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The characterization of an Fc receptor cell population in the maturing bursa of fabricius
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
23,470-477
(1982)
The Characterization of an Fc Receptor Cell Population the Maturing Bursa of Fabricius*
in
SUSAN A. DARBY AND PIERSON J. VAN ALTEN Department of Anatomy. The University of Illinois at the Medical Center, Chicago, Illinois 60680 An antibody-coated erythrocyte assay was used to identify the presence of cells with Fc receptors (FcR) within the bursa of embryonic chicks. The greatest percentage of FcR cells was found at 16 days and from then on there was a steady decrease until hatching. Organ culturing of 16-day embryonic bursae resulted in an increase in the percentage of FcR cells and the percentage was further increased when T-cell mitogens were added to the organ cultures. Using cell separation procedures, detection of surface immunoglobulin, and staining for nonspecific esterase indicated the FcR cells were not B cells but most likely macrophages. When embryonic bursal cells were tested for fibronectin-mediated phagocytosis, we observed cells which were able to ingest gelatin-coated latex. We conclude that the FcR cell population in the embryonic bursa consists primarily of monocytic macrophages.
INTRODUCTION
Recent investigations have suggested that the receptor for the Fe fragment of the immunoglobulin molecule is expressed on the surface of macrophages (I), monocytes (2), neutrophils (3), and lymphocytes (4); all are present in mammalians. Investigators (5) tested avian cells and reported that monocytes, heterophils, and subpopulations of lymphoid cells had Fc receptors (FcR). From studies on embryonic bursal tissue they observed that a significant number of cells, which they presumed to be lymphocytes, had FcR. The number of cells with FcR peaked around Day 17 and declined to a low level by hatching, at which time the number of spleen and bone marrow FcR cells increased and continued increasing until about 6 weeks of age. They concluded that bursal FcR cells were B lymphocytes that migrated from the bursa to the spleen and bone marrow. Others (6), however, reported that splenic FcR cells from normal and hormonally bursectomized chickens could develop independently of the bursa and that most appeared to be monocytic macrophages. They also observed that more than 50% of embryonic bursal FcR cells were positive for nonspecific esterases, indicating they were monocytic macrophages. FcR cells also have been identified in intraembryonic mesenchyme of 69-hr chick embryos and they resembled heterophilic promyelocytes, macrophages, and mononuclear cells (7). Interestingly, these particular FcR cells developed long before migrating stem cells colonized the bursa. This investigation further examines the ontogeny of FcR cells in the embryonic bursa. Studies were performed in vitro to observe changes in the maturation of this population of cells during development, alone and with exogenous materials present. Further investigations were done to more fully characterize and identify, *’ This article is dedicated to Robert A. Good on the occasion of his 60th birthday. 470 0090-1229/82/050470-08$01.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
Fc
RECEPTOR
morphologically and functionally, entiation of this organ,
CELL
IN
THE
BURSA
471
the various bursal cells present during differ-
MATERIAL AND METHODS Animals. Fertile, outbred White Leghorn chicken eggs were obtained from Spafas, Inc. (Roanoke, Ill.) and were incubated in a forced air, automatically rotating incubator at 39°C in 80% relative humidity. Adult chickens derived from these eggs were utilized for the production of antisera to sheep red blood cells (SRBC). Cell preparations. Chicken cells were isolated by teasing out the bursa from embryonic, newly hatched chicks, or from organ-cultured bursae. Following removal, the bursae from 10 to 12 embryos were placed into sterile plastic petri dishes containing RPM1 1640 medium (Gibco, Grand Island, N.Y.). A single-cell suspension was prepared by gently homogenizing these organs in a glass tissue homogenizer, washing three times in RPM1 1640 and resuspending to the final necessary concentration of mononuclear white cells per milliliter. Preparation of antisera. Chicken antisera to SRBC were prepared by intramuscularly injecting normal White Leghorn chickens once a week for 4 weeks with 0.5 ml of a 20% suspension of washed SRBC. One week after the last injection, the chickens were bled and the serum was obtained. The serum was heated at 56°C for 30 min to inactivate the complement; the agglutination titer was determined; and then the serum was stored in small aliquots at -20°C until used. Erythrocyte-antibody (EA) rosette assay. Bursal cells with FcR were detected by a SRBC rosette technique (5). Briefly, a 4% washed SRBC suspension was incubated with an equal volume of anti-SRBC antisera (at a just subagglutinating dilution) for 1 hr at 37°C with constant agitation. Following incubation, the EA complexes were washed three times in physiologic saline and resuspended to 0.5%. A 5-ml aliquot of the EA suspension was incubated with 0.5 ml of bursal cell suspension (5 x 10Vml) for 1 hr at 4°C with constant agitation. The cells were resuspended, toluidine blue added, and then the cells were placed on a hemocytometer for counting. At least 200 cells were counted and those with at least three SRBC attached were scored as rosettes. Spontaneous rosetting, in which SRBC were without antisera, was used as a control for the assay. Separation of EA-rosetting cells. Rosette-forming bursal cells were separated from nonrosetting by layering onto a Ficoll-Paque solution. Centrifugation was carried out at 800 g for 15 min to separate the rosetted cells, which formed a pellet, from the nonrosetting cells, which remained at the interface. Organ culture system. Bursae were removed aseptically from 16-day embryos, cut into small pieces, and placed epithelial surface up onto sterile stainless-steel organ culture grids in sterile petri dishes which then were filled with a sufficient amount of Trowell’s medium to moisten the grid surface, thus allowing good gaseous exchange and a supply of nutrients (8). Organ-cultured bursae were incubated at 37°C in a water-saturated atmosphere of 5% CO, and 95% ambient air for either 3 or 5 days with no mitogen, or for 5 days with concanavalin A (Con A, 5 &ml), phytohemagglutinin-P (PHA, 5 pi/ml), or lipopolysaccharide (LPS, 5 xidml). lnhibition of rosette formation. Fc fragments, used to inhibit rosette formation, were prepared by digesting purified chicken IgG with mercuripapain, hydrolysiz-
