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Early embryonic blood cells collect antigens and induce immunotolerance in the hatched chicken
Early embryonic blood cells collect antigens and induce immunotolerance in the hatched chicken G. j. Wu,* F. Yuan,* M. H. Du,† H. T. Han,* L. Q. Lu,* L. Yan,* W. X. Zhang,* X. P. Wang,* P. Sun,* and Z. D. Li*1 *Department of Biochemistry and Molecular Biology, College of Biology Science, and State Key Laboratory for Agrobiotechnology, China Agricultural University, 100193 Beijing, China; and †Beijing Center for Physical and Chemical Analysis, 100089 Beijing, China were depleted in chickens whose early embryo cells had been loaded with BSA, as evidenced by a significant decrease in anti-BSA antibody after challenge with BSA when the chickens were 3 wk old. In addition, by direct injection of BSA to embryonic d 3 embryo blood, the hatched chickens had decreased amounts of anti-trinitrophenol antibody after the chickens were challenged with trinitrophenol-BSA, indicating that the helper function of BSA-specific T cells was impaired. In conclusion, these observations suggest that some early embryo blood cells possibly collect and store antigen for the establishment of self-tolerance before the maturation of B and T cells.
Key words: chicken embryo, immunological tolerance, blood cell, endocytosis 2010 Poultry Science 89:457–463 doi:10.3382/ps.2009-00437
INTRODUCTION
guchi, 2004). Thus, antigens in the microenvironment during lymphocyte development are thought to be important for the establishment of self-tolerance. Selfantigens may be expressed in the thymus (Anderson et al., 2002; Kyewski and Derbinski, 2004) and reach the thymus via the blood circulation (Kyewski et al., 1984; Volkmann et al., 1997) but can also be transported to the thymus from the periphery by dendritic cells (DC), an efficient type of antigen-presenting cell (Bonasio et al., 2006). We have previously injected BSA or casein into embryonic blood at 65 to 70 h of embryogenesis and found that a considerable number of the injected chickens become tolerant to BSA or casein, respectively (Zhao et al., 2006). However, in another study, the protein injected was found to be cleared quickly from embryonic serum (Wu and Li, 2009). Thymus anlage takes place in the chicken embryo at approximately embryonic d (E)5, and the first thymocyte progenitors colonize the thymus at E6.5 (jotereau and Le Douarin, 1982). The B cells are first found in the para-aortic foci and yolk sac at E5 and undergo rapid proliferative expansion within the bursal epithelial follicles up to the time of hatching
Self-tolerance can be established via several mechanisms. Transgenic mice, in which most newly generated B cells express an antibody against an antigen with strong cross-linking capacity that is present in the bone marrow, have no B cells specific for that antigen in the peripheral immune system (Nemazee and Buerki, 1989; Hartley et al., 1991; Chen et al., 1995; Goodnow et al., 1995). In the thymus, immature T cells that encounter cognate MHC-peptide complexes are eliminated by clonal deletion, a key process leading to central tolerance (Palmer, 2003). The presence of an agonist peptide in the thymus can also result in the differentiation of CD4+CD25+ regulatory T cells that impose tolerance in the periphery by dampening the response of conventional lymphocytes (jordan et al., 2001; Saka-
©2010 Poultry Science Association Inc. Received September 4, 2009. Accepted October 8, 2009. 1 Corresponding author: [email protected]
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ABSTRACT Earlier experimental data in our laboratory showed that introduction of an exogenous protein into early chicken embryonic blood leads to immunotolerance of hatched chicken to that protein. However, the underlying mechanism is yet unknown. In the present study, we show that the blood cells collecting circulating antigen might contribute to the establishment of immunotolerance. In this experiment, most of the chicken embryo blood cells took up injected fluorescein isothiocyanate-BSA at approximately embryonic d 3. At the same stage, 1 μL of embryo blood was taken out and incubated with BSA. After being loaded with BSA in vitro and washed, these cells were injected back into the original embryo. The BSA-specific lymphocytes
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(Sayegh et al., 2000; Ratcliffe, 2006). For these reasons, it appears that other mechanism was preferred by the early embryo than circulating the injected protein to the thymus or the bursa alone, to induce tolerance to T cells or B cells, and some early embryonic blood cells might play the primary role. In this study, we found that the early embryo blood cells have a high ability to take up injected protein in vivo and in vitro. We loaded embryo blood cells with BSA in vitro and then injected the cells back into the embryo. Most of the hatched chickens appeared to have partial tolerance to BSA. It seems to be the blood cells that carry circulating antigen in early embryo blood to thymus or bursa and induce negative selection for maturing lymphocytes.
MATERIALS AND METHODS Bovine serum albumin (A7030), ovalbumin (OVA, A5378), fluorescein isothiocyanate (FITC)-BSA (A9771), and 2,4,6-trinitrobenzenesulfonic acid (P2297) were purchased from Sigma-Aldrich (St. Louis, MO). The preparation of trinitrophenol (TNP)-BSA and TNP-OVA was modified from the methods of Bondada and Robertson (2003). Briefly, 50 mg of OVA (or 80 mg of BSA) was dissolved in 2.5 mL of 0.1 M NaHCO3 slowly added to a 317-µL solution containing 95 µL of 5% 2,4,6-trinitrobenzenesulfonic acid under stirring. The mixture was covered with aluminum foil and incubated overnight at 4°C with gentle stirring. The mixture was then dialyzed 5 times against 2 L of saline, the absorbance was measured in a spectrophotometer at optical density (OD) 280 nm and OD 340 nm, and the ratio of TNP to protein was calculated. For OVA, the ratio was 1.98, and for BSA, the ratio was 4.20.
