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,§
*
Department of Clinical Pathology, Nihon University School of Dentistry at Matsudo, Chiba, Japan;
Biogen Institute, Cambridge, MA 02142;
Immunobiology Vaccine Center and Departments of Oral Biology and Microbiology, University of Alabama Medical Center, Birmingham, AL 35294;
§
Department of Mucosal Immunology, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; and
¶
F. Hoffmann La-Roche, Basel, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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double knockout (TNF/LT--/-) mice, which lack
both Peyer’s patches and MLN, were orally immunized with OVA plus
cholera toxin, neither mucosal IgA nor serum IgG anti-OVA Abs were
induced. On the other hand, LTßR-Ig- and TNF receptor 55-Ig-treated
normal adult mice elicited OVA- and cholera toxin B subunit-specific
mucosal IgA responses, indicating that both LT-ß and TNF/LT-
pathways do not contribute for class switching for IgA Ab responses.
These results show that the MLN plays a more important role than had
been appreciated until now for the induction of both mucosal and
systemic Ab responses after oral immunization. Further, organized
Peyer’s patches are not a strict requirement for induction of mucosal
IgA Ab responses in the gastrointestinal tract. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Recently, several studies have shown that TNF and lymphotoxin (LT)-
both play important roles for development of spleen and peripheral
lymph nodes (LN) including Peyer’s patches (9, 10, 11, 12, 13, 14). For
example, targeted knockout of LT- or TNF/LT- disrupts the
development of LN and Peyer’s patches and results in an altered
splenic architecture characterized by the absence of distinct T and B
cell areas (15, 16, 17). Further, mice that express a soluble
LT-ß receptor (LTßR) and human IgG1 chimeric protein result in
anatomic abnormalities affecting splenic architecture
(18). Recently, an interesting study showed that mice
heterozygous for both LT- and LT-ß, but not either LT- or
LT-ß alone, specifically lack only Peyer’s patches
(19), indicating that there is a gene dosage effect for
Peyer’s patch development.
Studies with LTßR-Ig fusion protein (LTßR-Ig) have shown that
administration of LTßR-Ig to mice during gestation disrupted Peyer’s
patches and LN development in the progeny (20, 21, 22). Thus,
to investigate the role of Peyer’s patches in the induction of mucosal
IgA Ab responses in vivo, a novel strategy of in utero treatment with
LTßR-Ig fusion protein, which physiologically prevents Peyer’s patch
development without affecting associated mesenteric lymph nodes (MLN),
was used in this study. By varying the gestational day of LTßR-Ig
injection, it was shown that the genesis of LN and Peyer’s patches is
sequential (20, 21). LTßR-Ig treatment on gestational
days 14 and 17 generates mice that lack Peyer’s patches but retain
MLN. The novel results obtained in this study show that oral
immunization of Peyer’s patch null (PP null) mice with OVA and the
mucosal adjuvant cholera toxin (CT) resulted in OVA-specific mucosal
IgA responses, whereas TNF and LT- double knockout
(TNF/LT--/-) mice, which lack both Peyer’s
patches and MLN, fail to respond to oral OVA with mucosal S-IgA Abs in
the gastrointestinal tract.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Timed pregnant C57BL/6 mice were purchased from The Jackson
Laboratory Animal Resources Center (Bar Harbor, ME). TNF and LT-
double knockout (TNF/LT--/-; 129 x
C57BL/6) mice were produced as described previously (15).
These mice were maintained and bred in the experimental facility under
pathogen-free conditions in the Immunobiology Vaccine Center at
University of Alabama (Birmingham, AL) and the Research Institute for
Microbial Diseases at Osaka University (Osaka, Japan).
Fusion proteins
Proteins comprised of the extracellular domain of either murine LTßR, TNF receptor 55 (TNF-R55) or LFA-3 (which does not bind murine CD2) fused to the hinge, CH2, and CH3 domains of human IgG1 (LTßR-Ig, TNF-R55-Ig, and LFA-3-Ig, respectively) were used in our studies as described elsewhere (20, 23, 24).
