Immunology 1977 32 581
T- and B-cell surface markers on rabbit lymphocytes NICOLE SABOLOVIC, D. SABOLOVICI,* & A. MARIE GUILMIN Laboratoire du Centre de MWdecine Preventive, Vandoeuvre-les-Nancy, Laboratoire de Biochimie Pharmacologique de l'Universite de Nancy I and * INSERM U95, Plateau de Brabois, Vandoeuvre-les-Nancy, France
Received 5 August 1976;
accepted for publication 13 August 1976
respond to lipopolysaccharide (LPS) in vitro but are stimulated by soluble Con A (Sell & Sheppard, 1973) or phytohaemagglutinin (Sell, Rowe & Gell, 1965; Sell, Hughes & Mascari, 1970). They suggested that most rabbit peripheral blood lymphocytes may be T cells bearing immunoglobulins on their cell surfaces. These results are intriguing to say the least, because it is generally conceded that B cells are Ig-bearing cells (Pernis, Forni & Amante, 1970; Froland, Natvig & Berdal, 1971; Unanue, Grey, Rabellino, Campbell & Schmidtke, 1971) and that T cells or subpopulation of T cells could in some circumstances possess Ig determinants otherwise absent from their surfaces (Goldschneider & Cogen, 1973; Roelants, Ryden, Hagg & Loor, 1974; Grey, Kubo & Cerottini, 1972). The purpose of this study was to distinguish these populations in terms of known T- and B-cell characteristics comparing the Ig determinants (by a fluorescence method) with the cell surface charges (by an analytical cell electrophoresis method) and in vitro mitogen responsiveness. Other rabbit cell surface markers were also utilized: namely the rosette-forming activity with rat red cells (Braganza, Stathopoulos, Davies, Elliott & Kerbel, 1975), homologous red cell rosettes (Wilson, Gurner & Coombs 1975) and cell refringency (Pompidou & Schramm 1971). We repeated these examinations with cyclo-
Summary. Rabbit thymus, appendix, blood and lymph nodes were characterized using immunological tests: rosette-forming ability with homologous or heterologous red blood cells, surface immunoglobulins, analytical cell-electrophoresis, cell refringency and in vitro mitogen responsiveness. The experiments were conducted on normal rabbits and cyclophosphamide-treated rabbits. Moreover, we tried to separate slow and high mobility cells by free-flow electrophoresis into fractions. These experiments suggest that rabbit lymphocyte behaviour is exceptional among mammals in that the majority of their lymphocytes appeared as B cells and that, in comparison with mouse and man, the proportions of these cells are inversed. INTRODUCTION Sell & Gell (1965) reported the first clear evidence that immunoglobulin (Ig) determinants are present on the surface of most peripheral blood lymphocytes in the rabbit. Jones, Marcuson & Roitt (1970) found that up to 85 per cent of blood lymphocytes possess Ig determinants. Other authors indicated that rabbit peripheral blood lymphocytes and thymocytes do not Correspondence: Dr Nicole Sabolovid, Laboratoire du Centre de MWdecine Preventive, 2, avenue du Doyen Jacques Parisot, 54500 Vandoeuvre-l1s-Nancy, France. 0o
581
582
Nicole Sabolovic, D. Sabolovic' & A. Marie Guilmin
phosphamide-treated rabbits. Cyclophosphamide is known to decrease the number of lymphocytes in man and mouse and to preferentially affect B cells (Dumont, 1974). Moreover, we tried to separate T and B lymphocytes from peripheral blood by means of cell-surface charge differences using free-flow preparative electrophoresis (Donner, 1976).
MATERIALS AND METHODS Donors 'Fauve de Bourgogne' male rabbits, approximately 2 kg in weight, were used throughout the experiments.
