Journal of Immunological Methods, 21 (1978) 211--216 © Elsevier/North-Holland Biomedical Press
211
K I N E T I C S O F I M M U N O G L O B U L I N P R O D U C T I O N BY C U L T U R E D HUMAN PERIPHERAL BLOOD LYMPHOCYTES
SANDRA M. McLACHLAN, BERNARD REES SMITH and REGINALD HALL Departments of Medicine and Clinical Biochemistry, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K. (Received 25 November 1977, accepted 16 January 1978)
A study of immunoglobulin synthesis by human peripheral blood lymphocytes cultured in Marbrook flasks is described. Maximal amounts of immunoglobulin were produced by 3 weeks and most of this was synthesised between 6 and 21 days. In the region of 90% of the immunoglobulin was secreted into the medium. Pokeweed mitogen (PWM) stimulated cultures produced about 10 times as much immunoglobulin as cells cultured in medium only. After 21 days stimulated cultures produced about 150 pg of IgG, 125 pg of IgM and 50 pg of IgA per 107 cells.
INTRODUCTION P e r i p h e r a l b l o o d is t h e o n l y readily available s o u r c e o f h u m a n l y m p h o c y t e s a n d c o n s e q u e n t l y m a n y studies o f t h e i m m u n e s y s t e m in m a n are r e s t r i c t e d to investigations using t h e s e cells. R e c e n t l y , i m p r o v e d m e t h o d s have b e e n d e s c r i b e d f o r t h e c u l t u r e o f l y m p h o c y t e s and o f these, M a r b r o o k ' s t e c h n i q u e a p p e a r s t o be t h e m o s t successful ( M a r b r o o k , 1 9 6 7 ; Maizels a n d Dresser, 1 9 7 7 ; N o r t h a n d Maizels, 1977). C o n s e q u e n t l y , we h a v e used t h e M a r b r o o k s y s t e m t o c u l t u r e h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s and in this p a p e r we d e s c r i b e a s t u d y o f t h e kinetics o f IgA, IgG a n d IgM p r o d u c t i o n b y t h e cell cultures. MATERIALS AND METHODS L y m p h o c y t e separation and culture H e p a r i n i s e d b l o o d (100 m l ) f r o m n o r m a l d o n o r s was d i l u t e d w i t h an e q u a l v o l u m e o f Earle's b a l a n c e d salt s o l u t i o n (EBSS), l a y e r e d on F i c o l l - - H y p a q u e a n d c e n t r i f u g e d at 4 0 0 X g f o r 30 m i n ( B C y u m , 1976). A f t e r washing 3 t i m e s w i t h EBSS, t h e cells w e r e r e s u s p e n d e d in c u l t u r e m e d i u m and c o u n t e d ; recoveries o f l y m p h o c y t e s averaged 85% a n d t h e viability, assessed b y t r y p a n b l u e e x c l u s i o n , was g r e a t e r t h a n 95%. T h e cells w e r e i n c u b a t e d in 1 m l v o l u m e s at densities o f 107/ml in M a r b r o o k flasks w i t h an o u t e r reservoir o f 25 ml. T h e c u l t u r e m e d i u m was R P M I 1 6 4 0 , b u f f e r e d w i t h NaHCO3 a n d
212 supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 pg/ml) and 10% heat-inactivated foetal calf serum (Flow Laboratories, Lot 428136). Cultures were maintained at 37°C in a mixture of 5% CO2 in air. At the end of the culture period the cell suspensions were removed from the inner chambers, centrifuged at 200 × g, and the culture supernatants separated from the cell pellets. Both supernatants and cell pellets were then stored at --70°C. Cell pellets were extracted after freezing and thawing by homogenisation in culture medium followed by centrifugation at 100,000 × g for 30 min. The supernatants, termed the 'soluble cell extracts', were collected and analysed. Peripheral blood lymphocytes from 10 normal donors were studied. The cells from some donors were cultured with medium alone; other cultures were stimulated with pokeweed mitogen (PWM, Grand Island, 10 pl/ml). Triplicate flasks were usually harvested at the following time intervals: 0, 3, 6, 10, 21 and 28 days.
