Immune capacity of the chicken bursectomized at 60 hr of incubation: Production of the immunoglobulins and specific antibodies

CLINICAL

IMMUNOLOGY

AND

IMMUNOPATHOLOGY

23,459-469

(1982)

Immune Capacity of the Chicken Bursectomized Incubation: Production of the lmmunoglobulins Specific Antibodies’s* KAISA GRANFORS, CLAUDE MARTIN, OLLI LASSILA, RAIMO AULI TOIVANEN, AND PAAVO TOIVANEN Departments Institut

of Medical d’Embryologie

Microbiology du CNRS

at 60 hr of and

SUVITAIVAL,

and Medicine, Turku University, Turku, et du CollPge de France. Nogent-sur-Marne,

Finland, and France

Chickens bursectomized (tailectomized) at 60 hr of incubation were tested for antibody and immunoglobulin production at the age of l-6 months. They were immunized at the age of 5 weeks with HGG, Bruce/la, and SRBC, at the age of 10 weeks with diphtheriatetanus-pertussis vaccine and levan, and at the age of 16 weeks with Escherichia coli LPS and Salmonella. Each antigen was administered intraperitoneally three times, with one-week intervals. Four of the eight animals studied showed virtually no antibody response of IgM, IgG, or IgA class against any of the antigens studied. Four chickens showed a normal or intermediate response indicating an incomplete bursectomy. The bursectomized birds, including those without any specific antibody response had good amounts of IgM, IgG, and IgA in the serum. Our findings indicate that the bursa of Fabricius is essential for activation of the V gene repertoire. Some specificities, the nature of which still remain undetermined, may develop extrabursally, and the same applies for the switch from IgM to IgG and IgA production.

INTRODUCTION

The roots of our study are in the Pink House, the former home of Robert A. Good. When the two senior authors of this paper arrived in Minneapolis in 1969, the laboratories of the Variety Club Heart Hospital were so filled with people and activity that the only place to find a small unoccupied comer was a few blocks away in the Pink House. There, guided by the Saturday morning rounds, we started our endeavors into the world of lymphocyte differentiation and were committed to use chicken as an experimental model. We carry happy memories from those years and feel proud to have been trained by Bob Good. The experiments carried out in the Pink House (l-4) were crucial for the later definition of bursal and postbursal stages of the avian lymphoid development (5-7). They have now led our studies into the prebursal B-cell differentiation and into the sources of the most primitive hemopoietic stem cells (S- 1 l), a problem often discussed in the immunobiology seminars on the basis of David Camp and other pioneering recipients of allogeneic bone marrow (12, 13). We have recently demonstrated that prebursal (as well as prethymic) stem cells exist in the intraembryonic mesenchyme of 7-day-old chick embryos (14- 16), and that at 2 days of incubation the stem cells for the lymphoid system are found in the embryo proper i Dedicated to Robert A. Good on the occasion of his 60th birthday. ? This study was supported by a grant from the Finnish Cancer Union. 459 0090-1229/82/050459-11$01.00/O Copyright Au rights

@ 1982 by Academic Press, Inc. of reproduction in any form reserved.

460

GRANFORS

ET

AL.

(17-20). From the 2nd to 7th day of incubation they migrate from the embryo into the yolk sac (21). The groups of Fitzsimmons (22-24) and JankoviC (25-27) have described a technique for surgical removal of the bursal primordia at 52-72 hr of incubation. Uniformly both groups also described humoral immune activity in the bursectomized chickens, including production of IgM, of agglutinating antibodies against sheep and guinea pig erythrocytes, as well as of direct and indirect plaque-forming cells using guinea pig erythrocytes as the antigen. If confirmed, these findings would indicate an extrabursal differentiation site for the prebursal stem cells. On the other hand, Ewert and Cooper (28) did not find surface immunoglobulinpositive cells in the spleen, bone marrow, or thymus of such embryonically bursectomized birds when studied at the age of 14 days posthatching. Detailed studies of the specificity of the potential immune response and particularly of the antibody repertoire of the embryonically bursectomized birds have so far been lacking. To answer these questions we have started a series of experiments using surgical removal of the early bursal anlage, and our experience on the production of immunoglobulins and specific antibodies by such bursectomized chickens is reported here. MATERIALS