Fertilized Eggs and Chickens Fertilized eggs of the Jingbai 938 strain, a White Leghorn (Gallus domesticus) strain purchased from the Experimental Station of the China Agricultural University, were incubated at 37.8°C in 70% RH up to hatching. The hatched chickens were bred in the laboratory. To demonstrate the uptake of antigen by embryo blood cells, on the one hand, FITC-BSA was injected into stage 18 to 19 (Hamburger and Hamilton stage, at about E3) embryonic blood via the aorta and 2 to 3 µL of blood was removed from the embryo after 2 h of injection. On the other hand, 5 to 6 µL of normal embryonic blood was diluted in 45 µL of warm RPMI1640 containing 50 µg of FITC-BSA and incubated at 37°C in 5% CO2. After 3 h, the blood cells were washed 3 times in 200 µL of cold PBS (pH 7.4) and centrifuged at 200 × g for 8 min. The washed cells were analyzed by confocal microscopy (200× magnified, Nikon D-Eclipse C1, Nikon, Tokyo, Japan).
Preparation of Serum Samples Chicken blood collected immediately before immunization (in the preimmunized group) or 10 d after the first immunization was placed at 37°C for 2 to 4 h to allow a clot to form and was then placed overnight at 4°C to allow the clot to retract. Serum was collected from the clotted blood by centrifugation at 10,000 ×
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Antigens
In the first experiment, 3 groups of birds were used: the quail-BSA group, self-BSA group, and nontreated group. For the quail-BSA group, quail embryonic blood cells were used, to eliminate the possibility of interference from BSA leaking from any broken BSA-loaded blood cells (early quail embryonic blood cells can also take up antigen in vitro, data not shown). For the selfBSA group, the chicken’s own embryonic blood cells were used, to prove whether the early embryonic blood cells collecting antigen could induce immunotolerance of the hatched chicken. One microliter of embryo blood taken from stage 17 to 19 embryo (Hamburger and Hamilton stage, at about E3 for chicken or 42 h of incubation for quail) was diluted in 50 µL of RPMI-1640 containing 500 µg of BSA and was incubated for 3 h at 37°C in 5% CO2 and then washed 4 times in 200 µL of warm PBS (pH 7.4). Washed cells were suspended in 1 to 2 µL of the last wash solution until injection. For the eggs of the quail-BSA group, we removed 1 µL of blood at 68~70 h of embryogenesis (at stage 17 to 19) and injected it with quail embryo blood cells loaded with BSA, at about 78~80 h of embryogenesis, and for the self-BSA group, 1 µL of blood was drawn at 68 to 70 h of embryogenesis and the blood cells were loaded with BSA in vitro and injected back into the embryo after washing at ~78 to 80 h of embryogenesis. Eggs continued to be incubated as before. Embryos in the nontreated group were normal and not treated. When the hatched chickens were 3 wk old, each chicken was treated with 100 µg of BSA emulsified in Freund’s complete adjuvant by dorsal hypodermic injection. In the second experiment, there were also 2 groups of birds treated. In the BSA-injected group, embryos were injected with 10 µg of BSA dissolved in 1 µL of PBS at 68 to 70 h of embryogenesis; in the PBS-injected group, PBS was injected instead of BSA. The humoral immune response requires an antigen-specific interaction between T and B cells (Lanzavecchia, 1985), and TNP is a hapten that has to be coupled to a carrier protein to trigger an immune response. When the hatched chickens were 3 wk old, they were treated with 50 µg of TNP-BSA emulsified in Freund’s complete adjuvant by dorsal hypodermic injection to evaluate the BSA-specific helper T cells by detecting anti-TNP antibodies. In the above 2 experiments, both the drawing of blood from the embryo and injection into the embryo were via the aorta.
BLOOD CELLS TAKE UP ANTIGEN TO INDUCE IMMUNOTOLERANCE
g for 10 min. All serum samples were stored at −20°C until analysis.
ELISA
Statistical Analysis The data were analyzed by SPSS 13.0 (SPSS Inc., Chicago, IL) with the 1-sample K-S program, and all of the data in each group were normally distributed (P > 0.3, data not shown). The comparison of each 2 groups was accomplished with the independent sample t-test. We considered comparisons to be statistically distinct at P < 0.05.
RESULTS Early Embryo Blood Cells Take Up Injected Proteins In Vivo and In Vitro We injected 10 µg of FITC-BSA into chicken embryo blood via the aorta at about E3. Two hours postinjection, most of the blood cells had taken up the FITCBSA (Figure 1a and 1b). The embryo blood cells also took up FITC-BSA in vitro (Figure 1c and 1d), and there was no difference between these cells and the cells treated in vivo.
Embryo Blood Cells Collecting Antigen to Establish Self-Tolerance Three groups of eggs were used in the first experiment: quail-BSA, self-BSA, and nontreated. The quailBSA group was used as a negative control for tolerance, and the self-BSA group was used to show that early embryo cells take up BSA and induce immune tolerance to BSA in the hatched chicken. The treatment of the eggs in these 2 groups was similar, except that in the quail-BSA group, the chicken embryo’s own blood cells were replaced with quail embryo blood cells extracted at the same stage of development and loaded with BSA (quail embryo blood cells have a similar ability to take up peptides in vitro; data not shown). The quail-BSA group was used to evaluate the effect of peptides derived from BSA and leaked from loaded cells. Because quail blood cells have different MHC with chicken, they cannot present peptides to chicken T cells in the process of selection in thymus, so we choose early quail embryonic cells instead of chicken blood cells in the quail-BSA group. Seven, 12, and 17 hatched chickens in quail-BSA, self-BSA, and nontreated, respectively, were obtained and immunized with BSA at 3 wk of age. Ten days after the first immunization, the antibody level of anti-BSA in the self-BSA group was 0.498 ± 0.336 (mean ± SD) and significantly lower than 1.123 ± 0.490 (mean ± SD) that was the level in the quail-BSA group (P = 0.0017) (Figure 2), but not significantly lower than 0.791 ± 0.514 (mean ± SD) that was the level in the nontreated group (P = 0.0575), possibly as a result of small sample size. The above result indicated that the embryonic blood cells loaded with BSA in vitro impaired the immune response of BSA in the hatched chickens.
