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Structural, functional relationship of thymus derived cells of the lizard Calotes versicolor
DEVELOPMENTAL AND COMPARATIVE IMMUNOLOGY, Vol. 12, pp. 603-610, 0145-305X/88 $3.00 + .00 Printed in the USA Copyright (c) 1988 Pergamon Press plc All rights reserved
STRUCTURAL,
1988
FUNCTIONAL RELATIONSHIP OF THYMUS DERIVED CELLS OF THE LIZARD CALOTES VERSICOLOR
M. MANICKASUNDARI AND RM. PITCHAPPAN Department of Immunology, School of Biological Sciences, Madural Kamaraj Unlveslty, Madural - 625 021, India
ABSTRACT:
Structural and functional characteristics of the lymphoid cells of Calotes verslcolor were studied using thymocytes and splenocytes isolated on a buoyant density gradient and adherence column. The isolated cells were treated with anti- Calotes thymocytes serum (ATS) and their residual capacity to effect antigen specific migration inhibition and antibody production assesed. Results revealed that antigen specific migration inhibition effector cells could be enriched in less dense nylon wool-adherent fraction and 95% of this fraction were sensitive to ATS. Antigen specific plaque forming cells (PFC) could also be enriched in the same fraction but they were not susceptible to ATS.
INTRODUCTION It is known in mammalian and avian systems that both T and B lymphocytes are heterogeneous in density distribution profile and these density sub-populatlons differ in biological functions (1-5). Further, more dense splenic T cells have been shown to be more effective helper cells (5). A minor sub-populatlon of thymocytes with a light density possess surface antigenic characteristic of peripheral T cells (1,6,7). A thymocyte antigen, B2A2, detected using a monoclonal antibody has been reported to be absent in the peripheral T cells but are present on large less dense and functlonally active recent thymus migrants in the spleen (8). In human system also an antigen T4, was reported on large mature thymocytes (9). Thus it has been suggested that the density sub-populatlons may differ in their structural and biological functions. The results presented here report the biophysical characteristics of the lizard lymphoid cells and demonstrate the use of density gradient and nylon wool column separation methods in delineating T and B cells of the lizard C. versicolor.
MATERIALS AND METHODS Animals: Adult garden lizards Calotes versicolor of both sexes weighing 40 to 60 gms obtained from local animal suppliers were used in the present study. 603
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Immunization and experimental design: Lizards were immunized with one tenth of a million washed sheep erythrocytes intramuscularly, their spleens dissected seventh day post immunization and splenocytes subjected to discontinuous gradient and nylon wool column separations. The resultant fractions were subjected to ATS and complement treatment. Subsequently these were tested for antigen specific migration inhibition (MI). For analysing the biophysical characteristics of antibody producing cells, lizards were immunized with I00 million sheep erythrocytes Intraperltoneally, their splenocytes obtained on fourteenth day post immunization and the biophysical separation carried out. The resultant cell fractions were assessed for plaque forming cells (PFC) and their susceptibility to ATS. Since the experimental designs were quite laborlus, each experiment was repeated twice. The results were found to be concordant. Only the results of representative experiments are presented here. Nylon wool column fractlonatlon: Isolation of adherent from non-adherent splenocytes was carried out following the method of Daniloves et al. (I0) and as adopted earlier in this laboratory (11). Miniature nylon wool columns prepared by packing drinking straws of 2.5 mm diameter and 6 cm height with 35 mg of scrubbed nylon fibre each to a height of 4 cm (Nylon fibre 3 Denier, 3.81 cm, type 200 Fenwal lab, USA code No. 4c2906) were utilized (11). Discontlnous ~radlent separation: Discontlnous gradient of 5 to 30% Ficoll (5% step) in medium TC 199 + 5% FCS was prepared by layering 1.5 ml of lighter fluid over the denser, one after another to a 15 ml cylindrical tube. The gradient was equilibrated at 4°C for I/2 hour and I00 million cells in 1 ml were layered on to the top of the gradient. It was spun at 2500 rpm (1100 g at interface) for 45 minutes at 4°C in a refrigerated swing out centrifuge. At the end of centrlfugatlon, fractions were collected from each interface using Pasteur pipettes, washed twice with Ca ++ and Mg ++ free PBS and suspended in L-15 medium supplemented with 5% homologous serum. Cells from different fractions were studied for different characteristics as described below. Mi~ratlon inhibition test: The migration inhibition assay method as described by David and David (12) was followed. An indirect migration inhibition test was used. In short one million splenocytes (either unseparated or nylon wool and gradient separated) obtained from immunized lizards (effector cells) were mixed with three million splenocytes obtained from unimmunlzed lizards (indicator cells), packed into capillaries and MI assay carried out in the