AND METHODS

Fertilized eggs from the lines P (genotype B2B2) and V (genotype B15B15) kept at the Department of Medical Microbiology, Turku University, were used. The incubation of the eggs and the care of the birds was as described previously (5). Bursectomy. Bursectomy at 60 hr of incubation was carried out according to the previously described techniques (22-26) using microsurgery scissors. It was performed under aseptic conditions by ablation of the tail bud caudally to the leg buds. The operation was carried out just before the amniotic membrane begins to cover the caudal part. Now, after an experience of more than 1500 operations about 10% of our operated embryos hatch. Antigens and immunizations. The chickens were immunized at the age of 5 weeks with human gammaglobulin (HGG), Brucella, and sheep red blood cells (SRBC), at the age of 10 weeks with diphtheria-tetanus-pertussis vaccine and levan, and at the age of 10 weeks with Escherichia coli lipopolysaccharide (LPS) and Salmonella. The origin and amount of the antigens given at a time were as follows: HGG (Finnish Red Cross Blood Transfusion Service, Helsinki), 1 mg, formalin-killed Brucella abortus, 2 x 105 bacteria, SRBC, 1 ml of 5% suspension, commercially licensed diphtheria-tetanus-pertussis vaccine (The Central Public Health Laboratory, Helsinki; 50 Lflml diphtheria toxoid, 10 LE/ml tetanus toxoid, and log Bordetella pertussis bacteria/ml), 1 ml of 1:20 dilution, native levan prepared from Corynebacterium levaniformis as described previously (29, 30), 100 pg, E. coli LPS (lipopolysaccharide W, E. coli 055:B5, Difco Laboratories, Detroit, Mich.), 10 pg, formalin-killed Salmonella pullorum, lo6 bacteria. Each antigen was administered intraperitoneally three times, with one-week intervals. Blood samples from a wing vein were collected into heparinized Chickens.

BURSECTOMY

AT

60 hr OF

INCUBATION

461

syringes 7 days after the first and the second stimulation and 5 days after the third stimulation. The plasmas were stored at -20°C. Determination of antibodies. Direct agglutinins to SRBC were measured by microtitration using 25-~1 volumes of the plasma to be titrated and 0.25% SRBC. One percent fetal bovine serum in saline was used as a diluent. The plates were incubated for 1 hr at 37°C and then for 20 hr at 4°C before reading. IgM, IgG, and IgA class antibodies against all other antigens were quantitated by an enzyme-linked immunosorbent assay (ELISA), which is a modification of our radioimmunoassay method (31). Details of ELISA for antibodies against HGG, Brucella, diphtheria and tetanus toxoids, and Bordetella have been described elsewhere (32-36). The antigen was adsorbed onto polystyrene microtiter plates (Linbro/Titertek, Linbro Division, Flow Laboratories, Hamden, Conn.), 75~1 samples of serum dilutions were added to the plates and incubated at 37°C for 2 hr. The plates were washed three times with phosphate-buffered saline, and 75 ~1 of alkaline phosphatase-conjugated sheep anti-chicken CL,anti-chicken y, or antichicken (Y was added to the plates. After incubation overnight at room temperature the plates were washed again, and 75 ~1 of fresh substrate, 2 mg of pnitrophenylphosphate (Sigma Chemical Co., St. Louis, MO.) per milliliter was added to the plates. The mixture was incubated for 30 min at 37°C and 125 ~1 of 1 N NaOH was added to stop the reaction. The optical density of the colored product was measured in a photometer (TiterteWMultiskan Flow Laboratories, Hamden, Conn.) at a wavelength of 405 nm. The same procedure was applied for the ELISA with levan, E. coli LPS, and Salmonella as the antigen. The results are presented as percentages of the concentration in hyperimmune reference sera. For the coating of the plates levan and E. coli LPS were dissolved in 0.05 M carbonate buffer, pH 9.6, and other antigens in phosphate-buffered saline. The optimal concentrations used for the coating of the plates were determined to be for Brucella lo6 bacteria/ml, HGG 0.1 mg/ml, levan 50 Fg/ml, E. cofi LPS 10 &ml, and Salmonella 2 x 105 bacteria/ml. Diphtheria and tetanus antigens were the same as those used in the vaccine, but not supplemented with aluminium phosphate (Orion Diagnostica, Helsinki). The purity of diphtheria and tetanus toxoids was 1875 and 2340 LfYmg protein N, respectively. As Bordetella antigens in ELISA, formalin-killed Bordetellu pertussis bacteria were used (Per-vaccin, batch 1176, Orion Diagnostica, Helsinki). The antigen concentrations in ELISA were for diphtheria toxoid 4.7 Lf/ml, for tetanus toxoid 3.5 LE/ml, and for Bordetella pertussis 57 x 106 bacteria/ml. Determination of immunoglobulins. Total IgM, IgG, and IgA concentrations were measured by ELISA. Polystyrene microplates were coated with (NH&SO, precipitated preparations of unabsorbed sheep antisera against chicken IgM, IgG, or IgA. For coating 100 ~1 of solution with concentration of 10 pg protein/ml was used. Samples (75 ~1) of plasma diluted 1:5000 for IgG determinations and 1: 100 for IgM and IgA determinations were added to the plates and incubated at 37°C for 2 hr. The plates were washed three times as earlier and 75 ~1 of alkaline phosphatase-conjugated, purified sheep anti-chicken p, anti-chicken y, or antichicken (Y was added. The rest of the procedure was carried out as described

