Immunology, 1975, 29, 307.
The Bursal Origin of an Immunocompetent Cell for Antibody Formation in the Chicken H. MATSUDA, T. BABA AND Y. BITO Department of Animal Microbiology, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka, Japan
(Received 21st November 1974; accepted for publication 10th January 1975)
Summary. Surgically bursectomized and irradiated chickens were given bursal, splenic, bone marrow or thymic cells taken from syngeneic donors, together with killed Brucella abortus and Salmonella pullorum. Blood samples were taken from those chickens 7 days later, and the serum agglutinin titres were determined. The cells of any lymphoid organ taken from 28-day-old chickens were more effective in restoring antibody response than those from 18-day-old ones. The restorative capacities of the bursa and splenic cells were greater than those of the bone marrow and thymic cells. On the other hand, splenic, bone marrow or thymic cells taken from bursa-less chickens, and lymphoid cells taken from normal chickens but treated with anti-bursa serum in the presence of complement, were virtually incapable of restoring the immune response. Bursal, splenic or bone marrow cells taken from neonatally thymectomized chickens, and bursal or thymic cells treated with anti-thymus serum were effective, being comparable with the corresponding cells taken from normal chickens or treated with normal sera, in restoring the suppressed immune response in chickens devoid of the lymphoid system. These facts clearly indicate that the primary and central agent crucial for development of the humoral immune response against the two bacterial antigens tested is the bursa. It is strongly suggested from the results of adoptive immunization using intrabursally primed cells that the cells recognizing Brucella abortus exist within the bursa of 4-day-old chicken. INTRODUCTION It is generally accepted that the bursa of Fabricius functions centrally in the development of the humoral immune system in the chicken. This conclusion stems from the following evidence. (A) Excision of the bursa just before or after hatching results in suppressed antibody response to subsequent immunization. The extent of the suppression varies considerably, depending upon the antigen and the time of bursectomy (Matsuda and Bito, 1973). (B) Syngeneic chickens made immunoincompetent by X-ray irradiation or cyclophosphamide treatment when transplanted with bursal cells taken from normal chickens regain function in the humoral immune system (Yamaguchi, Suzuki and Ohkuma, 1970; Gilmour, Theis and Thorbecke, 1970; Toivanen, Toivanen, Correspondence: Dr H. Matsuda, Department of Animal Microbiology, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka, Japan.
307
308 H. Matsuda, T. Baba and Y. Bito Linna and Good, 1972a; Toivanen, Toivanen and Good, 1972b; Weinbaum, Gilmour and Thorbecke, 1973). (C) Bursal lymphocytes specifically bind E. coli inhabiting the cloaca. The binding, detectable on agar plates as bacterial adherent colonies, is blocked by prior treatment of the lymphocytes with anti-chicken y-globulin serum (Waltenburgh and Van Alten, 1972). (D) Direct introduction of sheep erythrocytes into the cloaca or the bursal duct markedly increases the plaque-forming cells against sheep erythrocytes in the bursa (Van Alten and Meuwissen, 1972). (E) IgM-containing cells appear in the bursal follicles of 14-day embryos, while IgG-containing cells appear at the time of hatching (Kincade and Cooper, 1971). In other lymphoid tissues such as the spleen and thymus, immunoglobulin-containing cells are not detectable so early. In spite of general agreement on the immunologically central function of the bursa, the problem is complex. Cooper and co-workers (Cooper, Schwartz and Good, 1966) found morphologically restored germinal centres and plasma cells and functionally restored non-specific immunoglobulin production in the bursectomized chicken after intraperitoneal (i.p.) injection of dispersed autologous bursal cells, but no restoration of the specific antibody response. Ivanyi and co-workers (Ivanyi, Murgatroyd and Lydyard, 1972a) found no restoration of the antibody production to human serum albumin in similar experiments. Autologous implants of bursa failed to restore the impaired immunoglobulin response by the bursectomized chicken (Thompson and Cooper, 1971). On the other hand, immunoincompetent chickens induced by irradiation produced specific antibodies to Brucella abortus and sheep erythrocytes injected intraperitoneally, upon receiving thymus cells taken from normal chickens aged 4-10 weeks, together with antigen (Gilmour et al., 1970). Ivanyi et al. (1972a) succeeded in restoring humoral immunocompetence by intravenous (i.v.) injection of bone marrow cells taken from 6- to 8-week-old chickens. Bursa-less chickens induced by cyclophosphamide treatment produced antibody upon receiving thymus or bone marrow cells taken from normal chickens aged 14-17 weeks or 10-17 weeks, respectively, followed by antigen (Toivanen et al., 1972a). Similar results were obtained by transferring thymus or bone marrow cells taken from 3-5-17-week-old chickens together with antigen (Toivanen et al., 1972b). These findings put in question the immunologically central nature of bursa cells. Attempts were made, therefore, to answer this and other questions by immunizing chickens transplanted with lymphoid cells taken from bursectomized or thymectomized chickens, or other chickens with lymphoid cells treated with anti-bursa or anti-thymus immune serum. Such experiments clearly indicated that the bursa is the sole central organ responsible for humoral immunocompetence against the bacterial antigens used, at least with respect to titratable antibody production. MATERIALS AND METHODS Chickens Chickens used were White Leghorn inbred strain Anthony, homozygous at the major histocompatibility B locus, and of blood type BABA, BKBK and BAB.K BABA,BKBK and BABK chickens were used as donors, and BABK chickens as recipients exclusively. Recipient chickens were subjected to surgical bursectomy and whole body irradiation 2 and 3 days after hatching, respectively. They received 500 R at a dose rate 50 R per minute at a distance of 55 cm. The X-ray irradiation unit was run at 200 KV, 20 mA, and with a 0 3 mm Cu- and 0 5 mm Al-filter.
Bursal Origin of Immunocompetent Cell
309
Preparation of cell suspensions Cell suspensions were prepared from bursa, spleen, thymus and bone marrow of donors at different ages. The organs were dissected into cold medium 199; the tissues were cut into pieces with scissors. The suspension was filtered through layers of gauze after agitation with a pipette to break up the cell aggregates. The cells were washed three times by centrifugation at 600-700 rev/minute for 10 minutes. The viability of the cells was examined by trypan blue dye exclusion.
Cell transfer and immunization Four-day-old recipient chickens were injected intraperitoneally with 1-3 x 108 cells from donors in a volume of 0 5 ml together with 1-5 mg of killed Brucella abortus (BA) and Salmonella pullorum (SP). Antibody titration The serum was taken a week after immunization. Agglutinins against the two antigens were measured by microtitration with serial 2-fold dilutions of serum in 25-PI volumes. The titre was expressed as log2 of the reciprocal of the highest dilution giving positive agglutination. The mean titres are shown in the tables.
Antilymphocyte sera Rabbits were injected with doses of 3 x 108 bursal or thymic cells taken from 11- or 12-day-old chickens. The first dose was mixed with Freund's complete adjuvant and the mixture given subcutaneously. Booster injections were given intravenously a month later at 2-week intervals. The animals were bled 7 days after the last injection. The antiserum was inactivated by heating at 560 for 30 minutes and absorbed at 40 for 30 minutes first with 0.5 ml of packed erythrocytes and then with 109 thymic or bursal cells taken from 11-dayold chickens, per millilitre of the serum. Satisfactorily high specificity of the antiserum was obtained after absorbing three or more times. Cytotoxicity test The cytotoxicity test was performed by the following procedure. 0.05 ml of each of serial 2-fold dilutions of each antiserum was incubated with 0 05 ml of a cell suspension (5-10 x 106 cells per millilitre) and 0 05 ml complement (1: 4 diluted guinea-pig fresh serum) in wells in an haemagglutination tray for 45 minutes at 370. After incubation each mixture received 0.1 ml of a 0-16 per cent trypan blue solution and was examined for viability. Passive haemagglutination test (PHa T) Formalin-treated sheep erythrocytes were coated with chicken y-globulin by the method of Uemura, Sakaguchi and Riemann (1973). Treatment of donor cells with antilymphocyte sera To 1 ml of a mixture ofequal volumes of a 8-32-fold dilution of an antiserum and complement (fresh guinea-pig serum diluted to 1: 5), were added 2 5 x 108 cells; the mixture was incubated at 370 for 45 minutes. After sensitization, the cells were washed twice with cold medium 199 and examined for viability.
