Acta histochem. 90, 103-108 (1991) Gustav Fischer Verlag Jena
Department of Bacteriology, Saitama Medical School, Saitama, Japan
Age-related changes of J-chain-positive cells in chickens By OSAMU MORIYA and YOICHI ICHIKAWA With 3 Figures (Received April 3, 1989 - Accepted September 10,1990)
Summary Localizations of J-chain-positive cells (JPC) were examined in chicken lymphatic tissues before and after hatching. The cells containing J chain were first detected in medullary areas of the bursa of FABRICIUS during the embryonic stage. These positive cells were partly detected in the developing small lymphatic follicles: perphaps on newly differentiating precursor B-cells. In addition to these lymphatic follicles, connective tissue of bursal fold were also detected as J-chain positive. Although similar localizations of JPC were again observed in hatched chickens, positive areas of follicular medulla were strongly stained for fluorescence with corresponding antisera than that of embryonic ones. These data may reflect differences in the physiology of lymphocytes in respect to functional development. JPC localizations were next compared between the B-cell subpopulations, tJ.-(tJPC) and c¥-chainpositive cells (c¥PC). The J-chains detectable in the IgM molecules were also found in follicular medulla. However, these follicles were almost found to be negative for J-chains detectable in the c¥PC before hatching. Any strong stainings for J-chain in the c¥PC were, moreover, not be observed in bursa after hatching. The tJ.PC localizations in hatched chickens were roughly equal with the pattern of JPC localization. These analyses revealed the presence of the cells having the chains of both tJ. and J. The results together with other recent studies further shown that bursal Jchain can be partly detected in newly differentiated lymphatic follicles lacking IgM-producing and suggest the possible presence of B-cell-differentiation sequence of Ig-J+ -+ IgM+J+ -+ IgA +J+.
1. Introduction KAJI and PARKHOUSE (1975) have reported a role of i-chain in IgM polymerization. They have further noted the role of i-chain in IgG synthesis. Thus, there have been many studies implicating i-chain in B-cell differentiation (MATSUUCHI et al. 1986; MORIYA 1983; MORIYA and ICHIKAWA 1984a, b). The appearances of i-chain positive cells (lPC) at different stages of B-cell differentiation were thought that these i-chain might be participate in the 1st step of Ig synthesis, although their role(s) has not yet been explained exactly (KAJI and PARKHOUSE 1975; MORIYA and ICHIKAWA 1984a, b). Till now, many important informations on an early stage of B-cell differentiation have been studied throughout the investigations in chickens (GLICK et al. 1956; COOPER et al. 1966). The bursa of Fabricius is the first site of Ig synthesis in chickens and this lymphatic tissue has been shown to control the B-cell differentiation (COOPER et al. 1969). B-cells contained Ig components either on their surface or in cytoplasm have been observed by immunofluorescent technique, an easy procedure to identify these Ig components (KINCADE and COOPER 1971). In this study, we have studied immunohistochemically the i-chain localizations in chicken bursa in relation to the stage of B-cell differentiation.
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2. Materials and methods Animals: White Leghorn chickens were obtained as hatching eggs from the Saitama Prefectural Poultry Experiment Station. Adult chickens were maintained in our facility under standard conditions and were used at 6 weeks of age. Anti~hicken !-I-chain serum: Chicken IgM was purified from pooled sera by the method of LEBACQVERHEYDEN (1979). The IgM material was then purified by sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis (SDS-PAGE; LAEMMLIE 1971). Japanese white rabbits were immunized intramuscularly and intraperitoneally with purified !-I-chains in Freund's complete adjuvant (Difco Lab., Detroit) and boosted 3 weeks later. Booster injections were repeated intraperitoneally once every 10 d. Rabbit IgG fraction reactive toward chicken !-I-chain was prepared from the immune sera by precipitation twice with 18 % NazS04 and further purified by column chromatographies (DEAE cellulose: Serva, Germany, and Sephacryl-300, Pharmacw Uppsala, Sweden). Anti-J-chain-serum: Antisera to chicken J-chain were obtained by immunization of rabbits with J chains purified from either chicken or human IgM by alkaline urea-PAGE (REISFELD and SMALL 1964). Immunofluorescence: Fixations of the bursa of Fabricius taken from the 20th d of incubation and 6 weeks after hatching were carried out with 2.0 % glutaraldehyde in 100 mmolll cacodylate buffer at pH = 7.3 for 4h at 8°C. Some spleens were also prepared before and after hatching as the lymphatic tissue containing B-cells of matured type. These tissues were washed in phosphate buffered saline (PBS; pH = 7.2) and sliced at about 45 !-1m thickness using cryostat (Electro freeze, Komatsu Electronics, Tokyo), mounted on glass slides, incubated with each antibodies for 12 h at 8°C. These slides were rinsed wih cold PBS and incubated with fluorescein-conjugated goat anti-rabbit IgG (Miles Scientific, IL). After such treatments, the slides were again rinsed with PBS and covered with micro-cover glass. Observations were made using a Nikon fluorescent microscope. For the detection of total Jchains, the sections were treated with 8 molll urea in glycine-HCl buffer (PH = 3.2) for 3 h at 4°C before immunostaining (NAGURA et al. 1979). J-chains hidden in the IgM molecules were also observed after the reaction with rabbit antisera to human J-chain in their native conformations (without urea modification). Similarly, antisera specific to chicken J-chain were used for the observations of J-chains detectable in the IgA molecules. These antisera did not cross-react each other with the J-chains containing IgA or IgM molecules in their native conformations (MORIYA and ICHIKAWA 1991). Negative immunochemical controls were performed by incubation of specimens with FITC-labelled goat anti-rabbit IgG without the rabbit antibodies specific for J-chain (primary antibody). In these controls, no positive reactions were observed.
