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doi:10.2141/ jpsa.011078
Copyright Ⓒ 2012, Japan Poultry Science Association.
Effects of Exogenous Proteins Injection into the Bursa of
Fabricius on Humoral Immunity in Neonatal Chickens
Fang Yuan, Guojin Wu, Junshuang Gao, Xiaoyan Tang and Zandong Li
State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology,
College of Biological Science, China Agricultural University, Beijing, 100193, China
Early researches have shown that the bursa of Fabricius is a critical organ for development of B lymphocytes in
birds. Exogenous proteins from the gut lumen or the environment are taken up by the follicle-associated epithelium
and enter into the bursal follicle. Not all of the B lymphocytes in the bursal follicle mature and emigrate to the
periphery and antigen-dependent selection for B lymphocytes may occur in the bursa. However, the actual impact of
antigens in the bursa on B cells is not clear. In this study, dinitrophenyl (DNP) or 2,4,6-trinitrophenyl (TNP) coupled
bovine serum albumin (BSA) was injected into the bursa of one-day-old chickens, a time when most B lymphocytes in
the bursal follicles begun to emigrate from the medulla to the cortex. Immunohistochemical analysis showed that
DNP-BSA was only distributed in the bursal medulla. By injecting TNP-BSA into the bursa of one-day-old chickens,
and immunizing the chickens with TNP-HSA 3 weeks later, we found that injection of TNP-BSA increased anti-TNP
titers in the sera of chickens after immunization. Taken together, the results suggest that the bursa is the site of B cellantigen interaction and is capable of causing Ag-specific B cell emigration and increasing an antigen-specific immune
response at the B cell level.
Key words: B cell, bursa of Fabricius, chicken, exogenous protein, humoral immunity
J. Poult. Sci., 49: 124-129, 2012
Introduction
The bursa of Fabricius is the primary site for B cell
development in chickens (Ratcliffe and Paramithiotis, 1990;
Sayegh and Ratcliffe, 2000; Pike and Ratcliffe, 2002; Pike et
al., 2004). During bursal development, colonization of lymphoid precursors occurs between 7.5 embryonic development
day (E7. 5) and E14, and following this migration, B cell
surface immunoglobulin (SIg) begins to expand and as a
result, by E19 90% of bursal cells are SIg＋ (Lydyard et al.,
1976). The medullary anlage of bursal follicle emerges at
E11-E12 and is soon followed by formation of the follicleassociated epithelium (FAE) (E14-E15), with the first cortical cell appearing around the time of hatching (Bockman
and Cooper, 1973). The bursa is a gut-associated lymphoid
organ and a major trapping site for environmental antigens
(Ags) from the gut (Sorvari et al., 1975; Ekino et al., 1985;
Ekino et al., 1995). After hatching, environmental Ags are
Received: August 1, 2011, Accepted: January 10, 2012
Released Online Advance Publication: February 25, 2012
Correspondence: Prof. Z. Li, State Key Laboratory of Agrobiotechnology,
Department of Biochemistry and Molecular Biology, College of Biological
Science, China Agricultural University, No. 2 Yuanmingyuan West Road,
Beijing, 100193, China.
