Developmentaland ComparativeImmunology,Vol. 14, pp. 59-68, 1990 Printed in the USA. AlL rights reserved.
0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
CELLULAR REQUIREMENTS FOR LYMPHOKINE SECRETION BY RAINBOW TROUT SALMO GAIRDNERI LEUCOCYTES Susan Graham and Christopher J. Secombes Department of Zoology, Universityof Aberdeen, TiLlydroneAve., Aberdeen, AB9 2TN, UK (Submitted March 1989;Accepted May 1989) [~Abstract--The ability of different popula- lacking, due to difficulties encountered tions of rainbow trout blood leucocytesto pro- in thymectomizing young fish, the lack duce MAF following stimulation with Con of inbred strains, and the lack of conveA/PMA was assessed by the amount of NBT nient surface markers. That two lymreduction in target macrophages. The effect of phocyte populations exist in fish has varying lymphocyteor macrophage number on MAF production in the presence of a constant been suggested by studies looking at minumber of macrophages or iymphocytes re- togen responses and the hapten-carrier spectively, showed that in both cases MAF ac- effect (1-4), and indeed in these reactivity initially increased with increasing cell tions two functionally distinct populanumber and then plateaued. Macrophages tions have been separated by adherence alone did not produce MAF whereas some to nylon wool columns (5) or on the MAF activity was produced by macrophage- basis of nonimmune rosetting with heterdepleted lymphocytes, although significantly ologous erythrocytes (6). More conclulower than in the presence of macrophages. sive evidence that the populations preSeparation of leucocytes into sIg- and sIg + sent are similar to T and B cells of higher cells by panning showed that only sIg- lymvertebrates have been forthcoming over phocytes could produce MAF and that macrothe last few years from studies using anphages were necessaryas accessorycells. These results support the contention that fish lym- tibodies to lymphocyte surface deterphocytes can be divided into sIg- T cells and minants to enrich or deplete for these putative lymphocyte populations. sIg+ B cells. Monoclonal antibodies (MoAbs) to []Keywords--Lymphokine secretion; Cellular serum IgM of a range of teleost fish have requirements; Rainbow trout; Panning; slg + been raised that react with only a and sIg- cells; MAF; Macrophage-depletion; subpopulation (30-40%) of blood lymLymphocyte-depletion. phocytes (7-9) suggesting that surface Ig (slg) can be used as a marker for BIntroduction like cells. By selectively enriching for s l g + or s l g - lymphocytes by "panIt is generally agreed that the earliest ning" with these MoAbs the different manifestations of the specific immune populations have been separated for system occur in fish. Their ability to functional studies in vitro. Such studies produce antibody and reject allografts have shown that slg+ lymphocytes redemonstrates the possession of both hu- spond to LPS whereas s l g - lymphomoral and cell-mediated immunity. cytes respond to Con A, although the reHowever, formal proof for the existence sponse to LPS is not completely reof T and B l y m p h o c y t e s in fish is moved (8,10). In the mixed leucocyte response (MLR) only s l g - cells were Address correspondence to Dr. Christopher found to undergo proliferation, but both J. Secombes, Department of Zoology, Uni- slg + and s l g - lymphocytes could act as stimulator cells (11). In addition, versity of Aberdeen, Tillydrone Avenue, Aberdeen, U.K. s l g + lymphocytes were found to be
59
60
n e c e s s a r y for a n t i - h a p t e n plaqueforming cell (PFC) responses to thymusindependent antigens in contrast to PFC responses to thymus-dependent antigens which required both sIg+ and s I g lymphocytes (12). By combining panning with anti-lg MoAbs and specific adherence to hapten or carrier molecules it has been possible to demonstrate that both hapten-responsive slg + and carrier r e s p o n s i v e s l g - l y m p h o c y t e s are needed for the anti-hapten response to a thymus-dependent antigen (12). The requirement for macrophages or monocytes in these responses has also been investigated since they can be removed by adherence to baby hamster kidney cell microexudate-coated plates or by passing cells through a Sephadex G-10 column. Mitogen and MLR responses by s l g - cells were dependent upon the presence of macrophages (10,11), as was the anti-hapten PFC response to thymus-dependent and -independent antigens (12). Only the LPS response by slg + cells has been shown to occur in the absence of macrophages to date (10). Antibodies to determinants on T-like cells have also been used to investigate lymphocyte heterogeneity in fish. For instance, antibrain serum, thought to react with Thy-l-like antigens present on T cells and nervous tissue, can selectively kill PHA responsive cells but not LPS responsive cells (6). More recently a MoAb has been produced that specifically reacts with s I g - channel catfish lymphocytes and has been used to isolate those cells that provided helper activity for antibody synthesis to a thymusdependent antigen (13). Although the thymus-dependency of these populations has still to be ascertained, such data provide compelling evidence for the existence of T and B cells in fish. Further proof for functions such as the production of lymphokine and the generation of cytotoxic cells will strengthen this conclusion. To this end
S. Graham and C. J. Secombes
we have recently optimized the production and detection of a macrophage activating factor (MAF) secreted from rainbow trout (Salmo gairdneri) leucocytes (14). In this article we report on the secretion of MAF by slg + and s l g lymphocytes separated using an antitrout MoAb (I/14), and on the accessory cell requirements.
Materials and Methods
The Production and Detection of MAFContaining Supernatants MAF containing supernatants were produced as described by Graham & Secombes (14). Briefly, leucocyte suspensions were adjusted to 5 x 106 cells per ml in L-15 medium (L-15) containing I% Penicillin/Streptomycin (P/S), 5 x 10 -5 M 2-mercaptoethanol (2-ME), and l ml seeded into 24 well microtitre plates (MTP, Nunc). They were then pulsed with 10 ug/ml Concanavalin A (Con A, Difco) and 5 ng/ml phorbol myristate acetate (PMA, Sigma), previously shown to be optimal for MAF production (14), for 3h at 18°C. After this time the cells were gently washed five times with sterile phosphate buffered saline (PBS) to remove any residual Con A and PMA, and then cultured in L-15 containing 1% P/S, 10% foetal calf serum (FCS) and 5 x 10 -5 M 2-ME for 48h. The supernarants were then harvested and stored at -20°C until use. MAF activity was detected using purified head kidney (HK) macrophages as targets. These cells were isolated using Percoll density gradients followed by adherence to 96 well MTP as previously described (15,16). Following a 48h incubation with the MAF containing supernatants the respiratory burst activity of triplicate wells of macrophages was assessed by their ability to reduce nitroblue tetrazolium (NBT). The macrophage monolayers were covered with
Lymphokine secretion by trout
100 ~1 of 1 mg/ml NBT containing 1 ixg/ml PMA, to trigger the respiratory burst, for 30 min before being fixed in methanol. After washing in 70% methanol the reduced formazan within the macrophages was solubilized with KOH/ DMSO (17) and the optical density determined at 620 nm in a multiscan spectrophotometer. All results were exp r e s s e d per l0 s m a c r o p h a g e s after counting nuclei released with 0.1M citric acid, Tween 20 and 0.05% crystal violet in control wells. In some cases the results were expressed as a stimulation index by dividing the mean optical density reading from macrophages incubated with active supernatants with that from macrophages incubated with control supernatants obtained from leucocytes not stimulated with Con A/PMA.
