphokine fraction as the common source of active material for the 'unknowns', and prepared the 'standards' by diluting the 'unknowns' in the ratio 1.33 : 1. By simultaneous assay of both solutions, each at high and low dose levels, spaced at constant log-dose intervals within the most usefully linear part of a pre-determined dose-response curve, true and observed potency ratios were compared. In common with the assays described by Williams et al. (1973) and Morley et al. (1972), the present method uses the same number (4) of observations within doses, i.e. 16 capillaries in the 4 point assay. Essential differences however are (a) the method and unit of response measurement (i.e. micrometer measurement of migration zone diameters in millimetres); (b) the sources of inhibitory test material (mostly from HeLa cell culture supernatants); (c) the source of target cells (mainly from continuous lymphoid cell lines); and (d) the expression of potencies of 'unknown' samples as ratios of their inhibitory power, to that of solutions of Concanavalin A (Con A) of known concentration. The objects of the present work were (1) to achieve continuity of results by measuring and expressing them in terms of an external calibrating reagent or 'reference preparation', for which purpose Con A, being a readily purified and well characterised protein (Agrawal and Goldstein, 1967), having migration inhibitory properties, was examined; (2) to make use of the readily available cells from subcultures of continuous lymphoid lines (LCL) as capillary test cells, thereby simplifying the tedious and time-consuming preparation of peritoneal exudate cells, the conventional alternative.
2. Materials and methods 2.1. Tissue culture media
RPMI 1640, or Eagle's MEM, were supplemented with foetal calf serum (FCS not inactivated by heat treatment), 10% 15% v/v, to match the concentration
E.L. Fenton, M.M. Taylor, Assay Jor migration inhibition factors
125
already present in the sample to be assayed. Media and FCS were from Weltcome Reagents Ltd., Beckenham, Kent, U.K. Antibiotics (penicillin, 20,000 units, and streptomycin, 10,000 rig) were added to each 100 ml of supplemented RPMI 1640. Eagle's MEM already containing these concentrations of antibiotics. 2.2. Test cells.for capillaries
LCL cells were from 2 - 4 day subcultures of either line 1788 (RPMI) or line 7 (Wellcome) in RPMI 1640 medium, supplemented with FCS. Both lines were maintained at our laboratories (see Moore et al., 1967). Guinea-pig macrophages (peritoneal exudate cells - PEC) were prepared by the oil-induction method described by Remold et al. (1970). 2.3. Inhibitory test materials HeLa supernatants were from 3 - 4 day subcultures of HeLa cells, prepared and maintained in Eagle's MEM, supplemented with 10% FCS, as described by Gey et al. (1952). Culture suspensions were centrifuged for 20 min at 250g, the supernatants were filtered through Millipore membranes (0.22 ~m) and distributed into aliquots which were stored frozen at - 2 0 °C for use in subsequent experiments. Human peripheral lymphocyte supernatants. Cells were prepared by Ficoll-Triosil sedimentation (20 rain at 1000g) as described by Perper et al. (1968) and were cultured at concentrations of 2.5 × 10 6 viable lymphocytes per ml, in 1 oz bottles, loosely screw capped, each containing 6 ml of serum-free RPMI medium, with or without Con A (see below, and also Maini et al., 1973). Cultures were harvested after 8 and 48 hr incubation at 37°C, in 5% CO: in air. Samples and their notation were: Cell suspensions + 167" ttg Con A per ml. Centrifuged after incubation to provide 'P' samples. Cell suspensions without Con A were divided evenly. To one portion, 167 ttg of Con A per ml was added to provide 'R' samples. The other portion, without Con A, provided 'C' samples. Cultures were then centrifuged (10 min at 250 g) and filtered through Millipore membranes (0.22/~m). ('P' = pre-incubated; 'R' = reconstituted; 'C' = control). All samples were finally treated as described by Horvat et al. (1972) to remove Con A prior to assay. Con A was prepared as described by Agrawal and Goldstein (1967). The product was de-salted and lyophilised in small glass containers for use as the 'reference
* This concentration was found to be optimal in preliminary experiments.
126
E.L. Fenton, M.M. Taylor, Assay ¢br migration inhibition factors
preparation'. In one experiment, a comparison was made between this laboratory preparation and a commercial product (Calbiochem. Los Angeles, U.S.A.). 2.4. 'R eJbrence preparations' For each assay, a solution was prepared by dissolving a freshly weighed amount of the lyophilised Con A (Mettler H-20, 5 place balance) in the appropriate serumsupplemented medium (see Experimental section). From the resulting solution 'high' and 'low' dose levels were prepared by further diluting with the medium. 2.5. Maximow tissue culture" slides were obtained from Wray Optical Co., Bromley, Kent, U.K. 2. 6. Cleaning o f glassware Immersion in 5% v/v aqueous Decon 75 (Decon Laboratories, Brighton, Sussex, U.K.), followed by thorough washing in tap water, then in distilled water, and heat sterilising in an air oven at 180°C for 1 hr was adopted as standard procedure throughout. Drummond 'Microcap' disposable capillaries, 20 microlitre capacity (Shandon Scientific Ltd., London, U.K.) were cut in halves and sterilised in the air oven without pre-washing. 2. Z Preparation o f capillaries containing test cells Cells were centrifuged from their original medium, resuspended and washed in 3 X 10 ml Hank's balanced salt solution (HBSS), then in 1 X 10 ml of tissue culture medium supplemented with 10-15% v/v FCS, and identical in this and ill other respects to the medium in which the MIF and the Con A were diluted for assay. Each of the above centrifugations was for 10 min at 220g. The cell pellet was finally suspended in 4 times its judged volmne of the serum-supplemented medium, and, within 1 hr, capillaries were filled with the suspension as described by Bloom and Bennett (1966). One end of each capillary containing approx. 9 X l0 s viable cells (trypan blue dye exclusion) in 10 /al was sealed with 'Cristaseal' (Hawksley and Sons Ltd., Lancing, Sussex, U.K.) and, within 15 rain of filling, the capillaries were centrifuged for 5 rain at 100 g (excluding acceleration and deceleration times, each of approx. 1 rain). Timing was adjusted so that not more than 1 hr elapsed between making the final (4 vol) suspension and completion of the filling sequence. Attention has been drawn by Maini et al. (1973) to the importance of observing certain similar essential precautions in preparing capillaries with suspensions of human leucocytes, and by Morley et al. (1972) to the same requirements with guinea-pig peritoneal exudate cells. Satisfactory zones of migration were obtained from test cells drawn from the lines maintained during 40 passages. Reduced zone diameters and irregular profiles were however encountered from cell samples drawn after higher passage levels.
E.L. Fenton, M.M. Taylor, Assay ]or migration inhibition factors
127
2.8. Slide assembly and incubation Each capillary w a s cut just below the cell-liquid interface and two sections (each approx. 3 m m long) were attached, sealed ends together, to the fiat surface of a Maximow slide with a small dab of silicone grease, and the capillaries were pressed down to ensure contact with the glass surface. A second slide was placed on top with its concave well downwards, then moved slightly sideways and the cavity filled with the appropriate serum-supplemented medium (see fig. 1). The completed assembly was closed with firm hand pressure, applied to produce a sliding action.
Fig. 1. Method of filling a Maximow slide assembly with serum-supplemented medium containing test or reference dilution.