3. Results As shown in Fig. 1 a, total J-chain-positive cells (JPC) could be observed with considerable level in the bursal medulla after hatching. By the 20th d of embryo, such JPCs were detected within the lymphatic follicular area in bursa similarly with the adult ones (Fig. 1 b). The immunofluorescent stainings of the JPC detected in the embryonic bursa were, however, weaker as compared with the adult ones. Age-related differences exist in the bursa since the numbers of JPC seen within the medulla appeared to increase with age. Furthermore, cells containing J-chain were partly seen in the connective tissue cores in the same sections (Fig. I b). These data may reflect differences in the physiology of lymphocytes in respect of the functional development. When alternate sections of embryonic bursa were stained with anti-J, JPC could be detected in the developing lymphatic follicles: perhaps on newly differentiated bursal cells (Fig. I c). In the next observations of JPC using anti-J-chain antibodies which are not cross-reactive to Jchain in the Ig moleculs of either IgA or IgM in their native conformations, JPC belonged to the f.,tpositive cells (f.,tPC) were again detected within the medullary areas of bursal follicles (Fig. 2a). Combined with the results of the JPC localizations, the immunofluorescent pattern of f.,tPC detected in the medullary areas of lymphatic follicles suggested that many cells expressing IgM were mainly localized in the follicular medulla, although some positive cells were scantily shown in the cortical areas as determined by the fluorescent staining for f.,t-chain with corresponding antisera (Fig. 2b). JPC detectable in the cy-positive cells (CYPC) could not be observed in the embryonic bursa. These positive cells were detected as weak fluorescent stainings in bursa after hatching and strong stainings were not observed in this study (data not presented).
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Fig. I. Immunofluorescent staining for J-chain in bursa. a. Reaction to anti-J antibody in bursal lymphatic follicles of hatched chickens . Cryostat sections fixed in glutaraldehyde, modified with 8 molll urea in glycine-HCl buffer, incubated with rabbit anti-J-chain were treated with fluorescein-conjugated goat anti-rabbit IgO. JPC are localized mainly in the medullary area of the bursa. They are labelled in a ring-like fashion . x 360. b. Detection of J-chains in the embryonic bursa (20 d old embryos). Moderate activities were detected in follicle. Note the cells detected as JPC among the connective tissue core of the bursal fold . x 360. c. JPC in the embryonic bursa. Note the positive cells in newly differentiated bursal follicle (20 d old); x 180.
Fig. 2. JPC subpopulations and IlPC in bursa. a. Section, not treated with 8 molll urea in glycine-HCI buffer, incubated with rabbit anti-human J-chain, in which then was visualized the J-chains detectable in the IgM molecules by reacting with fluorescein-conjugated goat anti-rabbit [gO in their native conformation. Note that J-chains binding with Il-chains are mainly observed in the medullary area. x 360. b. Localization of IlPC in bursa. Sections stained with rabbit anti-chicken Il were treated with goat anti-rabbit [gO (fluorescein-conjugated). Stronger stainings are seen in the medullary field than in the cortical area. x 360.
