Eur. J. Immunol. 1990.20: 913-917
Nurse cells of the bursa of Fabricius
913
Maant WuorelaO, S i a JalkanenO, Laun J. PelliniemiAand Paavo ToivanenO
Nurse cells of the bursa of Fabricius: do they exist?*
Department of Medical Microbiologyo and Laboratory of Electron MicroscopyA,Turku University, Turku
Cell-cell interactions in B lymphocyte development have so far been incompletely characterized, mostly due to lack of a special organ for B cell maturation in the mammalian species. Certain well-known lymphostromal interactions in the thymus have raised the question whether similar interactions with nurse cells would also operate in the development of B cells.We have tested this hypothesis in the chicken bursa of Fabricius, an organ specific for the B cell maturation. To identify possible nurse cells, with viable lymphocytes enclosed, the cells in the bursa of Fabricius were dispersed with collagenase and trypsin. Light and electron microscopic examination of bursa cell suspensions showed four types of aggregates, identified by low magnification light microscopy as potential nurse cell-like complexes. Electron microscopy revealed that all aggregates consisted of epithelial cells, and complexes of epithelial cells with lymphocytes enclosed were not observed.These findings indicate that interactions similar to those seen in the avian and mammalian thymus between epithelial nurse cells and T lymphocytes are not a part of the avian B cell differentiation process.
1 Introduction
2 Materials and methods
The role of cell-cell interactions in the maturation, homing and function of various lymphoid cells has become increasingly important as knowledge of the molecules mediating these interactions increases. The possible interactions in B lymphocyte maturation areincompletely understood, especially because the site where mammalian B cells acquire their immunological competence is not known. In a birds B lymphocytes mature in the bursa of Fabricius, a perianal organ within easy reach for experimentation.
2.1 Animals
In T lymphocyte development, there are significant interactions between maturing thymocytes and thymic stromal cells. The cells interacting with T lymphocytes include thymic macrophages, medullary dendritic cells and cortical reticular epithelial cells, which in cell suspensions of dispersed tissue form the so-called thymic nurse cells [l]. The molecular basis of these interactions as well as their exact role in the maturation process is still unclear.Wick and Oberhuber [2] have recently described the presence of nurse cell-like cellular complexes in the chicken bursa of Fabricius. They used light microscopy and the complexes were, therefore, not described in detail nor were the cellular components characterized. In the present study we have tried to verify the possible lymphocyte-epithelia1 cell interactions and thc cxistence of nurse cells in the chicken bursa of Fabricius.
Animals used in this study were MHC-homozygous H.B2 chicken (B haplotype B2). During the embryonic phase bursas were collected on days 9, 11, 13, 15, 17 and 19 of incubation ( 1 5 4 0 bursadage group). After hatching, the bursas were taken fom 0-14-week-old chickens at 1-week intervals (5-20 bursadage group).
2.2 Isolation of epithelial cells To isolate the epithelial cells the thymuses and the bursas of Fabricius were dissected out from chickens and gently teased apart from adjacent tissue into RPMI 1640 medium, pH 7.2 at 4°C. The isolated organs were minced with scissors or with glass homogenizers. Combinations of different enzyme concentrations, incubation times and temperatures were tested as indicated in Table 1. Each experiment was repeated two t o three times. The medium used in collagenase digestions was Hepes-buffered RPMI 1640, which contained 10% FCS and 5% L-glutamine. For trypsin treatment HBSS, pH 7.2, without Ca2+ and Mg2+ was used. When complete degradation of the tissue was achieved, the cells released into the SN were gently layered over FCS and large cells, and cellular complexes were collected after 15 min sedimentation. This enrichment procedure was repeated two to three times. 2.3 Immunocytochemistry, light and EM
[I 82191-
*
This work was supported by grants from the Sigrid JusClius Foundation, the Research and Science Foundation of Farmos, Finnish Academy and the Finnish Cultural Foundation.
