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Originally published online as doi:10.1189/jlb.0605342 on October 4, 2005

Published online before print October 4, 2005
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(Journal of Leukocyte Biology. 2005;78:1291-1300.)
© 2005 by Society for Leukocyte Biology

Colon lamina propria dendritic cells induce a proinflammatory cytokine response in lamina propria T cells in the SCID mouse model of colitis

Maureen L. Drakes1, Thomas G. Blanchard and Steven J. Czinn

Department of Pediatrics, Case Western Reserve University School of Medicine, Cleveland, Ohio

1 Correspondence: Case Western Reserve University School of Medicine, Department of Pediatrics, Rm. 737, Rainbow Babies and Children’s Hospital, 11100 Euclid Avenue, Cleveland, OH 44106. E-mail: mld19{at}cwru.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intestinal immune responses are normally regulated to maintain a state of immune balance. Dendritic cells (DC) are antigen-presenting cells, which induce immune responses against microbes and other stimuli and are key players in the regulation of tolerance in the gut. These cells influence the differentiation of cytokine responses in T cells, and in the gut, in particular, such interactions may be critical to the course of inflammatory bowel disease (IBD). Using the CD45RBhi CD4+ T cell-reconstituted severe combined immunodeficient mouse model of colitis, we investigated the ability of isolated colon DC to stimulate immune responses in syngeneic and allogeneic spleen CD4+ T cells, as well as in colon T cells isolated from the same tissue as DC in IBD mice. We found that the frequency of DC in IBD mice colons and spleens was elevated in comparison with control mice, but colon and spleen DC exhibited different phenotypic and functional properties. Colon DC stimulated significantly higher levels of interferon-{gamma} and interleukin-6 when cocultured with autologous colon T cells than in cocultures with syngeneic or allogeneic spleen T cells. These data suggest that in the IBD colon, DC-T cell interactions may create conditions with an abundance of proinflammatory cytokines, which favor the inflammatory state.

Key Words: T helper 1 cytokines • inflammatory bowel disease • intestinal inflammation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Several laboratories have reported an increase of mononuclear cells in the lamina propria of the colons of mice with inflammatory bowel disease (IBD) [1 2 3 ]. The inflammatory process is amplified and driven by the interaction of antigen-presenting cells and lymphocytes and by locally produced, soluble immunoregulatory mediators. Dendritic cells (DC) are antigen-processing cells recognized as crucial immune regulators [4 5 6 7 8 ]. These cells process antigen and present antigenic peptides to T cells, which subsequently mount an adaptive immune response against potentially pathogenic organisms. Furthermore, DC can be activated directly by pathogen-associated molecular patterns (PAMPs) to secrete regulatory cytokines, and hence, these cells bridge the innate and adaptive immune responses. Recognition of PAMPs is made possible through Toll-like receptors on DC cell surfaces [9 10 11 12 ].

Small numbers of DC are distributed throughout the body, including many sites in the intestine [13 14 15 ]. In the normal intestine, they sample luminal bacteria for presentation of antigen to T cells [16 , 17 ]. These cells play a significant role in maintaining immune tolerance to food particles, endogenous microbes, and other gut antigens [18 ]. Recent evidence indicates that cytokines produced by DC in the intestine may be important in IBD pathogenesis [1 , 19 ]. To address the problem of the immunological events occurring in the IBD gut, it is essential to create a model that mimics the cellular interactions occurring between cells at this site. In the literature, detailed, functional studies of gut lamina propria DC (LPDC) are lacking, primarily as a result of the difficulty in isolating these cells in sufficient numbers with high purity. In fact, a recent study performed to evaluate the function of colon DC has used limited numbers of cells or semipurified colonic DC populations [20 ]. We have adopted a novel approach to decipher the cellular mechanism of DC-T cell stimulation in the severe combined immunodeficient (SCID) mouse model of IBD. This model of IBD is established by the transfer of CD45RBhi CD4+ syngeneic donor T cells into SCID mouse recipients [21 , 22 ], which subsequently develop colitis in response to endogenous gut flora.

Using freshly isolated and purified colon DC and spleen DC, we investigated the ability of these cells to stimulate T helper cell type 1 (Th1) and Th2 cytokines in coculture with T cells, as well as their potential to induce T cell proliferation. We now demonstrate that the T cell-reconstituted SCID mouse model of colitis yields approximately a tenfold increase in DC numbers in the colon lamina propria in comparison with normal mice. When colon LPDC were used to stimulate autologous colon T cells retrieved from the same tissue of IBD mice, significantly higher interferon-{gamma} (IFN-{gamma}) and interleukin (IL)-6 levels were observed above the levels released with stimulation of LPDC-allogeneic T cells.

Our data support the hypothesis that the limited numbers of lamina propria mononuclear cells (LPMC) in unmanipulated control mice, combined with the rarity (low frequency) of DC in this population, limit the likelihood of significant DC-T cell interaction in vivo and hence, do not favor IBD pathology. We propose that the increased presence of these cells at a site in which they typically exist at low frequency provides conditions for persistent, robust interaction with T cells, generating substantial amounts of proinflammatory mediators, which disrupt the balance from tolerance to inflammation. The stimulation of colon T cells with colon DC and the cytokines elicited provides a crucial environment favoring the immune pathogenesis of IBD in the colons of mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Adult (8- to 12-week-old) female BALB/c (H-2d) and C.B-17 SCID mice were purchased from Taconic Laboratories (Germantown, NY) or bred and maintained in a specific, pathogen-free environment in the animal facility at the Case Western Reserve University School of Medicine (Cleveland, OH) in accordance with institutional guidelines. C57BL/6J (H-2b) mice were purchased from the Jackson Laboratories (Bar Harbor, ME).