Treatment protocols
Timed pregnant C57BL/6 mice were injected i.v. with 200 µg fusion protein on gestational days 14 and 17 with fusion protein of LTßR-Ig or LFA-3-Ig (see below) as described previously (20, 21). In some experiments, 6-wk-old C57BL/6 mice were given 100 µg LTßR-Ig, TNF-R55-Ig or control IgG per injection twice weekly beginning 3 days before the first immunization until 3 days after the last immunization.
Immunohistology of the small intestine
For immunohistological study, the jejunum and ileum were obtained from mice in each group for staining of Ab-containing cells (25). Briefly, the tissues were fixed in 5% glacial acetic acid in 95% ethanol at -20°C for 24 h before paraffin embedding. Serial tissue sections 5 µm thick were mounted on glass slides, and IgA-containing cells were visualized with FITC-labeled anti-mouse IgA mAb (PharMingen, San Diego, CA).
Immunization
OVA was purchased from Sigma (St. Louis, MO), and CT was obtained from List Biologic Laboratories (Campbell, CA). For s.c. immunization, 100 µg OVA were given with 1 µg CT as adjuvant on days 0 and 14. For oral immunization, mice were deprived of food for 2 h and then given a solution of sodium bicarbonate to neutralize stomach acidity before oral immunization (26, 27). Thirty minutes later, these mice were orally immunized by gastric intubation with 1 mg OVA in the presence of 10 µg CT as mucosal adjuvant. This oral immunization procedure was conducted on days 0, 7 and 14.
Detection of Ag-specific Ab isotype responses
Serum and fecal extracts were obtained as described previously
(26, 27). The Ab titers in serum and fecal extracts were
determined by an ELISA as described elsewhere (26, 27).
Briefly, plates were coated with OVA (1 mg/ml) or cholera toxin B
subunit (CT-B, 2 µg/ml) and blocked with 10% goat serum, and
analyses were performed in duplicate. Following incubation, the plates
were washed and peroxidase-labeled goat anti-mouse µ, 
, or
heavy chain-specific Abs (Southern Biotechnology Associates,
Birmingham, AL) were added to appropriate wells. Finally,
2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) with
H2O2 (Moss, Pasadena, MD)
was added for color development. Endpoint titers were expressed as the
reciprocal log2 of the last dilution that gave an
optical density at 414 nm of 0.1 greater than background after 15 min
of incubation.
Enzyme-linked immunospot assay for assessment of Ab-forming cells (AFCs)
Single-cell suspensions were obtained from intestinal lamina
propria of naive or orally immunized mice as previously described
(26, 27). The mononuclear cells were obtained at the
interface of the 40 and 75% layers of a discontinuous Percoll gradient
(Amersham Pharmacia Biotech, Piscataway, NJ). To assess numbers of
total and Ag-specific AFCs, an enzyme-linked immunospot assay was
performed as previously described (26, 28). Briefly,
96-well nitrocellulose plates (Millititer HA. Millipore, Bedford, MA)
were coated with goat anti-mouse Ig Ab (2 µg/ml), OVA (1 mg/ml),
or CT-B (5 µg/ml) and incubated for 20 h at 25°C; the plates
were then washed extensively and blocked with 10% goat serum. The
blocking solution was discarded, and lymphoid cell suspensions at
various dilutions were added to wells and incubated for 4 h at
37°C in 5% CO2 in moist air. The detection Abs
consisted of HRP-conjugated goat anti-mouse or heavy
chain-specific Abs (Southern Biotechnology Associates). After overnight
incubation, plates were washed with PBS and developed by addition to
each well of 3-amino-9-ethylcarbazole dissolved in 0.1 M sodium acetate
buffer containing H2O2
(Moss). Plates were incubated at room temperature for 15–20 min and
washed with water, and AFCs were counted with the aid of a
stereomicroscope.
