- Email: [email protected]
Duck lymphocytes—III. Transformation responses to some common mitogens
Comp. Immun. Microbiol. infect. Dis. Vol. 13, No. 1, pp. 13-23, 1990 Printed in Great Britain. All rights reserved
0147-9571/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
DUCK LYMPHOCYTES--III. TRANSFORMATION R E S P O N S E S TO SOME C O M M O N M I T O G E N S D. A. HIGGINS Department of Pathology, University of Hong Kong, Queen Mary Hospital, Hong Kong
(Received 8 November 1989) Abstract--The lymphocyte transformation (LT) test was performed using duck blood lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A), lentil lectin (LC), Roman snail lectin (HP), peanut agglutinin (PNA), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), horseshoe crab lectin (HSC), pokeweed mitogen (PWM) and E. coli lipopolysaccharide (LPS). Cells were cultured in microtitre trays, at 41.6°C, 8 x 105 cells in 200/tl medium ( = 4 × 106 cells/ml) supplemented with 10% pooled duck serum. Mitogens were added at final concentrations of 0.1-100 ,ug/ml and triplicate cultures at each concentration were harvested daily for scintillation counting 6 hr after addition of I pCi [~H]thymidine. Three patterns of response were observed. The responses to Con A, LC, HP and HSC were greatest at high mitogen concentrations (40-100 #g/ml) throughout the 7 days of culture. With PHA, PNA, WGA and LPS maximum stimulation was obtained at 3-5 days, at which time the cells were responding to lower concentrations of mitogen than were required at other times during the experiment. The response to BSS and PWM showed increasing sensitivity to lower concentrations of mitogen during the first 3 days of culture and then stimulated most strongly at 2-10 pg/ml in cultures harvested after 4-7 days. Cells from two ducks were cultured for 3 and 5 days with selected concentrations of these mitogens; the results confirmed the variation in response to different mitogens. It is possible that these patterns of response are the outcome of stimulating different populations of duck lymphocytes.
Key words: ducks, lymphocytes, transformation, mitogens, lectins. R6sum6--Le test de transformation de lymphocytes a 6t6 ex6cut6 fi l'aide de lymphocytes sanguins de canard et activ6 par les agents suivant: phytohemagglutinine (PHA), concanavaline A (Con A), lectine de lentille (HP), agglutinine d'arachide (PNA), lectine de grain de Bandeiraea simplicifolia (BSS), agglutinine de germe de bl6 (WGA), lectine de crabe des Moluques (HSC), pokeweed mitog6n (PWM) et lipo-polysaccharide d'E. coli (LPS). Les cellules onty 6t6 plac6es dans des bassins microtitre de culture, fi une temp6rature de 41,6°C, 8 × 105 cellules dans 200 pl demi-lieu ( = 4 x l06 cellules/ml) additionn/~es de sbrum de canard fi 10%. Les mitogens out 6t6 ajout6s fi des concentrations finales de 0,1-100 #g/ml et des cultures en triplicate de chacune des concentrations out 6t6 r6colt6es quotidiennement pour scintillation 6 heures apr6s l'ajout de 1/~Ci de [3H]thymidine. Trois r6actions furent observ6es. Les r6actions aux Con A, LC, HP et HSC &aient maximales avec le mitogene ~i haute concentration (40-100/~g/ml) pendant toute la dur6e (7 jours) de culture. Avec les agents PHA, PNA, WGA et LPS la stimulation maximum est survenue aux jours 3-5, :i des concentrations moindre de mitogene que celles qui avaient ~t~ appliqu~es a d'autre temps. La r6action au BSS et PWM a d6montr6 une sensibilit6 croissante fi des concentrations moindre de mitogen lors des trois premiers jours de l'exp6rience et atteignirent une stimulation maximum a 2-10 pg/ml dans des cultures r6colt~es apr6s 4-7 jours. Des cellules de deux canards furent raises en culture pour 3 et 5 jours avec une s61ection de concentrations de ces mitogenes; les r6sultats ont confirm6 les 6carts de r~action. I1 est possible que ces r6actions et 6chantillons de r6sultats soient consecutifs ~i la stimulation de diverses populations de lymphocytes de canards.
Mots-cl(fs: canards, lymphocytes, transformation, mitogene, lectines.
INTRODUCTION Although lymphocytes from the duck (Anas platyrhynchos) do not possess surface markers similar to those occurring on mammalian T a n d B cells, t h e y d o r e s p o n d t o m i t o g e n s i n 13
14
D . A . HIGGINS
lymphocyte transformation (LT) tests [1]. Their requirements for optimum response to phytohaemagglutinin (PHA) have been reported [2]. These optimum culture conditions have now been used to study the in vitro response of duck blood lymphocytes to several mitogens, with a view to identifying subpopulations of cells responding to different stimulants. MATERIALS AND METHODS
Ducks Ducks were a hybrid strain of White Pekin (Super M, Cherry Valley Farms Ltd, Lincoln, U.K.), 4-7 months old at the time of experiments. Mitogens (i) Phytohaemagglutinin (PHA) from Phaseolus vulgaris, sugar specificity N-acetyl-Dglucosamine(1-2)D-mannose, N-acetyl-D-galactosamine [3,4], was in two batches. That used as a test mitogen was from Sigma Chemical Company (L-8754, lot number 25F-9640); that used as a 3-day positive control was from Pharmacia AB, Uppsala, Sweden (code 17-0449-01; lot number 9004). (ii) Concanavalin A (Con A) from Canavalia ensiformis (17-0450-01, Pharmacia; lot number NH 04564), sugar specificity ~-D-mannose, a-D-glucose, N-acetyl-D-glucosamine [5]. (iii) Lentil lectin (LC) from Lens culinaris (L-5880, Sigma; lot number 114F-9670), sugar specificity a-D-glucose, ~-D-mannose, ~-D-fUcose [6]. (iv) Roman snail lectin (HP) from Helix pomatia (L-3382, Sigma; lot number 53F-9013), sugar specificity N-acetyl-~-D-galactosamine [7, 8]. (v) Peanut agglutinin (PNA) from Arachis hypogaea (L-0881, Sigma; lot number 83F-9620), sugar specificity D-galactosyl-/~(1-3)N-acetyl-D-galactosamine [9]. (vi) Bandeiraea simplicifolia seed (BSS) extract (L-2380, Sigma; lot number 84F-9690), sugar specificity ~-D-galactose, N-acetyl-~-D-galactosamine [10-12]. (vii) Wheat germ agglutinin (WGA) from Triticum vulgaris (L-9640, Sigma; lot number 76F-4031-1), sugar specificity (N-acetyl-D-glucosamine)2, N-acetyl-D-neuraminic acid, N-acetyl-D-galactosamine [13 15]. (viii) Horseshoe crab lectin (HSC) from Limulus polyphemus (L-9509, Sigma; lot number 17F-9540), sugar specificity N-acetyl-D-neuraminic acid [16]. (ix) Pokeweed mitogen (PWM) from Phytolacca americana (L-9379, Sigma; lot numbers 65F-9570 and 83F-9670), a mitogen consisting of at least five components with broad sugar-binding capabilities [17]. (x) Lipopolysaccharide (LPS) from Escherichia coli serotype 0111 :B4 (L-2630, Sigma; lot number 114F-4043). Blood collection, lymphocyte separation and LT test The techniques described previously [2] were followed exactly. Briefly, blood was collected by cardiac puncture into an equal volume of 0.1 M Na2HPO4-NaH2PO4 buffered saline, pH 7.2 (PBS) containing sodium heparin (10 IU/ml, Heparin Injection BP, Commonwealth Serum Laboratories). Lymphocytes were purified by centrifuging 3 ml of the blood-PBS mixture over 3 ml Ficoll 400/diatrizoate sodium solution, specific gravity 1.077 (Ficoll-Paque, Pharmacia) at 200 g for 25 min at 20°C. The lymphocyte layer was
Duck lymphocytes
15
collected and washed once in PBS and twice in Roswell Park Memorial Institute medium 1640 (RPMI), then adjusted to a concentration of ~ 10 x 106 cells/ml in RPMI. The LT test was set up in 96-well, flat-bottomed microculture trays (code no 167008, Nunc) using 80/tl of cell suspension, 100#l RPMI containing 20% heat-inactivated (56°C, 45 min) pooled adult duck serum (PDS), and 20/~l of RPMI containing mitogen at 10 × the desired final concentration; thus, the conditions achieved were 8 × 105 cells in 200#1 cultures supplemented with 10% PDS and mitogen. Trays were incubated at 41.6°C in a humid atmosphere containing 5% COs. Cultures were radiolabelled 6 hr prior to harvesting by adding 20/~1 RPMI containing 1/tCi of [3H]thymidine (TRK-120, Amersham). Cells were harvested by washing with distilled water onto glass-fibre mats (GF/C, Whatman) using a semi-automated harvester. Mats were dried at 37°C overnight, then the individual discs were separated and placed in glass scintillation vials containing 2 ml scintillant [10g 2,5-diphenyloxazole (Sigma) and 0.5g 1,4-bis-2-(4-methyl-5-phenyloxazolyl)benzene (Sigma) per litre of scintillation grade toluene (Fisher)]. Radioactivity was measured as counts per min (cpm) in Beckman LS-7000 or LS-5801 /~-scintillation counters and expressed as means of triplicate cultures; standard deviations are not reported in the Results section but were within _+20% of the means. Unstimulated, unlabelled (i.e. no mitogen, no [3H]thymidine) and unstimulated, labelled controls were included in each experiment. The stimulation index (SI) was calculated using the mean cpm values by the formula (mitogen-stimulated, [3HI-labelled) - (unstimulated, unlabelled) (unstimulated, [3HI-labelled) - (unstimulated, unlabelled)
Experiment plan Each bleeding yielded sufficient cells to allow two lectins to be examined in one experiment. Lectins were used at final concentrations of 0.1, 0.5, 1, 2, 5, 10, 20, 40 and 100 pg/ml. Cells were harvested daily up to 7 days of culture. All mitogens were examined at least twice and each time were tested in parallel to a different mitogen. If the patterns of lymphocyte responsiveness obtained with a mitogen on two separate occasions were different, the experiment was performed a third (or fourth) time to identify the most repeatable pattern. For each experiment a "positive control" was set up consisting of a titration of PHA (Pharmacia) harvested after 3 days of culture; if this was subsequently found to have not given satisfactory stimulation, the experiment was repeated on the basis that the cells might, on that occasion, have been unresponsive. Finally, blood lymphocytes from two ducks were examined, each on a separate occasion, for their response to selected concentrations of the panel of mitogens after 3 and 5 days of culture. RESULTS Duck blood lymphocytes cultured in the presence of 10% PDS responded in LT test to PHA, Con A, LC, HP, PNA, BSS, WGA, HSC, PWM and LPS (Tables 1-10). PHA, PNA, WGA and LPS showed a pattern of stimulation with maximum sensitivity (i.e. greatest response being elicited to lower concentrations of mitogen) developing after 3-5 days culture. The response to Con A, LC, HP and HSC was greatest at high mitogen concentrations (40-100 #g/ml) throughout the 7 days of culture. BSS and PWM provoked