61
proximately 1 ml of the cell suspension was put onto the column containing 6 ml sterile Sephadex G-10. The cells were allowed to penetrate the column before addition of 10 ml of medium to elute the nonadherent cells. After centrifugation the cells were resuspended in fresh medium and cultured overnight in a 25 cm z flask to remove any residual macrophages that did not a d h e r e to the column. The nonadherent lymphocytes were then adjusted to 1.6 x 105 to 107 cells per ml, and were added back to the constant number of macrophages previously set up. MAF-containing supernatants were then produced as described above.
Effect of Macrophage Number on MAF Production Effect of Lymphocyte Number on MAF Production The effect of varying lymphocyte numbers on the production of MAF was studied by isolating a constant number of macrophages and adding to them varying numbers of leucocytes extensively depleted of macrophages (as assessed by a lack of adherent cells), prior to addition of Con A/PMA. Blood leucocytes (I ml) obtained after centrifugation over a 51% Percoll density gradient, were seeded into 24 well MTP at 1 × 107 cells per ml L-15 containing 0.1% FCS and 1% P/S. After 2h at 18°C for adherence, the nonadherent cells were removed and the adherent cells were covered with 1 ml L-15, 5% FCS, 1% P/S. The nonadherent cells were then centrifuged at 400g for 10 min and the pellet of cells resuspended to 2 × 107 cells per ml in L-15, 10% FCS, 1% P/S. The lymphocytes were then extensively depleted of macrophages by passage through a Sephadex G-10 column prepared as described by Mishell and Mishell (18). Ap-
The effect of varying macrophage number on the production of MAF was determined by isolating different concentrations of macrophages and adding to them a constant number of leucocytes which had been extensively depleted of macrophages, prior to the addition of Con A/PMA. Blood leucocytes were seeded into 24 well MTP in two-fold dilutions from 1.96 x 105 to 5 x 107 cells per ml L-15, 0.1% FCS, 1% P/S to obtain varying macrophage numbers. Duplicate wells of each cell concentration were used to enable macrophage nuclei counts to be performed. The cells were allowed to adhere for 2h at 18°C after which the nonadherent cells were collected and the adherent macrophages were supplemented with L-15, 5% FCS, 1% P/S and placed at 18°C until use. The nonadherent ceils were then depleted extensively of macrophages as above and 5 x 10 6 viable iymphocytes were added back to each well of macrophages previously set up. MAF-containing supernatants were then produced as described earlier.
62
Separation of slg + and slg Lymphocytes by Panning Petri dishes (I0 cm, Nunc) were coated overnight at 4°C with 8 ml of control ascitic fluid, obtained using a clone against an unrelated antigen, or monoclonal anti-trout Ig (1/14) ascitic fluid diluted 1:200 in PBS. The plates were then washed twice with PBS and 8 ml L-15, 10% FCS was added for lh at 18°C before addition of blood leucocytes. The leucocytes were initially panned for lh on control ascitic fluid coated plates at 2 × 107 cells per dish, to remove adherent s l g - leucocytes, and subsequently were panned twice on 1-14 coated plates. The n o n a d h e r e n t cells obtained after the second 1-14 panning were termed s l g lymphocytes. After removal of the nonad h er en t cells from the 1-14 coated plates an intermediate wash was carried out before removal of the adherent slg + cells by pipetting medium onto the plate. The two batches of slg+ lymphocytes were pooled before use and both the slg+ and the s l g - lymphocytes were then cultured overnight in 25 cm 2 flasks. Finally the nonadherent cells from these flasks were added to isolated macrophages from the same fish (see below) and used to generate MAF-containing supernatants as above. In some experiments s l g - cells were stimulated with Con A/PMA in the absence of macrophages. While the panning was occurring blood leucocytes from the same fish were added to a 24 well MTP at 5 × 106 cells per well, to serve as a total leucocyte control and as a source of macr o p h a g e s for " a d d - b a c k " and macrophage-only controls. For this latter purpose the nonadherent cells w e r e removed after lh at 18°C and the adherent cells supplemented with L-15, 10% FCS until use.
Flow Cytofluorometric Analysis To confirm that the separation of lymphocytes into sIg+ and s I g - cells had
S. Graham and C. J. Secombes
been successful on each occasion that this experiment was performed, a subsample of the cells was analyzed by flow cytofluorometry. 5 × 105 to 10 6 cells per ml were incubated with 1/14 supernatant containing 0.1% sodium azide, 1% bovine serum albumin (BSA). After lh at 4°C the cells were washed three times by centrifugation and incubated for a further lh with sheep anti-mouse IgG/FITC diluted 1:40 in PBS, 0.1% sodium azide, 1% BSA. Finally the cells were washed three additional times in PBS, 0.1% sodium azide and were then analyzed in an EPICS-C flow cytometer (Coulter Electronics) with the argon laser tuned to 488 nm and PMT voltage at 1600 V. Graphs of log fluorescence intensity versus cell number were plotted for 10,000 cells. Results were analyzed using Student's two-tailed t-test.
Results
Effect of Lymphocyte Number on MAF Production The effect of varying l y m p h o c y t e number on MAF production, in the presence of a constant number of macrophages (3.2 × 105), is seen in Fig. la. In the absence of lymphocytes no MAF was produced by macrophages, as determined by the amount of NBT reduced by macrophages incubated with these supernatants in comparison with supernatants from cultures not stimulated with Con A/PMA (O.D. of 0.15). Low numbers of lymphocytes, 1.6-3.1 × 105 , also did not produce significant levels of MAE With larger numbers of lymphocytes significant MAF activity was seen, and i n c r e a s e d with cell number up to a maximum at 5 × 10 6 cells. The MAF activity appeared to plateau using more than 106 lymphocytes in that there were no significant differences in the amount of N BT reduction between these cell concentrations, but it was significantly higher in each case
Lymphokine secretion by trout 0.22
(e)
63
(b)
0.21
0.20
S ~
0.19
Q
o ~
o.18.
,D
0.17-
e
i,m
z
0.16-
0.15.
0.14"
0.13 0.1
1.0 Cell number (x 1 0 - e )
10
2"0
Cell number (x 10 - s )
Figure 1. The effect of varying lymphocyte (a) or macrophage (b) number on MAF production in the presence of a constant number of macrophages (3.2 × 10s) or lymphocytes (5 × 106), respectively. MAF activity was assessed by the amount of NBT reduction in target macrophages following incubation with the MAF-containing supernatants diluted 1:8. The results were expressed as means _+ standard errors of triplicate wells adjusted to 10s cells.
compared with the NBT reduction using supernatants produced with low lymphocyte numbers.
Effect of Macrophage Number on MAF Production
The effect of varying macrophage number on MAF production, in the presence of a constant number of lymphocytes (5 x 106), is seen in Fig. lb. In the apparent absence of macrophages and at every concentration of macrophages used for MAF production significantly increased NBT reduction was seen in comparison with supernatants from cultures not stimulated with Con A/PMA (O.D. of 0.10). As with lymphocyte number increasing MAF activity was seen with increasing numbers of macrophages, with the highest mean NBT reduction occurring with the largest macrophage number used (2.2 x 106). However, analysis of this data suggested that
a plateau had been reached at a macrophage number of approximately 3.6 × 105 cells, since no significant differences were seen between the points above this cell number (with the exception of 5.8 × 105 vs. 1.6 × 106 macrophages). Thus macrophage numbers below 105 appear to be suboptimal for MAF production.