310
H. Matsuda, T. Baba and
r.
Bito
RESULTS ADOPTIVE IMMUNIZATION WITH NORMAL LYMPHOID CELLS
Recipient chickens were bursectomized and irradiated 2 and 3 days after hatching, respectively. On the 4th day after hatching, they were injected i.p. with 1-3 x 108 donor cells taken from 28-day-old normal chickens together with the two antigens. Serum samples were taken 1 week later and titrated for the agglutinins. All four kinds of cells injected restored the suppressed antibody responses to both antigens; the extent of restoration differed from one kind of cell to another (Table 1). The bursal and splenic cells were the most active; the bone marrow cells the least active in adoptively transferring immunocompetence.
Transfer of lymphoid cells taken from 18-day-old chickens was attempted. Bursal and splenic cells induced agglutinin production in the recipients (Table 1). Both the proportion of agglutinin producers to total chickens tested and the agglutinin titre to SP were lower than with BA. Injection of bone marrow cells resulted in a response to BA alone, possibly associated with the relative delay in immune maturation to SP TABLE 1
ADOPTIVE IMMUNE RESPONSES
BY LYMPHOID CELLS FROM NORMAL CHICKENS
SP
BA
Age of donor
28
Number of responders/ total
Cell transferred (x 108)
Bursa
1 3
0.1* 28
Spleen
28
Bone marrow
28
Thymus
18
Bursa
18
Spleen
18
Bone marrow Thymus
1 3 1 3 1 3
--t
1 3
0.1*
18
1 3 1 3 1 3
--t 14 14 10 -
Bursa
Spleen Bursa
2 2 3
4/5 5/5 5/5 3/3 4/4 3/5 1/3 7/9 4/5 0/6 5/6 5/5 4/4 5/5 5/5 3/5 3/4 0/5 0/5
0/5 0/13 0/17 0/12
Mean + s.d. (log2 titre)
Number of responders/ total
2-0+0 8 4-6+0 9 34+1.1 2 0+1 0 3 3+0 5 1 3+0 6 10 1-6+0 7 2-4+0 8 2-6+0 7 3 0+ 1-2 3 0+ 1.0 24+2-3 2 6+0 7 2 0+0 0 2 0+1 0
4/5
-
5/5 4/5
3/3 4/4 2/5 1/3 4/9 3/5 0/6 2/6 2/5 3/4 2/5 3/5 0/5 0/5 0/5 0/5 0/5 0/13 0/17 0/12
0/10
-t
Mean + s.d. (log2 titre) 1 3+0 9 3-6+0 5 32+19 1-3+0 6 2-7+0-5 1 0+0 0 2.0 1-7+0 9 3 0+0-0 1 5+0 7 1-5+0 7
1i8+ 1-3 2-5+07 2-0+ 00
0/10
1-3 x 108 lymphoid cells from donor chickens together with mixed BA and SP antigens were injected i.p. into the individual recipient chickens bursectomized and irradiated 2 and 3 days after hatching, respectively, and serum agglutinin titres were measured 1 week later.
*
Intravenous injection was employed.
t No cell recipient.
Bursal Origin of Immunocompetent Cell 311 which is usually found on immunization at various ages. Thymus cells had no effect on immunocompetence. Lymphoid cells taken from 10- to 14-day-old chickens failed to restore the suppressed immune response. ADOPTIVE IMMUNIZATION WITH LYMPHOID CELLS FROM BURSECTOMIZED OR THYMECTOMIZED CHICKENS
Experiments with isotope-labelled cells have shown migration of bursal cells to the spleen (Warner, 1955; Durkin, Theis and Thorbecke, 1972; Hemmingsson and Linna, 1972) and to the thymus (Hemmingsson and Linna, 1972). Attempts were made, therefore, to find which of these lymphoid organs was the primary source of the immune cells, allowing the primitive stem cells to mature into immunocompetent cells. For this purpose, lymphoid TABLE 2 ADOPTIVE IMMUNE RESPONSES BY LYMPHOID CELLS FROM NEONATALLY BURSECTOMIZED OR THYMECTOMIZED CHICKENS
SP
BA
Cells Number of Treatment transferred responders/ (3 x 108) total SB SB SB ST ST ST
Spleen Bone marrow Thymus Bursa Spleen Bone marrow Bursa
-
Spleen Bone
-
Thymus --*
Mean + s.d. (log2 titre)
Number of
Mean + s.d.
responders/ total
(log2 titre)
0/4 0/4
0/4 0/4
-
0/5 4/4 3/4 1/3
4-5+005 3-3+0-5 2-0
0/5 4/4 2/4 1/3
3-8+009 3 0+ 00 10
5/5 4/4 1/3
4-6+009 3-3 + 0 5 0-3
5/5 4/4 1/3
3-6+ 0 5 2-7 + 0 5 07
4/5 0/6
2-4+0-8
3/5 0/6
1-7+0 9 -
-
marrow
3 x 108 lymphoid cells from 4-week-old donor chickens neonatally bursectomized or thymectomized together with both antigens were injected i.p. into the individual recipient chickens, and serum agglutinin titres were measured 1 week later. * No cell recipient.
cells taken from bursectomized or thymectomized chickens were implanted. Splenic, thymic and bone marrow cells of 4-week-old bursectomized chickens failed to restore the antibody response; whereas bursal, splenic and bone marrow cells from thymectomized ones were as effective as normal ones (Table 2). The activities of the bursal and splenic cells were very high, but that of the bone marrow cells was low. THE CYTOTOXIC PROPERTIES OF ANTI-BURSA (ABuS) OR ANTI-THYMUS (ATS) SERA TOWARDS LYMPHOID CELLS
Immune reconstitution with thymic and bone marrow cells appeared to be dependent upon the central function of the bursa. To confirm this, adoptive immunization with lymphoid cells treated with ABuS or ATS was attempted.
H. Matsuda, T. Baba and r. Bito
312 UU
a ) _Q
(
(a) 80 o 60CD
2040C~~~~~~~~~~~~~-
CL200 8
16
32
64
128 256 8 16 Antiserum dilution
32
64
128
256
FIG. 1. Cytotoxicity of rabbit ABuS to bursa and thymus cells from 11-day-old chickens. Rabbit ABuS was prepared by hyperimmunization with 11-day bursa cells, and absorbed with 11-day thymus cells. Serial dilutions of the antiserum were incubated with equal volumes of 11-day cell suspension (5-10 x 1 O6 cells per millilitre) and of complement for 45 minutes at 37°. Viability of cells was assayed by the trypan blue exclusion method. ABuS lot number 1 was used. (a) Unabsorbed ABuS. (b) ABuS absorbed with 11-day thymus cells. Cytotoxicity for bursa cells from 11-day-old chickens (o). Cytotoxicity for thymus cells from 11-day-old chickens (-).