Radioimmunoassay for IgG Human IgG was prepared from a pool (6 donors) of normal human serum b y precipitation in 1.6 M ammonium sulphate, followed by chromatography on Sephadex G-200 (Smith et al., 1969). The purified IgG was labelled with 12sI to a specific activity of about 1 pCi/pg using the iodine monochloride m e t h o d (McFarlane, 1958; Smith, 1971), run on Sephadex G-200 to remove aggregates and free 12sI and stored at --70°C until required. Antisera to human IgG were prepared in guinea pigs and coupled to cyanogen bromideactivated cellulose {Wide et al., 1974). Suspensions of the cellulose antiserum complex were incubated with test material for 2 h at room temperature in buffer (50 mM phosphate, pH 7.4, containing 10 mM EDTA, 0.2% bovine serum albumin, 0.02% NAN3, 80 mM NaC1 and 0.05% 'Tween'). Labelled IgG (40,000 cpm) was then added and the suspension rotary mixed for a further 24 h. After washing by centrifugation and resuspension in 150 mM NaC1 containing 0.2% Tween, the amount of [~2sI]IgG b o u n d to the cellulose-antiserum complex was determined. The quantity of IgG in test samples was estimated from a plot of % [~2sI]IgG b o u n d versus quantity of standard amounts of IgG added. The assay system could readily detect 3 ng of IgG and was completely specific for IgG in the concentration ranges of test materials studied.
Radioimmunoassay for IgM and IgA Human IgM was prepared from the serum of a patient with an IgM myeloma by chromatography on Sepharose 4B. Similarly, IgA (dimer) was prepared from the serum of a patient with an IgA myeloma by chromatography on Sephadex G-200. The IgM and IgA were labelled with 12sI to a specific activity of a b o u t 100 gCi/gg using a modification of the Marchalonis (1969) lactoperoxidase method (Mukhtar et al., 1975), run on Sepharose 4B
213 or G-200 to remove aggregates and free ~2sI and stored at --70°C until required. Labelled IgM was always used within a few days of iodination. Radioimmunoassays for IgM and IgA were carried out exactly as described for IgG and the methods were similarly sensitive and specific.
Stability of immunoglobulins in culture In order to investigate the stability of immunoglobulins in culture, 12sIlabelled IgG was added to a series of Marbrook flasks containing peripheral blood lymphocytes. After various periods of time, the culture supernatants were analysed by chromatography on a 90 cm × 2.5 cm column of Sephadex G-200 in 150 mM NaC1. Data analysis Estimates of the amounts of immunoglobulin produced by an individual donor at a particular time were based on 3 separate cultures except in a few cases when only duplicate samples were available. As these triplicates (or occasionally duplicates) were in reasonable agreement, an arithmetic mean was calculated for the a m o u n t of immunoglobulin produced by the cells from each donor at each time interval. Since variations in the values obtained from individual donors studied were considerable, the data from the group of donors were expressed as a geometric mean with 95% confidence limits of the standard error. RESULTS
Cultures stimulated with PWM Human peripheral blood lymphocytes cultured with PWM produced considerable amounts of IgA, IgM and IgG (Figs. 1 and 2). The kinetics of synthesis of all 3 immunoglobulin classes were similar and most of the immunoglobulins were produced between day 6 and day 21. Usually the amounts of IgG produced were greater than IgM and the amounts of IgM produced greater than IgA. About 90% of the immunoglobulins synthesised by the cells were found in the culture supernatants. A slight reduction in the concentrations of IgG and IgM in the culture supernatants and cell pellets was observed between day 21 and day 28. Unstimulated cultures L y m p h o c y t e s cultured in the absence of PWM produced about 10% of the amounts of immunoglobulins synthesised in the presence of PWM (Figs. 2 and 3). The kinetics of production, the relative amounts of IgA, IgM and IgG produced and the distribution of immunoglobulins between culture supernatants and cell extracts were similar to those with PWM. Stability of immunoglobulins in culture When 12SI-labelled IgG was incubated with PWM stimulated lymphocytes
214 IgG
IgM
A
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IgA
A
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IgG
20 ~ t 3
I 6
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B
~.....~ ................. I 0
.......... o i i
I 10
I I 28 0
o. ..........
o
IgM
..o--
I 3
I 6
I 10
. ...........
I 2?
J I 28 0
...~ .................
I 3
. .o .o ......
IgA
B
. . o .................. o . . ...o'"' = _ "........ ~
~. .......... ~
I 21
. " ..c*......... ........
I (
] ~0
~, . . . .
