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Keywords:

  • Murine colitis;
  • Colonic dendritic cell;
  • Mucosal T cell response

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

CD11c+ (F4/80 CD68) dendritic cells (DC) in the colonic lamina propria (cLP) of normal and immunodeficient (RAG1–/–) C57BL/6 (B6) mice show high surface expression of MHC class I/II molecules and CD1d, and low surface expression of CD40, CD80, CD86 costimulator molecules. CD4+ α β T cells from normal or MHC class II-deficient B6 micetransferred into congenic RAG1–/– hosts induce a progressive, lethal colitis. Concomitant with colitis development, DC in the inflamed cLP increase in number and up-regulate surface expression of CD1d, MHC class II molecules and CD40, CD80, CD86 costimulator molecules. cLP DC from non-transplanted (healthy) and transplanted (diseased) mice produce similar amounts of IL-12 p70 and IL-10 in response to CD40 signaling, but the inducible IL-12 p40 release is 5–15-fold higher in mice with colitis than in non-transplanted mice. Binding of IL-12 p40 to p19 generates IL-23. Freshly isolated cLP lymphocytes (cLPL) from transplanted, diseased mice express 3–10-fold more p19 transcripts than cLPL from non-transplanted, healthy mice. p19 expression by cLPL is further up-regulated in response to CD40 ligation. Freshly isolated cLP DC from transplanted mice with colitis (but not from non-transplanted controls) stimulate IFN-γ (but not IL-4 or IL-13) release by co-cultured NKT cells. Incolitis, DC accumulate in the cLP, show an activated surface phenotype, up-regulate IL-12 p40 and p19 expression, and ‘spontaneously’ stimulate NKT-like cells. cLP DC may be interesting targets for novel therapeutic approaches to modulate mucosal T cell responses in situ.

Abbreviations:
LP:

Lamina propria

LPL:

LP lymphocytes

cLP:

Colonic LP

IEL:

Intraepithelial lymphocytes

IEC:

Intestinal epithelial cell

mLN:

Mesenteric lymph nodes

PP:

Peyer's patch

KO:

Knockout

αGalCer:

α-Galactosyl ceramide

B6:

C57BL/6 mice

SCID:

Severe combined immunodeficiency

NKT cells:

T cells expressing the NK1 marker

IBD:

Inflammatory bowel disease

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

Limited data are available on the phenotype, maturation and migration of intestinal dendritic cells (DC) from Peyer's patches (PP) or the lamina propria (LP) (reviewed in 1). Mucosal DC can take up antigen from the gut 2 and present it to CD4+ and CD8+ T cells 3. DC spend only 2–3 days in the mucosa of the gut before migrating to mesenteric lymph nodes (mLN). Considerably more is known about DC in PP and mLN than about those in the LP of the gut 4. DC either infiltrate the intestinal LP diffusely, or form subepithelial aggregates scattered along the small and large intestine 58. It is unknown if DC within the intestinal LP can prime naïve T cells insitu, or restimulate memory/effector T cell responses.

The transfer of CD4+ α β T cells into immunodeficient, adoptive hosts induces inflammatory bowel disease (IBD) in the form of severe colitis (reviewed in 9). Key factors that drive pathogenic, often Th1-biased, mucosal T cell responses in this extensively studied model are still unknown 10, i.e. the inducing CD4+ T cell subset, the bacterial antigen recognized by them, the presenting cell stimulating the response, and the regulatory cells modulating the phenotype of the presenting and responding cells are not defined. Interest recently focused on the modulation of the DC phenotype by cells of the innate immune system, in particular NK 1113 and NKT cells 14, 15. We reported that pathogenic Th1 CD4+ T cell responses developing in transfer colitis require MHC-II/CD1d-independent CD4+ T cells that are apparently critical for the initiation of Th1 polarization of T cell responses in the gut mucosa 16.

A key cytokine driving Th1 polarization is IL-12, a p70 heterodimer containing the two subunits p35 and p40 17. IL-12 signaling depends on the high-affinity IL-12 receptor composed of the β1 and β2 subunits. Each receptor subunit independently exhibits low affinity for p40 or p35, i.e. the IL-12Rβ1 binds p40, while the IL-12Rβ2 binds p35 18. IL-12 seems to play a key role in inducing and/or sustaining intestinal inflammation 19, 20. IL-23 composed of the IL-12 p40 and the (p35-homologous) p19 subunit binds to the IL-12Rβ1 subunit associated with an unknown (presumably IL-12Rβ2-like) subunit 21. Signal transduction by IL-23 depends on STAT4. Different clearance of infections in p35–/– versus p40–/–, and IL-12Rβ1–/– versus IL-12Rβ2–/– knockout (KO) mice indicates that IL-23 plays a unique role in protective Th1 T cell responses in vivo2224. In contrast to IL-12, IL-23 appears to provide proliferative signals to memory T cells 21. As the lamina propria lymphocyte (LPL) population of the intestinal mucosa contains mainly memory T cells, and colitis-associated colonic LP (cLP) CD4+ T cells in the transfer colitis model have exclusively a memory phenotype, an abundance of potential targets for IL-23 is available in situ. Little is known about the role of this cytokine in IBD.

