SEARCH

SEARCH BY CITATION

Keywords:

  • chemokine receptor;
  • costimulatory molecules;
  • cytokines;
  • DC maturation;
  • T-cell stimulation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

CCR9 has been identified on T cells as a chemokine receptor that directs these cells to migrate to the intestine. CCR9 has also been reported on different cell types in the intestine, thymus, liver and peripheral blood. Little is reported concerning the presence of or functional implications of this chemokine receptor on myeloid dendritic cells (DC). In the host, DC encounter a multiplicity of antigenic stimuli to which they mount immune responses. In addition to intracellular and functional changes on sensing antigen, maturation of DC is typically reflected in the up-regulation of costimulatory molecules on DC. However, alterations in other surface markers may also be an indicator of DC activation. Using bone marrow-propagated DC these studies investigated cellular maturation in the presence of microbial stimuli and analyzed the relationship of CCR9 expression with DC maturation. Fractionation of DC into CCR9high and CCR9lowsubsets revealed a distinct ability of each subset to induce division in naïve CD4+ T cells. Our results suggest that DC expressing high levels of CCR9 are less activated/mature than DC expressing low levels of CCR9.


Abbreviations:
BM

bone marrow

CFSE

carboxyfluorescein diacetate succinimidyl ester

CpG

cytosine–phosphate–guanosine

DC

dendritic cells

ELISA

enzyme-linked immunosorbent assay

FITC

fluorescein isothiocyanate

FBS

fetal bovine serum

GM-CSF

granulocyte–macrophage colony-stimulating factor

GpC

guanosine–phosphate–cytosine

IBD

inflammatory bowel disease

IFN-γ

interferon-γ

IgA

immunoglobulin A

IgG

immunoglobulin G

IL

interleukin

LB

Luria broth

LPMC

lamina propria mononuclear cells

mAb

monoclonal antibody

MACS

magnetic antibody cell sorting

ODN

oligonucleotide

PCR

polymerase chain reaction

PerCP

peridinin chlorophyll protein

SCID

severe combined immunodeficient

TCR

T-cell receptor

Th1

T-helper 1

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Interactions between chemokines and chemokine receptors are definitive components of the regulation of the immune system and, as such, changes in the expression or in the function of these chemokines or chemokine receptors can be a prime indicator of disease activity. Several chemokines may be expressed at the site of inflammatory lesions and hence it is usually difficult to determine the relationship between a single chemokine and its influence on disease. More specific information is usually obtained by investigating the relationship between chemokine receptor function and disease.

The chemokine receptor CCR9 is the only known receptor for the chemokine CCL25 [thymus-expressed chemokine (TECK)]. Both molecules are found in the thymic cortex and in the small intestinal mucosa.1–6 CCR9, a receptor believed to direct the migration of T cells to the intestine, is widely expressed on most intraepithelial lymphocytes and lamina propria T cells, and may be regulated by, and also possibly regulate, inflammatory diseases of the intestine.6–9 In chronic inflammatory liver disease, CCR9 possibly mediates the trafficking of CCR9-expressing T cells from the gut to the liver, since, these cells were found infiltrating the livers of patients with chronic inflammatory liver disease.10 The CCR9 receptor is not unique to T cells and has been reported on immunoglobulin A (IgA)-secreting B cells in the small intestine11,12 and more recently on plasmacytoid dendritic cells (DC).13

Apart from its involvement in cellular migration, the significance of CCR9 on cells is not well understood. Furthermore, the regulation of T-cell expression of this receptor is influenced by a multiplicity of factors. For example, pre-T-cell receptor (TCR) signaling,3 retinoic acid14 and stimulation by DC from Peyers’ patches and the mesenteric lymph nodes15 resulted in CCR9 up-regulation. Conversely, persistently activated T-helper 1 (Th1) cells generated from umbilical cord blood exhibited down-regulation of CCR9 expression. When TCR cross-linking stimulation was removed, CCR9 expression was restored on these T cells.6 Studies on duodenal biopsy specimens from patients with celiac disease showed that in both epithelial and lamina propria cell suspensions, T cells from control volunteer subjects expressed higher levels of CCR9 in comparison with T cells from treated celiac disease patients. Furthermore, T cells from treated celiac disease patients also expressed higher levels of CCR9 than T cells from untreated patients, the latter of which hardly expressed CCR9,7 supporting the hypothesis that under conditions of immune activation, T-cell CCR9 expression was down-regulated.