Adoptive transfer of T cells and induction of colitis in SCID mice
Colitis/IBD was induced in SCID mice as described previously [21 , 22 ]. Briefly, spleen cells were prepared from donor BALB/c mice and labeled with Thy1 (midiMACS, Miltenyi Biotec, Auburn, CA) magnetic cell sorter (MACS) beads for the enrichment of T cells on MACS columns according to the manufacturer’s instructions. Recovered T cells were monoclonal antibody (mAb)-stained with CD4 phycoerythrin (PE; L3T4 GK.1.5, PharMingen, San Diego, CA) and CD45RB fluorescein isothiocyanate (FITC; C363.16A, PharMingen) on ice for 30 min. Cells were sorted on a FACSAria sorter (Becton Dickinson, San Jose, CA), collecting the 35% most double-positive T cells. SCID mice were reconstituted with 0.7–1 x 106 T cells by intravenous injection of cells in 400 µl phosphate-buffered saline (PBS) in the lateral tail vein.

Clinical observation of mice
Mice were weighed on a weekly basis and observed for signs of diarrhea. When soft stools were evident, mice were monitored every other day. Diarrhea was induced in T cell-reconstituted SCID mice within 5–12 weeks after T cell transfer. On sacrifice, colons and spleens were harvested for isolation of cells. Gross pathology revealed enlarged and thickened colons in colitic mice as compared with BALB/c or SCID mice controls. In addition, ~90% of colitic SCID mice exhibited splenomegaly.

Isolation and purification of LPDC
Digestion of colon tissue was performed on pooled colons from colitic SCID mice or BALB/c mice using a modification of an earlier method [23 ]. Briefly, each colon was flushed with 30 cc Hanks’ balanced salt solution (HBSS; Ca2+-, Mg2+-free), cut longitudinally, and the inner surface cleaned with a cell scraper. After mincing with scissors, release of epithelial cells from tissue was performed in HBSS supplemented with 5% fetal bovine serum (FBS; Gibco-BRL, Life Technologies, Grand Island, NY), EDTA (0.05 mM), Hepes buffer (0.6 mM), penicillin/streptomycin/fungizone (1.2%, Cambrex Bioscience, Walkersville, MD), and 15 µg/ml dithiothreitol at 37°C for 15 min. Samples were filtered through a 100-µm filter to remove epithelial cells from the tissue. A further disaggregation of tissue was performed in the HBSS buffer and then in RPMI medium (Gibco-BRL, Life Technologies), supplemented with 10% FBS, penicillin (100 units/ml)/streptomycin (100 µg/ml, Gibco-BRL, Life Technologies), and Hepes buffer, each time incubating tissue for 15 min. After filtering, tissue was digested in a solution of collagenase D (Boehringer Mannheim, Germany, 1.75 mg/ml) and DNase (Boehringer Mannheim, 0.05 mg/ml) in RPMI medium for 50 min at 37°C with intermittent pipetting. The suspension containing released LPMC was filtered, collected, and washed twice in RPMI medium with supplements. CD11c+ LPMC were fractionated with magnetic beads based on CD11c expression, and selection was performed on MACS columns (Miltenyi Biotec). Any residual epithelial cells were excluded from the recovered cell fraction in the final step of purification, in which cells were stained with CD11c PE mAb and sorted on a Becton Dickinson FACSAria to obtain a purity of typically >98% CD11c+ cells.

Isolation of spleen DC
Spleens were harvested from mice, pooled, and a single-cell suspension prepared by conventional means. In addition, to maximize the yield of DC from spleen tissue, the spleen capsules were cut into fine pieces, and collagenase digestion was performed as above for the isolation of LPDC. In some experiments, the entire spleen preparation was incubated for an additional 10 min in collagenase/DNase solution at 37°C to ensure that spleens and colons received enzyme exposure. We did not notice any variations in results, whether or not this additional collagenase treatment of spleens was included. Spleen CD11c+ cells were enriched on MACS columns, followed by cell sorting on a Becton Dickinson FACSAria, as described above for LPMC. The morphology of DC was determined on Giemsa-stained cytocentrifuge preparations.

DC phenotypes
To determine the expression levels of cell-surface antigens on DC, immunostaining was performed using a panel of fluorescent-labeled mAb. Briefly, DC were preincubated with the Fc receptor for immunoglobulin G (IgG)III/II antibody (CD16/CD32, PharMingen) for 10 min to block nonspecific antibody staining. Cells were resuspended at 2–5 x 105 cells in 100 µl staining buffer (PBS, 1% FBS, 0.1% sodium azide) in flow tubes. Cells were subsequently stained directly with mAb against CD11c PE (HL3) and CD80 (B7-1; 16-10A1), CD86 (B7-2; GL1), CD40 (3/23), CD8a (Ly-2, 53-6.7), CD11b (membrane-activated complex 1), CD4 (L3T4, H129.19), B220 (CD45R; RA3-6B2), or anti-major histocompatibility complex (MHC) class II, I-Ad (AMS-32.1; all FITC-conjugated, PharMingen). The appropriate species-specific IgG isotype controls were included in each experiment. Staining was performed for 30 min at 4°C, and cells were washed twice in staining buffer as before and resuspended in PBS. At least 10,000 events were acquired on a FACScan (Becton Dickinson), and analysis was performed using Cell Quest software.

T cell purification
LPMC were prepared from colons as described above. In experiments where T cells were required from this site, 40% of the LPMC was set aside for DC purification, and 60% was used for T cell purification. T cells were purified from LPMC or from spleen cell populations. In each case, unfractionated spleen cells or LPMC were purified using the CD4 MACS MicroBeads and purification column system (MACS, Miltenyi Biotec). This routinely resulted in a cell enrichment of ~95% CD4+ T cells from spleen cells. The purity of T cells from LPMC by this method was less efficient but was improved to 99% by including an additional step of cell labeling with CD4 PE mAb and flow sorting on a Becton Dickinson FACSAria. In experiments where comparisons were made between spleen and colon T cell responses, T cells from both sources were flow-sorted to obtain similar purity.

Stimulation of carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T cells with LPDC
T cells from syngeneic BALB/c and allogeneic C57BL6/J mice were purified from spleens using the CD4+ MACS system and were adjusted to a concentration of 7.5 x 106/ml. CFSE [24 , 25 ] was added at a final concentration of 0.5 µM in 5% FBS/PBS for 7 min. Excess CFSE dye was removed by washing cells three times in 5% FBS/PBS medium. Cells were adjusted to a concentration of 2 x 106/ml (100 µl vol) in RPMI medium with supplements and cultured with DC (1x106/ml, 100 µl vol) in 96-well flat-bottomed plates in a 5% CO2-humidified incubator for a period of 6 days of CFSE dye incorporation into dividing daughter cells. Cells were harvested, washed twice in flow staining buffer, and mAb-stained with CD4 peridinin chlorophyll protein (PerCP; L3T4; RM4-5). Dead cells were gated out, and greater than 25,000 events were collected in the live gate for analysis using Cell Quest software.

