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A methodological study of E-rosette formation using AET-treated sheep red blood cells
Journal o f Immunological Methods, 27 (1979) 61--74
61
© Elsevier/North-Holland Biomedical Press
A METHODOLOGICAL STUDY OF E-ROSETTE FORMATION USING AET-TREATED SHEEP RED BLOOD CELLS *
M. MADSEN and H.E. JOHNSEN Tissue Typing Laboratory, Blood Bank and Blood Grouping Laboratory, Aarhus Kommuneho~pital, DK-8000 Aarhus C, Denmark
(Received 4 August 1978, accepted 18 October 1978)
The influence of some of the well known technical variables on the E-rosette technique was examined using sheep red blood cells (SRBC) treated with 2-aminoethylisothiouronium bromide (AET). With AET treatment, E-rosette formation becomes less dependent on time and temperature and on the presence of serum. The mechanical stability of the rosettes is enhanced, and the number of SRBC attached to each rosette-forming lymphocyte (RFC) is markedly increased, leading to a sharper distinction between RFC and nonRFC. Ultimately, significantly more E-receptor carrying lymphocytes become detectable. Evidence is provided that the specificity of the E-rosette technique is unchanged after AET treatment of SRBC, in spite of the enhanced binding. A simple and reliable method for E-RFC identification is documented.
INTRODUCTION T h e E - r e c e p t o r f o r u n s e n s i t i z e d s h e e p red b l o o d cells ( S R B C ) on h u m a n l y m p h o c y t e s is r e g a r d e d as specific to t h y m u s derived T cells (MSller, 1973) a l t h o u g h its n a t u r e a n d its possible biological significance are still u n r e s o l v e d (Jondal, 1976). A p p a r e n t l y s i m p l e t o p e r f o r m , t h e E - r o s e t t e t e c h n i q u e has t h e d r a w b a c k o f b e i n g e x t r e m e l y sensitive t o m e t h o d o l o g i c a l v a r i a t i o n . This is m a i n l y d u e to t h e w e a k b i n d i n g b e t w e e n the E - r e c e p t o r and the S R B C . T h e f o l l o w i n g has b e e n c l a i m e d t o be critical f o r reliable binding: Ca) l y m p h o c y t e viability ( F r S l a n d , 1 9 7 2 ) , (b) s o u r c e a n d age o f S R B C ( B r o w n a n d G r e a v e s , 1 9 7 4 ) , (c) S R B C : l y m p h o c y t e r a t i o (Bach, 1 9 7 3 ) , (d) r o s e t t i n g m e d i u m ( B e n t w i c h et al., 1 9 7 3 ) , (e) i n c u b a t i o n t e m p e r a t u r e ( L a y et al., 1 9 7 1 ; M e n d e s e t al., 1 9 7 3 ) , (f) i n c u b a t i o n t i m e ( W H O R e p o r t , 1 9 7 4 ) , (g) f o r c e o f r e s u s p e n s i o n ( R o s s et al., 1 9 7 3 ; Heier, 1 9 7 4 ) . E f f o r t s h a v e b e e n m a d e to stabilize T cell-SRBC b i n d i n g (a) b y m o d i f y i n g t h e m e d i u m b y a d d i n g s e r u m ( B e n t w i c h et al., 1 9 7 3 ) or d e x t r a n ( B r o w n e t al., 1 9 7 5 ) , (b) b y t r e a t m e n t o f t h e S R B C w i t h n e u r a m i n i d a s e (Weiner et al.,
* This study was supported by grants from the Danish Medical Research Council.
62 1973), papain (Wilson et al., 1975) or bromelin (Florey and Peetroom, 1976), (c) by treatment of the SRBC with the urea derivative 2-aminoethylisothiouronium bromide (AET) (Kaplan and Clark, 1974). The aim of this report is to evaluate the influence of some of the abovementioned variables on the E-rosetting capacity of AET-treated SRBC as compared with untreated SRBC. An unequivocal stabilizing effect of AET is described. MATERIALS AND METHODS
Lymphocyte suspensions L y m p h o c y t e suspensions were prepared from defibrinated venous blood from healthy blood donors by a local modification of B5yum's gradient centrifugation m e t h o d (B5yum, 1968; JOrgensen and Lamm, 1974). The isolated lymphocytes were washed twice in Tris-buffered balanced salt solution (BSS) (Favour, 1964) and adjusted to 3 X 106 cells/ml. The use of defibrinated blood gives an average yield of lymphocytes of 59.9% (range 44--85%) or on average 1.0 X 106 lymphocytes per ml venous blood (Johnsen and Madsen, 1978). Monocyte contamination is on average 2.9% (range 0--5%), and there is no selective loss of l y m p h o c y t e subpopulations (Johnsen and Madsen, 1978). The viability was more than 95% as judged by the trypan blue exclusion method.
Human lymphoid cell lines The B cell lines Daudi, Raji, P3 HR-1, Namalwa, K562, BJAB, Ramos, BJAB HR1K and AW-Ramos maintained on RPMI 1640 medium with the addition of 8% foetal calf serum, were kindly supplied by Dr. J. Zeuthen, Institute of Human Genetics, University of Aarhus. Immediately before use the cells were washed twice in BSS.
Trypsin treatment of lymphocytes 6 X 106 lymphocytes in 2 ml BSS were mixed with I ml 0.5% (w/v) trypsin (Difco Laboratories, Detroit, MI) solution in BSS. The cells were incubated at 37°C for 45 min followed by 3 washes in BSS.
Treatment of lymphocytes with fluorescent anti-human immunoglobulin 1--2 X 104 lymphocytes were incubated at 4°C for 30 min in 100 pl of a 1 : 10 dilution in BSS of FITC-conjugated polyvalent rabbit anti-human immunoglobulin ([g) (Cat. No. F1009, Dakopatts, Copenhagen, Denmark). After incubation the cells were washed twice in cold BSS. For examination, the cells were placed under oil in a microchamber (Hamax, Moss, Norway) as used for l y m p h o c y t o t o x i c i t y testing (Kissmeyer-Nielsen and Thorsby, 1970). The fluorescent cells were examined in an inverted fluorescence microscope (Leitz, Diavert).
63 Serum Fetal calf serum (FCS) (Flow Laboratories, Irvine, Scotland, U.K.) was inactivated at 56°C for 30 min. Each batch was tested for antibodies against h u m a n erythrocytes and SRBC as well as l y m p h o c y t o t o x i c antibodies against h u m a n l y m p h o c y t e s (Kissmeyer-Nielsen and Kjerbye, 1967) and always f o u n d negative.
Sheep blood Sheep blood was drawn aseptically in acid citrate dextrose (ACD = NIH solution A} and used within 2 weeks. The animals used were rams selected at random except in a few cases where lambs were chosen. Preparation of SRBC suspensions Sheep red blood cells were washed 3 times in isotonic saline, and care was taken to remove the buffy coat. After washing, the SRBC were either treated with AET (vide infra) or used directly for preparation of a 1% suspension in BSS with 40% heat-inactivated FCS. This suspension of untreated SRBC was always used freshly prepared, and is designated Eunt~. A E T treatment of SRBC The m e t h o d described by Kaplan and Clark (1974) was used. A 140 mM solution of AET (Sigma, St. Louis, MO) was made by dissolving 0.5 g crystalline AET in 12.5 ml distilled water. The pH was adjusted to 9.0 with 10 M NaOH. Packed, washed SRBC from approx. 10 ml sheep blood was resuspended in 10 ml freshly prepared AET solution and incubated at 37°C for 15 min. After incubation SRBC were washed in isotonic saline 4 times. If any residual haemolysis was observed after 4 washes, further washing was performed until the supernatant was clear. Finally, a 1% suspension of AETtreated SRBC (EAET) w a s made in BSS with 40% FCS. EAET w e r e used within 1 week. Human and bovine erythrocytes were treated with AET in the same way. E-rosette test The procedure was as follows unless otherwise stated: 100 pl 1% EAE T (or 1% Euntr ) and 100 pl l y m p h o c y t e suspension were mixed in 70 mm X 10 mm plastic test tubes (Cat. No. 1090, Nunc, Roskilde, Denmark). After mixing the tubes were centrifuged at approx. 50 X g for 5 min and then stored at 4°C until counted, i.e. usually overnight. Immediately before counting, one drop of 0.1% toluidine blue in 0.9% saline was added to each tube, and the pellet was resuspended by gentle manual shaking. Counting was performed in a Bfirker-Tfirk h a e m o c y t o m e t e r (magnification, X 500}, and a total of 200 l y m p h o c y t e s was counted. L y m p h o c y t e s with three or more SRBC tightly bound were considered rosette-forming cells (RFC). All tests were done in duplicate.
