Immunology 1979 37 857
Ontogeny of spontaneous antigen-binding cells in developing chick embryos
0.
MORIYA & Y. ICHIKAWA Department of Bacteriology, Saitama Medical School, Moroyama, Iruma-gun,
Saitama, Japan
Acceptedfor publication 22 February 1979
has not been well understood. For fuller understanding of the functional development in embryonic immune system, detailed studies seemed to be required with respect to the development of various clones of ABC. Red blood cells provide a convenient source for natural antigens and the rosette assay is a reliable method for detecting lymphocytes capable of binding red blood cell surface antigens. In the studies presented here, we examined the number of ABC to various mammalian red blood cell antigens in both the bursa of Fabricius and the spleen of chick embryos, as a function of developmental stages by the use of rosette assay.
Summary. Bursal and splenic lymphocytes in developing chick embryos were examined for the number of antigen binding cells (ABC) which could form rosettes with red blood cells from sheep, guinea-pig, rabbit or dinitrophenylated sheep red blood cells (DNP-SRBC). The kinetics of the development of bursal ABC to rabbit red blood cells (RRBC) did not vary significantly with age of embryo examined, and its frequency was constantly high. The number ofcells which bind to SRBC, guinea-pig red blood cells (GPRBC) or DNP-SRBC was low in the bursa and increased in an approximately linear fashion with the age of embryo. With the exception of RRBC-ABC, similar results were obtained with splenic ABC. Furthermore, the frequencies of ABC were higher in the spleen than in the bursa. These events suggest the antigen-independent generation of clonal diversity during the developmental stage of chick embryos.
MATERIALS AND METHODS Chickens Babcock B-300 white Leghorn eggs were used. Eggs were obtained from Niizuma Hatchery, Chiba, and were incubated at 39.50 with relative humidity at 65%.
INTRODUCTION
Immunological competence is known to vary with age. During the embryonic stages, the number of immunoglobulin-bearing cells (IgBC) continues to increase with age of embryo (Albini & Wick, 1973). The development of antigen-binding cells (ABC), however, Correspondence: 0. Moriya, Department of Bacteriology,
Antigens Sheep rcd blood cells (SRBC) were obtained from Bio Test Lab., Tokyo. Rabbit red blood cells (RRBC) and guinea-pig red blood cells (GPRBC) were obtained from animals maintained in our institution. Dinitrophenylated SRBC (DNP-SRBC) were prepared by coupling SRBC with 2,4-dinitrofluorobenzene according to the method of Scheper & Oort (1974). To determine whether or not lymphocytes were bound specifically to antigen, 2,4-dinitrobenzene (DNP; Tokyo
Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun, Saitama, 350-04, Japan. 00 1 9-2805/79/0800-0857502.00 A) 1979 Blackwell Scientific Publications
857
858
0.
Moriya & Y. Ichikawa
Kasei, Tokyo) and red blood cells lysed by exposure to ammonium chloride were used. Lymphocyte suspension Chick embryos were killed by bleeding from the allantoic vein. The bursa and spleen were removed, cut with scissors, minced with a glass homogenizer in phosphate butfered saline (PBS; pH 7-2) and the lymphocyte suspension prepared as described previously (Moriya & Ichikawa, 1978). Anti-chicken immunoglobulin serum Anti-chicken immunoglobulin serum used for the rosette-inhibition test was raised in rabbits by injecting chicken immunoglobulin intraperitoneally and subcutaneously. The injections were given four times at weekly intervals, and the final injection was given 10 days later. Serum was collected 10 days after the last immunization. The specificity and the titre of the antisera were checked with immunoelectrophoresis and immunoprecipitation in agar. The antisera used contained antibodies both to chicken IgM and IgG and possibly also to IgA. The subagglutinating dilution of the rabbit anti-chicken immunoglobulin serum for a 1 -0% suspension of bursal cells in RPMI 1640 medium was 1:512. This dilution of the antiserum was subsequently used for pre-treatment of lymphocytes in all rosette-inhibition tests. Detection of antigen-binding cells To test antigen-binding cells (ABC) in foetal lymphocytes, the rosette assay of Tufveson, Bach & van Alten (1974) was used. Bursal or. splenic lymphocytes (2.5 x 106 cells) were mixed with red blood cells (2-5 x 107 cells) in 1-0 ml of PBS, and incubated for 30 min at 37°. Then the cell suspension was centrifuged at 100 g for 5 min, incubated for 2 h at 40, and the resulting cell pellets were then resuspended with a capillary pipette. One hundred microlitres of 7% glutaraldehyde were added to the mixed cell suspension, and incubated for 30 min at 40 for fixation. The rosette mixture was layered onto 2 ml of Ficoll-Isopaque (d= 1-077), and centrifuged for 20 min at 300 g at room temperature. The fractions containing rosettes and free lymphocytes were collected, and were put on a glass slide. The cells were fixed with absolute methanol for 2 min followed by staining with May-GrunwaldGiemsa solution. The frequency of ABC was examined by counting at least 106 cells under a light microscope at a magnification of 100. Lymphocytes binding more than 5 RBC were regarded as positive cells.