Cytokine determination in supernatants
DC were cultured with T cells as described above to determine cytokine release on stimulation. Cell culture supernatants were centrifuged for the removal of cells and stored at –70°C. Cytokine concentration in supernatants was measured by enzyme-linked immunosorbent assay (ELISA) for IL-10 and IFN-{gamma} using paired antibodies (PharMingen). For bioactive IL-12 p70, IL-12 p40/IL-23, and IL-6, Duoset ELISA kits DY419, DY499, and DY406, respectively, were used according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN).

Statistical analysis
Analysis of data was performed by Student’s t-test. A P value of <0.05 was indicative of significant differences.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Recovery of LPMC from T cell-reconstituted SCID mice
DC from the spleen and colon were isolated from reconstituted SCID mice with overt signs of colitis, such as diarrhea, weight loss, and hunched posture. Greater than 90% of these mice had enlarged colons and spleens, features consistent with the gross pathology typically observed in this model [22 , 26 ]. LPMC were isolated from the colons of colitic SCID mice, unmanipulated SCID mice, and BALB/c mice to compare the average number of cells between groups. There was, on average, a sixfold increase in the number of LPMC isolated from colitic SCID mice (n=22) compared with those obtained from control BALB/c mice (n=25; 15x106 cells/colon vs. 2.62x106 cells/colon, respectively). Unmanipulated SCID mice had 0.63 x 106 cells/colon. Increased numbers of LPMC in colitic mice are consistent with IBD pathology. As stated above, IBD mice also developed splenomegaly. Therefore, we investigated differences in the number of cells recovered from the spleens of unmanipulated and colitic SCID mice. Spleens from colitic SCID mice had over four times as many cells as those of unmanipulated SCID mice (n=4; 32.8x106 cells/spleen vs. 7.25x106 cells/spleen, respectively). Thus, the spleens and colons of colitic SCID mice developed significant increases in the cellularity of these organs compared with unmanipulated SCID mice.

Colonic DC from mice with IBD differ in morphology compared with spleen DC
The most frequently used DC identification marker for mouse cells is the CD11c antigen. Light scatter plots of sorted CD11c+ cells from colonic LPMC and spleen cell suspensions from colitic mice revealed different patterns of cellular distribution. Colonic CD11c+ cells are more variable in size and are generally larger than spleen CD11c+ cells (Fig. 1A and 1B ). Colonic DC had large nuclei, which were, in some cases, irregular-shaped, surrounded by a large volume of cytoplasm, with or without dendrites at their cell surfaces, as demonstrated by visual analysis of Giemsa-stained cells (Fig. 1A , inset). The morphology of these cells was characteristic of the classic mature and immature myeloid DC [27 , 28 ]. Spleen DC were predominantly smaller, and nuclei occupied almost the entire volume of these cells (Fig. 1B , inset). DC were rarely present in the LPMC preparations of BALB/c and unmanipulated SCID mice, as a result of the rarity of DC in healthy colons (see Fig. 2 ), but when observed, they had a similar appearance to those of the colonic DC of colitic mice (data not shown). There were no visual differences between splenic DC of BALB/c and colitic SCID mice (data not shown).



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  		Figure 1. Size heterogeneity of colon DC. Column-sorted CD11c+ cells obtained from T cell-reconstituted mice with colitis were mAb-labeled (CD11c PE) and sorted on a Becton Dickinson FACSAria. Recovered DC were then gated on live cells, and events were collected on a flow cytometer. Dot plots of side scatter (SSC-H) versus forward scatter (FSC-H) show the relative difference in size of these two cell populations. Insets show Giemsa-stained cytocentrifuge preparations of LPDC obtained from (A) colons and (B) spleens of mice. Original magnification, x400. Results are representative of four separate experiments.

 


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  		Figure 2. Phenotypic expression of DC. Single-cell preparations of colons and spleens from (A) colitic SCID and (B) BALB/c mice were obtained by collagenase digestion. Cells were mAb-stained with CD11c PE (FL2) and FITC-labeled antibodies. Dead cells were gated out, and at least 15,000 events were acquired for analysis. The upper-left quadrant of the dot plot shows single-positive CD11c+ cells, and the upper-right quadrant shows cells expressing dual markers as indicated. Results are representative of three separate experiments.

 
Expression of cell-surface markers on colonic DC
To investigate the differences in cell-surface antigen expression of spleen DC and LPDC, single-cell suspensions of collagenase-digested spleens and colons from reconstituted SCID and normal BALB/c mice were double-stained with a panel of mAb for CD11c and other DC subset markers for analysis by flow cytometry. Preparations of LPMC from colitic SCID mice typically resulted in ~15% CD11c+ cells (Fig. 2A ). In contrast, BALB/c LPMC typically stained less than 3% of CD11c+ cells (Fig. 2B) . LPDC of colitic mice expressed high levels of CD80, MHC class II I-Ad, and CD11b (Fig. 2A) with lower levels of CD8{alpha}, CD40, CD86, and B220 (data not shown). mAb staining of CD11c+-sorted LPMC revealed a population of cells, which predominantly expressed CD11b (Fig. 3A and 3B ), the latter, a marker usually found on myeloid DC [27 , 28 ].



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  		Figure 3. DC of spleens and colons exhibit different surface phenotypes. Cells obtained from collagenase-digested colons were stained with (A) isotype controls of hamster (H)IgG PE and of rat (R)IgG FITC. (B) Flow-sorted colon CD11c+ PE-labeled cells were stained with CD11b FITC to determine the expression of this marker on DC. (C) Similar dual staining for spleen DC of colitic mice and (D) of BALB/c mice. The distribution of CD11b varies on spleen and colon DC populations, as determined in four representative experiments.