MAF Production Using slg + and slg Lymphocytes
EPICS analysis of the adherent and nonadherent lymphocyte populations following panning with 1/14 M o A b showed that these populations were indeed slg + and s l g - , respectively (Fig. 2). Typically 40% of the blood leucocytes were slg+ before the separation, whereas after panning 70-97% of the adherent cells were slg + and 85-99% of the nonadherent cells were s l g - . Early experiments found that MAF activity could be detected in supernatants from
64
S. Graham and C. J. Secombes
6
tion indices of approximately 1.0). Supernatants from s l g - lymphocytes in the presence of macrophages also had significant MAF activity, as great or greater than the same number of unfractionated leucocytes, but those from s l g + lymphocytes in the presence of macrophages lacked MAF activity. In some experiments s l g - lymphocytes were incubated in the presence and absence of macrophages to see if they required accessory cells for the production of MAE In the presence of macrophages NBT reduction was increased significantly above control values (OD of 0.1) between dilutions of 1:8 to 1:128 (Fig. 4). In the absence of macrophages MAF activity was abolished almost completely and in this example NBT reduction was only increased significantly above control values at a dilution of 1:32. Comparison of NBT reduction in macrophages incubated with these supernatants with macrophages incubated with supernatants derived from s l g - cells in the presence of macrophages showed the former to reduce significantly decreased amounts of NBT.
b
I
IC .i
Figure 2. Flow cytofluorometric analysis of unfractionated leucocytes (a), and adherent (b) or nonadherent (c) cells following panning with monoclonal anti-trout Ig. The percentage of cells calculated as slg+ in each case was (a) 39.24%, (b) 96.03%, and (c) 0.84%. x axis, log fluorescence intensity, y axis, cell number.
both slg + and s l g - lymphocytes in the presence of macrophages, and that the purity of the slg+ cells was critical for the presence or absence of MAF activity when using these cells. In general, only when the purity of the slg+ cells exceeded 95% was MAF activity lost from the supernatants. Examples of such results obtained from two representative fish are shown in Fig. 3. In both cases, as above, supernatants from unfractionated leucocytes contained significant MAF activity (i.e., stimulation indices above 1.0), whereas those from macrophage-only controls had none (stimula-
Discussion The results of this study, looking at the cellular requirements for MAF production in rainbow trout, support the contention that fish lymphocytes can be divided into s l g - T cells and slg+ B cells. Only purified s l g - lymphocytes were able to secrete MAF following stimulation with Con A/PMA, providing accessory cells were present. Thus lymphokine secretion can be added to the functions of helper activity for thymusdependent antibody responses and responsiveness to Con A and allogeneic leucocytes already ascribed to these cells in this and other teleost species (8,10-13).
Lymphokine secretion by trout
2.5"
65
Fish 2
Fish 1
2.0"
i!
1.5"
-$ W 1.0'
0.5'
MAF dilution ( - l o g 2)
Figure 3. MAF production by unfractionated blood leucocytes (0), macrophages (O), s l g - lymphocytes plus macrophages (A) and slg + lymphocytes plus macrophages (A). MAF activity was assessed by the amount of NBT reduction in target macrophages following incubation with the MAF-containing supernatants The results were expressed as a stimulation index by dividing the mean optical density reading from macrophages incubated with test supernatants with that from macrophages incubated with control supernatants obtained from leucocytes not stimulated with Con A/PMA.
To demonstrate whether this teleost MAF was truly a lymphokine secreted from lymphocytes, experiments were carried out using varying numbers of macrophage-depleted leucocytes added to a constant number of macrophages and vice versa. Since salmonid blood leucocytes normally contain < 10% granulocytes (19), after macrophage-depletion predominantly lymphocytes and some thrombocytes will remain, and the major role of the latter is in blood coagulation and general hemostasis (20). In both experimental approaches MAF activity initially increased with increasing numbers of macrophages or lymphocytes and then appeared to plateau. Perhaps more significantly MAF activity was completely absent from macrophage cultures without lymphocytes, whereas in the presence of optimal lymphocyte
numbers even in the apparent absence of macrophages significant MAF activity was present. In addition the panning experiments showed that in the presence of optimal numbers of macrophages MAF activity was only found using s l g - lymphocytes and not slg+ lymphocytes. Thus s l g - lymphocytes and not macrophages or slg+ lymphocytes would appear to be the responding cells. It is interesting to note that at high concentrations of these lymphokinecontaining supernatants a suppressive effect upon NBT reduction was sometimes seen, as shown in Figs. 3 and 4. This could be due to a multiplicity of factors being present in the supernatant, some of which were suppressive. However, this effect could still be seen following partial purification of the MAF activity by HPLC (unpublished), and so
66
S. Graham and C. J. Secombes
0.28
T
0.24 A
E c
0.20
O ~N (O
O
0.16
c
O o
0.12
"O L.
0.08 Z
0.04'
0.00
!
!
i
2
3
4
i
|
•
5
6
7
MAF dilution (-log 2) Figure 4. The effect of macrophage depletion on MAF production by s l g - lymphocytes. MAF activity was assessed by the amount of NBT reduction in target macrophages following incubation with supernatants from s l g - lymphocytes (G) or s l g - lymphocytes plus macrophages (A). The results were expressed as means _+ standard errors of triplicate wells adjusted to 10s cells.