Ag-specific CD4+ T cell responses
CD4+ T cells from MLN and spleen cell suspensions were purified by use of the magnetic-activated cell sorter system (Miltenyi Biotec, Sunnyvale, CA) as previously described (29). Briefly, mononuclear cells were added to a nylon wool column (Polysciences, Warrington, PA) and incubated at 37°C for 1 h to remove adherent cells. The enriched CD4+ T cell populations were obtained by the incubation with biotinylated anti-CD8 (53-6.7), anti-Mac-1 (M1/70), and anti-B220 (RA3-6A2) mAbs followed by streptavidin-conjugated microbeads for magnetic-activated cell sorter. Two cycles of the above procedures yielded CD4+ T cell preparations that were >98% pure. Purified MLN CD4+ T cells (2 x 106 cells/ml) were cultured with T cell-depleted, irradiated splenic feeder cells (2.5 x 106 cells/ml) from naive mice in complete medium (RPMI 1640, Cellgro Mediatech, Washington, DC) containing 10% FCS, 1% L-glutamine, 50 µM 2-ME, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, and 40 µg/ml gentamicin. The cultures were supplemented with 10 U/ml mouse rIL-2 (Genzyme, Cambridge, MA). OVA (1 mg/ml) or CT-B (2 µg/ml) was added for restimulation of Ag-specific CD4+ T cells (30). The CD4+ T cell cultures were incubated for 4 days at 37°C in 5% CO2 in air. To measure cell proliferation, 1.0 µCi [3H]thymidine (DuPont New England Nuclear Products, Boston, MA) was added to individual culture wells 15 h before termination, and the uptake by dividing cells of cpm was determined by scintillation counting (30).
Analysis of cytokine responses
Cytokine levels in culture supernatants were determined by a
cytokine-specific ELISA as described previously (29, 30, 31).
Maxisorp immunoplates (NUNC, Naperville, IL) were coated with
monoclonal anti-IFN-, anti-IL-2, anti-IL-4, or
anti-IL-5 mAbs (PharMingen). After blocking, samples and serial
2-fold dilutions of standards were added to duplicate wells and
incubated overnight at 4°C. The wells were washed and incubated with
biotinylated anti-IFN-, anti-IL-2, anti-IL-4, or
anti-IL-5 mAbs (PharMingen). After incubation,
peroxidase-labeled anti-biotin Ab (Vector Laboratories, Burlingame,
CA) was added and developed with
2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid containing
H2O2 (Moss). Standard
curves were generated with mouse rIFN- (Genzyme), rIL-2 (Genzyme),
rIL-4 (Endogen, Boston, MA), and rIL-5 (Endogen).
Statistics
Data are expressed as the mean ± SEM and compared by the unpaired Mann Whitney U test. The results were analyzed using the Statview II statistical program (Abacus Concepts, Berkeley, CA) adapted for Macintosh computers.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the initial study, we sought to determine whether an organized
Peyer’s patch structure is a strict requirement for the induction of
Ag-specific mucosal IgA Ab responses. For this purpose, mice treated in
utero with LTßR-Ig fusion protein were used because previous studies
have shown that exposure to LTßR-Ig during gestation disrupted
Peyer’s patch but not MLN development in the progeny (20, 22). For direct comparison,
TNF/LT--/- mice, which lack both Peyer’s
patches and MLN, were also used. Immunohistochemical analysis revealed
that IgA+ plasma cells were dramatically reduced
in intestinal lamina propria of TNF/LT--/-
mice when compared with control, TNF/LT-+/+
mice (Fig. 1
, A and
B). These findings are in full agreement with a previous
study (15). In contrast, significant numbers of
IgA+ plasma cells were seen in PP null mice,
although the frequency of cells was slightly lower than those observed
in control, LFA-3-Ig-treated, or fusion protein-nontreated mice (Fig. 1, C–E). Our analyses of AFC responses supported the
immunohistochemical study and showed that slightly lower but
significant numbers of total IgA-producing cells were detected in
mononuclear cells isolated from intestinal lamina propria of PP null
mice when compared with control mice, whereas only small numbers of
total IgA-producing cells were detected in
TNF/LT--/- mice (Table I).