D. A. HIGGINS
16
Table 1. Time,lose transformation responses of duck blood lymphocytes to phytohaemagglutinin (PHA) Radioactivity" of Unstimulated controls
Cultures stimulated with PHA (#g/ml)
Time of culture
-~H
+3H
0.1
0.5
1
2
5
10
20
40
100
I day
332
451
429 (0.81)
549 (1.82)
340 (0.07)
403 (0.60)
411 (0.66)
642 (2.60)
806 (3.98)
1083 (6.31)
1(21.7) 1
2 days
246
437
355 (0.57)
463 (1,14)
270 (0,12)
342 (0.50)
868 (3 26)
1282 (5.42)
2105 (9,73)
2335 (10.9)
[(12,9) I
3 days
360
923
696 (0.60)
447 (0.15)
844 (0.86)
1115 (1.34)
2124 (3.13)
3698 (5.93)
~ [(7.86) I
4149 (6.73)
1787 (2.53)
4 days
96
284
206 (0.58)
180 (0.45)
547 (2.40)
1300 (6.40)
3554 (18.4)
6139 (32.1)
~ [(33.9) I
6378 (33.4)
848 (4.00)
5 days
169
343
296 (0.73)
311 (0.82)
446 (1.59)
534 (2.10)
1586 (8.14)
6467 (36.2)
~ [(46.9)[
5374 (29.9)
1065 (5.15)
6 days
268
852
425 (0.27)
400 (0.23)
479 (0.36)
368 (0.17)
1187 (1.57)
1564 (2.22)
~ 1(5.26) 1
3011 (4.70)
424 (0.27)
7 days
395
821
927 (1.25)
594 (0.47)
442 (0.11)
333 (-0.14)
566 (0.40)
~ [(3.03) I
952 (1.38)
1320 (2.17)
465 (0.16)
"As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
responses which showed increasing sensitivity (from 100/~g to 2-10 #g) during the first few days of culture, thereafter retaining maximum SI to these lower concentrations throughout the remainder of the experiment. In terms of the cpm and SI achieved, PHA, LC, PNA, BSS, WGA, HSC and PWM were considered strong mitogens for duck blood lymphocytes, while Con A, HP and LPS were considered weak mitogens. The response of blood lymphocytes from two ducks to selected concentrations of this range of mitogens (Table 11) confirmed that some mitogens were stronger stimulants than others and that the time course of the response to different mitogens varied. In addition there appeared
Table 2. Time~zlose transformation responses of duck blood lymphocytes to Concanavalin A (Con A) Radioactivity" of Unstimulated controls
Cultures stimulated with Con A (l~g/ml)
Time of culture
-3H
+ 3H
0.l
0.5
1
2
5
10
20
40
100
1 day
110
463
302 (0.54)
402 (0.83)
520 (1.16)
367 (0.73)
550 (1.25)
493 (1.08)
443 (0.94)
426 (0.89)
1(2.29)[
2 days
334
517
281 (-0.29)
834 (2.73)
438 (0.57)
506 (0.94)
407 (0.40)
467 (0.73)
602 (1.46)
1(27.5) 1
3 days
165
281
356 (1.65)
263 (0.84)
467 (2.60)
293 (1.10)
268 (0.89)
333 (1.45)
687 (4.50)
656 (4.23)
/ (9.77) [
4 days
196
325
271 (0.56)
206 (0.08)
226 (0.23)
329 (1.03)
299 (0.80)
378 (1.41)
561 (2.83)
465 (2.08)
I (7.10) [
5 days
229
327
541 (3.18)
256 (0.27)
433 (2.08)
297 (0,69)
500 (2.76)
297 (0.69)
716 (4.97)
533 (3.10)
[(5.89) 1
6 days
97
230
320 (I.68)
221 (0.93)
318 (1.66)
280 (I.37)
345 (I.86)
263 (1.25)
436 (2.55)
488 (2.94)
1(8.27) 1
7 days
408
773
917 (1.39)
572 (0.45)
759 (0.96)
1032 (1.71)
968 (1.53)
625 (0.59)
1038 (1.73)
1171 (2.09)
1(3,97) 1
236 (0.53)
"As mean cpm of tripticate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
17
Duck lymphocytes Table 3. Time-dose transformation responses of duck blood lymphocytes to lentil lectin (LC) Radioactivity ~ of Unstimulated controls
Cultures stimulated with LC (vg/ml)
Time of culture
-3H
+3H
0.1
0.5
1
2
5
10
20
40
100
1 day
206
412
362 (0.76)
314 (0.52)
537 (1.61)
451 (1.19)
508 (1.47)
586 (1.84)
969 (3.70)
1534 (6.45)
1(21.7) 1
2 days
253
1760
1743 (0.99)
1836 (1.05)
1744 (0.99)
2073 (1.21)
1887 (1.08)
1763 (1.00)
6910 (4.42)
12596 (8.19)
1(10.9) 1
3 days
150
537
572 (1.09)
559 (1.06)
569 (1.08)
839 (1.78)
783 (1.63)
770 (I.60)
1824 (4.32)
9360 (23.8)
1(211) 1
4 days
107
507
583 (1.19)
553 (I.II)
224 (0.29)
338 (0.58)
579 (I.18)
362 (0.64)
7617 (18.8)
19202 (47.7)
1(308) 1
5 days
243
581
477 (0.69)
551 (0.91)
604 (1.07)
721 (I.41)
537 (0.87)
640 (1.17)
5074 (14.3)
9110 (26.2)
1(96.3) 1
6 days
249
636
546 (0.77)
577 (0.85)
588 (0.87)
614 (0.94)
573 (0.84)
1148 (2.32)
4763 (11.7)
5566 (13,7)
1(31.8) 1
7 days
534
855
961 (1.33)
1300 (2.39)
1041 (1.58)
975 (1.37)
810 (0.86)
1048 (I.60)
4119 (11.2)
5399 (15,2)
1(24.2) 1
~As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
to be some individual variation between the two ducks, e.g. there was a similar response to PHA by cells from both ducks but cells from duck 1 responded more vigorously to LC, WGA and PWM than cells from duck 2 which responded more strongly to BSS. DISCUSSION Lectins are proteins and glycoproteins which occur throughout the plant and animal kingdoms. They bind specifically to sugars present as mono- or oligosaccharides within Table 4. Time,lose transformation responses of duck blood lymphocytes to Helix pomatia lectin (HP) Radioactivity a of Unstimulated controls
Cultures stimulated with HP (#g/ml)
Time of culture
-3H
+3H
0.1
0.5
I
2
5
10
20
40
100
1 day
124
260
293 (I.24)
183 (0.43)
474 (2.57)
487 (2.67)
515 (2.87)
583 (3.37)
~ 1(3.98) 1
578 (3.34)
572 (3.29)
2 days
287
1874
1795 (0.95)
1319 (0.65)
1753 (0.92)
2377 (1.32)
3604 (2.09)
2314 0.28)
~ 1(2.20)[
3770 (0.18)
1964 0.06)
3 days
252
797
973 0.32)
545 (0.54)
581 (0.60)
608 (0.65)
1541 (2.36)
902 (1.19)
1881 (2.99)
962 (1.30)
4 days
218
504
631 (1.44)
766 (1.92)
379 (0.56)
610 (1.37)
1800 (5.53)
685 (1.63)
1362 (4.00)
~ 1(6.19)[
1(3.19) 1 1667 (5.07)
5 days
242
919
612 (0.55)
760 (0.76)
446 (0.30)
726 (0.71)
1667 (2.10)
847 (0.89)
1146 (1.33)
1621 (2.04)
1(3.39) 1
6 days
168
447
451 (1.01)
411 (0.88)
510 (1.22)
657 (1.75)
963 (2.85)
1256 (3.90)
1161 (3.56)
~ [(6.89) 1
2066 (6.80)
7 days
444
679
636 (0.82)
580 (0.58)
749 (1.30)
814 (1.57)
1186 (3.16)
1226 (3.33)
1531 (4.62)
~ [(10.8) 1
1691 (5.31)
~As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day. CIMID 13/I--B
18
D. A. H1GG1NS Table 5. T i m e . l o s e transformation responses of duck blood lymphocytes to peanut agglutinin (PNA) Radioactivity" of Unstimulated controls
Time of culture
~H
Cultures stimulated with PNA (,ug/ml)
+3H
0.1
0.5
1
2
5
10
20
40
100
1 day
472
708
1046 (2.43)
1169 (2.95)
1116 (2.73)
1216 (3.15)
2066 (6.75)
1716 (5.27)
1963 (6.32)
~ 1(7.00)]
1996 (6.46)
2 days
321
849
998 (I.28)
6526 (11.7)
11893 (21.9)
14633 (27.1)
11253 (20.7)
6483 (11.6)
11520 (21,2)
~ t(31.4)]
11314 (20.8)
3 days
315
1832
1236 (0,61)
1628 (0.86)
5407 (3,36)
5402 (3.35)
5883 (3.67)
6563 (4.12)
~ 1(4.38) 1
5730 (3.57)
4955 (3.06)
4 days
435
1960
2357 (I.25)
2781 (1.53)
4735 (2,81)
4883 (2.90)
~ [(8,58) 1
5938 (3.59)
7665 (4.72)
9219 (5.74)
4335 (2.56)
5 days
694
3588
4541 (1.33)
5295 (1.59)
11649 (3.78)
14705 (4.84)
~ [(13.9)[
20584 (6.87)
33370 (11.3)
36695 (12.4)
30309 (10.2)
6 days
587
2190
2459 (I.17)
5194 (2.87)
3281 (1.68)
5277 (2.92)
18533 (11.2)
11272 (6.66)
36039 ~ (22.1) 1(44.5)[
29170 (17.8)
7 days
867
2469
2142 (0.97)
4997 (2.58)
3787 (1.82)
5166 (2.68)
19929 (11.9)
20989 (12.6)
19024 (11.3)
22154 (13.3)
~ ](16.1) 1
~As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
glycoproteins and glycolipids on the surface of animal cells. Some are mitogenic and, along with mitogens such as LPS which bind to cell membranes by more complex mechanisms, have played an important role in studies of lymphocyte biology. Transformation of lymphocytes is not only dependent upon the presence of appropriate receptors but also requires their distribution in a density which allows cross-linking by the mitogen. It follows that some cells possess receptors that will bind appropriate lectins without resulting in transformation [18, 19]. While LT need not be an outcome of lectin-cell interaction, it has the advantage that such a response is usually considered a property of immunologically Table 6. Time,lose transformation responses of duck blood lymphocytes to Bandeiraea simplicifolia seed lectin (BSS) Radioactivity" of Unstimulated controls
Cultures stimulated with BSS (~g/ml)
Time of culture
-3H
+3H
0.1
0,5
1
2
5
10
20
40
100
1 day
300
725
842 (1.27)
1307 (2.37)
1657 (3.19)
1456 (2.72)
1317 (2.39)
988 (I.62)
1389 (2.56)
1231 (2.19)
1(3.33) 1
2 days
532
1220
1624 (1.59)
4255 (5.37)
4581 (5.88)
6146 (8.16)
9097 (12.4)
8012 (10.9)
11662 (16.2)
~ 1(17.7) 1
12273 (17.1)
3 days
241
653
757 (1.25)
1215 (2.36)
7002 (16.4)
9460 (22.4)
19406 (46.5)
16581 (39.7)
22967 ~ (55.2) 1(56.2) 1
21576 (51,8)
4 days
351
1626
2900 (1.99)
4695 (3.41)
7281 (5.43)
10916 (8.28)
18714 [~ffff] (14.4) ](20.7) 1
20632 (15.9)
14679 (11.2)
10682 (8.10)
5 days
272
571
930 (2.02)
1006 (2.45)
4493 (14.1)
11109 (36.2)
12749 (41.7)
~ [(48.2) I
14314 (46.9)
12744 (41.7)
5631 (17.9)
6 days
189
593
579 (0.96)
3657 (0.86)
7394 (17.8)
~ ](21.6) 1
6700 (16.1)
5662 (13.5)
7898 (19.1)
5390 (12.9)
2482 (5.7)
7 days
412
1311
516 (0.11)
1055 (0.71)
4809 (4.89)
6471 (6.74)
~ ](8.53) 1
2565 (2.39)
4032 (4.03)
1412 (I.I I)
1317 (1.01)
gAs mean cpm of triplicate cultures: SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
19
Duck lymphocytes Table 7. Time-dose transformation responses of duck blood lymphocytes to wheat germ agglutinin (WGA) Radioactivity~ of Unstimulated controls
Cultures stimulated with WGA (,ug/ml)
Time of culture
-3H
+3H
0.1
0.5
1
2
5
10
20
40
100
I day
238
638
698 (1.15)
661 (1.06)
451 (0.53)
393 (0.39)
354 (0.29)
409 (0.43)
635 (0.99)
627 (0.97)
234 (-0.01)
2 days
147
279
689 (4.11)
903 (5.73)
800 (4.95)
845 (5.29)
1011 (6.54)
1019 (6.61)
1378 (9.32)
~ 1(20.1)]
851 (5.33)
3 days
425
707
448 (-0.92)
903 (0.69)
899 (0.68)
882 (0.62)
692 (-0.05)
955 (0.88)
3579 (10.2)
~ 636 1(26.9) 1 (-0.25)
4 days
248
362
482 (2.05)
1043 (6.97)
880 (5.54)
953 (6.18)
975 (6.38)
777 (4.64)
3716 (30.4)
~ ](59.9)]
346 (0.86)
5 days
265
312
192 (-1.55)
836 (12.1)
896 (13.4)
1648 (29.4)
492 (4.83)
1028 (16.2)
~ 1(107) 1
4320 (86.3)
263 (-0.04)
6 days
321
480
822 (3.15)
1322 (6.29)
3190 (18.0)
3700 (21.3)
2551 (14.0)
1975 (10.4)
~ 1(66.1) 1
5973 (35.5)
561 (1.51)
7 days
254
1050
372 (0.15)
1161 (1.14)
838 (0.73)
3134 (3.62)
1930 (2.10)
2096 (2.31)
3034 (3.49)
~ 1(13.3) 1
496 (0.30)
"As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day, SI ~ 2.0 not included.
