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Temperature dependence of antigen-specific rosette formation by lymphocytes from immunised mice
Journal o f Immunological Methods, 15 (1977) 291--297
291
© Elsevier/North-Holland Biomedical Press
TEMPERATURE DEPENDENCE OF ANTIGEN-SPECIFIC ROSETTE F O R M A T I O N BY L Y M P H O C Y T E S F R O M I M M U N I S E D MICE
M. WANSBROUGH-JONES *, D. MIRJAH, M. DRUGUET ** and M.B. PEPYS Department o f Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS, U.K.
(Received 16 November 1976, accepted by 29 November 1976)
Spleen cell suspensions from mice undergoing a secondary response to sheep erythrocytes (SRBC) contained about one tenth as many specific antigen-binding, rosette-forming cells (RFC) when they had been washed at 37°C instead of 4°C before rosetting. This difference was correlated with the presence of IgG anti-SRBC antibody in the serum, and the 37°C washings of immunised spleen cells could passively allergise non-immune spleen cells at 4°C for specific rosette formation which was inhibitable by anti-mouse F(ab)2 serum. The RFC from actively immunised mice were lymphocytes and not macrophages by morphological and cytochemical criteria. It is suggested that the 37°C-labile RFC are lymphocytes to which IgG antibodies bind in the cold. These data indicate that in the use of antigen-binding cell assays to monitor immunological responses, it is necessary to wash lymphocytes at 37°C before testing. INTRODUCTION F o l l o w i n g i m m u n i s a t i o n there is an increase in the n u m b e r o f specific a n t i g e n - b i n d i n g l y m p h o c y t e s , and one simple and w i d e l y used t e c h n i q u e by w h i c h this p h e n o m e n o n can be m o n i t o r e d is the f o r m a t i o n o f rosettes bet w e e n l y m p h o c y t e s and i n d i c a t o r e r y t h r o c y t e s . T h e i n d i c a t o r cells m a y e i t h e r themselves c o n s t i t u t e the specific antigen or t h e y m a y be passively sensitised with it. In a d d i t i o n to the specific B cell clones w h i c h are actively synthesising the a p p r o p r i a t e specific i m m u n o g l o b u l i n it has long been recognised t h a t non-specific cells m a y passively a d s o r b h u m o r a l a n t i b o d y and t h e r e b y f o r m rosettes (Bussard, 1 9 6 4 ) . 'Passive' rosettes can be p r o d u c e d b y i n c u b a t i o n o f spleen cells in vitro with a n t i b o d y - r i c h s e r u m , or b y the injection o f such s e r u m in vivo ( K a p p and Banacerraf, 1 9 7 2 ) . M a c r o p h a g e s are well k n o w n t o a d s o r b c y t o p h i l i c a n t i b o d y and f o r m a t i o n o f 'passive' rosettes and p a r t i c u l a r l y m i x e d rosettes, cells b i n d i n g t w o distinct antigens in d o u b l y i m m u n i s e d mice, has been s h o w n to be essentially a p r o p e r t y o f a d h e r e n t cells (Biozzi et al., 1 9 6 6 ; K a p p and Banacerraf, 1 9 7 2 ) . T h e d e m o n s t r a t i o n * Present address: Liver Unit, Kings College Hospital, London SW5, U.K. ** Present address: Hospital Edouard Herriot, Lyons, France.
292
that IgG on the surface of hum a n peripheral l y m p h o c y t e s , washed at 4°C, can be eluted by washing them at 37°C ( L obo et al., 1975; Kumagai et al., 1975; Pepys et al., 1976) raised the possibility that some rosette forming cells (RFC) might be l y m p h o c y t e s binding antigen by means of IgG antibody passively adsorbed in the cold. The effect was therefore studied on numbers of RFC in the spleens of immunised mice o; washing at 37°C before forming rosettes. M A T E R I A L S AND M E T H O D S
Animals Balb/c mice bred at the Royal Postgraduate Medical School were used between 3 and 12 m o n t h s of age.
Immunisation 5 × 108 sheep red bl ood cells (SRBC) in phosphate buffered saline pH 7.3 (PBS) were given by intraperitoneal injection. Mice received either one or f o ur doses within twelve m ont hs . All animals were sacrificed three days after the last dose.