ABuS was tested for cytotoxicity to lymphoid cells (Fig. 1). Unabsorbed ABuS at a final dilution of 1: 128 or less was highly cytotoxic in the presence of complement to 11-day bursal cells and at 1: 16 or less to 1 -day thymic cells. Such cross-reaction strongly suggests inclusion of the thymic cells in the bursa and bursal cells in the thymus. Thymic cellabsorbed ABuS at a dilution up to 1:64 specifically killed bursal cells, whereas it was innocuous to thymic cells taken from 11-day-old chickens. The cytotoxicity of ATS is shown in Fig. 2. Unabsorbed ATS was strongly toxic to thymic and bursal cells taken from 11-day-old chickens at a dilution up to 1:128 and 1: 64, respectively. ATS absorbed with bursal cells taken from 1 1- to 12-day-old chickens killed 100 per cent of thymic cells at a final dilution up to 1:64 but not bursal cells. Specificities of these absorbed antisera for 11- and 28-day-lymphoid cells were examined (Fig. 3). Both were highly specific for specific target cells taken from 11-day-old chickens, being strongly cytotoxic at a dilution up to 1: 64; but innocous to the cells to which the antiserum had been raised. They were slightly less specific to the lymphoid cells taken from 28-day-old chickens. ABuS and ATS showed about 85 and 75 per cent cytotoxicity to bursal and thymic cells, respectively, and about 20 and 50 per cent cytotoxicity to heterologous cells. The passive haemagglutination test, however, detected an anti-immunoglobulin titre of 1: 3200 in ABuS absorbed with thymic cells, and rabbit anti-chicken immunoglobulin serum exhibited about 50 per cent of the cytotoxicity for bursa cells from 21-day-old chickens (Fig. 4). ABuS, therefore, was further absorbed twice with chicken immunoglobulin. In ABuS absorbed with immunoglobulin anti-immunoglobulin antibody was not detected with the passive haemagglutination test, but the cytotoxicity of ABuS against bursal cells taken from 21- and 28-day-old chickens was not affected by absorption with normal chicken immunoglobulin. Preliminary experiments showed that the bursal cells gradually changed their antigenic specificity as the donor chickens grew. This finding suggested that the antigenicity of the
313
Bursal Origin of Immunocompetent Cell °°r
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c:
60k-
401-
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20k O' 8
16
32
64
128
I-64
251i6 8 16 Antiserum dilution
32
128
256
FIG. 2. Cytotoxicity of rabbit ATS to bursa and thymus cells from 11-day-old chickens. Rabbit ATS was prepared by hyperimmunization with 11-day thymus cells, and absorbed with 11-day bursa cells. Serial dilutions of the antiserum were incubated with equal volume of 11-day cell suspension (5-10 x 106 cells per millilitre) and of complement for 45 minutes at 37°. ABuS lot number 1 was used. (a) Unabsorbed ATS. (b) ATS absorbed with 1 -day bursa cells. Cytotoxicity for bursa cells from 11-day-old chickens (0). Cytotoxicity for thymus cells from 11-day-old chickens (0).
IOflO-
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-
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(b )
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a)
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*
8
16
32
64
128
-
-
.
L0
-
--
256 8 6 Antiserum dilution
- -
-
32
-
-0-
64
-
-0-
128
-
-o
256
FIG. 3. Cytotoxicities for 11- and 28-day lymphoid cells of specific ABuS and ATS absorbed with heterologous lymphoid cell antigen. Bursa and thymus cells from 11 -day-old and 28-day-old chickens were treated by ABuS absorbed with 1 1-day thymus cells or by ATS absorbed with 1 1-day bursa cells. ABuS lot number 1 was used. (a) ABuS absorbed with 1 1-day thymus cells. (b) ATS absorbed with 11 -day bursa cells. Cytotoxicity for 11 -day bursa cells (0- -a). Cytotoxicity for 28-day bursa cells (o0 ). Cytotoxicity for 11 -day thymus cells (---). Cytotoxicity for 28-day thymus cells (--*).
H. Matsuda, T. Baba and r. Bito
314
(a)
100
(b)
80
Z 60
20 20
01 4
8
16
32
64 4 8 Dilution of antisera
16
32
64
FIG. 4. Cytotoxicities for bursa cells of immunoglobulin-unabsorbed ABuS, immunoglobulin-absorbed ABuS or anti-chicken immunoglobulin serum. After ABuS (lot number 2) was absorbed with thymus cells, it was further absorbed twice with chicken immunoglobulin. Twenty-one- or 28-day bursa cells were treated with ABuS absorbed with thymus cells, ABuS absorbed with thymus cells and chicken immunoglobulin, and anti-chicken immunoglobulin serum, respectively. (a) 2 1-day bursa cells. (b) 28-day bursa cells. Cytotoxicity by immunoglobulin-unabsorbed ABuS (0). Cytotoxicity by immunoglobulin-absorbed ABuS (a). Cytotoxicity by anti-chicken immunoglobulin serum (A).
cell itself changed in the organ, or alternatively that the cells migrated from one organ to another. ADOPTIVE IMMUNIZATION WITH ABuS- OR ATS-TREATED LYMPHOID CELLS
Lymphoid cells taken from 4- or 8-week-old chickens and treated with specific antibursa serum, together with the test antigens, were injected into immunologically suppressed chickens. As shown in Table 3, the immunocompetence of all the cells was completely abolished by the treatment with ABuS in the presence of complement, only thymus cells inducing some antibody response. ABuS used in these experiments, however, contains anti-immunoglobulin antibody which could kill all cells possessing immunoglobulin on the surface in co-operation with complement. Thus, the splenic cells taken from 8-week-old donors and treated with immunoglobulin-absorbed ABuS, together with antigens, were injected intravenously into recipients. The treatment of donor cells with immunoglobulinabsorbed ABuS as well as with immunoglobulin-unabsorbed ABuS prevented the restoration of antibody responses (Table 4). Lymphoid cells treated with ATS or NRS were still able to induce immunocompetence (Table 4). These results show clearly that the ability of the splenic, bone marrow and thymic cells to restore immunoincompetence originates in the bursa. ADOPTIVE IMMUNIZATION BY BURSA CELLS FROM CHICKENS PRIMED INTRABURSALLY
Immunoincompetent recipients were i.v. injected with 1 x IO' bursa cells from 11-dayold donors primed intrabursally or intravenously in the newly hatched period. As shown in Table 5, implant with bursa cells from donors primed intrabursally led to the restoration of the immune response against BA antigen but not SP antigen. It is noticeable that 1 -day bursa cells are competent for transferring the immune responsiveness, if primed by direct contact with antigens, in contrast to the adoptive immunization using cells unprimed or primed by the intravenous route.
Bursal Origin of Immunocompetent Cell
315
TABLE 3 ADOPTIVE
IMMUNE RESPONSES BY LYMPHOID CELLS TREATED WITH
ABuS
BA
Age of donor (weeks) 4 4 8 8
4 4 8 8 8 8 8
Cell transferred (x 108)
Number of Treatment responders/ total
Bursa 1-5 ABuS 1-5 Thymus ABuS 1-5 ABuS Spleen Bone 1 5 ABuS marrow 1-5 NRS Bursa 1-5 NRS Thymus 1-5 NRS Spleen 1-5 NRS Bone marrow 0-5* ABuS Spleen 0.5* Ig-absorbed Spleen ABuS 0.5* NRS Spleen
-t
SP
Mean + s.d.
Number of
(log2 titre)
responders/
Mean + s.d. (log2 titre)
total
0/8 2/10 0/5 0/6
1-5+0 5
0/7 2/10 0/5 0/6
2-5+0-5
3/3 5/5 5/5 6/6
4 0+1.0 3 4+0-5 5 0+0 5 3.8+0 9
3/3 5/5 5/5 6/6
3-3+0-5 24+0-5 4-6+0 4 3 5+0 5
0/4 0/4
0/4 0/4
4/4
5-3+1-0
0/16
4/4
4-8+1 3
0/16
1 5 x 108 lymphoid cells from 4- or 8-week-old donor chickens were treated with ABuS absorbed with thymus cells and injected i.p. into the individual recipient chickens together with both antigens, and serum agglutinin titres were measured 1 week later. 5 x 107 spleen cells from 8-week-old donor chickens were treated with ABuS absorbed with thymus cells and chicken immunoglobulin and injected i.v. into the individual recipients together with both antigens, and serum agglutinin titres were measured 1 week later. NRS = normal rabbit serum. * Intravenous injection was employed. t No cell recipient.
TABLE 4 ADOPTIVE IMMUNE RESPONSES BY BURSA OR THYMUS CELLS TREATED WITH ATS
BA
Age of donor
(weeks) 4 4 4 4
Cell transferred (x 108)
Bursa Thymus Bursa Thymus - -*
1-5 1-5 1-5 1-5 -
Number of Treatment responders/ total
ATS ATS NRS NRS -
3/3 5/5 3/3 5/5
0/6
SP
Mean + s.d. (log2 titre) 3-3+0-5 3 2+0 4 4 0+1-0 3-4+0 5
Number of responders/ total 2/3 5/5 3/3 5/5
0/6
Mean + s.d.