the possibility that high levels of MAF actually inhibit the target cells also exists. The dependence upon accessory cells for the optimal production of MAF was also shown in this study. Thus with decreasing numbers of macrophages MAF production, as measured by NBT reduction, also decreased. The role of macrophages as accessory cells in immune responses of higher vertebrates is well established (21), and their importance in certain immune responses of fish is already known. In studies on the proliferative responses to Con A and allogeneic leucocytes (MLR) and in vitro antihapten PFC responses, monocytes/macrophages or factors released by them have b e e n s h o w n to be n e c e s s a r y (10-12,22,23). The production of at least
one lymphokine (MAF) by s l g - lymphocytes in fish can now also be included as accessory-cell-dependent. Interestingly, some MAF activity was detected in supernatants produced by stimulating lymphocytes in the absence of macrophages. This is similar to the results obtained with mitogenesis assays by Sizemore et al. (10) using the Sephadex G-10 macrophage depletion method, where a mitogenic response to both LPS and Con A of 65% and 10% of the maximum response respectively persisted after macrophage depletion (the former being considered accessory-cellindependent). Although the Sephadex method is quick and convenient for the removal of most macrophages, some cells which develop functions similar to macrophages have been reported in the
Lymphokine secretion by trout
67
filtered population (18). Thus it is possible that some macrophages were not removed using the depletion technique, and provided a limited accessory cell function. Alternatively, the presence of PMA in the present study may have partly overridden the need for accessory cells since another phorbol ester TPA has been shown to overcome the accessory cell requirement for Con A stimulation of thymocytes in channel catfish (24). Whatever the explanation, intact macrophages clearly provide most effective accessory signals. The plateauing of NBT reduction of target macrophages using more than 10 6 lymphocytes is in contrast to the studies of the mitogenic response of channel catfish lymphocytes to Con A and LPS where proliferation increased linearly with increasing lymphocyte number up to 5 x 105 (10) and was used as evidence that lymphocytes were the mitogen-responsive cells in this system. Had the study on mitogen responses used larger numbers of lymphocytes it is possible that a plateauing of the response would also have been seen. Alternatively, since lymphocyte proliferation in response to mitogens is a direct measurement, whereas in this study the level of lymphokine released is measured indirectly by its effect on target macrophages, the plateauing effect could be due to the
target cells becoming saturated and not being able to respond further. The panning experiments coupled with flow cytofluorometric analysis showed that it was crucial to have highly purified cell types in order to see any differences in MAF production. Contamination of the s l g + lymphocytes with >5% s l g - lymphocytes was often sufficient to detect MAF activity in the supernatants. Assuming s l g - lymphocytes are the responding cells from the a b o v e arguments, then clearly only small numbers are required in order to release enough MAF to stimulate large numbers of target macrophages. If this is the case then the plateauing effect of NBT reduction by target macrophages w o u l d not be surprising since the number of lymphocytes able to secrete sufficient MAF to stimulate all of the fixed number of target cells would soon be reached. Further studies are in progress to characterize this teleost lymphokine, to see if it is equivalent to known mammalian MAFs such as ~/-interferon. Acknowledgements--This w o r k was supported by S E R C grant No. GR/E/15413. The authors gratefully acknowledge the gift of the 1/14 h y b r i d o m a cells from P r o f e s s o r G. W. Wart, University of South Carolina. Thanks also go to Miss K. E. H e n d e r s o n and Mr. J. Milton for the E P I C S analysis.
References 1. Stolen, J. S.; Makela, O. Cartier preimmunization in the anti-hapten response of a marine fish. Nature. 254:718-719; 1975. 2. Yocum, D.; Cuchens, M.; Clem, L. W. The hapten-carrier effect in teleost fish. J. Immunol. 114:925-927; 1975. 3. Etlinger, H. M.; Hodgins, H. O.; Chiller, J. M. Evolution of the lymphoid system. I. Evidence for lymphocyte heterogeneity in rainbow trout revealed by the organ distribution of mitogenic responses. J. Immunol. 116:1547-1553; 1976. 4. Chilmonczyk, S. In vitro stimulation by mitogens of peripheral blood lymphocytes from rainbow trout (Salmo gairdneri). Ann. D'lmmunol. 129C:3-12; 1978.
5. Ruben, L. N.; Warr, G. W.; Decker, J. M.; Marchalonis, J. J. Phylogenetic origins of immune recognition: lymphoid heterogeneity and the hapten/carrier effect in the goldfish, Carassius auratus. Cell. Immunol. 31:266-283; 1977. 6. Cuchens, M. A.; Clem, L. W. Phylogeny of lymphocyte heterogeneity. II. Differential effects of temperature on fish T-like and B-like cells. Cell. Immunol. 34:219-230; 1977. 7. Lobb, C. J.; Clem, L. W. Fish lymphocytes differ in the expression of surface immunoglobulin. Dev. Comp. Immunol. 6:473-479; 1982. 8. Deluca, D.; Wilson, M.; Wart, G. W. Lymphocyte heterogeneity in the trout, Salmo gaird-
68
9.
10.
11.
12.
13.
14.
15. 16.
S. Graham and C. J. Secombes
neri, defined with monoclonal antibodies to lgM. Eur. J. Immunol. 13:546-551; 1983. Secombes, C. J.; van Groningen, J. J. M.; Eghefts, E. Separation of lymphocyte subpopulations in carp Cyprinus carpio L. by monoclonal antibodies: i m m u n o h i s t o c h e m i c a l studies. Immunology. 48:165-175; 1983. Sizemore, R. C.; Miller, N. W.; Cuchens, M. A.; Lobb, C. J.; Clem, L. W. Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro mitogenic responses of channel catfish leukocytes. J. Immunol. 133: 2920-2924; 1984. Miller, N. W.; Deuter, A.; Clem, L. W. Phylogeny of lymphocyte heterogeneity: the cellular requirements for the mixed leucocyte reaction with channel catfish. Immunology. 59: 123-128, 1986. Miller, N. W.; Sizemore, R. C.; Clem, L. W. Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro antibody response of channel catfish leukocytes. J. lmmunol. 134:2884-2888; 1985. Miller, N. W.; Bly, J. E.; van Ginkel, F.; Ellsaesser, C. E; Clem, L. W. Phylogeny of lymphocyte heterogeneity: identification and separation of functionally distinct subpopulations of channel catfish lymphocytes with monoclonal antibodies. Dev. Comp. Immunol. 11: 739-747; 1987. Graham, S.; Secombes, C. J. The production of a macrophage-activating factor from rainbow trout Salmo gairdneri leucocytes. Immunology. 65:293-297; 1988. Chung, S.; Secombes, C. J. Activation of rainbow trout macrophages. J. Fish. Biol. 31A:51-56; 1987. Chung, S.; Secombes, C. J. Analysis of events occurring within teleost macrophages during the respiratory burst. Comp. Biochem. Physiol. 89B:539-544; 1988.
17. Rook, G. A.; Steele, J.; Umar, S.; Dockrell, H. M. A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by 3,-interferon. J. lmmunol. Methods. 82:161167; 1985. 18. Mishell, R. I.; Mishell, B. B. Use of Sephadex G-10 to separate macrophages and lymphocytes. In: di Sabato, G.; Langone, J. J.; van Vunakis, H., eds. lmmunochemical techniques. Part G. Separation and characterization of lymphoid cells. London: Academic Press; 1984:p. 303-307. 19. Yasutake, W. T.; Wales, J. H. Microscopic anatomy of salmonids: an atlas. Washington, DC, United States Department of the Interior Fish and Wildlife Service Resource Publication 150; 1983. 20. Rowley, A. E; Hunt, T. C.; Page, M.; Mainwaring, G. Fish. In: Rowley, A. E; Ratcliffe, N. A., eds. Vertebrate blood cells. Cambridge: Cambridge University Press; 1988:p. 19-127. 21. Unanue, E. R.; Belier, D. I.; Yu, C. Y.; Allen, P. M. Antigen presentation: comments on its regulation and mechanisms. J. Immunol. 132: 1-5; 1984. 22. Smith, P. D.; Braun-Nesje, R. Cell-mediated immunity in the salmon: lymphocyte and macrophage stimulation, lymphocyte/macrophage interactions, and the production of lymphokine-like factors by stimulated lymphocytes. Dev. Comp. lmmunol. Supplement 2:233-238; 1982. 23. Clem, L. W.; Sizemore, R . C . ; EIIsaesser, C. E; Miller, N. W. Monocytes as accessory cells in fish immune responses. Dev. Comp. Immunol. 9:803-809; 1985. 24. Ellsaesser, C. E; Bly, J. E.; Clem, L. W. Phylogeny of l y m p h o c y t e heterogeneity: the thymus of the channel catfish. Dev. Comp. Immunol. 12:787-799; 1988.