 
As expected, there was also an increased frequency of CD11c-positive cells in the spleens of colitic mice as compared with BALB/c control mice, but this increase in DC was not as high as for the colon (Fig. 2) . It was therefore more difficult to observe the trends in phenotypes in unpurified spleen cells. It was, however, evident in the spleen cells from colitic mice that a significant portion of DC expressed costimulatory molecule CD80 and MHC class II I-Ad (Fig. 2A) . Sorted spleen DC, unlike colon DC, were predominantly CD11c+CD11b–, and a lesser portion of CD11c+ cells expressed dual markers (Fig. 3C) . Although CD11c+ cells were observed at low frequency in spleen cell preparations of control BALB/c mice (Fig. 2B) , we were still able to sort these cells by virtue of the high numbers of total cells present in each of these BALB/c spleens. We found that like spleen DC isolated from colitic mice, this population of cells was also predominantly CD11c+CD11b– (Fig. 3C and 3D) . LPDC and spleen DC expressed minimal levels of CD4 (not shown).

Stimulation of T cells by colonic DC results in production of Th1 cytokines
The cytokine balance in the intestine is a critical, determining factor between tolerance and immunity. We tested the ability of DC from colitic mice to stimulate spleen T cells by quantifying the cytokines released in DC-T cell cocultures. As the exogenous bacterial antigens that drive T cell-mediated inflammation in IBD are unknown, we performed allogeneic stimulation of T cells, a well-studied system used for investigating the immunostimulatory potential of DC. We focused on a range of proinflammatory cytokines, which are relevant to the IBD process or to inflammation in general, including IL-12, IFN-{gamma}, and IL-6 [29 30 31 32 33 ]. We also investigated the release of IL-10, a cytokine that ameliorates IBD in this model and is also known to be important in intestinal homeostasis [26 , 34 35 36 ]. DC stimulation of allogeneic C57BL6/J CD4+ spleen T cells (B6T) cells in culture resulted in the production of significant amounts of IFN-{gamma}, which peaked at Day 6 (Fig. 4A ). There was spontaneous production of IL-6 in DC cultures in the absence of T cells, but in two of four experiments, IL-6 release was enhanced in allogeneic DC-T cell cocultures above basal levels (Fig. 4B) . IL-12 p40/IL-23 was also produced on allogeneic stimulation but to a lesser extent (Fig. 4C) . IL-10 was only detected in one of our four coculture experiments and reached a level of 366 pg/ml (data not shown). IL-12 p70 was not released on DC-T cell stimulation (not shown).



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  		Figure 4. Stimulation of cytokines by colon DC. (A) Flow-sorted colon DC of T cell-reconstituted SCID mice with colitis were adjusted to a concentration of 1 x 106/ml, and 100 µl was added to 96-well flat-bottomed plates. Column-purified B6T were added (100 µl vol) at a concentration of 2 x 106/ml, and cultures were incubated at 37°C for 1, 3, or 6 days. Supernatants were collected and assayed for (A) IFN-{gamma}, (B) IL-6, and (C) IL-12 p40/IL-23 by ELISA. Error bars represent standard deviations over duplicate wells. P values indicate levels of significance above control DC. Cytokine production is representative of two to three independent experiments.

 
We also performed coculture experiments to determine if freshly isolated, colonic DC could stimulate syngeneic T cells from BALB/c mice. In a side-by-side analysis with allogeneic B6T cells, we observed the production of significant levels of IFN-{gamma} with both models (Fig. 5A ). Low levels of IL-6 and IL-12 p40/IL-23 were also produced but did not achieve significance (Fig. 5B and 5C) . In addition, we tested spleen DC for their ability to stimulate T cells, as they displayed a distinct phenotype and morphology from LPDC (Figs. 1 and 3) and thus, may have the ability to stimulate the release of different levels of cytokines. Spleen DC from colitic mice stimulated the production of higher levels of IFN-{gamma} than released in allogeneic and syngeneic LPDC-T cell cocultures (Fig. 5A) .



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  		Figure 5. Cytokine release in cocultures on stimulation with spleen and colon DC. Flow-sorted spleen or colon DC of T cell-reconstituted SCID mice with colitis were added to allogeneic B6 or syngeneic BALB/c spleen CD4+ T cells as described above, and cytokine released in supernatants after 6 days, quantitated by ELISA. Error bars represent standard deviations over duplicate wells. Statistical significance above DC cytokine release in medium is indicated. Results are representative of two separate experiments.

 
Colon DC are potent stimulators of IFN-{gamma} production by colon T cells of IBD mice
In addition to determining the ability of colon DC to stimulate allogeneic and syngeneic splenic T cell cocultures, we inves-tigated the ability of freshly isolated colon DC from colitic mice to stimulate autologous CD4+ T cells obtained from the diseased colons of these same mice. Because of the low percentage of T cells present in LPMC relative to epithelial cells and other cell types, it was necessary to purify these cells in two stages to ascertain optimum purity of T cells from this site. The T cells were first enriched using CD4 MACS columns and then subsequently purified by fluorescence-activated cell sorting using a CD4-specific PE-labeled mAb. In this section of our study, all T cell populations were prepared in like manner.

In our prior assays, we observed the ability of colonic DC to stimulate IFN-{gamma} production in syngeneic and allogeneic splenic T cell cocultures, the most potent response being noted with allogeneic cells (Figs. 4 and 5) . When autologous T cells from the diseased colons of sick mice were cocultured with DC obtained from the same tissue, there was a more rapid (not shown) and more robust release of IFN-{gamma} (Fig. 6A ). The level of IFN-{gamma} released in these autologous cocultures was significantly higher than in colon DC-allogeneic T cell cocultures (P=0.01). Thus, DC from colons of IBD mice are capable of inducing an even greater Th1 response than observed in an allogeneic system. IL-6 production in autologous colonic DC-T cell cocultures was also enhanced significantly above that obtained in colon DC-allogeneic B6T cell cocultures (Fig. 6B) . No significant increase was observed for IL-12 p40/IL-23 release. The levels of this cytokine were low in all cases. As measured by cytokine production, all T cells used in these studies appeared to be in a nonactivated state, with little or no spontaneous cytokine release (Fig. 6) .