(log2 titre) 2-5+0-5 2-3+0 4 3-3+0-5 2-4+0 5
1-5 x 108 bursa or thymus cells from 4-week-old chickens were treated with ATS absorbed with bursa cells and injected i.p. into the individual recipients together with both antigens, and serum agglutinin titres were measured I week later. * No cell recipient.
H. Matsuda, T. Baba and
316
r.
Bito
TABLE 5 ADOPTIVE IMMUNE RESPONSES BY PRIMED BURSA CELLS
SP
BA
Number of responders/ total
Group
Donor cells
1
Bursa cells from chickens intrabursally primed at 4 days of age Bursa cells from chickens intravenously primed at 4 days of age
4/5
-t
0/5
2
3
015
Number of Mean + s.d. of P value* responders/ total responders (log2 titre)
3-6+2-0
0-028
0/5
-
0/5
Mean + s.d. P value* of responders (log2 titre)
0/5
107 Bursa cells from 11-day-old chickens primed intrabursally or i.v. with both antigens were injected i.p. into the individual recipients together with both antigens, and serum agglutinin titres were measured 1 week later. * P value for difference from group 3 calculated by Mann-Whitney U-test. t No cell recipient.
DISCUSSION The present findings may be summarized as follows: (a) bursal and splenic cells from normal chickens were more efficient than bone marrow or thymic cells in restoring the immune response in immunoincompetent chickens; (b) splenic, thymic and bone marrow cells taken from neonatally bursectomized chickens did not restore immunocompetence; (c) bursal, splenic or bone marrow cells from thymectomized chickens restored the antibody response in immunologically incompetent recipients; (d) treating spleen cells with specific anti-bursa antiserum in the presence of complement deprived them of the ability to restore the immune response in agammaglobulinaemic chickens; (e) bursal and thymic cells treated with ATS were fully active in restoring the suppressed immune response. These findings clearly show that the bursa functions as the primary source of the immunocompetent cells. Failures in immune reconstitution of bursectomized chickens by bursal implants have been reported (Cooper et al., 1966; Ivanyi et al., 1972a; Thompson and Cooper, 1971). Most of these authors performed autologous implant of the bursal cells from neonatal chickens. On the other hand, Toivanen, Toivanen and Good (1972c) succeeded in restoring the function and morphology of the bursa-dependent lymphoid system by transferring bursal cells taken from embryo or 4-day-chickens. In their experiments, the rudimentary follicles underwent drastic changes on cyclophosphamide treatment, but were not obliterated. The bursa cells introduced intravenously repopulated them, resulting in morphological and functional reconstitution of the bursa. These results strongly suggest that maturation in the bursa of the uncommitted stem cells transferred from embryos or newly hatched chickens is crucial to their ability to restore the immune response in immunologically impaired recipient chickens. No rudiments of the bursa to which injected bursa cells could home remain after surgical bursectomy (SB). This may
Bursal Origin of Immunocompetent Cell 317 explain partially the failure to reconstitute with bursa stem cells taken from embryos or neonatal chickens. Our data show that none of the lymphoid cells taken from 14-dayold or younger chickens were able to restore the immune response in bursectomized and irradiated chickens, whereas, those taken from 18-day-old or older chickens did restore the immune response. In White Leghorns, the bursa reaches its maximal size, acquiring plicate structure, at 3-6 weeks of ages. Each plica consists of follicles filled with lymphoid cells (Glick, 1956, 1960). Toivanen et al. (1972a, c) reported that the older the donor of the bursa cells, the more active the immune reconstituting capacity. This may reflect progressive maturation of the bursal cells. The mechanism by which the bursa functions in development of the humoral immune system is not clear. It is, however, generally accepted that the bursal cell serves as a precursor of the antibody-forming cell, i.e. the bursal stem cell itself maturates through unknown steps into an antibody-forming cell upon exposure to an antigenic stimulus. This supposition comes from the following evidence. (A) IgM-containing cells appear in the bursal follicles of 14-day-old embryos and IgG-containing cells in those of newly hatched chicken (Kincade and Cooper, 1971; Choi and Good, 1972). In other lymphoid organs, immunoglobulin-containing cells do not appear during the embryonic or newly hatching periods (Thorbecke, Warner, Hochwald and Ohanian, 1968). The bursa is the only organ that contains many 'double producers', cells synthesizing both IgM and IgG with very high frequency (Kincade and Cooper, 1971). Treating the 13-day-old embryo with anti-yu antibodies followed by neonatal bursectomy resulted in suppression of both IgM and IgG syntheses; the same treatment on hatching day decreased only IgM production. This suggests that the IgG producers originate from the IgM producers within the bursa (Kincade, Lawton, Bockman and Cooper, 1970). (B) Direct introduction of antigen into the cloaca markedly increases plaque-forming cells (Van Alten and Meuwissen, 1972). (C) ABuS inhibited appearance of splenic plaque-forming cells of chickens immunized with sheep erythrocytes (Potworowski, Richer, Borduas and Forget, 1971; Ivanyi and Lydyard, 1972b). Studies on the peripheral distribution of bursa cells (Durkin, Theis and Thorbecke, 1972; Hemmingsson and Linna, 1972) strongly support this postulate. Direct administration of antigens into the cavity of the bursa demonstrates the immunocompetence of 11-day-old bursa cells. Similar priming elicited a significant augmentation of anti-BA antibody production following the usual immunization by the intravenous route (unpublished data). Antigen-binding cells were detected in the embryonic bursa (Dwyer and Warner, 1971). Direct injection of sheep erythrocytes into the cloaca markedly increased the number of plaque-forming cells against the antigen (Van Alten and Meuwissen, 1972). These facts suggest strongly the presence of antigen-recognizing or antigen-reactive cells in the bursa of young birds and proliferation of the cells in response to antigenic triggering. Our experiments suggest the existence, in the bursa of neonatal chickens, of antigen-recognizing cells against BA but not SP antigen. The cause of the difference between the response to BA and SP antigens is not clear, but may be related to the delay of immune maturity against SP antigen.
ACKNOWLEDGMENTS The authors are sincerely grateful to Professor K. Kondo and Dr Y. Fujio, Faculty of Agriculture, Nagoya University, for their generous gift of inbred chicken, strain Anthony,
318 H. Matsuda, T. Baba and r. Bito and to Dr A. Takeda and co-workers, Radiation Center of Osaka Prefecture, for irradiation of the chickens.
REFERENCES
CHOI, Y. S. and GOOD, R. A. (1972). 'Development of chicken lymphoid system. II. Synthesis of primordial immunoglobulin M by the bursa cells of chick embryo.'_J. exp. Med., 136, 8. COOPER, M. D., SCHWARTZ, M. L. and GOOD, R. A. (1966). 'Restoration of gamma globulin production in agammaglobulinemic chickens.' Science, 151, 471. DURKIN, H. G., THEIS, G. A. and THORBECKE, G. J. (1972). 'Bursa of Fabricius as site of origin of germinal center cells.' Nature: New Biology, 235, 118. DWYER, J. M. and WARNER, N. L. (1971). 'Antigen binding cells in embryonic chicken bursa and thymus.' Nature: New Biology, 229, 210. GILMOUR, D. G., THEIS, G. A. and THORBECKE, G. J. (1970). 'Transfer of antibody production with cells from bursa of Fabricius.'J. exp. Med., 132, 134. GLICK, B. (1956). 'Normal growth of the bursa of Fabricius in chickens.' Poultry Sci., 35, 843. GLICK, B. (1960). 'Growth of the bursa of Fabricius and its relationship to the adrenal gland in the White Pekin Duck, White Leghorn, outbred and inbred New Hampshire.' Poultry Sci., 39, 130. HEMMINGSSON, E. J. and LINNA, T. J. (1972). 'Ontogenic studies on lymphoid cell traffic in the chicken. I. Cell migration from the bursa of Fabricius.' Int. Arch. Allergy, 42, 693. IVANYI, J., MURGATROYD, L. B. and LYDYARD, P. M. (1972a). 'Bursal origin of bone marrow cells with competence for antibody formation.' Immunology, 23, 107. IVANYI, J. and LYDYARD, P. M. (1972b). 'Delineation of chicken lymphocyte populations by specific anti-thymus and anti-bursa sera.' Cell. Immunol., 5, 180. KINCADE, P. W. and COOPER, M. D. (1971). 'Development and distribution of immunoglobulin-containing cells in the chicken.'J. Immunol., 106, 371. KINCADE, P. W., LAWTON, A. R., BOCKMAN, D. E. and COOPER, M. D. (1970). 'Suppression of immunoglobulin G synthesis as a result of antibody-mediated suppression of immunoglobulin M synthesis in chickens.' Proc. nat. Acad. Sci. (Wash.), 67, 1918. MATSUDA, H. and Brro, Y. (1973). 'Different effects of bursectomy of chickens on immune response to Newcastle disease virus and Salmonella pullorum antigens.' Poultry Sci., 52, 1042.