64 RESULTS
A E T treatment o f S R B C F o r A E T t r e a t m e n t o f SRBC a 140 mM A E T solution was used r o u t i n e l y . T h e same p e r c e n t a g e s o f EAET-RFC were o b t a i n e d with this c o n c e n t r a t i o n as with 70 mM or 280 mM A E T . With the 70 mM A E T solution, h o w e v e r , the r o s e t t e s t e n d e d to be less dense t h a n w h e n the higher c o n c e n t r a t i o n s were used. N o qualitative d i f f e r e n c e s were observed b e t w e e n 140 mM and 280 mM. Similar findings are r e p o r t e d b y Pellegrino e t al. (1975). Age o f EAE T suspension No d i f f e r e n c e s in r o s e t t e - f o r m i n g c a p a c i t y were observed w h e n c o m p a r i n g EAE w suspensions o f 0, 5 and 7 days o f age. This m a d e it practicable t o prepare suspensions o n l y o n c e a w e e k and this has been standard p r o c e d u r e in this s t u d y . Kaplan and Clark ( 1 9 7 4 ) came to the same conclusion. S R B C : l y m p h o c y t e ratio Fig. 1 shows the e f f e c t o f the EAE T : l y m p h o c y t e ratio on the p e r c e n t a g e o f EAET-RFC. It is seen t h a t the same p e r c e n t a g e s are o b t a i n e d over a range o f ratios o f 9 : 1 t o 100 : 1, whereas a l o w e r ratio gives l o w e r counts. A 1% suspension o f SRBC was f o u n d to c o n t a i n a p p r o x . 2 × l 0 s SRBC per ml, giving a ratio o f a p p r o x . 70 : 1. This cell d e n s i t y was suitable for c o u n t i n g rosettes. The presence o f serum in the m e d i u m F o r the s t a n d a r d p r o c e d u r e 40% FCS was a d d e d to the SRBC suspension, which gives a serum c o n c e n t r a t i o n o f 20% in the final r o s e t t i n g m i x t u r e . Table 1 shows t h a t n o q u a n t i t a t i v e e f f e c t o f serum was f o u n d on the per-
%E~,ET- R FC
70 60 50 40
I
i
I
I
I
I
4
9
17
35
70
100
SRBC : lymphocyte ratio
Fig. 1. The p e r c e n t a g e o f EAET-RFC as a f u n c t i o n o f the SRBC : l y m p h o c y t e ratio. The results are given as m e a n s o f results o n 5 n o r m a l b l o o d d o n o r s , t h e vertical bars r e p r e s e n t ing +1 S.D.
65 TABLE 1 THE EFFECT OF SERUM IN THE ROSETTING MEDIUM ON THE PERCENTAGE OF EAET-RFC Lymphocyte donor 1 2
Final concentration of fetal calf serum 0%
10%
20%
50%
100%
64.5 77.0
61.6 73.3
62.0 72.9
60.0 73.8
63.6 73.9
P > 0.05. Student-Newman-Keuls test.
c e n t a g e o f EAET-RFC. H o w e v e r , a l t h o u g h this does n o t a p p e a r f r o m Table 1, the rosettes h a d a m o r e c o m p a c t a p p e a r a n c e w h e n the final serum c o n c e n t r a t i o n was 10% or m o r e . In c o n t r a s t , w h e n Euntr were used, serum signific a n t l y increased the p e r c e n t a g e s o f R F C as s h o w n in Table 2. A final serum c o n c e n t r a t i o n o f 20% was c h o s e n because we also use EAEw f o r s e p a r a t i o n o f T a n d B cells, and for this p u r p o s e 20% was f o u n d t o be o p t i m a l (to be published).
Requirements for close contact Close c o n t a c t b e t w e e n l y m p h o c y t e s and SRBC is necessary f o r r o s e t t e form a t i o n (Fig. 2). L y m p h o c y t e s and EAEw were m i x e d a n d allowed to s e d i m e n t s p o n t a n e o u s l y . It is seen t h a t m o r e t h a n 4 h o f s p o n t a n e o u s s e d i m e n t a t i o n is r e q u i r e d for o p t i m a l rosetting. If the m i x t u r e is c e n t r i f u g e d , h o w e v e r , the same p e r c e n t a g e s are o b t a i n e d as after s p o n t a n e o u s s e d i m e n t a t i o n f o r 20 h; and f u r t h e r , f r o m Fig. 3 it is seen t h a t o n c e close c o n t a c t is established b y c e n t r i f u g a t i o n at 50 X g, the p e r c e n t a g e o f EAET-RFC is m a x i m a l . F u r t h e r , f r o m Table 3 it appears t h a t the f o r c e o f c e n t r i f u g a t i o n is n o t critical. I t is c o n c ] u d e d t h a t close c o n t a c t is critical, and is a d e q u a t e l y p r o v i d e d b y cent r i f u g a t i o n at 50 X g f o r 5 min.
TABLE 2 THE EFFECT OF 20% FINAL CONCENTRATION OF FETAL CALF SERUM (FCS) ON THE PERCENTAGE OF Euntr-RFC Lymphocyte donor
With 20% FCS No FCS
A
B
C
D
E
50.5 42.5
78.1 70.5
74.0 60.9
67.5 59.4
59.0 47.4
P < 0.001. Paired t-test.
66 % EAET- R FC .80 .60
/
.4o
~j. , ,
0
1
2
3
4
20hours
Time of spontaneous sedimentation
Fig. 2. T h e p e r c e n t a g e o f EAET-RFC as a f u n c t i o n o f t i m e o f s p o n t a n e o u s s e d i m e n t a t i o n at 4 °C. T h e results f r o m 2 n o r m a l b l o o d d o n o r s are s h o w n . • i n d i c a t e s t h e p e r c e n t a g e s o b t a i n e d b y t h e s t a n d a r d p r o c e d u r e , i n c l u d i n g c e n t r i f u g a t i o n at 50 x g.
Time o f incubation It is recommended in the WHO Report (1974) that the centrifugation step is followed by overnight incubation at 4°C to ensure maximal rosetting. Indeed, it has been shown t h a t the number of l y m p h o c y t e s that form rosettes with untreated SRBC after centrifugation but w i t h o u t any incubation is only about half the number of RFC after overnight incubation at 4 ° C. This has given rise to the terms early (or active) and late (or total) rosettes, the former being regarded as a subset of human T cells (Wybran and Fudenberg, 1973; Yu, 1975). Fig. 3 shows the percentage of E A E T - R F C and E u n t r RFC as a function of time of incubation at 4 ° C. The percentage of EAE TRFC is maximal immediately after centrifugation, the individual rosettes, %E - RFC 80 .
40 20 I
I
!
I
0
½
1
2
-,,
|
20
I
48 hours
T i m e of incubation
Fig. 3. T h e p e r c e n t a g e o f EAET-RFC (O) a n d E u n t r - R F C (©) as a f u n c t i o n o f t i m e o f incub a t i o n at 4°C. T h e results are given as m e a n s o f 10 ( o ) or 8 (o) n o r m a l b l o o d d o n o r s respectively. T h e vertical bars r e p r e s e n t +-1 S.D. T h e values a t 0 h o f i n c u b a t i o n are t h e p e r c e n t a g e s o b t a i n e d i m m e d i a t e l y a f t e r c e n t r i f u g a t i o n , i.e. w i t h o u t a n y i n c u b a t i o n .
67
however, showing a tendency to become denser after 30 min of cold incubation. After 30 min neither quantitative nor qualitative changes were observed. In contrast, it is seen that the number Euntr-RFC immediately after centrifugation is approx, half the value after overnight incubation. Further, there is a significant increase of Eunt~-RFC even between 2 h and 20 h of cold incubation, which underlines the validity of the above-mentioned recommendation of the WHO R e p o r t (1974), when Euntr are used.
Temperature conditions for rosetting E-rosette formation is generally thought to be extremely temperature dependent (WHO Report, 1974). To investigate the temperature conditions for EAEw-rOsetting , l y m p h o c y t e s and EAE w w e r e stored separately at 0°C, 4°C, 20°C and 37°C, respectively. After the lymphocytes and E A E w had obtained the required temperatures, they were mixed and centrifuged, and then reincubated at the original temperature for 1 h, whereafter the rosettes were scored. Because of lack of time for counting, the mixtures at 4°C had to be stored overnight, however. In addition, l y m p h o c y t e s from each l y m p h o c y t e donor were mixed with EAE w at room temperature, incubated at 37°C for 15 min, then centrifuged and stored at 4°C overnight. This latter procedure is routinely used by several workers in accordance with the WHO R e p o r t (1974). All these procedures were performed in parallel with Euntr on the same l y m p h o c y t e suspensions. The results are seen in Fig. 4. The percentages of EAET-RFC at 0°C, 4°C and 20°C were equal and identical to the percentages obtained when the preincubation procedure recom-
%E-RFC 80 60 40 20
0
4
20
°C
Temperature of incubation
Fig. 4. T h e p e r c e n t a g e o f EAET-RFC (O) a n d E u n t r - R F C (o) in 10 n o r m a l b l o o d d o n o r s as a f u n c t i o n o f t h e t e m p e r a t u r e o f i n c u b a t i o n . EAE T or Euntr a n d l y m p h o c y t e s were m i x e d a f t e r r e a c h i n g t h e i n d i c a t e d t e m p e r a t u r e s , c e n t r i f u g e d a n d t h e n r e i n c u b a t e d at t h e original t e m p e r a t u r e for 1 h a n d t h e r o s e t t e s scored. T h e m i x t u r e s were s t o r e d o v e r n i g h t at 4°C. * i n d i c a t e s t h e p e r c e n t a g e s o b t a i n e d w h e n l y m p h o c y t e s a n d S R B C were m i x e d at r o o m t e m p e r a t u r e , p r e i n c u b a t e d at 37°C for 15 m i n , t h e n c e n t r i f u g e d a n d s t o r e d at 4°C overnight. T h e results are given as m e a n s , t h e vertical bars i n d i c a t i n g 1 S.D.