462

GRANFORS

ET

AL.

above for the ELISA of specific antibodies of IgM, IgG, and IgA class. The results were calculated in milligrams per milliliter using purified chicken IgM, IgG, and IgA as standards. Anti-heavy chuin sera. Sheep antisera to chicken immunoglobulin heavy chains were produced and purified as described earlier (3 I). The final preparations of anti-chicken p, anti-chicken y, and anti-chicken (Y used were free of any other antibodies or serum proteins. The specificity of the antisera was confirmed by ELISA inhibition experiments. The final preparations were used to measure the inhibitory capacity of purified chicken IgM, IgG, and IgA; serial dilutions of the immunoglobulins were preincubated with a suboptimal dilution of antibodies for 30 min at 37°C. The mixtures were then used in triplicate for ELISA with IgM, IgG, or IgA as antigens. Fifty percent inhibition of homologous antibodies was obtained by 0.3 pg/ml for IgG and IgA, and 1.3 pghnl for IgM. For the 50% inhibition of heterologous antibodies at least lo-fold higher quantities were required . Alkaline phosphatase conjugation of the antibodies was carried according to Engvall and Perlmann (37). Lymphocyte responses to phytohemagglutinin (PHA) and concanavalin A). To determine mitogen responses of peripheral blood lymphocytes,

blood microcultures

were used as described previously

A (Con

whole

(38).

RESULTS Production of Antibodies; Groups (Bx-1 , Bx-2)

Division

of Bursectomized

Chickens

into Two

Eight bursectomized chickens (out of 100) survived to the age of 5 weeks. At this age they were immunized, together with age-matched controls, with HGG, Brucella abortus, and SRBC, at the age of 10 weeks with diphtheria-tetanuspertussis vaccine and levan, and at the age of 16 weeks with E. coli LPS and Salmonella. Each antigen was administered intraperitoneally three times, with one-week intervals. When the results were analyzed, it appeared that the birds were clearly divided into two groups. Four chickens showed virtually no antibody response to any of the nine antigens used (group Bx-1) and four animals produced antibodies against all the antigens used, at least after the third immunization (group Bx-2). All the bursectomized birds appeared typically tailless as described by Fitzsimmons et al. (23). Except one in group Bx-2 (which died at 20 weeks of age), they all survived in good condition to the end of the experiment; they were killed and autopsied at the age of 23 - 26 weeks. Mean body weight (+SEM) of birds in the Bx-1 group was 1353 t 60 g (two females, two males), in the Bx-2 group, 1601 t 258 g (one female, two males), and in the control group, 1814 ? 112 g (five females, nine males). No remnants of the bursal tissues were observed at gross examination or by histology in any of the bursectomized or control birds, due to the physiological involution at this age. Detailed analysis of antibody production against diphtheria toxoid is shown in Table 1. The Bx-1 birds did not form antibodies at all against diphtheria toxoid,