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  		Figure 6. LPDC are potent stimulators of Th1 cytokines in lamina propria T cells of IBD mice. LPDC were added to self-T cells obtained from the colon of IBD mice or to allogeneic B6T cells in cocultures for 6 days, as described above. All CD4+ T cells were flow-sorted. Cytokine release in supernatants was measured by ELISA. Error bars represent standard deviations over duplicate wells. P values shown indicate levels of significance above control DC. In addition, colon DC-colon T cell cocultures showed significantly higher IFN-{gamma} release (P=.01) and IL-6 release (P<.01) above that of allogeneic cocultures. Results are representative of two independent experiments.

 
Limited capacity of LPDC to stimulate T cells
Although DC can influence the development of inflammation by influencing the T cell cytokine profile, the degree to which these cells stimulate T cell proliferation may also contribute to colitis. The ability of DC to induce proliferation in spleen CD4+ T cells was measured by CFSE inclusion in dividing T cells following coculture with DC. Colon DC were slightly more immunostimulatory for allogeneic B6T cells than for syngeneic BALB/c T cells, inducing division peaks above that observed for T cells alone (Fig. 7 ). However, colon DC were less immunostimulatory than spleen DC for allogeneic and syngeneic T cells.



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  		Figure 7. Colon DC are moderate stimulators of allogeneic CD4+ T cells. Colon and spleen DC of colitic SCID mice were sequentially purified on MACS columns and then by flow sorting. BALB/c (syngeneic) and C57BL6/J (allogeneic) T cells were purified on MACS columns and CFSE-labeled. Cultures (1x105 DC and 2x105 CD4+ T cells, 100 µl each) were set up in 96-well plates and cultured for 6 days. Cells were harvested and mAb-stained for CD4 PerCP (FL3). Gating was performed to exclude dead cells, and over 25,000 events were collected. Histograms show CD4+ PerCP-stained T cells, which were CFSE-labeled. Results are representative of three separate experiments.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The capacity of DC to regulate the immune response depends on the level of activity of these cells in response to environmental signals and other cell types. Although IBD can be associated with secondary pathology in systemic sites such as the spleen, our studies focused on the colons of IBD mice, as in this disease, there is a unique intestinal inflammation, and the genetic, immunologic, and environmental factors that influence the disease must be combined with the presence of specific, gut-colonizing bacteria for the disease to ensue [37 38 39 ].

Because of the ability of DC to direct T cell cytokine and proliferative responses, the interaction of these cells in the colon can be a determining factor of the magnitude of local inflammation and of disease activity. The relationship of these two cell types in IBD has not been addressed adequately as a result of practical limitations in isolating these two cell populations from the lamina propria. Using the CD45RBhi CD4+ T cell-reconstituted SCID model of colitis, our studies investigated several characteristics of colon DC, including the ability of these cells to induce responses in allogeneic T cells and more specifically, cytokine secretion profiles resulting from autologous colon DC-colon T cell stimulation. These data represent one of the first accounts to solidify the role of DC in promoting IBD pathogenesis.

In normal mice, colon DC may contribute to the regulation of immunologic homeostasis and the maintenance of tolerance in the gut. Limited numbers of LPMC combined with the low percentages of these cells in unmanipulated mice limit DC-T cell interaction and cytokine production and potential adverse pathology in these mice. The recovery of significantly increased numbers of LPMC from the colons of colitic mice in comparison with normal mice is of importance to the disease process, because of the increased likelihood of enhanced cellular interaction, T cell expansion, stimulation of the release of damaging Th1 cytokines, and recruitment of cells. Furthermore, the increased presence of CD11c+ cells to provoke a T cell response in these mice creates an environment in which T cells can elaborate a variety of cytokines and contribute to immune imbalance in the gut.

The DC isolated from the colons of sick mice displayed high levels of CD80 and MHC class II expression, which is consistent with the phenotype of mature/activated DC, which when expressing high levels of costimulatory molecule CD80 and MHC class II I-Ad, may be more efficient at presentation of antigens encountered and may easily trigger T cell activation. Increased costimulatory molecule expression on colon DC has also been observed by others using the SCID mouse model of colitis, as well as in other animal models of colitis and on DC in human IBD [1 , 19 , 40 , 41 ]. In contrast to the colonic DC, which were mostly CD11c+/CD11b+, DC isolated from the spleens of IBD mice were mainly of the CD11c+CD11b– subset and were morphologically similar to the plasmacytoid DC subset [42 ]. The obvious difference in morphology and in phenotype of colon and spleen DC may indicate that these cells are of different subsets. Alternatively, colon DC and spleen DC may be of the same lineage but at a different stage of differentiation, because of the microbial antigens to which these cells respond in the spleen or colon.

The allogeneic coculture assay is used widely as a measure of DC stimulatory capacity, particularly where the stimulating antigen is unknown. Stimulation of allogeneic T cells with DC from this IBD model resulted in the release of significantly high levels of IFN-{gamma} in supernatants. However, when colon DC were used to stimulate autologous colon T cells, the levels of IFN-{gamma} released exceeded those detected in allogeneic cocultures. Furthermore, a robust cytokine response in autologous cocultures occurred as early as Day 1, suggesting that IFN-{gamma} may be critical to early inflammation at this site. The potent response in this system may be a result of the presence of microbial antigens that copurify with the colon DC. The presence of high levels of IFN-{gamma} has been associated with the production of other Th1 cytokines such as tumor necrosis factor-{alpha} and IL-12 and worsening of IBD in mouse colitis [30 , 43 ].

IL-6 is also associated with inflammation in some disease models and in human and mouse IBD [29 , 33 ], and we noted significant production of IL-6 in our in vitro coculture autologous model. Interaction of IL-6 with the IL-6 receptor induces phosphorylation of the signal transducer and activator of transcription (STAT)-3 protein, which binds directly to target genes [44 ]. Activation of STAT-3 is associated with severe colitis in mice as with other diseases [32 , 45 ]. Overproduction of IL-6 resulting from colon DC-colon T cell interaction may be a contributing factor to colitis.

