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Relationship between migration inhibition and plaque-forming cell responses to sheep erythrocytes in the teleost, Tilapiamossambica
DEVELOPMENTAL
AND
COMPARATIVE
01~5-305X/79/010067-09502.00/0 Copyright
(c) 1979 P e r g a m o n
IMMUNOLOGY, Vol.
Printed P r e s s Ltd.
3, PP. 6 7 - 7 5 , in the U S A . All rights reserved.
1979.
RELATIONSHIP BETWEEN MIGRATION INHIBITION AND PLAQUE-FORMING CELL RESPONSES TO SHEEP ERYTHROCYTES IN THE TELEOST, TILAPIAMOSSAMBICA
S. Jayaraman @, R. Mohan** and VR. Muthukkaruppan Department of Immunology School of Biological Sciences Madurai University Madurai-625021, India
ABSTRACT Cell-mediated immunity to sheep erythrocytes (SRBC) in the teleost, Tilapia mossambica was assessed using the in vitro capillary migration inhibition (MI) assay and the humoral immune response by the plaque-forming cell (PFC) assay. Both PFC and MI responses were found to be inversely proportional to the amount of antigen injected. Cyclophosphamide administration immediately after immunization completely abrogated both PFC and MI responses. In contrast, injection of formalinized SRBC resulted in greater elevation of MI levels accompanied by the absence of PFC production. The possible regulatory role of antibody-producing cells in the MI response to SRBC is discussed.
INTRODUCTION The migration inhibition (MI) technique is a valid in vitro test in m s m ~ i s for measuring cell-mediated immunity ((241) to various antigens including sheep erythrocytes (SRBC) (1,2,3).
Recently, we have described the
This work was supported in part by a research grant from the University Grants Commission, India. @Recipient of Senior Research Fellowship under the above scheme. Supported by Junior Research Fellowship awarded by University Grants Commission. Correspondence:
VR. Muthukkaruppan, School of Biological Sciences, Madurai University, Madurai - 625 021, India. 67
68
TELEOST CELLULAR IMMUNITY
Vol.3, No. I
utility of this technique for assessing CMI in reptiles to skin allografts (4), and SRBC (5).
Similarly, in amphibians, MI assays have been used to
study CMI to PPD (6).
Earlier we showed in the lizard, Calotes versicolor
that low doses of SRBC induced high levels of MI, but high doses, induced low levels (5,7).
The present report describes CMI in a teleost fish,
Tilapia mossambica to SRBC and its relationship to the development of plaque-forming cells (PFC) under varying conditions. METHODS Fish Adult fish of both sexes weighing 50 to I00 g were collected mld maintained at 30°C in glass aquaria as described earlier (8). _Antigen and immunization Sheep blood was drawn in Alsever's solution from the jugular vein of a single sheep, washed three times with phosphate-buffered saline (PBS, pH 7.2) and appropriate dilutions were made shortly before use.
Rat red blood cells
(RRBC) were obtained by cardiac puncture from white rats. Formalinized SRBC (F-SRBC) were prepared by adding an 8% suspension of erythrocytes in PBS to an equal volume of 3% formaldehyde, followed by incubation for 18 hr at 37°C with constant and gentle agitation (9). Treated erythrocytes were washed and tested for antigenicity as described by Dennert and Tucker (9). Fishes were usually immunized by injecting them with appropriate amounts of antigen in PBS via the c o ~ o n
cardinal vein (8).
Dru 8 treatment Cyclophosphamide (CY) (kindly donated by Asta-Werks AG, Brackwede, Germany) was dissolved in physiological saline and injected intraperitoneally at a dose of 300 mg/kg body weight (i0). Plaque-formin~ cell assay Direct haemolysin producing cells were detected using the method described previously for Tilapia (8). for 1 hr.
The plaques were developed at 37°C
The viability of white blood cells (WBC) of the head-kidney was
judged by the ability to exclude 0.25% trypan blue dye. Serology Haemagglutinin (HA) antibodies against SRBC were detected in the ~mmunized sera using the method previously described (8).
Vol. 3, No. 1
TELEOST CELLULAR IMMUNITY
69
Migration inhibition assay The MI assay adapted for lizards modifications head-kidney
for the fish system.
Briefly,
cells was made in PBS.