68
mended in the WHO R e p o r t (1974} was used (Student-Newman-Keuls test). At 37°C, however, the number of EAET-RFC was reduced to half the values obtained at the lower temperatures. With Euntr the percentages at 4°C and 20°C were equal and a b o u t 10% higher (P < 0.05) than at 0°C, whereas virtually no rosettes were formed at 37°C. Fig. 4 also shows that preincubation at 37°C for 15 min gave significantly lower percentages (approx. 10%) than the 4°C and 20°C figures.
Resuspension As E-rosettes are fragile and easily disrupted (Heier, 1974), the resuspension procedure is critical. To investigate the mechanical stability of EAE Trosettes as compared with Eunt~-rosettes, mechanical resuspension was performed for 5 sec on a Whirlmixer ® (Fisons Scientific Apparatus, U.K.) using 5 different speeds. The results are depicted in Fig. 5, which shows the increased mechanical stability of EAEw-rOsettes, although it is seen that EAEw-rOsettes can be disrupted if the resuspension force is strong enough. It should be mentioned that even the lowest strength of mechanical resuspension was much stronger than the standard manual resuspension. Coun ting o f rosettes An essential feature of the increased affinity between the E-receptor and AET-treated SRBC is the increased number of SRBC attached to each lymp h o c y t e (Table 4). A b o u t 95% of the EAEw-rOsettes had a " m o r u l a " appearance with more than 20 EAE T per lymphocyte. Only occasional RFC were
,,o
100
er
N
80
g ~ 6o
~
n -"
2o m
M
1
2
3
4
L 5
Force of resuspension
Fig. 5. T h e m e c h a n i c a l s t a b i l i t y o f EAET-rosettes ( h a t c h e d b a r s ) a n d E u n t r - r o s e t t e s ( o p e n bars). M = usual m a n u a l r e s u s p e n s i o n . T h e figures 1--5 i n d i c a t e a r b i t r a r y increasing u n i t s o f a g i t a t i o n o n a W h i r l m i x e r ® used for m e c h a n i c a l r e s u s p e n s i o n f o r 5 sec. T h e results o f 2 n o r m a l b l o o d d o n o r s are given as relative p e r c e n t a g e s o f R F C to t h e values o b t a i n e d b y usual m a n u a l r e s u s p e n s i o n . +1 S.D.
69 TABLE 3 PERCENTAGE OF EAET-RFC AT D I F F E R E N T FORCES OF SEDIMENTATION ± 1 S.D. 4 normal blood donors. Sedimentation force (g)
%EAET-RFC ± S.D.
1a
2
50 b
200
400
700
64.4 -+ 15.8
66.1 ± 11.2
65.7 ± 14.4
65.9 ± 8.8
58.2 ± 9.0
56.9 ± 8.6
a Spontaneous sedimentation overnight. b Standard procedure. P > 0.05. Student-Newman-Keuls test.
f o u n d w i t h as f e w as 3 EAE T a t t a c h e d . E u n t r - R F C o n t h e o t h e r h a n d w e r e n e v e r f o u n d t o b e as h e a v i l y s t u d d e d w i t h S R B C as E A E T - R F C , a n d i n a l m o s t e v e r y p r e p a r a t i o n l y m p h o c y t e s w e r e f o u n d w i t h o n l y o n e o r t w o Euntr a t t a c h e d t o t h e m e m b r a n e , a r a r e f i n d i n g w i t h EAE w. T h i s f e a t u r e o f A E T treated SRBC provides a sharp distinction between E-RFC and non-RFC, which renders counting quick and reliable. The addition of toluidine blue allows for quick and reliable differentiation between SRBC and lymphocytes.
E-RFC in healthy blood Table 5 shows the result of a paired comparison of the percentages of E A E T - R F C a n d E u n t r - R F C i n 2 0 h e a l t h y b l o o d d o n o r s . I t is s e e n t h a t signific a n t l y m o r e E - R F C is d e t e c t e d u s i n g A E T t r e a t m e n t o f S R B C . I n 1 0 4 h e a l t h y b l o o d d o n o r s t h e m e a n p e r c e n t a g e o f E A E T - R F C was 7 3 . 6
TABLE 4 PERCENTAGE OF Euntr-RFC AND EAET-RFC IN 6 NORMAL DONORS WITH INDICATION OF THE NUMBER OF SRBC PER ROSETTE -+ 1S.D. NumberofSRBC
%Euntr-RFC -+ S.D. %EAET-RFC ± S.D.
3--4
5--9
/> 10 a
Total % RFC
5.6 ± 1.9 (8.8) b 0.7 ± 0.3 (0.9)
15.8 ± 4.3 (24.8) 3.2 ± 1.1 (4.1)
42.4 ± 5.3 (66.6) 74.0 ± 4.6 (95.1)
63.7 ± 9.3 77.8 ± 4.0
a All EAET-rosettes of this category were "morula"-like, see text. The figures in the parentheses indicate the distribution in relation to total % RFC.
b
70 TABLE 5 PAIRED COMPARISON OF T H E PERCENTAGES OF EAET-RFC AND Euntr-RFC IN 20 HEALTHY DONORS -+ 1 S.D. %EAET-RFC -+ S.D.
%Euntr-RFC -+ S.D.
70.9 ± 5.8
60.1 ± 6.7
P < 0.01. Paired t-test. w i t h a r a n g e o f 4 9 . 3 - - 9 1 . 1 % and a s t a n d a r d d e v i a t i o n o f 8.3%. Similar figures h a v e b e e n r e p o r t e d b y o t h e r a u t h o r s using A E T - t r e a t e d S R B C ( K a p l a n and Clark, 1 9 7 4 ; Pellegrino et al., 1 9 7 5 ; Stein, 1977). T h e p e r c e n t a g e s o f EAET-RFC as well as Euntr-RFC in n o r m a l b l o o d donors were normally distributed.
Reactivity o f EAE T with human B cell lines N o r o s e t t i n g was o b s e r v e d w i t h A E T - t r e a t e d S R B C a n d a n y o f the cult u r e d h u m a n B cell lines m e n t i o n e d a b o v e , i n d i c a t i n g t h a t A E T t r e a t m e n t d o e s n o t r e n d e r S R B C c a p a b l e o f f o r m i n g r o s e t t e s w i t h B cells.
Reactivity o f AET-treated human and bovine erythrocytes with human lymphocytes N e i t h e r h u m a n or b o v i n e e r y t h r o c y t e s are c a p a b l e o f r o s e t t i n g with h u m a n l y m p h o c y t e s ( W H O R e p o r t , 1974). A E T t r e a t m e n t o f such e r y t h r o c y t e s in the w a y d e s c r i b e d f o r S R B C did n o t a f f e c t this n o n - r e a c t i v i t y .
EAET-rosetting with trypsinized lymphocytes T h e E - r e c e p t o r is sensitive to t r y p s i n t r e a t m e n t ( J o n d a l e t al., 1 9 7 2 ) . T a b l e 6 s h o w s t h a t t r y p s i n i z e d l y m p h o c y t e s h a v e also a l m o s t lost t h e i r rosetring c a p a c i t y w i t h EAE w.
EAET-rosetting with anti-human Ig-treated lymphocytes In d o u b l e m a r k e r e x p e r i m e n t s , l y m p h o c y t e s w e r e t r e a t e d w i t h F I T C TABLE 6 THE EFFECT OF TRYPSIN TREATMENT OF LYMPHOCYTES ON PERCENTAGES OF EAET-RFC Ten normal donors.
%EAET-RFC Range
Untreated lymphocytes
Trypsinized lymphocytes
75 57--84
3 2--4
71 conjugated anti-human Ig as described under Materials and Methods and then used for EAEw-rOsetting. EAET-rOsettes were scored as described and, in addition, the preparations were examined by fluorescence microscopy. No inhibition of rosetting was f o u n d when the l y m p h o c y t e s were treated with anti-Ig, in agreement with Coombs et al. (1970) and Bentwich et al. (1973) who used untreated SRBC. There was usually no overlapping between fluorescent cells (i.e. B cells) and RFC. In a few preparations, however, 1--2% of the RFC were also fluorescent, although the rosettes were much less dense than the other morula-like rosettes in the same preparation. These findings are in accordance with Bentwich et al. (1973) who also f o u n d a minor overlapping using untreated SRBC, and with Kaplan and Clark (1974) who used AET-treated SRBC. DISCUSSION The present report demonstrates a substantial stabilizing effect of AET treatment of SRBC on the E-rosettes, confirming the findings of others (Kaplan and Clark, 1974; Pellegrino et al., 1975; Stein, 1977). The effect of AET in stabilizing the otherwise weak binding between the E-receptor and SRBC is shown by the markedly decreased influence of several of the technical variables which are k n o w n to affect the results of E-rosetting. The enhancing effect observed implies that significantly more E-receptor carrying l y m p h o c y t e s are detectable by EAET than by Euntr , viz. 70.9% VS. 60.1% in a paired comparison (Table 5), which is in accordance with the figures reported by Kaplan and Clark (1974) and Mahowald et al. (1977). Unambiguous differentiation between E-RFC and non-E-RFC is a requirem e n t for a reliable E-rosette technique. One of the main results of AET t r e a t m e n t of SRBC is a substantial increase of the number of SRBC attached to each RFC, giving the majority of the EAEw-rOsettes a morula-like appearance; the non-EAET-RFC are left totally free of SRBC (Table 4). In contrast, Eunt,-RFC have considerably less SRBC bound to each l y m p h o c y t e , and some cells have only 3 or 4 SRBC attached. Further, in almost every preparation some lymphocytes are found with one or two Euntr clearly attached, raising d o u b t whether those cells possess E-receptors or not. Conventionally, l y m p h o c y t e s with three or more SRBC firmly attached to the cell membrane are regarded as E-receptor carrying cells (WHO Report, 1974), although some investigators also consider l y m p h o c y t e s with even one SRBC bound as E-receptor positive (Bentwich et al., 1973). With AET treatment less equivocal criteria for E-receptor identification can be adopted, since only occasionally are EAEw-rOsettes f o u n d with few SRBC (see also Stein, 1977). Another consequence of AET t r e a t m e n t of SRBC is improved mechanical stability of E-rosettes (Fig. 5), also reported by Hokland et a17(1976). This is an important point, since E-rosettes are sensitive to the force of resuspension, and this is generally considered a critical step in the technique (Heier, 1974; WHO Report, 1974).