Lymphocytes preparation Lymphocytes obtained from the marginal vein or by cardiac puncture, were separated from the heparinized blood by careful layering on to FicollMetrizoate (Boyum, 1968) and centrifuging at room temperature for 20 min at 400 g. The lymphocytes were removed from the Ficoll-Isopaque interface and were washed three times in medium 199. Cell suspensions were obtained from the thymus, appendix and popliteal node. The tissues were cut into small pieces and the cells were separated with a Potter homogenizer, then washed three times in medium 199. Before this treatment, the appendix was washed ten times in NaCI. The cells were counted and the viability was tested with trypan blue. After several attempts, the study of spleen lymphocytes was abandoned because of contamination of the lymphocyte suspension with platelets and erythrocytes.
Analytical electrophoresis The lymphocytes were washed twice with cold NaCI 0-145 M (pH 7 2) and used immediately. The electrophoretic mobility measurements were carried out in a cylindrical apparatus, MARK I (Rank Brothers, Bottisham, Cambridge) with a capillary tube (100 x 2 mm) in a water bath at 250 (Sabolovi6, 1972; Sabolovi6, Dumont, Sabolovic, Chollet & Amiel, 1973; Sabolovi6, Sabolovi6, Dumont & Siest, 1974). Between 100 and 150 cells were scored in both directions reversing the polarity. The electrophoretic mobilities (EM) are expressed in ,um/s - I/v- I/cm and plotted on histograms.
Free-flow preparative electrophoresis Free-flow electrophoresis was performed with an FF 5 apparatus (Bender and Hobein, Munich). Cells were introduced continuously into a stream of buffer by which they were carried with defined velocity across an electric field (85 V/cm). They deviated according to the surface charge and were collected in centrifuge tubes, after which the number of cells was counted. The separations were performed at 6° with a buffer flow rate of 400 ml/h and the cell suspension (10 x 106/ml) was introduced at a rate of 2-3 ml/h. Separation buffer Triethanolamine 0 015 M Potassium acetate 0 004 M Glycine 0-24 M A glucose-saccharose solution was added to this buffer until an osmolarity of 290 milliosmoles was obtained. Electrode buffer Triethanolamine 0075 M Potassium acetate 004 M In both cases, the pH was adjusted to 7 4 with acetic acid. Test procedures for rosettes (RFC) Two tests were employed: (1) rosette formation with homologous erythrocytes (Wilson et al., 1975); (2) rosette formation with rat erythrocytes (Braganza et al., 1975). In both tests the erythrocytes were washed three times in saline solution and suspended at a 1 per cent concentration in medium 199. One million lymphocytes in 0-25 ml medium 199 were mixed with 025 ml erythrocyte suspension. Inactivated foetal calf serum (50 u1) previously heated at 560 for 30 min and absorbed twice with rabbit and rats erythrocytes was added. The mixture was left to stand at room temperature (approximately 200) for 15 min and after centrifugation for 5 min at 200 g the pellet was left at 4° overnight. The cell pellets were resuspended by gentle rocking. A total of 200 lymphocytes were counted per tube and RFC were scored as those cells with at least three red blood cells attached to the surface.
Immunofluorescence Fluorescein-conjugated sheep antiserum to rabbit
T- and B-cell surface markers
583 RESULTS
immunoglobulins was purchased from Wellcome Laboratories 3 X 106 cells were incubated with fluorescent antiserum at a dilution of 1/6. The lymphocytes were incubated at 40 for 30 min washed twice in Medium 199, and 200 cells were examined by fluorescence microscopy.
(1) Cell surface markers on lymphocytes from thymus, lymph nodes, blood and appendix of normal rabbits In this series of ten experiments, we tested cellsurface immunoglobulins by the immunofluorescence method, the capacity of resetting with homologous heterologous red cells, refringency and finally cell electrophoresis. The results as shown in Table 1 reveal a rather marked heterogeneity from one organ to another; Ig-positive cells were absent from thymus and mostly present in blood and appendix; homologous rosettes were found predominantly in the thymus, then in lymph nodes and to a lesser degree in blood and appendix. Heterologous rosettes (rat red cells) were present in small numbers in all organs or absent completely (sheep red cells). Cell refringency was elevated in the appendix and lymph node. Cell electrophoresis gave almost identical results in all organs: most cells had slow EM characteristics and only in some circumstances was a small percentage of rapidly migrating cells present that could be detected by this method. When the results of lymphocyte EM were plotted on histograms (Fig. 1), this dominant slow-moving population, and a small tail of less than 5 per cent of high EM cells could be visualized. This situation in rabbit lymphoid organs contrasts with mouse or human lymphocytes where a population of high EM is dominant. Lymphocytes in lymph nodes had a mean EM higher than that of lymphocytes in the blood, thymus or appendix. This is to be compared with the higher percentage of homologous rosetteforming cells (possible T-cell marker) and the lower percentage of Ig-bearing cells.