IL-23 is a cytokine composed of the IL-12 p40 subunit and a cytokine-like subunit related to IL-12 p35, p19. IL-23, like IL-12, induces proliferation and IFN-{gamma} production by human T cells, but unlike IL-12, IL-23 primarily stimulates memory rather than naïve T cells [46 , 47 ]. It has also been implicated in multiorgan inflammation, including the intestinal tract [48 ]. Recent studies reported that this cytokine was expressed in the terminal ileum of mice but not in the colon [13 ]. Colon DC-colon T cell stimulation released low but detectable levels of IL-12 p40/IL-23 in our system.

Several reports indicate that IL-10, a Th2 cytokine, is a characteristic product of intestinal DC from normal mice, where it contributes to intestinal homeostasis. It is associated with the improvement of mouse IBD and in general, with down-regulation of Th1 immune responses [26 , 34 35 36 , 49 ]. We did not observe significant levels of IL-10 in DC-T cell cultures, possibly as a result of the fact that the high levels of IFN-{gamma} released in these cocultures may contribute to the suppression of IL-10 production. The absence of IL-10 on the interaction of two critical cell types in the IBD colon, combined with the up-regulation of cytokines such as IFN-{gamma}, may provide an environment that favors immune reactivity at this site.

Chemoattractants and inflammatory mediators such as IFN-{gamma} released by constant stimulation of DC-T cells in the IBD gut may be a cue for the migration of other cells to the site. In addition, as we report, DC possess the ability to induce local stimulation and proliferation of T cells. This too may contribute to the cellular increase and resulting inflammation in the colon of colitic mice.

This is the first report about the ability of colon DC from an inflamed gut to stimulate autologous T cells from the same site and elaborate proinflammatory cytokines. We provide novel evidence that cells isolated from the colons of IBD mice induce a Th1 cytokine response, which is significantly greater than those obtained even in an allogeneic system. Our data suggest that colon DC activity may be crucial to the chronic inflammation observed in IBD. It is not certain at this stage whether cytokines such as IFN-{gamma} produced by this mechanism are responsible for initiating T cell proliferation in the gut, but it is most likely a factor in the immune-mediated damage at this site.

Although the presence of DC in spleens was less frequent than in the colons of IBD mice, the cytokine and proliferative responses induced by spleen DC suggest that DC, at sites distant from the gut, may contribute to the systemic component of inflammation in this model. In addition to the morphologic and phenotypic variations of these spleen and colon DC, spleen DC were potent stimulators of T cells, and colon DC were moderately immunostimulatory. This is consistent with recent studies in which semipurified colon DC of normal mice stimulated only a weak, allogeneic, mixed leukocyte reaction in Terasaki plate cultures [20 ]. Other investigators reported that spleen DC of normal mice had a greater T cell stimulatory ability than liver DC [50 ]. Thus, it is possible that there is an inherent, immunogenic potency in spleen DC in comparison with those from other sites. We have shown too that spleen DC induced higher levels of IFN-{gamma} in cocultures than obtained with LPDC-T cell cocultures. Furthermore, our own unpublished observations indicate that spleen DC, when stimulated with certain microbial antigens, release higher levels of IFN-{gamma} than colon DC (M. L. Drakes, unpublished observations). Future studies will determine the ability of cells from this systemic site to contribute to the overall disease state.

Earlier investigators have focused on DC-T cell interactions in the lymph nodes of mice as a mechanism of IBD inflammation [51 ]. Using both cell types isolated from the colons, we have highlighted the unique ability of colon DC of colitic mice to stimulate autologous T cells and suggest that in the gut, these cells play a crucial, immunoregulatory role in IBD pathogenesis.


   ACKNOWLEDGEMENTS
 
This study was supported by National Institutes of Health Grants DK-57756, DK-46461-11S1 and DK-046461. Fluorescence-activated cell sorting was performed at the Flow Cytometry Core Facility of the Comprehensive Cancer Center of Case Western Reserve University, supported by Grant P30 CA43703. The authors thank Dr. Melvin Berger of the Department of Pediatrics, Case Western Reserve University, for the kind use of his flow cytometer.