We compared the phenotype of cLP DC from normal and immunodeficient (RAG–/–) B6 mice, with that of cLP DC from transplanted RAG–/– mice with severe colitis. We investigated if DC accumulate in the inflamed cLP, and if they are activated in situ. We furthermore tested their ‘spontaneous’ and inducible cytokine expression pattern, and their ability to activate MHC class II-independent CD4+ TCRα β T cells.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

2.1 DC in the cLP

Colonic LP lymphocytes (cLPL) were isolated from immunocompetent B6 mice or immunodeficient RAG1–/– B6 mice. CD11b+ CD11c cLPL expressed the macrophage markers F4/80 (Fig. 1, subset A), while CD11c+ (CD11b+ or CD11b) cells were F4/80 CD68 (Fig. 1, subset B). The CD11c+ LPL were CD3, CD8β and CD19/ CD21. Hence, the large majority of CD11c+ LPL are DC, while CD11b+ CD11c F4/80+ CD68+ LPL are macrophages. Similar numbers of CD11c+ DC (5±0.2×105 per mouse) were isolated from the cLP of normal and RAG1–/– B6 mice raised under SPF or germ-free conditions (data not shown). The purified cLP DC populations analyzed in this study always contained >94% CD11c+ cells.

CD11c+ DC were diffusely distributed throughout the non-inflamed cLP in non-transplanted, immunocompetent (normal) or immunodeficient (RAG1–/–) B6 mice (Fig. 2A) but were rarely detected in the submucosa (Fig. 2C, data not shown). Most DC in the cLP were subepithelial. Only few F4/80+ macrophages were present in the cLP, but macrophages were found in the submucosa (Fig. 2B).

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Figure 1.  Identification of cLP DC. LPL were isolated from the colon of normal B6 mice and stained with labeled mAb specific for CD11c or CD11b, and mAb binding either surface F4/80, or cytoplasmic CD68. Histograms of CD11c+ (CD11b+ or CD11b) and CD11b+ CD11c cells from a representative examination (in which cLPL from four mice were pooled) are shown.

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Figure 2.  cLP DC and macrophages in normal mice, and mice with IBD. Untransplanted RAG1–/– mice (A–C) show a normal histology of the large intestine (A). Few F4/80+ macrophages are present in the submucosa and the cLP (B). CD11+ DC (C) are largely restricted to the LP. At 4 weeks after adoptive transfer of CD4+ T cells from β2m–/– donors (D–F), a mild colitis has developed comprising a submucosal edema, slightly enhanced epithelial proliferation, and a minimal inflammatory infiltrate of the mucosa (D). The number of macrophages (E) and DC (F) is mildly increased. Hosts transplanted with CD4+ T cells from Aβ–/– donors (G–I) show a severe colitis with a striking mucosal hyperplasia, a distortion of the crypt architecture, a nearly complete loss of goblet cells and a dense inflammatory infiltrate (G). In the LP of the mucosa, there is a marked increase in the number of macrophages (H) and DC (I). Representative data from three analyzed mice per group are shown.

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2.2 Surface phenotype of murine cLP DC

Surface expression of MHC class I/II and CD1d by freshly isolated cLP DC from normal or RAG1–/– B6 mice was high, while surface expression of CD40, CD80 and CD86 costimulator molecules was low (Fig. 3). Most cLP DC were CD11b+ but a small subset (1–10%) of cLP CD11c+ DC was CD11b CD8α+ (Fig. 3). This cLP CD8α+ DC subset was reproducibly lower (or undetectable) in cLPL populations from immunodeficient RAG1–/– B6 mice. A small but variable fraction of CD11c+ DC from the cLP expressed CD4 (Fig. 3). Similar numbers of cLP DC with a similar surface phenotype were recovered from SPF and germ-free B6 mice (data not shown). Hence, abundant CD11c+ DC are present in the cLP of normal and immunodeficient mice.

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Figure 3.  Surface phenotype of cLP DC from normal mice, and mice with IBD. cLP DC were isolated from normal, immunocompetent (N-B6) or immunodeficient (RAG–/– B6) mice, or from RAG–/– B6 mice transplanted with CD4+ T cells from either Aβ–/– B6 mice (developing severe IBD), or β2m–/– B6 mice (developing only mild IBD). The CD11c+ LPL were stained with labeled mAb binding CD11b, MHC class II (Ab), CD1d, CD40, CD86 or CD8α.