Lymphocyte refringence The existence of a peculiar refringence phenomenon was described by Pompidou & Schramm (1971) and Pompidou (1976). We used this technique with Zenker fixation for 10 min at 900. After mounting with Techniconmountingmedium, cells were observed by phase contrast microscope. The refringent positive cells appeared as brilliant stars in comparison to the negative cells which had a dull appearance. In vitro mitogen responsiveness
For the determination of mitogen responsiveness, lymphocytes were cultivated in RPMI 1640 Medium supplemented with antibiotics without serum at a concentration of 2 x 105 lymphocytes per ml. The cultures were done in triplicate and 10 jig, 25 4ug and 50 pg of PHA-P (Difco) and 2 ug, 5 /ug and 10 pg of Con A (Calbiochem) in 004 ml were added. Microplates were incubated at 370 in a humidified atmosphere of 95 per cent air, 5 per cent CO2. 24 h later, [3H]thymidine (1 puCi, sp. act. 27 Ci/mM) was introduced in 002 ml. The cultures were harvested after a 72 h period, filtered on glass fibre filters and dissolved in Triton X-100 (Packard) scintillation fluid.
Table 1. Results with blood and organs lymphocytes from normal rabbits Per cent lymphoid cell rosettes with
Lymphocytes origin Blood Thymus Popliteal lymph node Appendix
EM
Ig positive cells
("m/s 1/v -/cm)
(%)
Rat red cells
Homologous red cells
Sheep red cells
(%)
0-78 0-82
79
T- and B-cell surface markers on rabbit lymphocytes NICOLE SABOLOVIC, D. SABOLOVICI,* & A. MARIE GUILMIN Laboratoire du Centre de MWdecine Preventive, Vandoeuvre-les-Nancy, Laboratoire de Biochimie Pharmacologique de l'Universite de Nancy I and * INSERM U95, Plateau de Brabois, Vandoeuvre-les-Nancy, France
Received 5 August 1976;
accepted for publication 13 August 1976
respond to lipopolysaccharide (LPS) in vitro but are stimulated by soluble Con A (Sell & Sheppard, 1973) or phytohaemagglutinin (Sell, Rowe & Gell, 1965; Sell, Hughes & Mascari, 1970). They suggested that most rabbit peripheral blood lymphocytes may be T cells bearing immunoglobulins on their cell surfaces. These results are intriguing to say the least, because it is generally conceded that B cells are Ig-bearing cells (Pernis, Forni & Amante, 1970; Froland, Natvig & Berdal, 1971; Unanue, Grey, Rabellino, Campbell & Schmidtke, 1971) and that T cells or subpopulation of T cells could in some circumstances possess Ig determinants otherwise absent from their surfaces (Goldschneider & Cogen, 1973; Roelants, Ryden, Hagg & Loor, 1974; Grey, Kubo & Cerottini, 1972). The purpose of this study was to distinguish these populations in terms of known T- and B-cell characteristics comparing the Ig determinants (by a fluorescence method) with the cell surface charges (by an analytical cell electrophoresis method) and in vitro mitogen responsiveness. Other rabbit cell surface markers were also utilized: namely the rosette-forming activity with rat red cells (Braganza, Stathopoulos, Davies, Elliott & Kerbel, 1975), homologous red cell rosettes (Wilson, Gurner & Coombs 1975) and cell refringency (Pompidou & Schramm 1971). We repeated these examinations with cyclo-
Summary. Rabbit thymus, appendix, blood and lymph nodes were characterized using immunological tests: rosette-forming ability with homologous or heterologous red blood cells, surface immunoglobulins, analytical cell-electrophoresis, cell refringency and in vitro mitogen responsiveness. The experiments were conducted on normal rabbits and cyclophosphamide-treated rabbits. Moreover, we tried to separate slow and high mobility cells by free-flow electrophoresis into fractions. These experiments suggest that rabbit lymphocyte behaviour is exceptional among mammals in that the majority of their lymphocytes appeared as B cells and that, in comparison with mouse and man, the proportions of these cells are inversed. INTRODUCTION Sell & Gell (1965) reported the first clear evidence that immunoglobulin (Ig) determinants are present on the surface of most peripheral blood lymphocytes in the rabbit. Jones, Marcuson & Roitt (1970) found that up to 85 per cent of blood lymphocytes possess Ig determinants. Other authors indicated that rabbit peripheral blood lymphocytes and thymocytes do not Correspondence: Dr Nicole Sabolovid, Laboratoire du Centre de MWdecine Preventive, 2, avenue du Doyen Jacques Parisot, 54500 Vandoeuvre-l1s-Nancy, France. 0o