472
DARBY
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ALTEN
ing the IgG, and then adding recrystallized iodoacetamide (9). The Fc fragments were prepared in PBS, pH 7.2, at a concentration of 0.82 mg/ml. A OS-ml bursal cell suspension (5 x lo6 cells/ml) and an equal volume of the Fc solution were combined and incubated at room temperature for 30 min. The cell suspension was centrifuged, the supernatant withdrawn, the cell pellet resuspended in 0.5 ml of RPM1 1640 medium, and then the EA rosette assay was performed. Fluorescent staining of surface membrane Zg. Direct fluorescent labeling was performed by using a standard technique (10). Bursal cells, at a concentration of 4 x 106, were suspended in cold Hank’s media containing 10 mM sodium azide in a 50-~1 volume. This was combined with 50 ~1 of a 1: 16 dilution of FITC-conjugated rabbit anti-chicken Ig and incubated for 30 min at 4°C. The cells were then centrifuged, the supernatant was withdrawn, and the cells were resuspended in fresh media and washed. After the final wash, the cell pellet was resuspended in 100 ~1 of Hank’s with azide and the labeled cells counted. Nonspecific esterase staining. Identification of esterase-positive cells was performed using a reaction mixture consisting of 8.9 ml phosphate buffer, 0.6 ml hexazotized pararosaniline solution, and 0.5 ml cold a-naphthyl butyrate solution (11). Approximately 1 x lo6 bursal cells in suspension were cytocentrifuged onto glass slides and then air dried. These slides were placed into the reaction mixture at 37°C for 45 min. Following this, the slides were rinsed in distilled water, counterstained with 0.5% methyl green for 15 set, rinsed, and air dried. Cells were considered positive if multiple intensely red-stained granules were present in the cytoplasm. Nylon wool column cell separation. Bursal cells were separated into adherent and nonadherent populations with nylon wool (12). This was done by adding 2 ml of warmed RPM1 1640 media to 5-ml glass syringes containing nylon wool, then adding 2 ml of a cell suspension containing 1 x lo8 bursal cells plus 10% heatinactivated autologous serum, and layering on 1 more ml of warmed media. The columns were incubated for 1 hr at 37°C; then 10 ml of warmed media was added to each syringe to elute the nonadherent cells. Adherent cells were recovered from the nylon wool by vigorously agitating it in ice cold media and compressing the fibers with forceps. The two separate cell populations were washed two times and resuspended into appropriate concentrations. Phagocytosis assays. Phagocytosis by monolayer cultures of embryonic bursal cells was tested by tibronectin-mediated phagocytosis of 1251-labeled gelatincoated latex beads (lz51-g-latex) and by serum-opsonized SRBC. The fibronectin-mediated phagocytosis was performed according to the procedure recently detailed for chickens (13). The monolayers were prepared by incubating 1 ml of 6.25 x lo6 embryonic bursal cells in chamberslides for l-l’/2 hr at 37°C in an humidified atmosphere. Following this, the nonadherent cells and media were removed, washed, and to each chamber the following was added: 100 ~1 of Delbecco’s modified Eagles medium (DMEM) containing 10 units heparin, 50 ,~l human plasma fibronectin, 100 ~1 of 1251-g-latex beads, and 750 ~1 DMEM. These chambers were incubated for 2 hr at 37°C and then washed three times with saline to remove unincorporated latex beads. Following this, 1 ml of a 0.1 M NaOH solution was added to each chamber in order to solubilize the cells, and the chambers were incubated for 30 min at 37°C. The contents of the chambers were then collected, each chamber was rinsed with NaOH; and aliquots of the alkaline
Fc RECEPTOR
CELL
IN THE
473
BURSA
hydrolyzate were transferred to counting tubes and the radioactivity determined. Protein content of the solubilized media was determined and phagocytic activity expressed as counts per minute of lz51-g-latex incorporated/100 pg of cell protein. The ingestion of SRBC by bursal cells was investigated by taking 1 ml of 1 x 10” cells and mixing with 0.1 ml of 5% SRBC sensitized with chicken antibody to SRBC and incubated for 1 hr at 37°C. Noningested SRBC were lysed by adding 1 ml of distilled water for 2 min, followed by the addition of 2 ml of RPM1 1640. The cells were then examined by light microscopy for the presence of SRBC ingestion (14). RESULTS
Distribution
of Rosette-Forming
Bursal Cells during Embryonic
Development
When embryonic bursal cells were tested for the presence of Fc receptors it can be seen in Table 1 that EA rosette-forming cells (EA-RFC) were first detected at Day 14 and by Day 16 the percentage of RFC had reached its peak. It is also seen (Table 1) that from Day 16 to hatching, there was a steady decrease in the percentage of RFC with no further significant decrease by one week post hatching. When bursal cells of 16-day embryos were incubated with SRBC free of anti-SRBC, there were no rosettes observed. When isolated Fc fragments from normal chicken IgG were incubated with bursal cells prior to testing for rosette formation, nearly complete inhibition of EA rosette formation was obtained. This was most apparent when a concentration of 0.8 mg Fc fragment/l ml was used. Presence of EA Rosette-Forming
Cells after Organ Culture
For examining the maturation of EA-RFC during development in vitro, pieces of 16-day embryonic bursae were organ cultured for either 3 or 5 days. In Table 2 it is evident that there was a significant increase (P < 0.01) in the percentage of RFC when bursae were organ-cultured for 5 days relative to the percentage present in 16-day embryonic bursae. Further, organ-culturing bursae (16-day embryos) for 5 days resulted in a very striking increase in the percentage of RFC when compared to the percentage of RFC in 21-day bursae which had developed in vivo. The results presented in Table 2 illustrate the effect of the presence of mitogens (PHA, TABLE MEAN
PERCENTAGE
EMBRYONIC
CHICKS
AGED
Mean percentage EA-RFC 2 SE”
Age (days) 13 14 15 16 17 ” Standard
OF ROSETTE-FORMING
0.0 0.8 1.8 6.0 4.2 error.