0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc
CELLULAR REQUIREMENTS FOR LYMPHOKINE SECRETION BY RAINBOW TROUT SALMO GAIRDNERI LEUCOCYTES Susan Graham and Christopher J. Secombes Department of Zoology, Universityof Aberdeen, TiLlydroneAve., Aberdeen, AB9 2TN, UK (Submitted March 1989;Accepted May 1989) [~Abstract--The ability of different popula- lacking, due to difficulties encountered tions of rainbow trout blood leucocytesto pro- in thymectomizing young fish, the lack duce MAF following stimulation with Con of inbred strains, and the lack of conveA/PMA was assessed by the amount of NBT nient surface markers. That two lymreduction in target macrophages. The effect of phocyte populations exist in fish has varying lymphocyteor macrophage number on MAF production in the presence of a constant been suggested by studies looking at minumber of macrophages or iymphocytes re- togen responses and the hapten-carrier spectively, showed that in both cases MAF ac- effect (1-4), and indeed in these reactivity initially increased with increasing cell tions two functionally distinct populanumber and then plateaued. Macrophages tions have been separated by adherence alone did not produce MAF whereas some to nylon wool columns (5) or on the MAF activity was produced by macrophage- basis of nonimmune rosetting with heterdepleted lymphocytes, although significantly ologous erythrocytes (6). More conclulower than in the presence of macrophages. sive evidence that the populations preSeparation of leucocytes into sIg- and sIg + sent are similar to T and B cells of higher cells by panning showed that only sIg- lymvertebrates have been forthcoming over phocytes could produce MAF and that macrothe last few years from studies using anphages were necessaryas accessorycells. These results support the contention that fish lym- tibodies to lymphocyte surface deterphocytes can be divided into sIg- T cells and minants to enrich or deplete for these putative lymphocyte populations. sIg+ B cells. Monoclonal antibodies (MoAbs) to []Keywords--Lymphokine secretion; Cellular serum IgM of a range of teleost fish have requirements; Rainbow trout; Panning; slg + been raised that react with only a and sIg- cells; MAF; Macrophage-depletion; subpopulation (30-40%) of blood lymLymphocyte-depletion. phocytes (7-9) suggesting that surface Ig (slg) can be used as a marker for BIntroduction like cells. By selectively enriching for s l g + or s l g - lymphocytes by "panIt is generally agreed that the earliest ning" with these MoAbs the different manifestations of the specific immune populations have been separated for system occur in fish. Their ability to functional studies in vitro. Such studies produce antibody and reject allografts have shown that slg+ lymphocytes redemonstrates the possession of both hu- spond to LPS whereas s l g - lymphomoral and cell-mediated immunity. cytes respond to Con A, although the reHowever, formal proof for the existence sponse to LPS is not completely reof T and B l y m p h o c y t e s in fish is moved (8,10). In the mixed leucocyte response (MLR) only s l g - cells were Address correspondence to Dr. Christopher found to undergo proliferation, but both J. Secombes, Department of Zoology, Uni- slg + and s l g - lymphocytes could act as stimulator cells (11). In addition, versity of Aberdeen, Tillydrone Avenue, Aberdeen, U.K. s l g + lymphocytes were found to be
59
60
n e c e s s a r y for a n t i - h a p t e n plaqueforming cell (PFC) responses to thymusindependent antigens in contrast to PFC responses to thymus-dependent antigens which required both sIg+ and s I g lymphocytes (12). By combining panning with anti-lg MoAbs and specific adherence to hapten or carrier molecules it has been possible to demonstrate that both hapten-responsive slg + and carrier r e s p o n s i v e s l g - l y m p h o c y t e s are needed for the anti-hapten response to a thymus-dependent antigen (12). The requirement for macrophages or monocytes in these responses has also been investigated since they can be removed by adherence to baby hamster kidney cell microexudate-coated plates or by passing cells through a Sephadex G-10 column. Mitogen and MLR responses by s l g - cells were dependent upon the presence of macrophages (10,11), as was the anti-hapten PFC response to thymus-dependent and -independent antigens (12). Only the LPS response by slg + cells has been shown to occur in the absence of macrophages to date (10). Antibodies to determinants on T-like cells have also been used to investigate lymphocyte heterogeneity in fish. For instance, antibrain serum, thought to react with Thy-l-like antigens present on T cells and nervous tissue, can selectively kill PHA responsive cells but not LPS responsive cells (6). More recently a MoAb has been produced that specifically reacts with s I g - channel catfish lymphocytes and has been used to isolate those cells that provided helper activity for antibody synthesis to a thymusdependent antigen (13). Although the thymus-dependency of these populations has still to be ascertained, such data provide compelling evidence for the existence of T and B cells in fish. Further proof for functions such as the production of lymphokine and the generation of cytotoxic cells will strengthen this conclusion. To this end
S. Graham and C. J. Secombes
we have recently optimized the production and detection of a macrophage activating factor (MAF) secreted from rainbow trout (Salmo gairdneri) leucocytes (14). In this article we report on the secretion of MAF by slg + and s l g lymphocytes separated using an antitrout MoAb (I/14), and on the accessory cell requirements.
Materials and Methods
The Production and Detection of MAFContaining Supernatants MAF containing supernatants were produced as described by Graham & Secombes (14). Briefly, leucocyte suspensions were adjusted to 5 x 106 cells per ml in L-15 medium (L-15) containing I% Penicillin/Streptomycin (P/S), 5 x 10 -5 M 2-mercaptoethanol (2-ME), and l ml seeded into 24 well microtitre plates (MTP, Nunc). They were then pulsed with 10 ug/ml Concanavalin A (Con A, Difco) and 5 ng/ml phorbol myristate acetate (PMA, Sigma), previously shown to be optimal for MAF production (14), for 3h at 18°C. After this time the cells were gently washed five times with sterile phosphate buffered saline (PBS) to remove any residual Con A and PMA, and then cultured in L-15 containing 1% P/S, 10% foetal calf serum (FCS) and 5 x 10 -5 M 2-ME for 48h. The supernarants were then harvested and stored at -20°C until use. MAF activity was detected using purified head kidney (HK) macrophages as targets. These cells were isolated using Percoll density gradients followed by adherence to 96 well MTP as previously described (15,16). Following a 48h incubation with the MAF containing supernatants the respiratory burst activity of triplicate wells of macrophages was assessed by their ability to reduce nitroblue tetrazolium (NBT). The macrophage monolayers were covered with
Lymphokine secretion by trout
100 ~1 of 1 mg/ml NBT containing 1 ixg/ml PMA, to trigger the respiratory burst, for 30 min before being fixed in methanol. After washing in 70% methanol the reduced formazan within the macrophages was solubilized with KOH/ DMSO (17) and the optical density determined at 620 nm in a multiscan spectrophotometer. All results were exp r e s s e d per l0 s m a c r o p h a g e s after counting nuclei released with 0.1M citric acid, Tween 20 and 0.05% crystal violet in control wells. In some cases the results were expressed as a stimulation index by dividing the mean optical density reading from macrophages incubated with active supernatants with that from macrophages incubated with control supernatants obtained from leucocytes not stimulated with Con A/PMA.