72 The influence of serum in the rosetting medium has been disputed, but most investigators add FCS or absorbed human AB serum in accordance with the findings of Bentwich et al. (1973). In this report the stabilizing effect of FCS is reflected in an increased percentage of Euntr-RFC, b u t the effect of EAET-rosettes is only qualitative (Tables 1 and 2). The incubation temperature is critical for E-rosetting (Lay et al., 1971; Mendes et al., 1973), and all workers recommend incubation at 4°C to ensure maximal rosetting. In agreement with Mendes et al. (1973) we find optimal rosetting at 4°C and 20°C with Eunt~, the percentage at 0°C being significantly lower. At 37°C virtually no rosettes were found. With EAET, however, no differences were observed at between 0°C, 4°C and 20°C although the percentage was significantly lower at 37°C (Fig. 4). Lay et al. (1971) r e c o m m e n d preincubation of the samples at 37°C for 15 min before centrifugation and incubation at 4°C. With this procedure, however, significantly lower percentages o f Euntr-RFC were obtained (Fig. 4). A possible explanation of our findings with 37°C preincubation might be that E-receptors are shed at this temperature and do n o t regenerate at 4°C, as described recently by S~rmay et al. (1978). The peculiar temperature requirements of the E-rosette technique need further investigation. As further indication of increased binding force following AET treatment of SRBC, preincubation at 37°C showed no effect on rosetting capacity. Most authors use incubation at 4°C overnight, usually 18--20 h as recommended (WHO Report, 1974). In this study we find the percentage of E~n~R F C significantly higher after overnight incubation at 4°C than after 2 h incubation. Further, we also find a low percentage (approx. 30%) of EuntrR F C when resuspension and counting is performed immediately after centrifugation, i.e. without any incubation step. These rosettes correspond to " e a r l y " or " i m m e d i a t e " E-RFC. With EAET,however, the percentage of R F C is maximal immediately after centrifugation, although the rosettes are less dense than after 30 min of incubation at 4°C. No changes, neither quantitative nor qualitative, were observed after 30 min up to 48 h (Fig. 3). Close contact between l y m p h o c y t e s and EAET, obtained either by centrifugation at 50 × g or by spontaneous sedimentation overnight is clearly critical. As emphasized by Kaplan and Clark (1974) and Pellegrino et al. (1975} there is no indication that AET treatment changes the specificity of binding of SRBC to lymphocytes. In 9 different cultured B cell lines tested with EAET, rosettes were never found. Further, AET-treated human and bovine erythrocytes were n o t capable of rosetting with human lymphocytes. No overlapping between EAET-RFC and B cells (surface membrane Ig positive cells) was observed in most preparations investigated for both markers, only a few showing 1--2% fluorescent RFC. Finally, we found that treatment of the l y m p h o c y t e s with trypsin almost totally abrogated the EAEw-rOsette formation capacity, as reported for untreated SRBC (FrSland, 1972; Jondal et al., 1972). The mechanism of the action of AET on erythrocytes is n o t understood,
73 a l t h o u g h it is likely t h a t it reacts with thiol g r o u p s in the cell m e m b r a n e (Sirchia and F e r r o n e , 1 9 7 2 ) . I t s h o u l d be e m p h a s i z e d t h a t A E T is n o t a s u l p h h y d r y l c o m p o u n d , b u t at n e u t r a l and alkaline p H is c o n v e r t e d t o merc a p t o e t h y l g u a n i d i n e ( D o h e r t y et al., 1 9 5 7 ) , w h i c h m i g h t be e x p e c t e d t o have an a c t i o n on the e r y t h r o c y t e m e m b r a n e . Pellegrino et al. ( 1 9 7 5 ) have suggested t h e possibility t h a t thiol g r o u p s are o f crucial i m p o r t a n c e for E-rosette f o r m a t i o n . Whatever t h e m e c h a n i s m , A E T t r e a t m e n t o f SRBC has been s h o w n t o be a valuable m o d i f i c a t i o n o f t h e E - r o s e t t e t e c h n i q u e resulting in stable E-rosettes, and it m a y also have i m p o r t a n t i m p l i c a t i o n s for T and B cell separation. ACKNOWLEDGEMENTS Mrs. H. JCrgensen and Mrs. I.-L. T h o r s e n are t h a n k e d f o r e x p e r t technical assistance and Dr. J. Z e u t h e n for p r o v i d i n g the B cell lines. REFERENCES Bach, J.F., 1973, Transplant. Rev. 16,196. Bentwich, Z., S.D. Douglas, F.P. Siegal and H.G. Kunkel, 1973, Clin. Immunol. Immunopathol. 1, 511. BSyum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77. Brown, C.S., H. Halpern and H.H. Wortis, 1975, Clin. Exp. Immunol. 20, 505. Brown, G. and M.F. Greaves, 1974, Eur. J. Immunol. 4,302. Coombs, R.R.A., B.W. Gurner, A.B. Wilson, G. Holm and B. Lindgren, 1970, Int. Arch. Allergy 39,658. Doherty, D.G., R. Shapira and J. Burnett, 1957, J. Am. Chem. Soc. 79, 5667. Favour, C.B., 1964, in: Immunological Methods, ed. J.F. Ackroyd (Blackwell Scientific Publ., Oxford) p. 212. Florey, M.J. and F. Peetroom, 1976, J. Immunol. Methods 13, 201. Fr61and, S.S., 1972, Scand. J. Immunol. 1, 269. Heier, H.E., 1974, Scand. J. Immunol. 3,677. Hokland, P., M. Hokland and I. Heron, 1976, J. Immunol. Methods 13, 175. Johnsen, H.E. and M. Madsen, 1978, Scand. J. Immunol. 8,239. Jondal, M., 1976, Scand. J. Immunol. 5 (Suppl. 5), 69. Jondal, M., G. Holm and H. Wigzell, 1972, J. Exp. Med. 136, 207. Jctrgensen, F. and L.U. Lamm, 1974, Tissue Antigens 4,482. Kaplan, M.E. and C. Cark, 1974, J. Immunol. Methods 5, 131. Kissmeyer-Nielsen, F. and K.E. Kjerbye, 1967, in: Histocompatibility Testing, eds. E.S. Curtoni, P.L. Mattiuz and R.M. Tosi (Munksgaard, Copenhagen) pp. 381--383. Kissmeyer-Nielsen, F. and E. Thorsby, 1970, Transplant. Rev. 4,117. Lay, W.H., N.F. Mendes, C. Bianco and V. Nussenzweig, 1971, Nature 230, 531. Mahowald, M.L., B.S. Handwerger, E.M. Capertone and S.D. Douglas, 1977, J. Immunol. Methods 15, 239. Mendes, N.F., M.E.A. Tolnai, N.P.A. Silveira, R.B. Gilbertsen and R.S. Metzgar, 1973, J. Immunol. 111, 860. MSller, G. (ed.), 1973, Transplant. Rev. 16, 1. Pellegrino, M.A., S. Ferrone, M.P. Dierich and R.A. Reisfeld, 1975, Clin. Immunol. Immunopathol. 3,324.
74 Ross, G.D., E.M. Rabellino, M.J. Polley and H.M. Gray, 1973, J. Clin. Invest. 52, 377 S~rmay, G., L. Istv~n and J. Gergely, 1978, Immunology 34, 315. Sirchia, G. and S. Ferrone, 1972, Ser. Haematol. 5,137. Stein, G., 1977, J. Immunol. Methods 14,371. Weiner, M.S., C. Bianco and V. Nussenzweig, 1973, Blood 42,939. WHO Report, 1974, Scand. J. Immunol. 3,521. Wilson, A.B., D.G. Haegert and R.R.A. Coombs, 1975, Clin. Exp. Immunol. 22, 177. Wybran, J. and H.H. Fudenberg, 1973, J. Clin. Invest. 52, 1026. Yu, D.T.Y., 1975, J. Immunol. 115, 91.