2 + -rr ”
0.00 0.08 0.40 0.30 0.70
1 CELLS
IN THE BURSAE
13 DAYS TO 1 WEEK Age (days) 18 19 20 21 1 Week
OF
POSTHATCH Mean percentage EA-RFC 4 SE” 4.0 2.3 1.0 0.4 0.3
k -c 2 k +
0.40 0.30 0.30 0.08 0.20
474
DARBY AND VAN ALTEN
MEAN
PERCENTAGE BEFORE
OF AND
TABLE EA-RFC AFTER
2 FROM
ORGAN
EMBRYONIC
(days)
Days in culture
Treatment
16 21 16 16 16 16 16
0 0 3 5 5 5 5
No No No No PHA Con A LPS
Age
BURSAE
CULTURING
Mean percentage rosettes 2 SE” 6.0 0.4* 6.4 8.8h 12.9 10.7” 8.0
+ 0.33 2 0.08 2 0.22 i 0.70 + 0.25 t 0.51 ir 0.38
n Standard error. b P < 0.01 vs 16 days no treatment.
Con A, and LPS) when added to the culture on the numbers of EA-RFC. It can be seen that when PHA was added for 5 days, the percentage of RFC was significantly higher than when there was no exposure to PHA. Similarly, this was also true for Con A. However, when LPS was added to the bursal organ cultures, no significant difference was observed between the percentage of RFC present. Presence of EA Rosette-Forming Bursal cells from ldday ing to their adherence or population had 2.9% RFC these percentages of RFC decreased from the 6.0% Characterization
Cells after Nylon
Wool Column Separation
embryos were separated into two populations accordnonadherence to nylon wool fibers. The adherent cell while the nonadherent cells had only 0.9% RFC. From it can be seen that both populations were substantially usually obtained from bursae of 16-day embryos.
of EA Rosette-Forming
Cells
Since we were interested in characterizing the RFC cell population fluorescence microscopy, nonspecific esterase staining and the ability to phagocytize were used to characterize the various populations of cells found in the embryonic bursa. It was observed by using the fluorescent antibody technique that 97.8% of adult bursal lymphocytes had surface Ig while only 0.7% of adult thymic lymphocytes did (Table 3). From the results presented in Table 3 it is clearly evident that there was a marked increase in the percentage of bursal cells with surface Ig obtained from 21-day embryos compared to those of 16 days. Further, when immunofluorescence was performed on the non-RFC of FicollPaque-separated cells of 16-day embryonic bursae, we observed that 62% of the interface cells stained positive for surface Ig. These cells constitute only 88% of the total cell population (12% of cells are pelleted and 50% of those are RFC), therefore, it was determined that 54% of the total cell population actually stained for surface Ig. This number is comparable to the percentages of ldday embryonic bursal cells that have surface Ig (Table 3).
Fc
RECEPTOR
CELL
TABLE MEAN
PERCENTAGE
IN
THE
475
BURSA
3
OF SURFACE
Cell type
Ig-BEARING
CELLS
Mean percentage SIg f SE”