The Bursal Origin of an Immunocompetent Cell for Antibody Formation in the Chicken H. MATSUDA, T. BABA AND Y. BITO Department of Animal Microbiology, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka, Japan
(Received 21st November 1974; accepted for publication 10th January 1975)
Summary. Surgically bursectomized and irradiated chickens were given bursal, splenic, bone marrow or thymic cells taken from syngeneic donors, together with killed Brucella abortus and Salmonella pullorum. Blood samples were taken from those chickens 7 days later, and the serum agglutinin titres were determined. The cells of any lymphoid organ taken from 28-day-old chickens were more effective in restoring antibody response than those from 18-day-old ones. The restorative capacities of the bursa and splenic cells were greater than those of the bone marrow and thymic cells. On the other hand, splenic, bone marrow or thymic cells taken from bursa-less chickens, and lymphoid cells taken from normal chickens but treated with anti-bursa serum in the presence of complement, were virtually incapable of restoring the immune response. Bursal, splenic or bone marrow cells taken from neonatally thymectomized chickens, and bursal or thymic cells treated with anti-thymus serum were effective, being comparable with the corresponding cells taken from normal chickens or treated with normal sera, in restoring the suppressed immune response in chickens devoid of the lymphoid system. These facts clearly indicate that the primary and central agent crucial for development of the humoral immune response against the two bacterial antigens tested is the bursa. It is strongly suggested from the results of adoptive immunization using intrabursally primed cells that the cells recognizing Brucella abortus exist within the bursa of 4-day-old chicken. INTRODUCTION It is generally accepted that the bursa of Fabricius functions centrally in the development of the humoral immune system in the chicken. This conclusion stems from the following evidence. (A) Excision of the bursa just before or after hatching results in suppressed antibody response to subsequent immunization. The extent of the suppression varies considerably, depending upon the antigen and the time of bursectomy (Matsuda and Bito, 1973). (B) Syngeneic chickens made immunoincompetent by X-ray irradiation or cyclophosphamide treatment when transplanted with bursal cells taken from normal chickens regain function in the humoral immune system (Yamaguchi, Suzuki and Ohkuma, 1970; Gilmour, Theis and Thorbecke, 1970; Toivanen, Toivanen, Correspondence: Dr H. Matsuda, Department of Animal Microbiology, College of Agriculture, University of Osaka Prefecture, Sakai, Osaka, Japan.
307
308 H. Matsuda, T. Baba and Y. Bito Linna and Good, 1972a; Toivanen, Toivanen and Good, 1972b; Weinbaum, Gilmour and Thorbecke, 1973). (C) Bursal lymphocytes specifically bind E. coli inhabiting the cloaca. The binding, detectable on agar plates as bacterial adherent colonies, is blocked by prior treatment of the lymphocytes with anti-chicken y-globulin serum (Waltenburgh and Van Alten, 1972). (D) Direct introduction of sheep erythrocytes into the cloaca or the bursal duct markedly increases the plaque-forming cells against sheep erythrocytes in the bursa (Van Alten and Meuwissen, 1972). (E) IgM-containing cells appear in the bursal follicles of 14-day embryos, while IgG-containing cells appear at the time of hatching (Kincade and Cooper, 1971). In other lymphoid tissues such as the spleen and thymus, immunoglobulin-containing cells are not detectable so early. In spite of general agreement on the immunologically central function of the bursa, the problem is complex. Cooper and co-workers (Cooper, Schwartz and Good, 1966) found morphologically restored germinal centres and plasma cells and functionally restored non-specific immunoglobulin production in the bursectomized chicken after intraperitoneal (i.p.) injection of dispersed autologous bursal cells, but no restoration of the specific antibody response. Ivanyi and co-workers (Ivanyi, Murgatroyd and Lydyard, 1972a) found no restoration of the antibody production to human serum albumin in similar experiments. Autologous implants of bursa failed to restore the impaired immunoglobulin response by the bursectomized chicken (Thompson and Cooper, 1971). On the other hand, immunoincompetent chickens induced by irradiation produced specific antibodies to Brucella abortus and sheep erythrocytes injected intraperitoneally, upon receiving thymus cells taken from normal chickens aged 4-10 weeks, together with antigen (Gilmour et al., 1970). Ivanyi et al. (1972a) succeeded in restoring humoral immunocompetence by intravenous (i.v.) injection of bone marrow cells taken from 6- to 8-week-old chickens. Bursa-less chickens induced by cyclophosphamide treatment produced antibody upon receiving thymus or bone marrow cells taken from normal chickens aged 14-17 weeks or 10-17 weeks, respectively, followed by antigen (Toivanen et al., 1972a). Similar results were obtained by transferring thymus or bone marrow cells taken from 3-5-17-week-old chickens together with antigen (Toivanen et al., 1972b). These findings put in question the immunologically central nature of bursa cells. Attempts were made, therefore, to answer this and other questions by immunizing chickens transplanted with lymphoid cells taken from bursectomized or thymectomized chickens, or other chickens with lymphoid cells treated with anti-bursa or anti-thymus immune serum. Such experiments clearly indicated that the bursa is the sole central organ responsible for humoral immunocompetence against the bacterial antigens used, at least with respect to titratable antibody production. MATERIALS AND METHODS Chickens Chickens used were White Leghorn inbred strain Anthony, homozygous at the major histocompatibility B locus, and of blood type BABA, BKBK and BAB.K BABA,BKBK and BABK chickens were used as donors, and BABK chickens as recipients exclusively. Recipient chickens were subjected to surgical bursectomy and whole body irradiation 2 and 3 days after hatching, respectively. They received 500 R at a dose rate 50 R per minute at a distance of 55 cm. The X-ray irradiation unit was run at 200 KV, 20 mA, and with a 0 3 mm Cu- and 0 5 mm Al-filter.
Bursal Origin of Immunocompetent Cell
309
Preparation of cell suspensions Cell suspensions were prepared from bursa, spleen, thymus and bone marrow of donors at different ages. The organs were dissected into cold medium 199; the tissues were cut into pieces with scissors. The suspension was filtered through layers of gauze after agitation with a pipette to break up the cell aggregates. The cells were washed three times by centrifugation at 600-700 rev/minute for 10 minutes. The viability of the cells was examined by trypan blue dye exclusion.
Cell transfer and immunization Four-day-old recipient chickens were injected intraperitoneally with 1-3 x 108 cells from donors in a volume of 0 5 ml together with 1-5 mg of killed Brucella abortus (BA) and Salmonella pullorum (SP). Antibody titration The serum was taken a week after immunization. Agglutinins against the two antigens were measured by microtitration with serial 2-fold dilutions of serum in 25-PI volumes. The titre was expressed as log2 of the reciprocal of the highest dilution giving positive agglutination. The mean titres are shown in the tables.
