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Departments of
*
Molecular Immunology and
Research Administration, Immunex Corp., Seattle, WA 98101; and
Department of Immunology, University of Glasgow, Western Infirmary, Glasgow, Scotland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC are reported to be the most potent of APC, characterized by an apparent constitutive ability to present Ag to naive T cells in an immunostimulatory manner (4). DC are present in the different compartments of the gut, including the diffuse and organized lymphoid tissues (5, 6, 7, 8, 9). Previous functional studies have indicated that intestinal DC can be strongly immunogenic after the oral administration of Ags (10, 11, 12), suggesting that DC in the intestine may be similar to those in the periphery, with a preferential ability to promote active immunity.
Studies of DC function have been complicated, however, by the low frequency of DC in normal tissues. As a result, much of the past work studying DC function has used DC in vitro or DC transferred into recipient mice. Since it is well documented that costimulatory molecules such as CD80 and CD86 are up-regulated on DC following removal from the local tissue microenvironment (13, 14), the in vitro manipulation of DC may have altered the intrinsic functional properties of the DC used in those studies. Thus, the possibility that DC may play a role in the induction of mucosal tolerance in vivo may have been missed. One approach for circumventing these problems originates from the recent observation that treating mice with the hemopoietic growth factor Flt3 ligand (Flt3L) can dramatically increase the numbers of functionally mature DC in peripheral lymphoid tissues (15). Here we have investigated whether Flt3L treatment has a similar effect on the DC populations in the gut-associated lymphoid tissues (GALT) of mice and have examined how increasing the number of DC in vivo influences the induction of systemic immune tolerance following oral administration of protein Ag.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female C57BL/6 or BALB/c mice (6–10 wk of age) were obtained from Taconic Laboratories (Germantown, NY) and maintained in a specific pathogen-free facility at Immunex (Seattle, WA) in accordance with approved ethical guidelines. DO11.10 OVA TCR transgenic mice (16) were bred at the specific pathogen-free facility at Immunex.
In vivo treatment of mice with Flt3L
Flt3L-treated mice were injected i.p. once daily with purified CHO-derived human Flt3L (10 µg) for 10 to 12 days as indicated. Control mice were injected i.p. with either human IgG (10 µg; Sigma, St. Louis, MO) or PBS (100 µl) for the same period.
Cell isolations
Organized lymphoid tissues. Single cell suspensions were prepared from mesenteric lymph nodes, spleen, Peyer’s patches (PP), and popliteal lymph nodes (PLN) by teasing tissues apart in complete medium, followed by mashing through nylon mesh.
Lamina propria. Small intestine lamina propria (SILP) and large intestine lamina propria (LILP) cell suspensions were prepared from intestines opened longitudinally and cut into 1-cm segments. Tissues were incubated at 37°C in 1 mM EDTA in Ca2+- and Mg2+-free HBSS (Life Technologies, Gaithersburg, MD) for three sequential 15-min incubations to remove the epithelial layer. Denuded tissues were digested with collagenase (90 U/ml; Sigma) in Ca2+- and Mg2+-free HBSS/FCS, and the resulting suspension of cells was washed and passed over a prewet glass wool column before centrifugation over a discontinuous Percoll (Pharmacia, Piscataway, NJ) density gradient as previously described (17, 18). This procedure did not influence the cell populations obtained from identically treated LN and spleen cell preparations (data not shown).
Monoclonal Abs
The following mAbs were used: anti-CD11b (M1/70, rat IgG2b), anti-CD11c (HL3, hamster IgG), anti-CD19 (1D3, rat IgG2a), anti-CD40 (3/23, rat IgG2a), anti-CD80 (1G10, rat IgG2a), anti-CD86 (GL1, rat IgG2a), anti-I-Ab,d (25-9-17, mouse IgG2a), anti-CD3 (145.2C11, hamster IgG), anti-B220 (RA3-6B2, rat IgG2a), mouse IgG2a isotype control (G155-178), hamster IgG isotype control (G235-2356), rat IgG1 isotype control (R3-34), rat IgG2a isotype control (R35-95), and rat IgG2b isotype control (R35-38), all purchased from PharMingen (San Diego, CA).
Immunofluorescence analysis of frozen sections
Tissues were frozen in liquid nitrogen. Cryostat sections were cut at 6 µm, air-dried, and acetone fixed before staining using conjugated Abs. Sections were stained with anti-CD11c biotin and anti-I-Ab,d FITC mAbs in PBS/30% mouse serum, followed by streptavidin-Texas Red (Molecular Probes, Eugene, OR). Sections were visualized using a confocal laser scanning microscope (Molecular Dynamics, Sunnyvale, CA).
Flow cytometric analysis of isolated cells
Isolated cells were incubated at 4°C for 1 h in primary Ab at 5 µg/ml in the presence of 30% mouse serum, washed with PBS-BSA (2 mg/ml), then incubated with 40 µl of APC-labeled streptavidin (10 µg/ml; Molecular Probes) for an additional 1 h. Samples were washed three times, resuspended in PBS-BSA supplemented with 1% paraformaldehyde, and stored at 4°C until analysis on a FACStar flow cytometer (Becton Dickinson, San Jose, CA). Fifty thousand cells were analyzed per sample. To determine the proportion of OVA-specific CD4+ cells expressing the clonotypic TCR, cells were stained with mAb KJ1.26 FITC, which detects the clonotypic transgenic TCR and with anti-CD4 PE in 50 µl of blocking buffer containing 10 µg/ml anti-CD16 (PharMingen), 10% normal goat serum, and 1% normal mouse serum.