BSA-Specific T Helper Cells Were Impaired After Microinjection of BSA at E3 In the second experiment, there were 2 groups of birds treated. In the BSA-injected group, BSA (dissolved in PBS) was directly injected into the blood of the E3 embryo, and in the PBS-injected group, PBS instead of BSA was injected. Eighteen and 12 hatched chickens were obtained and immunized with TNP-BSA at 3 wk of age in the PBS-injected and BSA-injected groups, respectively. The humoral immune response requires an antigen-specific interaction between T and B cells (Lanzavecchia, 1985), and TNP is a hapten that has to be coupled to a carrier protein to trigger an immune response. In the present study, we coupled TNP to BSA and evaluated the functions of BSA-specific T helper cells by detecting anti-TNP antibodies. Ten days after the first immunization, the average level of anti-TNP antibody in the PBS-injected group was significantly higher than that in BSA-injected group (P = 0.015; Figure 3). The result indicated that the helper
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Specific antibodies against different antigens were detected by ELISA. The plates were coated with BSA or TNP-OVA dissolved in carbonate-bicarbonate buffer (pH 9.6) at 2 µg/mL at 4°C overnight to detect antibodies against BSA and TNP, respectively. The plates were then washed in PBS containing 0.05% (vol/vol) Tween-20 (PBST) and blocked in 1% gelatin dissolved in carbonate-bicarbonate buffer (pH 9.6) at 37°C for 1 to 2 h. After washing, serum samples diluted (diluted to 1:1,000 or 1:5,000 for detecting anti-BSA or antiTNP, respectively) in PBST containing 0.1% gelatin were added and incubated at 37°C for 1 to 2 h. Finally, horseradish peroxidase-conjugated rabbit anti-chicken IgG (1:400 dilution, kindly provided by Zhao Jixun of China Agricultural University) was added. After incubation at 37°C for 1 h, the plates were washed with PBST, incubated with 3,3′,5,5′-tetramethylbenzidine (substrate of horseradish peroxidase) and H2O2 at 37°C for 15 min, stopped with 2 M H2SO4, and data of the OD value were recorded by microplate reader (model 550, Bio-Rad, Hercules, CA) with a 450-nm filter. In the first experiment, anti-BSA antibodies in the serum samples of different groups were detected in a 96-well plate coated with BSA; in the second experiment, anti-TNP antibodies in the serum samples of different groups were detected in another 96-well plate coated with TNP-OVA.
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Figure 2. Immunotolerance induction by loading embryonic d 3 embryo blood cells with BSA. Anti-BSA antibodies in different groups of antiserum 10 d after the first immunization were detected with ELISA, and the average values are shown in the figure. The self-BSA group, with their own blood cells, was loaded with BSA and had significantly lower levels of antibody than the quail-BSA group, whose embryos were injected with quail embryo blood cells instead. The sera of the preimmunized group shown in the figure were collected from chickens immediately before immunization.
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Figure 1. Embryo blood cells at embryonic d 3 take up fluorescein isothiocyanate (FITC)-BSA. Confocal micrographs show that embryo blood cells both in vivo (a, b) and in vitro (c, d) can take up FITC-BSA. Scale bars: 10 μm. DIC = differential interference contrast microscope.
BLOOD CELLS TAKE UP ANTIGEN TO INDUCE IMMUNOTOLERANCE
function of T cells with respect to TNP was impaired and BSA-specific T helper cells were affected by the presence of BSA in the early embryo blood.
DISCUSSION
first of which begins in chicken embryos at E6.5, the second at E12, and the third at around E18 (Jotereau and Le Douarin, 1982; Coltey et al., 1987, 1989). Each wave of progenitor cell influx lasts for 1 or 2 d (Coltey et al., 1987, 1989), and after 9 or 12 d of differentiation, the γ/δ or α/β1 T cells migrate from the thymus to the periphery (Dunon et al., 1997). Therefore, negative selection for T cells occurs only after E6.5 in the chicken embryo. Bovine serum albumin was cleared rapidly from the circulation of the embryo after injection at E3 and was hardly detectable after E6 (Wu and Li, 2009). We proposed that antigen in the early embryo would be stored for the selection of immature T and B cells later because the maturation of T and B cells begins several days after injection. The functions of DC in immune regulation have been studied extensively, and it has been shown that DC transport antigens from the periphery to the thymus and induce clonal deletion of immature T cells in the adult mouse (Bonasio et al., 2006). In addition, they can retain an intact antigen for several days before converting it to immunogenic complexes (Turley et al., 2000). Although DC have rarely been studied in chickens (Miranda de Carvalho et al., 2006) and have not yet been identified in the early chicken embryo, we propose that a similar mechanism exists at this stage of development in chickens. To identify DC, we microinjected
Figure 3. Bovine serum albumin-specific T helper cells were impaired by microinjection of BSA at embryonic d 3. Anti-trinitrophenol (TNP) antibodies in different groups of antiserum 10 d after the first immunization were detected with ELISA and the average values are shown in the figure. The BSA-injected group, whose embryos were injected with BSA, had significantly lower levels of antibody than the PBS-injected group, whose embryos were injected with PBS instead. The sera of the preimmunized group shown in the figure were collected from chickens immediately before immunization.
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The induction of immunologic tolerance through the introduction of a xenogenic antigen into chicken embryos or neonatal chickens was studied more than 50 yr ago (Wolfe et al., 1957; Stevens et al., 1958). We have demonstrated that immunologic tolerance can be induced by microinjecting antigens into chicken embryos at an early stage of development (Zhao et al., 2006). However, the precise mechanism of how this immunologic tolerance is induced has rarely been researched and remains unknown. Both B and T lymphocytes contribute greatly to the establishment of self-tolerance. Encountering a corresponding antigen tends to result in the negative selection of immature B cells and immature T cells (Healy and Goodnow, 1998). In fact, the committed B cell is first found in the para-aortic foci and yolk sac at E5, and V gene rearrangement is first observed 2 to 3 d later (Sayegh et al., 2000). Therefore, negative selection for B cells in the chicken embryo can only occur at a later developmental stage. In addition, the chicken embryonic thymus is colonized by the influx of hematopoietic progenitors in 3 discrete waves, the
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vitro renders the chicken a more valuable model system in which to study the development of the immune system, especially during its early stages. In the present study, we show an important mechanism, which may also exist in mammals, that early embryonic blood cells collect antigen to induce immunotolerance in the adult animal.
ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (30671031).
REFERENCES Anderson, M. S., E. S. Venanzi, L. Klein, Z. Chen, S. P. Berzins, S. J. Turley, and H. von Boehmer. 2002. Projection of an immunological self shadow within the thymus by the aire protein. Science 298:1395–1401. Bonasio, R., M. L. Scimone, P. Schaerli, N. Grabie, A. H. Lichtman, and U. H. von Andrian. 2006. Clonal deletion of thymocytes by circulating dendritic cells homing to the thymus. Nat. Immunol. 7:1092–1100. Bondada, S., and D. A. Robertson. 2003. Assays for B lymphocyte function. Pages 333–334 in Current Protocols in Immunology. J. E. Colijan, A. M. Kruisbeek, D. M. Marguiles, E. M. Shevach, and W. Strober, ed. John Wiley and Sons Inc., New York, NY. Chen, C., Z. Nagy, M. Z. Radic, R. R. Hardy, D. Huszar, S. A. Camper, and M. Weigert. 1995. The site and stage of anti-DNA B-cell deletion. Nature 373:252–255. Coltey, M., R. P. Bucy, C. H. Chen, J. Cihak, U. Lösch, D. Char, N. M. Le Douarin, and M. D. Cooper. 1989. Analysis of the first two waves of thymus homing stem cells and their T cell progeny in chick-quail chimeras. J. Exp. Med. 170:543–557. Coltey, M., F. V. Jotereau, and N. M. Le Douarin. 1987. Evidence for a cyclic renewal of lymphocyte precursor cells in the embryonic chick thymus. Cell Differ. 22:71–82. Dunon, D., D. Courtois, O. Vainio, A. Six, C. H. Chen, M. D. Cooper, J. P. Dangy, and B. A. Imhof. 1997. Ontogeny of the immune system: γ/δ and α/β T cells migrate from thymus to the periphery in alternating waves. J. Exp. Med. 186:977–988. Goodnow, C. C., J. G. Cyster, S. B. Hartley, S. E. Bell, M. P. Cooke, J. I. Healy, and S. Akkaraju. 1995. Self-tolerance checkpoints in B lymphocyte development. Adv. Immunol. 59:279–368. Hartley, S. B., J. Crosbie, R. Brink, A. B. Kantor, A. Basten, and C. C. Goodnow. 1991. Elimination from peripheral lymphoid tissues of self-reactive B lymphocytes recognizing membrane-bound antigens. Nature 353:765–769. Healy, J. I., and C. C. Goodnow. 1998. Positive versus negative signaling by lymphocyte antigen receptors. Annu. Rev. Immunol. 16:645–670. Jordan, M. S., A. Boesteanu, A. J. Reed, A. L. Petrone, A. E. Holenbeck, M. A. Lerman, A. Naji, and A. J. Caton. 2001. Thymic selection of CD4+CD25+ regulatory T cells induced by an agonist self-peptide. Nat. Immunol. 2:301–306. Jotereau, F. V., and N. M. Le Douarin. 1982. Demonstration of a cyclic renewal of the lymphocyte precursor cells in the quail thymus during embryonic and perinatal life. J. Immunol. 129:1869– 1877. Kyewski, B., and J. Derbinski. 2004. Self-representation in the thymus: An extended view. Nat. Rev. Immunol. 4:688–698. Kyewski, B. A., C. G. Fathman, and H. S. Kaplan. 1984. Intrathymic presentation of circulating non-major histocompatibility complex antigens. Nature 308:196–199. Lanzavecchia, A. 1985. Antigen-specific interaction between T and B cells. Nature 314:537–539. Miranda de Carvalho, C., C. Bonnefont-Rebeix, D. Rigal, and L. Chabanne. 2006. Dendritic cells in different animal species: An overview. Pathol. Biol. 54:85–93.