In order to investigate the JPC localization in the spleen as a lymphatic tissue containing the matured type of lymphocytes, spleens were prepared before and after hatching. Histologically, JPC subpopulations were detected as bright fluorescent deposits and these positive cells were localized randomly in the spleens examined (Fig. 3a, b). The localization of !l+-PC was very similar to that obtained by the positive areas of t-tPC subpopulations and one could not find
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Fig. 3. Distribution of JPC subpopulations in the adult spleen. a. Splenic section, incubated with rabbit anti-human i-chain was treated with fluorescein-conjugated goat anti-rabbit IgO . Moderate and intense stainings for i-chain containing in the IgM molecules are apparent. x 360. b. Splenic section, incubated with rabbit anti-chicken i-chain in their native conformations was then treated with fluorescein-conjugated anti-rabbit IgO . Apparent stainings were scattered in the spleen. Their stainings are weaker than the ~-positive cells. x 360. c. Localization of f.lPC in spleen. Sections stained with rabbit anti-chicken f.l were treated with goat anti-rabbit IgO (fluorescein-conjugated) . Stronger stainings are seen. x 360.
any differences of positive cell localizations (Figs . 3a, c) . Characteristically, splenic f..lPC were more brilliant than the bursal ones (Fig. 3 c).
4. Discussion MORRISON and KOSHLAND (1972) and KAJI and PARKHOUSE (1975) suggested the contribution of i-chain on Igpolymerizations. Despite these suggestions , there are contradictory findings that i-chains are not necessary for Ig polymerizations (ESKELAND and CHRISTENSEN 1975; HABER and MESTECKY 1985). In chickens, higher frequencies of f.lPC were apparent in the 2 lymphatic tissues, bursa and spleen, than that observed in the cells containing i-chain (MaRlY A 1983). Therefore, it may be interesting to investigate the i-chain localizations in bursa of Fabricius at different developing stages with reference to synthesis of Ig isotypes. JPC were expressed mainly on medullary lymphocytes of the follicles, and scantily on non-lymphocytes among the connective tissue core in bursa. Histologically, these non-lymphocytes were detected as heterophils . Heterophils are unique to chickens and analogous to mammalian neutrophils (Fox and SOLOMON 1981) . At present, the chicken JPC, like mouse and human, are considered as 8-cell lineage (HABER and MESTECKY 1985; KAJI and PARHOUSE 1975). In this respect, we could not exactly explain the JPC detected on the heterophils. Chicken heterophils have been reported to have Ig receptors on their surface (EWALD et al. 1976) and the bursae were not washed before the treatment with glutaraldehyde for rapid fixation in histological studies. Ultrastructurally, the i-chains detected on the heterophils may be blood origin , therefore , are perhapes non-endogeneous (manuscript in contribution). For studying the JPC subpopulations, we prepared 2 different antisera reactive with i-chain specific for either IgA or IgM in their native conformations (MORIYA and ICHIKAWA 1991). Of course, total JPC can be detected after the treatment of specimens with urea in glycine-HCL buffer. These antibodies used, being perhaps reactive with different peptide sequences of the i-chain molecule recognized different antigenic determinants of i-chain. Anti-i sera prepared by immunizing rabbits with the i-chains taken from chicken IgM might be reactive with the chicken IgA, whereas another anti-i sera prepared by immunizing i-chain purified from human IgM might be reactive with
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I-chain hidden in the IgM molecules in their native conformations. Based on these reaction specificities of anti-I, we attempted to identify the JPC subpopulations in chickens. When the I-chains detectable in the flPC were tested, considerable number of JPC were found, especially in the spleen. Characteristically, when antisera reactive with the I-chains hidden in the IgA molecules were used, JPC were found with low level in number in the spleen and negligible in bursa before and after hatching. This finding implies that the cells expressing oJ was a minor population in chickens. During the embryogenesis, well-developed and developing lymphatic follicles were found. It is worthwhile to point out that I -chains were clearly shown to be expressed in bursal cells of different developing stages in the embryonic chickens. Small lymphatic follicles appeared morphologically as less-developed type than the large ones. Even in such stage of B-cell differentiation. I-chains were detected on both follicles of well-developed and immature ones. The detections of I-chains in an early developing stage of B-cells were fundamentally consistent with our previously study (MORIYA 1983; MORIYA and ICHIKAMA 1984a, b). Our observations of I-chain expressions on immature (less-developed) follicles were further in agreement with the finding of gene expression in an early stage of cell development described by MATSUUCHI et al. (1986). I-chains were expressed on distinct stage of pre-mature B-cells, and these cells were detected independently for the Ig secretions (MORIYA 1983). Thus, I-chains are not unique molecule to B-cells matured or precursor type in chickens and are expressed on other B-celiline in species as widely divergent (DAVIS and SHULMAN 1989). The immunological function of I-chain is, however, unknown as described above and this problem remain to be studied. The strong fluorescent staining for I -chain among the splenic cells after hatching but negligible stainings with anti-I sera in the embryonic ones, suggest a synchronous increase of I-chain synthesis with the level of the B-cell development. The detection of I-chain on differentiating small lymphatic follicles, which are at an immature stage during the course of B-cell differentiation, provides a new subpopulation of JPC. These small follicles may be a site of undergoing the first stage in bursal B-cell differentiation. It is now suggested that these JPC may be differentiated from Ig-negative- into Ig-positive JPC in the microenvironments of chicken lymphatic tissues.