In vivo BrdUrd labeling
Cell suspension analysis. For identification of cycling cells, mice were injected i.p. with 1 mg of BrdUrd (Sigma) twice (24 and 12 h) before tissue harvest. Single cell suspensions were prepared from each tissue, and cells were stained with PE-conjugated mAb to CD11c, B220, or CD3 surface Ags before fixing and permeabilizing them for BrdUrd-FITC staining. PE-labeled cells were resuspended in 0.15 M NaCl before being fixed in 95% ethanol (4°C 30 min) and permeabilized with 1% paraformaldehyde/0.01% Tween-20 (at room temperature for 30 min followed by 4°C for 30 min). Cells were then treated with 50 U/ml DNase I (Sigma) in 0.15 M NaCl/4.2 mM MgCl2 (at room temperature for 10 min, then at 4°C for 30 min), washed, and stained with anti-BrdUrd FITC mAb (Sigma) for 30 min at room temperature.
Immunohistologic analysis. For localization of cycling cells in tissues in situ by immunohistology, mice were injected i.p. once with 1 mg of BrdUrd 2 h before tissue harvest. Dissected tissues were formalin fixed, paraffin embedded, sectioned, and stained using the immunoperoxidase technique. Positive cells were identified by the presence of a brown reaction product.
DC activation in vivo
Mice treated with Flt3L for 10 days were injected i.p. with saline only or with 100 µg of LPS solubilized in 0.9% pyrogen-free NaCl. Tissues were harvested 6 h after LPS injection, and single cell suspensions were prepared. The expression of costimulatory molecules on the CD11c+ cells was analyzed by FACS.
Induction and assessment of oral tolerance
Mice were injected with PBS or Flt3L for 10 days before and for 2 days after oral administration of Ag given as a single feed of OVA (fraction V, Sigma) in 0.2 ml of saline by gavage. Ten days after OVA feeding, all mice were immunized s.c. in the footpad with 100 µg of OVA in 50 µl of adjuvant (Ribi adjuvant, Ribi Immunochemicals). Two weeks after immunization half of the mice were sacrificed, and the draining PLN were removed for assessment of Ag-specific proliferative capability (see below). After an additional 7 days, the remaining mice were assayed for systemic delayed-type hypersensitivity (DTH) responses by measuring the increase in footpad thickness 24 h after challenge with 100 µg of heat-aggregated OVA in 50 µl of saline as described previously (19). Mice were then exsanguinated, and Ag-specific serum Ab titers were measured (see below).
Ag-specific proliferation assay
Cells isolated from PLN were cultured in complete RPMI 1640 medium supplemented with 10% FCS, penicillin/streptomycin, and ß-ME at 37°C in a humidified 6% CO2 incubator at a density of 2 x 105 cells/well for 48 to 96 h. All cultures were performed in quadruplicate in 96-well flat-bottom plates in a total volume of 200 µl either alone or in the presence of 1 µg/ml Con A (Sigma) or 1 mg/ml OVA. Proliferation was assessed by addition of 1 µCi/well [3H]thymidine (Amersham, Aylesbury, U.K.) 18 h before harvesting. The amount of radioactivity incorporated into DNA was measured using a Matrix-96 cell harvester (Inotech, Lansing, MI) and a direct beta counter (Packard, Meridan, CT). The data are reported as the mean counts per minute ± 1 SEM of quadruplicate wells.
Analysis of Ag-specific serum Ab titers
Ninety-six-well ELISA plates (Maxisorp, Nunc, Naperville, IL) were coated overnight with 1 µg/well OVA in PBS at 4°C, quenched with PBS/5% FCS and washed with PBS/0.1% Tween-20. Serum samples were diluted in PBS/5% FCS (starting at 1/100), and threefold dilutions were made. Plates were incubated for 2 h at room temperature, washed, and incubated with alkaline phosphatase-conjugated anti-IgG (1/3000; Sigma), anti-IgG1 (1/2000; PharMingen), or anti-IgG2a (1/1000; PharMingen) detecting Abs for an additional 2 h at room temperature. Plates were washed again, and enzyme activity was detected with p-nitrophenyl phosphate disodium (Sigma). The amount of reaction product was assessed on an ELISA plate reader at an OD of 405 nm using the Deltasoft program (DeltaPoint, Monterey, CA).
Assessment of tolerance in transfer mice
For adoptive transfer of OVA TCR transgenic T cells, syngeneic BALB/c mice were injected i.v. with 2.5 x 106 clonotypic TCR+ (CD4+,KJ1.26+) transgenic cells from DO11.10 mice, essentially as previously described (20, 21). BALB/c mice were treated with Flt3L or PBS for 8 days before and for 2 days after adoptive transfer of transgenic cells. Mice were fed a single dose of OVA in 0.2 ml of saline 2 days after transfer and immunized s.c. in the footpad 5 days later with 100 µg of OVA in RIBI adjuvant. After an additional 4 days, draining PLN were removed, and the proportion of transgenic cells in individual mice was determined by FACS analysis. Isolated cells from individual mice were also assessed for Ag-specific proliferative capability as described above.
Statistical analysis
Student’s t test was used to compare data from different groups.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We first investigated whether treating mice with Flt3L could
expand DC in the intestine as well as in peripheral lymphoid tissues.
Flow cytometric analysis of both the organized lymphoid tissues (PP and
mesenteric lymph nodes) and the diffuse lymphoid tissues (small and
large intestine lamina propria) of the intestine revealed a large
preferential increase in cells expressing CD11c, an Ag expressed by
cells of the DC lineage (13, 22), following 10 days of Flt3L treatment
(Fig. 1
and Table I). The increase in
CD11c+ cells in the GALT was comparable to that
observed in the spleen (Fig. 1). Similar results were seen in C57BL/6
and BALB/c mice (data not shown).
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DC have been divided into putative subpopulations based upon the
relative coexpression of CD11c with other cell surface molecules,
notably CD11b (15, 23, 24, 25, 26, 27, 28).