L-15 Leibovitz medium
(4,5) was followed with suitable a single cell suspension of
The cells were washed twice with diluted
(3 parts medium and i part triple distilled water),
supplemented with 20% normal Tilapia serum (decomplemented min).
The capillary
Morganville,
at 56°C for 30
tubes (0.8 x 90 mm, Laboratory Diagnostics,
N.J. 07751, USA) were filled with head-kidney
at one end with modeling clay.
After centrifuging
Co., Inc.,
cells and sealed
the capillaries at 500 rpm
for 2 min, a nick was made with a diamond pencil just below the cell-fluid interface.
By applying gentle pressure,
the cell pellet was separated and
secured to the bottom of the migration chambers grease.
(4) with a dab of silicone
After sealing the migration chambers, medium was introduced with a
gentle force, using a 2 ml syringe fitted with a 20 gauge needle. Antigen consisted of 0.01 ml of 10% sonicated For specificity
experiments,
SRBC (5) per ml of medium.
intact erythrocytes were added directly to the
culture medium at a concentration of 0.0i ml of 1% erythrocytes medium
per ml of
(4,5).
The migration cultures were incubated at 37°C for 24 hr and the extent of migration drawn with a camera lucida and measured by planimetry
(4).
The
%MI was calculated using the formula: Migration area with antigen % MI = i00 - i00 x Migration area without antigen Statistics Statistical assessment
of the data was computed using the Student's
t-test. RESULTS Culture conditions Tests of antigen-containing dur~Ig
the
culture medium revealed no change in pH
period of incubation.
More than 80% of cells were viable as
determined by the trypan blue dye exclusion test, irrespective
of the
presence or absence of antigen in the medium during the incubation period. Dose-Dependency Different head-kidney
of Immune Responses dilutions of SRBC were given to fishes and five days later,
cells were assayed for PFC and MI responses.
Data in Fig. i
show that low antigenic stimuli induced fairly high levels of MI with an
70
TELEOST CELLULAR IMMUNITY
insignificant number of PFC.
Vol. 3, No. 1
As we increased the dose of SRBC, the PFC
number increased logarithmically.
S75
50
,,
-
4O
(~ 225
50
14.
20
\
150
Q. ,
@ !
I I
75 0
c
5
4
5
6
7
8
I I0
9
0
S RBC DOSE LOGIo
FIGURE 1 Dose dependency of PFC and MI responses to SRBC. Five days after immunization, assays were performed with head-kldney lymphocytes (WBC). Each point represents the mean of 5-7 animals (±S.E.). C denotes control values.
This increase in PFC production however, was accompanied by a suppression of the MI level, especially to a dose of 2.4 x 109 SRBC.
Thus, the present
experiment reveals an inverse relationship between PFC and MI responses in fishes. Modulation of Immune Responses We attempted to modulate the ~mmune responses to SRBC using two protocols:
one utilizing the alkylating agent, CY, and the other, F-SRBC.
Sallne-injected fishes did not produce either HA antibodies or PFC to SRBC (Table l); the MI level was also minimal.
However, when 2.4 x 109 SRBC were
given to fishes, appreciable HA titre and PFC numbers were detected with a low but significant level of MI (0.025 > P < 0.05).
If the same amount of
antigen was followed by an injection of 300 mg CY, apparently no antibodies and PFC were produced.
Besides, the M I response was also significantly
reduced (0.025 > P < 0.05) as a consequence of drug treatment.
In contrast,
the injection of 2.4 x 109 F-SRBC greatly enhanced the level of MI with a concomitant decrease in HA tltre and PFC number.
Vol. 3, No. 1
TELEOST CELLULAR IMMUNITY
71
TABLE i Modulation of T--,une Responses
Treatment
HA titre b (Mean ± S.E)
PFC/IO 6 WBC (Mean'± S.E)
1:0
0
(10) c
(17)
1:60 ± 15
320 ± 31
(8)
(7)
i:0
25 ± 15
Saline
SRBC
cyd SRBC +
(9) F_SRBC e
1:7
± 2
(7)
a
% MI (Mean ± S.E)
5.6 ±
2.0
(17) 14.3 ±
4.9
(7) 1.6 ±
(12)
0.8
(6)
9 ± 3
54.8 ± 11.7
(7)
(7)
apFC and MI assays were performed with head-kidney cells 5 days after treatment. bsera bled from fish were titrated against SRBC, ii days after immunization. CNumber of fish investigated is given in parentheses. d300 mg CY/kg was given 3 hr after immunization with 2.4 x 109 SRBC. e2.4 x 109 formalinized SRBC were injected intraperitoneally.