61
© Elsevier/North-Holland Biomedical Press
A METHODOLOGICAL STUDY OF E-ROSETTE FORMATION USING AET-TREATED SHEEP RED BLOOD CELLS *
M. MADSEN and H.E. JOHNSEN Tissue Typing Laboratory, Blood Bank and Blood Grouping Laboratory, Aarhus Kommuneho~pital, DK-8000 Aarhus C, Denmark
(Received 4 August 1978, accepted 18 October 1978)
The influence of some of the well known technical variables on the E-rosette technique was examined using sheep red blood cells (SRBC) treated with 2-aminoethylisothiouronium bromide (AET). With AET treatment, E-rosette formation becomes less dependent on time and temperature and on the presence of serum. The mechanical stability of the rosettes is enhanced, and the number of SRBC attached to each rosette-forming lymphocyte (RFC) is markedly increased, leading to a sharper distinction between RFC and nonRFC. Ultimately, significantly more E-receptor carrying lymphocytes become detectable. Evidence is provided that the specificity of the E-rosette technique is unchanged after AET treatment of SRBC, in spite of the enhanced binding. A simple and reliable method for E-RFC identification is documented.
INTRODUCTION T h e E - r e c e p t o r f o r u n s e n s i t i z e d s h e e p red b l o o d cells ( S R B C ) on h u m a n l y m p h o c y t e s is r e g a r d e d as specific to t h y m u s derived T cells (MSller, 1973) a l t h o u g h its n a t u r e a n d its possible biological significance are still u n r e s o l v e d (Jondal, 1976). A p p a r e n t l y s i m p l e t o p e r f o r m , t h e E - r o s e t t e t e c h n i q u e has t h e d r a w b a c k o f b e i n g e x t r e m e l y sensitive t o m e t h o d o l o g i c a l v a r i a t i o n . This is m a i n l y d u e to t h e w e a k b i n d i n g b e t w e e n the E - r e c e p t o r and the S R B C . T h e f o l l o w i n g has b e e n c l a i m e d t o be critical f o r reliable binding: Ca) l y m p h o c y t e viability ( F r S l a n d , 1 9 7 2 ) , (b) s o u r c e a n d age o f S R B C ( B r o w n a n d G r e a v e s , 1 9 7 4 ) , (c) S R B C : l y m p h o c y t e r a t i o (Bach, 1 9 7 3 ) , (d) r o s e t t i n g m e d i u m ( B e n t w i c h et al., 1 9 7 3 ) , (e) i n c u b a t i o n t e m p e r a t u r e ( L a y et al., 1 9 7 1 ; M e n d e s e t al., 1 9 7 3 ) , (f) i n c u b a t i o n t i m e ( W H O R e p o r t , 1 9 7 4 ) , (g) f o r c e o f r e s u s p e n s i o n ( R o s s et al., 1 9 7 3 ; Heier, 1 9 7 4 ) . E f f o r t s h a v e b e e n m a d e to stabilize T cell-SRBC b i n d i n g (a) b y m o d i f y i n g t h e m e d i u m b y a d d i n g s e r u m ( B e n t w i c h et al., 1 9 7 3 ) or d e x t r a n ( B r o w n e t al., 1 9 7 5 ) , (b) b y t r e a t m e n t o f t h e S R B C w i t h n e u r a m i n i d a s e (Weiner et al.,
* This study was supported by grants from the Danish Medical Research Council.
62 1973), papain (Wilson et al., 1975) or bromelin (Florey and Peetroom, 1976), (c) by treatment of the SRBC with the urea derivative 2-aminoethylisothiouronium bromide (AET) (Kaplan and Clark, 1974). The aim of this report is to evaluate the influence of some of the abovementioned variables on the E-rosetting capacity of AET-treated SRBC as compared with untreated SRBC. An unequivocal stabilizing effect of AET is described. MATERIALS AND METHODS
Lymphocyte suspensions L y m p h o c y t e suspensions were prepared from defibrinated venous blood from healthy blood donors by a local modification of B5yum's gradient centrifugation m e t h o d (B5yum, 1968; JOrgensen and Lamm, 1974). The isolated lymphocytes were washed twice in Tris-buffered balanced salt solution (BSS) (Favour, 1964) and adjusted to 3 X 106 cells/ml. The use of defibrinated blood gives an average yield of lymphocytes of 59.9% (range 44--85%) or on average 1.0 X 106 lymphocytes per ml venous blood (Johnsen and Madsen, 1978). Monocyte contamination is on average 2.9% (range 0--5%), and there is no selective loss of l y m p h o c y t e subpopulations (Johnsen and Madsen, 1978). The viability was more than 95% as judged by the trypan blue exclusion method.
Human lymphoid cell lines The B cell lines Daudi, Raji, P3 HR-1, Namalwa, K562, BJAB, Ramos, BJAB HR1K and AW-Ramos maintained on RPMI 1640 medium with the addition of 8% foetal calf serum, were kindly supplied by Dr. J. Zeuthen, Institute of Human Genetics, University of Aarhus. Immediately before use the cells were washed twice in BSS.
Trypsin treatment of lymphocytes 6 X 106 lymphocytes in 2 ml BSS were mixed with I ml 0.5% (w/v) trypsin (Difco Laboratories, Detroit, MI) solution in BSS. The cells were incubated at 37°C for 45 min followed by 3 washes in BSS.
Treatment of lymphocytes with fluorescent anti-human immunoglobulin 1--2 X 104 lymphocytes were incubated at 4°C for 30 min in 100 pl of a 1 : 10 dilution in BSS of FITC-conjugated polyvalent rabbit anti-human immunoglobulin ([g) (Cat. No. F1009, Dakopatts, Copenhagen, Denmark). After incubation the cells were washed twice in cold BSS. For examination, the cells were placed under oil in a microchamber (Hamax, Moss, Norway) as used for l y m p h o c y t o t o x i c i t y testing (Kissmeyer-Nielsen and Thorsby, 1970). The fluorescent cells were examined in an inverted fluorescence microscope (Leitz, Diavert).
63 Serum Fetal calf serum (FCS) (Flow Laboratories, Irvine, Scotland, U.K.) was inactivated at 56°C for 30 min. Each batch was tested for antibodies against h u m a n erythrocytes and SRBC as well as l y m p h o c y t o t o x i c antibodies against h u m a n l y m p h o c y t e s (Kissmeyer-Nielsen and Kjerbye, 1967) and always f o u n d negative.
Sheep blood Sheep blood was drawn aseptically in acid citrate dextrose (ACD = NIH solution A} and used within 2 weeks. The animals used were rams selected at random except in a few cases where lambs were chosen. Preparation of SRBC suspensions Sheep red blood cells were washed 3 times in isotonic saline, and care was taken to remove the buffy coat. After washing, the SRBC were either treated with AET (vide infra) or used directly for preparation of a 1% suspension in BSS with 40% heat-inactivated FCS. This suspension of untreated SRBC was always used freshly prepared, and is designated Eunt~. A E T treatment of SRBC The m e t h o d described by Kaplan and Clark (1974) was used. A 140 mM solution of AET (Sigma, St. Louis, MO) was made by dissolving 0.5 g crystalline AET in 12.5 ml distilled water. The pH was adjusted to 9.0 with 10 M NaOH. Packed, washed SRBC from approx. 10 ml sheep blood was resuspended in 10 ml freshly prepared AET solution and incubated at 37°C for 15 min. After incubation SRBC were washed in isotonic saline 4 times. If any residual haemolysis was observed after 4 washes, further washing was performed until the supernatant was clear. Finally, a 1% suspension of AETtreated SRBC (EAET) w a s made in BSS with 40% FCS. EAET w e r e used within 1 week. Human and bovine erythrocytes were treated with AET in the same way. E-rosette test The procedure was as follows unless otherwise stated: 100 pl 1% EAE T (or 1% Euntr ) and 100 pl l y m p h o c y t e suspension were mixed in 70 mm X 10 mm plastic test tubes (Cat. No. 1090, Nunc, Roskilde, Denmark). After mixing the tubes were centrifuged at approx. 50 X g for 5 min and then stored at 4°C until counted, i.e. usually overnight. Immediately before counting, one drop of 0.1% toluidine blue in 0.9% saline was added to each tube, and the pellet was resuspended by gentle manual shaking. Counting was performed in a Bfirker-Tfirk h a e m o c y t o m e t e r (magnification, X 500}, and a total of 200 l y m p h o c y t e s was counted. L y m p h o c y t e s with three or more SRBC tightly bound were considered rosette-forming cells (RFC). All tests were done in duplicate.