active cells, within a complex chain of events involving lymphocytes, accessory cells and their numerous soluble factors. In mammals and chickens it is now well established that some mitogens stimulate LT response among T cells while some stimulate B cells [20-22]. Some lectins stimulate several cell types because the specific receptor is widespread, e.g. HP binds predominantly to human T cells but it also binds to N K cells and some myeloma cell lines [23-26], the last observation suggesting that normal B cells might have receptors for HP at certain stages of maturation. Others, e.g. PWM, stimulate both T and B cells because they comprise Table 8. Time,lose transformation responses of duck blood lymphocytes to horseshoe crab lectin (HSC) Radioactivity~ of Unstimulated controls Time of culture
Cultures stimulated with HSC (,ug/ml)
+3H
0.1
0.5
1
2
5
10
20
40
100
1 day
168
927
988 (1.08)
935 (1.01)
726 (0.73)
1092 (1.22)
984 (1.07)
718 (0.72)
737 (0.75)
232 (0.08)
567 (0.53)
2 days
193
468
559 (1.33)
391 (0.72)
522 (1.20)
456 (0.96)
737 (I.98)
786 (2.16)
941 (2.72)
1403 (4.40)
] ~2o.1)]
3 days
226
671
365 (0.31)
710 (1.09)
501 (0.62)
569 (0.77)
628 (O.9O)
673 (I.00)
48O (0.57)
7O0 (1.06)
] ~26.6)j
4 days
208
486
491 (1.02)
433 (0.81)
591 (1.38)
675 (1.68)
1118 (3.27)
632 (1.52)
400 (0.69)
576 (1.32)
1(48.5) 1
5 days
255
629
562 (0.82)
1113 (2.29)
615 (0.96)
322 (0.18)
618 (O.97)
672 (1.1 I)
278 (O.O6)
809 (1.48)
1¢3.5) 1
6 days
227
327
552 (3.25)
700 (4.73)
484 (2.57)
601 (3.74)
563 (3.36)
848 (6.21)
764 (5.37)
928 (7.01)
102.8) 1
7 days
274
432
426 (0.96)
700 (2.70)
607 (2.11)
821 (3.46)
517 (1.54)
858 (3.70)
610 (2.13)
911 (4.03)
I ~4.90)[
3H
aAs mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day, SI (-2.0 not included.
D. A. HIGGINS
20
Table 9. Time~zlose transformation responses of duck blood lymphocytes to pokeweed mitogen (PWM) Radioactivitya of Unstimulated controls
Cultures stimulated with PWM (/ag/ml)
Time of culture
-3H
+3H
0.1
0.5
1
2
5
10
20
40
100
1 day
328
1067
860 (0.72)
592 (0.36)
379 (0.07)
631 (0.41)
588 (0.35)
772 (0.60)
538 (0.28)
755 (0.58)
844 (0.75)
2 days
307
721
1148 (2.03)
1545 (2.99)
1693 (3.35)
2416 (5.09)
3122 (6.80)
2938 (6.35)
3518 (7.76)
6143 (14.1)
1(15.0) 1
3 days
236
615
975 (I.95)
3422 (8,41)
4184 (10.4)
5096 (12.8)
5581 (14,1)
7318 (18.7)
7442 (19.0)
~ 1(29.2)[
6974 (17.8)
4 days
427
593
643 (1.30)
2666 (13.5)
5977 (33.4)
8696 (49,8)
12306 (71.6)
9637 (55.5)
13722 (80.1)
~ 1(90.5) 1
3906 (20,9)
5 days
443
661
915 (2.16)
3904 (15.9)
10125 (44.4)
13471 (59.8)
~ 1(306) 1
9870 (43.2)
16960 (75.8)
7745 (33,5)
2579 (980)
6 days
246
498
629 (I.52)
1889 (6.52)
47018 (186)
14767 (57.6)
~ 1(378)]
45210 (178)
20399 (80.0)
8636 (33.3)
t208 (3.82)
7 days
336
458
736 (3.28)
2598 (18.5)
5862 (45.3)
18354 (148)
~ 1(637) 1
19270 (155)
31709 (257)
4738 (36.1)
578 (I.98)
"As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day, SI ,~ 2.0 not included.
several mitogenic components each with different specificity [27]. Some lectins react with subsets of cells within larger populations recognised by other lectins. PHA binds to only a proportion of human T cells which bind Con A [28]. Human T helper cells bind Riotous communis agglutinin via sugars in the CD4 complex and suppressor cells bind WGA via the CD8 complex [29]. While the reaction of lectins with lymphocyte populations in man and mouse is rather well characterized [30] the limited knowledge of iectin binding to cells from many animals and birds indicates that individual lectins might not bind to the same cell Table 10. Time-dose transformation responses of duck blood lymphocytes to E. coli lipopolysaccharide (LPS) Radioactivity~ of Unstimulated controls Time of culture
Cultures stimulated with LPS (ug/ml)
-3H
+ 3H
0.1
0,5
1
2
5
10
20
40
100
I day
180
360
388 (1.15)
435 (1.42)
297 (0.65)
444 (1.47)
465 (I.58)
462 (1.57)
332 (0.84)
401 (1.23)
453 (I.52)
2 days
225
349
411 (1.50)
585 (2.90)
621 (3.19)
759 (4.31)
427 (1.63)
634 (3.30)
643 (3.37)
752 (4.25)
1(4.43)
3 days
155
284
711 (4.31)
603 (3~47)
826 (5.20)
943 (6.11)
~ ](8.22) 1
1054 (6.97)
751 (4.62)
723 (4.40)
750 (4.61)
4 days
269
389
960 (5.76)
1232 (8.02)
~ 1(9.92) 1
1205 (7.80)
1314 (8.71)
924 (5.46)
1275 (8.38)
1110 (7.10)
888 (5.16)
5 days
137
265
656 (4.05)
972 (6.52)
1628 (11.6)
1336 (9.37)
~ 1(10.8) 1
1209 (8.37)
1463 (10.4)
1101 (7.53)
1148 (7.90)
6 days
453
917
1049 (t.28)
1914 (3.I5)
1622 (2.52)
1440 (2.13)
2187 (3.74)
~ 1(3.9511
2208 (3.78)
1574 (2.41)
1502 (2.26t
7 days
678
1270
1195 (0.87)
1505 (I.40)
2101 (2.40)
1980 (2.20)
2041 (2.30)
~ 1(2.92) 1
2073 (2.36)
1524 (1.43)
1432 (1.27)
"As mean cpm of triplicate cultures: SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day, SI ( 2.0 not included.