References COOPER, M. D., CAIN, W. A., VAN ALTEN, P. J., and GOOD, R. A., Development and function of the immunoglobulin producing system. 1. Effect of bursectomy at different stages of development on germinal center, plasma cells, immunoglobulin and antibody production. Int. Arch. Allergy, app!. Immuno!. 35, 242-252 (1969). PETERSON, R. D. A., SOUTH, M. A., and GOOD, R. A., The function of the thymus and the bursa system in chicken. J. Exp. Med. 123,75-102 (1966). DAVIS, A. C., und SHULMAN, M. J., IgM-molecule requirements for its assembly and function. Immunol. Today, 10, 118-128 (1989). ESKELAND, T., and CHRISTENSEN, T. B., IgM molecules with and without J chain in serum after purification. Studies by ultracentrifugation, electrophoresis, and electron microscopy. Scand. J. Immunol. 4, 217-228 (1975). EWALD, S. M., FREEDMAN, L., and SANDERS, B. G., EA rosette forming lymphoid cells in chickens: Specificity of the Fc receptor and its relationship to other surface antigens. Immunology 31,847-854 (1976). Fox, A. J., and SOLOMON, J. B., Chicken non-lymphoid leukocytes. In: Avian Immunology (Eds. ROSE, M. E., PAYNL, L. N., and FREEDAMAN, B. M.) Clark Constable Edinburgh 1981, pp. 135-160. GLICK, B., CHANG, J. S., and JAAP, R. G., The bursa of Fabricius and antibody production. Poultry Sci. 35, 224-225 (1956). HABER, P. L., and MESTECKY, J., J chain expression in human cells producing IgG subclasses. Cell. Immuno!. 91, 515-519 (1985). KAJI, H., and PARKHOUSE, R. M. E., Control of J chain biosynthesis in relation to heavy and light chain synthesis, polymerization and secretion. J. Immuno!. 114, 1218-1220 (1975). - - Intracellular J chain in mouse plasmacytomas secreting IgA, IgM, and IgG. Nature 249, 45-47 (1974). KINCADE, P. W., and COOPER, M. D., Development and distribution of immunoglobulin containing cells in the chicken: An immunofluorescent analysis using purified antibodies to fl, y, and light chain. J. Immuno!. 106, 371-382 (1971). LAEMMLIE, U. K., Cleavageof structural protein during assembly of head of bacteriophage T4. Nature 227, 680-685 (1971). LEBACQ-VERHEYDEN, A.-M., Purification and testing class-specific anti-chicken immunoglobulin antibodies. J. Immuno!. Methods 25, 101-117 (1979).
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MATSUUCHI, L., CANN, G. M., and KOSHLAND, M. E., ImmunoglobulinJ chain gene from the mouse. Proc. Nat!., Acad. Sci. USA 83, 456-460 (1986). MORIYA, 0., Ontogeny of J chain representing cells in developing chick embryos. Cell. Immunol. 80, 78-83 (1983). - and ICHIKAWA, Y., J chain positive cells in bursectomized chicks. Immunol. Lett. 7,289-291 (1984a). - - Loss of J chain during primary inunune response in chicks. Clin. Exp. Imrnunol. 58, 719-723 (1984b). MORRISON, S. L., and KOSHLAND, M. E., Characterization of the J chain from polymeric immunoglobulins. Proc. Nat!. Acad. Sci. USA 69, 124-128 (1972). NAGURA, H., BRANDTz, G. P., NAKANE, P. K., and BROWN, W. R., Uitrastructurallocalization of J chain in human intestinal mucosa. J. Immunol. 123, 1044-1050 (1979). REISFELD, R. A., and SMALL, R. A., Electrophoretic heterogeneity of polypeptide chain of specific antibodies. Science 152, 1253-1255 (1964). Authors' adress: Dr. OSAMU MORIYA, Department of Bacteriology, Saitama Medical School, 38, Morohongo, Moroyama, Irurna-gun, Saitama, 350-04 Japan.
Acta histochemica. Verlag: Gustav Fischer Verlag Jena GmbH, Villengang 2, D-0-69OO Jena, Telefon 27332. Geschiiftsfiihrer: Johanna Schliiter und Bernd Rolle. Verantwortlich fur die Redaktion: Prof. Dr. Dr. Dr. h. c. Joachim-Hermann Scharf, PostschlieBfach 302, D-O4010 Halle. Satz, Druck und Buchbinderei: Druckhaus Jena GmbH. Anzeigenannahme: Gustav Fischer Verlag Jena, Anzeigenverwaltung, Villengang 2, D-0-6900 Jena; Telefon 27332, Telex 588676. Zur Zeit gilt Anzeigenpreisliste Nr. 1 vom 1. 1. 1990. AIle Rechte beim Verlag. Nachdruck (auch auszugsweise) nur mit Genehmigung des Verlages und des Verfassers sowie mit Quellenangabe gestattet. Printed in Germany.