581
582
Nicole Sabolovic, D. Sabolovic' & A. Marie Guilmin
phosphamide-treated rabbits. Cyclophosphamide is known to decrease the number of lymphocytes in man and mouse and to preferentially affect B cells (Dumont, 1974). Moreover, we tried to separate T and B lymphocytes from peripheral blood by means of cell-surface charge differences using free-flow preparative electrophoresis (Donner, 1976).
MATERIALS AND METHODS Donors 'Fauve de Bourgogne' male rabbits, approximately 2 kg in weight, were used throughout the experiments.
Lymphocytes preparation Lymphocytes obtained from the marginal vein or by cardiac puncture, were separated from the heparinized blood by careful layering on to FicollMetrizoate (Boyum, 1968) and centrifuging at room temperature for 20 min at 400 g. The lymphocytes were removed from the Ficoll-Isopaque interface and were washed three times in medium 199. Cell suspensions were obtained from the thymus, appendix and popliteal node. The tissues were cut into small pieces and the cells were separated with a Potter homogenizer, then washed three times in medium 199. Before this treatment, the appendix was washed ten times in NaCI. The cells were counted and the viability was tested with trypan blue. After several attempts, the study of spleen lymphocytes was abandoned because of contamination of the lymphocyte suspension with platelets and erythrocytes.
Analytical electrophoresis The lymphocytes were washed twice with cold NaCI 0-145 M (pH 7 2) and used immediately. The electrophoretic mobility measurements were carried out in a cylindrical apparatus, MARK I (Rank Brothers, Bottisham, Cambridge) with a capillary tube (100 x 2 mm) in a water bath at 250 (Sabolovi6, 1972; Sabolovi6, Dumont, Sabolovic, Chollet & Amiel, 1973; Sabolovi6, Sabolovi6, Dumont & Siest, 1974). Between 100 and 150 cells were scored in both directions reversing the polarity. The electrophoretic mobilities (EM) are expressed in ,um/s - I/v- I/cm and plotted on histograms.
Free-flow preparative electrophoresis Free-flow electrophoresis was performed with an FF 5 apparatus (Bender and Hobein, Munich). Cells were introduced continuously into a stream of buffer by which they were carried with defined velocity across an electric field (85 V/cm). They deviated according to the surface charge and were collected in centrifuge tubes, after which the number of cells was counted. The separations were performed at 6° with a buffer flow rate of 400 ml/h and the cell suspension (10 x 106/ml) was introduced at a rate of 2-3 ml/h. Separation buffer Triethanolamine 0 015 M Potassium acetate 0 004 M Glycine 0-24 M A glucose-saccharose solution was added to this buffer until an osmolarity of 290 milliosmoles was obtained. Electrode buffer Triethanolamine 0075 M Potassium acetate 004 M In both cases, the pH was adjusted to 7 4 with acetic acid. Test procedures for rosettes (RFC) Two tests were employed: (1) rosette formation with homologous erythrocytes (Wilson et al., 1975); (2) rosette formation with rat erythrocytes (Braganza et al., 1975). In both tests the erythrocytes were washed three times in saline solution and suspended at a 1 per cent concentration in medium 199. One million lymphocytes in 0-25 ml medium 199 were mixed with 025 ml erythrocyte suspension. Inactivated foetal calf serum (50 u1) previously heated at 560 for 30 min and absorbed twice with rabbit and rats erythrocytes was added. The mixture was left to stand at room temperature (approximately 200) for 15 min and after centrifugation for 5 min at 200 g the pellet was left at 4° overnight. The cell pellets were resuspended by gentle rocking. A total of 200 lymphocytes were counted per tube and RFC were scored as those cells with at least three red blood cells attached to the surface.