Spleen cells Suspensions were prepared by crushing spleens with a silicone rubber bung in a plastic petri dish, and then filtering the resulting suspension in phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) rapidly through a 2.0 ml column of c o t t o n wool which had previously been boiled and preincubated in 10% foetal calf serum in PBS. The filtered cells were then washed three times with PBS/BSA at 4°C. Cell suspensions prepared at 4°C were ke pt on ice t h r o u g h o u t except when being centrifuged at 4 ° C. Cell suspensions prepared at 37°C were first brought to that temperature in a water bath and then were centrifuged at 37°C in a warm centrifuge (L.T.E. Ltd., Oldham, Lancs). The PBS/BSA used for washing was also k ep t at 37 ° C.
Elution of immunoglobulin from spleen cells 1.5 ml of a spIeen cell suspension from a secondarily immunised mouse, prepared at 4°C and containing 34.5 × 106 cells per ml was incubated at 37°C for 20 min before centrifugation (200 g, 5 min, 37°C). As a control an identical aliquot of the same spleen cell suspension was prepared at 37°C and then washed at 37°C in parallel with the test suspension. The eluates which were cell-free were bot h placed at 4°C and used immediately.
Transfer of rosette-forming capacity The cell pellets f r om separate 0.1 ml volumes of a spleen cell suspension (17.5 × 106 cells per ml), prepared at 37°C from an unimmunised mouse were incubated at 4°C for 30 min with 0.1 ml volumes of the following: test
293
and control eluates as above, neat serum from a primarily immunised, a secondarily immunised, and an unimmunised mouse. The cells were then washed three times in PBS/BSA at 4 ° C before forming rosettes.
Rosette formation Rosettes were prepared, a's described elsewhere (McConnell et al., 1969) and c o u n t e d in a 10 t~l h a e m o c y t o m e t e r in which at least 25 × 10 ~ nucleated cells were scanned. Each cell suspension was tested in duplicate, and the results are presented as arithmetic means. Inhibition tests with anti-mouse F(ab)2 serum were p e r f o r m e d as described elsewhere (McConnell et al.,
1969). Cytochemical staining of spleen cells A drop of a preparation of resuspended rosetted cells was gently spread over a clean glass slide and air-dried. After fixation in 95% ethanol/formaldeh y d e (9 : 1) cells containing peroxidase were stained by the 2-2-diaminobenzidine reaction (Graham and Karnovsky, 1966) and counter-stained with m e t h y l green. These smears were examined under oil immersion (×1000) and the n u m b e r of peroxidase-positive RFC and the p r o p o r t i o n of peroxidasepositive and negative m o n o n u c l e a r cells were counted. Smears of spleen cell preparations were also stained for non-specific esterase (Li et al., 1973).
Surface immunoglo bulin bearing cells These were d et ect ed by direct i m m u n o f l u o r e s c e n c e using fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin ant i body. Staining and washing were p e r f o r m e d at 4 ° C.
Serum haemagglutinating antibody Total and IgG a n t i b o d y was titrated as described elsewhere (Pepys, 1974). RESULTS
Effect of washing of spleen cells at different temperatures Spleen cells from secondarily immunised mice washed at 37°C before formation o f rosettes cont ai ned only a b o u t one-tenth as m any RFC as the same cells washed at 4°C. This e f f e c t was n o t d e t e c t e d when spleen cells were tested f r o m mice 3 days after primary immunisation (table 1) and it correlated with the presence in the serum of IgG a n t i b o d y to SRBC.
Transfer of rosette-forming capacity Spleen cells f r om unimmunised mice f o r m e d a low background number' of rosettes with SRBC. However, after pr e i n cubat i on at 4°C with either the eluate of spleen cells f r om a secondarily immunised mouse, or serum from such an animal containing IgG a n t i b o d y , t hey acquired the ability to bind SRBC. The eluate of cells previously washed at 37°C, or serum lacking IgG
294 TABLE 1 E f f e c t o f washing t e m p e r a t u r e o n n u m b e r o f R F C in spleen. Mouse
Immunisation
Washing temperature (°C)
RFC/106 lymphocytes
S e r u m agglutination titre (--log 2 ) Total
1
Secondary
2
Secondary
3
Primary
4
Primary
4 37 4 37 4 37 4 37
54,650 4509 61,793 7877 2636 2423 1375 846
IgG
9
9
11
9
4
0
3
0
1 and 2) mice given 5 X 10 s SRBC i n t r a p e r i t o n e a l l y on 4 occasions, and killed t h r e e days after the last dose. 3 and 4) mice given a single i n t r a p e r i t o n e a l dose o f 5 X 10 s SRBC and killed 3 days later.
