Questioning the role of adenosine deaminase in the development of B lymphocytes in chicken bursa

Developmentaland ComparativeImmunology,Vol. 14, pp. 95-104, 1990 Printed in the USA. All rights reserved.

0145-305X/90 $3.00 + .00 Copyright © 1990 Pergamon Press plc

QUESTIONING THE ROLE OF ADENOSINE DEAMINASE IN THE DEVELOPMENT OF B LYMPHOCYTES IN CHICKEN BURSA *Sonia Senesi, *Giovanna Batoni, Francesco Bianchi, *Giulia Freer, Amelio Dolfi, *Mario Campa, and Mario Lupetti *Department of Biomedicine, Via S. Zeno, 39 and Chair of Histology and General Embryology, Via Roma, 55 University of Pisa, 56100 Pisa, Italy

(Submitted December 1988; Accepted January 1989)

Introduction

I-qAbstract--Adenosine deaminase, a purine metabolic enzyme, was studied in lymphoid tissues of the developing chicken in order to evaluate whether enzyme activity is related to development of the immune system in birds in the same way as for mammals, in which adenosine deaminase is essential for lymphocyte differentiation, especially for the T-cell lineage. Enzyme activity was assayed in thymocytes and bursal lymphocytes at different times during chicken development ranging from the 17th day of embryonic life up to the 50th day after hatching. Adenosine deaminase activity was significantly higher in the bursa than in the thymus, regardless of whether such an activity was expressed per mg protein or per 10a cells; moreover, no substantial difference in the relative levels of adenosine deaminase was observed in thymocytes at the various stages of thymus development studied. Significant changes in enzyme activity, however, were found in bursal lymphocytes in which different amounts of adenosine deaminase appeared to be related to definite stages of bursal development and to specific immunological responsiveness of B lymphocytes to intravenously injected antigens. Therefore, if adenosine deaminase does play a role in the functional maturation of the immune system in birds, such a role appears to be related to the differentiation of the B- rather than the T-cell lineage.

The finding that congenital deficiency of adenosine deaminase (ADA, adenosine aminohydrolase, EC 3.5.4.4) is related causally to one of the forms of severe combined immunodeficiency (1,2) has proven that ADA plays a key role in the functional m a t u r a t i o n o f the immune system, at least in mammals. ADA is a ubiquitous enzyme which is involved in the purine salvage pathway, catalyzing the irreversible hydrolytic deamination of adenosine and 2'-deoxyadenosine to their inosine derivatives. The main biological function of ADA in lymphocytes appears to be the p r o t e c t i o n .of these cells from the toxic effect of high concentrations o f ADA substrates, especially 2'-deoxyadenosine (3), and their metabolites, especially dADP and dATP, which are believed to be largely responsible for the observed decreased immune function (4). In mammals, the highest levels o f A D A a c t i v i t y are f o u n d in l y m p h o i d tissues, particularly in the thymus (5,6). Different amounts of ADA activity are related to distinct stages of T-cell differentiation: Higher ADA activity is found in i m m a t u r e c o r t i c a l t h y m o c y t e s , whereas lower levels are detectable in i m m u n o c o m p e t e n t medullary t h y m o cytes and peripheral T cells (5-9). These data, together with the finding that a potent inhibitor of ADA, 2'-deoxycoformycin, s u p p r e s s e s the m a t u r a t i o n o f

[~Keywords--Adenosine deaminase; B lymphocytes; Bursa of fabricius; Chicken.

Address correspondence to Prof. Mario Lupetti, % Istituto Anatomico, Via Roma, 55, 56100 Pisa, Italy. 95

96

precursor cells into T lymphocytes (10), are consistent with the notion that high levels of ADA are essential for T-cell maturation and that the enzyme activity decays as T-cell maturation proceeds. B lymphocytes seem to be less susceptible to specific ADA inhibition than T lymphocytes (11), but the biochemical mechanism by which B- and T-lymphocyte function and survival are affected differently by ADA deficiency is not yet completely understood. It has been suggested that the severe impairment of Bcell function observed in congenital ADA deficiency is the result of a primary defect in T-cell maturation, most likely in the T-helper subpopulation (11). Very little is known about the quantitative expression of ADA in the different stages of B-lymphocyte maturation. Since birds are the only class in which B-cell maturation takes place separately from that of the T-cell lineage, this study was undertaken to measure the levels of ADA activity in lymphoid tissue of the developing chicken, focusing our interest on bursal lymphocytes. The bursa of Fabricius is the single central lymphoid organ for the primary differentiation of B lymphocytes (12), in which lymphoid stem cells accumulate and mature before giving rise to a diversified population of B cells (13). The present paper clearly demonstrates that ADA activity is higher in bursal lymphocytes than in thymocytes and that the amount of ADA activity, which remains roughly constant during thymus development, changes markedly d u r i n g b u r s a l d e v e l o p m e n t . Such changes might be related to certain particular stages of bursal lymphocyte proliferation as well as to definite stages of B-lymphocyte maturation. Optimal conditions for the determination of the enzyme activity are also reported together with some molecular and kinetic properties of chicken ADA partially purified from bursal lymphocytes.