Specificity of Immune Responses The specificity of both PFC and Ml responses was determined.
To test
the specificity of PFC responses, head-kldney cells from fishes that had been injected with 2.4 x 109 SRBC were assayed.
For the MI assay~ cells were
harvested from fish that had been injected with 109 SRBC.
Data in Table 2
show that the SRBC-immun~zed head-kidney cells did not produce anti-RRBC plaques, showing the specificity of the phenomenon.
Likewise, the SRBC-
sensitized head-kidney cells were not appreciably inhibited (0.025 > P < 0.05) in the presence of intact RRBC in the medium.
72
TELEOST CELLULAR IMMUNITY
Vol. 3, No. 1
TABLE 2 a
Specificity of Immune Responses
Immunizing antigen
Test antigen
PFC/I06 WBCb (Mean ± S.E)
% Mlc (Mean ± S.E)
Saline
SRBC
0 (17)
5.6 ± 2.0 (17)
SRBC
SRBC
335 ± 86 (5) d
SRBC
RRBC
1.2
28.0 ± 13.5 (4)
± 0.8 (5)
7.8
± 4.0 (4)
aFive days after immunization assays were performed with head-kidney cells. bpFC assay was performed with head-kidney cells taken from fish immunized with 2.4 x 109 SRBC, in the presence of either SRBC or RRBC. ~ I assay was performed with head-kidney cells harvested from fish immunized with 109 SRBC, in the presence of either intact SRBC or RRBC (4,5). dNumbers in parentheses denote the number of fish used.
DISCUSSION In the present report, CMI to SRBC was assessed in a teleost fish, T i l a p i a m o s s a m b i c a using the MI technique.
Drossler and Ambrosius have also
used the MI technique to detect CMI to PPD in Bufo bufo (6) and we demonstrated its utility for assessing CMI to skin allografts and to SRBC in the lizard, Calotes versicolor (4,5).
The MI of head-kidney cells from
sensitized fish is a n in vitro expression of CMI to SRBC and not a nonspecific effect caused by culture conditions. the following observations:
This conclusion is based on
(i) the L-15 Leibovitz medium has already been
shown to be optimal for i_.nvitro assays in fishes (ii); (ii) no alteration in the pH of the medium with or without antigen after 24 hr of incubation at 37°C was observed;
(iii) the degree of viability was the same in both
cultures with or without antigen and (iv) the MI obtained with unsensitized head-kidney cells was negligible in the presence of antigen (Table i).
The
Vol. 3, No. 1
TELEOST CELLULAR IMMUNITY
73
possible inhibitory action of antibodies and antigen--antibody complexes can be ruled out since MI could be demonstrated with head-kidney cells obtained from fish that produced little or no PFC and HA antibodies after immunization with low doses of SRBC (Fig. i) or F-SRBC (Table i).
Besides,
the MI assay was used to evaluate CMI to scale allografts in fish (12). Hence, it is suggested that MI of SRBC-sensitized head-kidney cells is an in vitro correlate of CMI to SRBC in Tilapia mossambica. Like PFC formation, MI is also an antigen-specific event in fishes (Table 2).
In previous reports, the specificity of MI responses of lizards
was confirmed by suspending intact erythrocytes in the culture medium and allowing spleen cells to migrate from the capillary tubes (4,5).
Following
the same protocol, we found that the MI of sensitized head-kidney cells of Tilapia mossambica is a highly antigen-specific phenomenon as in other fish (12) and animal systems (1,4,5). Based on these observations, the relationship between MI and PFC responses was analyzed.
The dose-dependency profile of MI and PFC responses
denotes the existence of a mirror-image relationship between them when tested 5 days after immunization (Fig. i).
Humoral immune (HI) response
was low to a low dose of SRBC and high to a high dose of SRBC throughout a period of 21 days in Tilapia (i0).
Such an inverse relationship between
PFC and MI responses to SRBC has already been reported in the lizard, Calotes versicolor (5,7).
In mice, delayed-type hypersensitivity
(DTH) and
the HI response to SRBC were reported to be inversely proportional to the dose of antigen (13-15).