Inhibition of rosette formation Prior to incubation with RBC, lymphocytes were reacted with either rabbit anti-chicken immunoglobulin serum or normal rabbit serum for 60 min at 370, and then incubated for 20 min at 40 (Dresser & Greaves, 1973). The cells were washed three times with cold PBS and then incubated with RBC for rosette assay as described above. In another test for the binding specificity, lymphocytes (106 cells) were treated with lysed RBCs (105 cells) or DNP for 20 min at 370 before addition of RBC for rosette assay.
RESULTS Cell types binding to RBC Cells prepared by the Ficoll-Isopaque method did not separate lymphocytes accurately from other cell populations (>90%). Cells separated by Ficoll-Isopaque contained lymphocytes as a major population as described above and monocytes, macrophages, heteroTable 1. The identification of cell types which bind to RBC
Rosette formation to RBC
Cell type*
Lymphocytes Macrophage Monocytes Heterophils Red blood cells
SRBC DNP-SRBC RRBC GPRBC + + + + -
+ + + + -
+ + + +
-
+ + + +
-
* Cells were obtained from the 20th embryonic bursa and spleen
phils and erythrocytes were detected in these suspensions as minor populations. To determine which cell types had receptors for RBC in chickens, smears of ABC were stained with May-Grunwald-Giemsa and examined microscopically. Table I represents the cell types which bound to RBC. Lymphocytes, monocytes, macrophages and heterophils attached RBC. On the other hand, chicken RBC were never observed in binding. Frequency of ABC in bursa The frequency of bursal cells spontaneously binding to either SRBC, GPRBC, RRBC or DNP-SRBC was determined on the 14th, 16th, 18th and 20th
ABC in developing chick embryos Table 2. Relative frequency of ABC to SRBC, RRBC, GPRBC or DNP-SRBC in the bursa of Fabricius as a function of developmental stages No. of ABC/ l 05 cells (mean + SE*)
Indicator cell
Day 14
0-2+0-1 SRBC 43-0+6-5 RRBC NDt GPRBC DNP-SRBC 0-6+0-4
Day 16
Day 18
Day 20
4-1+0-8 9-5+ 13 12-9+15 36-8+5-9 32-7+4-1 28-8+3-7 6-9+1-3 12 0+1-7 18-4+2-9 9-3+1-7 12 9+±18 15-6+19
* Mean of triplicate and ten embryos were used in each group.
t Not detected.