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A). Because the presence or
absence of Peyer’s patches may lead to different kinetics for mucosal
IgA responses, we assessed OVA- and CT-B-specific fecal IgA Ab
responses at weekly intervals. The kinetics of IgA responses were
almost identical in PP null and control mice. Thus, OVA- or
CT-B-specific IgA responses appeared by day 14 and increased to maximum
levels by day 21. Although we continued to examine IgA levels until day
42, the responses did not change in either mouse group (data not
shown). The appearance of OVA-specific IgA Abs correlated with the
presence of Ag-specific IgA AFCs, because significant numbers of OVA-
and CT-B-specific IgA AFCs were detected in mononuclear cells isolated
from intestinal lamina propria of PP null mice given OVA and CT orally
(Fig. 2B). Analysis of serum Ab responses also revealed that
PP null mice given oral OVA plus CT underwent high levels of OVA- and
CT-B-specific IgM, IgG, and IgA Ab responses which were comparable with
those induced in LFA-3-Ig-treated or nontreated mice (Fig. 3). These findings demonstrate that
Ag-specific mucosal IgA responses were induced after oral immunization
of LTßR-Ig-treated mice lacking Peyer’s patches.
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Based on the significant Ag-specific mucosal IgA Ab responses in
the gastrointestinal tract of mice without organized Peyer’s patches,
it was important to determine the source of CD4+
T cells which provides help for these Ab responses.
CD4+ T cells from MLN and spleen of PP null mice
orally immunized with OVA and CT, when stimulated with either OVA or
CT-B, resulted in significant proliferative responses; however, these
responses were lower than those induced in LFA-3-Ig-treated or in the
fusion protein nontreated, control mice (Fig. 4). These results showed that both OVA-
and CT-B-specific CD4+ T cells were present in
MLN and spleen of mice that lack Peyer’s patches after oral
immunization with OVA and CT.
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and IL-2) cytokines (Fig. 5).
Similar cytokine patterns were observed with CD4+
T cells from spleen (data not shown). These results indicate that
despite a lack of Peyer’s patches, the MLN CD4+
T cells produced Th2-type cytokine responses for support of
OVA-specific mucosal IgA Ab responses in the intestine.
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Because Ag-specific CD4+ T cells were
induced in MLN in the absence of Peyer’s patches (Figs. 2
and 3), we
next addressed the possibility that MLN, but not Peyer’s patches,
are required for the induction of Ag-specific intestinal mucosal IgA Ab
responses. Thus, TNF/LT--/- mice, which have
been shown to lack peripheral LN including both MLN and Peyer’s
patches (15, 16, 17), were orally immunized with OVA plus CT
as mucosal adjuvant. Whereas TNF/LT-+/+
control mice underwent significant OVA- and CT-B-specific S-IgA Ab
responses in the small intestine, neither S-IgA anti-OVA nor
anti-CT-B Abs were detected in
TNF/LT--/- mice orally immunized with OVA
plus CT (Fig. 6A). Assessment
of AFC responses also revealed low numbers of OVA- or CT-B-specific IgA
AFCs in mononuclear cells isolated from intestinal lamina propria of
TNF/LT--/- mice, whereas high numbers of
Ag-specific IgA AFCs were detected in
TNF/LT-+/+ control mice given oral OVA
together with CT (Fig. 6B).
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-/- mice given oral OVA and CT,
although this immunization regimen elicited high levels of OVA- and
CT-B-specific IgG responses in TNF/LT-+/+
control mice (Fig. 7A).
Interestingly, however, when TNF/LT--/- mice
were immunized parenterally with OVA plus CT, identical levels of
Ag-specific serum IgG Ab responses were induced when compared with
TNF/LT-+/+ control mice (Fig. 7B).
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nor the LT-/ß pathway contribute to IgA
class switching
To assess the possibility that LT/ß and TNF/LT pathways
may affect class switching to IgA for subsequent Ab responses, 6-wk-old
C57BL/6 mice, which have a fully developed immune system, were treated
with LTßR-Ig or TNF-R55-Ig during the immunization period. Both
LTßR-Ig- and TNFR55-Ig-treated mice elicited OVA- and CT-B-specific
fecal IgA Abs (Fig. 8A).