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2.3 Cytokine release by colonic DC

Purified cLP or splenic CD11c+ DC from normal or RAG1–/– B6 mice were cultured in vitro for 1–4 days. No (or only low amounts of) IL-12 p40, IL-12 p70 or IL-10 were released by unstimulated DC (Fig. 4A). Hence, neither the preparative isolation, nor the culture conditions induced cytokine release in freshly isolated splenic or mucosal DC. DC produced cytokines in response to CD40 ligation. Splenic DC showed a high and sustained release of IL-12 p40 in response to CD40 ligation. This response was reproducibly lower in cLP DC (Fig. 4A). An early, but low, CD40-dependent release of IL-12 p70 by splenic and cLP DC was detectable in the first 24 h of incubation but subsided thereafter (Fig. 4A). The finding of high, sustained IL-12 p40 but only low and transient IL-12 p70 (p40/p35) release by DC after CD40 ligation suggests differential regulation of p40 and p35 expression. Splenic and cLP DC released comparable and substantial amounts of IL-10 in a delayed response to CD40 ligation that peaked at 48–50 h of culture (Fig. 4A). In addition, CD40 ligation stimulated TNF-α release by splenic and cLP DC (data not shown). Splenic and mucosal DC from nontransplanted, normal or RAG–/– B6 mice thus display a similar pattern of IL-10 and IL-12 p70 release when stimulated by CD40 ligation.

We studied the regulation of cytokine release by cLP DC from normal or RAG1–/– B6 mice by IL-12 and IL-10 in response to CD40-dependent signals. IL-10 did not induce release of IL-12 p70, and IL-12 p70 did not induce release of IL-10 by cLP DC (Fig. 5A; group nos. 3, 4). IL-12 p40 (but not the low IL-12 p70) release after CD40 ligation was suppressed by IL-10 (group 5). IL-10 release was not suppressed by exogenous IL-12 p70 (group 6). Neutralizing IL-10 (by anti-IL-10 antibody) only marginally enhanced IL-12 p40 or IL-12 p70 release by CD40-stimulated, mucosal DC (group 7). Endogenous IL-10 of stimulated DC is thus too low to down-modulate IL-12 p40 and IL-12 p70 release, although a suppressive effect of high doses of exogenous IL-10 on IL-12 p40 release by cLP DC was readily apparent. Adding anti-IL-12 p40 or anti-IL-12 p35 antibody to the cultures did not change the IL-10 release by CD40-stimulated DC (groups 8, 9). Similar data were obtained using purified cLP DC from immunocompetent or immunodeficient B6 mice (data not shown).

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Figure 4.  Kinetics of IL-12 and IL-10 release of splenic and colonic CD11c+ DC from normal B6 mice (A) or transplanted RAG–/– B6 mice with IBD (B) in response to CD40 ligation. Splenic and cLP CD11c+ DC were isolated from normal B6, or transplanted RAG–/– B6 mice with severe IBD. cLP DC pooled from four individual mice from each group were co-cultured at 5×104 cells/ml for up to 5 days with either irradiated J558 cells (5×104 cells/well) (control group), or irradiated, CD40L-expressing J558 transfectants (5×104 cells/well) (CD40L). At the indicated time points, supernatant was harvested for cytokine determination by ELISA. Mean values of triplicates of one out of three independent experiments are shown.

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Figure 5.  Regulation of IL-12 release by cLP DC. The cLPL were prepared from six individual mice per group and pooled. CD11c+ DC were isolated by MACS from these cLPL populations from either normal B6 mice (A), or transplanted RAG–/– B6 mice with colitis (B). CD11c+ cLP DC (5×104 cells/well) were co-cultured with either irradiated J558 cells (5×104 cells/well) (groups 1, 3, 4), or irradiated, CD40L-expressing J558 transfectants (5×104 cells/well) (groups 2, 5–9). In some groups, 20 ng/ml recombinant mouse IL-10 (groups 3, 5), 20 ng/ml recombinant mouse IL-12 p70 (groups 4, 6), neutralizing anti-IL-10 mAb JES5–2A5 (αIL10, group 7), anti-IL-12 p40 mAb C15.6 (αIL12p40, group 8), or anti-IL-12 p70 mAb 9A5 (αIL12p70, group 9) were added at a concentration of 20 μg/ml. IL-10, IL-12 p40 and IL-12 p70 were determined by ELISA in supernatants harvested after 36 h of culture. Mean values of triplicates (± SD) of one out of three independent experiments are shown. n.t., not tested.