embryonic days. The results presented in Table 2 demonstrate that the bursal ABC to SRBC was first detected on the 14th embryonic day and then increased in number with age. Similar appearance and increase was detected in the population of ABC to DNP-SRBC. In contrast, bursal ABC to GPRBC was not detected until the 16th embryonic day. Once these ABC were detected in the bursa, the number ofABC to GPRBC then increased rapidly as seen in the population ofABC to SRBC or DNP-SRBC from the 16th to 20th embryonic day. Bursal cells binding to RRBC were detected on the 14th embryonic day with higher frequency than those binding to either SRBC, DNP-SRBC or GPRBC. Furthermore, the frequency of RRBC-ABC was high on the 16th and 18th embryonic days, and was then comparable with other ABC on the 20th embryonic day. Frequency of ABC in spleen Table 3 shows the results obtained when splenic cells
859
were examined for the number of ABC. Splenic cells which could bind to SRBC, GPRBC or DNP-SRBC were first detected on the 16th embryonic day. On the other hand, the population of RRBC-ABC was detected in the spleen on the 14th embryonic day as was shown in bursa and its frequency decreased gradually with age of embryo examined and was lower on the 18th and 20th embryonic days. Although the development of splenic ABC to various RBC was the same as detected in the bursa, the appearance of splenic ABC shifted later when compared with bursal ABC. Furthermore, with the exception of the RRBC-ABC population, spleen contained approximately ten times more ABC than did the bursa during the embryonic stages.
Inhibition of antigen binding by anti-chicken immunoglobulim The numbers of bursal and splenic ABC collected on the 20th embryonic day exposed to either rabbit antichicken immunoglobulins or normal rabbit serum are shown in Table 4. The normal level of ABC treated with normal serum indicated the absence of suppressive effect on antigen binding in bursal cells. Reduction in the number of those treated with anti-chicken immunoglobulin serum, however, clearly indicated the presence of suppressive property in antigen binding (P < 0 005). A similar effect of anti-chicken immunoglobulin serum was detected in the population of splenic ABC (Table 4).
Inhibition of antigen binding by lysed RBC or free hapten The specificity of antigen binding was further investigated. Lymphocytes prepared from the spleens of 20 day embryos were exposed to lysed RBC or DNP for
Table 3. Relative frequency of ABC to SRBC, RRBC, GPRBC or DNP-SRBC in the spleen as a function of developmental stages No. of ABC/105 cells (mean ± SE*) Indicator cell
Day 14
SRBC NDt RRBC 61-2+11-2 ND GPRBC ND DNP-SRBC *
Day 16 57-2+6-4 47-3 +7-8 60-2+12-6
61-3+91
Day 18
Day 20
85-3 ± 8-6 78-3 + 7-9 44-5 ±7-2 39-0±6-2 91-6+17-4 109-8+19-7 113-0+150 122-4+12-6
Mean of triplicate and ten embryos were used in each group.
t Not detected.
0. Moriya & Y. Ichikawa
860
Table 4. Effect of pre-treatment of lymphocytes with anti-chicken immunoglobulin serum on the relative frequency of ABC in bursa of Fabricius and spleen on the 20th embryonic day
Inhibition with serum (%)
Source cell
Indicator cell
No. of ABC/105 cells Anti-Ig Normal
SRBC RRBC GPRBC DNP-SRBC Spleen SRBC RRBC GRPBC DNP-SRBC
Bursa
14 0 274 18 7 16 5 91-4 43 0 113-8 109-7
95-2 98-1 95-1 95-7 97 2 95 2 98 3 99-6
24 0 11-6 21 4 20 2 23-8 16 8 27-7 23-8
* Mean of triplicate and ten embryos were used in each group. Table 5. Effect of pre-treatment of splenic lymphocytes with lysed RBC or DNP on the relative frequency of ABC
Treatment
Indicator cell No. of ABC/b15 cells
SRBC Saline SRBC Lysed SRBC* SRBC Lysed RRBC* SRBC Lysed GPRBC* SRBC DNPI DNP-SRBC Saline DNP-SRBC DNP DNP-SRBC Lysed SRBC* RRBC Saline RRBC Lysed RRBC* RRBC Lysed SRBC* RRBC Lysed GPRBC* GPRBC Saline GPRBC Lysed GPRBC* GPRBC Lysed RRBC* GPRBC Lysed SRBC*
83 5 28 64-5 68-0
79.3 96 7 5-1 36 0 48-1 51 40 8 38-9 120 2 21 5 103-6 98 5
* 105 RBC were used. t DNP (1 0 mg/ml -)
was used in 0 1 ml of the solution for pre-treatment with lymphocytes.
20 min at 370 before addition of RBC for the rosette assay (Table 5). Binding of guinea-pig, rabbit and sheep RBC was completely inhibited by lysed RBC of each respective species (P
Ontogeny of spontaneous antigen-binding cells in developing chick embryos
0.