Correspondence: Maarit Wuorela, Department of Medical Microbiology, Turku University, SF-20520 Turku, Finland 0 VCH VerlagsgesellschaftmbH, D-6940 Weinheim, 1990
The epithelial cells were fixed in acetone and stained with routine hematoxylin, Giemsa and May-Griinwald-Giemsa methods. For immunocytochemical characterization, smears made of cell suspensions were fixed and incubated with anti-B-L and anti-Bu-la antibodies. Production and specificity of the antibodies have been described earlier [3, 41. The antigen-antibody complexes were detected with 0014-2980/90/0404-0913$02.50/0
914
Eur. J. Immunol. 1990. 20: 913-917
M. Wuorela, S. Jalkanen, L. J. Pelliniemi and I? Toivanen
Table 1. Enzymatic digestions of the chicken bursa of Fabricius
Enzyme') Trypsin type
Trypsin type Collagenase Trypsin type Collagenase
Trypsin tYPe
Collagenase Trypsin type Collagenase Collagenase Trypsin type
II
Concentration
Temperature
Incubation time
0.012% 0.025% 0.04% 0.05% 0.1% 0.15% 0.2% 0.25% 0.3% 0.35%
0 "C 4 "C 20 "C
15 min. 20 min 2 4 x .20 min 90 min
37 "C 40 "C
n~ 1.0 x
1 0 - 3 ~ 1.5 ~ x 10-3% 20 "C 37 "C 2.0 x 10-3% 2.5 x 1 0 - 3 ~ ~ 37 "C IV loo0 U/ml 1600 Ulml 37 "C II 0.1% 0.15% 0.2% 0.25% IV 100 U/ml1600 Ulml 37 "C n 0.25% IV 0.15 Ulml 37 "C I1 0.3% I1 7 U/ml 37 "C I1 16 U/ml 37 "C I1 0.3%
Collagenase Typsin type Collagenase
I1 I1 I1
Trypsin type
I1
32 Ulml 0.3% 10 U/ml 14 Ulml 16 U/ml21 Ulml 32 Ulml 0.3%
1 4 x 20min 1 4 x 20min 2 4 x 15 min
I 4 x 20min 2 4 x 20min
37 "C
24 h 1-2 x 30min 2-3 x 15min 15 h 30 min + 45 min 24 h
37 "C
1.5 h
37 "C 37 "C
FITC-labeled anti-mouse IgG (Miles-Yeda, Rehovot, Israel) and then studied under a fluorescence microscope (SM-Lux, Leitz, Wetzlar, FRG). For transmission EM, isolated cells were fixed with 5% glutaraldehyde (Merck, Darmstadt, FRG) in 0.16 M s-collidine-HC1buffer, pH 7.4, and postfixed with potassium ferrocyanide-osmium fixative [5]. The cells were dehydrated in graded concentrations of ethanol and embedded in epon (Glycidether 100; Merck). Thin sections were cut on a Ultracut E ultramicrotome (C, Reichert Optische Werke A.G., Wien, Austria), and stained with uranyl acetate and lead citrate [6]. Sections were examined with a JEM 100 C (JEOL Ltd., Tokyo, Japan) electron microscope.
a) All enzymes in the Table were purchased from Sigma (St. Louis, MO) and digestions included DNase at the following concentrations: DNAse I, Sigma; DNAse 11, Calbiochem (La Jolla, CA) and DNAse I type 11, Boehringer Mannheim (Mannheim, FRG), all at either 0.05%, 0.1% or 0.15%.
cell suspensions were larger than those obtained from the thymus. In the thymus, the nurse cell-like complexes predominated already immediately after enzyme treatment, while in the bursa of Fabricius, most of the cell clusters became spherical, nurse cell-like structures only after one night of cultivation (Fig. 1). Epithelial cells of the
2.4 I n vitro cell culture Isolated cells, cellular aggregates and lymphoepithelial complexes were cultured in multiwell trays (15-mm diameter wells) in a humidified incubator at 40 "C in 5% COz.The culture medium was full McCoy's medium supplemented with 2.5% normal chicken serum.
3 Results 3.1 Lymphoepitbelial complexes Epithelial cells could be isolated both from the thymus and the bursa of Fabricius with the same enzymatic digestion. Lymphocytes were seen in close contact with epithelial cells in both organs.They formed either nurse cell-like structures or more irregularly shaped aggregates composed of epithelial cells and lymphocytes. The complexes from the bursa
Figure 1. Epithelial cell aggregate and lymphocytes (L) from the bursa of Fabricius from 2-week-old chickens. The aggregates were isolated with enzyme digestion and cultured overnight. (MayGrunwald-Giemsa stain.)
Eur. J. Immunol. 1990. 20: 913-917
bursa of Fabricius had also released most of the lymphocytes attached to their surfaces into the culture medium during overnight cell culture. This was not the case in thymus cell suspension,where part of the lymphocytes were still in close contact with epithelial cells.