Immunofluorescence Fluorescein-conjugated sheep antiserum to rabbit
T- and B-cell surface markers
583 RESULTS
immunoglobulins was purchased from Wellcome Laboratories 3 X 106 cells were incubated with fluorescent antiserum at a dilution of 1/6. The lymphocytes were incubated at 40 for 30 min washed twice in Medium 199, and 200 cells were examined by fluorescence microscopy.
(1) Cell surface markers on lymphocytes from thymus, lymph nodes, blood and appendix of normal rabbits In this series of ten experiments, we tested cellsurface immunoglobulins by the immunofluorescence method, the capacity of resetting with homologous heterologous red cells, refringency and finally cell electrophoresis. The results as shown in Table 1 reveal a rather marked heterogeneity from one organ to another; Ig-positive cells were absent from thymus and mostly present in blood and appendix; homologous rosettes were found predominantly in the thymus, then in lymph nodes and to a lesser degree in blood and appendix. Heterologous rosettes (rat red cells) were present in small numbers in all organs or absent completely (sheep red cells). Cell refringency was elevated in the appendix and lymph node. Cell electrophoresis gave almost identical results in all organs: most cells had slow EM characteristics and only in some circumstances was a small percentage of rapidly migrating cells present that could be detected by this method. When the results of lymphocyte EM were plotted on histograms (Fig. 1), this dominant slow-moving population, and a small tail of less than 5 per cent of high EM cells could be visualized. This situation in rabbit lymphoid organs contrasts with mouse or human lymphocytes where a population of high EM is dominant. Lymphocytes in lymph nodes had a mean EM higher than that of lymphocytes in the blood, thymus or appendix. This is to be compared with the higher percentage of homologous rosetteforming cells (possible T-cell marker) and the lower percentage of Ig-bearing cells.
Lymphocyte refringence The existence of a peculiar refringence phenomenon was described by Pompidou & Schramm (1971) and Pompidou (1976). We used this technique with Zenker fixation for 10 min at 900. After mounting with Techniconmountingmedium, cells were observed by phase contrast microscope. The refringent positive cells appeared as brilliant stars in comparison to the negative cells which had a dull appearance. In vitro mitogen responsiveness
For the determination of mitogen responsiveness, lymphocytes were cultivated in RPMI 1640 Medium supplemented with antibiotics without serum at a concentration of 2 x 105 lymphocytes per ml. The cultures were done in triplicate and 10 jig, 25 4ug and 50 pg of PHA-P (Difco) and 2 ug, 5 /ug and 10 pg of Con A (Calbiochem) in 004 ml were added. Microplates were incubated at 370 in a humidified atmosphere of 95 per cent air, 5 per cent CO2. 24 h later, [3H]thymidine (1 puCi, sp. act. 27 Ci/mM) was introduced in 002 ml. The cultures were harvested after a 72 h period, filtered on glass fibre filters and dissolved in Triton X-100 (Packard) scintillation fluid.
Table 1. Results with blood and organs lymphocytes from normal rabbits Per cent lymphoid cell rosettes with
Lymphocytes origin Blood Thymus Popliteal lymph node Appendix
EM
Ig positive cells
("m/s 1/v -/cm)
(%)
Rat red cells
Homologous red cells
Sheep red cells
(%)
0-78 0-82
79