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2.4 Colonic DC in established transfer colitis

Adoptive transfer of splenic CD4+ α β T cells from normal or MHC class II-deficient (Aβ–/–) B6 donor mice into congenic RAG1–/– B6 hosts induces a severe colitis within 3–4 weeks post-transfer, while transplantation of CD4+ T cells from β2m–/– donors induces only mild colitis 16. We analyzed by immunohistology the in situ distribution and increase in numbers of CD11c+ DC and F4/80+ macrophages in the inflamed mucosa of the colon of transplanted mice. Transplantation of CD4+ T cells from β2m–/– donors induced mild colitic changes (Fig. 2D), associated with a slight mucosal hyperplasia with elongation of crypts, and a mild influx of leukocytes into the mucosa. Immunostaining showed that the numbers of F4/80+ macrophages (Fig. 2E) and CD11c+ DC (Fig. 2F) were only slightly increased in the lamina propria and the submucosa. In contrast, transfer of CD4+ T cells from Aβ–/– mice induced a severe colitis (Fig. 2G). The histopathology of the colon showed a marked hyperplasia of the mucosa, a nearly complete loss of goblet cells and a severe distortion of the crypt architecture with several crypt abscesses. The diseased animals showed a pronounced increase in the numbers of F4/80+ macrophages (Fig. 2H) and CD11c+ DC (Fig. 2I) within the inflamed cLP.

cLP CD11c+ DC were purified from transplanted RAG1–/– mice with colitis. Threefold higher numbers of DC (15±5×105 DC/mouse) were routinely recovered from the cLP of transplanted RAG–/– mice with IBD. These DC showed up-regulated CD1d, CD40, CD80 and CD86 surface expression when compared to cLP DC from non-transplanted (immunocompetent or immunodeficient) B6 mice (Fig. 3). The fraction of CD11b CD8α+ DC in the inflamed cLP was increased two- to threefold (Fig. 3). While cLP DC from IBD+ RAG–/– mice transplanted with CD4+ α β T cells from class II-deficient Aβ–/– B6 mice showed up-regulated surface expression of CD1d, CD40, CD80 and CD86, the cLP DC from RAG–/– mice transplanted with CD4+ α β T cells from class I-deficient β2m–/– B6 mice had no (or only mild) IBD and showed no up-regulation of CD40, CD80 or CD86 surface expression (Fig. 3). An ‘activated’ costimulatory phenotype of mature DC in the inflamed colonic lamina propria thus correlated with disease activity.

2.5 Cytokine expression by cLP DC associated with colitis

CD11c+ DC purified from the inflamed cLP of transplanted RAG–/– mice and transferred into culture released low amounts of IL-12 p40 and p70 and IL-10 ‘spontaneously’ (Fig. 4B). Following CD40 ligation, colitis-associated DC produced abundant IL-12 p40 and IL-10 but low levels of IL-12 p70 (Fig. 4B). The kinetics of this inducible IL-12 and IL-10 release differed from that described above for cLP DC from normal mice. Both cytokines increasingly accumulated in supernatants of cultures of cLP DC from mice with colitis over a 5-day culture period (Fig. 4B). The amounts of IL-12 p70 and IL-10 released by DC from the diseased mucosa in response to CD40 ligation were comparable to those released by an equal number of DC from the normal mucosa or the spleen, but the amount of IL-12 p40 released from colitis-associated DC was reproducibly higher in six independent experiments (Fig. 5A, B). Endogenous and exogenous IL-10 suppressed IL-12 p40 (but not IL-12 p70) release by DC from the inflamed mucosa (Fig. 5B, groups 5, 7). Neutralizing anti-IL-10 antibody selectively enhanced IL-12 p40 (but not IL-12 p70) release from the activated DC 10- to 20-fold (group 7). Adding αIL-12 p40 antibodies or anti-IL-12 p70 antibody had no reproducible effect on IL-10 release by cLP DC in most experiments although it partially suppressed IL-10 release in some experiments (groups 8, 9). cLP DC from the inflamed colon thus show up-regulation of costimulator molecules, a different kinetics of IL-12 and IL-10 release in response to CD40 ligation, and an enhanced susceptibility of IL-12 p40 release to suppression by IL-10.