Nurse cells of the bursa of Fabricius
915
3.2 Light microscopy Histological organization of cultured cellular aggregates both from the thymus and the bursa of Fabricius resembled that of the so-called nurse cells (Fig. 1). Immunofluores-
Figure 2. Electron micrograph of an epithelial cell cluster from the bursa of Fabricius from 2-week-old chickens. The luminal surface (E) of epithelial cells shows a few microvilli (M), tight junctions (J), desmosomes (D) and a large accumulation of folded basement membrane (B) inside the cluster. The basal surface of epithelial cells is outlined by arrows.
916
M. Wuorela, S. Jalkanen, L. J. Pelliniemi and F? Toivanen
Eur. J. Immunol. 1990.20: 913-917
Nurse cells of the bursa of Fabricius
Eur. J. Immunol. 1990. 20: 913-917
cence showed thymic nurse cells and lymphocytes therein to be MHC class II+, as described earlier [7, 81. The spherical clusters of bursal cells had more ruffled surface than those from thymic origin, and there seemed to be some lymphocyte-like cells attached to the surface of some clusters. In these aggregates of bursal cells, only the attached B lymphocytes were class II+,whereas the epithelial component was negative.
917
apparently from this part of the bursa. The third type of clusters with both lumen and basement membrane inside the cluster were evidently from the junction between these two surface epithelia. The fourth type of aggregates consisting of various kinds of epithelial cells could arise from the interior parts of the follicle. The basement membrane gatherings seen inside these aggregates must derive from the junction between the cortex and the medulla of the follicle.
3.3 EM
Although the isolated epithelia cover all parts of the bursal follicle with a potential for the existence of nurse cells, no Ultrastructural analysis revealed that the spherical nurse nurse cell-like epithelial cells with intracytoplasmic lymcell-like cellular aggregates seen in light microscopy (Fig. 1) phocytes were seen in the aggregates from the bursa of in the bursa of Fabricius were composed of epithelial cells, Fabricius. However, with these enzyme digestions, we which were connected to each other by typical epithelial observed nurse cells in the chicken thymus as has been junctional complexes consisting of tight junctions and described by others [8, 15-17]. We conclude that interacdesmosomes (Fig. 2). Four types of aggregates were iden- tions between maturing lymphocytes and reticular epithetified, and in all of them the cells were morphologically lial cells similar to those seen in the thymus, are needed only in Tcell development, and are not a part of the B cell typical epithelial cells. differentiation process. Two types of aggregates had microvilli on luminal surface projecting outward from the aggregates, and junctional complexes along the apical parts of outermost cells. Some Excellent technical assistance by Paivi Valtanen, Marjo Vesanto, of these aggregates had internal interfollicular stromal Marjatta Leskenmaa, Sirpa From and Urpo Reunanen is gratefully spaces outlined with the basal surface and basement acknowledged. membrane of the epithelial cells. The space was tightly packed by accumulations of folded basement membranes Received January 2, 1990. (Fig. 2). Others consisted only of epithelial cells without basement membrane.The third type of aggregates had both lumen with microvilli and tight junctions, and areas of 5 References packed basement membrane inside the aggregate. The 1 Kyewski, B. A . , J. Exp. Med. 1987. 166: 520. fourth type of cell clusters had more variable cellular 2 Wick, G. and Oberhuber, G . , Eur. J. Immunol. 1986. 16: organization and islets of tightly compressed basement 855. membrane, but no signs of luminal surface (Fig. 3). No 3 Veromaa,T.,Vainio, O., Jalkanen, S., Eerola, E., Granfors, K . epithelial cells with intracytoplasmic lymphocytes were and Toivanen, P., Eur. J. Immunol. 1988. 18: 225. seen in the aggregates from the bursa of Fabricius. 4 Veromaa, T.,Vainio, O., Eerola, E. and Toivanen, I?, Hybri-
4 Discussion By using various enzyme digestions, we were able to isolate cellular clusters from the bursa of Fabricius, which in light microscopy resembled nurse cells. EM, however, showed the isolated clusters to consist of different types of epithelial cell aggregates without intracytoplasmic or interstitial lymphocytes. The four types of isolated epithelial cell aggregates were apparently derived from different parts of the bursal follicle as judged from the electron microscopic analyses describing the bursal ultrastructure [9-131. The aggregates with luminal surface but without basement membrane were from the follicle-associated epithelium (FAE), which is an epithelium without basement membrane, specialized in the uptake of environmental antigens into the bursal follicle [lo, 11, 141. The epithelium of the bursal lumen between FAE has a basement membrane, and the aggregates with microvilli on the outer surface and folded basement membrane inside the aggregate originated