CD11c+/CD11blow cells often coexpress
DEC205 and CD8
and represent a putative subset derived from lymphoid
precursors. Conversely, CD11c+/CD11bhigh cells
correspond to a putative myeloid-derived DC subset (15, 23, 24, 25, 26, 27, 28). Both
of these subpopulations are expanded in the peripheral tissues of
Flt3L-treated mice. To determine whether the same DC subpopulations can
be found in the GALT of Flt3L-treated animals, CD11c+ cells
from Flt3L-treated mice were analyzed for the expression of CD11b,
CD8, and DEC205. Similar patterns of expression for all three
markers by spleen and GALT-derived DC were observed, indicating that
there is no preferential bias toward expansion of individual
subpopulations in the gut following Flt3L treatment (Fig. 2).
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Having observed the increase in CD11c+ DC using
cells isolated from the intestinal lamina propria, we localized the
CD11c+ cells in frozen sections of small intestine using
immunofluorescence microscopy. Dual color staining was used to analyze
CD11c+ cells for expression of MHC class II, I-A (MHCII).
Occasional CD11c+/MHCII+ cells with DC
morphology were observed in sections of small intestine from control
PBS-treated mice (Fig. 3). This
population was greatly expanded in mice treated with Flt3L, with large
numbers of CD11c+/MHCII+ cells visible in the
lamina propria of both the villus and crypt regions (Fig. 3). DC were
also observed in the dome and interfollicular regions of PP from
control mice as previously described (9, 12), and these populations
were expanded following Flt3L treatment (not shown).
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Previous reports show that treating mice with Flt3L expands
CD11c+ DC in the spleen without influencing mature B
or T cell numbers (15). To determine whether this would also apply to
cells in the intestine, the absolute number of each of the various cell
types in the GALT was calculated. As expected, tissues from mice
treated with Flt3L contain significantly elevated numbers of DC (Table I
). There was a small, but not significant, increase in the number of B
cells in some tissues from Flt3L-treated mice compared with those from
PBS-treated mice and no change in the number of T cells (Table I).
To further analyze the effects of Flt3L treatment, mice were injected
with BrdUrd to label cycling cells, and two-color FACS analysis was
used to identify recently divided DC, B cells, or T cells (Fig. 4A).
There were few CD11c+ cells in SILP from PBS-treated mice,
none of which incorporated BrdUrd. In contrast, a much greater
proportion of BrdUrd+ CD11c+ cells was evident
in SILP from mice that were treated with Flt3L for 10 days before
BrdUrd injection; indeed, almost half the CD11c+ cells
detected were BrdUrd+. The relative proportion of B and T
cells was smaller in the Flt3L-treated mice, owing to the massive
increase in CD11c+ cells, but the absolute number of B and
T cells did not significantly differ between Flt3L-treated and control
mice. In both PBS- and Flt3L-treated mice, the proportion of dividing B
and T cells was low, and the absolute numbers of dividing B and T cells
were similar in the two groups. These data indicate that Flt3L
treatment preferentially stimulates CD11c+ DC to divide
without influencing mature B or T cells.
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B). In Flt3L-treated mice, there were many
BrdUrd+ cells obvious in the lamina propria in addition to
the labeled crypt epithelial cells (Fig. 4C). The
BrdUrd+ cells could be seen throughout the crypt-villus
axis of the lamina propria. These observations together with the FACS
data indicating that the BrdUrd+ cells were
CD11c+ DC suggest that DC in all regions of the mucosa can
be expanded by Flt3L. The overall gross architecture of the mucosal
tissues did not appear to be affected by Flt3L treatment. CD11c+ cells expanded by Flt3L are resting DC, but can be activated in vivo
To determine the activation status of CD11c+ DC
expanded in the gut by Flt3L treatment, CD11c+ cells were
analyzed for the expression of MHCII, CD80 (B7-1), and CD86 (B7-2).
Multicolor flow cytometry demonstrated that the majority of freshly
isolated CD11c+ cells from both GALT and spleen expressed
moderately high levels of MHCII (Fig. 5A). These
CD11c+/MHCII+ cells expressed little or no CD80
and only low levels of CD86 (Fig. 5A). This phenotype
is consistent with unactivated, resting DC (13, 14), suggesting that
Flt3L can expand DC in vivo without activation.
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B). CD11c+
cells isolated from Flt3L-treated mice injected with LPS exhibited
increased levels of MHCII, CD40, CD80, and CD86 compared with
CD11c+ cells from Flt3L-treated mice injected with saline
alone. Thus, the DC expanded by Flt3L are in a resting state, but can
be activated by appropriate inflammatory stimuli. Expansion of DC by Flt3L is associated with enhanced induction of oral tolerance
The massive expansion of DC in the GALT induced by Flt3L allowed
us to examine the potential role of DC in determining immune
responsiveness following oral administration of Ag. We decided to
investigate whether the increased numbers of DC would influence oral
tolerance induction using a well-established model in which mice are
fed soluble OVA before parenteral challenge (1, 19). As expected,
PBS-treated mice fed 25 mg of OVA before immunization with OVA plus
adjuvant displayed markedly reduced DTH responses when rechallenged
with OVA in vivo compared with saline-fed immunized control mice (Fig. 6A). Cells from the
draining LN of these animals also showed suppressed Ag-specific
proliferative reactivity after restimulation with OVA in vitro (Fig. 6B). T cell responses in vivo and in vitro were
essentially normal in PBS-treated mice fed 0.5 or 0.01 mg of OVA (Fig. 6). These results are consistent with many previous reports (1, 19, 30)
and illustrate the dose-dependent tolerogenic effects of OVA feeding on
T cell-dependent immune responses.
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). The
enhanced unresponsiveness induced by Flt3L treatment was illustrated
most clearly in mice that were fed low doses of Ag. Mice pretreated
with Flt3L before being fed 0.5 or 0.01 mg of OVA displayed
significantly decreased T cell responses, unlike PBS-treated animals
fed the same doses (Fig. 6, A and B). The
tolerance induced by low doses of fed OVA was generally of a magnitude
comparable to that seen in PBS-treated mice fed the highest dose
of Ag.