61
proximately 1 ml of the cell suspension was put onto the column containing 6 ml sterile Sephadex G-10. The cells were allowed to penetrate the column before addition of 10 ml of medium to elute the nonadherent cells. After centrifugation the cells were resuspended in fresh medium and cultured overnight in a 25 cm z flask to remove any residual macrophages that did not a d h e r e to the column. The nonadherent lymphocytes were then adjusted to 1.6 x 105 to 107 cells per ml, and were added back to the constant number of macrophages previously set up. MAF-containing supernatants were then produced as described above.
Effect of Macrophage Number on MAF Production Effect of Lymphocyte Number on MAF Production The effect of varying lymphocyte numbers on the production of MAF was studied by isolating a constant number of macrophages and adding to them varying numbers of leucocytes extensively depleted of macrophages (as assessed by a lack of adherent cells), prior to addition of Con A/PMA. Blood leucocytes (I ml) obtained after centrifugation over a 51% Percoll density gradient, were seeded into 24 well MTP at 1 × 107 cells per ml L-15 containing 0.1% FCS and 1% P/S. After 2h at 18°C for adherence, the nonadherent cells were removed and the adherent cells were covered with 1 ml L-15, 5% FCS, 1% P/S. The nonadherent cells were then centrifuged at 400g for 10 min and the pellet of cells resuspended to 2 × 107 cells per ml in L-15, 10% FCS, 1% P/S. The lymphocytes were then extensively depleted of macrophages by passage through a Sephadex G-10 column prepared as described by Mishell and Mishell (18). Ap-
The effect of varying macrophage number on the production of MAF was determined by isolating different concentrations of macrophages and adding to them a constant number of leucocytes which had been extensively depleted of macrophages, prior to the addition of Con A/PMA. Blood leucocytes were seeded into 24 well MTP in two-fold dilutions from 1.96 x 105 to 5 x 107 cells per ml L-15, 0.1% FCS, 1% P/S to obtain varying macrophage numbers. Duplicate wells of each cell concentration were used to enable macrophage nuclei counts to be performed. The cells were allowed to adhere for 2h at 18°C after which the nonadherent cells were collected and the adherent macrophages were supplemented with L-15, 5% FCS, 1% P/S and placed at 18°C until use. The nonadherent ceils were then depleted extensively of macrophages as above and 5 x 10 6 viable iymphocytes were added back to each well of macrophages previously set up. MAF-containing supernatants were then produced as described earlier.
62
Separation of slg + and slg Lymphocytes by Panning Petri dishes (I0 cm, Nunc) were coated overnight at 4°C with 8 ml of control ascitic fluid, obtained using a clone against an unrelated antigen, or monoclonal anti-trout Ig (1/14) ascitic fluid diluted 1:200 in PBS. The plates were then washed twice with PBS and 8 ml L-15, 10% FCS was added for lh at 18°C before addition of blood leucocytes. The leucocytes were initially panned for lh on control ascitic fluid coated plates at 2 × 107 cells per dish, to remove adherent s l g - leucocytes, and subsequently were panned twice on 1-14 coated plates. The n o n a d h e r e n t cells obtained after the second 1-14 panning were termed s l g lymphocytes. After removal of the nonad h er en t cells from the 1-14 coated plates an intermediate wash was carried out before removal of the adherent slg + cells by pipetting medium onto the plate. The two batches of slg+ lymphocytes were pooled before use and both the slg+ and the s l g - lymphocytes were then cultured overnight in 25 cm 2 flasks. Finally the nonadherent cells from these flasks were added to isolated macrophages from the same fish (see below) and used to generate MAF-containing supernatants as above. In some experiments s l g - cells were stimulated with Con A/PMA in the absence of macrophages. While the panning was occurring blood leucocytes from the same fish were added to a 24 well MTP at 5 × 106 cells per well, to serve as a total leucocyte control and as a source of macr o p h a g e s for " a d d - b a c k " and macrophage-only controls. For this latter purpose the nonadherent cells w e r e removed after lh at 18°C and the adherent cells supplemented with L-15, 10% FCS until use.
Flow Cytofluorometric Analysis To confirm that the separation of lymphocytes into sIg+ and s I g - cells had
S. Graham and C. J. Secombes
been successful on each occasion that this experiment was performed, a subsample of the cells was analyzed by flow cytofluorometry. 5 × 105 to 10 6 cells per ml were incubated with 1/14 supernatant containing 0.1% sodium azide, 1% bovine serum albumin (BSA). After lh at 4°C the cells were washed three times by centrifugation and incubated for a further lh with sheep anti-mouse IgG/FITC diluted 1:40 in PBS, 0.1% sodium azide, 1% BSA. Finally the cells were washed three additional times in PBS, 0.1% sodium azide and were then analyzed in an EPICS-C flow cytometer (Coulter Electronics) with the argon laser tuned to 488 nm and PMT voltage at 1600 V. Graphs of log fluorescence intensity versus cell number were plotted for 10,000 cells. Results were analyzed using Student's two-tailed t-test.
Results
Effect of Lymphocyte Number on MAF Production The effect of varying l y m p h o c y t e number on MAF production, in the presence of a constant number of macrophages (3.2 × 105), is seen in Fig. la. In the absence of lymphocytes no MAF was produced by macrophages, as determined by the amount of NBT reduced by macrophages incubated with these supernatants in comparison with supernatants from cultures not stimulated with Con A/PMA (O.D. of 0.15). Low numbers of lymphocytes, 1.6-3.1 × 105 , also did not produce significant levels of MAE With larger numbers of lymphocytes significant MAF activity was seen, and i n c r e a s e d with cell number up to a maximum at 5 × 10 6 cells. The MAF activity appeared to plateau using more than 106 lymphocytes in that there were no significant differences in the amount of N BT reduction between these cell concentrations, but it was significantly higher in each case
Lymphokine secretion by trout 0.22
(e)
63
(b)
0.21
0.20
S ~
0.19
Q
o ~
o.18.
,D
0.17-
e
i,m
z
0.16-
0.15.
0.14"
0.13 0.1
1.0 Cell number (x 1 0 - e )
10
2"0
Cell number (x 10 - s )
Figure 1. The effect of varying lymphocyte (a) or macrophage (b) number on MAF production in the presence of a constant number of macrophages (3.2 × 10s) or lymphocytes (5 × 106), respectively. MAF activity was assessed by the amount of NBT reduction in target macrophages following incubation with the MAF-containing supernatants diluted 1:8. The results were expressed as means _+ standard errors of triplicate wells adjusted to 10s cells.
compared with the NBT reduction using supernatants produced with low lymphocyte numbers.
Effect of Macrophage Number on MAF Production
The effect of varying macrophage number on MAF production, in the presence of a constant number of lymphocytes (5 x 106), is seen in Fig. lb. In the apparent absence of macrophages and at every concentration of macrophages used for MAF production significantly increased NBT reduction was seen in comparison with supernatants from cultures not stimulated with Con A/PMA (O.D. of 0.10). As with lymphocyte number increasing MAF activity was seen with increasing numbers of macrophages, with the highest mean NBT reduction occurring with the largest macrophage number used (2.2 x 106). However, analysis of this data suggested that
a plateau had been reached at a macrophage number of approximately 3.6 × 105 cells, since no significant differences were seen between the points above this cell number (with the exception of 5.8 × 105 vs. 1.6 × 106 macrophages). Thus macrophage numbers below 105 appear to be suboptimal for MAF production.