Duck lymphocytes
21
Table 11. Mitogen-induced transformation responses' of blood lymphocytes from two ducks after culture for 3 and 5 days Duck 1 Mitogen None (3H - ) (3H + ) PHA Con A LC HP PNA BSS WGA HSC PWM LPS
Concentration (#g/ml)
3 days
--40 100 40 100 40 100 20 100 5 10 I0 20 20 40 10 100 I 5 1 10
223 957 30722 (41.6) 52220 (70.8) 4724 (6.13) 463 (0.33) 24982 (33.7) 43245 (58.4) 749 (0.72) 1279 (1.44) 1084 (1.17) 1125 (I .23) 2350 (2.90) 3707 (4.75) 19548 (26.3) 12651 (16.9) 2559 (3.18) 4340 (5.61 ) 2932 (3.69) 47347 (64.2) 3446 (4.39) 5775(7.56)
Duck 2 5 days
528 949 2245 (4.08) 5628 (12.1) 1007 (1.14) 1764 (2.93) 1060 (1.26) 1785 (2.98) 1104 (1.37) 1019 (1.17) 821 (0.69) 1437 (2.16) 4746 (10.0) 1523 (2.36) 37815 (88.6) 105533 (249) 1736 (2.87) 5223 ( 11.1 ) 3600 (7.30) 14052 (32.1) 1266 (I.75) 2081 (3.69)
3 days
5 days
271 1041 24014 (30.8) 20103 (25.7) 9044 (11.4) 3970 (4.80) 3374 (4.03) 9796 (12.4) 1742 (I.91) 8842 (11.1) 6180 (7.67) 6639 (8.27) 10183 (12.9) 11027 (13.9) 4566 (5.58) 956 (0.89) 731 (0.59) 3478 (4.16) 8872 (11.2) 4904 (6.02) 10045 (12.7) 8030(10.1)
428 795 4865 (12.1) 15726 (41.7) 2976 (6.94) 1405 (2.66) 1451 (2.79) 4617 (11.4) 1349 (2.51) 2256 (4.98) 1357 (2.53) 1615 (3.23) 2244 (4.95) 1721 (3.52) 9147 (23.7) 6694 (17.1) 882 (1.24) 4256 (I 0,4) 2652 (6.06) 4752 (11.8) 3215 (7.59) 6200(15.7)
~rH]-thymidine uptake as mean cpm of triplicate cultures; SI of stimulated cultures in parentheses.
populations in different species [31]. Since duck lymphocyte subpopulations cannot be identified on the basis of receptors for erythrocytes or immunoglobulins, an attempt was made to demonstrate populations differing in responsiveness to mitogens chosen to reflect a wide range of sugar-binding specificities. It was assumed that: (i) functionally different lymphocyte populations must exist in the duck; (ii) different populations will possess different surface glycopeptides and glycolipids; and (iii) these populations will exhibit some individuality in the kinetics of their transformation responses. It was relevant therefore that three patterns of response were observed: that with maximum sensitivity at 3-5 days culture (to PHA, PNA, WGA and LPS); that in which high concentrations of mitogen gave maximum transformation throughout the experiment (to Con A, LC, HP and HSC); and that (to BSS and PWM) which attained and then sustained sensitivity to lower concentrations of mitogen. The pattern of response to PWM and BSS might result from the heterogeneity of cells responding to these lectins; PWM because of the complexity of the lectin and the resulting diversity of receptors, BSS because of the widespread distribution of the specific sugars. The response to HP was surprising in view of our previous observation that duck lymphocytes do not have receptors for fluorescein-labelled HP [1]. It is possible that HP binds primarily to glycoproteins in the PDS supplement and that the resulting complex is mitogenic; some of the lectins used, notably Con A, induced precipitation at concentrations above 100 ~g/ml. On mammalian lymphocytes, receptors for some lectins, e.g. HP and PNA, are often occluded by sialic acid which can be removed by neuraminidase. Previously we have shown that the percentage of duck lymphocytes staining with fluorescein-PNA was increased after treatment with Vibrio cholerae neuraminidase [1]. As an adjunct to the present study, LT was also attempted using HP and PNA on duck blood lymphocytes first treated with neuraminidase at 10 units/ml. The results were disappointing; the cells gave high background cpm and only a transient and slight increase in response to lectins. We have also attempted LT employing purified protein derivatives (PPD) from Mycobacterium
22
D.A. HIGGINS
t u b e r c u l o s i s , M . boris, M . a v i u m a n d M . j o h n e i . T h e e x p e r i m e n t s e x a m i n e d a n u m b e r o f v a r i a b l e s i n c l u d i n g cell c o n c e n t r a t i o n , flat- vs U - b o t t o m e d c u l t u r e trays a n d v a r i o u s m e d i u m s u p p l e m e n t s in a d d i t i o n to the t i m e - d o s e c o n d i t i o n s e m p l o y e d w i t h the m i t o g e n s d e s c r i b e d here. T h e results w e r e u n i v e r s a l l y n e g a t i v e i n d i c a t i n g t h a t P P D is n o t a m i t o g e n for lymphocytes from normal ducks. T h e r e s p o n s e s o f d u c k b l o o d l y m p h o c y t e s to the r a n g e o f m i t o g e n s u s e d in this w o r k m i g h t be a t t r i b u t a b l e to the a n a t i d i n e e q u i v a l e n t o f T cells, B cells a n d s u b p o p u l a t i o n s . O u r c u r r e n t studies are d e s i g n e d to c h a r a c t e r i z e the cells w h i c h r e s p o n d to t h e m i t o g e n s e m p l o y e d here; the results will be r e p o r t e d . Acknowledgements--This work was supported by grants from the University of Hong Kong Committee for Research and Conference Grants, the Pauline Chan Medical Research Fund, the Wing Lung Bank Medical Research Fund, the Wu Chung Medical Research Fund, the Medical Faculty Research Grant Fund, the Lee Wing Tat Medical Research Fund, the Sun Yat Sen Foundation Fund for Medical Research and Cherry Valley Farms Ltd. Dr L. Lalibert6 kindly translated the abstract. The technical assistance of Miss C. S. H. Teoh and Miss N. Chin and the secretarial assistance of Miss Y. Chow are also gratefully acknowledged.
REFERENCES 1. Higgins D. A. and Chung S. H. (1986) Duck lymphocytes. I. Purification and preliminary observations on surface markers. J. Immun. Meth. 86, 231 238. 2. Higgins D. A. and Teoh C. S. H. (1988) Duck lymphocytes. II. Culture conditions for optimum transformation response to phytohaemagglutinin. J. Immun. Meth. 106, 135 145. 3. Lis H. and Sharon N. (1973) The biochemistry of plant lectins (phytohemagglutinins). Ann. Rev. Biochem. 42, 541 574. 4. Sharon N. (1983) Lectin receptors as lymphocyte surface markers. Adv. Immun. 34, 213 298. 5. Allen A. K., Desai N. N. and Neuberger A. (1976) The purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of its properties with lectins of similar specificity. Biochem. J. 155, 127 135. 6. Kornfeld K., Reitman M. L. and Kornfeld R. (1981) The carbohydrate-binding specificity of pea and lentil lectins. Fucose is an important determinant. J. biol. Chem. 256, 6633 6640, 7. Hammarstr6m S. (1974) Structure, specificity, binding properties and some biological activities of a blood group A reactive hemagglutinin from the snail Helix pomatia. Ann. N.Y. Acad. Sci. 234, 183-195. 8. Hammarstr6m S. and Kabat E. A. (1971) Studies on specificity and binding properties of the blood group A reactive hemagglutinin from Helix pomatia. Biochemistry 10, 1684-1692. 9. Lotan R., Skutelsky E., Danon D. and Sharon N. (1975) The purification, composition and specificity of the anti-T lectin from peanut (Arachis hypogaea). J. biol. Chem. 250, 8518-8523. 10. Hayes C. E. and Goldstein I. J. (1974) An ~-D-galaetosyl-binding lectin from Bandeiraea simplic!folia seeds. J. biol. Chem. 249, 1904-1914. 11. Murphy L. A. and Goldstein I. J. (1977) Five ~-D-galacto-pyranosyl-binding isolectins from Bandeiraea simplicifolia seeds. J. biol. Chem. 252, 4739-4742. 12. Wood C., Kabat E. A., Murphy L. A. and Goldstein I. J. (1979) Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia. Archs Bioehem. Biophys. 198, 1-11. 13. Allen A. K., Neuberger A. and Sharon N. (1973) The purification, composition and specificity of wheat-germ agglutinin. Biochem. J. 131, 155-162. 14. Nagata Y. and Burger M, M. (1974) Wheat germ agglutinin. Molecular characteristics and specificity for sugar binding. J. biol. Chem. 249, 3116--3122. 15. Peters B. P., Ebisu S., Goldstein I. J. and Flashner M. (1979) Interaction of wheat germ agglutinin with sialic acid. Biochemistry 18, 5505 5511. 16. Cohen E., Rozenberg M. and Massaro E. J. (1974) Agglutinins of Limulus polyphemus (Horseshoe crab) and Birgus latra (Coconut crab). Ann. N.Y. Acad. Sci. 234, 28 32. 17. Waxdal M. J. (1978) Pokeweed mitogens. Meth. Enzym. 50, 354-361. 18. Dillner-Centerlind M.-L., Axelsson B., Hammarstr6m S., Hellstr6m H. and Perlmann P. (1980) Interaction of lectins with human T lymphocytes. Mitogenic properties, inhibitory effects, binding to the cell membrane and to isolated surface glycopeptides. Fur. J. lmmun. 10, 434-442. 19. Skoog V. T., Nilsson S. F. and Weber T. H. (1980) Characterization of human lymphocyte surface receptors for mitogenic and non-mitogenic substances. Scand. J. lmmun. 11, 369 376.
Duck lymphocytes
23
20. Greaves M. F., Roitt |. M. and Rose M. E. (1968) Effect of bursectomy and thymectomy on the responses of chicken peripheral blood lymphocytes to phytohaemagglutinin. Nature (Lond.) 220, 293-295. 21. Hovi T., Suni J., Hortling L. and Vaheri A. (1978) Stimulation of chicken lymphocytes by T- and B-cell mitogens. Cell. Immun. 39, 70-78. 22. Janossy G. and Greaves M. F. (1971) Lymphocyte activation. I. Response of T and B lymphocytes to phytomitogens. Clin. exp. Immun. 9, 483-498. 23. Hailer O., Gidlund M., Hellstr6m V., Hammerstr6m S. and Wigzell H. (1978) A new surface marker on mouse natural killer cells: receptors for Helix pomatia A hemagglutinin. Eur. J. Immun. 8, 765-771. 24. Hellstr6m U., Mellstedt H., Perlmann P., Holm G. and Pettersson D. (1976) Receptors for Helix pomatia A haemagglutinin on leukaemic lymphocytes from patients with chronic lymphocytic leukaemia (CLL). Clin. exp. Immun. 26, 196-203. 25. Hellstr6m U., Perlmann P., Robertsson E. S. and Hammarstr6m S. (1978) Receptors for Helix pomatia A haemagglutinin (HP) on a subpopulation of human B cells. Scand. J. lmmun. 7, 191-197. 26. Axelsson B., Kimura A., Hammarstr6m S., Wigzell H., Nilsson K. and Mellstedt H. (1978) Helix pomatia A hemagglutinin: selectivity of binding to lymphocyte surface glycoproteins on T cells and certain B cells. Eur. J. lmmun. 8, 757 764. 27. Waxdal M. J. and Basham T. Y. (1974) B- and T-cell stimulatory activities of multiple mitogens from pokeweed. Nature 251, 163-164. 28. Rawson A. J. and Daniele R. P. (1976) The separation of lymphocytes responsive only to concanavalin A from those also responsive to phytohemagglutinin. J. Retieuloendothelial Soe. 20, 421-427. 29. Boldt D. H. and Dorsey S. A. (1983) Interactions of lectins and monoclonal antibodies with human mononuclear cells. I. Specific inhibition of OKT4 and OKT8 binding by Ricinus communis agglutinin and wheat germ agglutinin. J. Immun. I30, 1646-1653. 30. Sharon N. (1980) Cell surface receptors for lectins: markers of murine and human lymphocyte subpopulations. In: Immunology 80 (Progress in Immunology IV) (Edited by Fougereau M. and Dausset J.), pp. 254 278. Academic Press, New York. 31. Higgins D. A. (1981) Markers for T and B lymphocytes and their application to animals. Vet. Bull. 51, 925-963.