Adult bursal
97.8 2 0.87
Adult thymic
0.7 + 0.08
16-Day embryonic bursal
50.9* k 1.12
16-Day nonadherent embryonic bursal
52.56 + 1.22
21-Day bursal
92.6 2 0.90
0 Standard error. b P < 0.01 vs 21 day.
By utilizing the nonspecific esterase stain, results indicated that 16-day embryonic bursal cells included a 4.4% population of cells positive for this stain. However, by 21 days, the number of positive staining cells had declined to 0.7%. This depletion is similar to the depletion of EA-RFC during the same time period. Experiments were performed to test the ability of bursal cells from 16day embryos to either phagocytize opsonized SRBC or fibronectin-treated gelatincoated latex beads. By light microscopy it was not possible to identify any cells that had ingested the SRBC. In contrast, when bursal cells from 16day embryos were incubated with fibronectin1251-g-latex beads, there was more than a 45fold increase in the amount of radioactivity incorporated into cells when compared to bursal cells incubated without fibronectin. That the radiolabeled beads were incorporated into the cells was demonstrated by the fact that the cells had been treated with trypsin (13) following incubation with the latex beads to remove the membrane-bound particles. DISCUSSION
In the present study the development of FcR cells in the bursa of Fabricius of the chick embryo was reexamined by observing rosette-forming cells. We demonstrated that FcR cells were first present in bursae of 1Cday embryos, reached their highest percentage at Day 16, and then steadily decreased by hatching. We also observed that the percentage of FcR cells increased when bursae were organ-cultured and they were further increased when T-cell mitogens (PHA and Con A) were added to the media. From these observations, it does not appear reasonable to assume the FcR cells were B cells as previously proposed (5). In further characterizing these FcR cells, the indirect evidence obtained also indicated they were not B lymphocytes. Detection of SIg by immunofluorescence
476
DARBY
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showed that there was an increase in the percentage of these cells between 16 and 21 days of incubation, similar to that seen by others (15). This was in direct contrast to the FcR cells whose percentages decreased during this time. When bursal cells were passed through nylon wool columns, it was found that the largest percentage of FcR cells were in the adherent population. This might be interpreted that they were B lymphocytes; however, previous investigators (12) showed that less than 50% of the splenic adherent cells have SIg so other adherent cells, i.e., macrophages must constitute part of this population. In contrast, when we incubated bursal cells with antibody-sensitized SRBC and then centrifuged them on Ficoll-Paque, all the FcR cells were removed from the interface while all the SIg-positive cells remained. Thus, we conclude that most of the ldday embryonic bursal cells bearing FcR were not B lymphocytes. It has been proposed that macrophages comprise most of the chicken splenic cell population having FcR (6). Our examination of suspensions of embryonic bursal cells with a nonspecific esterase stain, used to identify macrophages (ll), showed stained cells present and their percentages decreased between 16 and 21 days of incubation, similar to the FcR population. When we investigated whether bursal cells of ldday embryos were able to phagocytose antibody-coated SRBC, it was not possible to identify cells ingesting these particles although they did bind them. On the other hand, when we tested embryonic bursal cells for fibronectinmediated phagocytosis of gelatin-coated latex, ingestion of the latex beads occurred. In vivo evidence for phagocytosis by embryonic bursal cells has been demonstrated by electron microscopy (16). We observed that when latex beads in saline were placed into the bursal lumen of 16-day embryos the latex was transported by the follicle-associated epithelium and then found within macrophages present in the underlying follicle. Further, the lysosomes of the macrophages were observed fusing with the latex phagosomes. It is apparent from the esterase staining and from the phagocytic observations that functional macrophages were present in the bursa of embryonic chickens. We believe that embryonic bursal cells with FcR were mainly macrophages. The significance of macrophages in the developing bursa can only be speculated upon. Although FcR cells are not unique to the bursa, they have been observed in mesenchyme of 69-hr embryos; they seem to arise at a critical stage in ontogeny. They were not present at the initial time when there is the first influx of stem cells (Day 10) (17) or when the first cells with surface Ig appear (Day 13) (15), but appear at the time of intense lymphoid proliferation and follicle formation (18). Thus, the bursal macrophages could have a function similar to that proposed for thymocyte differentiation (19). It has been shown that when immature murine thymocytes were cultured on thymic macrophages, they were stimulated to develop immunocompetence (20). Likewise, it has been observed (2 1) that a physical interaction occurs between immature bursal lymphocytes and glass-adherent macrophages obtained from peripheral blood. The biological and developmental significance of the presence of macrophages during a critical phase in the development of the embryonic bursa remains uncertain. It is reasonable, however, to conclude that macrophages do not have a significant role in the initial phase of B-cell differentiation in the bursa when from
Fc RECEPTOR
CELL IN THE BURSA
477
ultrastructural studies (17) it has been shown that stem cells interact with epithelial cells. At the time when we and others (5) observed a marked increase in the percentage of FcR cells, two important events in the development of the bursa occur. One (15) is that the bursal lymphocytes in the late embryonic stages begin to switch from having mainly IgM on their membranes to cells with IgG. The other event (18) is that from 16 days until hatching bursal lymphocytes divide and rapidly form into large lymphoid follicles. Thus, either or both of these ontogenic changes may be dependent upon macrophages or their products. Although it would be tempting, using the murine in vitro models (22), to speculate on how macrophages affect the development of B lymphocytes in the bursa, it appears that only following further in vivo and in vitro investigations will it be possible to clarify the specific role of the macrophage in the ontogenesis of bursal lymphocytes. ACKNOWLEDGMENTS This work was supported by a research grant from USPHS Grant CA 20172
REFERENCES I. 2. 3. 4. S. 6. 7. 8. 9. 10. Il. 12. 13. 14. IS. 16. 17. 18. 19. 20. 21. 22.