MORIYA & Y. ICHIKAWA Department of Bacteriology, Saitama Medical School, Moroyama, Iruma-gun,
Saitama, Japan
Acceptedfor publication 22 February 1979
has not been well understood. For fuller understanding of the functional development in embryonic immune system, detailed studies seemed to be required with respect to the development of various clones of ABC. Red blood cells provide a convenient source for natural antigens and the rosette assay is a reliable method for detecting lymphocytes capable of binding red blood cell surface antigens. In the studies presented here, we examined the number of ABC to various mammalian red blood cell antigens in both the bursa of Fabricius and the spleen of chick embryos, as a function of developmental stages by the use of rosette assay.
Summary. Bursal and splenic lymphocytes in developing chick embryos were examined for the number of antigen binding cells (ABC) which could form rosettes with red blood cells from sheep, guinea-pig, rabbit or dinitrophenylated sheep red blood cells (DNP-SRBC). The kinetics of the development of bursal ABC to rabbit red blood cells (RRBC) did not vary significantly with age of embryo examined, and its frequency was constantly high. The number ofcells which bind to SRBC, guinea-pig red blood cells (GPRBC) or DNP-SRBC was low in the bursa and increased in an approximately linear fashion with the age of embryo. With the exception of RRBC-ABC, similar results were obtained with splenic ABC. Furthermore, the frequencies of ABC were higher in the spleen than in the bursa. These events suggest the antigen-independent generation of clonal diversity during the developmental stage of chick embryos.
MATERIALS AND METHODS Chickens Babcock B-300 white Leghorn eggs were used. Eggs were obtained from Niizuma Hatchery, Chiba, and were incubated at 39.50 with relative humidity at 65%.
INTRODUCTION
Immunological competence is known to vary with age. During the embryonic stages, the number of immunoglobulin-bearing cells (IgBC) continues to increase with age of embryo (Albini & Wick, 1973). The development of antigen-binding cells (ABC), however, Correspondence: 0. Moriya, Department of Bacteriology,
Antigens Sheep rcd blood cells (SRBC) were obtained from Bio Test Lab., Tokyo. Rabbit red blood cells (RRBC) and guinea-pig red blood cells (GPRBC) were obtained from animals maintained in our institution. Dinitrophenylated SRBC (DNP-SRBC) were prepared by coupling SRBC with 2,4-dinitrofluorobenzene according to the method of Scheper & Oort (1974). To determine whether or not lymphocytes were bound specifically to antigen, 2,4-dinitrobenzene (DNP; Tokyo
Saitama Medical School, 38 Morohongo, Moroyama, Iruma-gun, Saitama, 350-04, Japan. 00 1 9-2805/79/0800-0857502.00 A) 1979 Blackwell Scientific Publications
857
858
0.
Moriya & Y. Ichikawa
Kasei, Tokyo) and red blood cells lysed by exposure to ammonium chloride were used. Lymphocyte suspension Chick embryos were killed by bleeding from the allantoic vein. The bursa and spleen were removed, cut with scissors, minced with a glass homogenizer in phosphate butfered saline (PBS; pH 7-2) and the lymphocyte suspension prepared as described previously (Moriya & Ichikawa, 1978). Anti-chicken immunoglobulin serum Anti-chicken immunoglobulin serum used for the rosette-inhibition test was raised in rabbits by injecting chicken immunoglobulin intraperitoneally and subcutaneously. The injections were given four times at weekly intervals, and the final injection was given 10 days later. Serum was collected 10 days after the last immunization. The specificity and the titre of the antisera were checked with immunoelectrophoresis and immunoprecipitation in agar. The antisera used contained antibodies both to chicken IgM and IgG and possibly also to IgA. The subagglutinating dilution of the rabbit anti-chicken immunoglobulin serum for a 1 -0% suspension of bursal cells in RPMI 1640 medium was 1:512. This dilution of the antiserum was subsequently used for pre-treatment of lymphocytes in all rosette-inhibition tests. Detection of antigen-binding cells To test antigen-binding cells (ABC) in foetal lymphocytes, the rosette assay of Tufveson, Bach & van Alten (1974) was used. Bursal or. splenic lymphocytes (2.5 x 106 cells) were mixed with red blood cells (2-5 x 107 cells) in 1-0 ml of PBS, and incubated for 30 min at 37°. Then the cell suspension was centrifuged at 100 g for 5 min, incubated for 2 h at 40, and the resulting cell pellets were then resuspended with a capillary pipette. One hundred microlitres of 7% glutaraldehyde were added to the mixed cell suspension, and incubated for 30 min at 40 for fixation. The rosette mixture was layered onto 2 ml of Ficoll-Isopaque (d= 1-077), and centrifuged for 20 min at 300 g at room temperature. The fractions containing rosettes and free lymphocytes were collected, and were put on a glass slide. The cells were fixed with absolute methanol for 2 min followed by staining with May-GrunwaldGiemsa solution. The frequency of ABC was examined by counting at least 106 cells under a light microscope at a magnification of 100. Lymphocytes binding more than 5 RBC were regarded as positive cells.