E.L. Fenton, M.M. Taylor, Assay ]br migration inhibition fiTctors
128
Adhesion of the glass surfaces by the capillary action of the thin film of medium which spread between the slides during filling rendered wax-sealing unnecessary. The assemblies were incubated for 24 hr at 37°C. 2. 9. Measurement
of
migration zone responses
Zone diameters were measured with a micrometer fixed in a bracket attached to the stage of a Zeiss binocular microscope, viewing at constant magnification ( X 10) with substage illumination. Slide assemblies were placed between the micrometer spindle and the stage platform. The diameter to be measured and the micrometer spindle were focused together in the same plane by viewing through a small aperture (approx. 4 mm diameter) in a screen fixed immediately below the microscope objective. Two micrometer readings at right angles to each other were made on each zone and averaged to take into account any non-circularity present in the zones. Thus, each capillary provided one estimate, and 4 averages were obtained per dose level. 1.8 1"7 .-g 1-6 E 1.5-
E ~" 1'4im
~: 1.3
\
o 1.2 "B
\
1.1 "13 0)
1-0
m
0.9 t
0.8 o.125
o.25
0.5o
11o
Dose ( log scale} • : ConA, r e f e r e n c e p r e p a r a t i o n in ~ g / m l . • = HeLA test supernatant in relative dilutions.
Fig. 2. (Expt. 3.1 .) Log dose-responses from 8 point assay of HeLa test s u p e r n a t a n t with Con A as internal reference. Each point is tire mean of two cross-diameters.
E.L. Fenton, M.M. Taylor, Assay for migration inhibition factors
129
3. Experiments and results 3.1. Multi-point assays fi)r statistical analysis In preliminary experiments, separate dose-response curves from solutions of Con A and from HeLa cell supernatants were compared. With Con A, a usefully linear part of the curve was found for responses, plotted against log concentration between limits of approx. 0 . 1 - 1 . 0 / ~ g Con A per ml of medium, while HeLa supernatants from a number of 3 - 4 day subcultures behaved similarly between log concentrations representing dilutions of 1/ 1 to around 1 / 8 - 1 / 10. To examine the nature of the log dose-response lines between both substances, four log spaced doses of each were compared simultaneously, i.e. an 8 point, or 4 + 4 design. Capillary target cells were from LCL - line 7. Individual diameter readings from this experiment are illustrated in fig. 2 in order to show the array of responses and the regressions. In order to compare target cells from a different line (1788), a second assay, with 3 dilutions of Con A and 3 dilutions from the same pool of HeLa supernatant (6 point design), was carried out. Results from this experiment are tabulated (table 1). In these and all following experiments with HeLa supernatants, the diluent for 'unknowns' and for Con A was Eagle's MEM containing 10% v/v FCS. Preliminary analysis of variance on the raw data from both experiments showed that in neither assay did the mean squares attributable to slide assemblies differ significantly from those among the capillaries within them (p = 0.2>). The mean squares, pooled from both assays, were 0.0097 and 0.0089 for slide assemblies and capillaries within slides, respectively. These, against the pooled error variance, gave ' F ' ratios of 0.14 and 0.15: p = 0.2 > for both. Of further critical importance, the replication errors were not significantly correlated with their means (r ~ - 0.217;p = 0.616) and were homogeneous between assays over all treatments (X 2 = 7.96; d.f. = 7 ; p = 0.336). The data from both assays were next subjected to regression analysis (Emmens, 1948), treating each dose level, in the light o f the preliminary analyses, as having fourfold replication. Results of both analyses are shown in full in table 2, and may be summarised: 1) No significant deviations from linearity were encountered in either assay. The slopes for Con A and for HeLa were homogeneous in both assays. 2) 'Potency' estimates for the HeLa supernatant in terms of Con A were homogeneous over both assays, i.e. between capillary test cells from lines 7 and 1788 (X 2 = 0.055; d.f. = 1 ; p -- 0.78). The weighted mean 'potency' was 2.97 X Con A (limits of error = 2.50 3.54; p = 0.05). See table 2 for separate potency estimates and limits or error. In the light of the results from the 8 and 6 point designs, it was decided to test the ability of the simplest form of parallel line assay to estimate potencies of HeLa cell supernatants in terms of Con A. This was the 4 point assay, with equal numbers
130
E. L. Fenton, M.M. Taylor, Assay jbr migration inhibition factors
E.L. Fenton, MM. Taylor; Assay for migration inhibition factors
131
132
E.L. benton, M.M. Taylor, Assay jbr migration inhibition jhctors
of observations per dosage group, and equal spacing of log doses of both substances. It provided information on i) parallelism between the log dose-response lines; ii) relative potency, and iii) limits of error about the mean potency estimate, at a stated level of probability, using conventional methods of calculation (Emmens, 1948). Assuming non-significant departure from parallelism, quick approximations of relative potency (but no other information) may be obtained by solving the following equation for M: Log ratio of potency, M = ID/B where: I = log dose ratio (difference between log doses): identical for both substances D = difference in mean responses between preparations B = difference in mean responses between high and low doses, whence, Antilog M = relative potency. Alternatively, M may be obtained graphically by simple measurement of the difference between log doses on the abscissa (e.g. fig. 2) corresponding to response, preferably the mean response for all groups on the ordinate (see also, Morley et al., 1972).
3.2. Assay of HeLa cell supernatant at d(/'jbrent levels (4 point design) A collaborator was provided with a sample of supernatant from a 72 hr culture of Helm cells ('parent') and a sample of medium of the same composition as that used for culture ('control') and was invited to submit for assay a number of dilutions of one sample in the other. By request, only the 'parent' and 'control' samples were identified by labelling; the dilutions were coded. Seven coded samples were thus provided, and were assayed together with the two identified samples, and with Con A, all preparations at two dose levels. Capillaries were prepared from LCL 7 in the 'control' medium. From the potency estimates, the composition of each coded dilution was finally calculated and expressed as percentage by volume of 'parent' in 'control', for comparison with the true dilutions then revealed by the collaborator. The comparisons are shown in columns 10 and 11 of table 3. The differences between observed and true composition of the coded dilutions ranged from - 4 . 8 % to +6.8%.
3.3. Tests for reproducibiliO, o f the 4 point des•n A pool of HeLa cell supernatant (Sample A) was prepared, divided and stored frozen as described in section 2.3. On each of five separate test occasions, one bottle was allowed to thaw at room temperature and the contents divided between assays, (i) with LCL 7, and (ii) with LCL 1788 as target cells. The above series was repeated using a different pool of HeLa supernatant (Sample B) over a further five test occasions. In both series, the occasions, and the number of assays possible on each, were determined by the coincidence o f availa-
0.748 1.19 0.74 1.07 0.86 1.46 0.85 1.91 1.93
1.029 1.47 1.01 1.33 1.13 1.74 1.11 1.92 1.90
-
,
-
-
0.66
(0.913)
-
0.935
$2 dose) (0.454)
0.927 -0.897 -0.864 0.947 -0.930 -0.864 -0.033 c -0.033 c
-0.915
-
Combined S and U
Sa
U1 Uz (Dilutions for d o s e ) (1/1) (1/2) for
Slopes
Con A
HeLa s u p e r n a t a n t
(ug/ml
Derived d a t a
-0.265 -0.263
0.015
+0.005 -0.005 -0.015 -0.005 +0.005
0
0.746 0.186 0.797 0.285 0.579 0.116 0.603 0 -
(~g/ml)
U from S -
'Unknown' in terms o f Con A
Potency data Deviations from parallel
Assays o n seven ' c o d e d ' sample o f HeLa s u p e r n a t a n t (4 p o i n t design).
R e s p o n s e s ( z o n e d i a m e t e r s in m m ) a
3.2.