BURSECTOMY

TABLE IgM, IgG, AND IgA ANTIBODIES DIPHTHERIA-TETANUS-PERTUSSIS

463

AT 60 hr OF INCUBATION 1

AGAINST

DIPHTHERIA TOXOID AFTER IMMUNIZATION WITH VACCINE AT THE AGES OF 10 (I”), 11 (2”). AND 12 (3”) WEEKS”

Antibody isotype

Sample

kM

0

0.0

1” 2” 3”

0.0 0.0 0.0

3.5 2.0 4.0 35.8

Ifi 0.5 ” 0.9 k 0.7 k 21.5

7.9 7.8 19.6 41.5

k 2 ” +

1.5 1.5 4.4 8.3

1~6

0 1” 2 3”

0.0 0.0 0.1 t 0.0 0.1 * 0.1

0.2 0.2 9.8 26.0

k 0.0 -+ 0.0 + 4.4 i 4.6

1.9 1.5 14.6 25.0

2 k k +

0.8 0.5 3.4 3.1

I&

0 I” 2” 3”

0.0 0.0 0.0 0.0

0.1 0.4 3.9 61.8

rt i 2 t-

7.0 13.6 38.4 71.1

i 2.1 2 3.9 _f 8.4 + 7.4

Bx-1 (II = 4)

Bx-2 (n = 4)

0.1 0.2 12.6 20.1

o Zero (0) samples were collected immediately before the first immunization. SEM) are expressed as percentage of a standard serum.

Control (n = 14)

The results (mean 5

whereas Bx-2 birds showed anti-diphtheria responses which were not significantly less than those of the control animals (P > 0.05; Table 1). Exactly similar findings were made regarding antibody response against Brucella abortus (Table 2). Quite similar were also antibody responses against SRBC, Bordetella pertussis, levan, Salmonella pullorum, and E. coli LPS, with the only difference that Bx-2 birds showed responses equal to those of the control animals, whereas Bx-1 animals again did not have any significant formation of specific antibodies (Table 2). Antibody response of bursectomized animals to HGG (Table 3) was very similar to the response against the majority of the antigens used, with one exception. It appeared that small amounts of IgA class antibodies against HGG were detectable by our ELISA in the samples of all Bx-1 birds after the first, second, and third immunization. The same phenomenon was not observed regarding IgM or IgG class antibodies against HGG. Responses which can be more easily explained than the response against HGG occurred also against tetanus toxoid. In Bx-1 birds no responses were observed after the first and second immunization, whereas the mean values for the samples taken after the third immunization indicate a slight IgG and IgA class response (Table 2). When the responses of individual birds are analyzed, it appears that one out of the four Bx-1 birds shows a slight anti-tetanus response of all three immunoglobulin classes already after the second immunization, and this goes on particularly in the IgG and IgA class after the third immunization, representing most probably a specific antibody response (Table 4). Antibody responses of the three other birds remain at the background level (~1% of the hyperimmune reference serum).

GRANFORS

464

TABLE SRBC

AGGLUTININS, AND IgM, PLASMA SAMPLES TAKEN

8 8

Antigen

2

IgG, AND IgA ANTIBODIES 5 DAYS AFTER THE THIRD RESPECTIVE

Ageb (weeks)

ET AL.