2.6 Expression of p19 is up-regulated in cLPL from transplanted mice with IBD

The inducible IL-12 p40 release by cLP DC differed between normal mice and mice with IBD. IL-12 p40 associates with p19 to form the newly described cytokine IL-23 with IL-12-like activities 21. We tested if mucosal overexpression of IL-23 is found in IBD by quantitatively estimating p19 transcripts by real time RT-PCR in colon tissue, cLPL, and CD11c+ as well as CD11c cLPL (Fig. 6). A threefold increase of IL-23 p19 mRNA was detected in colon tissue from three RAG–/– mice with IBD reconstituted with CD4+ T cells from Aβ–/– donors (normalized ratio: 0.33±0.028) as compared to untransplanted RAG1–/– mice (normalized ratio: 0.11±0.016) (Fig. 6A). A similar picture emerged in the analysis of p19 transcripts in freshly isolated cLPL from non-transplanted versus transplanted, diseased RAG–/– mice (Fig. 6B). We further separated the CD11c+ (DC) and CD11c subsets (contining mainly T cells and macrophages) from the cLPL population of transplanted, diseased RAG–/– mice. Following CD40 ligation (by either CD40L-expressing J558/CD40L transfectants, or an anti-CD40 mAb), enhanced expression of p19 transcripts was found in both CD11c+ and CD11c cLPL subsets (Fig. 6C). Hence, DC and other cells from the inflamed cLP express more p19 than cLPL from normal mice, and this expression is up-regulated by CD40-dependent signals.

2.7 DC from the inflamed but not the normal cLP stimulate IFN-γ release by NKT cells

Activation of MHC class I-dependent CD4+ T cells seems a critical and essential event in triggering colitis in this model 16. We tested whether activated DC from the cLP of transplanted mice with IBD can activate NKT cells (Fig. 7). Co-culture of purified cLP DC from normal (or non-transplanted RAG–/–) B6 mice with purified, splenic CD4+ T cells from Aβ–/– mice did not stimulate IFN-γ, IL-13 or IL-4 release (group 1). When mucosal DC were pulsed with αGalCer, they presented the glycolipid to NKT cells and stimulated their IFN-γ, IL-13 or IL-4 release (group 2). A different picture emerged when DC purified from the inflamed cLPL of transplanted RAG–/– mice with IBD were co-cultured with NKT cells. These cLP DC triggered IFN-γ but neither IL-4, nor IL-13 release of co-cultured NKT cells (group 3). When cLP DC from mice with IBD were pulsed in addition with αGalCer, they stimulated IFN-γ but also IL-4 and IL-13 release by co-cultured NKT cells (group 4). Hence, cLP DC associated with IBD ‘spontaneously’ induce IFN-γ but not IL-4 and IL-13 release by co-cultured NKT cells, thus selectively promoting their Th1 differentiation.

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Figure 6.  Expression of IL-23 p19 by colonic lamina propria cells is up-regulated in IBD. RNA was extracted from colon tissue (A), freshly isolated cLPL (B), or fractionated CD11+ and CD11c subsets of freshly isolated cLPL (C) of either untransplanted, normal RAG–/– B6 mice (RAG–/–), or diseased RAG1–/– mice transplanted with CD4+ T cells from Aβ–/– donors (IBD+ RAG–/–). (C) Fractionated CD11c+ or CD11c cLPL from diseased RAG1–/– mice transplanted with CD4+ T cells from Aβ–/– donors were cultured for 18 h in vitro without any supplement (group 6), with irradiated J558 cells (groups 2, 4), with CD40L-expressing J558 transfectants (groups 3, 5), or with 3 μg/ml anti-CD40 mAb HM40–3 (cat. no. 09400D, PharMingen) (group 7). In the control group 1, only non-transfected J558 cells (that are known to express p19) were cultured. Relative IL-23 p19 mRNA expression is given as the mean ratio (n=3 in A and B, n=1 in C) (+ SEM) between a calibrator RNA and the RNA of the sample to be analyzed.

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Figure 7.  CD11c+ DC from the inflamed cLP of transplanted mice with colitis stimulate IFN-γ release by co-cultured NKT cells. cLP DC were isolated from normal RAG–/– B6 mice, or transplanted RAG–/– B6 mice with colitis. These DC were either non-pulsed, or pulsed with αGalCer and washed. DC were co-cultured with freshly isolated, splenic CD4+ CD3+ responder T cells from Aβ–/– donors (NKT cells). Cytokine (IFN-γ, IL-4, IL-13) release during the 18 h co-culture was determined by ELISA. Mean values of triplicates (+ SD) of one out of three independent experiments are shown.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

3.1 A putative role for IL-23 in the pathogenesis of IBD

Th1 polarization of mucosal T cell responses driven by IL-12 is considered a key factor in the pathogenesis of the analyzed transfer colitis. CD40-mediated signals are potent stimuli for IL-12 release by DC 2529. Expression of IL-12 p70 by splenic and cLP DC from normal mice showed an early peak that subsided after 24 h, while p40 release was sustained (Fig. 4A). Furthermore, the amount of p70 released by cLP DC from transplanted mice with IBD was not increased, while their p40 release was increased by more than tenfold (Fig. 4B). p40 homodimers bind to IL-12Rβ1 with low affinity but do not signal. They are therefore considered functional antagonists to p70 in mice because they inhibit IFN-γ release from spleen cells, CTL priming, IgG2/IgG3 antibody responses and allospecific delayed-type hypersensitivity reactions 3032. Suppression of IL-12(-like) bioactivity by excess IL-12 p40 was not observed in the inflamed cLP. It was therefore of interest to search for expression of the alternative p40-binding subunit, i.e. p19, which is known to drive Th1 immune responses as the p40/p19 heterodimer IL-23.