0147-9571/90 $3.00 + 0.00 Copyright © 1990 Pergamon Press plc
DUCK LYMPHOCYTES--III. TRANSFORMATION R E S P O N S E S TO SOME C O M M O N M I T O G E N S D. A. HIGGINS Department of Pathology, University of Hong Kong, Queen Mary Hospital, Hong Kong
(Received 8 November 1989) Abstract--The lymphocyte transformation (LT) test was performed using duck blood lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A), lentil lectin (LC), Roman snail lectin (HP), peanut agglutinin (PNA), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), horseshoe crab lectin (HSC), pokeweed mitogen (PWM) and E. coli lipopolysaccharide (LPS). Cells were cultured in microtitre trays, at 41.6°C, 8 x 105 cells in 200/tl medium ( = 4 × 106 cells/ml) supplemented with 10% pooled duck serum. Mitogens were added at final concentrations of 0.1-100 ,ug/ml and triplicate cultures at each concentration were harvested daily for scintillation counting 6 hr after addition of I pCi [~H]thymidine. Three patterns of response were observed. The responses to Con A, LC, HP and HSC were greatest at high mitogen concentrations (40-100 #g/ml) throughout the 7 days of culture. With PHA, PNA, WGA and LPS maximum stimulation was obtained at 3-5 days, at which time the cells were responding to lower concentrations of mitogen than were required at other times during the experiment. The response to BSS and PWM showed increasing sensitivity to lower concentrations of mitogen during the first 3 days of culture and then stimulated most strongly at 2-10 pg/ml in cultures harvested after 4-7 days. Cells from two ducks were cultured for 3 and 5 days with selected concentrations of these mitogens; the results confirmed the variation in response to different mitogens. It is possible that these patterns of response are the outcome of stimulating different populations of duck lymphocytes.
Key words: ducks, lymphocytes, transformation, mitogens, lectins. R6sum6--Le test de transformation de lymphocytes a 6t6 ex6cut6 fi l'aide de lymphocytes sanguins de canard et activ6 par les agents suivant: phytohemagglutinine (PHA), concanavaline A (Con A), lectine de lentille (HP), agglutinine d'arachide (PNA), lectine de grain de Bandeiraea simplicifolia (BSS), agglutinine de germe de bl6 (WGA), lectine de crabe des Moluques (HSC), pokeweed mitog6n (PWM) et lipo-polysaccharide d'E. coli (LPS). Les cellules onty 6t6 plac6es dans des bassins microtitre de culture, fi une temp6rature de 41,6°C, 8 × 105 cellules dans 200 pl demi-lieu ( = 4 x l06 cellules/ml) additionn/~es de sbrum de canard fi 10%. Les mitogens out 6t6 ajout6s fi des concentrations finales de 0,1-100 #g/ml et des cultures en triplicate de chacune des concentrations out 6t6 r6colt6es quotidiennement pour scintillation 6 heures apr6s l'ajout de 1/~Ci de [3H]thymidine. Trois r6actions furent observ6es. Les r6actions aux Con A, LC, HP et HSC &aient maximales avec le mitogene ~i haute concentration (40-100/~g/ml) pendant toute la dur6e (7 jours) de culture. Avec les agents PHA, PNA, WGA et LPS la stimulation maximum est survenue aux jours 3-5, :i des concentrations moindre de mitogene que celles qui avaient ~t~ appliqu~es a d'autre temps. La r6action au BSS et PWM a d6montr6 une sensibilit6 croissante fi des concentrations moindre de mitogen lors des trois premiers jours de l'exp6rience et atteignirent une stimulation maximum a 2-10 pg/ml dans des cultures r6colt~es apr6s 4-7 jours. Des cellules de deux canards furent raises en culture pour 3 et 5 jours avec une s61ection de concentrations de ces mitogenes; les r6sultats ont confirm6 les 6carts de r~action. I1 est possible que ces r6actions et 6chantillons de r6sultats soient consecutifs ~i la stimulation de diverses populations de lymphocytes de canards.
Mots-cl(fs: canards, lymphocytes, transformation, mitogene, lectines.
INTRODUCTION Although lymphocytes from the duck (Anas platyrhynchos) do not possess surface markers similar to those occurring on mammalian T a n d B cells, t h e y d o r e s p o n d t o m i t o g e n s i n 13
14
D . A . HIGGINS
lymphocyte transformation (LT) tests [1]. Their requirements for optimum response to phytohaemagglutinin (PHA) have been reported [2]. These optimum culture conditions have now been used to study the in vitro response of duck blood lymphocytes to several mitogens, with a view to identifying subpopulations of cells responding to different stimulants. MATERIALS AND METHODS
Ducks Ducks were a hybrid strain of White Pekin (Super M, Cherry Valley Farms Ltd, Lincoln, U.K.), 4-7 months old at the time of experiments. Mitogens (i) Phytohaemagglutinin (PHA) from Phaseolus vulgaris, sugar specificity N-acetyl-Dglucosamine(1-2)D-mannose, N-acetyl-D-galactosamine [3,4], was in two batches. That used as a test mitogen was from Sigma Chemical Company (L-8754, lot number 25F-9640); that used as a 3-day positive control was from Pharmacia AB, Uppsala, Sweden (code 17-0449-01; lot number 9004). (ii) Concanavalin A (Con A) from Canavalia ensiformis (17-0450-01, Pharmacia; lot number NH 04564), sugar specificity ~-D-mannose, a-D-glucose, N-acetyl-D-glucosamine [5]. (iii) Lentil lectin (LC) from Lens culinaris (L-5880, Sigma; lot number 114F-9670), sugar specificity a-D-glucose, ~-D-mannose, ~-D-fUcose [6]. (iv) Roman snail lectin (HP) from Helix pomatia (L-3382, Sigma; lot number 53F-9013), sugar specificity N-acetyl-~-D-galactosamine [7, 8]. (v) Peanut agglutinin (PNA) from Arachis hypogaea (L-0881, Sigma; lot number 83F-9620), sugar specificity D-galactosyl-/~(1-3)N-acetyl-D-galactosamine [9]. (vi) Bandeiraea simplicifolia seed (BSS) extract (L-2380, Sigma; lot number 84F-9690), sugar specificity ~-D-galactose, N-acetyl-~-D-galactosamine [10-12]. (vii) Wheat germ agglutinin (WGA) from Triticum vulgaris (L-9640, Sigma; lot number 76F-4031-1), sugar specificity (N-acetyl-D-glucosamine)2, N-acetyl-D-neuraminic acid, N-acetyl-D-galactosamine [13 15]. (viii) Horseshoe crab lectin (HSC) from Limulus polyphemus (L-9509, Sigma; lot number 17F-9540), sugar specificity N-acetyl-D-neuraminic acid [16]. (ix) Pokeweed mitogen (PWM) from Phytolacca americana (L-9379, Sigma; lot numbers 65F-9570 and 83F-9670), a mitogen consisting of at least five components with broad sugar-binding capabilities [17]. (x) Lipopolysaccharide (LPS) from Escherichia coli serotype 0111 :B4 (L-2630, Sigma; lot number 114F-4043). Blood collection, lymphocyte separation and LT test The techniques described previously [2] were followed exactly. Briefly, blood was collected by cardiac puncture into an equal volume of 0.1 M Na2HPO4-NaH2PO4 buffered saline, pH 7.2 (PBS) containing sodium heparin (10 IU/ml, Heparin Injection BP, Commonwealth Serum Laboratories). Lymphocytes were purified by centrifuging 3 ml of the blood-PBS mixture over 3 ml Ficoll 400/diatrizoate sodium solution, specific gravity 1.077 (Ficoll-Paque, Pharmacia) at 200 g for 25 min at 20°C. The lymphocyte layer was
Duck lymphocytes
15
collected and washed once in PBS and twice in Roswell Park Memorial Institute medium 1640 (RPMI), then adjusted to a concentration of ~ 10 x 106 cells/ml in RPMI. The LT test was set up in 96-well, flat-bottomed microculture trays (code no 167008, Nunc) using 80/tl of cell suspension, 100#l RPMI containing 20% heat-inactivated (56°C, 45 min) pooled adult duck serum (PDS), and 20/~l of RPMI containing mitogen at 10 × the desired final concentration; thus, the conditions achieved were 8 × 105 cells in 200#1 cultures supplemented with 10% PDS and mitogen. Trays were incubated at 41.6°C in a humid atmosphere containing 5% COs. Cultures were radiolabelled 6 hr prior to harvesting by adding 20/~1 RPMI containing 1/tCi of [3H]thymidine (TRK-120, Amersham). Cells were harvested by washing with distilled water onto glass-fibre mats (GF/C, Whatman) using a semi-automated harvester. Mats were dried at 37°C overnight, then the individual discs were separated and placed in glass scintillation vials containing 2 ml scintillant [10g 2,5-diphenyloxazole (Sigma) and 0.5g 1,4-bis-2-(4-methyl-5-phenyloxazolyl)benzene (Sigma) per litre of scintillation grade toluene (Fisher)]. Radioactivity was measured as counts per min (cpm) in Beckman LS-7000 or LS-5801 /~-scintillation counters and expressed as means of triplicate cultures; standard deviations are not reported in the Results section but were within _+20% of the means. Unstimulated, unlabelled (i.e. no mitogen, no [3H]thymidine) and unstimulated, labelled controls were included in each experiment. The stimulation index (SI) was calculated using the mean cpm values by the formula (mitogen-stimulated, [3HI-labelled) - (unstimulated, unlabelled) (unstimulated, [3HI-labelled) - (unstimulated, unlabelled)