Antibody isotype

SRBC Brucella

AGAINST SIX IMMUNIZATION

OTHER ANTIGENS WITH THE

IN

ANTIGENS

Bx-1 (n = 4)

Bx-2 (n = 4)

W IgG &A

o.o* o.o** 0 ()**r o.o*

11.5 9.7 4.2 7.4

c 4 2 2

0.3 3.1 2.7 1.7

Control (n = 14) 10.8 37.8 19.5 21.6

t 0.4 t 10.0 -c 6.9 t 5.1

13

Bordetella

Ii@ W &A

0.6 + 0.5* 1.0 2 0.4* 0.2 ‘- 0.1**

57.0 5 15.5 29.8 -t 0.8 65.2 -t 20.2

79.2 I 7.2 28.3 t 2.6 62.5 f 10.2

13

Tetanus

IN W kA

1.0 2 1.0* 4.8 k 4.4** 5.0 ” 5.0*

52.8 + 17.1 47.0 -t 17.7 66.8 t 14.6

48.5 -t 3.9 36.5 + 4.6 65.7 1: 6.3

13

Levan

hM IS &A

0.8 2 0.F’” O.Ob 0.5 t 0.5***

37.0 -t 21.3 55.2 t 17.0 41.2 t- 19.7

41.8 5 7.7 47.2 t- 8.5 36.2 t 8.3

18

Salmonella

kM kG &A

0.2 + 0.1 0.3 t 0.1*** 0.8 + 0.8

31.8 2 22.9 14.2 + 9.6 24.5 !z 19.6

34.7 ? 9.8 20.5 + 4.0 28.9 i 8.6

18

LPS

I&f kG 1.d

0.4 rt 0.2 o.o** 0 o*v*

27.5 t 24.2 10.0 t- 7.7 26.3 t- 24.6

16.0 2 7.2 9.5 t 2.4 11.1 % 4.9

a Mean values f SEM are given: log, titers for the SRBC agglutinins and percentage of a reference serum for the other antibodies. * Age of the animals when the sample was taken. * P < 0.001 in comparison with the control group, by Student’s t test ** P < 0.01.

**VP < 0.05.

Production of Immunoglobulins

All three immunoglobulins, IgM, IgG, and IgA, were detected in the plasma samples taken from the Bx-1 birds at the age of 5, 10, and 18 weeks. The concentrations were significantly lower than those in the simultaneous samples of Bx-2 or control birds (Table 5). The mean immunoglobulin concentrations observed in the samples of Bx-2 birds were at the same level as those in the control animals. PHA and Con A Responses

To evaluate the thymic function of embryonically bursectomized birds, lymphocyte responses to PHA and Con A by peripheral blood lymphocytes were determined at the age of 22 weeks. The results obtained (Table 6) indicate quite normal PHA and Con A responses in Bx-1 and Bx-2 birds; the differences observed are statistically insignificant (P > 0.05).

BURSECTOMY IgM, IgG,

AND

IgA

ANTIBODIES THE

Antibody isotype

AGES

TABLE 3 HGG AFTER IMMUNIZATION (lo), 6 (2”), AND 7 (3”) WEEKS~

AGAINST OF 5

Bx-1 (n = 4)

Sample

IN

465

AT 60 hr OF INCUBATION WITH

Bx-2 (n = 4)

HGG

AT

Control (n = 14)

1” 2” 3”

0.2 0.2 0.2 0.2

+ k 2 +

0.1 0.1* 0.2 0.2*

0.3 28.3 15.0 34.6

+ 2 * k

0.2 10.3 5.0 5.6

0.2 41.8 10.6 40.2

2 + ? ”

W

0 1” 2 3”

0.5 0.4 1.2 2.7

2 0.0 2 0.1* + ox* Ifr 2.4

0.4 7.2 18.2 15.8

k ? k +

0.0 2.3 4.8 2.5

0.4 9.5 10.4 18.7

? 0.0 + 1.2 -+ 1.3 + 8.3

kid

0 1” 2 3”

0.4 6.9 6.2 3.9

+ -t k f

0.5 13.4 26.4 19.8

+ + t ir

0.2 3.8 4.1 2.7

0.3 23.3 20.3 31.0

-e 0.1 + 33. + 1.6 2 4.0

0

0.1 2.1* 1.9** 1.5*

rcZero (0) samples were collected immediately before the first immunization. SEM) are expressed as percentage of a standard serum. * P c 0.01 in comparison with the control group. .‘-* P < 0.001.