(E-mail: [email protected])
transported along the bursal duct from the intestinal lumen to
the bursal lumen, across the FAE, and into the medulla of the
bursal follicle (Sorvari and Sorvari, 1977; Toivanen et al.,
1987). Starting around the time of hatching, B cells in the
epithelial buds begin to segregate to form the outer cortex,
which surrounds the inner medulla. Most bursal cell proliferation occurs within the cortex (Reynolds, 1987; Ekino,
1993; Paramithiotis and Ratcliffe, 1994; Yasuda et al., 2002)
and mature B cells start to immigrate to the periphery (Paramithiotis and Ratcliffe, 1993; Ratcliffe and Jacobsen,
1994). Significant levels of B cell death occurs in the bursa,
especially after hatching (Motyka and Reynolds, 1991;
Paramithiotis et al., 1995). When the bursal duct is ligated,
the normal progression of post-hatching B cell development
is compromised (Sayegh and Ratcliffe, 2000). The medullary portion of bursal follicles readily takes up small inert
particles after oral or intracloacal administration to chickens
(Schaffner et al., 1974). Dinitrophenyl-bovine serum albumin (DNP-BSA) applied to the cloacal lips of 3-week-old
chickens was rapidly transported to the bursal lumen by the
anal sucking reflex, and resided in the medulla for up to 5
days (Sayegh et al., 2000). In addition, the complete Ag
recognition unit is not present in the pro- or pre-B cell receptor (BCR) (Fuentes-Panana et al., 2004). Bursal B cells
expressing SIg＋ represent a second checkpoint during chick-
Yuan et al.: The Immunological Function of Cloaca-derived Proteins
en B-cell development (Pike et al., 2004). Ag-dependent
positive selection results in the development and movement
of B cells from the medulla to the cortex (Arakawa et al.,
2002). Since the bursal lumen is connected to the gut lumen
by the bursal duct, this provides a mechanism by which
bursal B cells develop in response to materials derived from
the gut after hatching (Ratcliffe, 2006). Brucella bacteria
and sheep red blood cells (SRBCs) have been applied to
bursa at different times for immunological study (Sorvari et
al., 1975; Sorvari and Sorvari, 1977; Ekino et al., 1985).
However, there is little information available in the literature
on the effect of soluble Ags in the bursa on B cells at the
level of a single specific B cell clone and humoral immunity.
In this study, to study the effect of Ags in the bursa on B
cells, we injected Ags into the bursa of one-day-old chickens
and examined its effect on humoral immunity at the B cell
level.
Materials and Methods
Fertilized Eggs and Chickens
Fertilized eggs from the White Leghorn (Gallus domesticus) strain Jingbai 938, were purchased from the Experimental Station of the China Agricultural University, and
incubated at 37.6℃ in a forced air incubator at 53-63% relative humidity with 90°tilting once every hour for up to 1
day after hatching (P-008B Biotype, Showa Furanki, Saitama, Japan).
Preparation of Antigens
BSA, ovalbumin (OVA), human serum albumin (HSA),
DNP-BSA and TNP were purchased from Sigma-Aldrich (St
Louis, MO). TNP-BSA, TNP-OVA, and TNP-HSA were
prepared according to the method of Bondada and Robertson
(2003), but with slight modifications as described by Wu et
al. (2010). Briefly, 50 mg of OVA (or 80 mg of BSA or
HSA) was dissolved in 2.5 ml of 0.1 M NaHCO3 buffer (pH
9.5) and slowly added to 222 μl of sterile deionized water
containing 95 μl of 5% TNP with stirring. The mixture was
then covered with aluminum foil and incubated overnight at
4℃ with gentle stirring. The mixture was then dialyzed five
times against 2 l of saline and the absorbance measured in a
spectrophotometer at 280 nm and 340 nm. The ratio of TNP
to protein was then calculated. The ratios were: 6.75 for
OVA, 3.54 for BSA, and 4.69 for HSA.
Foreign Antigens Injection into the Cloacal Lips
DNP-BSA or TNP-BSA (600 μg per chick) was applied to
the cloacal lips of one- day-old chickens. The bursa of Fabricius were embedded in paraffin after injection of DNPBSA after 1 h, 6 h, 1 d, 2 d, 3 d, 4 d, 8 d and 9 d respectively
and DNP-BSA compartmentalization within the bursa was
examined in paraffin-embedded tissue sections by immunohistochemistry. We measured the immune response to TNPBSA injected in one-day-old chickens by immunizing with
TNP-HSA plus HSA after 3 weeks. Because TNP is a hapten and can be recognized by a single specific B cell clone,
we immunized chickens with TNP-HSA instead of TNPBSA when the chicken was 3 weeks old. In this way, by
using different carrier proteins (BSA and HSA) for TNP
125
when chickens were 1 day or 3 weeks old, we could evaluate
the effect of TNP on anti-TNP B cell clones. We used HSA
as a reference for the immune response in this study. The
levels of anti-TNP and anti-HSA antibodies were determined
by ELISA.
Immunohistochemistry
The distribution of DNP-BSA in the bursa of Fabricius
was detected with a polyclonal rabbit anti-DNP antibody followed with a goat anti-rabbit-horseradish peroxidase conjugate and detected using the ABC kit (Vector Labs, Burlingame, CA) followed by counter-staining with hematoxylin.