presence or absence of antigen as described earlier (11). Membrane immunofluorescence assay: The method described by Pernls et al, (13) was followed. In short, one million splenocytes suspended in 50 microlitre of L-15 medium supplemented with 2% BSA were incubated with rabbit anti-llzard Ig (specific for IgM) for 30 minutes at 4°C, washed thrice and then treated with FITC conjugated goat anti-rabblt IgG (Cappell, USA) for further 30 minutes at 4°C (14). Cells were washed and suspended in a medium containing nine parts glycerol and one part 0.5M carbonate buffer pH 9.5. Two hundred mononuclear cells per slide were counted twice using Zelss-Jena, Fluoval microscope and average obtained. Plaque formln~ cell assay: Cunnlngham and Szenberga's plaque forming cell technique as adopted for Calotes system (15) was utilized to detect the number of hemolysin producing cells in the spleens of immunized lizards. The reaction mixture containing 0.06 ml of 30% foetal calf serum, 0.01 ml of 10% SRBC, 0.01 ml of Calotes complement and 0.01 ml of effector cells were fed to monolayers
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between two microscopic slides, edges sealed with paraffin, incubated for 30 mln. at 37°C and plaques of hemolysis counted using a low power stereo microscope
RESULTS Figure 1 presents the results on the susceptibility of various fractions of gradient separated unimmunized lizards thymocytes and splenocytes to anti-thymocyte serum (ATS). It is evident from Figure i that at the operational dilution of ATS used here all the (100% cytotoxlc index) thymocytes, but only 40% splenocytes were susceptible to it. While these cells were fractlonated based on density, a few interesting points emerged. All the thymocytes banding at density 1.081 gm/ml and 1.096 were susceptible to ATS (Fig. IA & B). Splenocytes banding at these densities were however not susceptible (Fig. IC & ID). On the contrary, majority of the splenocytes banding at d. 1.066 and 1.051 were susceptible to ATS. This clearly brought out the density differences between the unimmunized lizard splenocytes and thymocytes with reference to their susceptibility to ATS in question. The histograms in Figure 1 presents the total number of cells recovered in each density fraction and the total number of ATS susceptible cells in each one of them. All the thymocytes banding at d. 1.066 were susceptible to unabsorbed ATS (Fig. IA) but only 50% of them were susceptible to absorbed ATS. The ATS activity directed against a common antigenic determinant to liver and thymocytes seems to have been absorbed by the liver powder: The residual activity thereby detected a membrane antigenic determinant present both on high dense thymocytes and less dense (blasts) splenocytes. Table i presents the results on the distribution of cells in the unseparated, less dense (LD='I.066 gm/ml) to 1.08 gm/ml) fractions of normal lizard splenocytes. consisted more of ATS sensitive cells (70%) whereas more of Slg +ve cells(60%). This suggests that these enriched based on their buoyant density.
ATS sensitive and Slg +ve and high dense (HD=1.067 The less dense fraction the high dense consisted two types of cells can be
Table 2 presents the results on the distribution of migration inhibition effector cells in these fractions and the susceptibility of these cells to ATS and complement treatment. The unseparated splenocytes effected a 58% MI; but the HD effected only 34%. However upon treating these cells with ATS, this function was abrogated. This experiment revealed that the MI mediating effector cells could be enriched in LD fraction and ATS could abolish their MI function. These MI effector cells identified in different density fractions may belong to different stages of cell-cycle in their maturatlonal process. It is clear from the results presented here and in our earlier paper (ii) that nylon wool abherence column and density gradient analysis could enrich both ATS sensitive and SIg+ve cells and they are correlated with effector functions. However, the enrichment were not absolute. Hence the two techniques, one considering the buoyant density and another adherent properties of cells at different stages of maturation were coupled with an aim of isolating pure populations of functional subsets. Figure 2 presents evident from figure
the results on migration inhibition effector cells. It is 2A that the non-adherent less dense fraction consisted of
606
FUNCTION OF THYMUS DERIVED CELLS
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Flg.l. Density distribution profile of normal lizard thymocytes (Flg IA, B) and splenocytes (Fig. IC, D) and their susceptibility to ATS: Thymocytes and splenocytes of fifteen normal lizards were pooled and subjected to dlscontlnous Isopycnlc gradient separation: the histogram presents the total number of cells obtained ~n each fraction and the hatchedarea represents the ATS sensitive cells among them. The graph presents the ATS susceptibility of each fraction as % cytotoxlc index. Fig IA & IB are the results on thymocytes; IC and ID on splenocytes; IA and IC using unabsorbed ATS and IB and ID using liver absorbed ATS. U = Unseparated lymphoid cells; %CI = Cytotoxlc index %.