Department of Bacteriology, Saitama Medical School, Saitama, Japan
Age-related changes of J-chain-positive cells in chickens By OSAMU MORIYA and YOICHI ICHIKAWA With 3 Figures (Received April 3, 1989 - Accepted September 10,1990)
Summary Localizations of J-chain-positive cells (JPC) were examined in chicken lymphatic tissues before and after hatching. The cells containing J chain were first detected in medullary areas of the bursa of FABRICIUS during the embryonic stage. These positive cells were partly detected in the developing small lymphatic follicles: perphaps on newly differentiating precursor B-cells. In addition to these lymphatic follicles, connective tissue of bursal fold were also detected as J-chain positive. Although similar localizations of JPC were again observed in hatched chickens, positive areas of follicular medulla were strongly stained for fluorescence with corresponding antisera than that of embryonic ones. These data may reflect differences in the physiology of lymphocytes in respect to functional development. JPC localizations were next compared between the B-cell subpopulations, tJ.-(tJPC) and c¥-chainpositive cells (c¥PC). The J-chains detectable in the IgM molecules were also found in follicular medulla. However, these follicles were almost found to be negative for J-chains detectable in the c¥PC before hatching. Any strong stainings for J-chain in the c¥PC were, moreover, not be observed in bursa after hatching. The tJ.PC localizations in hatched chickens were roughly equal with the pattern of JPC localization. These analyses revealed the presence of the cells having the chains of both tJ. and J. The results together with other recent studies further shown that bursal Jchain can be partly detected in newly differentiated lymphatic follicles lacking IgM-producing and suggest the possible presence of B-cell-differentiation sequence of Ig-J+ -+ IgM+J+ -+ IgA +J+.
1. Introduction KAJI and PARKHOUSE (1975) have reported a role of i-chain in IgM polymerization. They have further noted the role of i-chain in IgG synthesis. Thus, there have been many studies implicating i-chain in B-cell differentiation (MATSUUCHI et al. 1986; MORIYA 1983; MORIYA and ICHIKAWA 1984a, b). The appearances of i-chain positive cells (lPC) at different stages of B-cell differentiation were thought that these i-chain might be participate in the 1st step of Ig synthesis, although their role(s) has not yet been explained exactly (KAJI and PARKHOUSE 1975; MORIYA and ICHIKAWA 1984a, b). Till now, many important informations on an early stage of B-cell differentiation have been studied throughout the investigations in chickens (GLICK et al. 1956; COOPER et al. 1966). The bursa of Fabricius is the first site of Ig synthesis in chickens and this lymphatic tissue has been shown to control the B-cell differentiation (COOPER et al. 1969). B-cells contained Ig components either on their surface or in cytoplasm have been observed by immunofluorescent technique, an easy procedure to identify these Ig components (KINCADE and COOPER 1971). In this study, we have studied immunohistochemically the i-chain localizations in chicken bursa in relation to the stage of B-cell differentiation.