-
0.48
0.61 0.15 0.64 0.23 0.47 0.09
0.05)
-
-
0.90 0.23 0.99 0.35 0.72 0.15 0.75
L i m i t s of error (/9 =
100 b 25.0 106.8 38.2 77.6 15.5 80.8 0 _ b
_
Observed
Ux ~- 0,
100 29.0 100 43.0 79.0 14.0 79.0 0 _
True
'Unknown' (percentage b y vol o f ' p a r e n t ' in 'control')
C o m p o s i t i o n of
a E a c h e n t r y under U1, U2, SI, S 2 is the mean o f 4 cross d i a m e t e r s , m e a s u r e d as described in the t e x t . b B o t h samples were i d e n t i f i e d b y the c o l l a b o r a t o r prior to the e x p e r i m e n t . c Samples 7 and ' C o n t r o l ' are seen to have v i r t u a l l y zero slope ( - 0 . 0 3 3 ) , h e n c e t h e y d e v i a t e from parallelism w i t h C o n A ( - 0 . 2 6 3 - 0.265). Since U2 the p o t e n c y of b o t h samples is zero.
Con A 'Parent' (HeLa supernatant C o d e d samples 1 C o d e d samples 2 C o d e d samples 3 C o d e d samples 4 C o d e d samples 5 C o d e d samples 6 C o d e d samples 7 'Control' medium
Preparation (samples)
Table 3 Results o f e x p e r i m e n t
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-Between cell lines in capillaries B e t w e e n test o c c a s i o n s Residual (replication) Total
Source o f v a r i a t i o n
0.0002 0.0172 0.1685 0.1859
squares
Sum of
A
1 4 10 15
d.f.
Hela s u p e r n a t a n t s
Table 5 A n a l y s e s o f variance on d a t a s h o w n in table 4.
0.0002 0.0043 0.01685 -
P
0 . 0 1 2 0.2 > 0.26 0.2 > -
Mean square F
0.0069 0.0488 0.0977 0.1534
Sum o f squares 1 4 9 14
d.f.
0.0069 0.0122 0.0109 -
Mean square
0.2 >
0.2> 1.12
P
0.63
F
%0
3"
2~
136
E.L. Fenton, M.M. Taylor, Assay ]or mi,~ration inhibition jactors
bility, and on the quantities, of subcultures from lines 7 and 1788 on any one day. Generally, line 1788 was available in greater quantity at any one time than was line 7, hence the majority of comparisons were between single (2 + 2) assays with line 7 and replicates (2 + 2) on line 1788. Separate Con A solutions, prepared as described in section 2.4, were used in each assay. In all, 31 assays were carried out during a period of several weeks. The resuls from both HeLa supernatants (A and B) are shown in table 4 and their analyses in table 5. The results of these experiments may be summarised: i) No evidence of systematic deterioration with time of storage (6 weeks) at - 2 0 ° C was evident in either supernatant A or B. ii) In neither experiment were the mean squares between cell lines or test occasions significant against the residual (replication) error (see later in Discussion). The pooled error variances gave limits of error (at the 5% level of significance) of -+ 14% and + 19% for supernatants A and B respectively (both cell lines combined).
3.4. Comparison between different sources of Con A (4 point design/ The Con A used in all previously reported experiments was a saltfree lyophilised powder, prepared in the laboratory by the method of Agrawal and Goldstein (1967). In order to compare this material with Con A from a different source, it was tested simultaneously with a sample purchased from Calbiochem USA. Both samples and a HeLa cell supernatant were set up together and the potency of the HeLa supernatant was expressed in terms of each Con A preparation. The Calbiochem Con A preparation contained salt, therefore its protein content was determined by measurement of absorbance at 280 nm (Unicam SP500 spectrophotometer) and adjusted to equal that of the laboratory preparation before allocating the doses. Two doses of each Con A sample (1.0 and 0.1 ~g/ml) and two equally spaced doses (1/1 and 1/10 dilutions) of the HeLa supernatant were thus compared (2 + 2 design). The difference between potency ratio estimates was 10 11%: each estinaate falling within the 95% confidence limits of the other. No significant deviations from parallelism between the log dose-response lines were encountered; the slopes for all three substances were homogeneous (X 2 = 0.027; 2 d.f.; P = 0 . 9 8 - 0 . 9 9 ) . It was concluded that, given equivalent protein concentration in the Con A solutions, either could be used satisfactorily (see table 6 for results).
3.5. Comparison between responses o f (i) lymphoM cell line cells, and (ii) guineapig peritoneal exudate cells to inhibitor in supernatants from Con-A stimulated human lymphoc:vte cultures Culture supernatants, prepared as described under Materials and methods, were harvested after 8 and 48 hr incubation and were treated to produce 'C', ' R ' and 'P' samples (see section 2.3.).
E.L. Fenton, MM. Taylor, Assay for migration inhibition factors
137
-~
1.03 + 4.8% (N = 4) 1.03 ? 5.4% (N = 4) 0.82 f 5.2% (N = 4)
28.0 f 2.9% (N = 4) 28.8 f 9.5% (N = 3) 14.7 ? 14.2% (N = 3)
1.09 + 4.6 % (N = 4) 1.06 f 8.9 % (N = 4) 0.84 i: 2.39% (N = 4)
39.3 + 3.8% (N = 4) 37.1 r 14.1% (iv = 4) 30.0 f 9.8% (N = 4)
of
RPM1 1640 me-
Diameter (mm by micrometer f coeff. variation (LCL 7)
in serum-free
supernatants
Area (arbitrary units) by planimetry f coeff. of variation (G.P.PEC)
of
cultures
of mitogen-freed
Diameter (mm) by micrometer t coeff. variation (LCL 7)
lymphocyte
by aliquots
Area (arbitrary units) by planimetry + coeff. of variation (G.P.PEC)
peripheral
cells and LCL-cells
48 hr
at 37°C of human
exudate
8 hr
of incubation
of guinea-pig peritoneal lymphocyte cultures.
G.P.PEC = Guinea-pig peritoneal exudate cells used in capillaries for area measurements. LCL 7 = Cells from lymphoid continuous line 7 used in capillaries for diameter measurements. Coefficient of variation = standard deviation expressed as percentage of mean. N = Number of observations for each sample, assayed at single dose level. See table 8 for analyses on the above data.
C (Control R (Re-constituted) P (Prc-incubated) -__ .~
Samples
Period dium
Table I Comparison between inhibition from Con A-stimulated human
1 5 1 5
R-P(MIF'effect) Replication
1 6
R-P ( M I F effect) Replication
C-R (Con A effect Replication
1 6
d.f.
3.010 0.059
0.0119 0.0343
1.0153 0.1819
0.0903 0.149
Mean square
50.5 -
0.35 -
5.6 -
0.61 -
F
Area m e a s u r e m e n t s GPE cells in capillaries
C-R (Con A effect) Replication
S o u r c e of v a r i a t i o n
0.001 > < 0 . 0 1 -
0.2 > -
0.1 > < 0.05 -
0.2 > -
P
1 6
1 6
1 6
1 6
d.f.
0.088 0.0025
0 0.0029
0.0968 0.00473
0.0018 0.0058
Mean square
32.1 -
0 -
20.4 -
0.31
F
Diameter measurements LCL 7 cells in capillaries
0.001 > < 0 . 0 1 -
N o t sig. -
0.001 > < 0.01
0.2 > -
In b o t h 8 a n d 4 8 hr harvests, d i a m e t e r m e a s u r e m e n t s on line 7 cells s h o w h i g h l y significant effects for i n h i b i t i o n d u e to MIF against non-significant effects w h i c h can be a t t r i b u t e d to residual C o n A in the samples. The same h o l d s for the 4 8 hr harvest, m e a s u r e d by areas on GPE cells, b u t in the 8 hr harvest, the MIF e f f e c t j u s t fails to reach significance at the 5% level.