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f :'
Days in culture
Fig. 1. Geometric m e a n (o) and 95% confidence limits of the standard error (©) of i m m u n o g l o b u l i n production by peripheral blood l y m p h o c y t e s cultured with PWM in Marbrook flasks over 28 days. The data were obtained from 6 experiments, each of which used cells from a different donor. A: i m m u n o g l o b u l i n s present in the supernatant. B: i m m u n o g l o b u l i n s present in the soluble cell extract.
I00F
~
~
Pg
immunoglobulin produced per
0
I
I
I
I
I
•
107 ceils 10
B
I 0
I
I
I
I
J
3
6
10
21
28
Days in culture
Fig. 2. Time-course of i m m u n o g l o b u l i n production by peripheral blood l y m p h o c y t e s from a typical individual donor. A: cultures stimulated with PWM. B: cells cultured in m e d i u m only. ©, IgG; "%IgM; A, IgA.
215
r
201 -/ 15
A
IgG P ...................
A
IgM
A
IgA
,o ........ .o
..." .P'"" -o ........... 0
fmmunoglobulin producer per 10 7 cells
I 4F 2 I--
I
I
1
L
I
(
I
L
'" "'o . . . . I
I
I
..o,'""°
I
I
.....
.................................. I I I
o
1
B IgG ~...~..¢.....o L 0
I 3
I 6
................. I 10
B ,o ....... I 21
IgM
,o ~ . - - o I L I I 28 0 3 6
B
IgA
.o ................. I 10
o ......... .o --;"~'"o'"'"~ i I I I I 21 28 0 3 6
................. I 10
.o .......... ,o I I 71 2~
Days in culture
Fig. 3. Geometric mean (e) and 95% confidence limits of the standard error (o) of immunoglobulin production by peripheral blood lymphocytes cultured in medium only in Marbrook flasks over 28 days. The data were obtained from 6 experiments, each of which used cells from a different donor. A: immunoglobulins present in the culture supernatant. B: immunoglobulins present in the soluble cell extract.
in Marbrook flasks, 12sI was f o u n d to diffuse into the m edi um reservoir at a rate of less than 2% per week. Analysis of labelled material from the culture chambers on Sephadex G-200 indicated that, even after 4 weeks, this consisted o f 94% IgG m o n o m e r and 6% aggregate.
Examination o f cultured cells Studies with fluorescein-conjugated antisera showed t hat the cultures contained IgG, IgA and IgM secreting plasma cells. Examination with t rypan blue indicated t ha t the n u m b e r of viable cells recoverable, expressed as a percentage o f the total n u m b e r seeded, fell from 100% at day 0 t o 3 0 - - 4 0 % at day 10. This was similar to the value obtained by Maizels and Dresser (1977) for murine splenocytes cultured in Marbrook flasks. By 21 days only 10% o f the original cells could be recovered from the cultures. DISCUSSION The results indicate t h a t Marbrook flasks provide an envi ronm ent in which peripheral blood l y m p h o c y t e s pr oduce considerable amounts of immunoglobulin. There was extensive variation b et w een individual donors particularly in the presence of PWM and this was in agreement with the phenom e n o n o f high and low responders to PWM r e p o r t e d by Keightley et al. (1976). Slightly mo r e IgG was pr oduced than IgM and t he amounts of IgA p r o d u c e d were in the region of 30% of the amounts of IgG. Most of the immunoglobulins synthesised in culture were secreted into the m edi um and studies with 12SI-labelled IgG suggested t hat very little degradation of secreted i mmu n o g lo b u lin occurred over the periods of culture used. Th e system o f culture described in this paper is particularly suitable for