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2. Materials and methods Animals: White Leghorn chickens were obtained as hatching eggs from the Saitama Prefectural Poultry Experiment Station. Adult chickens were maintained in our facility under standard conditions and were used at 6 weeks of age. Anti~hicken !-I-chain serum: Chicken IgM was purified from pooled sera by the method of LEBACQVERHEYDEN (1979). The IgM material was then purified by sodium dodecyl sulfate-containing polyacrylamide gel electrophoresis (SDS-PAGE; LAEMMLIE 1971). Japanese white rabbits were immunized intramuscularly and intraperitoneally with purified !-I-chains in Freund's complete adjuvant (Difco Lab., Detroit) and boosted 3 weeks later. Booster injections were repeated intraperitoneally once every 10 d. Rabbit IgG fraction reactive toward chicken !-I-chain was prepared from the immune sera by precipitation twice with 18 % NazS04 and further purified by column chromatographies (DEAE cellulose: Serva, Germany, and Sephacryl-300, Pharmacw Uppsala, Sweden). Anti-J-chain-serum: Antisera to chicken J-chain were obtained by immunization of rabbits with J chains purified from either chicken or human IgM by alkaline urea-PAGE (REISFELD and SMALL 1964). Immunofluorescence: Fixations of the bursa of Fabricius taken from the 20th d of incubation and 6 weeks after hatching were carried out with 2.0 % glutaraldehyde in 100 mmolll cacodylate buffer at pH = 7.3 for 4h at 8°C. Some spleens were also prepared before and after hatching as the lymphatic tissue containing B-cells of matured type. These tissues were washed in phosphate buffered saline (PBS; pH = 7.2) and sliced at about 45 !-1m thickness using cryostat (Electro freeze, Komatsu Electronics, Tokyo), mounted on glass slides, incubated with each antibodies for 12 h at 8°C. These slides were rinsed wih cold PBS and incubated with fluorescein-conjugated goat anti-rabbit IgG (Miles Scientific, IL). After such treatments, the slides were again rinsed with PBS and covered with micro-cover glass. Observations were made using a Nikon fluorescent microscope. For the detection of total Jchains, the sections were treated with 8 molll urea in glycine-HCl buffer (PH = 3.2) for 3 h at 4°C before immunostaining (NAGURA et al. 1979). J-chains hidden in the IgM molecules were also observed after the reaction with rabbit antisera to human J-chain in their native conformations (without urea modification). Similarly, antisera specific to chicken J-chain were used for the observations of J-chains detectable in the IgA molecules. These antisera did not cross-react each other with the J-chains containing IgA or IgM molecules in their native conformations (MORIYA and ICHIKAWA 1991). Negative immunochemical controls were performed by incubation of specimens with FITC-labelled goat anti-rabbit IgG without the rabbit antibodies specific for J-chain (primary antibody). In these controls, no positive reactions were observed.
3. Results As shown in Fig. 1 a, total J-chain-positive cells (JPC) could be observed with considerable level in the bursal medulla after hatching. By the 20th d of embryo, such JPCs were detected within the lymphatic follicular area in bursa similarly with the adult ones (Fig. 1 b). The immunofluorescent stainings of the JPC detected in the embryonic bursa were, however, weaker as compared with the adult ones. Age-related differences exist in the bursa since the numbers of JPC seen within the medulla appeared to increase with age. Furthermore, cells containing J-chain were partly seen in the connective tissue cores in the same sections (Fig. I b). These data may reflect differences in the physiology of lymphocytes in respect of the functional development. When alternate sections of embryonic bursa were stained with anti-J, JPC could be detected in the developing lymphatic follicles: perhaps on newly differentiated bursal cells (Fig. I c). In the next observations of JPC using anti-J-chain antibodies which are not cross-reactive to Jchain in the Ig moleculs of either IgA or IgM in their native conformations, JPC belonged to the f.,tpositive cells (f.,tPC) were again detected within the medullary areas of bursal follicles (Fig. 2a). Combined with the results of the JPC localizations, the immunofluorescent pattern of f.,tPC detected in the medullary areas of lymphatic follicles suggested that many cells expressing IgM were mainly localized in the follicular medulla, although some positive cells were scantily shown in the cortical areas as determined by the fluorescent staining for f.,t-chain with corresponding antisera (Fig. 2b). JPC detectable in the cy-positive cells (CYPC) could not be observed in the embryonic bursa. These positive cells were detected as weak fluorescent stainings in bursa after hatching and strong stainings were not observed in this study (data not presented).
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Fig. I. Immunofluorescent staining for J-chain in bursa. a. Reaction to anti-J antibody in bursal lymphatic follicles of hatched chickens . Cryostat sections fixed in glutaraldehyde, modified with 8 molll urea in glycine-HCl buffer, incubated with rabbit anti-J-chain were treated with fluorescein-conjugated goat anti-rabbit IgO. JPC are localized mainly in the medullary area of the bursa. They are labelled in a ring-like fashion . x 360. b. Detection of J-chains in the embryonic bursa (20 d old embryos). Moderate activities were detected in follicle. Note the cells detected as JPC among the connective tissue core of the bursal fold . x 360. c. JPC in the embryonic bursa. Note the positive cells in newly differentiated bursal follicle (20 d old); x 180.
Fig. 2. JPC subpopulations and IlPC in bursa. a. Section, not treated with 8 molll urea in glycine-HCI buffer, incubated with rabbit anti-human J-chain, in which then was visualized the J-chains detectable in the IgM molecules by reacting with fluorescein-conjugated goat anti-rabbit [gO in their native conformation. Note that J-chains binding with Il-chains are mainly observed in the medullary area. x 360. b. Localization of IlPC in bursa. Sections stained with rabbit anti-chicken Il were treated with goat anti-rabbit [gO (fluorescein-conjugated). Stronger stainings are seen in the medullary field than in the cortical area. x 360.