It seems highly likely therefore, that the inverse
relationship between MI and the HI response to SRBC is a phenomenon traceable through phylogeny. According to one interpretation, both functional thymus-derived (T-) and bone-marrow-bursa-equivalent
(B-) cells co-exist as distinct entities
and take part in the HI response of fishes (16,17).
In Tilapia mossambica,
the existence of functional T- and B-cells has also been demonstrated recently (in preparation).
Whereas long-term (2-3 months) adult thymectomy of fishes
greatly abrogated both the primary and secondary HI response to SRBC, the anti-polyvinyl pyrrolidone response was not affected, indicating the existence of both a T-dependent and T-independent immune system in Tilapia. Besides, MI had been equated as the T-cell response in fishes (12).
To
understand the probable modulation of T-cell function (MI) by B-cells, F-SRBC and CY were used.
Formalinized erythrocytes have been shown to
induce T-helper cells specifically without the generation of PFC in mice
74
TELEOST CELLULAR IMMUNITY
(9) and lizards (5) as well as in Tilapia (i0).
Vol. 3, No. 1
Injection of 2.4 x 109
SRBC resulted in the production of large numbers of PFC with a minimal level of MI (Fig. l: Table I). an enhanced MI response (Table i).
with
However, the same amount of F-SRBC evoked greatly decreased HA and PFC responses
That the enhanced CMI was accompanied by a low B-cell response
suggests that in fishes, the induction of functional T- and B-cells is causatively related.
It is probable that the activation of B-cells by
large doses of native SRBC might lead to the formation of antigen-antibody complexes that could inhibit the activity of functlolml T-cells (MI) as in mice (13). The data with CY treatment seem to contradict this hypothesis. abolished both CMI and the HI response to SRBC (Table i).
CY
In addition to
abolishing functional B-cell activity CY administration causes prolonged survival of scale allografts and damages T-helper activity in Tilapia mossambica (10).
Hence, CY administration probably does not result in
enhanced MI levels.
We suggest, therefore, that functional B-cells may exert
a regulatory role on the functional T-cell response (MI) in fishes, but this conclusion awaits detailed studies of immune responses in fishes.
ACKNOWLEDGEMENTS S. Jayaraman expresses thanks to Dr. E.L. Cooper for critically reading the manuscript and to Dr. P.W. Askenase for the generous gift of capillary tubes.
REFERENCES i.
BLOOM B.R. In vitro approaches to the mechanism of cell-mediated immune reactions. Advan. Immunol. 13, 10l, 1971.
2.
PICK, E., KREJCI, J. and TURK, J.L. Interaction between sensitized lymphocytes and antigen in vitro. III. Release of soluble mediators by particulate antigen. Immunol. 22, 25, 1972.
3.
GORDON, J. and YU. H. Relationship of T cells involved in cell-mediated immunity and antibody synthesis. Nature New Biol. 244, 21, 1973.
.
JAYARAMAIq, S. and MUTHUKI~%RUPPAN, VR. In vitro correlate of transplantation immunity: spleen cell migration inhibition in the lizard, Calotes versicolor. Dev. Comp. Immunol. i, 133, 1977.
.
JAYARAMAN, S. and MUTHUKKARUPPAN, VR. Detection of cell-medlated immunity to sheep erythrocytes by the capillary migration inhibition technique in the lizard, Calotes versicolor. Immunol., IN PRESS, 1977.
Vol. 3, No. 1
TELEOST CELLULAR IMMUNITY
75
.
DROSSLER, K. and AMBROSIUS, H. Speziflsche Zellvermittlte Immunitat bai Froschlurchen. II. Verzogeste Ubesempsindllchkeit belt der Erdkrote (Bufo bufo i.) gagen BCG. Acta Biol. Mad. Germ. 29, 441, 1972.
.
JAYARAMAN, S. and MUTHUKKARUPPAN, VR. Influence of route and dose of antigen on the migration inhibition and plaque-formlng cell response to sheep erythrocytes in the lizard, Calotes versicolor. Immunol., IN PRESS, 1977.
8.
SAILENDRI, K. and MUTHUKKARUPPAN, VR. The immune response of the teleost, Tilapia mossambica to soluble and cellular antigens. ~. Exp. Zool. 191, 371, 1975.
9.