48 hr harvest
8 hr harvest
Con Astimulated cultures
Incubation period o f
Table 8 Analyses of variance o n d a t a s h o w n in table 7.
140
E.L. Fenton, M.M. Taylor, Assay for migration inhibition factors
The six samples so produced were each sub-divided for migration-inhibition tests using (i) GPE-cells with area measurements by planimetry on projected images, and (ii) LCL 7 cells, with diameter measurements by micrometer. The amounts of each sample available were such that only the effects of single dose levels could be measured by either method, thereby restricting the experimental design to single level comparisons, without the benefits of multi-dose replication or of calibration with Con A. In this comparison, it was required to show the efficiency of the Sephadex procedure (Horvat et al., 1972) in removal of residual Con A, in order to avoid any overlapping of inhibitory effects in the test. The observed inhibitory effects (table 7) have therefore been isolated as those due to possible contamination with Con A (C-R effects) and those due to MIF (R-P effects), and the significance of the differences between them are shown in the analyses in table 8. The results of the experiment are summarised at the foot of table 8; conclusions and further comments appear in the Discussion.
4. Discussion It is well recognised that a satisfactory assay for a biologically active substance should give an unbiased estimate of potency, and of the limits of error, at a stated probability within which the estimate falls. Estimates of potency are conventionally made by simultaneous comparison between the responses of the test material and those of a defined biological standard (Emmens, 1948). In a comprehensive article on the measurement of lymphokines, Morley et al. (1972) describe their work on the development and potential use of material working standards for three lymphokine activities, one of which was macrophage migration inhibition. In our introductory section, we have already drawn attention to any similarities to, and essential differences between, the above work and our own. Additionally, we would stress that in no way is Con A to be regarded either as a standard for MIF activity, or as a substitute for material working standards from lymphokine sources. Bearing this in mind, we use the term 'reference preparation' throughout the present paper to describe Con A in its role as a reagent against which to calibrate our assays, mainly for process work, associated with fractionation and purification of migration inhibitory substances. The four point assay employing only two dose levels of each substance supplies evidence about the significance of difference between substances (potency ratio); it permits calculation of regression equations (slopes) for the individual lines and tests for departure from parallelism between them. It provides no test for curvature of regression, although any observed departure from parallelism may, indirectly, indi-
E.L. Fenton, M.M. Taylor, Assay for migration inhibition Jactors
14 l
cate curvature in one or both lines. Tests for curvature require the inclusion of more than two points on each line, hence the eight and six point assays were conducted as preliminaries (3.1.), to validate the use of the simpler four point design. The preliminary analyses of variance on the multi-point assays showed no significant differences either between the errors attributable to slide assemblies or between the capillaries within them, using the four fold replication, and the method of obtaining the four mean responses, d~scribed in section 2.9. On the question of replication, it is sometimes overlooked that increased replication for enhanced reliability of results is, in general, better directed to increasing the number of solutions initially prepared, rather than merely to replicate the number of response observations made. Limits of error of approx. +- 23% at the 95% significance level were calculated for seven potency estimates with a single test-cell line over a wide range of concentrations of HeLa inhibitor (.3.2.), and limits of approx. - 14% and -+ 19% for 16 and 15 estimates respectively over a more restricted range of inhibitor concentrations using test-cells from two different lines (3.3.), and should be compared with other published data. Results from similar numbers of observations for fair comparison were, however, not readily found in the literature, although limits of 1 . 0 6 - 1 . 9 0 about a mean potency ratio of 1.42 (i.e. 25.4% to +33.8%) are quoted by Morley et al. (1972) for a singJe 2 + 2 assay on macrophage MIF. The choice of inhibitory test material used in the present work (mainly from HeLa cultures) was dictated by its ready availability compared to that of the Con A-induced human MIF featured in the final experiment (3.5.), in which information was sought on the sensitivity of LCL cells to inhibition by this type of material. Despite the limitations imposed by the restricted amounts of human M | F available, it is concluded that migration of the LCL test cells was inhibited by the human MIF, and that the observed inhibitions were not due to contamination of the samples with residual Con A. In the early experiments, attempts to trace out the peripheries of migration zones from LCL cells had to be abandoned because, unlike guinea-pig PE cells, the projected images were of insufficient contrast for good reproducibility. Under the microscope, however, the zones were brightly illuminated and their diameters could be measured rapidly with good reproducibility using a micrometer as described. Preliminary d o s e - r e s p o n s e curves plotted from the d i a m e t e r - l o g dose relationships were only slightly affected by arithmetic transformation to areas. Therefore, diameter measurements were used, untransformed, throughout the present work. Comparisons of precision between diameter and area measurements may be made from the standard deviations shown in table 7 (expt. 3.5.), bearing in mind that, within such comparison, two different test-cell species are represented. It will of course be obvious that, once selected from a preliminary dose response curve, a response parameter should be tested in multi-point assays without transformation. The suitability or otherwise, of choice will be reflected in the parallel situation displayed between the log d o s e - r e s p o n s e lines of the substances compared.
142
E.L. benton, M.M. Taylor, Assay jor migration inhibition jactors
Acknowledgements T h e a u t h o r s gratefully a c k n o w l e d g e the c o - o p e r a t i o n o f colleagues at these laboratories: Mr. C.J. B u r m a n for p u r i f y i n g t h e Con A and providing the guinea-pig m a c r o p h a g e s , Dr. K.H. F a n t e s for the c o d e d samples a n d for helpful discussions, Dr. A.M. W h i t a k e r ( D e p t . o f V i r o l o g y ) for s u p p l y o f c u l t u r e d cells, Mr. P.A. Y o u n g ( B i o m e t r i c s D e p t . ) for assistance w i t h statistical analyses a n d for advice, a n d Miss A n n e Kane for t h e line diagrams.
References Agrawal, B.B.L. and I.J. Goldstein, 1967, Biochim. Biophys. Acta 147,262. Bennett, B. and B.R. Bloom, 1968, Proc. Natl. Acad. Sci. U.S. 59,756. Bloom, B.R. and B. Bennett, 1966, Science 153, 80. David, J.R., 1966, Proc. Natl. Acad. Sci. U.S. 56, 72. Dumonde, D.C., R.A. Wolstencroft, G.S. Panayi, M. Matthew, J. Morley and W.T. Howson, 1969, Nature 224, 38. Dumont, F., D. Sabolovic, D. Oth and C. Burg, 1972, Experientia 28, 1354. Emmens, C.W., 1948, in: Principles of biological assay, ed. C.W. Emmens (Chapman and Hall Ltd., Lond.). George, M. and J.H. Vaughan, 1962, Proc. Soc. Exptl. Biol. Med. 111,514. Gey, G.O., W.D. Cottman and M.T. Kubicek, 1952, Cancer Res. 12,264. Glade, P.R., 1971, In-vitro methods in cell mediated immunity, ed. B.R. Bloom and P.R. Glade (Academic Press, New York) p. 29. Glade, P.R. and K. Hirschhorn, 1970, Amer. J. Pathol., 60,483. Horvat, M., K. Havemmm, C.P. Sodomann and S. Burger, 1972, Intern, Arch. Allergy Appl. Immunol. 43,446. Maini, R.N., L.M. Roffe, I.T. Magrath and D.C. Dumonde, 1973, Intern. Arch. Allergy Appl. Immunol. 45,308. Moore, G.E., R.E. Gerner and tt.A. Franklin, 1967, J. Amer. Med. Assoc. 199, 87. Morley, J., R.A. Wolstencroft and D.C. Dumonde, 1972, in: Handbook of Experimental hnmunology, ed. D.M. Weir (Blackwell, Oxford and Edinburgh), Ch. 28. Perper, R.J., T.W. Zee and M.W. Mickelson, 1968, J. Lab. Clin. Med. 72,842. Remold, H.G., A.B. Katz, E. Haber and J.R. David, 1970, Cell. lmmunol. 1,133. Ruddle, N.tt. and B.ft. Waksman, 1968, J. Exptl. Med. 128, 1237. Ward, P.A., H.G. Remold and J.R. David, 1969, Science 163, 1079. Williams, T.J., J. Morley and R.A. Wolstencroft, 1973, J. Immunol. Methods 2, 137.