64 RESULTS
A E T treatment o f S R B C F o r A E T t r e a t m e n t o f SRBC a 140 mM A E T solution was used r o u t i n e l y . T h e same p e r c e n t a g e s o f EAET-RFC were o b t a i n e d with this c o n c e n t r a t i o n as with 70 mM or 280 mM A E T . With the 70 mM A E T solution, h o w e v e r , the r o s e t t e s t e n d e d to be less dense t h a n w h e n the higher c o n c e n t r a t i o n s were used. N o qualitative d i f f e r e n c e s were observed b e t w e e n 140 mM and 280 mM. Similar findings are r e p o r t e d b y Pellegrino e t al. (1975). Age o f EAE T suspension No d i f f e r e n c e s in r o s e t t e - f o r m i n g c a p a c i t y were observed w h e n c o m p a r i n g EAE w suspensions o f 0, 5 and 7 days o f age. This m a d e it practicable t o prepare suspensions o n l y o n c e a w e e k and this has been standard p r o c e d u r e in this s t u d y . Kaplan and Clark ( 1 9 7 4 ) came to the same conclusion. S R B C : l y m p h o c y t e ratio Fig. 1 shows the e f f e c t o f the EAE T : l y m p h o c y t e ratio on the p e r c e n t a g e o f EAET-RFC. It is seen t h a t the same p e r c e n t a g e s are o b t a i n e d over a range o f ratios o f 9 : 1 t o 100 : 1, whereas a l o w e r ratio gives l o w e r counts. A 1% suspension o f SRBC was f o u n d to c o n t a i n a p p r o x . 2 × l 0 s SRBC per ml, giving a ratio o f a p p r o x . 70 : 1. This cell d e n s i t y was suitable for c o u n t i n g rosettes. The presence o f serum in the m e d i u m F o r the s t a n d a r d p r o c e d u r e 40% FCS was a d d e d to the SRBC suspension, which gives a serum c o n c e n t r a t i o n o f 20% in the final r o s e t t i n g m i x t u r e . Table 1 shows t h a t n o q u a n t i t a t i v e e f f e c t o f serum was f o u n d on the per-
%E~,ET- R FC
70 60 50 40
I
i
I
I
I
I
4
9
17
35
70
100
SRBC : lymphocyte ratio
Fig. 1. The p e r c e n t a g e o f EAET-RFC as a f u n c t i o n o f the SRBC : l y m p h o c y t e ratio. The results are given as m e a n s o f results o n 5 n o r m a l b l o o d d o n o r s , t h e vertical bars r e p r e s e n t ing +1 S.D.
65 TABLE 1 THE EFFECT OF SERUM IN THE ROSETTING MEDIUM ON THE PERCENTAGE OF EAET-RFC Lymphocyte donor 1 2
Final concentration of fetal calf serum 0%
10%
20%
50%
100%
64.5 77.0
61.6 73.3
62.0 72.9
60.0 73.8
63.6 73.9
P > 0.05. Student-Newman-Keuls test.
c e n t a g e o f EAET-RFC. H o w e v e r , a l t h o u g h this does n o t a p p e a r f r o m Table 1, the rosettes h a d a m o r e c o m p a c t a p p e a r a n c e w h e n the final serum c o n c e n t r a t i o n was 10% or m o r e . In c o n t r a s t , w h e n Euntr were used, serum signific a n t l y increased the p e r c e n t a g e s o f R F C as s h o w n in Table 2. A final serum c o n c e n t r a t i o n o f 20% was c h o s e n because we also use EAEw f o r s e p a r a t i o n o f T a n d B cells, and for this p u r p o s e 20% was f o u n d t o be o p t i m a l (to be published).
Requirements for close contact Close c o n t a c t b e t w e e n l y m p h o c y t e s and SRBC is necessary f o r r o s e t t e form a t i o n (Fig. 2). L y m p h o c y t e s and EAEw were m i x e d a n d allowed to s e d i m e n t s p o n t a n e o u s l y . It is seen t h a t m o r e t h a n 4 h o f s p o n t a n e o u s s e d i m e n t a t i o n is r e q u i r e d for o p t i m a l rosetting. If the m i x t u r e is c e n t r i f u g e d , h o w e v e r , the same p e r c e n t a g e s are o b t a i n e d as after s p o n t a n e o u s s e d i m e n t a t i o n f o r 20 h; and f u r t h e r , f r o m Fig. 3 it is seen t h a t o n c e close c o n t a c t is established b y c e n t r i f u g a t i o n at 50 X g, the p e r c e n t a g e o f EAET-RFC is m a x i m a l . F u r t h e r , f r o m Table 3 it appears t h a t the f o r c e o f c e n t r i f u g a t i o n is n o t critical. I t is c o n c ] u d e d t h a t close c o n t a c t is critical, and is a d e q u a t e l y p r o v i d e d b y cent r i f u g a t i o n at 50 X g f o r 5 min.
TABLE 2 THE EFFECT OF 20% FINAL CONCENTRATION OF FETAL CALF SERUM (FCS) ON THE PERCENTAGE OF Euntr-RFC Lymphocyte donor
With 20% FCS No FCS
A
B
C
D
E
50.5 42.5
78.1 70.5
74.0 60.9
67.5 59.4
59.0 47.4
P < 0.001. Paired t-test.
66 % EAET- R FC .80 .60
/
.4o
~j. , ,
0
1
2
3
4
20hours
Time of spontaneous sedimentation
Fig. 2. T h e p e r c e n t a g e o f EAET-RFC as a f u n c t i o n o f t i m e o f s p o n t a n e o u s s e d i m e n t a t i o n at 4 °C. T h e results f r o m 2 n o r m a l b l o o d d o n o r s are s h o w n . • i n d i c a t e s t h e p e r c e n t a g e s o b t a i n e d b y t h e s t a n d a r d p r o c e d u r e , i n c l u d i n g c e n t r i f u g a t i o n at 50 x g.
Time o f incubation It is recommended in the WHO Report (1974) that the centrifugation step is followed by overnight incubation at 4°C to ensure maximal rosetting. Indeed, it has been shown t h a t the number of l y m p h o c y t e s that form rosettes with untreated SRBC after centrifugation but w i t h o u t any incubation is only about half the number of RFC after overnight incubation at 4 ° C. This has given rise to the terms early (or active) and late (or total) rosettes, the former being regarded as a subset of human T cells (Wybran and Fudenberg, 1973; Yu, 1975). Fig. 3 shows the percentage of E A E T - R F C and E u n t r RFC as a function of time of incubation at 4 ° C. The percentage of EAE TRFC is maximal immediately after centrifugation, the individual rosettes, %E - RFC 80 .
40 20 I
I
!
I
0
½
1
2
-,,
|
20
I
48 hours
T i m e of incubation
Fig. 3. T h e p e r c e n t a g e o f EAET-RFC (O) a n d E u n t r - R F C (©) as a f u n c t i o n o f t i m e o f incub a t i o n at 4°C. T h e results are given as m e a n s o f 10 ( o ) or 8 (o) n o r m a l b l o o d d o n o r s respectively. T h e vertical bars r e p r e s e n t +-1 S.D. T h e values a t 0 h o f i n c u b a t i o n are t h e p e r c e n t a g e s o b t a i n e d i m m e d i a t e l y a f t e r c e n t r i f u g a t i o n , i.e. w i t h o u t a n y i n c u b a t i o n .
67
however, showing a tendency to become denser after 30 min of cold incubation. After 30 min neither quantitative nor qualitative changes were observed. In contrast, it is seen that the number Euntr-RFC immediately after centrifugation is approx, half the value after overnight incubation. Further, there is a significant increase of Eunt~-RFC even between 2 h and 20 h of cold incubation, which underlines the validity of the above-mentioned recommendation of the WHO R e p o r t (1974), when Euntr are used.
Temperature conditions for rosetting E-rosette formation is generally thought to be extremely temperature dependent (WHO Report, 1974). To investigate the temperature conditions for EAEw-rOsetting , l y m p h o c y t e s and EAE w w e r e stored separately at 0°C, 4°C, 20°C and 37°C, respectively. After the lymphocytes and E A E w had obtained the required temperatures, they were mixed and centrifuged, and then reincubated at the original temperature for 1 h, whereafter the rosettes were scored. Because of lack of time for counting, the mixtures at 4°C had to be stored overnight, however. In addition, l y m p h o c y t e s from each l y m p h o c y t e donor were mixed with EAE w at room temperature, incubated at 37°C for 15 min, then centrifuged and stored at 4°C overnight. This latter procedure is routinely used by several workers in accordance with the WHO R e p o r t (1974). All these procedures were performed in parallel with Euntr on the same l y m p h o c y t e suspensions. The results are seen in Fig. 4. The percentages of EAET-RFC at 0°C, 4°C and 20°C were equal and identical to the percentages obtained when the preincubation procedure recom-
%E-RFC 80 60 40 20
0
4
20
°C
Temperature of incubation
Fig. 4. T h e p e r c e n t a g e o f EAET-RFC (O) a n d E u n t r - R F C (o) in 10 n o r m a l b l o o d d o n o r s as a f u n c t i o n o f t h e t e m p e r a t u r e o f i n c u b a t i o n . EAE T or Euntr a n d l y m p h o c y t e s were m i x e d a f t e r r e a c h i n g t h e i n d i c a t e d t e m p e r a t u r e s , c e n t r i f u g e d a n d t h e n r e i n c u b a t e d at t h e original t e m p e r a t u r e for 1 h a n d t h e r o s e t t e s scored. T h e m i x t u r e s were s t o r e d o v e r n i g h t at 4°C. * i n d i c a t e s t h e p e r c e n t a g e s o b t a i n e d w h e n l y m p h o c y t e s a n d S R B C were m i x e d at r o o m t e m p e r a t u r e , p r e i n c u b a t e d at 37°C for 15 m i n , t h e n c e n t r i f u g e d a n d s t o r e d at 4°C overnight. T h e results are given as m e a n s , t h e vertical bars i n d i c a t i n g 1 S.D.