30 15 0
• US 1.051
66
81
DENSITY
• 96
0 1.051
66
81
96
GM/ML
TABLE 2
TABLE 1
Distribution of migration inhibition effector cells in density f~actlons and their susceptibility to ATS
Slg +, ATS + cell in density fractions of normal lizard splenocytes
Splenocytes
Splenocytes
Total # Slg ATS
US
1000# 466 301
US
LD
HD
Treatment
18& 6 12
838 500 147
No antigen 6.0a + antigen 2.5 % MI 58.4
Cells are In 1 x i00,000; # = cells loaded; & = recoverd; Slg,surface Immunoglobulln;ATS, antlthymocyte serum sensitive; US, unseparated; LD, less dense (1.066 gm/ml); HD, hlgh dense (1.067 - 1.08 gm/ml)
nll
LESS DENSE
HIGH DENSE
nll
ATS
nll
ATS
10.7 2.8 73.6
7.1 7.1 0.0
4.9 3.2 33.8
6.2 5.9 4.1
Splenocytes obtained from llzards immunized wlth SRBC for CMI were subjected to discontinuous gradient separation. Less dense and high dense populations were subjected to ATS and complement treatment and their residual capacity to mediate MI assessed in the absence or presence of antigen, a = area of migration In square mm ; %MI = migration inhlbtlon In %.
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@
1.~0
¢o 0
8O
95
_J IJJ L)
40
m lO
-
÷
-
÷
-
÷
ATS LD NON
-
÷
-
÷
ATS HD ADHERENT
-
+
-
+
-
+
ATS
-
+
ATS
LD
HD
ADHERENT
Figure 2. Adherent and density distribution ~rofiles effector cells and their susceptibility to ATS.-
of migration
inhibition
Splenocytes obtained from lizards immunized for CMI and subjected to nylon wool column were further separated into less dense and high dense population in a discontinuous isopycnic gradient. These cells were treated with ATS and complement and their property to elicit migration inhibition assessed in the absence or presence of antigen. Firgure 2A presents the total number of splenocytes recovered in each fraction (open column) and the ATS sensitive cells in each fraction (solid bars). Figure 2B presents the area of migration of these cells in the absence (-) or presence (+) of antigen, sonlcated sheep erythrocytes. ATS denotes the migration after ATS and complement treatment. Other legends as in earlier figures.
95% ATS sensitive cells: it was only 32% in unseparated and 29% in non-adherent high dense fractions. Thus these two techniques coupled together seem to isolate majority of the ATS sensitive cells in non-adherent less dense fraction as a pure population. Figure 2B presents the results on the effector functions of these cells. In the presence of antigen, unseparated cells mediated an MI of 62%. The non-adherent LD cells again mediated 61% MI: of the 140 million cells processed, 50 million were obtained in this fraction. The non-adherent HD mediated 22% MI. The adherent LD cells mediated 45% MI: but there was only 3.2 million cells harvested in this fraction and 47% of it was ATS sensitive. The MI effector functions of all these cells were abrogated by ATS treatment (MI = 0). It is thus obvious that majority of the MI mediating ATS sensitive effector cells were enriched and isolated in non-adherent LD fraction: presemably they are the lymphoid cells of thymic lineage involved in MI response.
608
FUNCTION OF THYMUS DERIVED CELLS
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130
@
80 ©o
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.i
40 ._J U,.I
o
BB 140
O
O
100
v-
O n "
2o .
0 L D U
NON
HD ADHERENT
Figure 3. Adherent and density di~trlbutlon cells and their susceptlblllty to ATS-.