Berken, A., and Benacerraf, B., J. Exp. Med. 123, 119, 1966. LoBugho, A. F., Contran, R. S., and Jandl, J. H., Science 158, 1582, 1967. Messner, R. P., and Jelinek, J., f. C/in. Invest. 49, 2165, 1970. Basten, A., Miller, J. F. A. P., Sprent, J., and Pye, J.,J. Exp. Med. 135, 610. 1972. Ewald, S., Freedman, L., and Sanders, B., Cell. Inzmunol. 23, 158. 1976. Duncan, R., and McArthur, W., J. Immunol. 120, 1014, 1978. Nowak, J. S., Lassila, 0.. Granfors, K., and Toivanen, P., J. Immunol. 123, 1417, 1979. Waltenbaugh, C. R., Sachs, H. G., and Van Alten, P. J., De~~elop. Camp. Immrrnol. 1,353. 1977. Kubo, R. T., and Benedict, A. A., J. Immunol. 102, 1523, 1969. Mbller, G..J. Exp. Med. 114, 415, 1961. Koski. I. R. Poplack, D. G., and Blaese, R. M.,ln “In Vitro Methods in Cell Mediated and Tumor Immunity” (B. R. Bloom and J. R. David, Eds.), pp. 359-362, Academic Press, New York, 1976. Lamont. S. J., and Van Alten, P. J., J. Imnnrno/. Methods 40, 181, 1981. Marquette, D.. Molnar, J., Yamada, K., Schlesinger, D., Darby. S.. and Van Alten, P. J.. Mol. Cell. Biochem. 36, 147. 1981. Sabat, T.. Hsia, W. C., Stanisz, M.. El-Domeiri, A., and Van Alten, P. J., J. Immunol. Methods 14, 103, 1977. Lydyard, P. M., Grossi, C. E., and Cooper, M. D., J. Exp. Med. 144, 79. 1976. Beezhold, D. H., Sachs, H. G., and Van Alten, P. J., in preparation, 1982. Edwards, J., Murphy, R., and Cho, Y., Anat. Rec. 181, 735, 1974. Bockman, D. E., and Cooper. M. D., In “Phylogeny of Thymus and Bone Marrow-Bursa Cells” (R. K. Wright and E. L. Cooper, Eds.), pp. 277-285. Elsevier/North-Holland, Amsterdam, 1976. Beller, D. I.. and Unanue, E. R., .I. Immunol. 121, 1861, 1978. Beller, D. I., and Unanue, E. R., J. Immrrnol. 118, 1780. 1976. Duncan, R., and McArthur, W.. Immrrnology 38, 741, 1979. Hoffman. M. K., J. Immunol. 125, 2076, 1980.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
23,470-477
(1982)
The Characterization of an Fc Receptor Cell Population the Maturing Bursa of Fabricius*
in
SUSAN A. DARBY AND PIERSON J. VAN ALTEN Department of Anatomy. The University of Illinois at the Medical Center, Chicago, Illinois 60680 An antibody-coated erythrocyte assay was used to identify the presence of cells with Fc receptors (FcR) within the bursa of embryonic chicks. The greatest percentage of FcR cells was found at 16 days and from then on there was a steady decrease until hatching. Organ culturing of 16-day embryonic bursae resulted in an increase in the percentage of FcR cells and the percentage was further increased when T-cell mitogens were added to the organ cultures. Using cell separation procedures, detection of surface immunoglobulin, and staining for nonspecific esterase indicated the FcR cells were not B cells but most likely macrophages. When embryonic bursal cells were tested for fibronectin-mediated phagocytosis, we observed cells which were able to ingest gelatin-coated latex. We conclude that the FcR cell population in the embryonic bursa consists primarily of monocytic macrophages.
INTRODUCTION
Recent investigations have suggested that the receptor for the Fe fragment of the immunoglobulin molecule is expressed on the surface of macrophages (I), monocytes (2), neutrophils (3), and lymphocytes (4); all are present in mammalians. Investigators (5) tested avian cells and reported that monocytes, heterophils, and subpopulations of lymphoid cells had Fc receptors (FcR). From studies on embryonic bursal tissue they observed that a significant number of cells, which they presumed to be lymphocytes, had FcR. The number of cells with FcR peaked around Day 17 and declined to a low level by hatching, at which time the number of spleen and bone marrow FcR cells increased and continued increasing until about 6 weeks of age. They concluded that bursal FcR cells were B lymphocytes that migrated from the bursa to the spleen and bone marrow. Others (6), however, reported that splenic FcR cells from normal and hormonally bursectomized chickens could develop independently of the bursa and that most appeared to be monocytic macrophages. They also observed that more than 50% of embryonic bursal FcR cells were positive for nonspecific esterases, indicating they were monocytic macrophages. FcR cells also have been identified in intraembryonic mesenchyme of 69-hr chick embryos and they resembled heterophilic promyelocytes, macrophages, and mononuclear cells (7). Interestingly, these particular FcR cells developed long before migrating stem cells colonized the bursa. This investigation further examines the ontogeny of FcR cells in the embryonic bursa. Studies were performed in vitro to observe changes in the maturation of this population of cells during development, alone and with exogenous materials present. Further investigations were done to more fully characterize and identify, *’ This article is dedicated to Robert A. Good on the occasion of his 60th birthday. 470 0090-1229/82/050470-08$01.00/O Copyright All rights
0 1982 by Academic Press, Inc. of reproduction in any form reserved.