216 the study of a n t i b o d y p r o d u c t i o n in hum an a u t o i m m u n e diseases. As evidence o f this, we have recently d e m o n s t r a t e d the p r o d u c t i o n of thyroidstimulating autoantibodies by Marbrook cultures of peripheral blood lymphocytes from patients with Graves' disease (McLachlan et al., 1977). In addition, we have also shown t ha t similar cultures of peripheral blood lymphocytes from patients with Hashimoto's disease produce thyroglobulin and microsomal antibodies (McGregor et al., in press). It would appear therefore that under the appropriate culture conditions, h u m a n peripheral blood l y m p h o c y t e s can synthesise immunoglobulins with specific an tib o d y activity. Consequently it should be possible to use this t y p e of system to investigate the i m m une cell defects involved in the develo p m e n t o f h u ma n a u t o i m m u n e disease. ACKNOWLEDGEMENTS We would like to t hank Professor A.L. Latner, Professor J. Owen, Dr. J. North, Dr. T. Platts-Mills, Dr. N. Wilcox, Mr. B. Lowe and Mr. D. Weightman for useful discussions and Mrs. Alison Scaife for technical assistance. We are most grateful to Dr. P. Riches and Mr. B. Lowe for generous gifts of m y e l o m a serum. The work was supported by grants from the Medical Research Council, t h e North of England Cancer Research Campaign, the Wellcome Trust, the League o f Friends of the Royal Victoria Infirmary, the Ernest and Minnie Dawson Cancer Trust and the Research Com m i t t ee of the Newcastle Area Health A u t h o r i t y (Teaching). REFERENCES B~byum, A., 1976, Scand. J. Immunol. 5, Suppl. 5, 9. Keightley, R.G., M.D. Cooper and A. Lawton, 1976, J. Immunol. 117, 1538. Maizels, R.M. and D.W. Dresser, 1977, Immunology 32,793. Marbrook, J., 1967, Lancet ii, 1279. Marchalonis, J.J., 1969, Biochem. J. 113,299. McFarlane, A.S., 1958, Nature 182, 53. McGregor, A.M., S.M. McLachlan, B. Rees Smith, F. Clark and R.G. Hall, 1978, Immunology, in press. McLachlan, S.M., B. Rees Smith, V.B. Petersen, T.F. Davies and R. Hall, 1977, Nature 270,447. Mukhtar, E.D., B. Rees Smith, G.A. Pyle, R. Hall and P. Vice, 1975, Lancet i, 713. North, J.R. and R.M. Maizels, 1977, Immunology 32,771. Smith, B.R., 1971, Biochim. Biophys. Acta 229,649. Smith, B.R., D.S. Munro and K.J. Dorrington, 1969, Biochim. Biophys. Acta 188, 89. Wide, L., S.J. Nillius, O. Gemzell and P. Roos, 1974, Acta Endocr. Suppl. 174, 1.
211
K I N E T I C S O F I M M U N O G L O B U L I N P R O D U C T I O N BY C U L T U R E D HUMAN PERIPHERAL BLOOD LYMPHOCYTES
SANDRA M. McLACHLAN, BERNARD REES SMITH and REGINALD HALL Departments of Medicine and Clinical Biochemistry, University of Newcastle upon Tyne, Newcastle upon Tyne, U.K. (Received 25 November 1977, accepted 16 January 1978)
A study of immunoglobulin synthesis by human peripheral blood lymphocytes cultured in Marbrook flasks is described. Maximal amounts of immunoglobulin were produced by 3 weeks and most of this was synthesised between 6 and 21 days. In the region of 90% of the immunoglobulin was secreted into the medium. Pokeweed mitogen (PWM) stimulated cultures produced about 10 times as much immunoglobulin as cells cultured in medium only. After 21 days stimulated cultures produced about 150 pg of IgG, 125 pg of IgM and 50 pg of IgA per 107 cells.
INTRODUCTION P e r i p h e r a l b l o o d is t h e o n l y readily available s o u r c e o f h u m a n l y m p h o c y t e s a n d c o n s e q u e n t l y m a n y studies o f t h e i m m u n e s y s t e m in m a n are r e s t r i c t e d to investigations using t h e s e cells. R e c e n t l y , i m p r o v e d m e t h o d s have b e e n d e s c r i b e d f o r t h e c u l t u r e o f l y m p h o c y t e s and o f these, M a r b r o o k ' s t e c h n i q u e a p p e a r s t o be t h e m o s t successful ( M a r b r o o k , 1 9 6 7 ; Maizels a n d Dresser, 1 9 7 7 ; N o r t h a n d Maizels, 1977). C o n s e q u e n t l y , we h a v e used t h e M a r b r o o k s y s t e m t o c u l t u r e h u m a n p e r i p h e r a l b l o o d l y m p h o c y t e s and in this p a p e r we d e s c r i b e a s t u d y o f t h e kinetics o f IgA, IgG a n d IgM p r o d u c t i o n b y t h e cell cultures. MATERIALS AND METHODS L y m p h o c y t e separation and culture H e p a r i n i s e d b l o o d (100 m l ) f r o m n o