A role of IL-23 in the pathogenesis of colitis has not been considered. Ubiquitous expression of the p19 subunit of IL-23 in multiple tissues of transgenic mice induces systemic inflammation (with infiltrates of lymphocytes and macrophages in skin, lung, liver and the digestive tract), increase in serum concentrations of proinflammatory cytokines (TNF-α, IL-1), runting and death before 3 months of age 33. IL-23 induces strong proliferation of mouse memory CD4+ CD45RBlow T cells, a unique activity of IL-23 that is not found for IL-12 21. IL-23 is secreted by activated DC 21. p19 expression was enhanced in LPL from transplanted mice with IBD. IL-23 is thus an interesting candidate for inducing pathogenic mucosal Th1 memory T cell responses.

3.2 Selective activation of Th1 NKT cells by cLP DC from mice with IBD

MHC class II, CD1d and CD40 surface expression was higher in cLP than splenic DC. We have reported that MHC class I-dependent CD4+ T cells are required to induce transfer colitis 16. Freshly isolated cLP DC from mice with colitis induced IFN-γ (but not IL-4 or IL-13) release by CD4+ T cells from MHC class II-deficient mice. This was not observed when NKT cells were co-cultured with cLP DC freshly prepared from normal (healthy), syngeneic mice. The cLP DC from mice with IBD can thus target in situ regulatory T cells to locally induceand/or support Th1-biased T cell responses.

3.3 Murine cLP DC

The preparative isolation of murine cLP DC is difficult and their yield is limited. Purifying CD11c+ DC from LPL by MACS, we obtained 0.5±0.2×105 cLP DC per mouse from normal and immunodeficient (RAG1–/–) B6 mice. We detected <0.5% F4/80+ macrophages in the purified CD11c+ cLP DC population. For flow cytometry (FCM) and functional assays, cLP DC from three to four individual mice were pooled in most experiments. The differences in surface phenotype of cLP DC from normal and diseased mice and their inducible cytokine release profile make it unlikely that the isolation procedure triggered activation of DC.

Within the cLP DC population, the CD11b+ subset comprised 60–75% of the cells, while the CD8α+ DC subset represented only 1–10% of the cLP DC. A cLP DC subset of 20–30% was CD11b and CD8α, i.e. ‘double negative’ (DN). In the IBD-associated, Th1-like CD4+ T cell response in the cLP, CD8α+ DC increased innumber representing 15–25% of the mucosal DC population. Changes in the cLP DC population during the pathogenesis of colitis could result from changes in their recruitment, differentiation and/or activation.

3.4 The role of cLP DC in IBD

We have identified two novel features of cLP DC manifest in IBD that have the potential to decisively influence the outcome of the disease. These are the induction of Th1-promoting, regulatory(MHC class II-independent) CD4+ T cells, and the expression of the Th1-promoting cytokine IL-23. Both effects of DC can have a major influence on the induction, maintenance and progressionof the pathogenic T cell response in situ. It remains to be shown if these observation from a preclinical model point to a pathophysiology also prevalent in human IBD.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

4.1 Mice

C57BL/6J (B6) mice, B6 MHC-II–/– (Aα–/– or Aβ–/–) mice 34, 35, C57BL/6J-Rag1tm1Mom (RAG1–/–) mice 36 (Jackson Labs; stock no. 2216), and B6 MHC-I–/– (β2m–/–) mice 37 were used. Mice were bred and kept under SPF conditions in the animal facility of Ulm University. RAG1–/– mice were transplanted at 8–12 weeks of age.

4.2 CD4+ T cells used for adoptive transfer

Transplantation of RAG1–/– mice with purified splenic CD4+ T cells has been described 16. The purity of the positively selected CD4+ populations was always >98%. We injected 5×105 CD4+ cells intraperitoneally into RAG–/– B6 mice. The transplanted mice were weighed and their clinical condition was monitoredat biweekly intervals.