Assessment of AFC responses also revealed high numbers of OVA- and
CT-B-specific AFCs in mononuclear cells isolated from the lamina
propria (Fig. 8B). Interestingly, mucosal IgA responses in
LTßR-Ig-treated, but not TNF-R55-Ig-treated, mice were significantly
lower than those induced in control mice (Fig. 8). These findings
indicate that neither the TNF/LT nor the LT/ß pathway
contribute to class switches to IgA and thus emphasize the importance
of the MLN for the induction of Ag-specific mucosal as well as systemic
T and B cell responses under conditions in which organized Peyer’s
patches have been physiologically deleted.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-/- mice, which
lack both Peyer’s patches and MLN, failed to elicit intestinal IgA
Abs. These results suggest that immunocompetent T and B cells in MLN
possess the ability to extravasate into intestinal lamina propria for
subsequent IgA immune responses despite the lack of an organized
Peyer’s patch network. It is now well established that the Peyer’s patch is a major inductive site for Ag-specific IgA Ab responses in the small intestine. The importance of Peyer’s patches in the induction of mucosal Ab responses has been shown in the rabbit model where the lamina propria of intestine contains IgA-secreting plasma cells that arose from precursors in the Peyer’s patch (3). Further, Ag-specific IgA precursors from Peyer’s patches repopulated in the lamina propria with differentiation into IgA-producing plasma cells (33). In addition, introduction of Ags into the surgical constructed intestinal loops that bore a Peyer’s patch resulted in the appearance of S-IgA Abs after immunization of Ag, whereas no mucosal responses were detected in intestinal loops that lacked visible Peyer’s patches (34). These studies clearly show that Peyer’s patches are a major source of precursors for IgA-producing cells in the small intestine. However, our results now show that oral immunization of mice treated with LTßR-Ig in utero that lack the development of Peyer’s patches also undergo Ag-specific IgA Ab responses in the intestinal lumen. This interesting outcome could be trivially explained by the presence of Peyer’s patches after LTßR-Ig treatment in utero and are sufficient to support the induction of Ag-specific IgA Abs because of insufficient maternal-fetal transport of LTßR-Ig. However, a previous study has shown that the progeny of mice that received a single injection of as little as 1 µg of LTßR-Ig on day 14 of gestation, and which had a plasma concentration below the levels of detection by ELISA lacked peripheral, but not mesenteric, LN (20). In the present study, mice were treated with 200 µg fusion protein on gestational days 14 and 17. Thus, it is likely that all Peyer’s patches in the mice used in this study are depleted. Indeed, the small intestinal tissue of all mice was carefully examined for visible patches, and no evidence for Peyer’s patches were seen in offspring of LTßR-Ig-treated mice.
A more plausible explanation would be that the small intestine has a
compensatory system for induction of mucosal IgA Abs when Peyer’s
patches are absent such as would occur during the early stages of
development. Our results showed that OVA- as well as CT-B-specific
CD4+ T cells secreting Th2-type (e.g., IL-4 and
IL-5) cytokines were induced in the MLN of PP null mice. Because oral
administration of CT has been shown to induce Th2-type cytokine
responses to coadministered protein in mucosal inductive tissues, e.g.,
the Peyer’s patches with subsequent intestinal IgA Ab responses
(27, 32), it is likely that MLN act as inductive tissues
in the absence of an organized intestine-associated lymphoreticular
tissue. To this end, lymphocytes in MLN may migrate into the intestinal
lamina propria and commit to Ag-specific effector functions. It is well
established that the MLN drain lymphocytes from the intestine and it
was shown that MLN possess features of both mucosal and peripheral
lymphoid tissue in terms of lymphocyte trafficking (35, 36). In this regard, high endothelial venules in MLN clonally
express high levels of both mucosal addressin cell adhesion molecule-1
and peripheral LN addressin which are major mucosal and peripheral LN
addressins, respectively (37). Further, mucosal as well as
peripheral node cell lines have been shown to bind to high endothelial
venules of MLN in vitro (38). In addition, in vivo homing
studies have shown that
4ß7high
CD44high cells traffic into the MLN at higher
frequencies than into peripheral LN (39). Thus, MLN are
the most likely source of lymphocytes destined to home into the
intestinal lumen in the absence of Peyer’s patches. In support of
this, our results showed that significant numbers of total
IgA-producing cells were seen in the intestinal lamina propria of PP
null mice, whereas only low numbers of total IgA-producing cells were
detected in TNF/LT--/- mice. These results
suggest that MLN are a major source of immunocompetent cells that
repopulate the intestinal mucosal areas and can compensate for
immunological inductive functions if Peyer’s patches are absent.