doma 1988. 7: 41. Karnovsky, M. J., J. Cell. Biol. (abstract) 1971. 284: 146. Pelliniemi, L. J., Anat. Embryol. (Berl.) 1975. 147: 19. Wekerle, H. and Ketelsen, U.-I?, Nature 1980. 283: 402. Kyewski, B. A . and Kaplan, H. S., J. Immunol. 1982. 128: 2287. 9 Holbrook, K. A . , Perkins,W. D. and Glick, B., J. Reticuloendothel. SOC. 1974. 16: 300. 10 Naukkarinen, A . , Arstila, A . U. and Sorvari,T. E., Anat. Rec. 1978. 191: 415. 11 Olah, I., Glick, B., McCorkle, F. and Stinson, R., Dev. Comp. Immunol. 1979. 3: 101. 12 Glick, B. and Olah, I., Anat. Rec. 1982. 204: 341. 13 Freeman, B. M. (Ed.), Physiology and biochemistry of the domestic fowl, Vol. 5., Academic Press, London 1984, p. 277. 14 Sorvari, T., Sorvari, R . , Ruotsalainen, €?,Toivanen, A . and Toivanen, €?, Nature 1975. 253: 217. 15 Wekerle, H., Ketelsen, U.-€?and Ernst, M., J. Exp. Med. 1980. 151: 925. 16 Boyd, R. L., Oberhuber, G., Hala, K. and Wick, G., J. Immunol. 1984. 132: 718. 17 Andrews, l? and Boyd, R. L., Eur. J. Immunol. 1985. 15: 36. 5 6 7 8
4 Figure 3. Electron micrograph of the central region of an epithelial cell aggregate from the bursa of Fabriciusfrom a 2-week-old chicken.
The aggregate consists of diverse epithelial cells and accumulations of tightly packed, folded basement membrane (B) apparently originating from the medulla-cortex junction of the follicle. The basal surface of epithelial cells is outlined by arrows.
Nurse cells of the bursa of Fabricius
913
Maant WuorelaO, S i a JalkanenO, Laun J. PelliniemiAand Paavo ToivanenO
Nurse cells of the bursa of Fabricius: do they exist?*
Department of Medical Microbiologyo and Laboratory of Electron MicroscopyA,Turku University, Turku
Cell-cell interactions in B lymphocyte development have so far been incompletely characterized, mostly due to lack of a special organ for B cell maturation in the mammalian species. Certain well-known lymphostromal interactions in the thymus have raised the question whether similar interactions with nurse cells would also operate in the development of B cells.We have tested this hypothesis in the chicken bursa of Fabricius, an organ specific for the B cell maturation. To identify possible nurse cells, with viable lymphocytes enclosed, the cells in the bursa of Fabricius were dispersed with collagenase and trypsin. Light and electron microscopic examination of bursa cell suspensions showed four types of aggregates, identified by low magnification light microscopy as potential nurse cell-like complexes. Electron microscopy revealed that all aggregates consisted of epithelial cells, and complexes of epithelial cells with lymphocytes enclosed were not observed.These findings indicate that interactions similar to those seen in the avian and mammalian thymus between epithelial nurse cells and T lymphocytes are not a part of the avian B cell differentiation process.
1 Introduction
2 Materials and methods
The role of cell-cell interactions in the maturation, homing and function of various lymphoid cells has become increasingly important as knowledge of the molecules mediating these interactions increases. The possible interactions in B lymphocyte maturation areincompletely understood, especially because the site where mammalian B cells acquire their immunological competence is not known. In a birds B lymphocytes mature in the bursa of Fabricius, a perianal organ within easy reach for experimentation.
2.1 Animals
In T lymphocyte development, there are significant interactions between maturing thymocytes and thymic stromal cells. The cells interacting with T lymphocytes include thymic macrophages, medullary dendritic cells and cortical reticular epithelial cells, which in cell suspensions of dispersed tissue form the so-called thymic nurse cells [l]. The molecular basis of these interactions as well as their exact role in the maturation process is still unclear.Wick and Oberhuber [2] have recently described the presence of nurse cell-like cellular complexes in the chicken bursa of Fabricius. They used light microscopy and the complexes were, therefore, not described in detail nor were the cellular components characterized. In the present study we have tried to verify the possible lymphocyte-epithelia1 cell interactions and thc cxistence of nurse cells in the chicken bursa of Fabricius.