r m a l d o n o r s was d i l u t e d w i t h an e q u a l v o l u m e o f Earle's b a l a n c e d salt s o l u t i o n (EBSS), l a y e r e d on F i c o l l - - H y p a q u e a n d c e n t r i f u g e d at 4 0 0 X g f o r 30 m i n ( B C y u m , 1976). A f t e r washing 3 t i m e s w i t h EBSS, t h e cells w e r e r e s u s p e n d e d in c u l t u r e m e d i u m and c o u n t e d ; recoveries o f l y m p h o c y t e s averaged 85% a n d t h e viability, assessed b y t r y p a n b l u e e x c l u s i o n , was g r e a t e r t h a n 95%. T h e cells w e r e i n c u b a t e d in 1 m l v o l u m e s at densities o f 107/ml in M a r b r o o k flasks w i t h an o u t e r reservoir o f 25 ml. T h e c u l t u r e m e d i u m was R P M I 1 6 4 0 , b u f f e r e d w i t h NaHCO3 a n d
212 supplemented with L-glutamine (2 mM), penicillin (50 U/ml), streptomycin (50 pg/ml) and 10% heat-inactivated foetal calf serum (Flow Laboratories, Lot 428136). Cultures were maintained at 37°C in a mixture of 5% CO2 in air. At the end of the culture period the cell suspensions were removed from the inner chambers, centrifuged at 200 × g, and the culture supernatants separated from the cell pellets. Both supernatants and cell pellets were then stored at --70°C. Cell pellets were extracted after freezing and thawing by homogenisation in culture medium followed by centrifugation at 100,000 × g for 30 min. The supernatants, termed the 'soluble cell extracts', were collected and analysed. Peripheral blood lymphocytes from 10 normal donors were studied. The cells from some donors were cultured with medium alone; other cultures were stimulated with pokeweed mitogen (PWM, Grand Island, 10 pl/ml). Triplicate flasks were usually harvested at the following time intervals: 0, 3, 6, 10, 21 and 28 days.
Radioimmunoassay for IgG Human IgG was prepared from a pool (6 donors) of normal human serum b y precipitation in 1.6 M ammonium sulphate, followed by chromatography on Sephadex G-200 (Smith et al., 1969). The purified IgG was labelled with 12sI to a specific activity of about 1 pCi/pg using the iodine monochloride m e t h o d (McFarlane, 1958; Smith, 1971), run on Sephadex G-200 to remove aggregates and free 12sI and stored at --70°C until required. Antisera to human IgG were prepared in guinea pigs and coupled to cyanogen bromideactivated cellulose {Wide et al., 1974). Suspensions of the cellulose antiserum complex were incubated with test material for 2 h at room temperature in buffer (50 mM phosphate, pH 7.4, containing 10 mM EDTA, 0.2% bovine serum albumin, 0.02% NAN3, 80 mM NaC1 and 0.05% 'Tween'). Labelled IgG (40,000 cpm) was then added and the suspension rotary mixed for a further 24 h. After washing by centrifugation and resuspension in 150 mM NaC1 containing 0.2% Tween, the amount of [~2sI]IgG b o u n d to the cellulose-antiserum complex was determined. The quantity of IgG in test samples was estimated from a plot of % [~2sI]IgG b o u n d versus quantity of standard amounts of IgG added. The assay system could readily detect 3 ng of IgG and was completely specific for IgG in the concentration ranges of test materials studied.
Radioimmunoassay for IgM and IgA Human IgM was prepared from the serum of a patient with an IgM myeloma by chromatography on Sepharose 4B. Similarly, IgA (dimer) was prepared from the serum of a patient with an IgA myeloma by chromatography on Sephadex G-200. The IgM and IgA were labelled with 12sI to a specific activity of a b o u t 100 gCi/gg using a modification of the Marchalonis (1969) lactoperoxidase method (Mukhtar et al., 1975), run on Sepharose 4B
213 or G-200 to remove aggregates and free ~2sI and stored at --70°C until required. Labelled IgM was always used within a few days of iodination. Radioimmunoassays for IgM and IgA were carried out exactly as described for IgG and the methods were similarly sensitive and specific.