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FITC-BSA into the blood of embryos at E3; unexpectedly, almost all of the blood cells exhibited the ability to take up FITC-BSA (Figure 1). By removing blood cells from the early embryos and incubating them with FITC-BSA, we found that these early embryo blood cells had similar abilities to take up the antigen in vitro (Figure 1). Therefore, we loaded the blood cells with BSA in vitro and reinjected the BSA-loaded cells into the original embryos to prove whether the early embryonic blood cells collecting antigen could induce immunotolerance of the hatched chicken. To eliminate the possibility of interference from BSA leaking from any broken BSA-loaded blood cells, we used blood cells from quail embryos at the same stage in place of the chicken embryo blood cells because early quail embryonic blood cells can also take up antigen in vitro. We found that the hatched chickens in the self-BSA group had significantly weaker immune responses to BSA than those in the quail-BSA group (Figure 2), which suggests that the ability of early chicken embryo blood cells to take up protein contributes to the establishment of self-tolerance. However, there was a lower antibody level in the nontreated group, in which the embryos were normal and not treated, than in the quail-BSA group, with unknown reason, and chickens in the selfBSA group had weaker immune responses to BSA than those in the nontreated group, but the difference was not significant. This might be the result of comparative small sample size, and further research is needed. In the second experiment, we established that BSAspecific T-cell tolerance can be induced by injecting BSA at approximately E3, but whether this is accomplished through the uptake of BSA by early embryo cells can only be established by further research. And BSA-specific T helper cells seemed to not be significantly impaired by loading E3 embryo blood cells in vitro in the self-BSA group (data not shown), possibly because in the BSA-injected group, all of the embryo blood cells had the opportunity to take up BSA, whereas only 1/10 to 1/20 of the cells in the self-BSA group could have done so (in our experience, chicken embryos at approximately E3 have approximately 10 to 20 µL of blood). However, most of the early embryonic blood cells rapidly degraded the assimilated protein, and only a few cells could retain the protein for several days (data not show). Whether these cells played the primary role in the process of self-tolerance as mouse DC needs further research. In summary, we used the chicken as a model to study the establishment of self-tolerance and found that early embryo blood cells that collect antigens contribute to the development of adaptive immunity and self-tolerance. Because genetic modification is more easily conducted in the mouse than in the chicken, most current immunologic studies are carried out in the mouse. However, the ease of manipulation of chicken embryos in
BLOOD CELLS TAKE UP ANTIGEN TO INDUCE IMMUNOTOLERANCE Nemazee, D. A., and K. Buerki. 1989. Clonal deletion of B lymphocytes in a transgenic mouse bearing anti-MHC class I antibody genes. Nature 337:562–566. Palmer, E. 2003. Negative selection—Clearing out the bad apples from the T-cell repertoire. Nat. Rev. Immunol. 3:383–391. Ratcliffe, M. J. H. 2006. Antibodies, immunoglobulin genes and the bursa of Fabricius in chicken B cell development. Dev. Comp. Immunol. 30:101–118. Sakaguchi, S. 2004. Naturally arising CD4 regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annu. Rev. Immunol. 22:531–562. Sayegh, C. E., S. L. Demaries, K. A. Pike, J. E. Friedman, and M. J. H. Ratcliffe. 2000. The chicken B-cell receptor complex and its role in avian B-cell development. Immunol. Rev. 175:187–200. Stevens, K. M., H. C. Pietryk, and J. L. Ciminera. 1958. Acquired immunological tolerance to a protein antigen in chickens. Br. J. Exp. Pathol. 39:1–7. Turley, S. J., K. Inaba, W. S. Garrett, M. Ebersold, J. Unternaehrer, R. M. Steinman, and I. Mellman. 2000. Transport of peptide-
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MHC class II complexes in developing dendritic cells. Science 288:522–527 Volkmann, A, T Zal, and B. Stockinger. 1997. Antigen-presenting cells in the thymus that can negatively select MHC class II-resticted T cells recognizing a circulating self antigen. J. Immunol. 158:693–706. Wolfe, H. R., C. Tempelis, A. Mueller, and S. Reibel. 1957. Precipitin production in chickens. XVII. The effect of massive injections of bovine serum albumin at hatching on subsequent antibody production. J. Immunol. 79:147–153. Wu, G., and Z. Li. 2009. Rapid clearance of circulating protein by early chicken embryo blood cells. J. Exp. Biol. 212:2176–2182. Zhao, C., C. Song, X. Wang, Z. Li, J. Sha, H. Han, Y. Zhang, and Z. Li. 2006. Induction of immunological tolerance in chickens inoculated with xenogeneic antigens at an early stage of embryonic development. Dev. Comp. Immunol. 30:431–440.
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Key words: chicken embryo, immunological tolerance, blood cell, endocytosis 2010 Poultry Science 89:457–463 doi:10.3382/ps.2009-00437
INTRODUCTION
guchi, 2004). Thus, antigens in the microenvironment during lymphocyte development are thought to be important for the establishment of self-tolerance. Selfantigens may be expressed in the thymus (Anderson et al., 2002; Kyewski and Derbinski, 2004) and reach the thymus via the blood circulation (Kyewski et al., 1984; Volkmann et al., 1997) but can also be transported to the thymus from the periphery by dendritic cells (DC), an efficient type of antigen-presenting cell (Bonasio et al., 2006). We have previously injected BSA or casein into embryonic blood at 65 to 70 h of embryogenesis and found that a considerable number of the injected chickens become tolerant to BSA or casein, respectively (Zhao et al., 2006). However, in another study, the protein injected was found to be cleared quickly from embryonic serum (Wu and Li, 2009). Thymus anlage takes place in the chicken embryo at approximately embryonic d (E)5, and the first thymocyte progenitors colonize the thymus at E6.5 (jotereau and Le Douarin, 1982). The B cells are first found in the para-aortic foci and yolk sac at E5 and undergo rapid proliferative expansion within the bursal epithelial follicles up to the time of hatching
Self-tolerance can be established via several mechanisms. Transgenic mice, in which most newly generated B cells express an antibody against an antigen with strong cross-linking capacity that is present in the bone marrow, have no B cells specific for that antigen in the peripheral immune system (Nemazee and Buerki, 1989; Hartley et al., 1991; Chen et al., 1995; Goodnow et al., 1995). In the thymus, immature T cells that encounter cognate MHC-peptide complexes are eliminated by clonal deletion, a key process leading to central tolerance (Palmer, 2003). The presence of an agonist peptide in the thymus can also result in the differentiation of CD4+CD25+ regulatory T cells that impose tolerance in the periphery by dampening the response of conventional lymphocytes (jordan et al., 2001; Saka-
©2010 Poultry Science Association Inc. Received September 4, 2009. Accepted October 8, 2009. 1 Corresponding author: [email protected]
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ABSTRACT Earlier experimental data in our laboratory showed that introduction of an exogenous protein into early chicken embryonic blood leads to immunotolerance of hatched chicken to that protein. However, the underlying mechanism is yet unknown. In the present study, we show that the blood cells collecting circulating antigen might contribute to the establishment of immunotolerance. In this experiment, most of the chicken embryo blood cells took up injected fluorescein isothiocyanate-BSA at approximately embryonic d 3. At the same stage, 1 μL of embryo blood was taken out and incubated with BSA. After being loaded with BSA in vitro and washed, these cells were injected back into the original embryo. The BSA-specific lymphocytes
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(Sayegh et al., 2000; Ratcliffe, 2006). For these reasons, it appears that other mechanism was preferred by the early embryo than circulating the injected protein to the thymus or the bursa alone, to induce tolerance to T cells or B cells, and some early embryonic blood cells might play the primary role. In this study, we found that the early embryo blood cells have a high ability to take up injected protein in vivo and in vitro. We loaded embryo blood cells with BSA in vitro and then injected the cells back into the embryo. Most of the hatched chickens appeared to have partial tolerance to BSA. It seems to be the blood cells that carry circulating antigen in early embryo blood to thymus or bursa and induce negative selection for maturing lymphocytes.