DENNERT, G. and TUCKER, D.F. Selective priming of T cells by chemically altered cell antigens. J. Exp. Med. 136, 656, 1972.
i0.
MOHAN, R. Studies on the humoral and cell-mediated immune responses in a teleost, Tilapia mossambica. Ph.D. Thesis. Madural University, India, 1977.
ii.
ETLINGER H.M., HODGINS, H.O. and 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, 1947, 1976.
12.
Mc KINNEY, E.C., ORTIZ, G., LEE, J.C., SIGEL, M.M., LOPEZ, D.M., EPSTEIN, R.S. and McLEOD, T.F. LympHocytes of fish: multipotential or specialised? In: Phylogeny of Thymus and Bone Marrow-Bursa Cells. R.K. Wright and E.L. Cooper, Elsevler/North-Holland Biomedical Press, Amsterdam, p. 73, 1976.
13.
MACKANESS, G.B., LAGRANGE, P.H. and ISHIBASHI, T. Feedback inhibition of specifically sensitized lymphocytes. ~. Exp. Med. 139, 543, 1974.
14.
KERCKHAERT, J.A.M. Influence of cyclophosphamide on the delayed hypersensitivity in the mouse after intraperitoneal ~mmunization. Ann. d'I,,nunol. (Inst. Pasteur). 125C, 559, 1974.
15.
TAMURA, S.I. and EGASHIRA, Y. Cellular and humoral immune responses in mice. II. Effect of intraperltoneal or subcutaneous injection of carrier on anti-hapten antibody and delayed hypersensitivity responses. Immunol. 28, 909, 1975.
16.
STOLEN, J.S. and M ~ L A , O. responses of a marine fish.
17.
YOCUM, D., CUCHENS, M. and CLEM, L.W. The hapten-carrier effect in teleost fish. J. Immunol. 114, 925, 1975.
18.
SAILENDRI, K. Studies on the development of lymphoid organ and immune responses in the teleost, Tilapia mossamblca. Ph.D. Thesis, Madurai University, India, 1973.
Carrier preimmnlzation in the antlhapten Nature 254, 718, 1975.
AND
COMPARATIVE
01~5-305X/79/010067-09502.00/0 Copyright
(c) 1979 P e r g a m o n
IMMUNOLOGY, Vol.
Printed P r e s s Ltd.
3, PP. 6 7 - 7 5 , in the U S A . All rights reserved.
1979.
RELATIONSHIP BETWEEN MIGRATION INHIBITION AND PLAQUE-FORMING CELL RESPONSES TO SHEEP ERYTHROCYTES IN THE TELEOST, TILAPIAMOSSAMBICA
S. Jayaraman @, R. Mohan** and VR. Muthukkaruppan Department of Immunology School of Biological Sciences Madurai University Madurai-625021, India
ABSTRACT Cell-mediated immunity to sheep erythrocytes (SRBC) in the teleost, Tilapia mossambica was assessed using the in vitro capillary migration inhibition (MI) assay and the humoral immune response by the plaque-forming cell (PFC) assay. Both PFC and MI responses were found to be inversely proportional to the amount of antigen injected. Cyclophosphamide administration immediately after immunization completely abrogated both PFC and MI responses. In contrast, injection of formalinized SRBC resulted in greater elevation of MI levels accompanied by the absence of PFC production. The possible regulatory role of antibody-producing cells in the MI response to SRBC is discussed.
INTRODUCTION The migration inhibition (MI) technique is a valid in vitro test in m s m ~ i s for measuring cell-mediated immunity ((241) to various antigens including sheep erythrocytes (SRBC) (1,2,3).
Recently, we have described the
This work was supported in part by a research grant from the University Grants Commission, India. @Recipient of Senior Research Fellowship under the above scheme. Supported by Junior Research Fellowship awarded by University Grants Commission. Correspondence:
VR. Muthukkaruppan, School of Biological Sciences, Madurai University, Madurai - 625 021, India. 67
68
TELEOST CELLULAR IMMUNITY
Vol.3, No. I
utility of this technique for assessing CMI in reptiles to skin allografts (4), and SRBC (5).
Similarly, in amphibians, MI assays have been used to
study CMI to PPD (6).
Earlier we showed in the lizard, Calotes versicolor
that low doses of SRBC induced high levels of MI, but high doses, induced low levels (5,7).