Stability of immunoglobulins in culture In order to investigate the stability of immunoglobulins in culture, 12sIlabelled IgG was added to a series of Marbrook flasks containing peripheral blood lymphocytes. After various periods of time, the culture supernatants were analysed by chromatography on a 90 cm × 2.5 cm column of Sephadex G-200 in 150 mM NaC1. Data analysis Estimates of the amounts of immunoglobulin produced by an individual donor at a particular time were based on 3 separate cultures except in a few cases when only duplicate samples were available. As these triplicates (or occasionally duplicates) were in reasonable agreement, an arithmetic mean was calculated for the a m o u n t of immunoglobulin produced by the cells from each donor at each time interval. Since variations in the values obtained from individual donors studied were considerable, the data from the group of donors were expressed as a geometric mean with 95% confidence limits of the standard error. RESULTS
Cultures stimulated with PWM Human peripheral blood lymphocytes cultured with PWM produced considerable amounts of IgA, IgM and IgG (Figs. 1 and 2). The kinetics of synthesis of all 3 immunoglobulin classes were similar and most of the immunoglobulins were produced between day 6 and day 21. Usually the amounts of IgG produced were greater than IgM and the amounts of IgM produced greater than IgA. About 90% of the immunoglobulins synthesised by the cells were found in the culture supernatants. A slight reduction in the concentrations of IgG and IgM in the culture supernatants and cell pellets was observed between day 21 and day 28. Unstimulated cultures L y m p h o c y t e s cultured in the absence of PWM produced about 10% of the amounts of immunoglobulins synthesised in the presence of PWM (Figs. 2 and 3). The kinetics of production, the relative amounts of IgA, IgM and IgG produced and the distribution of immunoglobulins between culture supernatants and cell extracts were similar to those with PWM. Stability of immunoglobulins in culture When 12SI-labelled IgG was incubated with PWM stimulated lymphocytes
214 IgG
IgM
A
o .o"
c~ ..." '......
.0," •""
.."
...'
A "'o
.:
:"
..."
.. ..-
p.*"
/
,
immunoglobulin produced
~-
~0 7 oe,,s
/
per
IgA
A
~...~...,o. _
.'..,.~.'.o- . . . ' ' ° . . . . . .
0 ~ ~)--'r40~
J
J
S
..- ,~
IgG
20 ~ t 3
I 6
..,o- ......
B
~.....~ ................. I 0
.......... o i i
I 10
I I 28 0
o. ..........
o
IgM
..o--
I 3
I 6
I 10
. ...........
I 2?
J I 28 0
...~ .................
I 3
. .o .o ......
IgA
B
. . o .................. o . . ...o'"' = _ "........ ~
~. .......... ~
I 21
. " ..c*......... ........
I (
] ~0
~, . . . .
I_ 2'
f :'
Days in culture
Fig. 1. Geometric m e a n (o) and 95% confidence limits of the standard error (©) of i m m u n o g l o b u l i n production by peripheral blood l y m p h o c y t e s cultured with PWM in Marbrook flasks over 28 days. The data were obtained from 6 experiments, each of which used cells from a different donor. A: i m m u n o g l o b u l i n s present in the supernatant. B: i m m u n o g l o b u l i n s present in the soluble cell extract.
I00F
~
~
Pg
immunoglobulin produced per
0
I
I
I
I
I
•
107 ceils 10
B
I 0
I
I
I
I
J
3
6
10
21
28
Days in culture
Fig. 2. Time-course of i m m u n o g l o b u l i n production by peripheral blood l y m p h o c y t e s from a typical individual donor. A: cultures stimulated with PWM. B: cells cultured in m e d i u m only. ©, IgG; "%IgM; A, IgA.
215
r
201 -/ 15
A
IgG P ...................
A
IgM
A
IgA
,o ........ .o
..." .P'"" -o ........... 0
fmmunoglobulin producer per 10 7 cells
I 4F 2 I--
I
I
1
L
I
(
I
L
'" "'o . . . . I
I
I
..o,'""°
I
I
.....
.................................. I I I
o
1
B IgG ~...~..¢.....o L 0
I 3
I 6
................. I 10
B ,o ....... I 21
IgM
,o ~ . - - o I L I I 28 0 3 6
B
IgA
.o ................. I 10
o ......... .o --;"~'"o'"'"~ i I I I I 21 28 0 3 6
................. I 10
.o .......... ,o I I 71 2~
Days in culture
Fig. 3. Geometric mean (e) and 95% confidence limits of the standard error (o) of immunoglobulin production by peripheral blood lymphocytes cultured in medium only in Marbrook flasks over 28 days. The data were obtained from 6 experiments, each of which used cells from a different donor. A: immunoglobulins present in the culture supernatant. B: immunoglobulins present in the soluble cell extract.
in Marbrook flasks, 12sI was f o u n d to diffuse into the m edi um reservoir at a rate of less than 2% per week. Analysis of labelled material from the culture chambers on Sephadex G-200 indicated that, even after 4 weeks, this consisted o f 94% IgG m o n o m e r and 6% aggregate.