MATERIALS AND METHODS Bovine serum albumin (A7030), ovalbumin (OVA, A5378), fluorescein isothiocyanate (FITC)-BSA (A9771), and 2,4,6-trinitrobenzenesulfonic acid (P2297) were purchased from Sigma-Aldrich (St. Louis, MO). The preparation of trinitrophenol (TNP)-BSA and TNP-OVA was modified from the methods of Bondada and Robertson (2003). Briefly, 50 mg of OVA (or 80 mg of BSA) was dissolved in 2.5 mL of 0.1 M NaHCO3 slowly added to a 317-µL solution containing 95 µL of 5% 2,4,6-trinitrobenzenesulfonic acid under stirring. The mixture was covered with aluminum foil and incubated overnight at 4°C with gentle stirring. The mixture was then dialyzed 5 times against 2 L of saline, the absorbance was measured in a spectrophotometer at optical density (OD) 280 nm and OD 340 nm, and the ratio of TNP to protein was calculated. For OVA, the ratio was 1.98, and for BSA, the ratio was 4.20.
Fertilized Eggs and Chickens Fertilized eggs of the Jingbai 938 strain, a White Leghorn (Gallus domesticus) strain purchased from the Experimental Station of the China Agricultural University, were incubated at 37.8°C in 70% RH up to hatching. The hatched chickens were bred in the laboratory. To demonstrate the uptake of antigen by embryo blood cells, on the one hand, FITC-BSA was injected into stage 18 to 19 (Hamburger and Hamilton stage, at about E3) embryonic blood via the aorta and 2 to 3 µL of blood was removed from the embryo after 2 h of injection. On the other hand, 5 to 6 µL of normal embryonic blood was diluted in 45 µL of warm RPMI1640 containing 50 µg of FITC-BSA and incubated at 37°C in 5% CO2. After 3 h, the blood cells were washed 3 times in 200 µL of cold PBS (pH 7.4) and centrifuged at 200 × g for 8 min. The washed cells were analyzed by confocal microscopy (200× magnified, Nikon D-Eclipse C1, Nikon, Tokyo, Japan).
Preparation of Serum Samples Chicken blood collected immediately before immunization (in the preimmunized group) or 10 d after the first immunization was placed at 37°C for 2 to 4 h to allow a clot to form and was then placed overnight at 4°C to allow the clot to retract. Serum was collected from the clotted blood by centrifugation at 10,000 ×
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Antigens
In the first experiment, 3 groups of birds were used: the quail-BSA group, self-BSA group, and nontreated group. For the quail-BSA group, quail embryonic blood cells were used, to eliminate the possibility of interference from BSA leaking from any broken BSA-loaded blood cells (early quail embryonic blood cells can also take up antigen in vitro, data not shown). For the selfBSA group, the chicken’s own embryonic blood cells were used, to prove whether the early embryonic blood cells collecting antigen could induce immunotolerance of the hatched chicken. One microliter of embryo blood taken from stage 17 to 19 embryo (Hamburger and Hamilton stage, at about E3 for chicken or 42 h of incubation for quail) was diluted in 50 µL of RPMI-1640 containing 500 µg of BSA and was incubated for 3 h at 37°C in 5% CO2 and then washed 4 times in 200 µL of warm PBS (pH 7.4). Washed cells were suspended in 1 to 2 µL of the last wash solution until injection. For the eggs of the quail-BSA group, we removed 1 µL of blood at 68~70 h of embryogenesis (at stage 17 to 19) and injected it with quail embryo blood cells loaded with BSA, at about 78~80 h of embryogenesis, and for the self-BSA group, 1 µL of blood was drawn at 68 to 70 h of embryogenesis and the blood cells were loaded with BSA in vitro and injected back into the embryo after washing at ~78 to 80 h of embryogenesis. Eggs continued to be incubated as before. Embryos in the nontreated group were normal and not treated. When the hatched chickens were 3 wk old, each chicken was treated with 100 µg of BSA emulsified in Freund’s complete adjuvant by dorsal hypodermic injection. In the second experiment, there were also 2 groups of birds treated. In the BSA-injected group, embryos were injected with 10 µg of BSA dissolved in 1 µL of PBS at 68 to 70 h of embryogenesis; in the PBS-injected group, PBS was injected instead of BSA. The humoral immune response requires an antigen-specific interaction between T and B cells (Lanzavecchia, 1985), and TNP is a hapten that has to be coupled to a carrier protein to trigger an immune response. When the hatched chickens were 3 wk old, they were treated with 50 µg of TNP-BSA emulsified in Freund’s complete adjuvant by dorsal hypodermic injection to evaluate the BSA-specific helper T cells by detecting anti-TNP antibodies. In the above 2 experiments, both the drawing of blood from the embryo and injection into the embryo were via the aorta.
BLOOD CELLS TAKE UP ANTIGEN TO INDUCE IMMUNOTOLERANCE
g for 10 min. All serum samples were stored at −20°C until analysis.
ELISA
Statistical Analysis The data were analyzed by SPSS 13.0 (SPSS Inc., Chicago, IL) with the 1-sample K-S program, and all of the data in each group were normally distributed (P > 0.3, data not shown). The comparison of each 2 groups was accomplished with the independent sample t-test. We considered comparisons to be statistically distinct at P < 0.05.