ELISA
Twenty-seven 1-day-old chickens were used in this experiment. These animals were divided into two groups. In
the experimental group, 20 chickens had TNP-BSA (600 μg
per chicken) injected into the bursa of Fabricius. In the control group, seven chickens remained untreated. After 3
weeks, both groups of chickens were immunized with TNPHSA and HSA, and the levels of anti-TNP and anti-HSA
specific antibodies in the sera were determined by ELISA
according to the method of Wu et al. (2010).
We obtained peripheral blood by venipuncture on the
eighth and the tenth day after the first immunization. Sera
were separated by coagulation at 37℃ for 1 h followed by
centrifuging at 4℃ at 10,000 g for 10 min. We then used
ELISA to detect TNP or HSA specific antibodies. Briefly,
96 well plates (Sigma-Aldrich) were coated with 2 μg/ml
TNP-OVA or HSA dissolved in carbonate-bicarbonate buffer
(pH 9.6) and incubated at 4℃ overnight. The plates were
then washed with phosphate buffer (pH 7.4) containing 0.05
% Tween-20 (PBST), and the remaining binding sites
blocked with 1% gelatin dissolved in carbonate-bicarbonate
buffer (pH 9.6) for 1 h at 37℃. The plates were washed
again with PBST. Serum was diluted in 0.1% gelatin/PBST
(pH 7.4) and incubated at 37℃ for 1.5 h. The plates were
washed again, and incubated with secondary antibodies goat
anti-chicken IgG and IgM labeled peroxides (Southern Biotechnology Associates, Birmingham, AL) at 37℃ for 1 h. 3,
3′
, 5, 5′
-tetramethylbenzidine (TMB) was used for color
development. The levels of anti-TNP or anti-HSA in the sera
were calculated from the absorbance at 450 nm as measured
by microplate reader (Model 550, Bio-Rad, Hercules, CA).
All animal care and handling procedures were performed
in accordance with the guidelines of the Animal Care and
approved by the Committee of the China Agricultural University.
Statistical Analysis
Differences in the levels of anti-TNP and anti-HSA antibodies in the sera of the experimental and control groups
were analyzed using SPSS software (v13. 0; SPSS Inc.,
Chicago, IL) and an independent sample t-test. A P value of
＜0.05 was considered significantly different.
Results
Distribution of Antigens within the Bursa of Fabricius
The reverse sucking reflex of the bursal lumen rapidly
126
Journal of Poultry Science, 49 (2)
Fig. 1. The distribution of DNP-BSA injected into the
bursa of chickens. DNP-BSA distributed in the bursal
medulla 1 h (a), 6 h (b), 1 d (c), 2 d (d), 3 d (e), 4 d (f), 8 d
(g) and 9 d (h) after DNP-BSA deposition on the cloacal lips
at one-day-old. DNP-BSA was detected with a polyclonal
rabbit anti-DNP antibody followed with a goat anti-rabbit
horseradish peroxidase conjugate and detected using the
ABC kit (Vector Labs) followed by counter-staining with
hematoxylin. m: medulla; c: cortex; FAE: follicle-associated
epithelium; brown color indicate positive position, indicated
by black arrows. Magnification: ×400. Bars indicate 100
μm.
transported DNP-BSA applied to the cloacal lips across the
FAE to the bursal follicles. The DNP-BSA was confined to
the medulla from 1 h to 8 d (Fig. 1). We detected low levels
of Ag in the medulla at 1 h and high levels of Ag were found
in the medulla of bursal follicle at 6 h (Fig. 1b), which
persisted up to the 8th day after injection (Fig. 1g). We did
not detected Ags on the 9th day, nor in the bursal cortex.
Foreign Antigens Injected into the Bursa of One-day-old
Chickens Increased the Specific Humoral Immune Response
We injected TNP-BSA (600 μg per chicken) into chicken
cloacal lips of the bursa of Fabricius. After 3 weeks these
chickens, and non-treated controls, were immunized with
TNP-HSA plus HSA, and the levels of anti-TNP and antiHSA specific antibodies in the sera measured by ELISA.