Experiment plan Each bleeding yielded sufficient cells to allow two lectins to be examined in one experiment. Lectins were used at final concentrations of 0.1, 0.5, 1, 2, 5, 10, 20, 40 and 100 pg/ml. Cells were harvested daily up to 7 days of culture. All mitogens were examined at least twice and each time were tested in parallel to a different mitogen. If the patterns of lymphocyte responsiveness obtained with a mitogen on two separate occasions were different, the experiment was performed a third (or fourth) time to identify the most repeatable pattern. For each experiment a "positive control" was set up consisting of a titration of PHA (Pharmacia) harvested after 3 days of culture; if this was subsequently found to have not given satisfactory stimulation, the experiment was repeated on the basis that the cells might, on that occasion, have been unresponsive. Finally, blood lymphocytes from two ducks were examined, each on a separate occasion, for their response to selected concentrations of the panel of mitogens after 3 and 5 days of culture. RESULTS Duck blood lymphocytes cultured in the presence of 10% PDS responded in LT test to PHA, Con A, LC, HP, PNA, BSS, WGA, HSC, PWM and LPS (Tables 1-10). PHA, PNA, WGA and LPS showed a pattern of stimulation with maximum sensitivity (i.e. greatest response being elicited to lower concentrations of mitogen) developing after 3-5 days culture. The response to Con A, LC, HP and HSC was greatest at high mitogen concentrations (40-100 #g/ml) throughout the 7 days of culture. BSS and PWM provoked
D. A. HIGGINS
16
Table 1. Time,lose transformation responses of duck blood lymphocytes to phytohaemagglutinin (PHA) Radioactivity" of Unstimulated controls
Cultures stimulated with PHA (#g/ml)
Time of culture
-~H
+3H
0.1
0.5
1
2
5
10
20
40
100
I day
332
451
429 (0.81)
549 (1.82)
340 (0.07)
403 (0.60)
411 (0.66)
642 (2.60)
806 (3.98)
1083 (6.31)
1(21.7) 1
2 days
246
437
355 (0.57)
463 (1,14)
270 (0,12)
342 (0.50)
868 (3 26)
1282 (5.42)
2105 (9,73)
2335 (10.9)
[(12,9) I
3 days
360
923
696 (0.60)
447 (0.15)
844 (0.86)
1115 (1.34)
2124 (3.13)
3698 (5.93)
~ [(7.86) I
4149 (6.73)
1787 (2.53)
4 days
96
284
206 (0.58)
180 (0.45)
547 (2.40)
1300 (6.40)
3554 (18.4)
6139 (32.1)
~ [(33.9) I
6378 (33.4)
848 (4.00)
5 days
169
343
296 (0.73)
311 (0.82)
446 (1.59)
534 (2.10)
1586 (8.14)
6467 (36.2)
~ [(46.9)[
5374 (29.9)
1065 (5.15)
6 days
268
852
425 (0.27)
400 (0.23)
479 (0.36)
368 (0.17)
1187 (1.57)
1564 (2.22)
~ 1(5.26) 1
3011 (4.70)
424 (0.27)
7 days
395
821
927 (1.25)
594 (0.47)
442 (0.11)
333 (-0.14)
566 (0.40)
~ [(3.03) I
952 (1.38)
1320 (2.17)
465 (0.16)
"As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
responses which showed increasing sensitivity (from 100/~g to 2-10 #g) during the first few days of culture, thereafter retaining maximum SI to these lower concentrations throughout the remainder of the experiment. In terms of the cpm and SI achieved, PHA, LC, PNA, BSS, WGA, HSC and PWM were considered strong mitogens for duck blood lymphocytes, while Con A, HP and LPS were considered weak mitogens. The response of blood lymphocytes from two ducks to selected concentrations of this range of mitogens (Table 11) confirmed that some mitogens were stronger stimulants than others and that the time course of the response to different mitogens varied. In addition there appeared
Table 2. Time~zlose transformation responses of duck blood lymphocytes to Concanavalin A (Con A) Radioactivity" of Unstimulated controls
Cultures stimulated with Con A (l~g/ml)
Time of culture
-3H
+ 3H
0.l
0.5
1
2
5
10
20
40
100
1 day
110
463
302 (0.54)
402 (0.83)
520 (1.16)
367 (0.73)
550 (1.25)
493 (1.08)
443 (0.94)
426 (0.89)
1(2.29)[
2 days
334
517
281 (-0.29)
834 (2.73)
438 (0.57)
506 (0.94)
407 (0.40)
467 (0.73)
602 (1.46)
1(27.5) 1
3 days
165
281
356 (1.65)
263 (0.84)
467 (2.60)
293 (1.10)
268 (0.89)
333 (1.45)
687 (4.50)
656 (4.23)
/ (9.77) [
4 days
196
325
271 (0.56)
206 (0.08)
226 (0.23)
329 (1.03)
299 (0.80)
378 (1.41)
561 (2.83)
465 (2.08)
I (7.10) [
5 days
229
327
541 (3.18)
256 (0.27)
433 (2.08)
297 (0,69)
500 (2.76)
297 (0.69)
716 (4.97)
533 (3.10)
[(5.89) 1
6 days
97
230
320 (I.68)
221 (0.93)
318 (1.66)
280 (I.37)
345 (I.86)
263 (1.25)
436 (2.55)
488 (2.94)
1(8.27) 1
7 days
408
773
917 (1.39)
572 (0.45)
759 (0.96)
1032 (1.71)
968 (1.53)
625 (0.59)
1038 (1.73)
1171 (2.09)
1(3,97) 1
236 (0.53)
"As mean cpm of tripticate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
17
Duck lymphocytes Table 3. Time-dose transformation responses of duck blood lymphocytes to lentil lectin (LC) Radioactivity ~ of Unstimulated controls
Cultures stimulated with LC (vg/ml)
Time of culture
-3H
+3H
0.1
0.5
1
2
5
10
20
40
100
1 day
206
412
362 (0.76)
314 (0.52)
537 (1.61)
451 (1.19)
508 (1.47)
586 (1.84)
969 (3.70)
1534 (6.45)
1(21.7) 1
2 days
253
1760
1743 (0.99)
1836 (1.05)
1744 (0.99)
2073 (1.21)
1887 (1.08)
1763 (1.00)
6910 (4.42)
12596 (8.19)
1(10.9) 1
3 days
150
537
572 (1.09)
559 (1.06)
569 (1.08)
839 (1.78)
783 (1.63)
770 (I.60)
1824 (4.32)
9360 (23.8)
1(211) 1
4 days
107
507
583 (1.19)
553 (I.II)
224 (0.29)
338 (0.58)
579 (I.18)
362 (0.64)
7617 (18.8)
19202 (47.7)
1(308) 1
5 days
243
581
477 (0.69)
551 (0.91)
604 (1.07)
721 (I.41)
537 (0.87)
640 (1.17)
5074 (14.3)
9110 (26.2)
1(96.3) 1
6 days
249
636
546 (0.77)
577 (0.85)
588 (0.87)
614 (0.94)
573 (0.84)
1148 (2.32)
4763 (11.7)
5566 (13,7)
1(31.8) 1
7 days
534
855
961 (1.33)
1300 (2.39)
1041 (1.58)
975 (1.37)
810 (0.86)
1048 (I.60)
4119 (11.2)
5399 (15,2)
1(24.2) 1
~As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
to be some individual variation between the two ducks, e.g. there was a similar response to PHA by cells from both ducks but cells from duck 1 responded more vigorously to LC, WGA and PWM than cells from duck 2 which responded more strongly to BSS. DISCUSSION Lectins are proteins and glycoproteins which occur throughout the plant and animal kingdoms. They bind specifically to sugars present as mono- or oligosaccharides within Table 4. Time,lose transformation responses of duck blood lymphocytes to Helix pomatia lectin (HP) Radioactivity a of Unstimulated controls
Cultures stimulated with HP (#g/ml)
Time of culture
-3H
+3H
0.1
0.5
I
2
5
10
20
40
100
1 day
124
260
293 (I.24)
183 (0.43)
474 (2.57)
487 (2.67)
515 (2.87)
583 (3.37)
~ 1(3.98) 1
578 (3.34)
572 (3.29)
2 days
287
1874
1795 (0.95)
1319 (0.65)
1753 (0.92)
2377 (1.32)
3604 (2.09)
2314 0.28)
~ 1(2.20)[
3770 (0.18)
1964 0.06)
3 days
252
797
973 0.32)
545 (0.54)
581 (0.60)
608 (0.65)
1541 (2.36)
902 (1.19)
1881 (2.99)
962 (1.30)
4 days
218
504
631 (1.44)
766 (1.92)
379 (0.56)
610 (1.37)
1800 (5.53)
685 (1.63)
1362 (4.00)
~ 1(6.19)[
1(3.19) 1 1667 (5.07)
5 days
242
919
612 (0.55)
760 (0.76)
446 (0.30)
726 (0.71)
1667 (2.10)
847 (0.89)
1146 (1.33)
1621 (2.04)
1(3.39) 1
6 days
168
447
451 (1.01)
411 (0.88)
510 (1.22)
657 (1.75)
963 (2.85)
1256 (3.90)
1161 (3.56)
~ [(6.89) 1
2066 (6.80)
7 days
444
679
636 (0.82)
580 (0.58)
749 (1.30)
814 (1.57)
1186 (3.16)
1226 (3.33)
1531 (4.62)
~ [(10.8) 1
1691 (5.31)
~As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day. CIMID 13/I--B
18
D. A. H1GG1NS Table 5. T i m e . l o s e transformation responses of duck blood lymphocytes to peanut agglutinin (PNA) Radioactivity" of Unstimulated controls
Time of culture
~H
Cultures stimulated with PNA (,ug/ml)
+3H
0.1
0.5
1
2
5
10
20
40
100
1 day
472
708
1046 (2.43)
1169 (2.95)
1116 (2.73)
1216 (3.15)
2066 (6.75)
1716 (5.27)
1963 (6.32)
~ 1(7.00)]
1996 (6.46)
2 days
321
849
998 (I.28)
6526 (11.7)
11893 (21.9)
14633 (27.1)
11253 (20.7)
6483 (11.6)
11520 (21,2)
~ t(31.4)]
11314 (20.8)
3 days
315
1832
1236 (0,61)
1628 (0.86)
5407 (3,36)
5402 (3.35)
5883 (3.67)
6563 (4.12)
~ 1(4.38) 1
5730 (3.57)
4955 (3.06)
4 days
435
1960
2357 (I.25)
2781 (1.53)
4735 (2,81)
4883 (2.90)
~ [(8,58) 1
5938 (3.59)
7665 (4.72)
9219 (5.74)
4335 (2.56)
5 days
694
3588
4541 (1.33)
5295 (1.59)
11649 (3.78)
14705 (4.84)
~ [(13.9)[
20584 (6.87)
33370 (11.3)
36695 (12.4)
30309 (10.2)
6 days
587
2190
2459 (I.17)
5194 (2.87)
3281 (1.68)
5277 (2.92)
18533 (11.2)
11272 (6.66)
36039 ~ (22.1) 1(44.5)[
29170 (17.8)
7 days
867
2469
2142 (0.97)
4997 (2.58)
3787 (1.82)
5166 (2.68)
19929 (11.9)
20989 (12.6)
19024 (11.3)
22154 (13.3)
~ ](16.1) 1
~As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
glycoproteins and glycolipids on the surface of animal cells. Some are mitogenic and, along with mitogens such as LPS which bind to cell membranes by more complex mechanisms, have played an important role in studies of lymphocyte biology. Transformation of lymphocytes is not only dependent upon the presence of appropriate receptors but also requires their distribution in a density which allows cross-linking by the mitogen. It follows that some cells possess receptors that will bind appropriate lectins without resulting in transformation [18, 19]. While LT need not be an outcome of lectin-cell interaction, it has the advantage that such a response is usually considered a property of immunologically Table 6. Time,lose transformation responses of duck blood lymphocytes to Bandeiraea simplicifolia seed lectin (BSS) Radioactivity" of Unstimulated controls
Cultures stimulated with BSS (~g/ml)
Time of culture
-3H
+3H
0.1
0,5
1
2
5
10
20
40
100
1 day
300
725
842 (1.27)
1307 (2.37)
1657 (3.19)
1456 (2.72)
1317 (2.39)
988 (I.62)
1389 (2.56)
1231 (2.19)
1(3.33) 1
2 days
532
1220
1624 (1.59)
4255 (5.37)
4581 (5.88)
6146 (8.16)
9097 (12.4)
8012 (10.9)
11662 (16.2)
~ 1(17.7) 1
12273 (17.1)
3 days
241
653
757 (1.25)
1215 (2.36)
7002 (16.4)
9460 (22.4)
19406 (46.5)
16581 (39.7)
22967 ~ (55.2) 1(56.2) 1
21576 (51,8)
4 days
351
1626
2900 (1.99)
4695 (3.41)
7281 (5.43)
10916 (8.28)
18714 [~ffff] (14.4) ](20.7) 1
20632 (15.9)
14679 (11.2)
10682 (8.10)
5 days
272
571
930 (2.02)
1006 (2.45)
4493 (14.1)
11109 (36.2)
12749 (41.7)
~ [(48.2) I
14314 (46.9)
12744 (41.7)
5631 (17.9)
6 days
189
593
579 (0.96)
3657 (0.86)
7394 (17.8)
~ ](21.6) 1
6700 (16.1)
5662 (13.5)
7898 (19.1)
5390 (12.9)
2482 (5.7)
7 days
412
1311
516 (0.11)
1055 (0.71)
4809 (4.89)
6471 (6.74)
~ ](8.53) 1
2565 (2.39)
4032 (4.03)
1412 (I.I I)
1317 (1.01)
gAs mean cpm of triplicate cultures: SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day.