In contrast to this, a previous study has suggested that both Peyer’s
patches and intestinal lamina propria are sources of immediate
precursors of IgA plasma cells (40). Furthermore, a recent
study has reported that the common mucosal immune system
(CMIS)-independent B-1 lineage of IgA precursor cells developed in the
intestinal lamina propria, whereas CMIS-dependent B-2 lineage IgA
precursor cells mainly resided in Peyer’s patches (41).
These studies imply that B-1 cells in intestinal lamina propria may
contribute to the induction of Ag-specific mucosal IgA Ab responses in
the absence of Peyer’s patches. However, almost no
IgA+ cells were found in intestinal lamina
propria of TNF/LT--/- when compared with PP
null mice. These results clearly indicate that although
CMIS-independent IgA precursor cells (e.g., B-1 cells) may support
mucosal IgA responses in the absence of Peyer’s patches, at a minimum
the presence of MLN appear necessary for localization of those cells
into the intestinal lamina propria.
Our results showed that neither mucosal IgA nor serum IgG Ab responses
were induced in TNF/LT--/- mice orally
immunized with OVA plus CT. Previous studies have shown that
TNF/LT--/- as well as
LT--/- mice manifest an aberrant spleen
structure with small white pulp follicles that fail to segregate into T
and B cell zones and fail to generate clusters of follicular dendritic
cells (FDC) or germinal centers (15, 16, 17, 42, 43). Further,
it was reported that a functional splenic microarchitecture, including
the presence of primary and secondary lymphoid follicles that contain
FDC, is thought to be required for a mature T cell-dependent B cell
response and is associated with B cell Ig class switching, affinity
maturation, and development of Ab-secreting cells
(44, 45, 46). In support of this, impaired IgG responses are
seen in LT--/- mice after s.c. immunization
with keyhole limpet hemocyanin adsorbed to alum or in response to
immunization with viral Ag (16). Further,
LT--/- mice immunized with low doses of the
T cell-dependent Ag 4-hydroxy-3-nitrophenyl-OVA adsorbed to alum
exhibit impaired production of high affinity
anti-4-hydroxy-3-nitrophenyl IgG Abs, whereas those
LT--/- mice were able to generate high
affinity anti-4-hydroxy-3-nitrophenyl IgG Abs after immunization
with high doses of 4-hydroxy-3-nitrophenyl-OVA adsorbed to alum
(42). Moreover, reconstitution of irradiated wild-type
mice with bone marrow from LT--/- mice
resulted in loss of FDC clusters and germinal centers and impairment of
Ag-specific IgG responses (11). Taken together, these
studies imply that impairment of serum IgG and mucosal IgA Ab responses
in TNF/LT--/- mice may be due to a failure
of Ig class switching and development of Ab-secreting cells.
To assess the possibility that LT/ß and TNF/LT pathways may
affect class switching to IgA for subsequent Ab responses, young adult
C57BL/6 mice, which have a fully developed immune system, were treated
with LTßR-Ig or TNFR55-Ig during the immunization period. Our results
showed that both LTßR-Ig-treated and TNFR55-Ig-treated adult mice
developed mucosal IgA and serum IgG Ab responses when those mice
were immunized orally with OVA plus CT. Further, s.c. immunization of
TNF/LT--/- mice elicited significant OVA-
and CT-B-specific serum IgG Ab responses. These results indicate that
neither LT/ß nor TNF/LT signaling are required for IgA class
switching. In this regard, TNF/LT--/- mice
show variable IgG responses after i.p. immunization with SRBC but
retained immunity to vesicular stomatitis virus after an infectious
challenge (15). Further, recent studies have shown that
TNF--/- mice undergo significant IgG Ab
responses when immunized with DNP-keyhole limpet hemocyanin adsorbed to
alum; however, SRBC-specific Ab responses were impaired
(47). These studies suggest that the variability of Ab
response depends on the immunization protocol and on the nature of Ags
used. Thus, when appropriate Ags are given systemically, IgG responses
can be induced without peripheral LN in the systemic compartment. In
this study, we used CT as adjuvant for s.c. immunization. It is well
established that CT is a strong adjuvant for enhancement of Ab
responses to coadministered protein Ags when given systemically or
mucosally (27, 32, 48). Therefore, although
TNF/LT-/- and PP null mice have disrupted
peripheral LN development and splenic microarchitecture, they can
undergo systemic Ab responses after immunization with CT as adjuvant.