The numbers of chickens left (three or four chickens died
after immunization) are shown in Fig.2, in which P＜0.05
indicates a significant difference between the experimental
group and control group (t-test). The data are expressed as
the mean±SE. Eight days after the first immunization, the
level of anti-TNP IgG in the experimental group was significantly higher than that in the control group (1.173±0.149 vs.
0.623±0.163, Fig. 2a). However, the levels of anti-HSA
IgG were not significantly different (0.827±0.107 vs. 0.475
±0.153, Fig. 2b). Ten days after the first immunization, the
anti-TNP IgG in the experimental group was significantly
higher than that in the control group (0.658±0.108 vs. 0.328
±0.071, Fig. 2c), but the level of anti-HSA IgG was not
significantly different (0.700±0.093 vs. 0.5±0.125, Fig.
2d). The results for anti-TNP and anti-HSA IgM antibodies
were similar to those of the anti IgG antibodies (Fig. 2).
Eight days after the first immunization, the level of anti-TNP
IgM in the experimental group was significantly higher than
that in the control group (1.310±0.123 vs. 0.839±0.147,
Fig. 2e). However, the levels of anti-HSA IgM were not
significantly different (1.432±0.126 vs. 1.128±0.218, Fig.
2f). Ten days after the first immunization, the anti-TNP IgM
levels in the experimental group were significantly higher
than those in the control group (1.428±0.103 vs. 1.019±
153, Fig. 2g), but the levels of anti-HSA IgM were not
significantly different (1.130±0.091 vs. 0.966±0.134, Fig.
2h). The results above suggest that injection of TNP-BSA
into the bursa 1 day after hatching increased the immune
response to TNP in 3-week-old chickens and at the same
time, there was no influence on the response to HSA, indicating that specific anti-TNP B clones not only emigrated,
but also were able to elicit an increased immune response.
Discussion
Here, we study the effect of soluble Ag injected into the
bursal on B cells and humoral immunity at the level of a
single B cell clone in chickens at one-day-old, a time when
the immune system is not completely developed and most B
cells begin to emigrate towards the periphery. Therefore, we
designed experiments based on the anticipation that the
soluble Ag may induce immune tolerance in the bursa. First,
we injected foreign Ag DNP-BSA to detect the distribution
of the DNP-BSA in the bursa. Second, we injected TNPBSA into the bursa of Fabricius to detect a single B cell clone
response to TNP. In the first experiment, we used DNPBSA substitute TNP-BSA to detect the distribution of the Ag.
TNP is a hapten that can be recognized by a single specific B
cell clone, and TNP-coupled proteins are usually used to
study the function of B cells; however, due to the lack of a
commercial anti-TNP antibody, TNP was hard to detect.
Therefore, we used DNP, a substance that has a very similar
molecular structure and size to TNP, and for which a
commercial antibody is available. The results show that the
FAE was able to take up the DNP-BSA and locate it in the
follicular medulla, where it persisted up to 8 days after
Yuan et al.: The Immunological Function of Cloaca-derived Proteins
Fig. 2. Twenty chickens were injected with TNP-BSA in the bursa at one-day-old.
Serum anti-TNP and anti-HSA antibodies were detected by ELISA on the 8th and 10th
day after the first immunization. Eight days after the first immunization, anti-TNP IgG
bound at a dilution of 1:400 (a), anti-HSA IgG bound at a dilution of 1:400 (b), anti-TNP
IgM bound at a dilution of 1:400 (e) and anti-HSA IgM bound at a dilution of 1:80 (f).
Ten days after the first immunization, anti-TNP IgG bound at a dilution of 1:2000 (c),
anti-HSA IgG bound at a dilution of 1:400 (d), anti-TNP IgM bound at a dilution of 1:400
(g) and anti-HSA IgM bound at a dilution of 1:80 (h). Bars represent the average optical
density at 450 nm and error bars represent the standard errors of the mean (SE). P＜0.05
indicates a statistically significant difference between experimental group and control
group (t-test), and the letter n indicates the numbers of chickens in each group.