L D
HD
ADHERENT
profiles
of
antibody
producing
Splenocytes obtained from lizards immunized for humeral immunity were subjected to nylon wool column and discontinuous gradient separation; subsequently these fractions were treated with ATS & complement and the number of antl-sheep erythrocyte plaque forming cells were enumerated. Figure 3A presents the total number of splenocytes recovered in each fraction (open clumn) and the ATS sensitive cells in them (solid bars). Open columns in Figure 3B presents the total number of plaque forming cells in each fraction and the solid bars, after ATS & complement treatment. Legends as in earlier figures. Efforts were also made to isolate antigen (SRBC) specific plaque forming cells (PFC) by these procedures. When the splenocytes obtained from lizards immunized for humeral immunity were subjected to these biophysical procedures, out of 130 million cells loaded, 66 million could be harvested in non-adherent LD fraction: but only 10% of it was sensitive to ATS (FIG. 3A). This is in contrast to the 95% sensitivity of the non-adherent LD, MI mediating cells (Fig. 2A). When the presence of anti SRBC plaque forming cells (PFC) were assessed in these fractions, again 104,000 PFC out of 140,000 PFC loaded were harvested in non-adherent LD fraction (Fig. 3B). The harvest in other fractions were only of the order of 10,000. When these splenocyte fractions were treated, with ATS and complement, there was no substantial reduction in the PFC count in any of these fractions (Fig. 2B) suggesting their insensitivity to ATS.
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DISCUSSION Two important points emerged from these experiments: firstly majority of the migration inhibition effector cells as well as the PFCs were harvested in non-adherent LD fraction. Secondly the less dense non-adhrent population of splenocyte effectlng MI but not PFC were sensitive to ATS. Since these effector cells were procured from immunized lizards during their earlier (exponential) phase of the immune responses presumably they were of less dense non-adherent cells: (cf.16) however the membrane antigenic markers of MI mediating cells were that of thymus derived lineage as identified by ATS. The heterologous ATS in question thus seems to identify a splenic non-adherent less dense functional T-cells of the lizard reaffirming the existence of a separate thymlc lineage in Calotes verslcolor. The conclusions that can be drawn from these experiments are that ATS and complement is able to kill cells involved in mlgratlon-inhlbltion, but not antibody-releaslng cells and these "T" and "B" cells (at the given stage of differentiation) are found in the same nylon wool non-adherent, low density fractions. The nylon wool adherence and buoyant density thus do not appear to separate the functional "T" and "B" (lymphoblasts) cells of the lizards clearly, but antibodies to cell surface determinants, do.
ACKNOWLEDGEMENTS The grant (7/201 (B2)/ 80-81-EMR-1) from Council of Scientific and Industrial Research, India to this study is gratefully acknowledged. We thank Ms. H a r i h a r a n and Subash Chandar f o r p r e p a r i n g the m a n u s c r i p t f o r p u b l i c a t i o n .
REFERENCES I.
Shortman, K.D., Cerottinl, J.C. and Brunner, K.T. The separation of sub populations of T and B lymphocytes. Eur. J. Immunol., 2, 313, 1972.
2. Shortman, K.D., Byrd, W.J., Cerottinl, J.C. and Brunner, K.T. Characterization and separation of mouse lymphocyte subpopnlatlons responding to phytohemagglutinln and pokeweed mltogens. Cell. Immunol., 6, 25, 1973. 3.
Tammlnen, P., Tolvanen, of chicken thymus cells.
A and Tolvanen, P. Density gradient Eur. J. Immunol., 3, 521, 1973.
separation
4.
All, F.M.K., May, A., Mc Laren, G.P., Jacobs, A. A two step procedure for obtaining normal peripheral blood T lymphocytes using continous equilibrium density gradient centrlfugation on percol. J. Immunol. Methods., 49, 185, 1982.
5. Gorcyznskl, R.M., Miller, R.G. and Phillps, R.A. Identification by density separation of antigen specific surface receptors on the progenitors of antibody producing cells. Immunology. 20, 693, 1971. 6.
Paplernik, M. Study of thymic lymphocyte subpopulations in neonatal mice compared to adult mice. Ann. Immunol. (Inst. Pasteur) 127, 957, 1976.
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FUNCTION OF THYMUS DERIVED CELLS
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7.
Singh, U and Owen, J.J.T. Studies on subpopulatlons of foetal thymocytes separated on Ficoll Hypaque density gradients. Dev. Comp. Immunol., 3, 543, 1979.