Animals used in this study were MHC-homozygous H.B2 chicken (B haplotype B2). During the embryonic phase bursas were collected on days 9, 11, 13, 15, 17 and 19 of incubation ( 1 5 4 0 bursadage group). After hatching, the bursas were taken fom 0-14-week-old chickens at 1-week intervals (5-20 bursadage group).
2.2 Isolation of epithelial cells To isolate the epithelial cells the thymuses and the bursas of Fabricius were dissected out from chickens and gently teased apart from adjacent tissue into RPMI 1640 medium, pH 7.2 at 4°C. The isolated organs were minced with scissors or with glass homogenizers. Combinations of different enzyme concentrations, incubation times and temperatures were tested as indicated in Table 1. Each experiment was repeated two t o three times. The medium used in collagenase digestions was Hepes-buffered RPMI 1640, which contained 10% FCS and 5% L-glutamine. For trypsin treatment HBSS, pH 7.2, without Ca2+ and Mg2+ was used. When complete degradation of the tissue was achieved, the cells released into the SN were gently layered over FCS and large cells, and cellular complexes were collected after 15 min sedimentation. This enrichment procedure was repeated two to three times. 2.3 Immunocytochemistry, light and EM
[I 82191-
*
This work was supported by grants from the Sigrid JusClius Foundation, the Research and Science Foundation of Farmos, Finnish Academy and the Finnish Cultural Foundation.
Correspondence: Maarit Wuorela, Department of Medical Microbiology, Turku University, SF-20520 Turku, Finland 0 VCH VerlagsgesellschaftmbH, D-6940 Weinheim, 1990
The epithelial cells were fixed in acetone and stained with routine hematoxylin, Giemsa and May-Griinwald-Giemsa methods. For immunocytochemical characterization, smears made of cell suspensions were fixed and incubated with anti-B-L and anti-Bu-la antibodies. Production and specificity of the antibodies have been described earlier [3, 41. The antigen-antibody complexes were detected with 0014-2980/90/0404-0913$02.50/0
914
Eur. J. Immunol. 1990. 20: 913-917
M. Wuorela, S. Jalkanen, L. J. Pelliniemi and I? Toivanen
Table 1. Enzymatic digestions of the chicken bursa of Fabricius
Enzyme') Trypsin type
Trypsin type Collagenase Trypsin type Collagenase
Trypsin tYPe
Collagenase Trypsin type Collagenase Collagenase Trypsin type
II
Concentration
Temperature
Incubation time
0.012% 0.025% 0.04% 0.05% 0.1% 0.15% 0.2% 0.25% 0.3% 0.35%
0 "C 4 "C 20 "C
15 min. 20 min 2 4 x .20 min 90 min
37 "C 40 "C
n~ 1.0 x
1 0 - 3 ~ 1.5 ~ x 10-3% 20 "C 37 "C 2.0 x 10-3% 2.5 x 1 0 - 3 ~ ~ 37 "C IV loo0 U/ml 1600 Ulml 37 "C II 0.1% 0.15% 0.2% 0.25% IV 100 U/ml1600 Ulml 37 "C n 0.25% IV 0.15 Ulml 37 "C I1 0.3% I1 7 U/ml 37 "C I1 16 U/ml 37 "C I1 0.3%
Collagenase Typsin type Collagenase
I1 I1 I1
Trypsin type
I1
32 Ulml 0.3% 10 U/ml 14 Ulml 16 U/ml21 Ulml 32 Ulml 0.3%
1 4 x 20min 1 4 x 20min 2 4 x 15 min
I 4 x 20min 2 4 x 20min
37 "C
24 h 1-2 x 30min 2-3 x 15min 15 h 30 min + 45 min 24 h
37 "C
1.5 h
37 "C 37 "C
FITC-labeled anti-mouse IgG (Miles-Yeda, Rehovot, Israel) and then studied under a fluorescence microscope (SM-Lux, Leitz, Wetzlar, FRG). For transmission EM, isolated cells were fixed with 5% glutaraldehyde (Merck, Darmstadt, FRG) in 0.16 M s-collidine-HC1buffer, pH 7.4, and postfixed with potassium ferrocyanide-osmium fixative [5]. The cells were dehydrated in graded concentrations of ethanol and embedded in epon (Glycidether 100; Merck). Thin sections were cut on a Ultracut E ultramicrotome (C, Reichert Optische Werke A.G., Wien, Austria), and stained with uranyl acetate and lead citrate [6]. Sections were examined with a JEM 100 C (JEOL Ltd., Tokyo, Japan) electron microscope.