Inhibition of rosette formation Prior to incubation with RBC, lymphocytes were reacted with either rabbit anti-chicken immunoglobulin serum or normal rabbit serum for 60 min at 370, and then incubated for 20 min at 40 (Dresser & Greaves, 1973). The cells were washed three times with cold PBS and then incubated with RBC for rosette assay as described above. In another test for the binding specificity, lymphocytes (106 cells) were treated with lysed RBCs (105 cells) or DNP for 20 min at 370 before addition of RBC for rosette assay.
RESULTS Cell types binding to RBC Cells prepared by the Ficoll-Isopaque method did not separate lymphocytes accurately from other cell populations (>90%). Cells separated by Ficoll-Isopaque contained lymphocytes as a major population as described above and monocytes, macrophages, heteroTable 1. The identification of cell types which bind to RBC
Rosette formation to RBC
Cell type*
Lymphocytes Macrophage Monocytes Heterophils Red blood cells
SRBC DNP-SRBC RRBC GPRBC + + + + -
+ + + + -
+ + + +
-
+ + + +
-
* Cells were obtained from the 20th embryonic bursa and spleen
phils and erythrocytes were detected in these suspensions as minor populations. To determine which cell types had receptors for RBC in chickens, smears of ABC were stained with May-Grunwald-Giemsa and examined microscopically. Table I represents the cell types which bound to RBC. Lymphocytes, monocytes, macrophages and heterophils attached RBC. On the other hand, chicken RBC were never observed in binding. Frequency of ABC in bursa The frequency of bursal cells spontaneously binding to either SRBC, GPRBC, RRBC or DNP-SRBC was determined on the 14th, 16th, 18th and 20th
ABC in developing chick embryos Table 2. Relative frequency of ABC to SRBC, RRBC, GPRBC or DNP-SRBC in the bursa of Fabricius as a function of developmental stages No. of ABC/ l 05 cells (mean + SE*)
Indicator cell
Day 14
0-2+0-1 SRBC 43-0+6-5 RRBC NDt GPRBC DNP-SRBC 0-6+0-4
Day 16
Day 18
Day 20
4-1+0-8 9-5+ 13 12-9+15 36-8+5-9 32-7+4-1 28-8+3-7 6-9+1-3 12 0+1-7 18-4+2-9 9-3+1-7 12 9+±18 15-6+19
* Mean of triplicate and ten embryos were used in each group.
t Not detected.