S. Senesi et al.

Materials and Methods

Animals Fertile eggs of the Hubbard strain were obtained from a local hatchery and i n c u b a t e d in our l a b o r a t o r y . After hatching, the chicks received food and water ad libitum. Five separate sets of about 150 eggs were used throughout this study.

Preparation of Cell Suspensions and Whole Cell Homogenates from Bursa and Thymus Uusually, bursa and thymus lobes were taken aseptically from 5-6 chicks killed by cervical dislocation. From the 17th day of embryonic life up to the first two weeks after hatching, 10-20 chicks were sacrificed to obtain cellular suspensions thick enough to allow all the analytical assays. Bursae and thymuses were washed in sterile phosphate-buffered saline (PBS, pH 7.4) and pooled. Neither bursae nor thymuses were ever stored below 0°C since a loss of ADA activity was observed in the homogenates obtained from thawed organs. Thymuses were minced with sterile scissors, pressed through a steel strainer in cold PBS and filtered through a layer of nylon gauze to remove clumps. The cell suspensions were washed twice with the same buffer at 500 x g for 5 min. At this stage, cell viability was more than 96%, as assessed by nigrosine dye exclusion. All the procedures were carried out rapidly at 0-4°C. Bursal cell suspensions were prepared essentially as described for thymocytes. In some experiments, however, bursal cell suspensions were enriched in lymphocytes by two separate procedures: (1) the gravity-sedimentation method described by Ratech et al. (14), by which the whole bursal cell suspensions were simply allowed to

ADA activity in chicken bursa

sediment spontaneously for 5 min to give supernatant cells with less than 1% contamination by epithelial cells; (2) filtration through a glass-wool column, as described by Julius et al. (15), to remove macrophage-like cells. Attempts were also made to separate cortical from medullary lymphocytes following the procedure described by Grossi et al. (16) at particular stages of bursal development, namely the 6th, 20th, 30th, and 45th day of chicken life. All cell suspensions described above were diluted to a final concentration of about 2.0 x 10s ceils/ml with cold 50 mM Tris. HCI (pH 7.0) and homogenized at 0-4°C by sonication with a BP-10 Blackstone Ultrasonic, Sheffield, PA, U.S.A. Sonication for 30-60 s, in cycles of 15 s, was usually effective in disrupting more than 99% of the cells, as seen at the light microscope. When the whole cell homogenates were not immediately assayed for ADA activity and protein content, they were stored at 2 - 4 ° C , at the most for one month, without any loss of ADA activity.

97

Partial Purification of ADA from Bursal Lymphocytes The crude homogenate was prepared as described above using 10 bursae taken from 1-month-old chickens. The homogenate (162 ml, 583.2 mg protein) was adjusted to pH 5.0 with 1.0 M Trisacetate (pH 3.5) while constantly mixed in the cold. The pH-5.0 homogenate (242 ml) was centrifuged at 46,000 x g for 1 h and the clear supernatant (230 ml, 138 mg protein) was subjected to ammonium sulfate fractionation between 30 and 80% saturation. The precipitate was then dissolved in a minimal volume of 0.05 M Tris • HCI buffer (pH 7.0) (33 ml, 115.5 mg protein) and chromatographed on a Sephadex G-100 column (90 x 5.5 cm) equilibrated with the same buffer. The flow rate was 40 ml • h-~ and 5-ml fractions were collected. Active fractions were pooled (81 ml, 84.5 mg protein) and used for the determination of some molecular and kinetic properties of chicken ADA.

Animal Immunization and Antibody Production Analytical Assays ADA was measured at 37°C during the linear zero-order portion of the curve by the spectrophotometric method described by Kalchar (17), assuming an absorption coefficient between adenosine and inosine of 8.11 cm- l . m M - 1 at 265 nm. The mixture was made up of 50 mM Tris • HCI (pH 7.5), 56 p~M adenosine (Sigma Chemical Co., St. Louis, MO) and various amounts of enzyme preparation not exceeding 0.1 ml in a final volume of 1.0 ml. Specific activity is expressed as nmol of inosine formed/ rain per mg protein or per l0 a cells. Protein content was determined according to the method of Lowry et al. (18) using bovine serum albumin (Sigma Chemical Co.) as a standard.