4.3 Isolation of spleen cells and cLP cells

Mice were killed by cervical dislocation. Single-cell suspensions were aseptically prepared from the spleen and the cLP. LPL were isolated as described 3840. CD11c+ cells were enriched to >98% purity by positive MACS selection using CD11c MicroBeads (cat. no. 130-052-001, Miltenyi Biotec, Bergisch Gladbach, Germany).

4.4 Flow cytometry

Cells were suspended in PBS/0.3% w/v bovine serum albumin (BSA) supplemented with 0.1% w/v sodium azide. Nonspecific binding of antibodies to Fc receptor was blocked by preincubating cells with the mAb 2.4G2 (BD-PharMingen, Hamburg, Germany; cat. no. 01241D) directed against the FcγRIII/II CD16/CD32 (1 μg mAb/106 cells/100 μl). Cells were incubated with 0.5 μg/106 cells of the relevant mAb for 30 min at 4°C, and washed. In most experiments, cells were subsequently incubated with a second-step reagent for 10 min at 4°C. Three-color FCM analyses wereperformed on a FACSCalibur (Becton Dickinson, Mountain View, CA). The following reagents and mAb were obtained from PharMingen: PE-conjugated anti-CD3ϵ mAb 145-2C11 (cat. no. 01085B), FITC- andPE-conjugated anti-CD4 mAb GK1.5 (cat. no. 09424D and 09425B), biotinylated anti-CD4 mAb RM4–5 (cat. no. 01062D), FITC-conjugated anti-CD8α mAb 53–6.7 (cat. no. 01351), PE-conjugated anti-NK1.1mAb PK136 (cat. no. 01295B), PE-conjugated anti-CD1d mAb 1B1 (cat. no. 553846), PE-conjugated and biotinylated anti-H-2Kb mAb AF6–88.5 (cat. no. 06105A and 06102D), FITC-conjugated and biotinylated anti-I-Ab mAb (cat. no. 06045A and 06042D), biotinylated anti-CD40 mAb M1/70 (cat. no. 09662D), FITC-conjugated anti-CD80 mAb 16–10A1 (cat. no. 09604D), biotinylated anti-CD86 mAb GL1 (cat. no. 09272D), and FITC-conjugated anti-CD11b mAb M1/70 (cat. no. 01714A). PE-conjugated anti-F4/80 mAb A3–1 (cat. no. MCA497PE) and FITC-conjugated anti-CD68 mAb FA-11 (cat. no. MCA1957F) were obtained from Serotec (Düsseldorf, Germany). SA-Red670 (cat. no. 19543–024) was obtained from Gibco-BRL (Berlin, Germany).

4.5 Cell cultures

CD11c+ DC (5×104 DC/well) were cultured for 1–4 days in 200 μl flat-bottom microwells in RPMI 1640 medium supplemented with 5% FCS. For CD40 ligation, we used either10,000 rad-irradiated CD40L-expressing J558 transfectants (5×104 cells/well), or 3 μg/ml of the anti-CD40 mAb HM40–3 (cat. no. 09400D, PharMingen). Supernatants were collected from DC cultures at the indicated time points for cytokine determination. Cytokine production by DC was blocked with 20 μg/ml anti-IL-10 mAb JES5–2A5 (cat. no. 554422), anti-IL-12 p40 mAb C15.6 (cat. no. 18491U), anti-IL-12 p70 mAb 9A5 (cat. no. 20011D), or isotype control mAb A110–2 (cat. no. 11191D) or B81–3 (cat. no. 11221D). To some DC cultures, IL-10 (cat. no. 210–10, PeproTech) or IL12 p70 (cat. no. 19361V, PharMingen) were added at 20 ng/ml.

4.6 Cytokine determination by ELISA

Cytokines released into culture supernatants were detected by a double-sandwich ELISA. For detection and capture, the following mAb (from PharMingen) were used: mAb R4–6A2 (cat. no. 18181D) and biotinylated mAb XMG1.2 (cat. no. 18112D) were used for IFN-γ detection, mAb BVD4–1D11 (cat. no. 18031D) and biotinylated mAb BVD6–24G2 (cat. no. 18042D) were used for IL-4 detection; mAb 9A5 (cat. no. 20011D) and biotinylated mAb C17.8 (cat. no. 18482D) were used for IL-12 p70 detection; mAb JES5–2A5 (cat. no. 554422) and biotinylated mAb SXC-1 (cat. no. 554423) were used for IL-10 detection; mAb C15.6 (cat. no. 18491U) and biotinylated mAb C17.8 (cat. no. 18482D) were used for IL-12 p40 detection; non-conjugated (cat. no. MAB413) and biotinylated mAb 38213.11 (cat. no. BAF413) were used for IL-13 detection (R&D systems, Wiesbaden). Extinction was analyzed at 405/490 nm on a TECAN micro plate-ELISA reader (TECAN, Crailsheim, Germany) using the EasyWin software (TECAN, Crailsheim, Germany).