Received June 24, 2005; revised July 29, 2005; accepted August 4, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Karlis, J., Penttila, I., Tran, T. B., Jones, B., Nobbs, S., Zola, H., Flesch, I. E. (2004) Characterization of colonic and mesenteric lymph node dendritic cell subpopulations in a murine adoptive transfer model of inflammatory bowel disease Inflamm. Bowel Dis. 10,834-847[CrossRef][Medline]
  2. Leithauser, F., Krajina, T., Trobonjaca, Z., Reimann, J. (2002) Early events in the pathogenesis of a murine transfer colitis Pathobiology 70,156-163[CrossRef][Medline]
  3. Leithauser, F., Trobonjaca, Z., Moller, P., Reimann, J. (2001) Clustering of colonic lamina propria CD4(+) T cells to subepithelial dendritic cell aggregates precedes the development of colitis in a murine adoptive transfer model Lab. Invest. 81,1339-1349[Medline]
  4. Banchereau, J., Steinman, R. M. (1998) Dendritic cells and the control of immunity Nature 392,245-252[CrossRef][Medline]
  5. Hart, D. N. (1997) Dendritic cells: unique leukocyte populations which control the primary immune response Blood 90,3245-3287[Free Full Text]
  6. Lanzavecchia, A., Sallusto, F. (2001) Regulation of T cell immunity by dendritic cells Cell 106,263-266[CrossRef][Medline]
  7. Liu, Y. J. (2001) Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity Cell 106,259-262[CrossRef][Medline]
  8. Mellman, I., Steinman, R. M. (2001) Dendritic cells: specialized and regulated antigen processing machines Cell 106,255-258[CrossRef][Medline]
  9. Barton, G. M., Medzhitov, R. (2002) Control of adaptive immune responses by Toll-like receptors Curr. Opin. Immunol. 14,380-383[CrossRef][Medline]
  10. Fujii, S., Liu, K., Smith, C., Bonito, A. J., Steinman, R. M. (2004) The linkage of innate to adaptive immunity via maturing dendritic cells in vivo requires CD40 ligation in addition to antigen presentation and CD80/86 costimulation J. Exp. Med. 199,1607-1618[Abstract/Free Full Text]
  11. Rescigno, M., Borrow, P. (2001) The host-pathogen interaction: new themes from dendritic cell biology Cell 106,267-270[CrossRef][Medline]
  12. Rescigno, M., Granucci, F., Ricciardi-Castagnoli, P. (2000) Molecular events of bacterial-induced maturation of dendritic cells J. Clin. Immunol. 20,161-166[CrossRef][Medline]
  13. Becker, C., Wirtz, S., Blessing, M., Pirhonen, J., Strand, D., Bechthold, O., Frick, J., Galle, P. R., Autenrieth, I., Neurath, M. F. (2003) Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells J. Clin. Invest. 112,693-706[CrossRef][Medline]
  14. Iwasaki, A., Kelsall, B. L. (2000) Localization of distinct Peyer’s patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3{alpha}, MIP-3ß, and secondary lymphoid organ chemokine J. Exp. Med. 191,1381-1394[Abstract/Free Full Text]
  15. Kelsall, B. L., Strober, W. (1996) Distinct populations of dendritic cells are present in the subepithelial dome and T cell regions of the murine Peyer’s patch J. Exp. Med. 183,237-247[Abstract/Free Full Text]
  16. Rescigno, M., Urbano, M., Valzasina, B., Francolini, M., Rotta, G., Bonasio, R., Granucci, F., Kraehenbuhl, J. P., Ricciardi-Castagnoli, P. (2001) Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria Nat. Immunol. 2,361-367[CrossRef][Medline]
  17. Stagg, A. J., Hart, A. L., Knight, S. C., Kamm, M. A. (2003) The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria Gut 52,1522-1529[Abstract/Free Full Text]
  18. Bilsborough, J., Viney, J. L. (2004) Gastrointestinal dendritic cells play a role in immunity, tolerance, and disease Gastroenterology 127,300-309[CrossRef][Medline]
  19. Krajina, T., Leithauser, F., Moller, P., Trobonjaca, Z., Reimann, J. (2003) Colonic lamina propria dendritic cells in mice with CD4+ T cell-induced colitis Eur. J. Immunol. 33,1073-1083[CrossRef][Medline]
  20. Rigby, R. J., Knight, S. C., Kamm, M. A., Stagg, A. J. (2005) Production of interleukin (IL)-10 and IL-12 by murine colonic dendritic cells in response to microbial stimuli Clin. Exp. Immunol. 139,245-256[CrossRef][Medline]
  21. Morrissey, P. J., Charrier, K., Braddy, S., Liggitt, D., Watson, J. D. (1993) CD4+ T cells that express high levels of CD45RB induce wasting disease when transferred into congenic severe combined immunodeficient mice. Disease development is prevented by cotransfer of purified CD4+ T cells J. Exp. Med. 178,237-244[Abstract/Free Full Text]
  22. Powrie, F., Leach, M. W., Mauze, S., Caddle, L. B., Coffman, R. L. (1993) Phenotypically distinct subsets of CD4+ T cells induce or protect from chronic intestinal inflammation in C. B-17 scid mice Int. Immunol. 5,1461-1471[Abstract/Free Full Text]
  23. Van der Heijden, P. J., Stok, W. (1987) Improved procedure for the isolation of functionally active lymphoid cells from the murine intestine J. Immunol. Methods 103,161-167[CrossRef][Medline]
  24. Lyons, A. B. (2000) Analyzing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution J. Immunol. Methods 243,147-154[CrossRef][Medline]
  25. Suchin, E. J., Langmuir, P. B., Palmer, E., Sayegh, M. H., Wells, A. D., Turka, L. A. (2001) Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question J. Immunol. 166,973-981[Abstract/Free Full Text]
  26. Asseman, C., Mauze, S., Leach, M. W., Coffman, R. L., Powrie, F. (1999) An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation J. Exp. Med. 190,995-1004[Abstract/Free Full Text]
  27. Drakes, M. L., Lu, L., Subbotin, V. M., Thomson, A. W. (1997) In vivo administration of flt3 ligand markedly stimulates generation of dendritic cell progenitors from mouse liver J. Immunol. 159,4268-4278[Abstract]
  28. Lu, L., Woo, J., Rao, A. S., Li, Y., Watkins, S. C., Qian, S., Starzl, T. E., Demetris, A. J., Thomson, A. W. (1994) Propagation of dendritic cell progenitors from normal mouse liver using granulocyte/macrophage colony-stimulating factor and their maturational development in the presence of type-1 collagen J. Exp. Med. 179,1823-1834[Abstract/Free Full Text]