A similar pattern of enhanced tolerance induction in Flt3L-treated mice
was seen when humoral immunity was examined. Ag-specific total IgG,
IgG1, and IgG2a levels in the sera of PBS-treated mice fed 25 mg of OVA
were significantly decreased compared with those in immunized mice,
while feeding low doses (0.5 or 0.01 mg) had no effect (Fig. 7). In contrast, humoral immune responses
were significantly decreased in mice that were fed all doses of OVA
after treatment with Flt3L (Fig. 7). Notably, the levels of both IgG1
and IgG2a isotypes were decreased to similar extents in these mice,
suggesting that Th2- and Th1-dependent immune responses were equally
diminished in the Flt3L-treated OVA-fed mice.
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To evaluate further the ability of Flt3L-mediated DC expansion to enhance the induction of tolerance in specific T cells by feeding Ag, we used an adoptive transfer model in which OVA-specific transgenic T cells from D011.10 mice are transferred into normal syngeneic BALB/c hosts. Previous studies have shown this to be an appropriate means of studying peripheral and oral tolerance of T cells in a relatively physiologic manner (20, 21), and transgenic T cells can be easily detected with the clonotypic Ab (KJ1.26) to the transgenic TCR.
When adoptively transferred PBS-treated mice were fed saline and
immunized with OVA in adjuvant, there was a marked expansion of
transgenic cells in the draining LN. The relative proportion and the
absolute number of transgenic T cells were significantly decreased in
PBS-treated mice fed 25 mg of OVA before immunization compared with
saline-fed PBS-treated mice (Fig. 8). The expansion of
transgenic T cells in Flt3L-treated adoptive transfer mice fed saline
before immunization was comparable to that of PBS-treated mice fed
saline. As in PBS-treated mice, the relative proportion and the
absolute number of transgenic cells were decreased in Flt3L-treated
mice fed 25 mg of OVA before immunization (Fig. 8). The major
difference between PBS-treated and Flt3L-treated mice was most evident
in animals fed a low dose of OVA. Feeding 1 mg of OVA to PBS-treated
mice did not prevent the expansion of Ag-specific T cells, whereas
feeding 1 mg of OVA to Flt3L-treated mice did result in a decrease in
both the relative proportion and absolute number of transgenic T cells
(Fig. 8).
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The reduced expansion of transgenic T cells in OVA fed
Flt3L-treated adoptive transfer mice was accompanied by enhanced
functional tolerance (Fig. 9). LN cells
from both PBS- and Flt3L-treated mice that were fed saline before
immunization proliferated vigorously in response to OVA stimulation in
vitro, whereas cells isolated from PBS- and Flt3L-treated mice fed 25
mg of OVA showed significantly decreased proliferative responses.
Again, the differences between PBS- and Flt3L-treated mice were most
evident in animals fed a low dose of Ag. Cells from the Flt3L-treated
mice fed 1 mg of OVA had significantly decreased Ag-specific
proliferative capability compared with equivalently fed PBS-treated
mice and saline-fed animals (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The way in which intestinally derived Ags are processed and presented to T cells is likely to be a critical factor in determining how the mucosal immune system can distinguish pathogens from harmless Ags. Ag presentation in the intestine has been the focus of many studies, and the gut contains many different types of conventional APC, including DC, B cells, and macrophages, as well as other putative APC, such as MHCII-expressing epithelial cells (reviewed in 31 . The relative contributions of these different APC types are likely to be an important factor in determining whether active immunity or tolerance is induced at any particular time. The data presented here show clearly that increasing the numbers of DC in vivo, by treating mice with Flt3L, enhances the induction of tolerance in two models induced by feeding soluble protein. In the first, we found that conventional mice fed OVA developed a more profound systemic tolerance when treated with Flt3L before feeding, and this was particularly notable in mice fed low doses of Ag. In the second model, we used a recently characterized adoptive transfer system to assess directly the behavior of Ag-specific T cells following the induction of oral tolerance (21). In these experiments we confirmed that the expansion of Ag-specific transgenic T cells in response to systemic challenge is reduced after oral tolerance induction (21) and observed that the effect is augmented by Flt3L.
At first sight these results are not consistent with the classical assumption that DC have an apparent constitutive ability to activate T cells or with previous findings that intestinal DC isolated from protein-fed mice can be potently immunogenic (11). These discrepancies may reflect the fact that most previous studies have used isolated DC, employing protocols likely to induce costimulatory molecule expression (13, 14). Since a role for DC in the induction of tolerance may have been overlooked previously due to the use of inadvertently activated cells, we decided to explore the function of DC under physiologic conditions in situ. As we confirm here, the DC expanded by Flt3L in vivo are apparently in a resting state, with low or no expression of CD80, CD86. However, these resting DC remain fully responsive to inflammatory stimuli, as administration of LPS to Flt3L-treated mice induces significant increases in the expression of MHCII, CD80, CD86, and CD40 on the DC. These cells are thus in a good position to regulate the nature of the immune response to intestinal Ags depending on the presence or the absence of appropriate inflammatory signals. We are currently testing the conjecture that the enhanced tolerance associated with expansion of DC will be reversed by converting the DC from a resting phenotype to an activated phenotype. Our hypothesis that DC may be involved in oral tolerance induction is further supported by studies which show that targeting DC with Abs in vivo does allow these cells to present Ag in a tolerogenic as well as an immunogenic fashion (34). In addition, it has been previously suggested that donor-derived hepatic DC may be involved in the maintenance of donor-specific unresponsiveness in liver allograft recipients (35, 36).