4.7 Assessment of p19 mRNA expression

Expression of IL-23 p19 mRNA was determined by real time RT-PCR and relative quantification using G6PDH as a reference gene 41. Ten slices of 25 μm each were cut off frozen tissue blocks from the cecum, transverse and sigmoid colon. Tissue was collected in a FastRNA Tube (cat. no. 6040–601, BIO 101 Inc., Carlsbad, CA) and mechanically homogenized in TrizolTM reagent (cat. no. 15596–026, Life Technologies, Karlsruhe, Germany) by a FastPrep 120 device (Bio 101 Inc). RNA from tissue homogenates or cell pellets was extracted according to the manufacturer's instructions of the Trizol reagent. RNA was annealed to 1 pmol of an oligo(dt)15 primer, denatured at 80°C and reverse-transcribed with 200 U Super ScriptTM reverse transcriptase (cat. no. 18053–017, Life Technologies) in 25 μl of a reaction mix containing buffer supplied by the manufacturer, 10 mM DTT, 40 U ribonuclease inhibitor (RNAsinTM, cat. no. N2111, Promega, Mannheim, Germany), and 1 mM of each dNTP. The RT-reaction product (1 μl of a 1:8 dilution) was submitted to a real time PCR using the LightCycler-DNA Master SYBR Green I kit (cat. no. 2158817, Roche Diagnostics, Mannheim, Germany). PCR was carried out at an annealing temperature of 55°C and an MgCl2 concentration of 4 mM. Primer sequences were 5′-CCAGCGGGACATATGAATCT-3′ (p19 left), 5′-AGGCTCCCCTTTGAAGATGT-3′ (p19 right), 5′-CATGAGTCAGACAGGCTGGA-3′ (G6PDH left), 5′-CAGCACCATGAGATTCTGGA-3′ (G6PDH right). PCR was carriedout in the LightCycler instrument (cat. no. 2011468, Roche Diagnostics). For the construction of standard curves, PCR products were ligated into a PCR-cloning vector (pGEM-T-Vector-System, cat. no.A3610, Promega). Inserts were amplified by conventional DNA-PCR using standard M13 Primers, gel purified (QIAquick gel extraction kit, cat. no. 28104, Qiagen, Hilden, Germany) and diluted over fourranges of magnitude with five replicates per dilution step to cover the CT values found at amplification of cDNA samples. Quantification was performed using the LightCycler Relative Quantification Software version 1.0 (cat. no. 3158527, Roche Diagnostics) by calculating the ratio between the sample of interest and a calibrator RNA.

4.8 Histopathology and immunofluorescence staining

Sections (2 μm thick) were cut with a microtome from paraffin blocks. These were submitted to automatic hematoxylineosin (H&E) staining. Frozen tissue was cut into 2-μm sections, mounted and acetone-fixed for 2 min at room temperature. mAb used for immunohistology were biotin-labeled anti-CD11c clone HL3 (cat. no. 09702D; PharMingen) and biotin labeled anti-F4/80 clone A3–1 (cat.no. MCA497B; Biozol Diagnostika, München, Germany). Cryosections were incubated with the appropriate antibodies diluted 1:10 for 1 h, and washed twice in PBS buffer. Binding of these biotin-labeledantibodies was detected using Cy3-conjugated streptavidin (cat. no. 016-160-084; Dianova, Hamburg, Germany). In negative control stainings, isotype-matched mAb to an irrelevant target were used. These mAb were from the same species as the primary antibody and applied at identical concentration. The control mAb for CD11c was biotin-conjugated Armenian hamster IgG1, λ clone G235-2356 (cat. no. 11122C; PharMingen), and biotin-conjugated rat IgG2b, κ clone A95-1 (cat. no. 11182C, PharMingen) was used as a control for F4/80. All incubation steps were carried out at room temperature in PBS. Immunofluorescence stainings were counterstained with 0.3 μg/ml 4,6-diamidino-2-phenylindole (DAPI, cat. no. D9542; Sigma-Aldrich, Deisenhofen, Germany), and embedded in Vectashield medium (cat. no. H1000; Vector, Burlingame, CA).

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods
  7. Acknowledgements

The expert technical assistance of Beate Wotschke and Anja Müller is gratefully acknowledged. J.R. greatly appreciates the helpful comments of Drs. D. Rennick (DNAX, Palo Alto, CA) and M. J. Wick (Göteborg, Sweden). This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG Re549/9–1) to J.R., and the University of Ulm (Interdisziplinäres Zentrum für Klinische Forschung A7) to F.L.

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