A MODIFIED, QUANTITATIVE ASSAY FOR SUBSTANCES TI-IAT INHIBIT THE MIGRATION OF MACROPHAGES AND OTHER LEUCOCYTES: THE USE OF CONCANAVALIN A AS A REFERENCE REAGENT E.L. FENTON and Morag M. TAYLOR Department of Biological Chemistry, The Wellcome Research Laboratories, Langley Court, Beckenham, Kent, U.K.
Received 27 August 1974,
accepted 15 October 1974
Supernatants from long term cultures of HeLa cells and from short term, Con A-stimulated human peripheral lymphocyte cultures, are shown to inhibit the migration of lymphoid cells from continuous lines (LCL), in the capillary tube assay. Con A is used to calibrate parallel line assays on the HeLa inhibitor; data are given on the precision and reproducibility of the method. LCL cells and guinea-pig macrophages are compared as capillary test cells for inhibition by supernatants from human lymphocyte cultures.
1. Introduction When sensitised lymphoid cells of animal or human origin are cultured with sensitising antigen, or when lymphoid cells are incubated with certain plant lectins in tissue culture medium under defined conditions, a number of soluble factors, the 'mediators of cellular i m m u n i t y ' , or lymphokines, are liberated into the medium (Bloom and Bennett, 1966; David, 1966; Bennett and Bloom, 1968; Ruddle and Waksman, 1968; Dumonde et al., 1969; Ward et al., 1969). Interest in these factors arises from their possible role in augmenting the immune response, and among them, the macrophage migration inhibition factor (abbr M1F) has been extensively studied. This factor is characterised by its ability to limit the extent to which macrophages migrate from capillary tubes in tissue culture medium, a property which formed the basis for several well-known and widely used test systems (George and Vaughan, 1962; Bloom and Bennett, 1966; David, 1966). Generally, these methods employ macrophages from guinea-pig peritoneal exudates as migration test cells in the capillaries; the alternative and often more convenient use of human lymphoid cells from continuously maintained lines was suggested by Glade and Hischhorn (1970) and Glade (1971). 123
124
E.L. Fenton, M.M. Taylor, Assay Jor m~,ration inhibition factors
In general, the test cells migrate from capillaries during incubation in a closed environment of tissue culture medium and form zones on fiat glass surfaces of specially constructed chambers. Many reproductions of such zones are to be found in the literature (Bloom and Bennett, 1966; David, 1966; Dumont et al., 1972). In the earlier methods, indices of inhibition for 'unknowns' were simply expressed as percentages of controls, and among the recent improvements are notably those of Williams et al. (1973) and of Morley et al. (1972). Both feature the application of accepted principles of biological assay in the field of lymphocyte mediators by the use of the 4 point parallel line design (i.e. 2 doses each of an 'unknown' and of a 'standard') in the assay of MIF. Morley et al. (1972) extended the 4 point assay to the measurement of increased vascular permeability and to lymphocyte mitogenic activity in addition to MIF. Both groups of workers used a partially purified guinea-pig lymphokine fraction as the common source of active material for the 'unknowns', and prepared the 'standards' by diluting the 'unknowns' in the ratio 1.33 : 1. By simultaneous assay of both solutions, each at high and low dose levels, spaced at constant log-dose intervals within the most usefully linear part of a pre-determined dose-response curve, true and observed potency ratios were compared. In common with the assays described by Williams et al. (1973) and Morley et al. (1972), the present method uses the same number (4) of observations within doses, i.e. 16 capillaries in the 4 point assay. Essential differences however are (a) the method and unit of response measurement (i.e. micrometer measurement of migration zone diameters in millimetres); (b) the sources of inhibitory test material (mostly from HeLa cell culture supernatants); (c) the source of target cells (mainly from continuous lymphoid cell lines); and (d) the expression of potencies of 'unknown' samples as ratios of their inhibitory power, to that of solutions of Concanavalin A (Con A) of known concentration. The objects of the present work were (1) to achieve continuity of results by measuring and expressing them in terms of an external calibrating reagent or 'reference preparation', for which purpose Con A, being a readily purified and well characterised protein (Agrawal and Goldstein, 1967), having migration inhibitory properties, was examined; (2) to make use of the readily available cells from subcultures of continuous lymphoid lines (LCL) as capillary test cells, thereby simplifying the tedious and time-consuming preparation of peritoneal exudate cells, the conventional alternative.
2. Materials and methods 2.1. Tissue culture media
RPMI 1640, or Eagle's MEM, were supplemented with foetal calf serum (FCS not inactivated by heat treatment), 10% 15% v/v, to match the concentration
E.L. Fenton, M.M. Taylor, Assay Jor migration inhibition factors
125
already present in the sample to be assayed. Media and FCS were from Weltcome Reagents Ltd., Beckenham, Kent, U.K. Antibiotics (penicillin, 20,000 units, and streptomycin, 10,000 rig) were added to each 100 ml of supplemented RPMI 1640. Eagle's MEM already containing these concentrations of antibiotics. 2.2. Test cells.for capillaries
LCL cells were from 2 - 4 day subcultures of either line 1788 (RPMI) or line 7 (Wellcome) in RPMI 1640 medium, supplemented with FCS. Both lines were maintained at our laboratories (see Moore et al., 1967). Guinea-pig macrophages (peritoneal exudate cells - PEC) were prepared by the oil-induction method described by Remold et al. (1970). 2.3. Inhibitory test materials HeLa supernatants were from 3 - 4 day subcultures of HeLa cells, prepared and maintained in Eagle's MEM, supplemented with 10% FCS, as described by Gey et al. (1952). Culture suspensions were centrifuged for 20 min at 250g, the supernatants were filtered through Millipore membranes (0.22 ~m) and distributed into aliquots which were stored frozen at - 2 0 °C for use in subsequent experiments. Human peripheral lymphocyte supernatants. Cells were prepared by Ficoll-Triosil sedimentation (20 rain at 1000g) as described by Perper et al. (1968) and were cultured at concentrations of 2.5 × 10 6 viable lymphocytes per ml, in 1 oz bottles, loosely screw capped, each containing 6 ml of serum-free RPMI medium, with or without Con A (see below, and also Maini et al., 1973). Cultures were harvested after 8 and 48 hr incubation at 37°C, in 5% CO: in air. Samples and their notation were: Cell suspensions + 167" ttg Con A per ml. Centrifuged after incubation to provide 'P' samples. Cell suspensions without Con A were divided evenly. To one portion, 167 ttg of Con A per ml was added to provide 'R' samples. The other portion, without Con A, provided 'C' samples. Cultures were then centrifuged (10 min at 250 g) and filtered through Millipore membranes (0.22/~m). ('P' = pre-incubated; 'R' = reconstituted; 'C' = control). All samples were finally treated as described by Horvat et al. (1972) to remove Con A prior to assay. Con A was prepared as described by Agrawal and Goldstein (1967). The product was de-salted and lyophilised in small glass containers for use as the 'reference
* This concentration was found to be optimal in preliminary experiments.