MAF Production Using slg + and slg Lymphocytes
EPICS analysis of the adherent and nonadherent lymphocyte populations following panning with 1/14 M o A b showed that these populations were indeed slg + and s l g - , respectively (Fig. 2). Typically 40% of the blood leucocytes were slg+ before the separation, whereas after panning 70-97% of the adherent cells were slg + and 85-99% of the nonadherent cells were s l g - . Early experiments found that MAF activity could be detected in supernatants from
64
S. Graham and C. J. Secombes
6
tion indices of approximately 1.0). Supernatants from s l g - lymphocytes in the presence of macrophages also had significant MAF activity, as great or greater than the same number of unfractionated leucocytes, but those from s l g + lymphocytes in the presence of macrophages lacked MAF activity. In some experiments s l g - lymphocytes were incubated in the presence and absence of macrophages to see if they required accessory cells for the production of MAE In the presence of macrophages NBT reduction was increased significantly above control values (OD of 0.1) between dilutions of 1:8 to 1:128 (Fig. 4). In the absence of macrophages MAF activity was abolished almost completely and in this example NBT reduction was only increased significantly above control values at a dilution of 1:32. Comparison of NBT reduction in macrophages incubated with these supernatants with macrophages incubated with supernatants derived from s l g - cells in the presence of macrophages showed the former to reduce significantly decreased amounts of NBT.
b
I
IC .i
Figure 2. Flow cytofluorometric analysis of unfractionated leucocytes (a), and adherent (b) or nonadherent (c) cells following panning with monoclonal anti-trout Ig. The percentage of cells calculated as slg+ in each case was (a) 39.24%, (b) 96.03%, and (c) 0.84%. x axis, log fluorescence intensity, y axis, cell number.
both slg + and s l g - lymphocytes in the presence of macrophages, and that the purity of the slg+ cells was critical for the presence or absence of MAF activity when using these cells. In general, only when the purity of the slg+ cells exceeded 95% was MAF activity lost from the supernatants. Examples of such results obtained from two representative fish are shown in Fig. 3. In both cases, as above, supernatants from unfractionated leucocytes contained significant MAF activity (i.e., stimulation indices above 1.0), whereas those from macrophage-only controls had none (stimula-
Discussion The results of this study, looking at the cellular requirements for MAF production in rainbow trout, support the contention that fish lymphocytes can be divided into s l g - T cells and slg+ B cells. Only purified s l g - lymphocytes were able to secrete MAF following stimulation with Con A/PMA, providing accessory cells were present. Thus lymphokine secretion can be added to the functions of helper activity for thymusdependent antibody responses and responsiveness to Con A and allogeneic leucocytes already ascribed to these cells in this and other teleost species (8,10-13).
Lymphokine secretion by trout
2.5"
65
Fish 2
Fish 1
2.0"
i!
1.5"
-$ W 1.0'
0.5'
MAF dilution ( - l o g 2)
Figure 3. MAF production by unfractionated blood leucocytes (0), macrophages (O), s l g - lymphocytes plus macrophages (A) and slg + lymphocytes plus macrophages (A). MAF activity was assessed by the amount of NBT reduction in target macrophages following incubation with the MAF-containing supernatants The results were expressed as a stimulation index by dividing the mean optical density reading from macrophages incubated with test supernatants with that from macrophages incubated with control supernatants obtained from leucocytes not stimulated with Con A/PMA.
To demonstrate whether this teleost MAF was truly a lymphokine secreted from lymphocytes, experiments were carried out using varying numbers of macrophage-depleted leucocytes added to a constant number of macrophages and vice versa. Since salmonid blood leucocytes normally contain < 10% granulocytes (19), after macrophage-depletion predominantly lymphocytes and some thrombocytes will remain, and the major role of the latter is in blood coagulation and general hemostasis (20). In both experimental approaches MAF activity initially increased with increasing numbers of macrophages or lymphocytes and then appeared to plateau. Perhaps more significantly MAF activity was completely absent from macrophage cultures without lymphocytes, whereas in the presence of optimal lymphocyte
numbers even in the apparent absence of macrophages significant MAF activity was present. In addition the panning experiments showed that in the presence of optimal numbers of macrophages MAF activity was only found using s l g - lymphocytes and not slg+ lymphocytes. Thus s l g - lymphocytes and not macrophages or slg+ lymphocytes would appear to be the responding cells. It is interesting to note that at high concentrations of these lymphokinecontaining supernatants a suppressive effect upon NBT reduction was sometimes seen, as shown in Figs. 3 and 4. This could be due to a multiplicity of factors being present in the supernatant, some of which were suppressive. However, this effect could still be seen following partial purification of the MAF activity by HPLC (unpublished), and so
66
S. Graham and C. J. Secombes
0.28
T
0.24 A
E c
0.20
O ~N (O
O
0.16
c
O o
0.12
"O L.
0.08 Z
0.04'
0.00
!
!
i
2
3
4
i
|
•
5
6
7
MAF dilution (-log 2) Figure 4. The effect of macrophage depletion on MAF production by s l g - lymphocytes. MAF activity was assessed by the amount of NBT reduction in target macrophages following incubation with supernatants from s l g - lymphocytes (G) or s l g - lymphocytes plus macrophages (A). The results were expressed as means _+ standard errors of triplicate wells adjusted to 10s cells.