embryonic days. The results presented in Table 2 demonstrate that the bursal ABC to SRBC was first detected on the 14th embryonic day and then increased in number with age. Similar appearance and increase was detected in the population of ABC to DNP-SRBC. In contrast, bursal ABC to GPRBC was not detected until the 16th embryonic day. Once these ABC were detected in the bursa, the number ofABC to GPRBC then increased rapidly as seen in the population ofABC to SRBC or DNP-SRBC from the 16th to 20th embryonic day. Bursal cells binding to RRBC were detected on the 14th embryonic day with higher frequency than those binding to either SRBC, DNP-SRBC or GPRBC. Furthermore, the frequency of RRBC-ABC was high on the 16th and 18th embryonic days, and was then comparable with other ABC on the 20th embryonic day. Frequency of ABC in spleen Table 3 shows the results obtained when splenic cells
859
were examined for the number of ABC. Splenic cells which could bind to SRBC, GPRBC or DNP-SRBC were first detected on the 16th embryonic day. On the other hand, the population of RRBC-ABC was detected in the spleen on the 14th embryonic day as was shown in bursa and its frequency decreased gradually with age of embryo examined and was lower on the 18th and 20th embryonic days. Although the development of splenic ABC to various RBC was the same as detected in the bursa, the appearance of splenic ABC shifted later when compared with bursal ABC. Furthermore, with the exception of the RRBC-ABC population, spleen contained approximately ten times more ABC than did the bursa during the embryonic stages.
Inhibition of antigen binding by anti-chicken immunoglobulim The numbers of bursal and splenic ABC collected on the 20th embryonic day exposed to either rabbit antichicken immunoglobulins or normal rabbit serum are shown in Table 4. The normal level of ABC treated with normal serum indicated the absence of suppressive effect on antigen binding in bursal cells. Reduction in the number of those treated with anti-chicken immunoglobulin serum, however, clearly indicated the presence of suppressive property in antigen binding (P < 0 005). A similar effect of anti-chicken immunoglobulin serum was detected in the population of splenic ABC (Table 4).
Inhibition of antigen binding by lysed RBC or free hapten The specificity of antigen binding was further investigated. Lymphocytes prepared from the spleens of 20 day embryos were exposed to lysed RBC or DNP for
Table 3. Relative frequency of ABC to SRBC, RRBC, GPRBC or DNP-SRBC in the spleen as a function of developmental stages No. of ABC/105 cells (mean ± SE*) Indicator cell
Day 14
SRBC NDt RRBC 61-2+11-2 ND GPRBC ND DNP-SRBC *
Day 16 57-2+6-4 47-3 +7-8 60-2+12-6
61-3+91
Day 18
Day 20
85-3 ± 8-6 78-3 + 7-9 44-5 ±7-2 39-0±6-2 91-6+17-4 109-8+19-7 113-0+150 122-4+12-6
Mean of triplicate and ten embryos were used in each group.
t Not detected.
0. Moriya & Y. Ichikawa
860
Table 4. Effect of pre-treatment of lymphocytes with anti-chicken immunoglobulin serum on the relative frequency of ABC in bursa of Fabricius and spleen on the 20th embryonic day
Inhibition with serum (%)
Source cell
Indicator cell
No. of ABC/105 cells Anti-Ig Normal
SRBC RRBC GPRBC DNP-SRBC Spleen SRBC RRBC GRPBC DNP-SRBC
Bursa
14 0 274 18 7 16 5 91-4 43 0 113-8 109-7
95-2 98-1 95-1 95-7 97 2 95 2 98 3 99-6
24 0 11-6 21 4 20 2 23-8 16 8 27-7 23-8
* Mean of triplicate and ten embryos were used in each group. Table 5. Effect of pre-treatment of splenic lymphocytes with lysed RBC or DNP on the relative frequency of ABC
Treatment
Indicator cell No. of ABC/b15 cells
SRBC Saline SRBC Lysed SRBC* SRBC Lysed RRBC* SRBC Lysed GPRBC* SRBC DNPI DNP-SRBC Saline DNP-SRBC DNP DNP-SRBC Lysed SRBC* RRBC Saline RRBC Lysed RRBC* RRBC Lysed SRBC* RRBC Lysed GPRBC* GPRBC Saline GPRBC Lysed GPRBC* GPRBC Lysed RRBC* GPRBC Lysed SRBC*
83 5 28 64-5 68-0
79.3 96 7 5-1 36 0 48-1 51 40 8 38-9 120 2 21 5 103-6 98 5
* 105 RBC were used. t DNP (1 0 mg/ml -)
was used in 0 1 ml of the solution for pre-treatment with lymphocytes.
20 min at 370 before addition of RBC for the rosette assay (Table 5). Binding of guinea-pig, rabbit and sheep RBC was completely inhibited by lysed RBC of each respective species (P