127
128
Journal of Poultry Science, 49 (2)
injection. This finding is consistent with results of Sayegh et
al. (2000) who showed that foreign Ags from the gut lumen
located in the medulla of the bursa, implying that the active
site of locally derived exogenous proteins was the medulla
of bursal follicles. In the second experiment, to detect a single B cell clone response to TNP, we injected TNP-BSA into
the bursa of Fabricius of one-day-old chickens, and the
chickens were immunized with TNP-HSA plus HSA 3 weeks
later. Interestingly, we observed a statistical difference in
anti-TNP antibody titers but not in anti-HSA antibody titers
between inoculated and control chickens. This may be because (a) the dose of the Ag is not enough to induce any B
cell tolerance, (b) the follicular medulla of the bursa is not
the location of B-cell negative selection, or (c) the time we
choose is not suitable for negative selection. According to
the reasons above, it was difficult to induce tolerance at the B
cell level and further studies are needed to address this problem.The results from this study are similar to those reported
by Arakawa et al., (2002) who indicated Ag-dependent
positive selection results in the development and movement
of B cells from the medulla to the cortex. Interestingly, although there was no statistical difference in anti-HSA titers,
the experimental group titers were slightly higher than the
control group, and the reason might be that carrier BSA and
HSA share some similar epitopes. To prove that the statistical difference of anti-TNP titers was not because of carrier
protein BSA, two groups of one-day-old chickens were injected with an equal dose of TNP-BSA or BSA in the bursa.
After 3 weeks, all chickens were immunized with TNP-HSA
plus HSA. The result indicated that anti-TNP titers in TNPBSA was significantly higher than that in the BSA group
(data not show). Moreover, as CD4＋ T cells are not distributed in the medulla (Yasuda et al., 2002), we can confirm
that specific Ags in the bursal medulla promoted B cell
emigration, and not infected T cells in the cortex. The immunohistochemistry and ELISA analysis suggested that Ags
might drive specific B cell emigration, which may provide a
theoretical basis for immunization of neonatal chickens with
the pathogenic epitopes of bacteria and viruses in the bursa,
allowing an efficient immune response when exposed to such
Ags in the future. The present study further supports the
finding of Sorvari et al. (1975) who reported that chickens
can be immunized by applying Brucella bacteria to the bursa.
Previous reports have shown that intra-follicular redistribution of bursal B cells represents a second checkpoint during
chicken B cell development (Pike et al., 2004). Ag-dependent positive selection results in the migration of Ag-specific
B cells from the medulla to the cortex (Pike and Ratcliffe,
2002), and surface IgG＋ cells are localized to the medulla of
bursal follicles after hatching (Ekino et al., 1995). Therefore, we may speculate that Ags in the medulla of bursa
follicle may bind their specific SIg on B cells, and promote
specific B cell emigration to the cortex for proliferation and
then to the peripheral immune organ, thus enhancing the antigen specific humoral immune response upon a second exposure to the same Ag.
Furthermore, two distinct populations of peripheral B cells
have been defined as major populations of short-lived B cells
comprising a diverse repertoire that have not encountered the
foreign Ag in the bursa, and minor populations of B cells
exposed to environmental Ag within the bursa, acquiring a
longer-lived state (Sorvari et al., 1975). B cell populations
clonally expanded in the bursa microenvironment and positively selected by environmental Ags may acquire the longerlived state and respond to environmental Ags in the periphery
after emigrating from the bursa (Arakawa et al., 2002). We
deduce that the Ag-dependent B cell immigrated to the
periphery after interacting with specific Ags in the bursa and
became the longer-lived B cell state, with an ability to mount
a fast immune respond upon a second exposure to the Ag.
In the present study, we show that injection of soluble T
cell-dependent hapten increased the immune response to
hapten at the level of the single B cell clone upon exposure to
Ag within 3 weeks. It is well known that exogenous proteins
from the gut lumen or the environment are taken up by the
FAE and enter into the bursal follicle. This study shows that
the FAE takes up exogenous proteins into the medulla of the
bursal follicle, providing a theoretical basis for intestinal immunity and furthering our understanding of bursal immunization.
Acknowledgments
This work was supported by a grant from the Natural
Science Foundation of China (30671031) and the State 863
High-technology R&D Project of China (2007AA100504).
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