a) All enzymes in the Table were purchased from Sigma (St. Louis, MO) and digestions included DNase at the following concentrations: DNAse I, Sigma; DNAse 11, Calbiochem (La Jolla, CA) and DNAse I type 11, Boehringer Mannheim (Mannheim, FRG), all at either 0.05%, 0.1% or 0.15%.
cell suspensions were larger than those obtained from the thymus. In the thymus, the nurse cell-like complexes predominated already immediately after enzyme treatment, while in the bursa of Fabricius, most of the cell clusters became spherical, nurse cell-like structures only after one night of cultivation (Fig. 1). Epithelial cells of the
2.4 I n vitro cell culture Isolated cells, cellular aggregates and lymphoepithelial complexes were cultured in multiwell trays (15-mm diameter wells) in a humidified incubator at 40 "C in 5% COz.The culture medium was full McCoy's medium supplemented with 2.5% normal chicken serum.
3 Results 3.1 Lymphoepitbelial complexes Epithelial cells could be isolated both from the thymus and the bursa of Fabricius with the same enzymatic digestion. Lymphocytes were seen in close contact with epithelial cells in both organs.They formed either nurse cell-like structures or more irregularly shaped aggregates composed of epithelial cells and lymphocytes. The complexes from the bursa
Figure 1. Epithelial cell aggregate and lymphocytes (L) from the bursa of Fabricius from 2-week-old chickens. The aggregates were isolated with enzyme digestion and cultured overnight. (MayGrunwald-Giemsa stain.)
Eur. J. Immunol. 1990. 20: 913-917
bursa of Fabricius had also released most of the lymphocytes attached to their surfaces into the culture medium during overnight cell culture. This was not the case in thymus cell suspension,where part of the lymphocytes were still in close contact with epithelial cells.
Nurse cells of the bursa of Fabricius
915
3.2 Light microscopy Histological organization of cultured cellular aggregates both from the thymus and the bursa of Fabricius resembled that of the so-called nurse cells (Fig. 1). Immunofluores-
Figure 2. Electron micrograph of an epithelial cell cluster from the bursa of Fabricius from 2-week-old chickens. The luminal surface (E) of epithelial cells shows a few microvilli (M), tight junctions (J), desmosomes (D) and a large accumulation of folded basement membrane (B) inside the cluster. The basal surface of epithelial cells is outlined by arrows.
916
M. Wuorela, S. Jalkanen, L. J. Pelliniemi and F? Toivanen
Eur. J. Immunol. 1990.20: 913-917
Nurse cells of the bursa of Fabricius
Eur. J. Immunol. 1990. 20: 913-917
cence showed thymic nurse cells and lymphocytes therein to be MHC class II+, as described earlier [7, 81. The spherical clusters of bursal cells had more ruffled surface than those from thymic origin, and there seemed to be some lymphocyte-like cells attached to the surface of some clusters. In these aggregates of bursal cells, only the attached B lymphocytes were class II+,whereas the epithelial component was negative.
917
apparently from this part of the bursa. The third type of clusters with both lumen and basement membrane inside the cluster were evidently from the junction between these two surface epithelia. The fourth type of aggregates consisting of various kinds of epithelial cells could arise from the interior parts of the follicle. The basement membrane gatherings seen inside these aggregates must derive from the junction between the cortex and the medulla of the follicle.