8.
Scollay, R., Wilson, A and Shortman, K. Thymus cell migration: Analysis of thymus emigrants with markers that distinguish medullary thymocytes from peripheral T cells. J. Immunol., 132, 1089, 1984.
9.
Appleyard, J., Scornlc, J.C., Braylan, R.C. and Benson, N.A. Distinctive expression of the T4 antigen in normal and stimulated lymphocytes. Cell Immunol., 76, 171, 1983.
I0.
Daniloves, J.A., Ayob, G and Terasakl, P.I. B lymphocyte isolation by thrombln-nylon wool. In: Histocompatlbillty Testing (Ed. P.I. Terasakl), ULCA Tissue Typing Laboratory, Los Angeles, California, pp. 287, 1980.
11. Manlckasundarl, M., Selvaraj, P. and Pitchappan, RM. (1984) Studies on T cells of the lizard, Calotes verslcolor Nylon wool adherent and non-adherent populations of the spleen. Dev. Comp. Immunol., 8, 367, 1984. 12. David, J.R. and David, R. Assay for inhibition of macrophage migration. In: In-vltro methods in cell-medlated immunity (Eds. B.R. Bloom and P.R. Glade), Academic Press, New York, pp. 249, 1971. 13. Pernls, B., Fornl, L. and Amantl, L. Immunoglobulln spots on the surface of rabbit lymphocytes. J. Exp. Med., 132, I001, 1970. 14. Natarajan, K. Studies on Bone-marrow/Bursa equlvalent-Derived cells in the garden lizard Calotes verslcolor. Ph.D Thesis, Madural KamaraJ University, India. 1983. 15.
Kanakamblka, P. and Muthukkaruppan, VII. The immune response to sheep erythrocytes in the lizard Calotes verslcolor. J. Immunol., 109, 415, 1972.
16. Kraft, N. and Shortman, K. Differentiation of antibody forming cells in toad spleen. Study using density and sedimentation velocity cell separation J. Cell. Biol., 52, 438, 1972. Received: April, 1987 Accepted: March, 1988
STRUCTURAL,
1988
FUNCTIONAL RELATIONSHIP OF THYMUS DERIVED CELLS OF THE LIZARD CALOTES VERSICOLOR
M. MANICKASUNDARI AND RM. PITCHAPPAN Department of Immunology, School of Biological Sciences, Madural Kamaraj Unlveslty, Madural - 625 021, India
ABSTRACT:
Structural and functional characteristics of the lymphoid cells of Calotes verslcolor were studied using thymocytes and splenocytes isolated on a buoyant density gradient and adherence column. The isolated cells were treated with anti- Calotes thymocytes serum (ATS) and their residual capacity to effect antigen specific migration inhibition and antibody production assesed. Results revealed that antigen specific migration inhibition effector cells could be enriched in less dense nylon wool-adherent fraction and 95% of this fraction were sensitive to ATS. Antigen specific plaque forming cells (PFC) could also be enriched in the same fraction but they were not susceptible to ATS.
INTRODUCTION It is known in mammalian and avian systems that both T and B lymphocytes are heterogeneous in density distribution profile and these density sub-populatlons differ in biological functions (1-5). Further, more dense splenic T cells have been shown to be more effective helper cells (5). A minor sub-populatlon of thymocytes with a light density possess surface antigenic characteristic of peripheral T cells (1,6,7). A thymocyte antigen, B2A2, detected using a monoclonal antibody has been reported to be absent in the peripheral T cells but are present on large less dense and functlonally active recent thymus migrants in the spleen (8). In human system also an antigen T4, was reported on large mature thymocytes (9). Thus it has been suggested that the density sub-populatlons may differ in their structural and biological functions. The results presented here report the biophysical characteristics of the lizard lymphoid cells and demonstrate the use of density gradient and nylon wool column separation methods in delineating T and B cells of the lizard C. versicolor.