TABLE 2 T r a n s f e r o f r o s e t t e f o r m i n g capacity. Preincubation a
RFC/106 lymphocytes
PBS/BSA Test eluate b C o n t r o l eluate e IgG anti-SRBC s e r u m d IgM anti-SRBC s e r u m e Control serum f Test eluate and anti-F(ab)2 s e r u m g
429 6,105 495 14,400 433 462 450
a Spleen cells f r o m an u n i m m u n i s e d m o u s e were washed at 37°C b e f o r e i n c u b a t i o n at 4°C as s h o w n . T h e y were t h e n w a s h e d at 4°C b e f o r e f o r m i n g rosettes. b Eluate o f spleen cells f r o m s e c o n d a r i l y i m m u n i s e d m o u s e washed at 4°C and t h e n incub a t e d at 37°C. c Eluate o f spleen cells f r o m s e c o n d a r i l y i m m u n i s e d m o u s e w a s h e d at 37°C t h e n incub a t e d at 37°C. d S e r u m f r o m secondarily i m m u n i s e d m o u s e with a --log 2 9 titre o f IgG anti-SRBC antibody. e S e r u m from a primarily i m m u n i s e d m o u s e with a - - l o g : 3 titre o f IgM anti-SRBC antibody, f S e r u m f r o m an u n i m m u n i s e d m o u s e . g A n t i - m o u s e F(ab)2 s e r u m was a d d e d to spleen cells p r e i n c u b a t e d with test eluate. The cells were i n c u b a t e d at 4°C for 15 m i n b e f o r e f o r m i n g rosettes.
3
a
content
923 339
13 3
936 342
Total
from mice after
Peroxidase positive
cells
Nucleated
Peroxidase negative
cells and RFC
of spleen
1.4 0.8
’
of nucleated
SRBC.
0 0
Peroxidase positive
cells forming
doses of 5 X 10’
50 31
Peroxidase negative
RFC
immunisation.
% positive
secondary
a Rosetted spleen cells from 2 different mice immunised with 4 intraperitoneal b RFC identified in fixed preparations. ’ RFC per lo6 lymphocytes counted in suspension before smearing. Percentage
1 2
Experiment
Peroxidase
TABLE
rosettes
shown
54,650 59,823
’
in brackets.
(5.5%) (6.0%)
RFC per lo6 lymphocytes
296
antibody, could not confer rosette-forming capacity (table 2). The rosettes in these different preparations were inhibited by anti-F(ab)2 serum.
Effect of washing at 37~C on the proportion of immunoglobulin bearing cells in the spleen Surface immunoglobulin was detected on 36 to 41% of spleen cells irrespective of the washing temperature. Identity of the RFC No RFC contained peroxidase in the smears of rosetted spleen cell suspensions and the proportion of peroxidase-positive cells was considerably less than the proportion of RFC (table 3). About half as many cells contained non-specific esterase as contained peroxidase, and they were all large cells morphologically distinct from lymphocytes. DISCUSSION
The present findings indicate that in mice undergoing a secondary response to SRBC the majority of the RFC detected in spleen cell suspensions prepared in the cold are non-specific cells which have been passively allergised, probably with IgG antibodies. RFC all had the morphology of lymphocytes and none contained peroxidase, a marker for monocytes. Our spleen cell suspensions, which has been c o t t o n wool filtered, contained very few mononuclear phagocytes (identified by the presence of intracellular colloidal gold after in vivo intravenous injection), and there were always less cells containing either peroxidase or non-specific esterase (another monocyte/macrophage marker), than there were RFC. We extend earlier studies by demonstrating elution of antibody from 'passive' RFC and its uptake on fresh non-specific cells. Furthermore we have shown that, at least in Balb/c mice immunised with SRBC, a large number of the acceptor cells are