RESULTS Early Embryo Blood Cells Take Up Injected Proteins In Vivo and In Vitro We injected 10 µg of FITC-BSA into chicken embryo blood via the aorta at about E3. Two hours postinjection, most of the blood cells had taken up the FITCBSA (Figure 1a and 1b). The embryo blood cells also took up FITC-BSA in vitro (Figure 1c and 1d), and there was no difference between these cells and the cells treated in vivo.
Embryo Blood Cells Collecting Antigen to Establish Self-Tolerance Three groups of eggs were used in the first experiment: quail-BSA, self-BSA, and nontreated. The quailBSA group was used as a negative control for tolerance, and the self-BSA group was used to show that early embryo cells take up BSA and induce immune tolerance to BSA in the hatched chicken. The treatment of the eggs in these 2 groups was similar, except that in the quail-BSA group, the chicken embryo’s own blood cells were replaced with quail embryo blood cells extracted at the same stage of development and loaded with BSA (quail embryo blood cells have a similar ability to take up peptides in vitro; data not shown). The quail-BSA group was used to evaluate the effect of peptides derived from BSA and leaked from loaded cells. Because quail blood cells have different MHC with chicken, they cannot present peptides to chicken T cells in the process of selection in thymus, so we choose early quail embryonic cells instead of chicken blood cells in the quail-BSA group. Seven, 12, and 17 hatched chickens in quail-BSA, self-BSA, and nontreated, respectively, were obtained and immunized with BSA at 3 wk of age. Ten days after the first immunization, the antibody level of anti-BSA in the self-BSA group was 0.498 ± 0.336 (mean ± SD) and significantly lower than 1.123 ± 0.490 (mean ± SD) that was the level in the quail-BSA group (P = 0.0017) (Figure 2), but not significantly lower than 0.791 ± 0.514 (mean ± SD) that was the level in the nontreated group (P = 0.0575), possibly as a result of small sample size. The above result indicated that the embryonic blood cells loaded with BSA in vitro impaired the immune response of BSA in the hatched chickens.
BSA-Specific T Helper Cells Were Impaired After Microinjection of BSA at E3 In the second experiment, there were 2 groups of birds treated. In the BSA-injected group, BSA (dissolved in PBS) was directly injected into the blood of the E3 embryo, and in the PBS-injected group, PBS instead of BSA was injected. Eighteen and 12 hatched chickens were obtained and immunized with TNP-BSA at 3 wk of age in the PBS-injected and BSA-injected groups, respectively. The humoral immune response requires an antigen-specific interaction between T and B cells (Lanzavecchia, 1985), and TNP is a hapten that has to be coupled to a carrier protein to trigger an immune response. In the present study, we coupled TNP to BSA and evaluated the functions of BSA-specific T helper cells by detecting anti-TNP antibodies. Ten days after the first immunization, the average level of anti-TNP antibody in the PBS-injected group was significantly higher than that in BSA-injected group (P = 0.015; Figure 3). The result indicated that the helper
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Specific antibodies against different antigens were detected by ELISA. The plates were coated with BSA or TNP-OVA dissolved in carbonate-bicarbonate buffer (pH 9.6) at 2 µg/mL at 4°C overnight to detect antibodies against BSA and TNP, respectively. The plates were then washed in PBS containing 0.05% (vol/vol) Tween-20 (PBST) and blocked in 1% gelatin dissolved in carbonate-bicarbonate buffer (pH 9.6) at 37°C for 1 to 2 h. After washing, serum samples diluted (diluted to 1:1,000 or 1:5,000 for detecting anti-BSA or antiTNP, respectively) in PBST containing 0.1% gelatin were added and incubated at 37°C for 1 to 2 h. Finally, horseradish peroxidase-conjugated rabbit anti-chicken IgG (1:400 dilution, kindly provided by Zhao Jixun of China Agricultural University) was added. After incubation at 37°C for 1 h, the plates were washed with PBST, incubated with 3,3′,5,5′-tetramethylbenzidine (substrate of horseradish peroxidase) and H2O2 at 37°C for 15 min, stopped with 2 M H2SO4, and data of the OD value were recorded by microplate reader (model 550, Bio-Rad, Hercules, CA) with a 450-nm filter. In the first experiment, anti-BSA antibodies in the serum samples of different groups were detected in a 96-well plate coated with BSA; in the second experiment, anti-TNP antibodies in the serum samples of different groups were detected in another 96-well plate coated with TNP-OVA.
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Figure 2. Immunotolerance induction by loading embryonic d 3 embryo blood cells with BSA. Anti-BSA antibodies in different groups of antiserum 10 d after the first immunization were detected with ELISA, and the average values are shown in the figure. The self-BSA group, with their own blood cells, was loaded with BSA and had significantly lower levels of antibody than the quail-BSA group, whose embryos were injected with quail embryo blood cells instead. The sera of the preimmunized group shown in the figure were collected from chickens immediately before immunization.
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Figure 1. Embryo blood cells at embryonic d 3 take up fluorescein isothiocyanate (FITC)-BSA. Confocal micrographs show that embryo blood cells both in vivo (a, b) and in vitro (c, d) can take up FITC-BSA. Scale bars: 10 μm. DIC = differential interference contrast microscope.
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function of T cells with respect to TNP was impaired and BSA-specific T helper cells were affected by the presence of BSA in the early embryo blood.