  29. Ito, H., Takazoe, M., Fukuda, Y., Hibi, T., Kusugami, K., Andoh, A., Matsumoto, T., Yamamura, T., Azuma, J., Nishimoto, N., Yoshizaki, K., Shimoyama, T., Kishimoto, T. (2004) A pilot randomized trial of a human anti-interleukin-6 receptor monoclonal antibody in active Crohn’s disease Gastroenterology 126,989-996[CrossRef][Medline]
  30. Powrie, F., Leach, M. W., Mauze, S., Menon, S., Caddle, L. B., Coffman, R. L. (1994) Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells Immunity 1,553-562[CrossRef][Medline]
  31. Simpson, S. J., Shah, S., Comiskey, M., de Jong, Y. P., Wang, B., Mizoguchi, E., Bhan, A. K., Terhorst, C. (1998) T cell-mediated pathology in two models of experimental colitis depends predominantly on the interleukin 12/signal transducer and activator of transcription (Stat)-4 pathway, but is not conditional on interferon {gamma} expression by T cells J. Exp. Med. 187,1225-1234[Abstract/Free Full Text]
  32. Suzuki, A., Hanada, T., Mitsuyama, K., Yoshida, T., Kamizono, S., Hoshino, T., Kubo, M., Yamashita, A., Okabe, M., Takeda, K., Akira, S., Matsumoto, S., Toyonaga, A., Sata, M., Yoshimura, A. (2001) CIS3/SOCS3/SSI3 plays a negative regulatory role in STAT3 activation and intestinal inflammation J. Exp. Med. 193,471-481[Abstract/Free Full Text]
  33. Yamamoto, M., Yoshizaki, K., Kishimoto, T., Ito, H. (2000) IL-6 is required for the development of Th1 cell-mediated murine colitis J. Immunol. 164,4878-4882[Abstract/Free Full Text]
  34. Fuss, I. J., Boirivant, M., Lacy, B., Strober, W. (2002) The interrelated roles of TGF-ß and IL-10 in the regulation of experimental colitis J. Immunol. 168,900-908[Abstract/Free Full Text]
  35. Liu, H., Hu, B., Xu, D., Liew, F. Y. (2003) CD4+CD25+ regulatory T cells cure murine colitis: the role of IL-10, TGF-ß, and CTLA4 J. Immunol. 171,5012-5017[Abstract/Free Full Text]
  36. Iwasaki, A., Kelsall, B. L. (1999) Freshly isolated Peyer’s patch, but not spleen, dendritic cells produce interleukin 10 and induce the differentiation of T helper type 2 cells J. Exp. Med. 190,229-239[Abstract/Free Full Text]
  37. Kuhn, R., Lohler, J., Rennick, D., Rajewsky, K., Muller, W. (1993) Interleukin-10-deficient mice develop chronic enterocolitis Cell 75,263-274[CrossRef][Medline]
  38. Sellon, R. K., Tonkonogy, S., Schultz, M., Dieleman, L. A., Grenther, W., Balish, E., Rennick, D. M., Sartor, R. B. (1998) Resident enteric bacteria are necessary for development of spontaneous colitis and immune system activation in interleukin-10-deficient mice Infect. Immun. 66,5224-5231[Abstract/Free Full Text]
  39. Taurog, J. D., Richardson, J. A., Croft, J. T., Simmons, W. A., Zhou, M., Fernandez-Sueiro, J. L., Balish, E., Hammer, R. E. (1994) The germfree state prevents development of gut and joint inflammatory disease in HLA-B27 transgenic rats J. Exp. Med. 180,2359-2364[Abstract/Free Full Text]
  40. Ikeda, Y., Akbar, F., Matsui, H., Onji, M. (2001) Characterization of antigen-presenting dendritic cells in the peripheral blood and colonic mucosa of patients with ulcerative colitis Eur. J. Gastroenterol. Hepatol. 13,841-850[CrossRef][Medline]
  41. Vuckovic, S., Florin, T. H., Khalil, D., Zhang, M. F., Patel, K., Hamilton, I., Hart, D. N. (2001) CD40 and CD86 upregulation with divergent CMRF44 expression on blood dendritic cells in inflammatory bowel diseases Am. J. Gastroenterol. 96,2946-2956[CrossRef][Medline]
  42. Nakano, H., Yanagita, M., Gunn, M. D. (2001) CD11c(+)B220(+)Gr-1(+) cells in mouse lymph nodes and spleen display characteristics of plasmacytoid dendritic cells J. Exp. Med. 194,1171-1178[Abstract/Free Full Text]
  43. Magram, J., Connaughton, S. E., Warrier, R. R., Carvajal, D. M., Wu, C. Y., Ferrante, J., Stewart, C., Sarmiento, U., Faherty, D. A., Gately, M. K. (1996) IL-12-deficient mice are defective in IFN {gamma} production and type 1 cytokine responses Immunity 4,471-481[CrossRef][Medline]
  44. Harroch, S., Revel, M., Chebath, J. (1994) Interleukin-6 signaling via four transcription factors binding palindromic enhancers of different genes J. Biol. Chem. 269,26191-26195[Abstract/Free Full Text]
  45. Syed, V., Ulinski, G., Mok, S. C., Ho, S. M. (2002) Reproductive hormone-induced, STAT3-mediated interleukin 6 action in normal and malignant human ovarian surface epithelial cells J. Natl. Cancer Inst. 94,617-629[Abstract/Free Full Text]
  46. Aggarwal, S., Ghilardi, N., Xie, M. H., de Sauvage, F. J., Gurney, A. L. (2003) Interleukin-23 promotes a distinct CD4 T cell activation state characterized by the production of interleukin-17 J. Biol. Chem. 278,1910-1914[Abstract/Free Full Text]
  47. Parham, C., Chirica, M., Timans, J., Vaisberg, E., Travis, M., Cheung, J., Pflanz, S., Zhang, R., Singh, K. P., Vega, F., To, W., Wagner, J., O’Farrell, A. M., McClanahan, T., Zurawski, S., Hannum, C., Gorman, D., Rennick, D. M., Kastelein, R. A., de Waal Malefyt, R., Moore, K. W. (2002) A receptor for the heterodimeric cytokine IL-23 is composed of IL-12Rß1 and a novel cytokine receptor subunit, IL-23R J. Immunol. 168,5699-5708[Abstract/Free Full Text]
  48. Wiekowski, M. T., Leach, M. W., Evans, E. W., Sullivan, L., Chen, S. C., Vassileva, G., Bazan, J. F., Gorman, D. M., Kastelein, R. A., Narula, S., Lira, S. A. (2001) Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation, runting, infertility, and premature death J. Immunol. 166,7563-7570[Abstract/Free Full Text]
  49. Enk, A. H., Angeloni, V. L., Udey, M. C., Katz, S. I. (1993) Inhibition of Langerhans cell antigen-presenting function by IL-10. A role for IL-10 in induction of tolerance J. Immunol. 151,2390-2398[Abstract]
  50. Pillarisetty, V. G., Shah, A. B., Miller, G., Bleier, J. I., DeMatteo, R. P. (2004) Liver dendritic cells are less immunogenic than spleen dendritic cells because of differences in subtype composition J. Immunol. 172,1009-1017[Abstract/Free Full Text]
  51. Malmstrom, V., Shipton, D., Singh, B., Al-Shamkhani, A., Puklavec, M. J., Barclay, A. N., Powrie, F. (2001) CD134L expression on dendritic cells in the mesenteric lymph nodes drives colitis in T cell-restored SCID mice J. Immunol. 166,6972-6981[Abstract/Free Full Text]



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