lymphocytes and that the phenomenon is temperature-dependent. In the original studies of Biozzi et al. (1966), the absence of any rosette formation by l y m p h o c y t e s from normal mice after in vitro incubation at 4°C with h y p e r i m m u n e serum could be attributable to the technique used. The temperature at which the cells were washed after cold incubation, and before rosetting, was n o t specified, and if it was room temperature rather than 4°C passive allergisation would be greatly diminished. Also their rosetring technique with cells in the presence of 5--10% homologous normal serum being permitted to settle for 16 h is in marked contrast to the centrifugation-resuspension m e t h o d of Coombs (McConnell et al., 1969) used by us. The effect of washing spleen cells at 37°C before rosetting suggests that the passive uptake of antibody is an in vitro artefact which occurs as the cells are processed in the cold. However, in contrast to human peripheral
297 b l o o d l y m p h o c y t e s ( K u m a g a i et al., 1 9 7 5 ; L o b o e t al., 1 9 7 5 ; P e p y s et al., 1 9 7 6 ) washing m u r i n e spleen cells at 3 7 ° C r a t h e r t h a n 4°C did n o t r e d u c e t h e n u m b e r o f cells s t a i n e d b y f l u o r e s c e n t a n t i - i m m u n o g l o b u l i n a n t i b o d i e s . This suggests t h a t the g r e a t m a j o r i t y o f the m u r i n e cells which bind antib o d y in t h e cold, a n d are t h e r e b y able t o f o r m r o s e t t e s , p r o b a b l y are B cells w h i c h a l r e a d y h a v e stable s u r f a c e i m m u n o g l o b u l i n of their own. I t w o u l d n o n e t h e l e s s be i n t e r e s t i n g t o c o m p a r e w i t h a p p r o p r i a t e r e a g e n t s the class d i s t r i b u t i o n o f i m m u n o g l o b u l i n on m u r i n e cells w a s h e d at 4°C a n d 37°C, p a r t i c u l a r l y as the m a j o r i t y o f h u m a n p e r i p h e r a l l y m p h o c y t e s which bind IgG in the c o l d are n o t B cells ( L o b o a n d H o r w i t z , 1976). I t is clear t h a t t h e s a m e p r e c a u t i o n s o f p r e w a s h i n g at 3 7 ° C a n d / o r the use o f F(ab)2 r e a g e n t s ( W i n c h e s t e r et al., 1 9 7 5 ; P e p y s et al., 1 9 7 6 ) as are necessary f o r precise e v a l u a t i o n o f s u r f a c e i m m u n o g l o b u l i n s t r u c t u r e s on h u m a n l y m p h o c y t e s are also r e q u i r e d in mice. ACKNOWLEDGEMENTS We wish t o t h a n k Dr. M.F. G r e a v e s f o r h e l p f u l discussions a n d t h e gift o f f l u o r e s c i n a t e d g o a t a n t i - m o u s e Ig a n t i b o d y . REFERENCES Biozzi, G., C. Stiffel, D. Mouton, M. Liacopoulos-Briot, C. Decreusefond and Y. Bouthillier, 1966, Ann. Inst. Pasteur 110, 7. Bussard, A. 1964, Acad. Sci. Paris 259, 3411. Graham, R.C. and M.J. Karnovsky, 1966, J. Histochem. Cytochem. 14, 291. Kapp, J. and B. Benacerraf, 1972, Eur. J. Immunol. 2 467. Kumagai, K., T. Abo, T. Sekizaka and M. Sasaki, 1975, J. Immunol. 115,982. Li, C.Y., K.W. Lain and L.T. Yam, 1973, J. Histochem. Cytochem. 21, 1. Lobo, P.I., F.B. Westervelt and D.A. Horwitz, 1975, J. Immunol. 114, 116. McConnell, I., A. Munro, B.W. Gurner and R.R.A. Coombs, 1969, Int. Arch. Allergy Appl. Immunol. 35, 209. Pepys, M.B., 1974, J. Exp. Med. 140, 126. Pepys, M.B., C. Sategna-Guidetti, D.D. Mirjah, M.K. Wansbrough-Jones and A.C. Dash, 1976, Clin. Exp. Immunol. 26, 91. Winchester, R.J., S.M. Fu, T. Hoffman and H.G. Kunkel, 1975, J. Immunol. 114, 1210.