In order to investigate the JPC localization in the spleen as a lymphatic tissue containing the matured type of lymphocytes, spleens were prepared before and after hatching. Histologically, JPC subpopulations were detected as bright fluorescent deposits and these positive cells were localized randomly in the spleens examined (Fig. 3a, b). The localization of !l+-PC was very similar to that obtained by the positive areas of t-tPC subpopulations and one could not find
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Fig. 3. Distribution of JPC subpopulations in the adult spleen. a. Splenic section, incubated with rabbit anti-human i-chain was treated with fluorescein-conjugated goat anti-rabbit IgO . Moderate and intense stainings for i-chain containing in the IgM molecules are apparent. x 360. b. Splenic section, incubated with rabbit anti-chicken i-chain in their native conformations was then treated with fluorescein-conjugated anti-rabbit IgO . Apparent stainings were scattered in the spleen. Their stainings are weaker than the ~-positive cells. x 360. c. Localization of f.lPC in spleen. Sections stained with rabbit anti-chicken f.l were treated with goat anti-rabbit IgO (fluorescein-conjugated) . Stronger stainings are seen. x 360.
any differences of positive cell localizations (Figs . 3a, c) . Characteristically, splenic f..lPC were more brilliant than the bursal ones (Fig. 3 c).
4. Discussion MORRISON and KOSHLAND (1972) and KAJI and PARKHOUSE (1975) suggested the contribution of i-chain on Igpolymerizations. Despite these suggestions , there are contradictory findings that i-chains are not necessary for Ig polymerizations (ESKELAND and CHRISTENSEN 1975; HABER and MESTECKY 1985). In chickens, higher frequencies of f.lPC were apparent in the 2 lymphatic tissues, bursa and spleen, than that observed in the cells containing i-chain (MaRlY A 1983). Therefore, it may be interesting to investigate the i-chain localizations in bursa of Fabricius at different developing stages with reference to synthesis of Ig isotypes. JPC were expressed mainly on medullary lymphocytes of the follicles, and scantily on non-lymphocytes among the connective tissue core in bursa. Histologically, these non-lymphocytes were detected as heterophils . Heterophils are unique to chickens and analogous to mammalian neutrophils (Fox and SOLOMON 1981) . At present, the chicken JPC, like mouse and human, are considered as 8-cell lineage (HABER and MESTECKY 1985; KAJI and PARHOUSE 1975). In this respect, we could not exactly explain the JPC detected on the heterophils. Chicken heterophils have been reported to have Ig receptors on their surface (EWALD et al. 1976) and the bursae were not washed before the treatment with glutaraldehyde for rapid fixation in histological studies. Ultrastructurally, the i-chains detected on the heterophils may be blood origin , therefore , are perhapes non-endogeneous (manuscript in contribution). For studying the JPC subpopulations, we prepared 2 different antisera reactive with i-chain specific for either IgA or IgM in their native conformations (MORIYA and ICHIKAWA 1991). Of course, total JPC can be detected after the treatment of specimens with urea in glycine-HCL buffer. These antibodies used, being perhaps reactive with different peptide sequences of the i-chain molecule recognized different antigenic determinants of i-chain. Anti-i sera prepared by immunizing rabbits with the i-chains taken from chicken IgM might be reactive with the chicken IgA, whereas another anti-i sera prepared by immunizing i-chain purified from human IgM might be reactive with
I -chain-positive cells in chickens
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I-chain hidden in the IgM molecules in their native conformations. Based on these reaction specificities of anti-I, we attempted to identify the JPC subpopulations in chickens. When the I-chains detectable in the flPC were tested, considerable number of JPC were found, especially in the spleen. Characteristically, when antisera reactive with the I-chains hidden in the IgA molecules were used, JPC were found with low level in number in the spleen and negligible in bursa before and after hatching. This finding implies that the cells expressing oJ was a minor population in chickens. During the embryogenesis, well-developed and developing lymphatic follicles were found. It is worthwhile to point out that I -chains were clearly shown to be expressed in bursal cells of different developing stages in the embryonic chickens. Small lymphatic follicles appeared morphologically as less-developed type than the large ones. Even in such stage of B-cell differentiation. I-chains were detected on both follicles of well-developed and immature ones. The detections of I-chains in an early developing stage of B-cells were fundamentally consistent with our previously study (MORIYA 1983; MORIYA and ICHIKAMA 1984a, b). Our observations of I-chain expressions on immature (less-developed) follicles were further in agreement with the finding of gene expression in an early stage of cell development described by MATSUUCHI et al. (1986). I-chains were expressed on distinct stage of pre-mature B-cells, and these cells were detected independently for the Ig secretions (MORIYA 1983). Thus, I-chains are not unique molecule to B-cells matured or precursor type in chickens and are expressed on other B-celiline in species as widely divergent (DAVIS and SHULMAN 1989). The immunological function of I-chain is, however, unknown as described above and this problem remain to be studied. The strong fluorescent staining for I -chain among the splenic cells after hatching but negligible stainings with anti-I sera in the embryonic ones, suggest a synchronous increase of I-chain synthesis with the level of the B-cell development. The detection of I-chain on differentiating small lymphatic follicles, which are at an immature stage during the course of B-cell differentiation, provides a new subpopulation of JPC. These small follicles may be a site of undergoing the first stage in bursal B-cell differentiation. It is now suggested that these JPC may be differentiated from Ig-negative- into Ig-positive JPC in the microenvironments of chicken lymphatic tissues.