Sheep red blood cells (SRBC), obtained from a single donor and stored in Alsever's solution, were washed twice with PBS before use. Chicks to be tested for antibody production against SRBC at different stages of development were immunized with 1.0 x 108 SRBC given intravenously in 0.25 ml of PBS. Four days after immunization, the chicks were sacrificed. Spleen or bursa cells were suspended in Eagle's medium (Flow Laboratories, UK) and the direct plaque-forming cells (PFC) determined by the Jerne assay (19). Blood samples (1.0 ml) were collected from the same animals before sacrificing; sera were assessed for agglutinin titers individually, using the same antigen employed for animal immunization.

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S. Senesi et al.

Statistical Analysis

bursa or thymus collection. An 80% decrease in enzyme activity was also found in crude homogenates and in partially purified enzyme preparations after more than 3-4 cycles of freezing-thawing. On the other hand, the enzyme activity was preserved for months by simply storing the enzyme preparation at 4°C in media buffered between pH 5.0-7.5. . The pH-stability studies of the enzyme, which were carried out as described for mouse lymphocyte A D A (20), s h o w e d t h a t the chicken enzyme is stable in alkaline-, neutral-, and acid-buffered media; in alkaline media between pH 8.5 and 11.0 a loss of activity ranging from 12 to 90%, respectively, was observed. . The range of optimum pH for ADA determination turned out to be very narrow (7.2-7.7) and a pH value of 7.5 was used routinely in the ADA assays. . Differently from mammals, adenosine was found to be the best substrate for chicken ADA, followed by 2'-deoxyadenosine and 2,6 diaminopurine riboside. The Michaelis-Menten plot gave a Km

Data are expressed as a mean _ standard deviation (SD). Two-factor analysis of variance and the Student-NewmanKeuls test were used to evaluate the results statistically.

Results

Assay Conditions and Kinetic Properties of Chicken Lymphocyte ADA Preliminary experiments were designed to establish optimal conditions for the determination of ADA activity in lymphoid tissues of developing chickens together with some molecular and kinetic properties of the enzyme activity partially purified from bursal lymphocytes (Fraction IV of Table 1). These studies showed that: . An appreciable loss of ADA activity occurred when intact organs (both bursa and t h y m u s ) were stored below 0°C. Freezing determines a rapid decay of ADA activity, ranging from 30 to 60%, as compared to the amount of ADA found in the corresponding homogenates prepared immediately after

Table 1. Partial purification of chicken ADA from bursal lymphocytes.

Fraction h Crude homogenate Ih pH 5.0 fraction IIh 3 0 - 8 0 % ammonium sulfate fraction IV: G-100 filtration

Volume (ml)

Total Protein (mg)

Total Activity (nmol/min)

Specific Activity (nmol/min per mg)

162

583.2

459.5

0.78

1,0

230

138.0

5962.9

43.20

54.8

33

115.5

5785.1

50.08

63.5

81

84.5

4394.5

217.02

275.4

Purification factor a

Crude homogenate from 10 bursae (Fraction I) was adjusted to pH 5.0 and centrifuged; the clear supernatant (Fraction II) was subjected to ammonium sulfate fractionation (Fraction III) and filtered through a Sephadex G-100 column (Fraction IV). a Purification factor is expressed as a ratio between the specific activity found in the fraction and that found in the crude homogenate.

ADA activity in chicken bursa

99

value of 1.8 x 10 -5 M for chicken ADA, with adenosine as a substrate, which is in agreement with that reported for the chicken duodenal enzyme (21). . Gel filtration through a G-100 Sephadex column gave 36,000 as an apparent Mr of the enzyme activity extracted from bursal lymphocytes. Such a Mr value corresponds to that found for the lowMr form of the ADA molecule in mammalian tissues. To establish whether ADA is also present in a high-Mr form in chicken lymphocytes, as reported in the case of chicken liver (22), crude homogenates of lymphocytes were filtered through a Sephadex G-200 column, under the conditions described by Nishihara et al. (23): Only one peak of ADA activity

was r e c o v e r e d at the effluent volume corresponding to the lowMr form of the enzyme molecule.