126
E.L. Fenton, M.M. Taylor, Assay ¢br migration inhibition factors
preparation'. In one experiment, a comparison was made between this laboratory preparation and a commercial product (Calbiochem. Los Angeles, U.S.A.). 2.4. 'R eJbrence preparations' For each assay, a solution was prepared by dissolving a freshly weighed amount of the lyophilised Con A (Mettler H-20, 5 place balance) in the appropriate serumsupplemented medium (see Experimental section). From the resulting solution 'high' and 'low' dose levels were prepared by further diluting with the medium. 2.5. Maximow tissue culture" slides were obtained from Wray Optical Co., Bromley, Kent, U.K. 2. 6. Cleaning o f glassware Immersion in 5% v/v aqueous Decon 75 (Decon Laboratories, Brighton, Sussex, U.K.), followed by thorough washing in tap water, then in distilled water, and heat sterilising in an air oven at 180°C for 1 hr was adopted as standard procedure throughout. Drummond 'Microcap' disposable capillaries, 20 microlitre capacity (Shandon Scientific Ltd., London, U.K.) were cut in halves and sterilised in the air oven without pre-washing. 2. Z Preparation o f capillaries containing test cells Cells were centrifuged from their original medium, resuspended and washed in 3 X 10 ml Hank's balanced salt solution (HBSS), then in 1 X 10 ml of tissue culture medium supplemented with 10-15% v/v FCS, and identical in this and ill other respects to the medium in which the MIF and the Con A were diluted for assay. Each of the above centrifugations was for 10 min at 220g. The cell pellet was finally suspended in 4 times its judged volmne of the serum-supplemented medium, and, within 1 hr, capillaries were filled with the suspension as described by Bloom and Bennett (1966). One end of each capillary containing approx. 9 X l0 s viable cells (trypan blue dye exclusion) in 10 /al was sealed with 'Cristaseal' (Hawksley and Sons Ltd., Lancing, Sussex, U.K.) and, within 15 rain of filling, the capillaries were centrifuged for 5 rain at 100 g (excluding acceleration and deceleration times, each of approx. 1 rain). Timing was adjusted so that not more than 1 hr elapsed between making the final (4 vol) suspension and completion of the filling sequence. Attention has been drawn by Maini et al. (1973) to the importance of observing certain similar essential precautions in preparing capillaries with suspensions of human leucocytes, and by Morley et al. (1972) to the same requirements with guinea-pig peritoneal exudate cells. Satisfactory zones of migration were obtained from test cells drawn from the lines maintained during 40 passages. Reduced zone diameters and irregular profiles were however encountered from cell samples drawn after higher passage levels.
E.L. Fenton, M.M. Taylor, Assay ]or migration inhibition factors
127
2.8. Slide assembly and incubation Each capillary w a s cut just below the cell-liquid interface and two sections (each approx. 3 m m long) were attached, sealed ends together, to the fiat surface of a Maximow slide with a small dab of silicone grease, and the capillaries were pressed down to ensure contact with the glass surface. A second slide was placed on top with its concave well downwards, then moved slightly sideways and the cavity filled with the appropriate serum-supplemented medium (see fig. 1). The completed assembly was closed with firm hand pressure, applied to produce a sliding action.
Fig. 1. Method of filling a Maximow slide assembly with serum-supplemented medium containing test or reference dilution.
E.L. Fenton, M.M. Taylor, Assay ]br migration inhibition fiTctors
128
Adhesion of the glass surfaces by the capillary action of the thin film of medium which spread between the slides during filling rendered wax-sealing unnecessary. The assemblies were incubated for 24 hr at 37°C. 2. 9. Measurement
of
migration zone responses
Zone diameters were measured with a micrometer fixed in a bracket attached to the stage of a Zeiss binocular microscope, viewing at constant magnification ( X 10) with substage illumination. Slide assemblies were placed between the micrometer spindle and the stage platform. The diameter to be measured and the micrometer spindle were focused together in the same plane by viewing through a small aperture (approx. 4 mm diameter) in a screen fixed immediately below the microscope objective. Two micrometer readings at right angles to each other were made on each zone and averaged to take into account any non-circularity present in the zones. Thus, each capillary provided one estimate, and 4 averages were obtained per dose level. 1.8 1"7 .-g 1-6 E 1.5-
E ~" 1'4im
~: 1.3
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o 1.2 "B
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Fig. 2. (Expt. 3.1 .) Log dose-responses from 8 point assay of HeLa test s u p e r n a t a n t with Con A as internal reference. Each point is tire mean of two cross-diameters.
E.L. Fenton, M.M. Taylor, Assay for migration inhibition factors
129
3. Experiments and results 3.1. Multi-point assays fi)r statistical analysis In preliminary experiments, separate dose-response curves from solutions of Con A and from HeLa cell supernatants were compared. With Con A, a usefully linear part of the curve was found for responses, plotted against log concentration between limits of approx. 0 . 1 - 1 . 0 / ~ g Con A per ml of medium, while HeLa supernatants from a number of 3 - 4 day subcultures behaved similarly between log concentrations representing dilutions of 1/ 1 to around 1 / 8 - 1 / 10. To examine the nature of the log dose-response lines between both substances, four log spaced doses of each were compared simultaneously, i.e. an 8 point, or 4 + 4 design. Capillary target cells were from LCL - line 7. Individual diameter readings from this experiment are illustrated in fig. 2 in order to show the array of responses and the regressions. In order to compare target cells from a different line (1788), a second assay, with 3 dilutions of Con A and 3 dilutions from the same pool of HeLa supernatant (6 point design), was carried out. Results from this experiment are tabulated (table 1). In these and all following experiments with HeLa supernatants, the diluent for 'unknowns' and for Con A was Eagle's MEM containing 10% v/v FCS. Preliminary analysis of variance on the raw data from both experiments showed that in neither assay did the mean squares attributable to slide assemblies differ significantly from those among the capillaries within them (p = 0.2>). The mean squares, pooled from both assays, were 0.0097 and 0.0089 for slide assemblies and capillaries within slides, respectively. These, against the pooled error variance, gave ' F ' ratios of 0.14 and 0.15: p = 0.2 > for both. Of further critical importance, the replication errors were not significantly correlated with their means (r ~ - 0.217;p = 0.616) and were homogeneous between assays over all treatments (X 2 = 7.96; d.f. = 7 ; p = 0.336). The data from both assays were next subjected to regression analysis (Emmens, 1948), treating each dose level, in the light o f the preliminary analyses, as having fourfold replication. Results of both analyses are shown in full in table 2, and may be summarised: 1) No significant deviations from linearity were encountered in either assay. The slopes for Con A and for HeLa were homogeneous in both assays. 2) 'Potency' estimates for the HeLa supernatant in terms of Con A were homogeneous over both assays, i.e. between capillary test cells from lines 7 and 1788 (X 2 = 0.055; d.f. = 1 ; p -- 0.78). The weighted mean 'potency' was 2.97 X Con A (limits of error = 2.50 3.54; p = 0.05). See table 2 for separate potency estimates and limits or error. In the light of the results from the 8 and 6 point designs, it was decided to test the ability of the simplest form of parallel line assay to estimate potencies of HeLa cell supernatants in terms of Con A. This was the 4 point assay, with equal numbers
130
E. L. Fenton, M.M. Taylor, Assay jbr migration inhibition factors
E.L. Fenton, MM. Taylor; Assay for migration inhibition factors
131
132
E.L. benton, M.M. Taylor, Assay jbr migration inhibition jhctors
of observations per dosage group, and equal spacing of log doses of both substances. It provided information on i) parallelism between the log dose-response lines; ii) relative potency, and iii) limits of error about the mean potency estimate, at a stated level of probability, using conventional methods of calculation (Emmens, 1948). Assuming non-significant departure from parallelism, quick approximations of relative potency (but no other information) may be obtained by solving the following equation for M: Log ratio of potency, M = ID/B where: I = log dose ratio (difference between log doses): identical for both substances D = difference in mean responses between preparations B = difference in mean responses between high and low doses, whence, Antilog M = relative potency. Alternatively, M may be obtained graphically by simple measurement of the difference between log doses on the abscissa (e.g. fig. 2) corresponding to response, preferably the mean response for all groups on the ordinate (see also, Morley et al., 1972).