DISCUSSION
first of which begins in chicken embryos at E6.5, the second at E12, and the third at around E18 (Jotereau and Le Douarin, 1982; Coltey et al., 1987, 1989). Each wave of progenitor cell influx lasts for 1 or 2 d (Coltey et al., 1987, 1989), and after 9 or 12 d of differentiation, the γ/δ or α/β1 T cells migrate from the thymus to the periphery (Dunon et al., 1997). Therefore, negative selection for T cells occurs only after E6.5 in the chicken embryo. Bovine serum albumin was cleared rapidly from the circulation of the embryo after injection at E3 and was hardly detectable after E6 (Wu and Li, 2009). We proposed that antigen in the early embryo would be stored for the selection of immature T and B cells later because the maturation of T and B cells begins several days after injection. The functions of DC in immune regulation have been studied extensively, and it has been shown that DC transport antigens from the periphery to the thymus and induce clonal deletion of immature T cells in the adult mouse (Bonasio et al., 2006). In addition, they can retain an intact antigen for several days before converting it to immunogenic complexes (Turley et al., 2000). Although DC have rarely been studied in chickens (Miranda de Carvalho et al., 2006) and have not yet been identified in the early chicken embryo, we propose that a similar mechanism exists at this stage of development in chickens. To identify DC, we microinjected
Figure 3. Bovine serum albumin-specific T helper cells were impaired by microinjection of BSA at embryonic d 3. Anti-trinitrophenol (TNP) antibodies in different groups of antiserum 10 d after the first immunization were detected with ELISA and the average values are shown in the figure. The BSA-injected group, whose embryos were injected with BSA, had significantly lower levels of antibody than the PBS-injected group, whose embryos were injected with PBS instead. The sera of the preimmunized group shown in the figure were collected from chickens immediately before immunization.
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The induction of immunologic tolerance through the introduction of a xenogenic antigen into chicken embryos or neonatal chickens was studied more than 50 yr ago (Wolfe et al., 1957; Stevens et al., 1958). We have demonstrated that immunologic tolerance can be induced by microinjecting antigens into chicken embryos at an early stage of development (Zhao et al., 2006). However, the precise mechanism of how this immunologic tolerance is induced has rarely been researched and remains unknown. Both B and T lymphocytes contribute greatly to the establishment of self-tolerance. Encountering a corresponding antigen tends to result in the negative selection of immature B cells and immature T cells (Healy and Goodnow, 1998). In fact, the committed B cell is first found in the para-aortic foci and yolk sac at E5, and V gene rearrangement is first observed 2 to 3 d later (Sayegh et al., 2000). Therefore, negative selection for B cells in the chicken embryo can only occur at a later developmental stage. In addition, the chicken embryonic thymus is colonized by the influx of hematopoietic progenitors in 3 discrete waves, the
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vitro renders the chicken a more valuable model system in which to study the development of the immune system, especially during its early stages. In the present study, we show an important mechanism, which may also exist in mammals, that early embryonic blood cells collect antigen to induce immunotolerance in the adult animal.
ACKNOWLEDGMENTS This work was financially supported by the Natural Science Foundation of China (30671031).
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FITC-BSA into the blood of embryos at E3; unexpectedly, almost all of the blood cells exhibited the ability to take up FITC-BSA (Figure 1). By removing blood cells from the early embryos and incubating them with FITC-BSA, we found that these early embryo blood cells had similar abilities to take up the antigen in vitro (Figure 1). Therefore, we loaded the blood cells with BSA in vitro and reinjected the BSA-loaded cells into the original embryos to prove whether the early embryonic blood cells collecting antigen could induce immunotolerance of the hatched chicken. To eliminate the possibility of interference from BSA leaking from any broken BSA-loaded blood cells, we used blood cells from quail embryos at the same stage in place of the chicken embryo blood cells because early quail embryonic blood cells can also take up antigen in vitro. We found that the hatched chickens in the self-BSA group had significantly weaker immune responses to BSA than those in the quail-BSA group (Figure 2), which suggests that the ability of early chicken embryo blood cells to take up protein contributes to the establishment of self-tolerance. However, there was a lower antibody level in the nontreated group, in which the embryos were normal and not treated, than in the quail-BSA group, with unknown reason, and chickens in the selfBSA group had weaker immune responses to BSA than those in the nontreated group, but the difference was not significant. This might be the result of comparative small sample size, and further research is needed. In the second experiment, we established that BSAspecific T-cell tolerance can be induced by injecting BSA at approximately E3, but whether this is accomplished through the uptake of BSA by early embryo cells can only be established by further research. And BSA-specific T helper cells seemed to not be significantly impaired by loading E3 embryo blood cells in vitro in the self-BSA group (data not shown), possibly because in the BSA-injected group, all of the embryo blood cells had the opportunity to take up BSA, whereas only 1/10 to 1/20 of the cells in the self-BSA group could have done so (in our experience, chicken embryos at approximately E3 have approximately 10 to 20 µL of blood). However, most of the early embryonic blood cells rapidly degraded the assimilated protein, and only a few cells could retain the protein for several days (data not show). Whether these cells played the primary role in the process of self-tolerance as mouse DC needs further research. In summary, we used the chicken as a model to study the establishment of self-tolerance and found that early embryo blood cells that collect antigens contribute to the development of adaptive immunity and self-tolerance. Because genetic modification is more easily conducted in the mouse than in the chicken, most current immunologic studies are carried out in the mouse. However, the ease of manipulation of chicken embryos in
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MHC class II complexes in developing dendritic cells. Science 288:522–527 Volkmann, A, T Zal, and B. Stockinger. 1997. Antigen-presenting cells in the thymus that can negatively select MHC class II-resticted T cells recognizing a circulating self antigen. J. Immunol. 158:693–706. Wolfe, H. R., C. Tempelis, A. Mueller, and S. Reibel. 1957. Precipitin production in chickens. XVII. The effect of massive injections of bovine serum albumin at hatching on subsequent antibody production. J. Immunol. 79:147–153. Wu, G., and Z. Li. 2009. Rapid clearance of circulating protein by early chicken embryo blood cells. J. Exp. Biol. 212:2176–2182. Zhao, C., C. Song, X. Wang, Z. Li, J. Sha, H. Han, Y. Zhang, and Z. Li. 2006. Induction of immunological tolerance in chickens inoculated with xenogeneic antigens at an early stage of embryonic development. Dev. Comp. Immunol. 30:431–440.
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