MATERIALS AND METHODS Animals: Adult garden lizards Calotes versicolor of both sexes weighing 40 to 60 gms obtained from local animal suppliers were used in the present study. 603
604
FUNCTION OF THYMUS DERIVED CELLS
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Immunization and experimental design: Lizards were immunized with one tenth of a million washed sheep erythrocytes intramuscularly, their spleens dissected seventh day post immunization and splenocytes subjected to discontinuous gradient and nylon wool column separations. The resultant fractions were subjected to ATS and complement treatment. Subsequently these were tested for antigen specific migration inhibition (MI). For analysing the biophysical characteristics of antibody producing cells, lizards were immunized with I00 million sheep erythrocytes Intraperltoneally, their splenocytes obtained on fourteenth day post immunization and the biophysical separation carried out. The resultant cell fractions were assessed for plaque forming cells (PFC) and their susceptibility to ATS. Since the experimental designs were quite laborlus, each experiment was repeated twice. The results were found to be concordant. Only the results of representative experiments are presented here. Nylon wool column fractlonatlon: Isolation of adherent from non-adherent splenocytes was carried out following the method of Daniloves et al. (I0) and as adopted earlier in this laboratory (11). Miniature nylon wool columns prepared by packing drinking straws of 2.5 mm diameter and 6 cm height with 35 mg of scrubbed nylon fibre each to a height of 4 cm (Nylon fibre 3 Denier, 3.81 cm, type 200 Fenwal lab, USA code No. 4c2906) were utilized (11). Discontlnous ~radlent separation: Discontlnous gradient of 5 to 30% Ficoll (5% step) in medium TC 199 + 5% FCS was prepared by layering 1.5 ml of lighter fluid over the denser, one after another to a 15 ml cylindrical tube. The gradient was equilibrated at 4°C for I/2 hour and I00 million cells in 1 ml were layered on to the top of the gradient. It was spun at 2500 rpm (1100 g at interface) for 45 minutes at 4°C in a refrigerated swing out centrifuge. At the end of centrlfugatlon, fractions were collected from each interface using Pasteur pipettes, washed twice with Ca ++ and Mg ++ free PBS and suspended in L-15 medium supplemented with 5% homologous serum. Cells from different fractions were studied for different characteristics as described below. Mi~ratlon inhibition test: The migration inhibition assay method as described by David and David (12) was followed. An indirect migration inhibition test was used. In short one million splenocytes (either unseparated or nylon wool and gradient separated) obtained from immunized lizards (effector cells) were mixed with three million splenocytes obtained from unimmunlzed lizards (indicator cells), packed into capillaries and MI assay carried out in the presence or absence of antigen as described earlier (11). Membrane immunofluorescence assay: The method described by Pernls et al, (13) was followed. In short, one million splenocytes suspended in 50 microlitre of L-15 medium supplemented with 2% BSA were incubated with rabbit anti-llzard Ig (specific for IgM) for 30 minutes at 4°C, washed thrice and then treated with FITC conjugated goat anti-rabblt IgG (Cappell, USA) for further 30 minutes at 4°C (14). Cells were washed and suspended in a medium containing nine parts glycerol and one part 0.5M carbonate buffer pH 9.5. Two hundred mononuclear cells per slide were counted twice using Zelss-Jena, Fluoval microscope and average obtained. Plaque formln~ cell assay: Cunnlngham and Szenberga's plaque forming cell technique as adopted for Calotes system (15) was utilized to detect the number of hemolysin producing cells in the spleens of immunized lizards. The reaction mixture containing 0.06 ml of 30% foetal calf serum, 0.01 ml of 10% SRBC, 0.01 ml of Calotes complement and 0.01 ml of effector cells were fed to monolayers
Vol.
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between two microscopic slides, edges sealed with paraffin, incubated for 30 mln. at 37°C and plaques of hemolysis counted using a low power stereo microscope
RESULTS Figure 1 presents the results on the susceptibility of various fractions of gradient separated unimmunized lizards thymocytes and splenocytes to anti-thymocyte serum (ATS). It is evident from Figure i that at the operational dilution of ATS used here all the (100% cytotoxlc index) thymocytes, but only 40% splenocytes were susceptible to it. While these cells were fractlonated based on density, a few interesting points emerged. All the thymocytes banding at density 1.081 gm/ml and 1.096 were susceptible to ATS (Fig. IA & B). Splenocytes banding at these densities were however not susceptible (Fig. IC & ID). On the contrary, majority of the splenocytes banding at d. 1.066 and 1.051 were susceptible to ATS. This clearly brought out the density differences between the unimmunized lizard splenocytes and thymocytes with reference to their susceptibility to ATS in question. The histograms in Figure 1 presents the total number of cells recovered in each density fraction and the total number of ATS susceptible cells in each one of them. All the thymocytes banding at d. 1.066 were susceptible to unabsorbed ATS (Fig. IA) but only 50% of them were susceptible to absorbed ATS. The ATS activity directed against a common antigenic determinant to liver and thymocytes seems to have been absorbed by the liver powder: The residual activity thereby detected a membrane antigenic determinant present both on high dense thymocytes and less dense (blasts) splenocytes. Table i presents the results on the distribution of cells in the unseparated, less dense (LD='I.066 gm/ml) to 1.08 gm/ml) fractions of normal lizard splenocytes. consisted more of ATS sensitive cells (70%) whereas more of Slg +ve cells(60%). This suggests that these enriched based on their buoyant density.