0.0 6.5 2.9 7.0

The results (mean ?

DISCUSSION

The method of surgical removal of the bursal anlage before it is colonized by stem cells has not been widely used, due to technical difficulties. However, after some practice we have been able to increase the hatchability of operated embryos to a level which permits meaningful experiments with sufficient numbers of birds. In the present material, the birds were allowed to grow for 6 months. At this age the bursa has already spontaneously involuted, and therefore the completeness of the procedure could not be evaluated by autopsy. The antibody responses in the four birds of the group Bx-2 suggest that in these individuals some bursal follicles may have developed. It is also possible that the same applies to one bird (No. 808, TABLE IgM, IgG,

AND

Chicken Sample 0 I’ 2” 3”

IgA

ANTIBODIES

663

kM

I&

Id

0.0 0.0 0.0 0.0

0.0 0.0 0.0 1.0

0.0 0.0 0.0 0.2

n Immunizations were percentage of a standard For antibody response

kM 0.4 0.0 0.0 0.0

4

AGAINST TETANUS BIRDS OF GROUP Bx-1”

TOXOID

Chicken

Chicken

698

kG

Lg.4

0.0 0.0 0.0 1.0

0.0 0.0 0.7 0.0

I&f 0.0 0.0 0.0 0.0

carried out at the ages of 10 (I”), 11 (2”), and serum. in the control birds see Table 2.

IN FOUR

INDIVIDUAL

806

Chicken

I&

kid

0.0 0.0 0.0 0.0

0.0 0.0 0.3 0.0

12 (3”) weeks.

kM 0.0 0.0 6.0 4.0 The results

808

IIs

II@

0.0 0.0 7.0 18.0

0.0 0.0 1.7 20.0

are expressed

as

466

GRANFORS

CONCENTRATION

k

TABLE 5 OF PLASMA IMMUNOGLOBULINS GROUPS OF CHICKENS

(mgml) THREE

Age of chickens (weeks)

ET AL.

Bx-I (n = 4)

Bx-2 (n =4)

IN THE

Control (n = 14)

W

5 10 18

0.17 + 0.05 0.05 t 0.02** 0.49 + 0.24*

0.24 2 0.08 0.12 2 0.02 0.45 2 0.06*

0.24 k 0.03 0.20 2 0.02 1.34 2 0.05

IgG"

5 10 18

0.22 2 0.12 0.87 k 0.17 0.77 k 0.25

0.47 2 0.15 0.90 + 0.47 1.37 2 0.23

0.51 + 0.08 1.05 ? 0.23 1.56 t 0.21

&A

5 10 18

0.33 2 0.16 0.13 +- 0.05*** 0.17 f 0.08

0.54 2 0.14 0.31 k 0.05 0.93 k 0.25

0.38 t 0.04 0.33 2 0.04 1.43 k 0.32

(1 Concentrations of IgG found by us in the control animals are considerably lower than the mean values reported by others (3.1- 12.6 mg/ml; 62-64). At present, we have no explanation discrepancy and are studying whether it depends on technical factors (analysis, storage) or chickens. However, whatever the explanation is it hardly can affect the conclusions presented paper. * P < 0.001 in comparison with the control group. ** P < 0.01. *** P < 0.05.

normal for this on the in this

Table 4) of group Bx-I producing tetanus antibodies but not others. IgA and IgG class antibodies against HGG were also found in Bx-1 birds. However, since no evidence was observed for the production of IgM antibodies against HGG in these birds, we believe that these findings in the ELISA are rather due to some nonspecific binding of sheep anti-chicken (Y by HGG than to the occurrence of specific antibodies against HGG.