Examination o f cultured cells Studies with fluorescein-conjugated antisera showed t hat the cultures contained IgG, IgA and IgM secreting plasma cells. Examination with t rypan blue indicated t ha t the n u m b e r of viable cells recoverable, expressed as a percentage o f the total n u m b e r seeded, fell from 100% at day 0 t o 3 0 - - 4 0 % at day 10. This was similar to the value obtained by Maizels and Dresser (1977) for murine splenocytes cultured in Marbrook flasks. By 21 days only 10% o f the original cells could be recovered from the cultures. DISCUSSION The results indicate t h a t Marbrook flasks provide an envi ronm ent in which peripheral blood l y m p h o c y t e s pr oduce considerable amounts of immunoglobulin. There was extensive variation b et w een individual donors particularly in the presence of PWM and this was in agreement with the phenom e n o n o f high and low responders to PWM r e p o r t e d by Keightley et al. (1976). Slightly mo r e IgG was pr oduced than IgM and t he amounts of IgA p r o d u c e d were in the region of 30% of the amounts of IgG. Most of the immunoglobulins synthesised in culture were secreted into the m edi um and studies with 12SI-labelled IgG suggested t hat very little degradation of secreted i mmu n o g lo b u lin occurred over the periods of culture used. Th e system o f culture described in this paper is particularly suitable for
216 the study of a n t i b o d y p r o d u c t i o n in hum an a u t o i m m u n e diseases. As evidence o f this, we have recently d e m o n s t r a t e d the p r o d u c t i o n of thyroidstimulating autoantibodies by Marbrook cultures of peripheral blood lymphocytes from patients with Graves' disease (McLachlan et al., 1977). In addition, we have also shown t ha t similar cultures of peripheral blood lymphocytes from patients with Hashimoto's disease produce thyroglobulin and microsomal antibodies (McGregor et al., in press). It would appear therefore that under the appropriate culture conditions, h u m a n peripheral blood l y m p h o c y t e s can synthesise immunoglobulins with specific an tib o d y activity. Consequently it should be possible to use this t y p e of system to investigate the i m m une cell defects involved in the develo p m e n t o f h u ma n a u t o i m m u n e disease. ACKNOWLEDGEMENTS We would like to t hank Professor A.L. Latner, Professor J. Owen, Dr. J. North, Dr. T. Platts-Mills, Dr. N. Wilcox, Mr. B. Lowe and Mr. D. Weightman for useful discussions and Mrs. Alison Scaife for technical assistance. We are most grateful to Dr. P. Riches and Mr. B. Lowe for generous gifts of m y e l o m a serum. The work was supported by grants from the Medical Research Council, t h e North of England Cancer Research Campaign, the Wellcome Trust, the League o f Friends of the Royal Victoria Infirmary, the Ernest and Minnie Dawson Cancer Trust and the Research Com m i t t ee of the Newcastle Area Health A u t h o r i t y (Teaching). REFERENCES B~byum, A., 1976, Scand. J. Immunol. 5, Suppl. 5, 9. Keightley, R.G., M.D. Cooper and A. Lawton, 1976, J. Immunol. 117, 1538. Maizels, R.M. and D.W. Dresser, 1977, Immunology 32,793. Marbrook, J., 1967, Lancet ii, 1279. Marchalonis, J.J., 1969, Biochem. J. 113,299. McFarlane, A.S., 1958, Nature 182, 53. McGregor, A.M., S.M. McLachlan, B. Rees Smith, F. Clark and R.G. Hall, 1978, Immunology, in press. McLachlan, S.M., B. Rees Smith, V.B. Petersen, T.F. Davies and R. Hall, 1977, Nature 270,447. Mukhtar, E.D., B. Rees Smith, G.A. Pyle, R. Hall and P. Vice, 1975, Lancet i, 713. North, J.R. and R.M. Maizels, 1977, Immunology 32,771. Smith, B.R., 1971, Biochim. Biophys. Acta 229,649. Smith, B.R., D.S. Munro and K.J. Dorrington, 1969, Biochim. Biophys. Acta 188, 89. Wide, L., S.J. Nillius, O. Gemzell and P. Roos, 1974, Acta Endocr. Suppl. 174, 1.