the possibility that high levels of MAF actually inhibit the target cells also exists. The dependence upon accessory cells for the optimal production of MAF was also shown in this study. Thus with decreasing numbers of macrophages MAF production, as measured by NBT reduction, also decreased. The role of macrophages as accessory cells in immune responses of higher vertebrates is well established (21), and their importance in certain immune responses of fish is already known. In studies on the proliferative responses to Con A and allogeneic leucocytes (MLR) and in vitro antihapten PFC responses, monocytes/macrophages or factors released by them have b e e n s h o w n to be n e c e s s a r y (10-12,22,23). The production of at least
one lymphokine (MAF) by s l g - lymphocytes in fish can now also be included as accessory-cell-dependent. Interestingly, some MAF activity was detected in supernatants produced by stimulating lymphocytes in the absence of macrophages. This is similar to the results obtained with mitogenesis assays by Sizemore et al. (10) using the Sephadex G-10 macrophage depletion method, where a mitogenic response to both LPS and Con A of 65% and 10% of the maximum response respectively persisted after macrophage depletion (the former being considered accessory-cellindependent). Although the Sephadex method is quick and convenient for the removal of most macrophages, some cells which develop functions similar to macrophages have been reported in the
Lymphokine secretion by trout
67
filtered population (18). Thus it is possible that some macrophages were not removed using the depletion technique, and provided a limited accessory cell function. Alternatively, the presence of PMA in the present study may have partly overridden the need for accessory cells since another phorbol ester TPA has been shown to overcome the accessory cell requirement for Con A stimulation of thymocytes in channel catfish (24). Whatever the explanation, intact macrophages clearly provide most effective accessory signals. The plateauing of NBT reduction of target macrophages using more than 10 6 lymphocytes is in contrast to the studies of the mitogenic response of channel catfish lymphocytes to Con A and LPS where proliferation increased linearly with increasing lymphocyte number up to 5 x 105 (10) and was used as evidence that lymphocytes were the mitogen-responsive cells in this system. Had the study on mitogen responses used larger numbers of lymphocytes it is possible that a plateauing of the response would also have been seen. Alternatively, since lymphocyte proliferation in response to mitogens is a direct measurement, whereas in this study the level of lymphokine released is measured indirectly by its effect on target macrophages, the plateauing effect could be due to the
target cells becoming saturated and not being able to respond further. The panning experiments coupled with flow cytofluorometric analysis showed that it was crucial to have highly purified cell types in order to see any differences in MAF production. Contamination of the s l g + lymphocytes with >5% s l g - lymphocytes was often sufficient to detect MAF activity in the supernatants. Assuming s l g - lymphocytes are the responding cells from the a b o v e arguments, then clearly only small numbers are required in order to release enough MAF to stimulate large numbers of target macrophages. If this is the case then the plateauing effect of NBT reduction by target macrophages w o u l d not be surprising since the number of lymphocytes able to secrete sufficient MAF to stimulate all of the fixed number of target cells would soon be reached. Further studies are in progress to characterize this teleost lymphokine, to see if it is equivalent to known mammalian MAFs such as ~/-interferon. Acknowledgements--This w o r k was supported by S E R C grant No. GR/E/15413. The authors gratefully acknowledge the gift of the 1/14 h y b r i d o m a cells from P r o f e s s o r G. W. Wart, University of South Carolina. Thanks also go to Miss K. E. H e n d e r s o n and Mr. J. Milton for the E P I C S analysis.
References 1. Stolen, J. S.; Makela, O. Cartier preimmunization in the anti-hapten response of a marine fish. Nature. 254:718-719; 1975. 2. Yocum, D.; Cuchens, M.; Clem, L. W. The hapten-carrier effect in teleost fish. J. Immunol. 114:925-927; 1975. 3. Etlinger, H. M.; Hodgins, H. O.; Chiller, J. M. Evolution of the lymphoid system. I. Evidence for lymphocyte heterogeneity in rainbow trout revealed by the organ distribution of mitogenic responses. J. Immunol. 116:1547-1553; 1976. 4. Chilmonczyk, S. In vitro stimulation by mitogens of peripheral blood lymphocytes from rainbow trout (Salmo gairdneri). Ann. D'lmmunol. 129C:3-12; 1978.
5. Ruben, L. N.; Warr, G. W.; Decker, J. M.; Marchalonis, J. J. Phylogenetic origins of immune recognition: lymphoid heterogeneity and the hapten/carrier effect in the goldfish, Carassius auratus. Cell. Immunol. 31:266-283; 1977. 6. Cuchens, M. A.; Clem, L. W. Phylogeny of lymphocyte heterogeneity. II. Differential effects of temperature on fish T-like and B-like cells. Cell. Immunol. 34:219-230; 1977. 7. Lobb, C. J.; Clem, L. W. Fish lymphocytes differ in the expression of surface immunoglobulin. Dev. Comp. Immunol. 6:473-479; 1982. 8. Deluca, D.; Wilson, M.; Wart, G. W. Lymphocyte heterogeneity in the trout, Salmo gaird-
68
9.
10.
11.
12.
13.
14.
15. 16.
S. Graham and C. J. Secombes
neri, defined with monoclonal antibodies to lgM. Eur. J. Immunol. 13:546-551; 1983. Secombes, C. J.; van Groningen, J. J. M.; Eghefts, E. Separation of lymphocyte subpopulations in carp Cyprinus carpio L. by monoclonal antibodies: i m m u n o h i s t o c h e m i c a l studies. Immunology. 48:165-175; 1983. Sizemore, R. C.; Miller, N. W.; Cuchens, M. A.; Lobb, C. J.; Clem, L. W. Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro mitogenic responses of channel catfish leukocytes. J. Immunol. 133: 2920-2924; 1984. Miller, N. W.; Deuter, A.; Clem, L. W. Phylogeny of lymphocyte heterogeneity: the cellular requirements for the mixed leucocyte reaction with channel catfish. Immunology. 59: 123-128, 1986. Miller, N. W.; Sizemore, R. C.; Clem, L. W. Phylogeny of lymphocyte heterogeneity: the cellular requirements for in vitro antibody response of channel catfish leukocytes. J. lmmunol. 134:2884-2888; 1985. Miller, N. W.; Bly, J. E.; van Ginkel, F.; Ellsaesser, C. E; Clem, L. W. Phylogeny of lymphocyte heterogeneity: identification and separation of functionally distinct subpopulations of channel catfish lymphocytes with monoclonal antibodies. Dev. Comp. Immunol. 11: 739-747; 1987. Graham, S.; Secombes, C. J. The production of a macrophage-activating factor from rainbow trout Salmo gairdneri leucocytes. Immunology. 65:293-297; 1988. Chung, S.; Secombes, C. J. Activation of rainbow trout macrophages. J. Fish. Biol. 31A:51-56; 1987. Chung, S.; Secombes, C. J. Analysis of events occurring within teleost macrophages during the respiratory burst. Comp. Biochem. Physiol. 89B:539-544; 1988.
17. Rook, G. A.; Steele, J.; Umar, S.; Dockrell, H. M. A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by 3,-interferon. J. lmmunol. Methods. 82:161167; 1985. 18. Mishell, R. I.; Mishell, B. B. Use of Sephadex G-10 to separate macrophages and lymphocytes. In: di Sabato, G.; Langone, J. J.; van Vunakis, H., eds. lmmunochemical techniques. Part G. Separation and characterization of lymphoid cells. London: Academic Press; 1984:p. 303-307. 19. Yasutake, W. T.; Wales, J. H. Microscopic anatomy of salmonids: an atlas. Washington, DC, United States Department of the Interior Fish and Wildlife Service Resource Publication 150; 1983. 20. Rowley, A. E; Hunt, T. C.; Page, M.; Mainwaring, G. Fish. In: Rowley, A. E; Ratcliffe, N. A., eds. Vertebrate blood cells. Cambridge: Cambridge University Press; 1988:p. 19-127. 21. Unanue, E. R.; Belier, D. I.; Yu, C. Y.; Allen, P. M. Antigen presentation: comments on its regulation and mechanisms. J. Immunol. 132: 1-5; 1984. 22. Smith, P. D.; Braun-Nesje, R. Cell-mediated immunity in the salmon: lymphocyte and macrophage stimulation, lymphocyte/macrophage interactions, and the production of lymphokine-like factors by stimulated lymphocytes. Dev. Comp. lmmunol. Supplement 2:233-238; 1982. 23. Clem, L. W.; Sizemore, R . C . ; EIIsaesser, C. E; Miller, N. W. Monocytes as accessory cells in fish immune responses. Dev. Comp. Immunol. 9:803-809; 1985. 24. Ellsaesser, C. E; Bly, J. E.; Clem, L. W. Phylogeny of l y m p h o c y t e heterogeneity: the thymus of the channel catfish. Dev. Comp. Immunol. 12:787-799; 1988.