3.2. Assay of HeLa cell supernatant at d(/'jbrent levels (4 point design) A collaborator was provided with a sample of supernatant from a 72 hr culture of Helm cells ('parent') and a sample of medium of the same composition as that used for culture ('control') and was invited to submit for assay a number of dilutions of one sample in the other. By request, only the 'parent' and 'control' samples were identified by labelling; the dilutions were coded. Seven coded samples were thus provided, and were assayed together with the two identified samples, and with Con A, all preparations at two dose levels. Capillaries were prepared from LCL 7 in the 'control' medium. From the potency estimates, the composition of each coded dilution was finally calculated and expressed as percentage by volume of 'parent' in 'control', for comparison with the true dilutions then revealed by the collaborator. The comparisons are shown in columns 10 and 11 of table 3. The differences between observed and true composition of the coded dilutions ranged from - 4 . 8 % to +6.8%.
3.3. Tests for reproducibiliO, o f the 4 point des•n A pool of HeLa cell supernatant (Sample A) was prepared, divided and stored frozen as described in section 2.3. On each of five separate test occasions, one bottle was allowed to thaw at room temperature and the contents divided between assays, (i) with LCL 7, and (ii) with LCL 1788 as target cells. The above series was repeated using a different pool of HeLa supernatant (Sample B) over a further five test occasions. In both series, the occasions, and the number of assays possible on each, were determined by the coincidence o f availa-
0.748 1.19 0.74 1.07 0.86 1.46 0.85 1.91 1.93
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Assays o n seven ' c o d e d ' sample o f HeLa s u p e r n a t a n t (4 p o i n t design).
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a E a c h e n t r y under U1, U2, SI, S 2 is the mean o f 4 cross d i a m e t e r s , m e a s u r e d as described in the t e x t . b B o t h samples were i d e n t i f i e d b y the c o l l a b o r a t o r prior to the e x p e r i m e n t . c Samples 7 and ' C o n t r o l ' are seen to have v i r t u a l l y zero slope ( - 0 . 0 3 3 ) , h e n c e t h e y d e v i a t e from parallelism w i t h C o n A ( - 0 . 2 6 3 - 0.265). Since U2 the p o t e n c y of b o t h samples is zero.
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Preparation (samples)
Table 3 Results o f e x p e r i m e n t
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136
E.L. Fenton, M.M. Taylor, Assay ]or mi,~ration inhibition jactors
bility, and on the quantities, of subcultures from lines 7 and 1788 on any one day. Generally, line 1788 was available in greater quantity at any one time than was line 7, hence the majority of comparisons were between single (2 + 2) assays with line 7 and replicates (2 + 2) on line 1788. Separate Con A solutions, prepared as described in section 2.4, were used in each assay. In all, 31 assays were carried out during a period of several weeks. The resuls from both HeLa supernatants (A and B) are shown in table 4 and their analyses in table 5. The results of these experiments may be summarised: i) No evidence of systematic deterioration with time of storage (6 weeks) at - 2 0 ° C was evident in either supernatant A or B. ii) In neither experiment were the mean squares between cell lines or test occasions significant against the residual (replication) error (see later in Discussion). The pooled error variances gave limits of error (at the 5% level of significance) of -+ 14% and + 19% for supernatants A and B respectively (both cell lines combined).
3.4. Comparison between different sources of Con A (4 point design/ The Con A used in all previously reported experiments was a saltfree lyophilised powder, prepared in the laboratory by the method of Agrawal and Goldstein (1967). In order to compare this material with Con A from a different source, it was tested simultaneously with a sample purchased from Calbiochem USA. Both samples and a HeLa cell supernatant were set up together and the potency of the HeLa supernatant was expressed in terms of each Con A preparation. The Calbiochem Con A preparation contained salt, therefore its protein content was determined by measurement of absorbance at 280 nm (Unicam SP500 spectrophotometer) and adjusted to equal that of the laboratory preparation before allocating the doses. Two doses of each Con A sample (1.0 and 0.1 ~g/ml) and two equally spaced doses (1/1 and 1/10 dilutions) of the HeLa supernatant were thus compared (2 + 2 design). The difference between potency ratio estimates was 10 11%: each estinaate falling within the 95% confidence limits of the other. No significant deviations from parallelism between the log dose-response lines were encountered; the slopes for all three substances were homogeneous (X 2 = 0.027; 2 d.f.; P = 0 . 9 8 - 0 . 9 9 ) . It was concluded that, given equivalent protein concentration in the Con A solutions, either could be used satisfactorily (see table 6 for results).
3.5. Comparison between responses o f (i) lymphoM cell line cells, and (ii) guineapig peritoneal exudate cells to inhibitor in supernatants from Con-A stimulated human lymphoc:vte cultures Culture supernatants, prepared as described under Materials and methods, were harvested after 8 and 48 hr incubation and were treated to produce 'C', ' R ' and 'P' samples (see section 2.3.).
E.L. Fenton, MM. Taylor, Assay for migration inhibition factors
137
-~
1.03 + 4.8% (N = 4) 1.03 ? 5.4% (N = 4) 0.82 f 5.2% (N = 4)
28.0 f 2.9% (N = 4) 28.8 f 9.5% (N = 3) 14.7 ? 14.2% (N = 3)
1.09 + 4.6 % (N = 4) 1.06 f 8.9 % (N = 4) 0.84 i: 2.39% (N = 4)
39.3 + 3.8% (N = 4) 37.1 r 14.1% (iv = 4) 30.0 f 9.8% (N = 4)
of
RPM1 1640 me-
Diameter (mm by micrometer f coeff. variation (LCL 7)
in serum-free
supernatants
Area (arbitrary units) by planimetry f coeff. of variation (G.P.PEC)
of
cultures
of mitogen-freed
Diameter (mm) by micrometer t coeff. variation (LCL 7)
lymphocyte
by aliquots
Area (arbitrary units) by planimetry + coeff. of variation (G.P.PEC)
peripheral
cells and LCL-cells
48 hr
at 37°C of human
exudate
8 hr
of incubation
of guinea-pig peritoneal lymphocyte cultures.