Antilymphocyte sera Rabbits were injected with doses of 3 x 108 bursal or thymic cells taken from 11- or 12-day-old chickens. The first dose was mixed with Freund's complete adjuvant and the mixture given subcutaneously. Booster injections were given intravenously a month later at 2-week intervals. The animals were bled 7 days after the last injection. The antiserum was inactivated by heating at 560 for 30 minutes and absorbed at 40 for 30 minutes first with 0.5 ml of packed erythrocytes and then with 109 thymic or bursal cells taken from 11-dayold chickens, per millilitre of the serum. Satisfactorily high specificity of the antiserum was obtained after absorbing three or more times. Cytotoxicity test The cytotoxicity test was performed by the following procedure. 0.05 ml of each of serial 2-fold dilutions of each antiserum was incubated with 0 05 ml of a cell suspension (5-10 x 106 cells per millilitre) and 0 05 ml complement (1: 4 diluted guinea-pig fresh serum) in wells in an haemagglutination tray for 45 minutes at 370. After incubation each mixture received 0.1 ml of a 0-16 per cent trypan blue solution and was examined for viability. Passive haemagglutination test (PHa T) Formalin-treated sheep erythrocytes were coated with chicken y-globulin by the method of Uemura, Sakaguchi and Riemann (1973). Treatment of donor cells with antilymphocyte sera To 1 ml of a mixture ofequal volumes of a 8-32-fold dilution of an antiserum and complement (fresh guinea-pig serum diluted to 1: 5), were added 2 5 x 108 cells; the mixture was incubated at 370 for 45 minutes. After sensitization, the cells were washed twice with cold medium 199 and examined for viability.
310
H. Matsuda, T. Baba and
r.
Bito
RESULTS ADOPTIVE IMMUNIZATION WITH NORMAL LYMPHOID CELLS
Recipient chickens were bursectomized and irradiated 2 and 3 days after hatching, respectively. On the 4th day after hatching, they were injected i.p. with 1-3 x 108 donor cells taken from 28-day-old normal chickens together with the two antigens. Serum samples were taken 1 week later and titrated for the agglutinins. All four kinds of cells injected restored the suppressed antibody responses to both antigens; the extent of restoration differed from one kind of cell to another (Table 1). The bursal and splenic cells were the most active; the bone marrow cells the least active in adoptively transferring immunocompetence.
Transfer of lymphoid cells taken from 18-day-old chickens was attempted. Bursal and splenic cells induced agglutinin production in the recipients (Table 1). Both the proportion of agglutinin producers to total chickens tested and the agglutinin titre to SP were lower than with BA. Injection of bone marrow cells resulted in a response to BA alone, possibly associated with the relative delay in immune maturation to SP TABLE 1
ADOPTIVE IMMUNE RESPONSES
BY LYMPHOID CELLS FROM NORMAL CHICKENS
SP
BA
Age of donor
28
Number of responders/ total
Cell transferred (x 108)
Bursa
1 3
0.1* 28
Spleen
28
Bone marrow
28
Thymus
18
Bursa
18
Spleen
18
Bone marrow Thymus
1 3 1 3 1 3
--t
1 3
0.1*
18
1 3 1 3 1 3
--t 14 14 10 -
Bursa
Spleen Bursa
2 2 3
4/5 5/5 5/5 3/3 4/4 3/5 1/3 7/9 4/5 0/6 5/6 5/5 4/4 5/5 5/5 3/5 3/4 0/5 0/5
0/5 0/13 0/17 0/12
Mean + s.d. (log2 titre)
Number of responders/ total
2-0+0 8 4-6+0 9 34+1.1 2 0+1 0 3 3+0 5 1 3+0 6 10 1-6+0 7 2-4+0 8 2-6+0 7 3 0+ 1-2 3 0+ 1.0 24+2-3 2 6+0 7 2 0+0 0 2 0+1 0
4/5
-
5/5 4/5
3/3 4/4 2/5 1/3 4/9 3/5 0/6 2/6 2/5 3/4 2/5 3/5 0/5 0/5 0/5 0/5 0/5 0/13 0/17 0/12
0/10
-t
Mean + s.d. (log2 titre) 1 3+0 9 3-6+0 5 32+19 1-3+0 6 2-7+0-5 1 0+0 0 2.0 1-7+0 9 3 0+0-0 1 5+0 7 1-5+0 7
1i8+ 1-3 2-5+07 2-0+ 00
0/10
1-3 x 108 lymphoid cells from donor chickens together with mixed BA and SP antigens were injected i.p. into the individual recipient chickens bursectomized and irradiated 2 and 3 days after hatching, respectively, and serum agglutinin titres were measured 1 week later.
*
Intravenous injection was employed.
t No cell recipient.
Bursal Origin of Immunocompetent Cell 311 which is usually found on immunization at various ages. Thymus cells had no effect on immunocompetence. Lymphoid cells taken from 10- to 14-day-old chickens failed to restore the suppressed immune response. ADOPTIVE IMMUNIZATION WITH LYMPHOID CELLS FROM BURSECTOMIZED OR THYMECTOMIZED CHICKENS
Experiments with isotope-labelled cells have shown migration of bursal cells to the spleen (Warner, 1955; Durkin, Theis and Thorbecke, 1972; Hemmingsson and Linna, 1972) and to the thymus (Hemmingsson and Linna, 1972). Attempts were made, therefore, to find which of these lymphoid organs was the primary source of the immune cells, allowing the primitive stem cells to mature into immunocompetent cells. For this purpose, lymphoid TABLE 2 ADOPTIVE IMMUNE RESPONSES BY LYMPHOID CELLS FROM NEONATALLY BURSECTOMIZED OR THYMECTOMIZED CHICKENS
SP
BA
Cells Number of Treatment transferred responders/ (3 x 108) total SB SB SB ST ST ST
Spleen Bone marrow Thymus Bursa Spleen Bone marrow Bursa
-
Spleen Bone
-
Thymus --*
Mean + s.d. (log2 titre)
Number of
Mean + s.d.
responders/ total
(log2 titre)
0/4 0/4
0/4 0/4
-
0/5 4/4 3/4 1/3
4-5+005 3-3+0-5 2-0
0/5 4/4 2/4 1/3
3-8+009 3 0+ 00 10
5/5 4/4 1/3
4-6+009 3-3 + 0 5 0-3
5/5 4/4 1/3
3-6+ 0 5 2-7 + 0 5 07
4/5 0/6
2-4+0-8
3/5 0/6
1-7+0 9 -
-
marrow
3 x 108 lymphoid cells from 4-week-old donor chickens neonatally bursectomized or thymectomized together with both antigens were injected i.p. into the individual recipient chickens, and serum agglutinin titres were measured 1 week later. * No cell recipient.
cells taken from bursectomized or thymectomized chickens were implanted. Splenic, thymic and bone marrow cells of 4-week-old bursectomized chickens failed to restore the antibody response; whereas bursal, splenic and bone marrow cells from thymectomized ones were as effective as normal ones (Table 2). The activities of the bursal and splenic cells were very high, but that of the bone marrow cells was low. THE CYTOTOXIC PROPERTIES OF ANTI-BURSA (ABuS) OR ANTI-THYMUS (ATS) SERA TOWARDS LYMPHOID CELLS
Immune reconstitution with thymic and bone marrow cells appeared to be dependent upon the central function of the bursa. To confirm this, adoptive immunization with lymphoid cells treated with ABuS or ATS was attempted.
H. Matsuda, T. Baba and r. Bito
312 UU
a ) _Q
(
(a) 80 o 60CD
2040C~~~~~~~~~~~~~-
CL200 8
16
32
64
128 256 8 16 Antiserum dilution
32
64
128
256
FIG. 1. Cytotoxicity of rabbit ABuS to bursa and thymus cells from 11-day-old chickens. Rabbit ABuS was prepared by hyperimmunization with 11-day bursa cells, and absorbed with 11-day thymus cells. Serial dilutions of the antiserum were incubated with equal volumes of 11-day cell suspension (5-10 x 1 O6 cells per millilitre) and of complement for 45 minutes at 37°. Viability of cells was assayed by the trypan blue exclusion method. ABuS lot number 1 was used. (a) Unabsorbed ABuS. (b) ABuS absorbed with 11-day thymus cells. Cytotoxicity for bursa cells from 11-day-old chickens (o). Cytotoxicity for thymus cells from 11-day-old chickens (-).