ATS sensitive and Slg +ve and high dense (HD=1.067 The less dense fraction the high dense consisted two types of cells can be
Table 2 presents the results on the distribution of migration inhibition effector cells in these fractions and the susceptibility of these cells to ATS and complement treatment. The unseparated splenocytes effected a 58% MI; but the HD effected only 34%. However upon treating these cells with ATS, this function was abrogated. This experiment revealed that the MI mediating effector cells could be enriched in LD fraction and ATS could abolish their MI function. These MI effector cells identified in different density fractions may belong to different stages of cell-cycle in their maturatlonal process. It is clear from the results presented here and in our earlier paper (ii) that nylon wool abherence column and density gradient analysis could enrich both ATS sensitive and SIg+ve cells and they are correlated with effector functions. However, the enrichment were not absolute. Hence the two techniques, one considering the buoyant density and another adherent properties of cells at different stages of maturation were coupled with an aim of isolating pure populations of functional subsets. Figure 2 presents evident from figure
the results on migration inhibition effector cells. It is 2A that the non-adherent less dense fraction consisted of
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Flg.l. Density distribution profile of normal lizard thymocytes (Flg IA, B) and splenocytes (Fig. IC, D) and their susceptibility to ATS: Thymocytes and splenocytes of fifteen normal lizards were pooled and subjected to dlscontlnous Isopycnlc gradient separation: the histogram presents the total number of cells obtained ~n each fraction and the hatchedarea represents the ATS sensitive cells among them. The graph presents the ATS susceptibility of each fraction as % cytotoxlc index. Fig IA & IB are the results on thymocytes; IC and ID on splenocytes; IA and IC using unabsorbed ATS and IB and ID using liver absorbed ATS. U = Unseparated lymphoid cells; %CI = Cytotoxlc index %.
30 15 0
• US 1.051
66
81
DENSITY
• 96
0 1.051
66
81
96
GM/ML
TABLE 2
TABLE 1
Distribution of migration inhibition effector cells in density f~actlons and their susceptibility to ATS
Slg +, ATS + cell in density fractions of normal lizard splenocytes
Splenocytes
Splenocytes
Total # Slg ATS
US
1000# 466 301
US
LD
HD
Treatment
18& 6 12
838 500 147
No antigen 6.0a + antigen 2.5 % MI 58.4
Cells are In 1 x i00,000; # = cells loaded; & = recoverd; Slg,surface Immunoglobulln;ATS, antlthymocyte serum sensitive; US, unseparated; LD, less dense (1.066 gm/ml); HD, hlgh dense (1.067 - 1.08 gm/ml)
nll
LESS DENSE
HIGH DENSE
nll
ATS
nll
ATS
10.7 2.8 73.6
7.1 7.1 0.0
4.9 3.2 33.8
6.2 5.9 4.1
Splenocytes obtained from llzards immunized wlth SRBC for CMI were subjected to discontinuous gradient separation. Less dense and high dense populations were subjected to ATS and complement treatment and their residual capacity to mediate MI assessed in the absence or presence of antigen, a = area of migration In square mm ; %MI = migration inhlbtlon In %.
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@
1.~0
¢o 0
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95
_J IJJ L)
40
m lO
-
÷
-
÷
-
÷
ATS LD NON
-
÷
-
÷
ATS HD ADHERENT
-
+
-
+
-
+
ATS
-
+
ATS
LD
HD
ADHERENT
Figure 2. Adherent and density distribution ~rofiles effector cells and their susceptibility to ATS.-
of migration
inhibition
Splenocytes obtained from lizards immunized for CMI and subjected to nylon wool column were further separated into less dense and high dense population in a discontinuous isopycnic gradient. These cells were treated with ATS and complement and their property to elicit migration inhibition assessed in the absence or presence of antigen. Firgure 2A presents the total number of splenocytes recovered in each fraction (open column) and the ATS sensitive cells in each fraction (solid bars). Figure 2B presents the area of migration of these cells in the absence (-) or presence (+) of antigen, sonlcated sheep erythrocytes. ATS denotes the migration after ATS and complement treatment. Other legends as in earlier figures.