The present report describes CMI in a teleost fish,
Tilapia mossambica to SRBC and its relationship to the development of plaque-forming cells (PFC) under varying conditions. METHODS Fish Adult fish of both sexes weighing 50 to I00 g were collected mld maintained at 30°C in glass aquaria as described earlier (8). _Antigen and immunization Sheep blood was drawn in Alsever's solution from the jugular vein of a single sheep, washed three times with phosphate-buffered saline (PBS, pH 7.2) and appropriate dilutions were made shortly before use.
Rat red blood cells
(RRBC) were obtained by cardiac puncture from white rats. Formalinized SRBC (F-SRBC) were prepared by adding an 8% suspension of erythrocytes in PBS to an equal volume of 3% formaldehyde, followed by incubation for 18 hr at 37°C with constant and gentle agitation (9). Treated erythrocytes were washed and tested for antigenicity as described by Dennert and Tucker (9). Fishes were usually immunized by injecting them with appropriate amounts of antigen in PBS via the c o ~ o n
cardinal vein (8).
Dru 8 treatment Cyclophosphamide (CY) (kindly donated by Asta-Werks AG, Brackwede, Germany) was dissolved in physiological saline and injected intraperitoneally at a dose of 300 mg/kg body weight (i0). Plaque-formin~ cell assay Direct haemolysin producing cells were detected using the method described previously for Tilapia (8). for 1 hr.
The plaques were developed at 37°C
The viability of white blood cells (WBC) of the head-kidney was
judged by the ability to exclude 0.25% trypan blue dye. Serology Haemagglutinin (HA) antibodies against SRBC were detected in the ~mmunized sera using the method previously described (8).
Vol. 3, No. 1
TELEOST CELLULAR IMMUNITY
69
Migration inhibition assay The MI assay adapted for lizards modifications head-kidney
for the fish system.
Briefly,
cells was made in PBS.
L-15 Leibovitz medium
(4,5) was followed with suitable a single cell suspension of
The cells were washed twice with diluted
(3 parts medium and i part triple distilled water),
supplemented with 20% normal Tilapia serum (decomplemented min).
The capillary
Morganville,
at 56°C for 30
tubes (0.8 x 90 mm, Laboratory Diagnostics,
N.J. 07751, USA) were filled with head-kidney
at one end with modeling clay.
After centrifuging
Co., Inc.,
cells and sealed
the capillaries at 500 rpm
for 2 min, a nick was made with a diamond pencil just below the cell-fluid interface.
By applying gentle pressure,
the cell pellet was separated and
secured to the bottom of the migration chambers grease.
(4) with a dab of silicone
After sealing the migration chambers, medium was introduced with a
gentle force, using a 2 ml syringe fitted with a 20 gauge needle. Antigen consisted of 0.01 ml of 10% sonicated For specificity
experiments,
SRBC (5) per ml of medium.
intact erythrocytes were added directly to the
culture medium at a concentration of 0.0i ml of 1% erythrocytes medium
per ml of
(4,5).
The migration cultures were incubated at 37°C for 24 hr and the extent of migration drawn with a camera lucida and measured by planimetry
(4).
The
%MI was calculated using the formula: Migration area with antigen % MI = i00 - i00 x Migration area without antigen Statistics Statistical assessment
of the data was computed using the Student's
t-test. RESULTS Culture conditions Tests of antigen-containing dur~Ig
the
culture medium revealed no change in pH
period of incubation.
More than 80% of cells were viable as
determined by the trypan blue dye exclusion test, irrespective
of the
presence or absence of antigen in the medium during the incubation period. Dose-Dependency Different head-kidney
of Immune Responses dilutions of SRBC were given to fishes and five days later,
cells were assayed for PFC and MI responses.
Data in Fig. i
show that low antigenic stimuli induced fairly high levels of MI with an
70
TELEOST CELLULAR IMMUNITY
insignificant number of PFC.
Vol. 3, No. 1
As we increased the dose of SRBC, the PFC
number increased logarithmically.
S75
50
,,
-
4O
(~ 225
50
14.
20
\
150
Q. ,
@ !
I I
75 0
c
5
4
5
6
7
8
I I0
9
0
S RBC DOSE LOGIo
FIGURE 1 Dose dependency of PFC and MI responses to SRBC. Five days after immunization, assays were performed with head-kldney lymphocytes (WBC). Each point represents the mean of 5-7 animals (±S.E.). C denotes control values.