References COOPER, M. D., CAIN, W. A., VAN ALTEN, P. J., and GOOD, R. A., Development and function of the immunoglobulin producing system. 1. Effect of bursectomy at different stages of development on germinal center, plasma cells, immunoglobulin and antibody production. Int. Arch. Allergy, app!. Immuno!. 35, 242-252 (1969). PETERSON, R. D. A., SOUTH, M. A., and GOOD, R. A., The function of the thymus and the bursa system in chicken. J. Exp. Med. 123,75-102 (1966). DAVIS, A. C., und SHULMAN, M. J., IgM-molecule requirements for its assembly and function. Immunol. Today, 10, 118-128 (1989). ESKELAND, T., and CHRISTENSEN, T. B., IgM molecules with and without J chain in serum after purification. Studies by ultracentrifugation, electrophoresis, and electron microscopy. Scand. J. Immunol. 4, 217-228 (1975). EWALD, S. M., FREEDMAN, L., and SANDERS, B. G., EA rosette forming lymphoid cells in chickens: Specificity of the Fc receptor and its relationship to other surface antigens. Immunology 31,847-854 (1976). Fox, A. J., and SOLOMON, J. B., Chicken non-lymphoid leukocytes. In: Avian Immunology (Eds. ROSE, M. E., PAYNL, L. N., and FREEDAMAN, B. M.) Clark Constable Edinburgh 1981, pp. 135-160. GLICK, B., CHANG, J. S., and JAAP, R. G., The bursa of Fabricius and antibody production. Poultry Sci. 35, 224-225 (1956). HABER, P. L., and MESTECKY, J., J chain expression in human cells producing IgG subclasses. Cell. Immuno!. 91, 515-519 (1985). KAJI, H., and PARKHOUSE, R. M. E., Control of J chain biosynthesis in relation to heavy and light chain synthesis, polymerization and secretion. J. Immuno!. 114, 1218-1220 (1975). - - Intracellular J chain in mouse plasmacytomas secreting IgA, IgM, and IgG. Nature 249, 45-47 (1974). KINCADE, P. W., and COOPER, M. D., Development and distribution of immunoglobulin containing cells in the chicken: An immunofluorescent analysis using purified antibodies to fl, y, and light chain. J. Immuno!. 106, 371-382 (1971). LAEMMLIE, U. K., Cleavageof structural protein during assembly of head of bacteriophage T4. Nature 227, 680-685 (1971). LEBACQ-VERHEYDEN, A.-M., Purification and testing class-specific anti-chicken immunoglobulin antibodies. J. Immuno!. Methods 25, 101-117 (1979).
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MATSUUCHI, L., CANN, G. M., and KOSHLAND, M. E., ImmunoglobulinJ chain gene from the mouse. Proc. Nat!., Acad. Sci. USA 83, 456-460 (1986). MORIYA, 0., Ontogeny of J chain representing cells in developing chick embryos. Cell. Immunol. 80, 78-83 (1983). - and ICHIKAWA, Y., J chain positive cells in bursectomized chicks. Immunol. Lett. 7,289-291 (1984a). - - Loss of J chain during primary inunune response in chicks. Clin. Exp. Imrnunol. 58, 719-723 (1984b). MORRISON, S. L., and KOSHLAND, M. E., Characterization of the J chain from polymeric immunoglobulins. Proc. Nat!. Acad. Sci. USA 69, 124-128 (1972). NAGURA, H., BRANDTz, G. P., NAKANE, P. K., and BROWN, W. R., Uitrastructurallocalization of J chain in human intestinal mucosa. J. Immunol. 123, 1044-1050 (1979). REISFELD, R. A., and SMALL, R. A., Electrophoretic heterogeneity of polypeptide chain of specific antibodies. Science 152, 1253-1255 (1964). Authors' adress: Dr. OSAMU MORIYA, Department of Bacteriology, Saitama Medical School, 38, Morohongo, Moroyama, Irurna-gun, Saitama, 350-04 Japan.
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