On the other hand, oral immunization of
TNF/LT-/-, but not PP null, mice with OVA
plus CT failed to elicit serum IgG Ab responses. Because
TNF/LT-/- mice are capable of developing
systemic Ab responses without peripheral LN in the systemic compartment
when CT is used as adjuvant, impairment of serum IgG Abs in those mice
is likely due to a lack of MLN. Thus, the MLN plays a critical role not
only for intestinal mucosal IgA but also for systemic IgG Ab responses
when Ag is given orally.
Ag-specific intestinal mucosal IgA Abs as well as
CD4+ T cell responses in MLN of PP null mice were
lower than those of control mice. Further, treatment of young adult
mice with LTßR-Ig but not TNF-R55-Ig during the immunization period
resulted in reduced Ag-specific mucosal IgA Ab responses. In this
regard, it has been shown that blocking of the LT-ß, but not the
TNF/LT, pathway in adult mice inhibits germinal center formation in
the spleen and impairment of Ig production in response to immunization
with sheep erythrocytes (14). Our results also showed that
LTßR-Ig- but not TNF-R55-Ig-treated adult mice failed to develop
germinal centers in Peyer’s patches (unpublished data). These studies
together with our results suggest that LTß but not TNF/LT
signaling is required for maintenance of peripheral immune organs
including Peyer’s patches and that organized Peyer’s patches are
required for maximum S-IgA Ab responses in the gastrointestinal
tract.
In summary, our present study has demonstrated that oral immunization
with OVA plus CT of PP null mice undergo Ag-specific mucosal IgA Ab
responses. It was further shown that OVA-specific Th2-type
CD4+ T cells are induced in MLN and spleen. In
contrast, neither mucosal IgA nor serum IgG anti-OVA Abs were
induced in TNF/LT--/- mice, which lack both
Peyer’s patches and MLN. Taken together, our findings provide
important new evidence that the mucosal immune system possesses a
compensatory inductive mechanism for the maintenance of the common
mucosal immune system even under the deficient organogenesis of
Peyer’s patches. Thus, the MLN plays an important role for both
mucosal and systemic immunity when Ags are administered orally to PP
null mice. Finally, our findings suggest that organized Peyer’s
patches may not be an essential element for induction of mucosal IgA Ab
responses in the gastrointestinal tract.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Masafumi Yamamoto, Department of Clinical Pathology, Nihon University School of Dentistry at Matsudo 2-870-1 Sakaecho-Nishi, Matsudo, Chiba 271-8587, Japan.
3 Abbreviations used in this paper: S-IgA, secretory IgA; CMIS, common mucosal immune system; CT, cholera toxin; CT-B, cholera toxin B subunit; LT--/-, lymphotoxin- gene-disrupted; LTßR, LT-ß receptor; LTßR-Ig, fusion protein of lymphotoxin-ß receptor and Ig; PP null, Peyer’s patch null; TNF/LT- -/-, TNF and LT- double gene knockout; LN, lymph nodes; TNF-R55, TNF receptor 55; AFCs, Ab-forming cells; FDC, follicular dendritic cells.
Received for publication June 22, 1999. Accepted for publication March 6, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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  M. Gohda, J. Kunisawa, F. Miura, Y. Kagiyama, Y. Kurashima, M. Higuchi, I. Ishikawa, I. Ogahara, and H. Kiyono Sphingosine 1-Phosphate Regulates the Egress of IgA Plasmablasts from Peyer's Patches for Intestinal IgA Responses J. Immunol., April 15, 2008; 180(8): 5335 - 5343. [Abstract] [Full Text] [PDF]
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), LFA3-Ig-treated
(
), and nontreated (
) C57BL/6 mice were orally immunized with 1
mg OVA plus 10 µg CT on days 0, 7, and 14. Fecal samples were
collected at day 21 and examined for OVA- or CT-B-specific Ab responses
by ELISA. Mononuclear cells were isolated at day 21, and Ag-specific
AFC responses were examined by ELISPOT. The results are expressed as
the mean ± SEM from five mice per group and from a total of three
experiments. *, p < 0.05 when compared with
control mice.