Fc
RECEPTOR
morphologically and functionally, entiation of this organ,
CELL
IN
THE
BURSA
471
the various bursal cells present during differ-
MATERIAL AND METHODS Animals. Fertile, outbred White Leghorn chicken eggs were obtained from Spafas, Inc. (Roanoke, Ill.) and were incubated in a forced air, automatically rotating incubator at 39°C in 80% relative humidity. Adult chickens derived from these eggs were utilized for the production of antisera to sheep red blood cells (SRBC). Cell preparations. Chicken cells were isolated by teasing out the bursa from embryonic, newly hatched chicks, or from organ-cultured bursae. Following removal, the bursae from 10 to 12 embryos were placed into sterile plastic petri dishes containing RPM1 1640 medium (Gibco, Grand Island, N.Y.). A single-cell suspension was prepared by gently homogenizing these organs in a glass tissue homogenizer, washing three times in RPM1 1640 and resuspending to the final necessary concentration of mononuclear white cells per milliliter. Preparation of antisera. Chicken antisera to SRBC were prepared by intramuscularly injecting normal White Leghorn chickens once a week for 4 weeks with 0.5 ml of a 20% suspension of washed SRBC. One week after the last injection, the chickens were bled and the serum was obtained. The serum was heated at 56°C for 30 min to inactivate the complement; the agglutination titer was determined; and then the serum was stored in small aliquots at -20°C until used. Erythrocyte-antibody (EA) rosette assay. Bursal cells with FcR were detected by a SRBC rosette technique (5). Briefly, a 4% washed SRBC suspension was incubated with an equal volume of anti-SRBC antisera (at a just subagglutinating dilution) for 1 hr at 37°C with constant agitation. Following incubation, the EA complexes were washed three times in physiologic saline and resuspended to 0.5%. A 5-ml aliquot of the EA suspension was incubated with 0.5 ml of bursal cell suspension (5 x 10Vml) for 1 hr at 4°C with constant agitation. The cells were resuspended, toluidine blue added, and then the cells were placed on a hemocytometer for counting. At least 200 cells were counted and those with at least three SRBC attached were scored as rosettes. Spontaneous rosetting, in which SRBC were without antisera, was used as a control for the assay. Separation of EA-rosetting cells. Rosette-forming bursal cells were separated from nonrosetting by layering onto a Ficoll-Paque solution. Centrifugation was carried out at 800 g for 15 min to separate the rosetted cells, which formed a pellet, from the nonrosetting cells, which remained at the interface. Organ culture system. Bursae were removed aseptically from 16-day embryos, cut into small pieces, and placed epithelial surface up onto sterile stainless-steel organ culture grids in sterile petri dishes which then were filled with a sufficient amount of Trowell’s medium to moisten the grid surface, thus allowing good gaseous exchange and a supply of nutrients (8). Organ-cultured bursae were incubated at 37°C in a water-saturated atmosphere of 5% CO, and 95% ambient air for either 3 or 5 days with no mitogen, or for 5 days with concanavalin A (Con A, 5 &ml), phytohemagglutinin-P (PHA, 5 pi/ml), or lipopolysaccharide (LPS, 5 xidml). lnhibition of rosette formation. Fc fragments, used to inhibit rosette formation, were prepared by digesting purified chicken IgG with mercuripapain, hydrolysiz-
472
DARBY
AND
VAN
ALTEN
ing the IgG, and then adding recrystallized iodoacetamide (9). The Fc fragments were prepared in PBS, pH 7.2, at a concentration of 0.82 mg/ml. A OS-ml bursal cell suspension (5 x lo6 cells/ml) and an equal volume of the Fc solution were combined and incubated at room temperature for 30 min. The cell suspension was centrifuged, the supernatant withdrawn, the cell pellet resuspended in 0.5 ml of RPM1 1640 medium, and then the EA rosette assay was performed. Fluorescent staining of surface membrane Zg. Direct fluorescent labeling was performed by using a standard technique (10). Bursal cells, at a concentration of 4 x 106, were suspended in cold Hank’s media containing 10 mM sodium azide in a 50-~1 volume. This was combined with 50 ~1 of a 1: 16 dilution of FITC-conjugated rabbit anti-chicken Ig and incubated for 30 min at 4°C. The cells were then centrifuged, the supernatant was withdrawn, and the cells were resuspended in fresh media and washed. After the final wash, the cell pellet was resuspended in 100 ~1 of Hank’s with azide and the labeled cells counted. Nonspecific esterase staining. Identification of esterase-positive cells was performed using a reaction mixture consisting of 8.9 ml phosphate buffer, 0.6 ml hexazotized pararosaniline solution, and 0.5 ml cold a-naphthyl butyrate solution (11). Approximately 1 x lo6 bursal cells in suspension were cytocentrifuged onto glass slides and then air dried. These slides were placed into the reaction mixture at 37°C for 45 min. Following this, the slides were rinsed in distilled water, counterstained with 0.5% methyl green for 15 set, rinsed, and air dried. Cells were considered positive if multiple intensely red-stained granules were present in the cytoplasm. Nylon wool column cell separation. Bursal cells were separated into adherent and nonadherent populations with nylon wool (12). This was done by adding 2 ml of warmed RPM1 1640 media to 5-ml glass syringes containing nylon wool, then adding 2 ml of a cell suspension containing 1 x lo8 bursal cells plus 10% heatinactivated autologous serum, and layering on 1 more ml of warmed media. The columns were incubated for 1 hr at 37°C; then 10 ml of warmed media was added to each syringe to elute the nonadherent cells. Adherent cells were recovered from the nylon wool by vigorously agitating it in ice cold media and compressing the fibers with forceps. The two separate cell populations were washed two times and resuspended into appropriate concentrations. Phagocytosis assays. Phagocytosis by monolayer cultures of embryonic bursal cells was tested by tibronectin-mediated