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© Elsevier/North-Holland Biomedical Press
TEMPERATURE DEPENDENCE OF ANTIGEN-SPECIFIC ROSETTE F O R M A T I O N BY L Y M P H O C Y T E S F R O M I M M U N I S E D MICE
M. WANSBROUGH-JONES *, D. MIRJAH, M. DRUGUET ** and M.B. PEPYS Department o f Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS, U.K.
(Received 16 November 1976, accepted by 29 November 1976)
Spleen cell suspensions from mice undergoing a secondary response to sheep erythrocytes (SRBC) contained about one tenth as many specific antigen-binding, rosette-forming cells (RFC) when they had been washed at 37°C instead of 4°C before rosetting. This difference was correlated with the presence of IgG anti-SRBC antibody in the serum, and the 37°C washings of immunised spleen cells could passively allergise non-immune spleen cells at 4°C for specific rosette formation which was inhibitable by anti-mouse F(ab)2 serum. The RFC from actively immunised mice were lymphocytes and not macrophages by morphological and cytochemical criteria. It is suggested that the 37°C-labile RFC are lymphocytes to which IgG antibodies bind in the cold. These data indicate that in the use of antigen-binding cell assays to monitor immunological responses, it is necessary to wash lymphocytes at 37°C before testing. INTRODUCTION F o l l o w i n g i m m u n i s a t i o n there is an increase in the n u m b e r o f specific a n t i g e n - b i n d i n g l y m p h o c y t e s , and one simple and w i d e l y used t e c h n i q u e by w h i c h this p h e n o m e n o n can be m o n i t o r e d is the f o r m a t i o n o f rosettes bet w e e n l y m p h o c y t e s and i n d i c a t o r e r y t h r o c y t e s . T h e i n d i c a t o r cells m a y e i t h e r themselves c o n s t i t u t e the specific antigen or t h e y m a y be passively sensitised with it. In a d d i t i o n to the specific B cell clones w h i c h are actively synthesising the a p p r o p r i a t e specific i m m u n o g l o b u l i n it has long been recognised t h a t non-specific cells m a y passively a d s o r b h u m o r a l a n t i b o d y and t h e r e b y f o r m rosettes (Bussard, 1 9 6 4 ) . 'Passive' rosettes can be p r o d u c e d b y i n c u b a t i o n o f spleen cells in vitro with a n t i b o d y - r i c h s e r u m , or b y the injection o f such s e r u m in vivo ( K a p p and Banacerraf, 1 9 7 2 ) . M a c r o p h a g e s are well k n o w n t o a d s o r b c y t o p h i l i c a n t i b o d y and f o r m a t i o n o f 'passive' rosettes and p a r t i c u l a r l y m i x e d rosettes, cells b i n d i n g t w o distinct antigens in d o u b l y i m m u n i s e d mice, has been s h o w n to be essentially a p r o p e r t y o f a d h e r e n t cells (Biozzi et al., 1 9 6 6 ; K a p p and Banacerraf, 1 9 7 2 ) . T h e d e m o n s t r a t i o n * Present address: Liver Unit, Kings College Hospital, London SW5, U.K. ** Present address: Hospital Edouard Herriot, Lyons, France.
292
that IgG on the surface of hum a n peripheral l y m p h o c y t e s , washed at 4°C, can be eluted by washing them at 37°C ( L obo et al., 1975; Kumagai et al., 1975; Pepys et al., 1976) raised the possibility that some rosette forming cells (RFC) might be l y m p h o c y t e s binding antigen by means of IgG antibody passively adsorbed in the cold. The effect was therefore studied on numbers of RFC in the spleens of immunised mice o; washing at 37°C before forming rosettes. M A T E R I A L S AND M E T H O D S
Animals Balb/c mice bred at the Royal Postgraduate Medical School were used between 3 and 12 m o n t h s of age.
Immunisation 5 × 108 sheep red bl ood cells (SRBC) in phosphate buffered saline pH 7.3 (PBS) were given by intraperitoneal injection. Mice received either one or f o ur doses within twelve m ont hs . All animals were sacrificed three days after the last dose.