68
mended in the WHO R e p o r t (1974} was used (Student-Newman-Keuls test). At 37°C, however, the number of EAET-RFC was reduced to half the values obtained at the lower temperatures. With Euntr the percentages at 4°C and 20°C were equal and a b o u t 10% higher (P < 0.05) than at 0°C, whereas virtually no rosettes were formed at 37°C. Fig. 4 also shows that preincubation at 37°C for 15 min gave significantly lower percentages (approx. 10%) than the 4°C and 20°C figures.
Resuspension As E-rosettes are fragile and easily disrupted (Heier, 1974), the resuspension procedure is critical. To investigate the mechanical stability of EAE Trosettes as compared with Eunt~-rosettes, mechanical resuspension was performed for 5 sec on a Whirlmixer ® (Fisons Scientific Apparatus, U.K.) using 5 different speeds. The results are depicted in Fig. 5, which shows the increased mechanical stability of EAEw-rOsettes, although it is seen that EAEw-rOsettes can be disrupted if the resuspension force is strong enough. It should be mentioned that even the lowest strength of mechanical resuspension was much stronger than the standard manual resuspension. Coun ting o f rosettes An essential feature of the increased affinity between the E-receptor and AET-treated SRBC is the increased number of SRBC attached to each lymp h o c y t e (Table 4). A b o u t 95% of the EAEw-rOsettes had a " m o r u l a " appearance with more than 20 EAE T per lymphocyte. Only occasional RFC were
,,o
100
er
N
80
g ~ 6o
~
n -"
2o m
M
1
2
3
4
L 5
Force of resuspension
Fig. 5. T h e m e c h a n i c a l s t a b i l i t y o f EAET-rosettes ( h a t c h e d b a r s ) a n d E u n t r - r o s e t t e s ( o p e n bars). M = usual m a n u a l r e s u s p e n s i o n . T h e figures 1--5 i n d i c a t e a r b i t r a r y increasing u n i t s o f a g i t a t i o n o n a W h i r l m i x e r ® used for m e c h a n i c a l r e s u s p e n s i o n f o r 5 sec. T h e results o f 2 n o r m a l b l o o d d o n o r s are given as relative p e r c e n t a g e s o f R F C to t h e values o b t a i n e d b y usual m a n u a l r e s u s p e n s i o n . +1 S.D.
69 TABLE 3 PERCENTAGE OF EAET-RFC AT D I F F E R E N T FORCES OF SEDIMENTATION ± 1 S.D. 4 normal blood donors. Sedimentation force (g)
%EAET-RFC ± S.D.
1a
2
50 b
200
400
700
64.4 -+ 15.8
66.1 ± 11.2
65.7 ± 14.4
65.9 ± 8.8
58.2 ± 9.0
56.9 ± 8.6
a Spontaneous sedimentation overnight. b Standard procedure. P > 0.05. Student-Newman-Keuls test.
f o u n d w i t h as f e w as 3 EAE T a t t a c h e d . E u n t r - R F C o n t h e o t h e r h a n d w e r e n e v e r f o u n d t o b e as h e a v i l y s t u d d e d w i t h S R B C as E A E T - R F C , a n d i n a l m o s t e v e r y p r e p a r a t i o n l y m p h o c y t e s w e r e f o u n d w i t h o n l y o n e o r t w o Euntr a t t a c h e d t o t h e m e m b r a n e , a r a r e f i n d i n g w i t h EAE w. T h i s f e a t u r e o f A E T treated SRBC provides a sharp distinction between E-RFC and non-RFC, which renders counting quick and reliable. The addition of toluidine blue allows for quick and reliable differentiation between SRBC and lymphocytes.
E-RFC in healthy blood Table 5 shows the result of a paired comparison of the percentages of E A E T - R F C a n d E u n t r - R F C i n 2 0 h e a l t h y b l o o d d o n o r s . I t is s e e n t h a t signific a n t l y m o r e E - R F C is d e t e c t e d u s i n g A E T t r e a t m e n t o f S R B C . I n 1 0 4 h e a l t h y b l o o d d o n o r s t h e m e a n p e r c e n t a g e o f E A E T - R F C was 7 3 . 6
TABLE 4 PERCENTAGE OF Euntr-RFC AND EAET-RFC IN 6 NORMAL DONORS WITH INDICATION OF THE NUMBER OF SRBC PER ROSETTE -+ 1S.D. NumberofSRBC
%Euntr-RFC -+ S.D. %EAET-RFC ± S.D.
3--4
5--9
/> 10 a
Total % RFC
5.6 ± 1.9 (8.8) b 0.7 ± 0.3 (0.9)
15.8 ± 4.3 (24.8) 3.2 ± 1.1 (4.1)
42.4 ± 5.3 (66.6) 74.0 ± 4.6 (95.1)
63.7 ± 9.3 77.8 ± 4.0
a All EAET-rosettes of this category were "morula"-like, see text. The figures in the parentheses indicate the distribution in relation to total % RFC.
b
70 TABLE 5 PAIRED COMPARISON OF T H E PERCENTAGES OF EAET-RFC AND Euntr-RFC IN 20 HEALTHY DONORS -+ 1 S.D. %EAET-RFC -+ S.D.
%Euntr-RFC -+ S.D.
70.9 ± 5.8
60.1 ± 6.7
P < 0.01. Paired t-test. w i t h a r a n g e o f 4 9 . 3 - - 9 1 . 1 % and a s t a n d a r d d e v i a t i o n o f 8.3%. Similar figures h a v e b e e n r e p o r t e d b y o t h e r a u t h o r s using A E T - t r e a t e d S R B C ( K a p l a n and Clark, 1 9 7 4 ; Pellegrino et al., 1 9 7 5 ; Stein, 1977). T h e p e r c e n t a g e s o f EAET-RFC as well as Euntr-RFC in n o r m a l b l o o d donors were normally distributed.
Reactivity o f EAE T with human B cell lines N o r o s e t t i n g was o b s e r v e d w i t h A E T - t r e a t e d S R B C a n d a n y o f the cult u r e d h u m a n B cell lines m e n t i o n e d a b o v e , i n d i c a t i n g t h a t A E T t r e a t m e n t d o e s n o t r e n d e r S R B C c a p a b l e o f f o r m i n g r o s e t t e s w i t h B cells.
Reactivity o f AET-treated human and bovine erythrocytes with human lymphocytes N e i t h e r h u m a n or b o v i n e e r y t h r o c y t e s are c a p a b l e o f r o s e t t i n g with h u m a n l y m p h o c y t e s ( W H O R e p o r t , 1974). A E T t r e a t m e n t o f such e r y t h r o c y t e s in the w a y d e s c r i b e d f o r S R B C did n o t a f f e c t this n o n - r e a c t i v i t y .
EAET-rosetting with trypsinized lymphocytes T h e E - r e c e p t o r is sensitive to t r y p s i n t r e a t m e n t ( J o n d a l e t al., 1 9 7 2 ) . T a b l e 6 s h o w s t h a t t r y p s i n i z e d l y m p h o c y t e s h a v e also a l m o s t lost t h e i r rosetring c a p a c i t y w i t h EAE w.
EAET-rosetting with anti-human Ig-treated lymphocytes In d o u b l e m a r k e r e x p e r i m e n t s , l y m p h o c y t e s w e r e t r e a t e d w i t h F I T C TABLE 6 THE EFFECT OF TRYPSIN TREATMENT OF LYMPHOCYTES ON PERCENTAGES OF EAET-RFC Ten normal donors.
%EAET-RFC Range
Untreated lymphocytes
Trypsinized lymphocytes
75 57--84
3 2--4
71 conjugated anti-human Ig as described under Materials and Methods and then used for EAEw-rOsetting. EAET-rOsettes were scored as described and, in addition, the preparations were examined by fluorescence microscopy. No inhibition of rosetting was f o u n d when the l y m p h o c y t e s were treated with anti-Ig, in agreement with Coombs et al. (1970) and Bentwich et al. (1973) who used untreated SRBC. There was usually no overlapping between fluorescent cells (i.e. B cells) and RFC. In a few preparations, however, 1--2% of the RFC were also fluorescent, although the rosettes were much less dense than the other morula-like rosettes in the same preparation. These findings are in accordance with Bentwich et al. (1973) who also f o u n d a minor overlapping using untreated SRBC, and with Kaplan and Clark (1974) who used AET-treated SRBC. DISCUSSION The present report demonstrates a substantial stabilizing effect of AET treatment of SRBC on the E-rosettes, confirming the findings of others (Kaplan and Clark, 1974; Pellegrino et al., 1975; Stein, 1977). The effect of AET in stabilizing the otherwise weak binding between the E-receptor and SRBC is shown by the markedly decreased influence of several of the technical variables which are k n o w n to affect the results of E-rosetting. The enhancing effect observed implies that significantly more E-receptor carrying l y m p h o c y t e s are detectable by EAET than by Euntr , viz. 70.9% VS. 60.1% in a paired comparison (Table 5), which is in accordance with the figures reported by Kaplan and Clark (1974) and Mahowald et al. (1977). Unambiguous differentiation between E-RFC and non-E-RFC is a requirem e n t for a reliable E-rosette technique. One of the main results of AET t r e a t m e n t of SRBC is a substantial increase of the number of SRBC attached to each RFC, giving the majority of the EAEw-rOsettes a morula-like appearance; the non-EAET-RFC are left totally free of SRBC (Table 4). In contrast, Eunt,-RFC have considerably less SRBC bound to each l y m p h o c y t e , and some cells have only 3 or 4 SRBC attached. Further, in almost every preparation some lymphocytes are found with one or two Euntr clearly attached, raising d o u b t whether those cells possess E-receptors or not. Conventionally, l y m p h o c y t e s with three or more SRBC firmly attached to the cell membrane are regarded as E-receptor carrying cells (WHO Report, 1974), although some investigators also consider l y m p h o c y t e s with even one SRBC bound as E-receptor positive (Bentwich et al., 1973). With AET treatment less equivocal criteria for E-receptor identification can be adopted, since only occasionally are EAEw-rOsettes f o u n d with few SRBC (see also Stein, 1977). Another consequence of AET t r e a t m e n t of SRBC is improved mechanical stability of E-rosettes (Fig. 5), also reported by Hokland et a17(1976). This is an important point, since E-rosettes are sensitive to the force of resuspension, and this is generally considered a critical step in the technique (Heier, 1974; WHO Report, 1974).