ADA Activity in the Thymus and Bursa of the Developing Chicken The profiles of ADA activity during thymus and bursa development (Fig. 1) clearly show that enzyme activity is higher in the bursa than in the thymus (19 < 0.001) at each time studied, particularly when expressed per 108 cells. ADA activity in bursal cells was found to be, on the average, 15 times higher than in thymocytes. Indeed, while no significant differences were observed in the level of ADA activity in thymocytes at various stages of thymus development, remarkable changes in the enzyme amount were found in bursal cells at definite

30

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40

4~5

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Figure 1. Lymphocyte ADA activity at different times of chicken development. Values are the mean +- SD of 5 separate experiments,

100

S. Senesi et al.

times of organ development, regardless of whether the enzyme activity was expressed per mg protein or per l0 s cells. As observed in Fig. l, changes in bursal lymphocyte ADA activity occur during embryonic life (p < 0.001), when a sharp decrease in enzyme activity was observed up to a few days after hatching, followed by a roughly symmetric peak of activity during the first two weeks of life, with a m a x i m u m a r o u n d the 5th-6th day (p < 0.001). The levels of ADA activity reported in Fig. 1 for the Hubbard strain were confirmed essentially for the Red and Grey strain, in which, however, the enzyme activity was assayed only during a short period of development (from the 17th day of embryonic life to the 12th day after hatching) and only in bursal lymphocytes (data not shown).

ADA activity with respect to those detected in the whole bursal cell suspensions (Table 2). These findings are in agreement with those reported by Ratech et al. (14) and indicate that epithelial ceils do not contribute to the value of ADA activity measured in the whole bursal cell suspensions. Again, when lymphocyte enrichment was achieved by filtering whole bursal cell suspensions through a glass-wool column, no substantial differences in enzyme activity were found in the homogenates of effluent cells as compared to the amount of ADA in the corresponding homogenates of whole bursal cell suspensions; on the contrary, the levels of ADA activity in the effluent cells were even higher than those detected in the supernatant cells obtained by gravity sedimentation (Table 2).

ADA Activity in Lymphocyte-enriched Bursal Cells

ADA Activity in Cortical and Medullary L ymphocytes

In those experiments in which ADA activity was measured in bursal cell suspensions enriched in lymphocytes by the gravity sedimentation method, no relevant differences were found in levels of

The determination of ADA activity in cortical and medullary bursal lymphocytes was carried out starting from the 6th day after hatching and performed only at a few stages of chicken develop-

Table 2. ADA activity in lymphocyte-enriched bursal cells. Enzyme Activity (nmol/min per 109 cells) a Bursal Lymphocytes Enriched by Days Before or After Hatching - 5 0 3 6 12 20

Whole Bursa Cell Suspension

Gravitysedimentation

Glass-wool filtration

280 (276- 286) 211 (200-222) 158 (169-147) 292 (285-299) 204 (196-212) 210 (198-232)

278 (268- 286) 198 (208-188) 144 (125-163) 280 (292-268) 194 (206-182) 196 (182-203)

302 (298--310) 228 (212- 244) 142 (140-144) 312 (292-332) 213 (202- 224) 221 (194-236)

a Values are the mean (ranges in parentheses)of three separate experiments using three separate organ pools (8-15 chicks).

ADA a c t i v i t y in c h i c k e n

bursa

101

ment (Table 3). No substantial differences in ADA amount were appreciated between cortical and medullary lymphocytes, with the exception of the sixth day of life when a significantly higher level of ADA activity was observed in cortical lymphocytes where the enzyme activity was even higher than that found in whole bursal cell suspensions (Fig. 1).

Anti-SRBC Agglutinins No dosable level of agglutinins was detected during the first week of life. At the beginning of the second week, a sudden increase in antibody response was observed. The agglutinin titer continued to rise until the fourth week, as from which it reached its maximum level (Fig. 2).

Table 3. A D A activity in cortical and medullary bursal lymphocytes. Enzyme Activity (nmol/min per mg) a Days After Hatching

Cortical Lymphocytes

6 20 30 45

39.5 13.4 12.8 9.2

Medullary Lymphocytes

-+ 6.5 b +_ 3.4 _+ 2.8 _+ 1.9

11.5 9.2 12.6 13.4

-+ 2.3 _+ 2.3 _+ 1.8 _+ 1.5

• Values are the mean ± SD of five separate experiments. For each experiment cortical and medullary lymphocytes were individually collected from at least three chicks and pooled after being separately homogenized. b At least p < 0.05 with respect to all the other groups.

Anti-SRBC PFC

PFC had the same behavior described for the agglutinins. In fact, before the 14th day of life, the PFC number per 10 6 spleen cells was undetectable. Subse-

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Figure 2. Antibody p r o d u c t i o n to SRBC of chickens immunized at various times after hatching. The response was evaluated as splenic PFC and serum agglutinins. Data represent the mean _+ SD of at least 7 animals.

102

quently, a progressive increase in the PFC response was observed until the chicks were about 1 month old, and no further increase was seen as from this age (Fig. 2).