phagocytosis of 1251-labeled gelatincoated latex beads (lz51-g-latex) and by serum-opsonized SRBC. The fibronectin-mediated phagocytosis was performed according to the procedure recently detailed for chickens (13). The monolayers were prepared by incubating 1 ml of 6.25 x lo6 embryonic bursal cells in chamberslides for l-l’/2 hr at 37°C in an humidified atmosphere. Following this, the nonadherent cells and media were removed, washed, and to each chamber the following was added: 100 ~1 of Delbecco’s modified Eagles medium (DMEM) containing 10 units heparin, 50 ,~l human plasma fibronectin, 100 ~1 of 1251-g-latex beads, and 750 ~1 DMEM. These chambers were incubated for 2 hr at 37°C and then washed three times with saline to remove unincorporated latex beads. Following this, 1 ml of a 0.1 M NaOH solution was added to each chamber in order to solubilize the cells, and the chambers were incubated for 30 min at 37°C. The contents of the chambers were then collected, each chamber was rinsed with NaOH; and aliquots of the alkaline
Fc RECEPTOR
CELL
IN THE
473
BURSA
hydrolyzate were transferred to counting tubes and the radioactivity determined. Protein content of the solubilized media was determined and phagocytic activity expressed as counts per minute of lz51-g-latex incorporated/100 pg of cell protein. The ingestion of SRBC by bursal cells was investigated by taking 1 ml of 1 x 10” cells and mixing with 0.1 ml of 5% SRBC sensitized with chicken antibody to SRBC and incubated for 1 hr at 37°C. Noningested SRBC were lysed by adding 1 ml of distilled water for 2 min, followed by the addition of 2 ml of RPM1 1640. The cells were then examined by light microscopy for the presence of SRBC ingestion (14). RESULTS
Distribution
of Rosette-Forming
Bursal Cells during Embryonic
Development
When embryonic bursal cells were tested for the presence of Fc receptors it can be seen in Table 1 that EA rosette-forming cells (EA-RFC) were first detected at Day 14 and by Day 16 the percentage of RFC had reached its peak. It is also seen (Table 1) that from Day 16 to hatching, there was a steady decrease in the percentage of RFC with no further significant decrease by one week post hatching. When bursal cells of 16-day embryos were incubated with SRBC free of anti-SRBC, there were no rosettes observed. When isolated Fc fragments from normal chicken IgG were incubated with bursal cells prior to testing for rosette formation, nearly complete inhibition of EA rosette formation was obtained. This was most apparent when a concentration of 0.8 mg Fc fragment/l ml was used. Presence of EA Rosette-Forming
Cells after Organ Culture
For examining the maturation of EA-RFC during development in vitro, pieces of 16-day embryonic bursae were organ cultured for either 3 or 5 days. In Table 2 it is evident that there was a significant increase (P < 0.01) in the percentage of RFC when bursae were organ-cultured for 5 days relative to the percentage present in 16-day embryonic bursae. Further, organ-culturing bursae (16-day embryos) for 5 days resulted in a very striking increase in the percentage of RFC when compared to the percentage of RFC in 21-day bursae which had developed in vivo. The results presented in Table 2 illustrate the effect of the presence of mitogens (PHA, TABLE MEAN
PERCENTAGE
EMBRYONIC
CHICKS
AGED
Mean percentage EA-RFC 2 SE”
Age (days) 13 14 15 16 17 ” Standard
OF ROSETTE-FORMING
0.0 0.8 1.8 6.0 4.2 error.
2 + -rr ”
0.00 0.08 0.40 0.30 0.70
1 CELLS
IN THE BURSAE
13 DAYS TO 1 WEEK Age (days) 18 19 20 21 1 Week
OF
POSTHATCH Mean percentage EA-RFC 4 SE” 4.0 2.3 1.0 0.4 0.3
k -c 2 k +
0.40 0.30 0.30 0.08 0.20
474
DARBY AND VAN ALTEN
MEAN
PERCENTAGE BEFORE
OF AND
TABLE EA-RFC AFTER
2 FROM
ORGAN
EMBRYONIC
(days)
Days in culture
Treatment
16 21 16 16 16 16 16
0 0 3 5 5 5 5
No No No No PHA Con A LPS
Age
BURSAE
CULTURING
Mean percentage rosettes 2 SE” 6.0 0.4* 6.4 8.8h 12.9 10.7” 8.0
+ 0.33 2 0.08 2 0.22 i 0.70 + 0.25 t 0.51 ir 0.38
n Standard error. b P < 0.01 vs 16 days no treatment.
Con A, and LPS) when added to the culture on the numbers of EA-RFC. It can be seen that when PHA was added for 5 days, the percentage of RFC was significantly higher than when there was no exposure to PHA. Similarly, this was also true for Con A. However, when LPS was added to the bursal organ cultures, no significant difference was observed between the percentage of RFC present. Presence of EA Rosette-Forming Bursal cells from ldday ing to their adherence or population had 2.9% RFC these percentages of RFC decreased from the 6.0% Characterization
Cells after Nylon
Wool Column Separation
embryos were separated into two populations accordnonadherence to nylon wool fibers. The adherent cell while the nonadherent cells had only 0.9% RFC. From it can be seen that both populations were substantially usually obtained from bursae of 16-day embryos.
of EA Rosette-Forming
Cells
Since we were interested in characterizing the RFC cell population fluorescence microscopy, nonspecific esterase staining and the ability to phagocytize were used to characterize the various populations of cells found in the embryonic bursa. It was observed by using the fluorescent antibody technique that 97.8% of adult bursal lymphocytes had surface Ig while only 0.7% of adult thymic lymphocytes did (Table 3). From the results presented in Table 3 it is clearly evident that there was a marked increase in the percentage of bursal cells with surface Ig obtained from 21-day embryos compared to those of 16 days. Further, when immunofluorescence was performed on the non-RFC of FicollPaque-separated cells of 16-day embryonic bursae, we observed that 62% of the interface cells stained positive for surface Ig. These cells constitute only 88% of the total cell population (12% of cells are pelleted and 50% of those are RFC), therefore, it was determined that 54% of the total cell population actually stained for surface Ig. This number is comparable to the percentages of ldday embryonic bursal cells that have surface Ig (Table 3).