How does increasing the number of DC lead to enhanced tolerance? One possibility is that there are simply more DC accessible to pick up the available Ag, and therefore, there is an increased probability that more Ag-specific T cells can interact with a tolerogenic Ag/APC complex. However, this still leaves the question of whether DC can be tolerogenic APC. There is accumulating evidence that it is the qualitative nature of the interaction between APC and Ag-specific T cells that determines whether tolerance is induced. It has been hypothesized that T cell tolerance occurs when APC expressing low levels of CD80/86 interact with naive T cells via the high affinity CTLA4 receptor in preference to the CD28 receptor, which delivers an activating stimulus after interacting with APC expressing high levels of CD80/86. This hypothesis is supported by a recent study demonstrating that peripheral tolerance can be prevented by using neutralizing Abs directed against CTLA4 (37). Our data show that DC in the intestine normally express minimal levels of CD80/86 and therefore may be potential candidates for presenting intestinally derived Ags to T cells in a tolerogenic fashion. Studies are currently underway investigating the roles of CD80/86 and CTLA4 interactions in the induction of oral tolerance.
The intestine clearly provides a unique environment allowing for the development of both tolerogenic and immunogenic responses, but whether this is regulated purely at the level of costimulatory molecule expression on a homogeneous population of DC is unclear. An alternative explanation might be that discrete subsets of intestinal DC are responsible for different functions. Recent studies of DC have identified at least two distinct lineages of cells derived from different precursors, and some evidence indicates that these populations might be functionally distinct (15, 23, 24, 25, 26, 27, 28). In our studies, all the putative lineages and subpopulations of DC were present in intestinal tissues of Flt3L-treated mice. Their proportions were relatively similar to those found elsewhere, and we were unable to identify any novel subsets of cells in the GALT, suggesting that the intestine is unlikely to harbor a unique regulatory DC population.
Although we observe enhanced tolerance associated with Flt3L treatment, the origin and the precise location of the DC involved in induction of oral tolerance remain to be clarified. Is there a population of tolerogenic DC present in the gut in normal mice, or are tolerogenic DC recruited de novo by Flt3L? Our immunohistologic analysis shows that the greatest increases in the numbers of CD11c+ cells in Flt3L-treated mice can be seen in the tissues of the intestine, and these cells are distributed throughout the mucosal compartments. Furthermore, a significant proportion of these mucosal DC appear to be dividing in situ in response to Flt3L treatment, suggesting that there might be a local expansion of DC from immature precursors. It is feasible, then, that a substantial amount of fed Ag associates preferentially with a locally derived population of tolerogenic DC in the GALT of Flt3L-treated mice. This is currently under investigation, and we have some preliminary observations that suggest that only APC from the lamina propria of Flt3L-treated mice are Ag loaded following OVA feeding, with little evidence of Ag loading of APC from other sites. These data appear consistent with previous reports suggesting that the intestinal lamina propria contains a population of APC with selective ability to induce tolerance (8). However, in view of the known ability of fed Ag to disseminate rapidly to the systemic circulation, a contribution to tolerogenic Ag presentation by extraintestinal DC cannot be excluded. It is important to note that Flt3L treatment does not alter the gross architecture of the gut, and there is no histologic evidence of mucosal pathology. In addition, Flt3L treatment does not appear to have any significant effect on the numbers of other mature cell types, confirming previous studies that mature B and T cells remain unaffected in Flt3L-treated animals (15).
The mechanisms by which oral tolerance is mediated in the presence of enhanced numbers of DC is unclear, and it is not known whether these are similar to the ones found normally or if novel mechanisms become involved. Currently, it is believed that feeding single high doses of protein might lead to T cell anergy/deletion, while feeding multiple low doses of protein leads to tolerance through active suppression of immune responsiveness mediated by the induction of regulatory cells and a shift in cytokine production (3). In confirmation of the results of previous studies using the adoptive transfer model, we observed reduced expansion of transgenic T cells in mice that had been tolerized with a high dose of Ag orally before systemic challenge, and those cells remaining exhibited functional tolerance. Similar results were evident in Flt3L-treated mice fed a single low dose of Ag, perhaps suggesting that the mechanistic process of tolerance induction is comparable in Flt3L-treated mice following a single feeding of either a high or a low dose of Ag. Although these findings could reflect either clonal deletion or anergy of Ag-specific T cells following contact with fed Ag, it is tempting to speculate that the marked decrease in the absolute numbers of T cells after the induction of tolerance indicates that deletion may be the dominant mechanism in Flt3L-treated mice. Proof of this idea will require direct assessment of apoptosis in Ag-specific T cells in Ag-fed mice. In addition to the hypothesis that clonal deletion and anergy of Ag-specific T cells can give rise to tolerance, there is a great deal of evidence suggesting that tolerance induced by feeding multiple low doses of Ag is mediated by regulatory cells that secrete cytokines such as IL-4, IL-10, and TGF-ß (3). Since we used only single dose feedings throughout this study, it is not clear whether the tolerance seen in Flt3L-treated mice following low dose feeding will be mediated by this type of active suppression. We are currently in the process of determining whether there is evidence for cytokine biasing and increased induction or recruitment of regulatory cells in Flt3L-treated mice following Ag feeding.
The ability to modulate oral tolerance induction with Flt3L in vivo highlights a potentially central role for DC in regulating mucosal immune responses. This has important implications for investigating how and where orally administered proteins are processed and presented to the immune system. Thus, it should now be possible to explore where the initial contact between APC and T cell occurs after Ag feeding, to determine the subsequent fate of Ag-reactive T cells, and to define the immunologic consequences of these interactions more precisely. These investigations will help in understanding the mechanisms of oral tolerance as well as increase our understanding of how the mucosal immune system discriminates between harmful and beneficial Ags. This information will assist vaccine design and aid in designing regimens aimed at applying mucosal tolerance as a treatment for human disease, where oral tolerance has been promoted as therapy for a number of clinical disorders, including rheumatoid arthritis, multiple sclerosis, and uveitis (reviewed in 3 .
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cells; Flt3L, Flt3 ligand; GALT, gut-associated lymphoid tissues; PP, Peyer’s patch; PLN, popliteal lymph nodes; SILP, small intestine lamina propria; LN, lymph node; PE, phycoerythrin; BrdUrd, bromodeoxyuridine; DTH, delayed-type hypersensitivity; MHCII, major histocompatibility complex class II.