Discussion The results of the present investigation show that ADA activity is higher in the bursa than in the thymus during chicken development, as already reported by Ratech et al. (14). In particular, bursal cells express more than 15 times as much ADA as thymus cells, and such a difference turned out to be smaller, but still statistically significant, when the enzyme specific activity was expressed per mg protein. In addition, remarkable changes in the quantitative expression of the enzyme activity were observed during the various stages of bursa d e v e l o p m e n t , whereas in the thymus, the level of ADA activity was constantly low and showed no significant change at any time tested. Particular care was taken to obtain bursal cell suspensions devoid of epithelial cells, since the latter are rich in ADA activity (24) and, what is more, the enzyme activity in mucosal epithelial cells of chicken intestine increases 11 times from day 17 of embryonation up to the time of hatching (24). By contrast, during such a period we observed a sharp decrease in ADA activity in the bursa. Moreover, no differences in the enzyme level were appreciated when ADA activity was measured in bursal cell suspensions depleted of macrophage-like cells. These findings, taken together, suggest that the ADA profile during bursa development depends almost exclusively on the amount of the enzyme activity present in bursal lymphocytes. It should be emphasized that no evidence is available concerning the role that ADA plays in the development of

S. Senesi et al.

the bird's immune system. Even in mammals, very little is known about a possible relationship between different levels of ADA activity and definite stages of B-cell maturation, whereas the enzyme has proved to be critically important for maturation of the T-cell lineage (5-11). However, since the bursa of Fabricius is a primary organ for B-cell differentiation and, different from mammalian species, chicken B-cell development proceeds through a series of ontogenetically restricted stages (13,25,29), it is tempting to correlate changes in ADA activity to definite stages of B-cell differentiation. It is well known that, during B-cell ontogeny, the bursal rudiment is colonized by pre-bursal stem cells between day 8 and 14 of embryonation (26-28) and, when such cells reach the bursal epithelium, they collect in an invagination of the basal part of the epithelium. These cells gradually increase in number, and a cellular mass is formed which, therefore, grows under the epithelial framework; only at a later stage does the epithelium surround the whole mesenchymatic mass which will give origin to the bursal lymphoid follicle (29). The prebursal stem cells rapidly divide (25), giving rise to a population of cells bearing IgM at their surface (30). By the time of hatching, 90% of cells are slg + bursal stem cells, which most likely represent the least "mature" bursal B-lymphocyte population (25,30). Since our data show that a sharp decrease in the level of ADA activity occurs during this first period of intrabursal B-lymphocyte development, the possibility exists that ADA activity decays as B-precursor cell maturation proceeds, leading to a population of diversified stem cells already committed to a particular Ig gene rearrangement (25,30,31). After hatching, ADA activity increases rapidly, reaching at day 6 a level comparable to that of the bursa at day 17 of embryonation; such an increase may be related to the development of an or-

ADA activity in chicken bursa

103

ganized lymphoid cell compartment, the follicular cortex, within the organ. Indeed, although the cortex begins to develop by about day 18 of embryonation, at this time very few cells are present in the cortex (25), which shortly after hatching appears as a mantle of lymphocytes external to the medullary compartment, from which it is separated by a layer of flattened epithelial cells (31). It has been suggested that cortex formation is due to medullary bursal stem cells which migrate from the medulla to the cortex where further cell proliferation occurs before being exported to the periphery (25). The expansion of the cell number within the cortex, especially at the beginning of its formation, might be due to cells which are more immature (16) and richer in ADA activity than most medullary lymphocytes at the same time. Indeed, at day 6 after hatching, we found that cortex lymphocytes do contain significantly more ADA activity than the medullary ones. Such a sharp difference in enzyme activity was not seen at the other stages of bursal development studied. Therefore, if the more immature bursal lymphocytes do prove

to be richer in ADA activity, it seems possible that, after a rapid expansion of immature cells within the cortex, cortical lymphocytes undergo further proliferative differentiation, prior to being exported to the periphery as mature antigen-reactive B cells (32). From about day 15 after hatching, the level of ADA was found largely stable over time and, as from then, mature reactive B cells appeared in the peripheral lymphoid tissues such as the spleen, where they can be detected, for instance, as plaque-forming cells. In conclusion, although the data presented in this paper suggest that ADA activity correlates with particular stages of intrabursal B-lymphocyte development, further evidence of the relationship between ADA and functional differentiation of B lymphocytes is needed before firmly establishing the role that the enzyme plays in the maturation of the immune system in birds.

Acknowledgements--This work was supported by grants from the "Ministero El. (Fondi 40% e 60%)", Roma.

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