Fc
RECEPTOR
CELL
TABLE MEAN
PERCENTAGE
IN
THE
475
BURSA
3
OF SURFACE
Cell type
Ig-BEARING
CELLS
Mean percentage SIg f SE”
Adult bursal
97.8 2 0.87
Adult thymic
0.7 + 0.08
16-Day embryonic bursal
50.9* k 1.12
16-Day nonadherent embryonic bursal
52.56 + 1.22
21-Day bursal
92.6 2 0.90
0 Standard error. b P < 0.01 vs 21 day.
By utilizing the nonspecific esterase stain, results indicated that 16-day embryonic bursal cells included a 4.4% population of cells positive for this stain. However, by 21 days, the number of positive staining cells had declined to 0.7%. This depletion is similar to the depletion of EA-RFC during the same time period. Experiments were performed to test the ability of bursal cells from 16day embryos to either phagocytize opsonized SRBC or fibronectin-treated gelatincoated latex beads. By light microscopy it was not possible to identify any cells that had ingested the SRBC. In contrast, when bursal cells from 16day embryos were incubated with fibronectin1251-g-latex beads, there was more than a 45fold increase in the amount of radioactivity incorporated into cells when compared to bursal cells incubated without fibronectin. That the radiolabeled beads were incorporated into the cells was demonstrated by the fact that the cells had been treated with trypsin (13) following incubation with the latex beads to remove the membrane-bound particles. DISCUSSION
In the present study the development of FcR cells in the bursa of Fabricius of the chick embryo was reexamined by observing rosette-forming cells. We demonstrated that FcR cells were first present in bursae of 1Cday embryos, reached their highest percentage at Day 16, and then steadily decreased by hatching. We also observed that the percentage of FcR cells increased when bursae were organ-cultured and they were further increased when T-cell mitogens (PHA and Con A) were added to the media. From these observations, it does not appear reasonable to assume the FcR cells were B cells as previously proposed (5). In further characterizing these FcR cells, the indirect evidence obtained also indicated they were not B lymphocytes. Detection of SIg by immunofluorescence
476
DARBY
AND
VAN
ALTEN
showed that there was an increase in the percentage of these cells between 16 and 21 days of incubation, similar to that seen by others (15). This was in direct contrast to the FcR cells whose percentages decreased during this time. When bursal cells were passed through nylon wool columns, it was found that the largest percentage of FcR cells were in the adherent population. This might be interpreted that they were B lymphocytes; however, previous investigators (12) showed that less than 50% of the splenic adherent cells have SIg so other adherent cells, i.e., macrophages must constitute part of this population. In contrast, when we incubated bursal cells with antibody-sensitized SRBC and then centrifuged them on Ficoll-Paque, all the FcR cells were removed from the interface while all the SIg-positive cells remained. Thus, we conclude that most of the ldday embryonic bursal cells bearing FcR were not B lymphocytes. It has been proposed that macrophages comprise most of the chicken splenic cell population having FcR (6). Our examination of suspensions of embryonic bursal cells with a nonspecific esterase stain, used to identify macrophages (ll), showed stained cells present and their percentages decreased between 16 and 21 days of incubation, similar to the FcR population. When we investigated whether bursal cells of ldday embryos were able to phagocytose antibody-coated SRBC, it was not possible to identify cells ingesting these particles although they did bind them. On the other hand, when we tested embryonic bursal cells for fibronectinmediated phagocytosis of gelatin-coated latex, ingestion of the latex beads occurred. In vivo evidence for phagocytosis by embryonic bursal cells has been demonstrated by electron microscopy (16). We observed that when latex beads in saline were placed into the bursal lumen of 16-day embryos the latex was transported by the follicle-associated epithelium and then found within macrophages present in the underlying follicle. Further, the lysosomes of the macrophages were observed fusing with the latex phagosomes. It is apparent from the esterase staining and from the phagocytic observations that functional macrophages were present in the bursa of embryonic chickens. We believe that embryonic bursal cells with FcR were mainly macrophages. The significance of macrophages in the developing bursa can only be speculated upon. Although FcR cells are not unique to the bursa, they have been observed in mesenchyme of 69-hr embryos; they seem to arise at a critical stage in ontogeny. They were not present at the initial time when there is the first influx of stem cells (Day 10) (17) or when the first cells with surface Ig appear (Day 13) (15), but appear at the time of intense lymphoid proliferation and follicle formation (18). Thus, the bursal macrophages could have a function similar to that proposed for thymocyte differentiation (19). It has been shown that when immature murine thymocytes were cultured on thymic macrophages, they were stimulated to develop immunocompetence (20). Likewise, it has been observed (2 1) that a physical interaction occurs between immature bursal lymphocytes and glass-adherent macrophages obtained from peripheral blood. The biological and developmental significance of the presence of macrophages during a critical phase in the development of the embryonic bursa remains uncertain. It is reasonable, however, to conclude that macrophages do not have a significant role in the initial phase of B-cell differentiation in the bursa when from
Fc RECEPTOR
CELL IN THE BURSA
477
ultrastructural studies (17) it has been shown that stem cells interact with epithelial cells. At the time when we and others (5) observed a marked increase in the percentage of FcR cells, two important events in the development of the bursa occur. One (15) is that the bursal lymphocytes in the late embryonic stages begin to switch from having mainly IgM on their membranes to cells with IgG. The other event (18) is that from 16 days until hatching bursal lymphocytes divide and rapidly form into large lymphoid follicles. Thus, either or both of these ontogenic changes may be dependent upon macrophages or their products. Although it would be tempting, using the murine in vitro models (22), to speculate on how macrophages affect the development of B lymphocytes in the bursa, it appears that only following further in vivo and in vitro investigations will it be possible to clarify the specific role of the macrophage in the ontogenesis of bursal lymphocytes. ACKNOWLEDGMENTS This work was supported by a research grant from USPHS Grant CA 20172
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