Spleen cells Suspensions were prepared by crushing spleens with a silicone rubber bung in a plastic petri dish, and then filtering the resulting suspension in phosphate buffered saline (PBS) containing 0.2% bovine serum albumin (BSA) rapidly through a 2.0 ml column of c o t t o n wool which had previously been boiled and preincubated in 10% foetal calf serum in PBS. The filtered cells were then washed three times with PBS/BSA at 4°C. Cell suspensions prepared at 4°C were ke pt on ice t h r o u g h o u t except when being centrifuged at 4 ° C. Cell suspensions prepared at 37°C were first brought to that temperature in a water bath and then were centrifuged at 37°C in a warm centrifuge (L.T.E. Ltd., Oldham, Lancs). The PBS/BSA used for washing was also k ep t at 37 ° C.
Elution of immunoglobulin from spleen cells 1.5 ml of a spIeen cell suspension from a secondarily immunised mouse, prepared at 4°C and containing 34.5 × 106 cells per ml was incubated at 37°C for 20 min before centrifugation (200 g, 5 min, 37°C). As a control an identical aliquot of the same spleen cell suspension was prepared at 37°C and then washed at 37°C in parallel with the test suspension. The eluates which were cell-free were bot h placed at 4°C and used immediately.
Transfer of rosette-forming capacity The cell pellets f r om separate 0.1 ml volumes of a spleen cell suspension (17.5 × 106 cells per ml), prepared at 37°C from an unimmunised mouse were incubated at 4°C for 30 min with 0.1 ml volumes of the following: test
293
and control eluates as above, neat serum from a primarily immunised, a secondarily immunised, and an unimmunised mouse. The cells were then washed three times in PBS/BSA at 4 ° C before forming rosettes.
Rosette formation Rosettes were prepared, a's described elsewhere (McConnell et al., 1969) and c o u n t e d in a 10 t~l h a e m o c y t o m e t e r in which at least 25 × 10 ~ nucleated cells were scanned. Each cell suspension was tested in duplicate, and the results are presented as arithmetic means. Inhibition tests with anti-mouse F(ab)2 serum were p e r f o r m e d as described elsewhere (McConnell et al.,
1969). Cytochemical staining of spleen cells A drop of a preparation of resuspended rosetted cells was gently spread over a clean glass slide and air-dried. After fixation in 95% ethanol/formaldeh y d e (9 : 1) cells containing peroxidase were stained by the 2-2-diaminobenzidine reaction (Graham and Karnovsky, 1966) and counter-stained with m e t h y l green. These smears were examined under oil immersion (×1000) and the n u m b e r of peroxidase-positive RFC and the p r o p o r t i o n of peroxidasepositive and negative m o n o n u c l e a r cells were counted. Smears of spleen cell preparations were also stained for non-specific esterase (Li et al., 1973).
Surface immunoglo bulin bearing cells These were d et ect ed by direct i m m u n o f l u o r e s c e n c e using fluorescein isothiocyanate-conjugated goat anti-mouse immunoglobulin ant i body. Staining and washing were p e r f o r m e d at 4 ° C.
Serum haemagglutinating antibody Total and IgG a n t i b o d y was titrated as described elsewhere (Pepys, 1974). RESULTS
Effect of washing of spleen cells at different temperatures Spleen cells from secondarily immunised mice washed at 37°C before formation o f rosettes cont ai ned only a b o u t one-tenth as m any RFC as the same cells washed at 4°C. This e f f e c t was n o t d e t e c t e d when spleen cells were tested f r o m mice 3 days after primary immunisation (table 1) and it correlated with the presence in the serum of IgG a n t i b o d y to SRBC.
Transfer of rosette-forming capacity Spleen cells f r om unimmunised mice f o r m e d a low background number' of rosettes with SRBC. However, after pr e i n cubat i on at 4°C with either the eluate of spleen cells f r om a secondarily immunised mouse, or serum from such an animal containing IgG a n t i b o d y , t hey acquired the ability to bind SRBC. The eluate of cells previously washed at 37°C, or serum lacking IgG
294 TABLE 1 E f f e c t o f washing t e m p e r a t u r e o n n u m b e r o f R F C in spleen. Mouse
Immunisation
Washing temperature (°C)
RFC/106 lymphocytes
S e r u m agglutination titre (--log 2 ) Total
1
Secondary
2
Secondary
3
Primary
4
Primary
4 37 4 37 4 37 4 37
54,650 4509 61,793 7877 2636 2423 1375 846
IgG
9
9
11
9
4
0
3
0
1 and 2) mice given 5 X 10 s SRBC i n t r a p e r i t o n e a l l y on 4 occasions, and killed t h r e e days after the last dose. 3 and 4) mice given a single i n t r a p e r i t o n e a l dose o f 5 X 10 s SRBC and killed 3 days later.