ABuS was tested for cytotoxicity to lymphoid cells (Fig. 1). Unabsorbed ABuS at a final dilution of 1: 128 or less was highly cytotoxic in the presence of complement to 11-day bursal cells and at 1: 16 or less to 1 -day thymic cells. Such cross-reaction strongly suggests inclusion of the thymic cells in the bursa and bursal cells in the thymus. Thymic cellabsorbed ABuS at a dilution up to 1:64 specifically killed bursal cells, whereas it was innocuous to thymic cells taken from 11-day-old chickens. The cytotoxicity of ATS is shown in Fig. 2. Unabsorbed ATS was strongly toxic to thymic and bursal cells taken from 11-day-old chickens at a dilution up to 1:128 and 1: 64, respectively. ATS absorbed with bursal cells taken from 1 1- to 12-day-old chickens killed 100 per cent of thymic cells at a final dilution up to 1:64 but not bursal cells. Specificities of these absorbed antisera for 11- and 28-day-lymphoid cells were examined (Fig. 3). Both were highly specific for specific target cells taken from 11-day-old chickens, being strongly cytotoxic at a dilution up to 1: 64; but innocous to the cells to which the antiserum had been raised. They were slightly less specific to the lymphoid cells taken from 28-day-old chickens. ABuS and ATS showed about 85 and 75 per cent cytotoxicity to bursal and thymic cells, respectively, and about 20 and 50 per cent cytotoxicity to heterologous cells. The passive haemagglutination test, however, detected an anti-immunoglobulin titre of 1: 3200 in ABuS absorbed with thymic cells, and rabbit anti-chicken immunoglobulin serum exhibited about 50 per cent of the cytotoxicity for bursa cells from 21-day-old chickens (Fig. 4). ABuS, therefore, was further absorbed twice with chicken immunoglobulin. In ABuS absorbed with immunoglobulin anti-immunoglobulin antibody was not detected with the passive haemagglutination test, but the cytotoxicity of ABuS against bursal cells taken from 21- and 28-day-old chickens was not affected by absorption with normal chicken immunoglobulin. Preliminary experiments showed that the bursal cells gradually changed their antigenic specificity as the donor chickens grew. This finding suggested that the antigenicity of the
313
Bursal Origin of Immunocompetent Cell °°r
-0--
(a )
*
e
e
(b)
\
80[-
0
0
.2
ca 0-
c:
60k-
401-
a1)
20k O' 8
16
32
64
128
I-64
251i6 8 16 Antiserum dilution
32
128
256
FIG. 2. Cytotoxicity of rabbit ATS to bursa and thymus cells from 11-day-old chickens. Rabbit ATS was prepared by hyperimmunization with 11-day thymus cells, and absorbed with 11-day bursa cells. Serial dilutions of the antiserum were incubated with equal volume of 11-day cell suspension (5-10 x 106 cells per millilitre) and of complement for 45 minutes at 37°. ABuS lot number 1 was used. (a) Unabsorbed ATS. (b) ATS absorbed with 1 -day bursa cells. Cytotoxicity for bursa cells from 11-day-old chickens (0). Cytotoxicity for thymus cells from 11-day-old chickens (0).
IOflO-
- -0- - -0-
-
(a )
*
(b )
.i x 0
0
60k
0 S' 0)
a)
40k 20 0-
*
8
16
32
64
128
-
-
.
L0
-
--
256 8 6 Antiserum dilution
- -
-
32
-
-0-
64
-
-0-
128
-
-o
256
FIG. 3. Cytotoxicities for 11- and 28-day lymphoid cells of specific ABuS and ATS absorbed with heterologous lymphoid cell antigen. Bursa and thymus cells from 11 -day-old and 28-day-old chickens were treated by ABuS absorbed with 1 1-day thymus cells or by ATS absorbed with 1 1-day bursa cells. ABuS lot number 1 was used. (a) ABuS absorbed with 1 1-day thymus cells. (b) ATS absorbed with 11 -day bursa cells. Cytotoxicity for 11 -day bursa cells (0- -a). Cytotoxicity for 28-day bursa cells (o0 ). Cytotoxicity for 11 -day thymus cells (---). Cytotoxicity for 28-day thymus cells (--*).
H. Matsuda, T. Baba and r. Bito
314
(a)
100
(b)
80
Z 60
20 20
01 4
8
16
32
64 4 8 Dilution of antisera
16
32
64
FIG. 4. Cytotoxicities for bursa cells of immunoglobulin-unabsorbed ABuS, immunoglobulin-absorbed ABuS or anti-chicken immunoglobulin serum. After ABuS (lot number 2) was absorbed with thymus cells, it was further absorbed twice with chicken immunoglobulin. Twenty-one- or 28-day bursa cells were treated with ABuS absorbed with thymus cells, ABuS absorbed with thymus cells and chicken immunoglobulin, and anti-chicken immunoglobulin serum, respectively. (a) 2 1-day bursa cells. (b) 28-day bursa cells. Cytotoxicity by immunoglobulin-unabsorbed ABuS (0). Cytotoxicity by immunoglobulin-absorbed ABuS (a). Cytotoxicity by anti-chicken immunoglobulin serum (A).
cell itself changed in the organ, or alternatively that the cells migrated from one organ to another. ADOPTIVE IMMUNIZATION WITH ABuS- OR ATS-TREATED LYMPHOID CELLS
Lymphoid cells taken from 4- or 8-week-old chickens and treated with specific antibursa serum, together with the test antigens, were injected into immunologically suppressed chickens. As shown in Table 3, the immunocompetence of all the cells was completely abolished by the treatment with ABuS in the presence of complement, only thymus cells inducing some antibody response. ABuS used in these experiments, however, contains anti-immunoglobulin antibody which could kill all cells possessing immunoglobulin on the surface in co-operation with complement. Thus, the splenic cells taken from 8-week-old donors and treated with immunoglobulin-absorbed ABuS, together with antigens, were injected intravenously into recipients. The treatment of donor cells with immunoglobulinabsorbed ABuS as well as with immunoglobulin-unabsorbed ABuS prevented the restoration of antibody responses (Table 4). Lymphoid cells treated with ATS or NRS were still able to induce immunocompetence (Table 4). These results show clearly that the ability of the splenic, bone marrow and thymic cells to restore immunoincompetence originates in the bursa. ADOPTIVE IMMUNIZATION BY BURSA CELLS FROM CHICKENS PRIMED INTRABURSALLY
Immunoincompetent recipients were i.v. injected with 1 x IO' bursa cells from 11-dayold donors primed intrabursally or intravenously in the newly hatched period. As shown in Table 5, implant with bursa cells from donors primed intrabursally led to the restoration of the immune response against BA antigen but not SP antigen. It is noticeable that 1 -day bursa cells are competent for transferring the immune responsiveness, if primed by direct contact with antigens, in contrast to the adoptive immunization using cells unprimed or primed by the intravenous route.
Bursal Origin of Immunocompetent Cell
315
TABLE 3 ADOPTIVE
IMMUNE RESPONSES BY LYMPHOID CELLS TREATED WITH
ABuS
BA
Age of donor (weeks) 4 4 8 8
4 4 8 8 8 8 8
Cell transferred (x 108)
Number of Treatment responders/ total
Bursa 1-5 ABuS 1-5 Thymus ABuS 1-5 ABuS Spleen Bone 1 5 ABuS marrow 1-5 NRS Bursa 1-5 NRS Thymus 1-5 NRS Spleen 1-5 NRS Bone marrow 0-5* ABuS Spleen 0.5* Ig-absorbed Spleen ABuS 0.5* NRS Spleen
-t
SP
Mean + s.d.