Received for publication October 31, 1997. Accepted for publication February 13, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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/ß+ and 
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/ T cells in the gut epithelium. Gut 31:841.This article has been cited by other articles:
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J. N. Samsom, A. P. J. van der Marel, L. A. van Berkel, J. M. L. M. van Helvoort, Y. Simons-Oosterhuis, W. Jansen, M. Greuter, R. L. H. Nelissen, C. M. L. Meeuwisse, E. E. S. Nieuwenhuis, et al. Secretory Leukoprotease Inhibitor in Mucosal Lymph Node Dendritic Cells Regulates the Threshold for Mucosal Tolerance J. Immunol., November 15, 2007; 179(10): 6588 - 6595. [Abstract] [Full Text] [PDF]
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 O. Pabst, G. Bernhardt, and R. Forster The impact of cell-bound antigen transport on mucosal tolerance induction J. Leukoc. Biol., October 1, 2007; 82(4): 795 - 800. [Abstract] [Full Text] [PDF]
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B. Corthesy Roundtrip Ticket for Secretory IgA: Role in Mucosal Homeostasis? J. Immunol., January 1, 2007; 178(1): 27 - 32. [Abstract] [Full Text] [PDF]
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 U. I. Chaudhry, S. C. Katz, T. P. Kingham, V. G. Pillarisetty, J. R. Raab, A. B. Shah, and R. P. DeMatteo In vivo overexpression of Flt3 ligand expands and activates murine spleen natural killer dendritic cells FASEB J, May 1, 2006; 20(7): 982 - 984. [Abstract] [Full Text] [PDF]
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 T. Worbs, U. Bode, S. Yan, M. W. Hoffmann, G. Hintzen, G. Bernhardt, R. Forster, and O. Pabst Oral tolerance originates in the intestinal immune system and relies on antigen carriage by dendritic cells J. Exp. Med., March 20, 2006; 203(3): 519 - 527. [Abstract] [Full Text] [PDF]
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A. J. Macpherson and K. Smith Mesenteric lymph nodes at the center of immune anatomy J. Exp. Med., March 20, 2006; 203(3): 497 - 500. [Abstract] [Full Text] [PDF]
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 L. H. Zeuthen, H. R. Christensen, and H. Frokiaer Lactic Acid Bacteria Inducing a Weak Interleukin-12 and Tumor Necrosis Factor Alpha Response in Human Dendritic Cells Inhibit Strongly Stimulating Lactic Acid Bacteria but Act Synergistically with Gram-Negative Bacteria. Clin. Vaccine Immunol., March 1, 2006; 13(3): 365 - 375. [Abstract] [Full Text] [PDF]
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J O Lindsay, K Whelan, A J Stagg, P Gobin, H O Al-Hassi, N Rayment, M A Kamm, S C Knight, and A Forbes Clinical, microbiological, and immunological effects of fructo-oligosaccharide in patients with Crohn's disease Gut, March 1, 2006; 55(3): 348 - 355. [Abstract] [Full Text] [PDF]
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J. N. Samsom, L. A. van Berkel, J. M. L. M. van Helvoort, W. W. J. Unger, W. Jansen, T. Thepen, R. E. Mebius, S. S. Verbeek, and G. Kraal Fc{gamma}RIIB Regulates Nasal and Oral Tolerance: A Role for Dendritic Cells J. Immunol., May 1, 2005; 174(9): 5279 - 5287. [Abstract] [Full Text] [PDF]
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K. Akadegawa, S. Ishikawa, T. Sato, J. Suzuki, H. Yurino, M. Kitabatake, T. Ito, T. Kuriyama, and K. Matsushima Breakdown of Mucosal Immunity in the Gut and Resultant Systemic Sensitization by Oral Antigens in a Murine Model for Systemic Lupus Erythematosus J. Immunol., May 1, 2005; 174(9): 5499 - 5506. [Abstract] [Full Text] [PDF]
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D. W. Smith and C. Nagler-Anderson Preventing Intolerance: The Induction of Nonresponsiveness to Dietary and Microbial Antigens in the Intestinal Mucosa J. Immunol., April 1, 2005; 174(7): 3851 - 3857. [Abstract] [Full Text] [PDF]
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 M. O'Keeffe, T. C. Brodnicki, B. Fancke, D. Vremec, G. Morahan, E. Maraskovsky, R. Steptoe, L. C. Harrison, and K. Shortman Fms-like tyrosine kinase 3 ligand administration overcomes a genetically determined dendritic cell deficiency in NOD mice and protects against diabetes development Int. Immunol., March 1, 2005; 17(3): 307 - 314. [Abstract] [Full Text] [PDF]
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A L Hart, K Lammers, P Brigidi, B Vitali, F Rizzello, P Gionchetti, M Campieri, M A Kamm, S C Knight, and A J Stagg Modulation of human dendritic cell phenotype and function by probiotic bacteria Gut, November 1, 2004; 53(11): 1602 - 1609. [Abstract] [Full Text] [PDF]
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B. J. Masten, G. K. Olson, D. F. Kusewitt, and M. F. Lipscomb Flt3 Ligand Preferentially Increases the Number of Functionally Active Myeloid Dendritic Cells in the Lungs of Mice J. Immunol., April 1, 2004; 172(7): 4077 - 4083. [Abstract] [Full Text] [PDF]
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K. Kataoka, J. R. McGhee, R. Kobayashi, K. Fujihashi, S. Shizukuishi, and K. Fujihashi Nasal Flt3 Ligand cDNA Elicits CD11c+CD8+ Dendritic Cells for Enhanced Mucosal Immunity J. Immunol., March 15, 2004; 172(6): 3612 - 3619. [Abstract] [Full Text] [PDF]