72 The influence of serum in the rosetting medium has been disputed, but most investigators add FCS or absorbed human AB serum in accordance with the findings of Bentwich et al. (1973). In this report the stabilizing effect of FCS is reflected in an increased percentage of Euntr-RFC, b u t the effect of EAET-rosettes is only qualitative (Tables 1 and 2). The incubation temperature is critical for E-rosetting (Lay et al., 1971; Mendes et al., 1973), and all workers recommend incubation at 4°C to ensure maximal rosetting. In agreement with Mendes et al. (1973) we find optimal rosetting at 4°C and 20°C with Eunt~, the percentage at 0°C being significantly lower. At 37°C virtually no rosettes were found. With EAET, however, no differences were observed at between 0°C, 4°C and 20°C although the percentage was significantly lower at 37°C (Fig. 4). Lay et al. (1971) r e c o m m e n d preincubation of the samples at 37°C for 15 min before centrifugation and incubation at 4°C. With this procedure, however, significantly lower percentages o f Euntr-RFC were obtained (Fig. 4). A possible explanation of our findings with 37°C preincubation might be that E-receptors are shed at this temperature and do n o t regenerate at 4°C, as described recently by S~rmay et al. (1978). The peculiar temperature requirements of the E-rosette technique need further investigation. As further indication of increased binding force following AET treatment of SRBC, preincubation at 37°C showed no effect on rosetting capacity. Most authors use incubation at 4°C overnight, usually 18--20 h as recommended (WHO Report, 1974). In this study we find the percentage of E~n~R F C significantly higher after overnight incubation at 4°C than after 2 h incubation. Further, we also find a low percentage (approx. 30%) of EuntrR F C when resuspension and counting is performed immediately after centrifugation, i.e. without any incubation step. These rosettes correspond to " e a r l y " or " i m m e d i a t e " E-RFC. With EAET,however, the percentage of R F C is maximal immediately after centrifugation, although the rosettes are less dense than after 30 min of incubation at 4°C. No changes, neither quantitative nor qualitative, were observed after 30 min up to 48 h (Fig. 3). Close contact between l y m p h o c y t e s and EAET, obtained either by centrifugation at 50 × g or by spontaneous sedimentation overnight is clearly critical. As emphasized by Kaplan and Clark (1974) and Pellegrino et al. (1975} there is no indication that AET treatment changes the specificity of binding of SRBC to lymphocytes. In 9 different cultured B cell lines tested with EAET, rosettes were never found. Further, AET-treated human and bovine erythrocytes were n o t capable of rosetting with human lymphocytes. No overlapping between EAET-RFC and B cells (surface membrane Ig positive cells) was observed in most preparations investigated for both markers, only a few showing 1--2% fluorescent RFC. Finally, we found that treatment of the l y m p h o c y t e s with trypsin almost totally abrogated the EAEw-rOsette formation capacity, as reported for untreated SRBC (FrSland, 1972; Jondal et al., 1972). The mechanism of the action of AET on erythrocytes is n o t understood,
73 a l t h o u g h it is likely t h a t it reacts with thiol g r o u p s in the cell m e m b r a n e (Sirchia and F e r r o n e , 1 9 7 2 ) . I t s h o u l d be e m p h a s i z e d t h a t A E T is n o t a s u l p h h y d r y l c o m p o u n d , b u t at n e u t r a l and alkaline p H is c o n v e r t e d t o merc a p t o e t h y l g u a n i d i n e ( D o h e r t y et al., 1 9 5 7 ) , w h i c h m i g h t be e x p e c t e d t o have an a c t i o n on the e r y t h r o c y t e m e m b r a n e . Pellegrino et al. ( 1 9 7 5 ) have suggested t h e possibility t h a t thiol g r o u p s are o f crucial i m p o r t a n c e for E-rosette f o r m a t i o n . Whatever t h e m e c h a n i s m , A E T t r e a t m e n t o f SRBC has been s h o w n t o be a valuable m o d i f i c a t i o n o f t h e E - r o s e t t e t e c h n i q u e resulting in stable E-rosettes, and it m a y also have i m p o r t a n t i m p l i c a t i o n s for T and B cell separation. ACKNOWLEDGEMENTS Mrs. H. JCrgensen and Mrs. I.-L. T h o r s e n are t h a n k e d f o r e x p e r t technical assistance and Dr. J. Z e u t h e n for p r o v i d i n g the B cell lines. REFERENCES Bach, J.F., 1973, Transplant. Rev. 16,196. Bentwich, Z., S.D. Douglas, F.P. Siegal and H.G. Kunkel, 1973, Clin. Immunol. Immunopathol. 1, 511. BSyum, A., 1968, Scand. J. Clin. Lab. Invest. 21 (Suppl. 97), 77. Brown, C.S., H. Halpern and H.H. Wortis, 1975, Clin. Exp. Immunol. 20, 505. Brown, G. and M.F. Greaves, 1974, Eur. J. Immunol. 4,302. Coombs, R.R.A., B.W. Gurner, A.B. Wilson, G. Holm and B. Lindgren, 1970, Int. Arch. Allergy 39,658. Doherty, D.G., R. Shapira and J. Burnett, 1957, J. Am. Chem. Soc. 79, 5667. Favour, C.B., 1964, in: Immunological Methods, ed. J.F. Ackroyd (Blackwell Scientific Publ., Oxford) p. 212. Florey, M.J. and F. Peetroom, 1976, J. Immunol. Methods 13, 201. Fr61and, S.S., 1972, Scand. J. Immunol. 1, 269. Heier, H.E., 1974, Scand. J. Immunol. 3,677. Hokland, P., M. Hokland and I. Heron, 1976, J. Immunol. Methods 13, 175. Johnsen, H.E. and M. Madsen, 1978, Scand. J. Immunol. 8,239. Jondal, M., 1976, Scand. J. Immunol. 5 (Suppl. 5), 69. Jondal, M., G. Holm and H. Wigzell, 1972, J. Exp. Med. 136, 207. Jctrgensen, F. and L.U. Lamm, 1974, Tissue Antigens 4,482. Kaplan, M.E. and C. Cark, 1974, J. Immunol. Methods 5, 131. Kissmeyer-Nielsen, F. and K.E. Kjerbye, 1967, in: Histocompatibility Testing, eds. E.S. Curtoni, P.L. Mattiuz and R.M. Tosi (Munksgaard, Copenhagen) pp. 381--383. Kissmeyer-Nielsen, F. and E. Thorsby, 1970, Transplant. Rev. 4,117. Lay, W.H., N.F. Mendes, C. Bianco and V. Nussenzweig, 1971, Nature 230, 531. Mahowald, M.L., B.S. Handwerger, E.M. Capertone and S.D. Douglas, 1977, J. Immunol. Methods 15, 239. Mendes, N.F., M.E.A. Tolnai, N.P.A. Silveira, R.B. Gilbertsen and R.S. Metzgar, 1973, J. Immunol. 111, 860. MSller, G. (ed.), 1973, Transplant. Rev. 16, 1. Pellegrino, M.A., S. Ferrone, M.P. Dierich and R.A. Reisfeld, 1975, Clin. Immunol. Immunopathol. 3,324.
74 Ross, G.D., E.M. Rabellino, M.J. Polley and H.M. Gray, 1973, J. Clin. Invest. 52, 377 S~rmay, G., L. Istv~n and J. Gergely, 1978, Immunology 34, 315. Sirchia, G. and S. Ferrone, 1972, Ser. Haematol. 5,137. Stein, G., 1977, J. Immunol. Methods 14,371. Weiner, M.S., C. Bianco and V. Nussenzweig, 1973, Blood 42,939. WHO Report, 1974, Scand. J. Immunol. 3,521. Wilson, A.B., D.G. Haegert and R.R.A. Coombs, 1975, Clin. Exp. Immunol. 22, 177. Wybran, J. and H.H. Fudenberg, 1973, J. Clin. Invest. 52, 1026. Yu, D.T.Y., 1975, J. Immunol. 115, 91.