G.P.PEC = Guinea-pig peritoneal exudate cells used in capillaries for area measurements. LCL 7 = Cells from lymphoid continuous line 7 used in capillaries for diameter measurements. Coefficient of variation = standard deviation expressed as percentage of mean. N = Number of observations for each sample, assayed at single dose level. See table 8 for analyses on the above data.
C (Control R (Re-constituted) P (Prc-incubated) -__ .~
Samples
Period dium
Table I Comparison between inhibition from Con A-stimulated human
1 5 1 5
R-P(MIF'effect) Replication
1 6
R-P ( M I F effect) Replication
C-R (Con A effect Replication
1 6
d.f.
3.010 0.059
0.0119 0.0343
1.0153 0.1819
0.0903 0.149
Mean square
50.5 -
0.35 -
5.6 -
0.61 -
F
Area m e a s u r e m e n t s GPE cells in capillaries
C-R (Con A effect) Replication
S o u r c e of v a r i a t i o n
0.001 > < 0 . 0 1 -
0.2 > -
0.1 > < 0.05 -
0.2 > -
P
1 6
1 6
1 6
1 6
d.f.
0.088 0.0025
0 0.0029
0.0968 0.00473
0.0018 0.0058
Mean square
32.1 -
0 -
20.4 -
0.31
F
Diameter measurements LCL 7 cells in capillaries
0.001 > < 0 . 0 1 -
N o t sig. -
0.001 > < 0.01
0.2 > -
In b o t h 8 a n d 4 8 hr harvests, d i a m e t e r m e a s u r e m e n t s on line 7 cells s h o w h i g h l y significant effects for i n h i b i t i o n d u e to MIF against non-significant effects w h i c h can be a t t r i b u t e d to residual C o n A in the samples. The same h o l d s for the 4 8 hr harvest, m e a s u r e d by areas on GPE cells, b u t in the 8 hr harvest, the MIF e f f e c t j u s t fails to reach significance at the 5% level.
48 hr harvest
8 hr harvest
Con Astimulated cultures
Incubation period o f
Table 8 Analyses of variance o n d a t a s h o w n in table 7.
140
E.L. Fenton, M.M. Taylor, Assay for migration inhibition factors
The six samples so produced were each sub-divided for migration-inhibition tests using (i) GPE-cells with area measurements by planimetry on projected images, and (ii) LCL 7 cells, with diameter measurements by micrometer. The amounts of each sample available were such that only the effects of single dose levels could be measured by either method, thereby restricting the experimental design to single level comparisons, without the benefits of multi-dose replication or of calibration with Con A. In this comparison, it was required to show the efficiency of the Sephadex procedure (Horvat et al., 1972) in removal of residual Con A, in order to avoid any overlapping of inhibitory effects in the test. The observed inhibitory effects (table 7) have therefore been isolated as those due to possible contamination with Con A (C-R effects) and those due to MIF (R-P effects), and the significance of the differences between them are shown in the analyses in table 8. The results of the experiment are summarised at the foot of table 8; conclusions and further comments appear in the Discussion.
4. Discussion It is well recognised that a satisfactory assay for a biologically active substance should give an unbiased estimate of potency, and of the limits of error, at a stated probability within which the estimate falls. Estimates of potency are conventionally made by simultaneous comparison between the responses of the test material and those of a defined biological standard (Emmens, 1948). In a comprehensive article on the measurement of lymphokines, Morley et al. (1972) describe their work on the development and potential use of material working standards for three lymphokine activities, one of which was macrophage migration inhibition. In our introductory section, we have already drawn attention to any similarities to, and essential differences between, the above work and our own. Additionally, we would stress that in no way is Con A to be regarded either as a standard for MIF activity, or as a substitute for material working standards from lymphokine sources. Bearing this in mind, we use the term 'reference preparation' throughout the present paper to describe Con A in its role as a reagent against which to calibrate our assays, mainly for process work, associated with fractionation and purification of migration inhibitory substances. The four point assay employing only two dose levels of each substance supplies evidence about the significance of difference between substances (potency ratio); it permits calculation of regression equations (slopes) for the individual lines and tests for departure from parallelism between them. It provides no test for curvature of regression, although any observed departure from parallelism may, indirectly, indi-
E.L. Fenton, M.M. Taylor, Assay for migration inhibition Jactors
14 l
cate curvature in one or both lines. Tests for curvature require the inclusion of more than two points on each line, hence the eight and six point assays were conducted as preliminaries (3.1.), to validate the use of the simpler four point design. The preliminary analyses of variance on the multi-point assays showed no significant differences either between the errors attributable to slide assemblies or between the capillaries within them, using the four fold replication, and the method of obtaining the four mean responses, d~scribed in section 2.9. On the question of replication, it is sometimes overlooked that increased replication for enhanced reliability of results is, in general, better directed to increasing the number of solutions initially prepared, rather than merely to replicate the number of response observations made. Limits of error of approx. +- 23% at the 95% significance level were calculated for seven potency estimates with a single test-cell line over a wide range of concentrations of HeLa inhibitor (.3.2.), and limits of approx. - 14% and -+ 19% for 16 and 15 estimates respectively over a more restricted range of inhibitor concentrations using test-cells from two different lines (3.3.), and should be compared with other published data. Results from similar numbers of observations for fair comparison were, however, not readily found in the literature, although limits of 1 . 0 6 - 1 . 9 0 about a mean potency ratio of 1.42 (i.e. 25.4% to +33.8%) are quoted by Morley et al. (1972) for a singJe 2 + 2 assay on macrophage MIF. The choice of inhibitory test material used in the present work (mainly from HeLa cultures) was dictated by its ready availability compared to that of the Con A-induced human MIF featured in the final experiment (3.5.), in which information was sought on the sensitivity of LCL cells to inhibition by this type of material. Despite the limitations imposed by the restricted amounts of human M | F available, it is concluded that migration of the LCL test cells was inhibited by the human MIF, and that the observed inhibitions were not due to contamination of the samples with residual Con A. In the early experiments, attempts to trace out the peripheries of migration zones from LCL cells had to be abandoned because, unlike guinea-pig PE cells, the projected images were of insufficient contrast for good reproducibility. Under the microscope, however, the zones were brightly illuminated and their diameters could be measured rapidly with good reproducibility using a micrometer as described. Preliminary d o s e - r e s p o n s e curves plotted from the d i a m e t e r - l o g dose relationships were only slightly affected by arithmetic transformation to areas. Therefore, diameter measurements were used, untransformed, throughout the present work. Comparisons of precision between diameter and area measurements may be made from the standard deviations shown in table 7 (expt. 3.5.), bearing in mind that, within such comparison, two different test-cell species are represented. It will of course be obvious that, once selected from a preliminary dose response curve, a response parameter should be tested in multi-point assays without transformation. The suitability or otherwise, of choice will be reflected in the parallel situation displayed between the log d o s e - r e s p o n s e lines of the substances compared.
142
E.L. benton, M.M. Taylor, Assay jor migration inhibition jactors
Acknowledgements T h e a u t h o r s gratefully a c k n o w l e d g e the c o - o p e r a t i o n o f colleagues at these laboratories: Mr. C.J. B u r m a n for p u r i f y i n g t h e Con A and providing the guinea-pig m a c r o p h a g e s , Dr. K.H. F a n t e s for the c o d e d samples a n d for helpful discussions, Dr. A.M. W h i t a k e r ( D e p t . o f V i r o l o g y ) for s u p p l y o f c u l t u r e d cells, Mr. P.A. Y o u n g ( B i o m e t r i c s D e p t . ) for assistance w i t h statistical analyses a n d for advice, a n d Miss A n n e Kane for t h e line diagrams.
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