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I. T. Tobagus, W. R. Thomas, and P. G. Holt Adjuvant Costimulation during Secondary Antigen Challenge Directs Qualitative Aspects of Oral Tolerance Induction, Particularly during the Neonatal Period J. Immunol., February 15, 2004; 172(4): 2274 - 2285. [Abstract] [Full Text] [PDF]
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C. Johansson and M. J. Wick Liver Dendritic Cells Present Bacterial Antigens and Produce Cytokines upon Salmonella Encounter J. Immunol., February 15, 2004; 172(4): 2496 - 2503. [Abstract] [Full Text] [PDF]
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B.-G. Xiao, X.-C. Wu, J.-S. Yang, L.-Y. Xu, X. Liu, Y.-M. Huang, B. Bjelke, and H. Link Therapeutic potential of IFN-{gamma}-modified dendritic cells in acute and chronic experimental allergic encephalomyelitis Int. Immunol., January 1, 2004; 16(1): 13 - 22. [Abstract] [Full Text] [PDF]
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A J Stagg, A L Hart, S C Knight, and M A Kamm The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria Gut, October 1, 2003; 52(10): 1522 - 1529. [Abstract] [Full Text] [PDF]
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R. Pabst, A. Luhrmann, I. Steinmetz, and T. Tschernig A Single Intratracheal Dose of the Growth Factor Fms-Like Tyrosine Kinase Receptor-3 Ligand Induces a Rapid Differential Increase of Dendritic Cells and Lymphocyte Subsets in Lung Tissue and Bronchoalveolar Lavage, Resulting in an Increased Local Antibody Production J. Immunol., July 1, 2003; 171(1): 325 - 330. [Abstract] [Full Text] [PDF]
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H. Beacock-Sharp, A. M. Donachie, N. C. Robson, and A. M. Mowat A role for dendritic cells in the priming of antigen-specific CD4+ and CD8+ T lymphocytes by immune-stimulating complexes in vivo Int. Immunol., June 1, 2003; 15(6): 711 - 720. [Abstract] [Full Text] [PDF]
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H. Kato, K. Fujihashi, R. Kato, T. Dohi, K. Fujihashi, Y. Hagiwara, K. Kataoka, R. Kobayashi, and J. R. McGhee Lack of oral tolerance in aging is due to sequential loss of Peyer's patch cell interactions Int. Immunol., February 1, 2003; 15(2): 145 - 158. [Abstract] [Full Text] [PDF]
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E. Williamson, J. M. Bilsborough, and J. L. Viney Regulation of Mucosal Dendritic Cell Function by Receptor Activator of NF-{kappa}B (RANK)/RANK Ligand Interactions: Impact on Tolerance Induction J. Immunol., October 1, 2002; 169(7): 3606 - 3612. [Abstract] [Full Text] [PDF]
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R. L. Jump and A. D. Levine Murine Peyer's Patches Favor Development of an IL-10-Secreting, Regulatory T Cell Population J. Immunol., June 15, 2002; 168(12): 6113 - 6119. [Abstract] [Full Text] [PDF]
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 M. L. Disis, K. Rinn, K. L. Knutson, D. Davis, D. Caron, C. dela Rosa, and K. Schiffman Flt3 ligand as a vaccine adjuvant in association with HER-2/neu peptide-based vaccines in patients with HER-2/neu-overexpressing cancers Blood, April 15, 2002; 99(8): 2845 - 2850. [Abstract] [Full Text] [PDF]
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T. Teshima, P. Reddy, K. P. Lowler, M. A. KuKuruga, C. Liu, K. R. Cooke, and J. L. M. Ferrara Flt3 ligand therapy for recipients of allogeneic bone marrow transplants expands host CD8alpha + dendritic cells and reduces experimental acute graft-versus-host disease Blood, March 1, 2002; 99(5): 1825 - 1832. [Abstract] [Full Text] [PDF]
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H. R. Christensen, H. Frokiar, and J. J. Pestka Lactobacilli Differentially Modulate Expression of Cytokines and Maturation Surface Markers in Murine Dendritic Cells J. Immunol., January 1, 2002; 168(1): 171 - 178. [Abstract] [Full Text] [PDF]
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 A. George-Chandy, K. Eriksson, M. Lebens, I. Nordstrom, E. Schon, and J. Holmgren Cholera Toxin B Subunit as a Carrier Molecule Promotes Antigen Presentation and Increases CD40 and CD86 Expression on Antigen-Presenting Cells Infect. Immun., September 1, 2001; 69(9): 5716 - 5725. [Abstract] [Full Text] [PDF]
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N. Etchart, P.-O. Desmoulins, K. Chemin, C. Maliszewski, B. Dubois, F. Wild, and D. Kaiserlian Dendritic Cells Recruitment and In Vivo Priming of CD8+ CTL Induced by a Single Topical or Transepithelial Immunization Via the Buccal Mucosa with Measles Virus Nucleoprotein J. Immunol., July 1, 2001; 167(1): 384 - 391. [Abstract] [Full Text] [PDF]
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T. De Smedt, E. Butz, J. Smith, R. Maldonado-Lopez, B. Pajak, M. Moser, and C. Maliszewski CD8{alpha}- and CD8{alpha}+ subclasses of dendritic cells undergo phenotypic and functional maturation in vitro and in vivo J. Leukoc. Biol., June 1, 2001; 69(6): 951 - 958. [Abstract] [Full Text] [PDF]
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A. L. Meyer, J. Benson, F. Song, N. Javed, I. E. Gienapp, J. Goverman, T. A. Brabb, L. Hood, and C. C. Whitacre Rapid Depletion of Peripheral Antigen-Specific T Cells in TCR-Transgenic Mice After Oral Administration of Myelin Basic Protein J. Immunol., May 1, 2001; 166(9): 5773 - 5781. [Abstract] [Full Text] [PDF]
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O. Alpan, G. Rudomen, and P. Matzinger The Role of Dendritic Cells, B Cells, and M Cells in Gut-Oriented Immune Responses J. Immunol., April 15, 2001; 166(8): 4843 - 4852. [Abstract] [Full Text] [PDF]