3.3 EM
Although the isolated epithelia cover all parts of the bursal follicle with a potential for the existence of nurse cells, no Ultrastructural analysis revealed that the spherical nurse nurse cell-like epithelial cells with intracytoplasmic lymcell-like cellular aggregates seen in light microscopy (Fig. 1) phocytes were seen in the aggregates from the bursa of in the bursa of Fabricius were composed of epithelial cells, Fabricius. However, with these enzyme digestions, we which were connected to each other by typical epithelial observed nurse cells in the chicken thymus as has been junctional complexes consisting of tight junctions and described by others [8, 15-17]. We conclude that interacdesmosomes (Fig. 2). Four types of aggregates were iden- tions between maturing lymphocytes and reticular epithetified, and in all of them the cells were morphologically lial cells similar to those seen in the thymus, are needed only in Tcell development, and are not a part of the B cell typical epithelial cells. differentiation process. Two types of aggregates had microvilli on luminal surface projecting outward from the aggregates, and junctional complexes along the apical parts of outermost cells. Some Excellent technical assistance by Paivi Valtanen, Marjo Vesanto, of these aggregates had internal interfollicular stromal Marjatta Leskenmaa, Sirpa From and Urpo Reunanen is gratefully spaces outlined with the basal surface and basement acknowledged. membrane of the epithelial cells. The space was tightly packed by accumulations of folded basement membranes Received January 2, 1990. (Fig. 2). Others consisted only of epithelial cells without basement membrane.The third type of aggregates had both lumen with microvilli and tight junctions, and areas of 5 References packed basement membrane inside the aggregate. The 1 Kyewski, B. A . , J. Exp. Med. 1987. 166: 520. fourth type of cell clusters had more variable cellular 2 Wick, G. and Oberhuber, G . , Eur. J. Immunol. 1986. 16: organization and islets of tightly compressed basement 855. membrane, but no signs of luminal surface (Fig. 3). No 3 Veromaa,T.,Vainio, O., Jalkanen, S., Eerola, E., Granfors, K . epithelial cells with intracytoplasmic lymphocytes were and Toivanen, P., Eur. J. Immunol. 1988. 18: 225. seen in the aggregates from the bursa of Fabricius. 4 Veromaa, T.,Vainio, O., Eerola, E. and Toivanen, I?, Hybri-
4 Discussion By using various enzyme digestions, we were able to isolate cellular clusters from the bursa of Fabricius, which in light microscopy resembled nurse cells. EM, however, showed the isolated clusters to consist of different types of epithelial cell aggregates without intracytoplasmic or interstitial lymphocytes. The four types of isolated epithelial cell aggregates were apparently derived from different parts of the bursal follicle as judged from the electron microscopic analyses describing the bursal ultrastructure [9-131. The aggregates with luminal surface but without basement membrane were from the follicle-associated epithelium (FAE), which is an epithelium without basement membrane, specialized in the uptake of environmental antigens into the bursal follicle [lo, 11, 141. The epithelium of the bursal lumen between FAE has a basement membrane, and the aggregates with microvilli on the outer surface and folded basement membrane inside the aggregate originated
doma 1988. 7: 41. Karnovsky, M. J., J. Cell. Biol. (abstract) 1971. 284: 146. Pelliniemi, L. J., Anat. Embryol. (Berl.) 1975. 147: 19. Wekerle, H. and Ketelsen, U.-I?, Nature 1980. 283: 402. Kyewski, B. A . and Kaplan, H. S., J. Immunol. 1982. 128: 2287. 9 Holbrook, K. A . , Perkins,W. D. and Glick, B., J. Reticuloendothel. SOC. 1974. 16: 300. 10 Naukkarinen, A . , Arstila, A . U. and Sorvari,T. E., Anat. Rec. 1978. 191: 415. 11 Olah, I., Glick, B., McCorkle, F. and Stinson, R., Dev. Comp. Immunol. 1979. 3: 101. 12 Glick, B. and Olah, I., Anat. Rec. 1982. 204: 341. 13 Freeman, B. M. (Ed.), Physiology and biochemistry of the domestic fowl, Vol. 5., Academic Press, London 1984, p. 277. 14 Sorvari, T., Sorvari, R . , Ruotsalainen, €?,Toivanen, A . and Toivanen, €?, Nature 1975. 253: 217. 15 Wekerle, H., Ketelsen, U.-€?and Ernst, M., J. Exp. Med. 1980. 151: 925. 16 Boyd, R. L., Oberhuber, G., Hala, K. and Wick, G., J. Immunol. 1984. 132: 718. 17 Andrews, l? and Boyd, R. L., Eur. J. Immunol. 1985. 15: 36. 5 6 7 8
4 Figure 3. Electron micrograph of the central region of an epithelial cell aggregate from the bursa of Fabriciusfrom a 2-week-old chicken.
The aggregate consists of diverse epithelial cells and accumulations of tightly packed, folded basement membrane (B) apparently originating from the medulla-cortex junction of the follicle. The basal surface of epithelial cells is outlined by arrows.