This increase in PFC production however, was accompanied by a suppression of the MI level, especially to a dose of 2.4 x 109 SRBC.
Thus, the present
experiment reveals an inverse relationship between PFC and MI responses in fishes. Modulation of Immune Responses We attempted to modulate the ~mmune responses to SRBC using two protocols:
one utilizing the alkylating agent, CY, and the other, F-SRBC.
Sallne-injected fishes did not produce either HA antibodies or PFC to SRBC (Table l); the MI level was also minimal.
However, when 2.4 x 109 SRBC were
given to fishes, appreciable HA titre and PFC numbers were detected with a low but significant level of MI (0.025 > P < 0.05).
If the same amount of
antigen was followed by an injection of 300 mg CY, apparently no antibodies and PFC were produced.
Besides, the M I response was also significantly
reduced (0.025 > P < 0.05) as a consequence of drug treatment.
In contrast,
the injection of 2.4 x 109 F-SRBC greatly enhanced the level of MI with a concomitant decrease in HA tltre and PFC number.
Vol. 3, No. 1
TELEOST CELLULAR IMMUNITY
71
TABLE i Modulation of T--,une Responses
Treatment
HA titre b (Mean ± S.E)
PFC/IO 6 WBC (Mean'± S.E)
1:0
0
(10) c
(17)
1:60 ± 15
320 ± 31
(8)
(7)
i:0
25 ± 15
Saline
SRBC
cyd SRBC +
(9) F_SRBC e
1:7
± 2
(7)
a
% MI (Mean ± S.E)
5.6 ±
2.0
(17) 14.3 ±
4.9
(7) 1.6 ±
(12)
0.8
(6)
9 ± 3
54.8 ± 11.7
(7)
(7)
apFC and MI assays were performed with head-kidney cells 5 days after treatment. bsera bled from fish were titrated against SRBC, ii days after immunization. CNumber of fish investigated is given in parentheses. d300 mg CY/kg was given 3 hr after immunization with 2.4 x 109 SRBC. e2.4 x 109 formalinized SRBC were injected intraperitoneally.
Specificity of Immune Responses The specificity of both PFC and Ml responses was determined.
To test
the specificity of PFC responses, head-kldney cells from fishes that had been injected with 2.4 x 109 SRBC were assayed.
For the MI assay~ cells were
harvested from fish that had been injected with 109 SRBC.
Data in Table 2
show that the SRBC-immun~zed head-kidney cells did not produce anti-RRBC plaques, showing the specificity of the phenomenon.
Likewise, the SRBC-
sensitized head-kidney cells were not appreciably inhibited (0.025 > P < 0.05) in the presence of intact RRBC in the medium.
72
TELEOST CELLULAR IMMUNITY
Vol. 3, No. 1
TABLE 2 a
Specificity of Immune Responses
Immunizing antigen
Test antigen
PFC/I06 WBCb (Mean ± S.E)
% Mlc (Mean ± S.E)
Saline
SRBC
0 (17)
5.6 ± 2.0 (17)
SRBC
SRBC
335 ± 86 (5) d
SRBC
RRBC
1.2
28.0 ± 13.5 (4)
± 0.8 (5)
7.8
± 4.0 (4)
aFive days after immunization assays were performed with head-kidney cells. bpFC assay was performed with head-kidney cells taken from fish immunized with 2.4 x 109 SRBC, in the presence of either SRBC or RRBC. ~ I assay was performed with head-kidney cells harvested from fish immunized with 109 SRBC, in the presence of either intact SRBC or RRBC (4,5). dNumbers in parentheses denote the number of fish used.
DISCUSSION In the present report, CMI to SRBC was assessed in a teleost fish, T i l a p i a m o s s a m b i c a using the MI technique.
Drossler and Ambrosius have also
used the MI technique to detect CMI to PPD in Bufo bufo (6) and we demonstrated its utility for assessing CMI to skin allografts and to SRBC in the lizard, Calotes versicolor (4,5).