Number of
(log2 titre)
responders/
Mean + s.d. (log2 titre)
total
0/8 2/10 0/5 0/6
1-5+0 5
0/7 2/10 0/5 0/6
2-5+0-5
3/3 5/5 5/5 6/6
4 0+1.0 3 4+0-5 5 0+0 5 3.8+0 9
3/3 5/5 5/5 6/6
3-3+0-5 24+0-5 4-6+0 4 3 5+0 5
0/4 0/4
0/4 0/4
4/4
5-3+1-0
0/16
4/4
4-8+1 3
0/16
1 5 x 108 lymphoid cells from 4- or 8-week-old donor chickens were treated with ABuS absorbed with thymus cells and injected i.p. into the individual recipient chickens together with both antigens, and serum agglutinin titres were measured 1 week later. 5 x 107 spleen cells from 8-week-old donor chickens were treated with ABuS absorbed with thymus cells and chicken immunoglobulin and injected i.v. into the individual recipients together with both antigens, and serum agglutinin titres were measured 1 week later. NRS = normal rabbit serum. * Intravenous injection was employed. t No cell recipient.
TABLE 4 ADOPTIVE IMMUNE RESPONSES BY BURSA OR THYMUS CELLS TREATED WITH ATS
BA
Age of donor
(weeks) 4 4 4 4
Cell transferred (x 108)
Bursa Thymus Bursa Thymus - -*
1-5 1-5 1-5 1-5 -
Number of Treatment responders/ total
ATS ATS NRS NRS -
3/3 5/5 3/3 5/5
0/6
SP
Mean + s.d. (log2 titre) 3-3+0-5 3 2+0 4 4 0+1-0 3-4+0 5
Number of responders/ total 2/3 5/5 3/3 5/5
0/6
Mean + s.d.
(log2 titre) 2-5+0-5 2-3+0 4 3-3+0-5 2-4+0 5
1-5 x 108 bursa or thymus cells from 4-week-old chickens were treated with ATS absorbed with bursa cells and injected i.p. into the individual recipients together with both antigens, and serum agglutinin titres were measured I week later. * No cell recipient.
H. Matsuda, T. Baba and
316
r.
Bito
TABLE 5 ADOPTIVE IMMUNE RESPONSES BY PRIMED BURSA CELLS
SP
BA
Number of responders/ total
Group
Donor cells
1
Bursa cells from chickens intrabursally primed at 4 days of age Bursa cells from chickens intravenously primed at 4 days of age
4/5
-t
0/5
2
3
015
Number of Mean + s.d. of P value* responders/ total responders (log2 titre)
3-6+2-0
0-028
0/5
-
0/5
Mean + s.d. P value* of responders (log2 titre)
0/5
107 Bursa cells from 11-day-old chickens primed intrabursally or i.v. with both antigens were injected i.p. into the individual recipients together with both antigens, and serum agglutinin titres were measured 1 week later. * P value for difference from group 3 calculated by Mann-Whitney U-test. t No cell recipient.
DISCUSSION The present findings may be summarized as follows: (a) bursal and splenic cells from normal chickens were more efficient than bone marrow or thymic cells in restoring the immune response in immunoincompetent chickens; (b) splenic, thymic and bone marrow cells taken from neonatally bursectomized chickens did not restore immunocompetence; (c) bursal, splenic or bone marrow cells from thymectomized chickens restored the antibody response in immunologically incompetent recipients; (d) treating spleen cells with specific anti-bursa antiserum in the presence of complement deprived them of the ability to restore the immune response in agammaglobulinaemic chickens; (e) bursal and thymic cells treated with ATS were fully active in restoring the suppressed immune response. These findings clearly show that the bursa functions as the primary source of the immunocompetent cells. Failures in immune reconstitution of bursectomized chickens by bursal implants have been reported (Cooper et al., 1966; Ivanyi et al., 1972a; Thompson and Cooper, 1971). Most of these authors performed autologous implant of the bursal cells from neonatal chickens. On the other hand, Toivanen, Toivanen and Good (1972c) succeeded in restoring the function and morphology of the bursa-dependent lymphoid system by transferring bursal cells taken from embryo or 4-day-chickens. In their experiments, the rudimentary follicles underwent drastic changes on cyclophosphamide treatment, but were not obliterated. The bursa cells introduced intravenously repopulated them, resulting in morphological and functional reconstitution of the bursa. These results strongly suggest that maturation in the bursa of the uncommitted stem cells transferred from embryos or newly hatched chickens is crucial to their ability to restore the immune response in immunologically impaired recipient chickens. No rudiments of the bursa to which injected bursa cells could home remain after surgical bursectomy (SB). This may
Bursal Origin of Immunocompetent Cell 317 explain partially the failure to reconstitute with bursa stem cells taken from embryos or neonatal chickens. Our data show that none of the lymphoid cells taken from 14-dayold or younger chickens were able to restore the immune response in bursectomized and irradiated chickens, whereas, those taken from 18-day-old or older chickens did restore the immune response. In White Leghorns, the bursa reaches its maximal size, acquiring plicate structure, at 3-6 weeks of ages. Each plica consists of follicles filled with lymphoid cells (Glick, 1956, 1960). Toivanen et al. (1972a, c) reported that the older the donor of the bursa cells, the more active the immune reconstituting capacity. This may reflect progressive maturation of the bursal cells. The mechanism by which the bursa functions in development of the humoral immune system is not clear. It is, however, generally accepted that the bursal cell serves as a precursor of the antibody-forming cell, i.e. the bursal stem cell itself maturates through unknown steps into an antibody-forming cell upon exposure to an antigenic stimulus. This supposition comes from the following evidence. (A) IgM-containing cells appear in the bursal follicles of 14-day-old embryos and IgG-containing cells in those of newly hatched chicken (Kincade and Cooper, 1971; Choi and Good, 1972). In other lymphoid organs, immunoglobulin-containing cells do not appear during the embryonic or newly hatching periods (Thorbecke, Warner, Hochwald and Ohanian, 1968). The bursa is the only organ that contains many 'double producers', cells synthesizing both IgM and IgG with very high frequency (Kincade and Cooper, 1971). Treating the 13-day-old embryo with anti-yu antibodies followed by neonatal bursectomy resulted in suppression of both IgM and IgG syntheses; the same treatment on hatching day decreased only IgM production. This suggests that the IgG producers originate from the IgM producers within the bursa (Kincade, Lawton, Bockman and Cooper, 1970). (B) Direct introduction of antigen into the cloaca markedly increases plaque-forming cells (Van Alten and Meuwissen, 1972). (C) ABuS inhibited appearance of splenic plaque-forming cells of chickens immunized with sheep erythrocytes (Potworowski, Richer, Borduas and Forget, 1971; Ivanyi and Lydyard, 1972b). Studies on the peripheral distribution of bursa cells (Durkin, Theis and Thorbecke, 1972; Hemmingsson and Linna, 1972) strongly support this postulate. Direct administration of antigens into the cavity of the bursa demonstrates the immunocompetence of 11-day-old bursa cells. Similar priming elicited a significant augmentation of anti-BA antibody production following the usual immunization by the intravenous route (unpublished data). Antigen-binding cells were detected in the embryonic bursa (Dwyer and Warner, 1971). Direct injection of sheep erythrocytes into the cloaca markedly increased the number of plaque-forming cells against the antigen (Van Alten and Meuwissen, 1972). These facts suggest strongly the presence of antigen-recognizing or antigen-reactive cells in the bursa of young birds and proliferation of the cells in response to antigenic triggering. Our experiments suggest the existence, in the bursa of neonatal chickens, of antigen-recognizing cells against BA but not SP antigen. The cause of the difference between the response to BA and SP antigens is not clear, but may be related to the delay of immune maturity against SP antigen.
ACKNOWLEDGMENTS The authors are sincerely grateful to Professor K. Kondo and Dr Y. Fujio, Faculty of Agriculture, Nagoya University, for their generous gift of inbred chicken, strain Anthony,
318 H. Matsuda, T. Baba and r. Bito and to Dr A. Takeda and co-workers, Radiation Center of Osaka Prefecture, for irradiation of the chickens.
REFERENCES
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