19
Duck lymphocytes Table 7. Time-dose transformation responses of duck blood lymphocytes to wheat germ agglutinin (WGA) Radioactivity~ of Unstimulated controls
Cultures stimulated with WGA (,ug/ml)
Time of culture
-3H
+3H
0.1
0.5
1
2
5
10
20
40
100
I day
238
638
698 (1.15)
661 (1.06)
451 (0.53)
393 (0.39)
354 (0.29)
409 (0.43)
635 (0.99)
627 (0.97)
234 (-0.01)
2 days
147
279
689 (4.11)
903 (5.73)
800 (4.95)
845 (5.29)
1011 (6.54)
1019 (6.61)
1378 (9.32)
~ 1(20.1)]
851 (5.33)
3 days
425
707
448 (-0.92)
903 (0.69)
899 (0.68)
882 (0.62)
692 (-0.05)
955 (0.88)
3579 (10.2)
~ 636 1(26.9) 1 (-0.25)
4 days
248
362
482 (2.05)
1043 (6.97)
880 (5.54)
953 (6.18)
975 (6.38)
777 (4.64)
3716 (30.4)
~ ](59.9)]
346 (0.86)
5 days
265
312
192 (-1.55)
836 (12.1)
896 (13.4)
1648 (29.4)
492 (4.83)
1028 (16.2)
~ 1(107) 1
4320 (86.3)
263 (-0.04)
6 days
321
480
822 (3.15)
1322 (6.29)
3190 (18.0)
3700 (21.3)
2551 (14.0)
1975 (10.4)
~ 1(66.1) 1
5973 (35.5)
561 (1.51)
7 days
254
1050
372 (0.15)
1161 (1.14)
838 (0.73)
3134 (3.62)
1930 (2.10)
2096 (2.31)
3034 (3.49)
~ 1(13.3) 1
496 (0.30)
"As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day, SI ~ 2.0 not included.
active cells, within a complex chain of events involving lymphocytes, accessory cells and their numerous soluble factors. In mammals and chickens it is now well established that some mitogens stimulate LT response among T cells while some stimulate B cells [20-22]. Some lectins stimulate several cell types because the specific receptor is widespread, e.g. HP binds predominantly to human T cells but it also binds to N K cells and some myeloma cell lines [23-26], the last observation suggesting that normal B cells might have receptors for HP at certain stages of maturation. Others, e.g. PWM, stimulate both T and B cells because they comprise Table 8. Time,lose transformation responses of duck blood lymphocytes to horseshoe crab lectin (HSC) Radioactivity~ of Unstimulated controls Time of culture
Cultures stimulated with HSC (,ug/ml)
+3H
0.1
0.5
1
2
5
10
20
40
100
1 day
168
927
988 (1.08)
935 (1.01)
726 (0.73)
1092 (1.22)
984 (1.07)
718 (0.72)
737 (0.75)
232 (0.08)
567 (0.53)
2 days
193
468
559 (1.33)
391 (0.72)
522 (1.20)
456 (0.96)
737 (I.98)
786 (2.16)
941 (2.72)
1403 (4.40)
] ~2o.1)]
3 days
226
671
365 (0.31)
710 (1.09)
501 (0.62)
569 (0.77)
628 (O.9O)
673 (I.00)
48O (0.57)
7O0 (1.06)
] ~26.6)j
4 days
208
486
491 (1.02)
433 (0.81)
591 (1.38)
675 (1.68)
1118 (3.27)
632 (1.52)
400 (0.69)
576 (1.32)
1(48.5) 1
5 days
255
629
562 (0.82)
1113 (2.29)
615 (0.96)
322 (0.18)
618 (O.97)
672 (1.1 I)
278 (O.O6)
809 (1.48)
1¢3.5) 1
6 days
227
327
552 (3.25)
700 (4.73)
484 (2.57)
601 (3.74)
563 (3.36)
848 (6.21)
764 (5.37)
928 (7.01)
102.8) 1
7 days
274
432
426 (0.96)
700 (2.70)
607 (2.11)
821 (3.46)
517 (1.54)
858 (3.70)
610 (2.13)
911 (4.03)
I ~4.90)[
3H
aAs mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day, SI (-2.0 not included.
D. A. HIGGINS
20
Table 9. Time~zlose transformation responses of duck blood lymphocytes to pokeweed mitogen (PWM) Radioactivitya of Unstimulated controls
Cultures stimulated with PWM (/ag/ml)
Time of culture
-3H
+3H
0.1
0.5
1
2
5
10
20
40
100
1 day
328
1067
860 (0.72)
592 (0.36)
379 (0.07)
631 (0.41)
588 (0.35)
772 (0.60)
538 (0.28)
755 (0.58)
844 (0.75)
2 days
307
721
1148 (2.03)
1545 (2.99)
1693 (3.35)
2416 (5.09)
3122 (6.80)
2938 (6.35)
3518 (7.76)
6143 (14.1)
1(15.0) 1
3 days
236
615
975 (I.95)
3422 (8,41)
4184 (10.4)
5096 (12.8)
5581 (14,1)
7318 (18.7)
7442 (19.0)
~ 1(29.2)[
6974 (17.8)
4 days
427
593
643 (1.30)
2666 (13.5)
5977 (33.4)
8696 (49,8)
12306 (71.6)
9637 (55.5)
13722 (80.1)
~ 1(90.5) 1
3906 (20,9)
5 days
443
661
915 (2.16)
3904 (15.9)
10125 (44.4)
13471 (59.8)
~ 1(306) 1
9870 (43.2)
16960 (75.8)
7745 (33,5)
2579 (980)
6 days
246
498
629 (I.52)
1889 (6.52)
47018 (186)
14767 (57.6)
~ 1(378)]
45210 (178)
20399 (80.0)
8636 (33.3)
t208 (3.82)
7 days
336
458
736 (3.28)
2598 (18.5)
5862 (45.3)
18354 (148)
~ 1(637) 1
19270 (155)
31709 (257)
4738 (36.1)
578 (I.98)
"As mean cpm of triplicate cultures; SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day, SI ,~ 2.0 not included.
several mitogenic components each with different specificity [27]. Some lectins react with subsets of cells within larger populations recognised by other lectins. PHA binds to only a proportion of human T cells which bind Con A [28]. Human T helper cells bind Riotous communis agglutinin via sugars in the CD4 complex and suppressor cells bind WGA via the CD8 complex [29]. While the reaction of lectins with lymphocyte populations in man and mouse is rather well characterized [30] the limited knowledge of iectin binding to cells from many animals and birds indicates that individual lectins might not bind to the same cell Table 10. Time-dose transformation responses of duck blood lymphocytes to E. coli lipopolysaccharide (LPS) Radioactivity~ of Unstimulated controls Time of culture
Cultures stimulated with LPS (ug/ml)
-3H
+ 3H
0.1
0,5
1
2
5
10
20
40
100
I day
180
360
388 (1.15)
435 (1.42)
297 (0.65)
444 (1.47)
465 (I.58)
462 (1.57)
332 (0.84)
401 (1.23)
453 (I.52)
2 days
225
349
411 (1.50)
585 (2.90)
621 (3.19)
759 (4.31)
427 (1.63)
634 (3.30)
643 (3.37)
752 (4.25)
1(4.43)
3 days
155
284
711 (4.31)
603 (3~47)
826 (5.20)
943 (6.11)
~ ](8.22) 1
1054 (6.97)
751 (4.62)
723 (4.40)
750 (4.61)
4 days
269
389
960 (5.76)
1232 (8.02)
~ 1(9.92) 1
1205 (7.80)
1314 (8.71)
924 (5.46)
1275 (8.38)
1110 (7.10)
888 (5.16)
5 days
137
265
656 (4.05)
972 (6.52)
1628 (11.6)
1336 (9.37)
~ 1(10.8) 1
1209 (8.37)
1463 (10.4)
1101 (7.53)
1148 (7.90)
6 days
453
917
1049 (t.28)
1914 (3.I5)
1622 (2.52)
1440 (2.13)
2187 (3.74)
~ 1(3.9511
2208 (3.78)
1574 (2.41)
1502 (2.26t
7 days
678
1270
1195 (0.87)
1505 (I.40)
2101 (2.40)
1980 (2.20)
2041 (2.30)
~ 1(2.92) 1
2073 (2.36)
1524 (1.43)
1432 (1.27)
"As mean cpm of triplicate cultures: SI of stimulated cultures in parentheses; boxes enclose the highest values obtained on each day, SI ( 2.0 not included.