TABLE 2 T r a n s f e r o f r o s e t t e f o r m i n g capacity. Preincubation a
RFC/106 lymphocytes
PBS/BSA Test eluate b C o n t r o l eluate e IgG anti-SRBC s e r u m d IgM anti-SRBC s e r u m e Control serum f Test eluate and anti-F(ab)2 s e r u m g
429 6,105 495 14,400 433 462 450
a Spleen cells f r o m an u n i m m u n i s e d m o u s e were washed at 37°C b e f o r e i n c u b a t i o n at 4°C as s h o w n . T h e y were t h e n w a s h e d at 4°C b e f o r e f o r m i n g rosettes. b Eluate o f spleen cells f r o m s e c o n d a r i l y i m m u n i s e d m o u s e washed at 4°C and t h e n incub a t e d at 37°C. c Eluate o f spleen cells f r o m s e c o n d a r i l y i m m u n i s e d m o u s e w a s h e d at 37°C t h e n incub a t e d at 37°C. d S e r u m f r o m secondarily i m m u n i s e d m o u s e with a --log 2 9 titre o f IgG anti-SRBC antibody. e S e r u m from a primarily i m m u n i s e d m o u s e with a - - l o g : 3 titre o f IgM anti-SRBC antibody, f S e r u m f r o m an u n i m m u n i s e d m o u s e . g A n t i - m o u s e F(ab)2 s e r u m was a d d e d to spleen cells p r e i n c u b a t e d with test eluate. The cells were i n c u b a t e d at 4°C for 15 m i n b e f o r e f o r m i n g rosettes.
3
a
content
923 339
13 3
936 342
Total
from mice after
Peroxidase positive
cells
Nucleated
Peroxidase negative
cells and RFC
of spleen
1.4 0.8
’
of nucleated
SRBC.
0 0
Peroxidase positive
cells forming
doses of 5 X 10’
50 31
Peroxidase negative
RFC
immunisation.
% positive
secondary
a Rosetted spleen cells from 2 different mice immunised with 4 intraperitoneal b RFC identified in fixed preparations. ’ RFC per lo6 lymphocytes counted in suspension before smearing. Percentage
1 2
Experiment
Peroxidase
TABLE
rosettes
shown
54,650 59,823
’
in brackets.
(5.5%) (6.0%)
RFC per lo6 lymphocytes
296
antibody, could not confer rosette-forming capacity (table 2). The rosettes in these different preparations were inhibited by anti-F(ab)2 serum.
Effect of washing at 37~C on the proportion of immunoglobulin bearing cells in the spleen Surface immunoglobulin was detected on 36 to 41% of spleen cells irrespective of the washing temperature. Identity of the RFC No RFC contained peroxidase in the smears of rosetted spleen cell suspensions and the proportion of peroxidase-positive cells was considerably less than the proportion of RFC (table 3). About half as many cells contained non-specific esterase as contained peroxidase, and they were all large cells morphologically distinct from lymphocytes. DISCUSSION
The present findings indicate that in mice undergoing a secondary response to SRBC the majority of the RFC detected in spleen cell suspensions prepared in the cold are non-specific cells which have been passively allergised, probably with IgG antibodies. RFC all had the morphology of lymphocytes and none contained peroxidase, a marker for monocytes. Our spleen cell suspensions, which has been c o t t o n wool filtered, contained very few mononuclear phagocytes (identified by the presence of intracellular colloidal gold after in vivo intravenous injection), and there were always less cells containing either peroxidase or non-specific esterase (another monocyte/macrophage marker), than there were RFC. We extend earlier studies by demonstrating elution of antibody from 'passive' RFC and its uptake on fresh non-specific cells. Furthermore we have shown that, at least in Balb/c mice immunised with SRBC, a large number of the acceptor cells are