The MI of head-kidney cells from
sensitized fish is a n in vitro expression of CMI to SRBC and not a nonspecific effect caused by culture conditions. the following observations:
This conclusion is based on
(i) the L-15 Leibovitz medium has already been
shown to be optimal for i_.nvitro assays in fishes (ii); (ii) no alteration in the pH of the medium with or without antigen after 24 hr of incubation at 37°C was observed;
(iii) the degree of viability was the same in both
cultures with or without antigen and (iv) the MI obtained with unsensitized head-kidney cells was negligible in the presence of antigen (Table i).
The
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TELEOST CELLULAR IMMUNITY
73
possible inhibitory action of antibodies and antigen--antibody complexes can be ruled out since MI could be demonstrated with head-kidney cells obtained from fish that produced little or no PFC and HA antibodies after immunization with low doses of SRBC (Fig. i) or F-SRBC (Table i).
Besides,
the MI assay was used to evaluate CMI to scale allografts in fish (12). Hence, it is suggested that MI of SRBC-sensitized head-kidney cells is an in vitro correlate of CMI to SRBC in Tilapia mossambica. Like PFC formation, MI is also an antigen-specific event in fishes (Table 2).
In previous reports, the specificity of MI responses of lizards
was confirmed by suspending intact erythrocytes in the culture medium and allowing spleen cells to migrate from the capillary tubes (4,5).
Following
the same protocol, we found that the MI of sensitized head-kidney cells of Tilapia mossambica is a highly antigen-specific phenomenon as in other fish (12) and animal systems (1,4,5). Based on these observations, the relationship between MI and PFC responses was analyzed.
The dose-dependency profile of MI and PFC responses
denotes the existence of a mirror-image relationship between them when tested 5 days after immunization (Fig. i).
Humoral immune (HI) response
was low to a low dose of SRBC and high to a high dose of SRBC throughout a period of 21 days in Tilapia (i0).
Such an inverse relationship between
PFC and MI responses to SRBC has already been reported in the lizard, Calotes versicolor (5,7).
In mice, delayed-type hypersensitivity
(DTH) and
the HI response to SRBC were reported to be inversely proportional to the dose of antigen (13-15).
It seems highly likely therefore, that the inverse
relationship between MI and the HI response to SRBC is a phenomenon traceable through phylogeny. According to one interpretation, both functional thymus-derived (T-) and bone-marrow-bursa-equivalent
(B-) cells co-exist as distinct entities
and take part in the HI response of fishes (16,17).
In Tilapia mossambica,
the existence of functional T- and B-cells has also been demonstrated recently (in preparation).
Whereas long-term (2-3 months) adult thymectomy of fishes
greatly abrogated both the primary and secondary HI response to SRBC, the anti-polyvinyl pyrrolidone response was not affected, indicating the existence of both a T-dependent and T-independent immune system in Tilapia. Besides, MI had been equated as the T-cell response in fishes (12).
To
understand the probable modulation of T-cell function (MI) by B-cells, F-SRBC and CY were used.
Formalinized erythrocytes have been shown to
induce T-helper cells specifically without the generation of PFC in mice
74
TELEOST CELLULAR IMMUNITY
(9) and lizards (5) as well as in Tilapia (i0).
Vol. 3, No. 1
Injection of 2.4 x 109
SRBC resulted in the production of large numbers of PFC with a minimal level of MI (Fig. l: Table I). an enhanced MI response (Table i).
with
However, the same amount of F-SRBC evoked greatly decreased HA and PFC responses
That the enhanced CMI was accompanied by a low B-cell response
suggests that in fishes, the induction of functional T- and B-cells is causatively related.
It is probable that the activation of B-cells by
large doses of native SRBC might lead to the formation of antigen-antibody complexes that could inhibit the activity of functlolml T-cells (MI) as in mice (13). The data with CY treatment seem to contradict this hypothesis. abolished both CMI and the HI response to SRBC (Table i).
CY
In addition to
abolishing functional B-cell activity CY administration causes prolonged survival of scale allografts and damages T-helper activity in Tilapia mossambica (10).
Hence, CY administration probably does not result in
enhanced MI levels.
We suggest, therefore, that functional B-cells may exert
a regulatory role on the functional T-cell response (MI) in fishes, but this conclusion awaits detailed studies of immune responses in fishes.
ACKNOWLEDGEMENTS S. Jayaraman expresses thanks to Dr. E.L. Cooper for critically reading the manuscript and to Dr. P.W. Askenase for the generous gift of capillary tubes.
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