95% ATS sensitive cells: it was only 32% in unseparated and 29% in non-adherent high dense fractions. Thus these two techniques coupled together seem to isolate majority of the ATS sensitive cells in non-adherent less dense fraction as a pure population. Figure 2B presents the results on the effector functions of these cells. In the presence of antigen, unseparated cells mediated an MI of 62%. The non-adherent LD cells again mediated 61% MI: of the 140 million cells processed, 50 million were obtained in this fraction. The non-adherent HD mediated 22% MI. The adherent LD cells mediated 45% MI: but there was only 3.2 million cells harvested in this fraction and 47% of it was ATS sensitive. The MI effector functions of all these cells were abrogated by ATS treatment (MI = 0). It is thus obvious that majority of the MI mediating ATS sensitive effector cells were enriched and isolated in non-adherent LD fraction: presemably they are the lymphoid cells of thymic lineage involved in MI response.
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BB 140
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Figure 3. Adherent and density di~trlbutlon cells and their susceptlblllty to ATS-.
L D
HD
ADHERENT
profiles
of
antibody
producing
Splenocytes obtained from lizards immunized for humeral immunity were subjected to nylon wool column and discontinuous gradient separation; subsequently these fractions were treated with ATS & complement and the number of antl-sheep erythrocyte plaque forming cells were enumerated. Figure 3A presents the total number of splenocytes recovered in each fraction (open clumn) and the ATS sensitive cells in them (solid bars). Open columns in Figure 3B presents the total number of plaque forming cells in each fraction and the solid bars, after ATS & complement treatment. Legends as in earlier figures. Efforts were also made to isolate antigen (SRBC) specific plaque forming cells (PFC) by these procedures. When the splenocytes obtained from lizards immunized for humeral immunity were subjected to these biophysical procedures, out of 130 million cells loaded, 66 million could be harvested in non-adherent LD fraction: but only 10% of it was sensitive to ATS (FIG. 3A). This is in contrast to the 95% sensitivity of the non-adherent LD, MI mediating cells (Fig. 2A). When the presence of anti SRBC plaque forming cells (PFC) were assessed in these fractions, again 104,000 PFC out of 140,000 PFC loaded were harvested in non-adherent LD fraction (Fig. 3B). The harvest in other fractions were only of the order of 10,000. When these splenocyte fractions were treated, with ATS and complement, there was no substantial reduction in the PFC count in any of these fractions (Fig. 2B) suggesting their insensitivity to ATS.
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DISCUSSION Two important points emerged from these experiments: firstly majority of the migration inhibition effector cells as well as the PFCs were harvested in non-adherent LD fraction. Secondly the less dense non-adhrent population of splenocyte effectlng MI but not PFC were sensitive to ATS. Since these effector cells were procured from immunized lizards during their earlier (exponential) phase of the immune responses presumably they were of less dense non-adherent cells: (cf.16) however the membrane antigenic markers of MI mediating cells were that of thymus derived lineage as identified by ATS. The heterologous ATS in question thus seems to identify a splenic non-adherent less dense functional T-cells of the lizard reaffirming the existence of a separate thymlc lineage in Calotes verslcolor. The conclusions that can be drawn from these experiments are that ATS and complement is able to kill cells involved in mlgratlon-inhlbltion, but not antibody-releaslng cells and these "T" and "B" cells (at the given stage of differentiation) are found in the same nylon wool non-adherent, low density fractions. The nylon wool adherence and buoyant density thus do not appear to separate the functional "T" and "B" (lymphoblasts) cells of the lizards clearly, but antibodies to cell surface determinants, do.
ACKNOWLEDGEMENTS The grant (7/201 (B2)/ 80-81-EMR-1) from Council of Scientific and Industrial Research, India to this study is gratefully acknowledged. We thank Ms. H a r i h a r a n and Subash Chandar f o r p r e p a r i n g the m a n u s c r i p t f o r p u b l i c a t i o n .
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