PHA

TABLE 6 AND CON A RESPONSES BY PERIPHERAL BLOOD LYMPHOCYTES OF BURSECTOMIZED (Bx-1. Bx-2) AND CONTROL ANIMALS, AT THE AGE OF 18 WEEKS”

Mitogen and dose (&ml)

Bx-1 (n = 4)

Bx-2 (n = 4)

Control (n = 14)

129 + 27 145 + 47 124 k 51

194 * 60 199 k 64 195 * 44

165 t 28 183 -t- 35 159 t 36

3.1 * 1.2 4.5 * 1.0 13.1 * 5.1

6.2 + 4.9 4.6 ‘- 1.1 26.1 2 10.5

5.4 t- 1.0 21.2 t 10.3 25.9 it 8.0

1.5 t 0.3

1.0 * 0.1

2.0 -r- 0.5

PHA 300 600 1200 Con

A

5 10 20 None u Mean

values

-t SEM

(cpm

x lo-*)

are given.

BURSECTOMY

AT

60 hr OF

INCUBATION

467

The most striking finding in the present experiments is the capacity of Bx-1 birds to produce immunoglobulins of all three classes. The most likely explanation is that the removal of the bursal anlage has been only partial. This explanation becomes improbable, however, on the basis of the complete incompetence of the three Bx-1 birds to produce specific antibodies against nine different antigens, and on the basis of our recent, similar experience with three birds autopsied at the age of 6 weeks. In these experiments, still in progress, we have observed no macroscopic or microscopic evidence for the existence of bursal remnants in spite of demonstrable IgM and IgA in the serum of birds bursectomized in the same way as in the present work. The existence of an extrabursal site for B-cell differentiation has been proposed by several investigators (26, 39-45). The suggestions have been based first of all on the findings with birds bursectomized hormonally or surgically in ova. These birds may be able to produce immunoglobulins, especially IgM, but with antibody activity against a limited number of antigens. Since they in most instances lack IgG, the bursa has been considered essential for the switch from IgM to IgG production (39, 42, 46, 47). Our observations do not support this possibility. Our findings suggest that the switch from IgM to IgG and IgA production may occur also in the absence of the bursa, but the resulting antibody repertoire is very small. It still remains to be established what the antibody specificities of these immunoglobulins are. Huang and Dreyer (48), using two-dimensional gel electrophoresis, demonstrated a limited diversity of CL,y, and light chains of chickens hormonally bursectomized on Day 11 of incubation. Three out of seventeen bursectomized birds responded with low titers to SRBC. The authors interpreted their findings to indicate that the generation of the immunoglobulin diversity was blocked by bursectomy at different points in different individual birds, a view which is in concordance with the theory of sequential development of the antigen-specific B-cell diversity (44, 45, 49, 50). When the prebursal stem cells do not find a proper microenvironment for further differentiation as is the case in the bursectomized embryos, they may become nonspecifically activated, due to a physiological occurrence of polyclonal B-cell activators, including bacterial products (51). Different polyclonal B-cell activators are known to activate B cells of different maturity degrees not only in the mouse but also in the chicken (52, 53). It has also become clear that this nonspecific activation may result in the expression of immunoglobulin classes other than IgM (5 1, 54). A possible localization of the extrabursal B-cell differentiation might be in the gut-associated lymphoid tissue, originally suggested as the mammalian analog of the avian bursa (50, 55). Recent experimental evidence in mammals has been provided by Reynolds (56). Accordingly, Bryant et al. (40) have demonstrated plasma cells in the gut lymphoepithelia of bursaless chickens lacking plasma cells in the spleen, and Befus et al. (58) have extended the extrabursal site of B-cell differentiation to include all mucosal-associated lymphoid tissues. Another possibility to consider is the bone marrow (59-61), where the postbursal cells also normally migrate after the physiological bursal involution (6). In conclusion, it seems likely in the light of the present findings that the bursa of

GRANFORS

468

ET AL.

Fabricius is necessary for the generation of antibody diversity, i.e., for the activation of the V gene repertoire. Some specificities, the nature of which still remain undetermined, also may develop extrabursally, and the same applies for the switch from IgM to IgG and IgA production. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.

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