lymphocytes and that the phenomenon is temperature-dependent. In the original studies of Biozzi et al. (1966), the absence of any rosette formation by l y m p h o c y t e s from normal mice after in vitro incubation at 4°C with h y p e r i m m u n e serum could be attributable to the technique used. The temperature at which the cells were washed after cold incubation, and before rosetting, was n o t specified, and if it was room temperature rather than 4°C passive allergisation would be greatly diminished. Also their rosetring technique with cells in the presence of 5--10% homologous normal serum being permitted to settle for 16 h is in marked contrast to the centrifugation-resuspension m e t h o d of Coombs (McConnell et al., 1969) used by us. The effect of washing spleen cells at 37°C before rosetting suggests that the passive uptake of antibody is an in vitro artefact which occurs as the cells are processed in the cold. However, in contrast to human peripheral
297 b l o o d l y m p h o c y t e s ( K u m a g a i et al., 1 9 7 5 ; L o b o e t al., 1 9 7 5 ; P e p y s et al., 1 9 7 6 ) washing m u r i n e spleen cells at 3 7 ° C r a t h e r t h a n 4°C did n o t r e d u c e t h e n u m b e r o f cells s t a i n e d b y f l u o r e s c e n t a n t i - i m m u n o g l o b u l i n a n t i b o d i e s . This suggests t h a t the g r e a t m a j o r i t y o f the m u r i n e cells which bind antib o d y in t h e cold, a n d are t h e r e b y able t o f o r m r o s e t t e s , p r o b a b l y are B cells w h i c h a l r e a d y h a v e stable s u r f a c e i m m u n o g l o b u l i n of their own. I t w o u l d n o n e t h e l e s s be i n t e r e s t i n g t o c o m p a r e w i t h a p p r o p r i a t e r e a g e n t s the class d i s t r i b u t i o n o f i m m u n o g l o b u l i n on m u r i n e cells w a s h e d at 4°C a n d 37°C, p a r t i c u l a r l y as the m a j o r i t y o f h u m a n p e r i p h e r a l l y m p h o c y t e s which bind IgG in the c o l d are n o t B cells ( L o b o a n d H o r w i t z , 1976). I t is clear t h a t t h e s a m e p r e c a u t i o n s o f p r e w a s h i n g at 3 7 ° C a n d / o r the use o f F(ab)2 r e a g e n t s ( W i n c h e s t e r et al., 1 9 7 5 ; P e p y s et al., 1 9 7 6 ) as are necessary f o r precise e v a l u a t i o n o f s u r f a c e i m m u n o g l o b u l i n s t r u c t u r e s on h u m a n l y m p h o c y t e s are also r e q u i r e d in mice. ACKNOWLEDGEMENTS We wish t o t h a n k Dr. M.F. G r e a v e s f o r h e l p f u l discussions a n d t h e gift o f f l u o r e s c i n a t e d g o a t a n t i - m o u s e Ig a n t i b o d y . REFERENCES Biozzi, G., C. Stiffel, D. Mouton, M. Liacopoulos-Briot, C. Decreusefond and Y. Bouthillier, 1966, Ann. Inst. Pasteur 110, 7. Bussard, A. 1964, Acad. Sci. Paris 259, 3411. Graham, R.C. and M.J. Karnovsky, 1966, J. Histochem. Cytochem. 14, 291. Kapp, J. and B. Benacerraf, 1972, Eur. J. Immunol. 2 467. Kumagai, K., T. Abo, T. Sekizaka and M. Sasaki, 1975, J. Immunol. 115,982. Li, C.Y., K.W. Lain and L.T. Yam, 1973, J. Histochem. Cytochem. 21, 1. Lobo, P.I., F.B. Westervelt and D.A. Horwitz, 1975, J. Immunol. 114, 116. McConnell, I., A. Munro, B.W. Gurner and R.R.A. Coombs, 1969, Int. Arch. Allergy Appl. Immunol. 35, 209. Pepys, M.B., 1974, J. Exp. Med. 140, 126. Pepys, M.B., C. Sategna-Guidetti, D.D. Mirjah, M.K. Wansbrough-Jones and A.C. Dash, 1976, Clin. Exp. Immunol. 26, 91. Winchester, R.J., S.M. Fu, T. Hoffman and H.G. Kunkel, 1975, J. Immunol. 114, 1210.