Duck lymphocytes
21
Table 11. Mitogen-induced transformation responses' of blood lymphocytes from two ducks after culture for 3 and 5 days Duck 1 Mitogen None (3H - ) (3H + ) PHA Con A LC HP PNA BSS WGA HSC PWM LPS
Concentration (#g/ml)
3 days
--40 100 40 100 40 100 20 100 5 10 I0 20 20 40 10 100 I 5 1 10
223 957 30722 (41.6) 52220 (70.8) 4724 (6.13) 463 (0.33) 24982 (33.7) 43245 (58.4) 749 (0.72) 1279 (1.44) 1084 (1.17) 1125 (I .23) 2350 (2.90) 3707 (4.75) 19548 (26.3) 12651 (16.9) 2559 (3.18) 4340 (5.61 ) 2932 (3.69) 47347 (64.2) 3446 (4.39) 5775(7.56)
Duck 2 5 days
528 949 2245 (4.08) 5628 (12.1) 1007 (1.14) 1764 (2.93) 1060 (1.26) 1785 (2.98) 1104 (1.37) 1019 (1.17) 821 (0.69) 1437 (2.16) 4746 (10.0) 1523 (2.36) 37815 (88.6) 105533 (249) 1736 (2.87) 5223 ( 11.1 ) 3600 (7.30) 14052 (32.1) 1266 (I.75) 2081 (3.69)
3 days
5 days
271 1041 24014 (30.8) 20103 (25.7) 9044 (11.4) 3970 (4.80) 3374 (4.03) 9796 (12.4) 1742 (I.91) 8842 (11.1) 6180 (7.67) 6639 (8.27) 10183 (12.9) 11027 (13.9) 4566 (5.58) 956 (0.89) 731 (0.59) 3478 (4.16) 8872 (11.2) 4904 (6.02) 10045 (12.7) 8030(10.1)
428 795 4865 (12.1) 15726 (41.7) 2976 (6.94) 1405 (2.66) 1451 (2.79) 4617 (11.4) 1349 (2.51) 2256 (4.98) 1357 (2.53) 1615 (3.23) 2244 (4.95) 1721 (3.52) 9147 (23.7) 6694 (17.1) 882 (1.24) 4256 (I 0,4) 2652 (6.06) 4752 (11.8) 3215 (7.59) 6200(15.7)
~rH]-thymidine uptake as mean cpm of triplicate cultures; SI of stimulated cultures in parentheses.
populations in different species [31]. Since duck lymphocyte subpopulations cannot be identified on the basis of receptors for erythrocytes or immunoglobulins, an attempt was made to demonstrate populations differing in responsiveness to mitogens chosen to reflect a wide range of sugar-binding specificities. It was assumed that: (i) functionally different lymphocyte populations must exist in the duck; (ii) different populations will possess different surface glycopeptides and glycolipids; and (iii) these populations will exhibit some individuality in the kinetics of their transformation responses. It was relevant therefore that three patterns of response were observed: that with maximum sensitivity at 3-5 days culture (to PHA, PNA, WGA and LPS); that in which high concentrations of mitogen gave maximum transformation throughout the experiment (to Con A, LC, HP and HSC); and that (to BSS and PWM) which attained and then sustained sensitivity to lower concentrations of mitogen. The pattern of response to PWM and BSS might result from the heterogeneity of cells responding to these lectins; PWM because of the complexity of the lectin and the resulting diversity of receptors, BSS because of the widespread distribution of the specific sugars. The response to HP was surprising in view of our previous observation that duck lymphocytes do not have receptors for fluorescein-labelled HP [1]. It is possible that HP binds primarily to glycoproteins in the PDS supplement and that the resulting complex is mitogenic; some of the lectins used, notably Con A, induced precipitation at concentrations above 100 ~g/ml. On mammalian lymphocytes, receptors for some lectins, e.g. HP and PNA, are often occluded by sialic acid which can be removed by neuraminidase. Previously we have shown that the percentage of duck lymphocytes staining with fluorescein-PNA was increased after treatment with Vibrio cholerae neuraminidase [1]. As an adjunct to the present study, LT was also attempted using HP and PNA on duck blood lymphocytes first treated with neuraminidase at 10 units/ml. The results were disappointing; the cells gave high background cpm and only a transient and slight increase in response to lectins. We have also attempted LT employing purified protein derivatives (PPD) from Mycobacterium
22
D.A. HIGGINS
t u b e r c u l o s i s , M . boris, M . a v i u m a n d M . j o h n e i . T h e e x p e r i m e n t s e x a m i n e d a n u m b e r o f v a r i a b l e s i n c l u d i n g cell c o n c e n t r a t i o n , flat- vs U - b o t t o m e d c u l t u r e trays a n d v a r i o u s m e d i u m s u p p l e m e n t s in a d d i t i o n to the t i m e - d o s e c o n d i t i o n s e m p l o y e d w i t h the m i t o g e n s d e s c r i b e d here. T h e results w e r e u n i v e r s a l l y n e g a t i v e i n d i c a t i n g t h a t P P D is n o t a m i t o g e n for lymphocytes from normal ducks. T h e r e s p o n s e s o f d u c k b l o o d l y m p h o c y t e s to the r a n g e o f m i t o g e n s u s e d in this w o r k m i g h t be a t t r i b u t a b l e to the a n a t i d i n e e q u i v a l e n t o f T cells, B cells a n d s u b p o p u l a t i o n s . O u r c u r r e n t studies are d e s i g n e d to c h a r a c t e r i z e the cells w h i c h r e s p o n d to t h e m i t o g e n s e m p l o y e d here; the results will be r e p o r t e d . Acknowledgements--This work was supported by grants from the University of Hong Kong Committee for Research and Conference Grants, the Pauline Chan Medical Research Fund, the Wing Lung Bank Medical Research Fund, the Wu Chung Medical Research Fund, the Medical Faculty Research Grant Fund, the Lee Wing Tat Medical Research Fund, the Sun Yat Sen Foundation Fund for Medical Research and Cherry Valley Farms Ltd. Dr L. Lalibert6 kindly translated the abstract. The technical assistance of Miss C. S. H. Teoh and Miss N. Chin and the secretarial assistance of Miss Y. Chow are also gratefully acknowledged.
REFERENCES 1. Higgins D. A. and Chung S. H. (1986) Duck lymphocytes. I. Purification and preliminary observations on surface markers. J. Immun. Meth. 86, 231 238. 2. Higgins D. A. and Teoh C. S. H. (1988) Duck lymphocytes. II. Culture conditions for optimum transformation response to phytohaemagglutinin. J. Immun. Meth. 106, 135 145. 3. Lis H. and Sharon N. (1973) The biochemistry of plant lectins (phytohemagglutinins). Ann. Rev. Biochem. 42, 541 574. 4. Sharon N. (1983) Lectin receptors as lymphocyte surface markers. Adv. Immun. 34, 213 298. 5. Allen A. K., Desai N. N. and Neuberger A. (1976) The purification of the glycoprotein lectin from the broad bean (Vicia faba) and a comparison of its properties with lectins of similar specificity. Biochem. J. 155, 127 135. 6. Kornfeld K., Reitman M. L. and Kornfeld R. (1981) The carbohydrate-binding specificity of pea and lentil lectins. Fucose is an important determinant. J. biol. Chem. 256, 6633 6640, 7. Hammarstr6m S. (1974) Structure, specificity, binding properties and some biological activities of a blood group A reactive hemagglutinin from the snail Helix pomatia. Ann. N.Y. Acad. Sci. 234, 183-195. 8. Hammarstr6m S. and Kabat E. A. (1971) Studies on specificity and binding properties of the blood group A reactive hemagglutinin from Helix pomatia. Biochemistry 10, 1684-1692. 9. Lotan R., Skutelsky E., Danon D. and Sharon N. (1975) The purification, composition and specificity of the anti-T lectin from peanut (Arachis hypogaea). J. biol. Chem. 250, 8518-8523. 10. Hayes C. E. and Goldstein I. J. (1974) An ~-D-galaetosyl-binding lectin from Bandeiraea simplic!folia seeds. J. biol. Chem. 249, 1904-1914. 11. Murphy L. A. and Goldstein I. J. (1977) Five ~-D-galacto-pyranosyl-binding isolectins from Bandeiraea simplicifolia seeds. J. biol. Chem. 252, 4739-4742. 12. Wood C., Kabat E. A., Murphy L. A. and Goldstein I. J. (1979) Immunochemical studies of the combining sites of the two isolectins, A4 and B4, isolated from Bandeiraea simplicifolia. Archs Bioehem. Biophys. 198, 1-11. 13. Allen A. K., Neuberger A. and Sharon N. (1973) The purification, composition and specificity of wheat-germ agglutinin. Biochem. J. 131, 155-162. 14. Nagata Y. and Burger M, M. (1974) Wheat germ agglutinin. Molecular characteristics and specificity for sugar binding. J. biol. Chem. 249, 3116--3122. 15. Peters B. P., Ebisu S., Goldstein I. J. and Flashner M. (1979) Interaction of wheat germ agglutinin with sialic acid. Biochemistry 18, 5505 5511. 16. Cohen E., Rozenberg M. and Massaro E. J. (1974) Agglutinins of Limulus polyphemus (Horseshoe crab) and Birgus latra (Coconut crab). Ann. N.Y. Acad. Sci. 234, 28 32. 17. Waxdal M. J. (1978) Pokeweed mitogens. Meth. Enzym. 50, 354-361. 18. Dillner-Centerlind M.-L., Axelsson B., Hammarstr6m S., Hellstr6m H. and Perlmann P. (1980) Interaction of lectins with human T lymphocytes. Mitogenic properties, inhibitory effects, binding to the cell membrane and to isolated surface glycopeptides. Fur. J. lmmun. 10, 434-442. 19. Skoog V. T., Nilsson S. F. and Weber T. H. (1980) Characterization of human lymphocyte surface receptors for mitogenic and non-mitogenic substances. Scand. J. lmmun. 11, 369 376.
Duck lymphocytes
23
20. Greaves M. F., Roitt |. M. and Rose M. E. (1968) Effect of bursectomy and thymectomy on the responses of chicken peripheral blood lymphocytes to phytohaemagglutinin. Nature (Lond.) 220, 293-295. 21. Hovi T., Suni J., Hortling L. and Vaheri A. (1978) Stimulation of chicken lymphocytes by T- and B-cell mitogens. Cell. Immun. 39, 70-78. 22. Janossy G. and Greaves M. F. (1971) Lymphocyte activation. I. Response of T and B lymphocytes to phytomitogens. Clin. exp. Immun. 9, 483-498. 23. Hailer O., Gidlund M., Hellstr6m V., Hammerstr6m S. and Wigzell H. (1978) A new surface marker on mouse natural killer cells: receptors for Helix pomatia A hemagglutinin. Eur. J. Immun. 8, 765-771. 24. Hellstr6m U., Mellstedt H., Perlmann P., Holm G. and Pettersson D. (1976) Receptors for Helix pomatia A haemagglutinin on leukaemic lymphocytes from patients with chronic lymphocytic leukaemia (CLL). Clin. exp. Immun. 26, 196-203. 25. Hellstr6m U., Perlmann P., Robertsson E. S. and Hammarstr6m S. (1978) Receptors for Helix pomatia A haemagglutinin (HP) on a subpopulation of human B cells. Scand. J. lmmun. 7, 191-197. 26. Axelsson B., Kimura A., Hammarstr6m S., Wigzell H., Nilsson K. and Mellstedt H. (1978) Helix pomatia A hemagglutinin: selectivity of binding to lymphocyte surface glycoproteins on T cells and certain B cells. Eur. J. lmmun. 8, 757 764. 27. Waxdal M. J. and Basham T. Y. (1974) B- and T-cell stimulatory activities of multiple mitogens from pokeweed. Nature 251, 163-164. 28. Rawson A. J. and Daniele R. P. (1976) The separation of lymphocytes responsive only to concanavalin A from those also responsive to phytohemagglutinin. J. Retieuloendothelial Soe. 20, 421-427. 29. Boldt D. H. and Dorsey S. A. (1983) Interactions of lectins and monoclonal antibodies with human mononuclear cells. I. Specific inhibition of OKT4 and OKT8 binding by Ricinus communis agglutinin and wheat germ agglutinin. J. Immun. I30, 1646-1653. 30. Sharon N. (1980) Cell surface receptors for lectins: markers of murine and human lymphocyte subpopulations. In: Immunology 80 (Progress in Immunology IV) (Edited by Fougereau M. and Dausset J.), pp. 254 278. Academic Press, New York. 31. Higgins D. A. (1981) Markers for T and B lymphocytes and their application to animals. Vet. Bull. 51, 925-963.