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S. J. Bell, R. Rigby, N. English, S. D. Mann, S. C. Knight, M. A. Kamm, and A. J. Stagg Migration and Maturation of Human Colonic Dendritic Cells J. Immunol., April 15, 2001; 166(8): 4958 - 4967. [Abstract] [Full Text] [PDF]
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 P. L. Ogra, H. Faden, and R. C. Welliver Vaccination Strategies for Mucosal Immune Responses Clin. Microbiol. Rev., April 1, 2001; 14(2): 430 - 445. [Abstract] [Full Text] [PDF]
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 K. Fujihashi, T. Dohi, P. D. Rennert, M. Yamamoto, T. Koga, H. Kiyono, and J. R. McGhee Peyer's patches are required for oral tolerance to proteins PNAS, March 13, 2001; 98(6): 3310 - 3315. [Abstract] [Full Text] [PDF]
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I. B. Kremer, M. P. Gould, K. D. Cooper, and F. P. Heinzel Pretreatment with Recombinant Flt3 Ligand Partially Protects against Progressive Cutaneous Leishmaniasis in Susceptible BALB/c Mice Infect. Immun., February 1, 2001; 69(2): 673 - 680. [Abstract] [Full Text] [PDF]
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 A. Hayday and J. L. Viney The Ins and Outs of Body Surface Immunology Science, October 6, 2000; 290(5489): 97 - 100. [Abstract] [Full Text]
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 K. M. SMITH, A. D. EATON, L. M. FINLAYSON, and P. GARSIDE Oral Tolerance Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): S175 - 178. [Abstract] [Full Text] [PDF]
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A. E. Morelli, M. A. Antonysamy, T. Takayama, H. Hackstein, Z. Chen, S. Qian, N. B. Zurowski, and A. W. Thomson Microchimerism, Donor Dendritic Cells, and Alloimmune Reactivity in Recipients of Flt3 Ligand-Mobilized Hemopoietic Cells: Modulation by Tacrolimus J. Immunol., July 1, 2000; 165(1): 226 - 237. [Abstract] [Full Text] [PDF]
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C. Desvignes, N. Etchart, J. Kehren, I. Akiba, J.-F. Nicolas, and D. Kaiserlian Oral Administration of Hapten Inhibits In Vivo Induction of Specific Cytotoxic CD8+ T Cells Mediating Tissue Inflammation: A Role for Regulatory CD4+ T Cells J. Immunol., March 1, 2000; 164(5): 2515 - 2522. [Abstract] [Full Text] [PDF]
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F.-P. Huang, N. Platt, M. Wykes, J. R. Major, T. J. Powell, C. D. Jenkins, and G. G. MacPherson A Discrete Subpopulation of Dendritic Cells Transports Apoptotic Intestinal Epithelial Cells to T Cell Areas of Mesenteric Lymph Nodes J. Exp. Med., February 7, 2000; 191(3): 435 - 444. [Abstract] [Full Text] [PDF]
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J L VINEY Dendritic cell subsets: the ultimate T cell differentiation decision makers? Gut, November 1, 1999; 45(5): 640 - 641. [Full Text] [PDF]
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E. Williamson, G. M. Westrich, and J. L. Viney Modulating Dendritic Cells to Optimize Mucosal Immunization Protocols J. Immunol., October 1, 1999; 163(7): 3668 - 3675. [Abstract] [Full Text] [PDF]
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Y. Chung, S.-Y. Chang, and C.-Y. Kang Kinetic Analysis of Oral Tolerance: Memory Lymphocytes Are Refractory to Oral Tolerance J. Immunol., October 1, 1999; 163(7): 3692 - 3698. [Abstract] [Full Text] [PDF]
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J. Sun, B. Dirden-Kramer, K. Ito, P. B. Ernst, and N. Van Houten Antigen-Specific T Cell Activation and Proliferation During Oral Tolerance Induction J. Immunol., May 15, 1999; 162(10): 5868 - 5875. [Abstract] [Full Text] [PDF]
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J. M. Benson, S. S. Stuckman, K. L. Cox, R. M. Wardrop, I. E. Gienapp, A. H. Cross, J. L. Trotter, and C. C. Whitacre Oral Administration of Myelin Basic Protein Is Superior to Myelin in Suppressing Established Relapsing Experimental Autoimmune Encephalomyelitis J. Immunol., May 15, 1999; 162(10): 6247 - 6254. [Abstract] [Full Text] [PDF]
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B. Pulendran, J.L. Smith, M. Jenkins, M. Schoenborn, E. Maraskovsky, and C.R. Maliszewski Prevention of Peripheral Tolerance by a Dendritic Cell Growth Factor: Flt3 Ligand as an Adjuvant J. Exp. Med., December 7, 1998; 188(11): 2075 - 2082. [Abstract] [Full Text] [PDF]
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) footpad response ± 1 SEM from groups of
five mice and are representative of three experiments from C57BL/6
mice. Similar results were observed in BALB/c mice. B,
OVA-specific proliferation in vitro. Draining LN cells isolated from
Ag-fed control mice exhibited significantly decreased proliferative
responses to OVA compared with those in saline-fed control mice (##
indicates p < 0.01). All groups of mice treated with
Flt3L and fed OVA displayed significantly decreased proliferative
responses compared with those in saline-fed Flt3L-treated mice (##
indicates p < 0.01). The Flt3L-treated OVA-fed mice
were significantly more tolerant than equivalently fed control mice (*
indicates p < 0.05; ** indicates p <
0.01). The data are reported as the mean counts per minute ± 1
SEM of quadruplicate wells and are representative of three experiments
from C57BL/6 mice. Similar results were observed in BALB/c mice.