DC are potent antigen-presenting cells.16–20 DC, although rare in most tissues, maintain immune tolerance, and the activation level of these cells can shift the balance between tolerance and disease.21 In disease, interactions between DC and other cells can perpetuate inflammation and may maintain the chronicity of the disease.22–24 We designed our studies to determine whether CCR9 expression correlated with the activation state of bone marrow-generated DC. Stimulation of DC with activation agents such as cytosine–phosphate–guanosine (CpG) and Escherichia coli up-regulated the costimulatory molecule expression of CD40 and CD86, and released pro-inflammatory cytokines in culture, whereas a significant decrease in the number of cells expressing CCR9 was observed. To elucidate a possible functional role of CCR9, we separated DC into two fractions for study. It was found that CCR9low and CCR9high DC subsets had a differential ability to induce naïve T cells to divide and that this interaction with T cells resulted in the production of a distinct cytokine profile. Our results provided a novel means of determining the maturation state of DC subsets according to the level of CCR9 expression on the surface of these cells, in which cells abundant in CCR9 may be associated with lowered DC function.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

Animals

Adult female BALB/c mice (8–12 weeks of age) were purchased from Jackson Laboratories (Bar Harbor, ME). C57BL/6J (H-2b) mice were purchased from Jackson Laboratories and spleen T cells from these mice were used as allogeneic responders. For the induction of colitis, adult female donor BALB/c mice (8–12-weeks of age) and C.B-17 severe combined immunodeficient (SCID) recipient mice were purchased from Taconic Laboratories (Germantown, NY). Mice were maintained in a specific pathogen-free environment in the animal facility at Case Western Reserve University, in accordance with Institutional guidelines.

Propagation of bone marrow-derived DC

Bone marrow cells were propagated from mice in RPMI-1640 containing 10% (vol/vol) fetal bovine serum (FBS; Life Technologies, Carlsbad, CA) and supplements in the presence of 7 ng/ml of recombinant mouse granulocyte–macrophage colony-stimulating factor (GM-CSF; R&D Systems, Minneapolis, MN) over 5 days, as described previously.25 Upon harvesting, the non-adherent cells released from clusters were collected for study. In most experiments, DC were further enriched using CD11c magnetic antibody cell sorting (MACS) beads and columns according to the miniMACS system procedure (Miltenyi Biotec, Auburn, CA).

Dendritic cell stimulation

Purified DC were cultured in 24-well plates at a concentration of 1 × 106 cells/ml or as indicated in the figure legends. Microbial stimulants, or an equivalent volume (10 μl) of control buffer, were added to the wells. DC were stimulated with CpG oligonucleotide [oligonucleotide (ODN) 1826, cat. no. tlrl-modn; InvivoGen, San Diego, CA], sequence 5′-tccatgacgttcctgacgtt-3′ (20 mer) used at a final concentration of 3 μm. In some experiments ODN 1826 control-tlrl-modnc was used as a control. In this sequence, CpG dinucleotide is replaced with guanosine–phosphate–cytosine (GpC) dinucleotide, and this molecule, unlike CpG, does not stimulate Toll-like receptor 9 (TLR9) on cells. Other stimuli used were Lactobacillus plantarum (strain number ATCC 1024), which was cultured in MRS broth (Becton Dickinson, Franklin Lakes, NJ) and used at a final concentration of 105 or 107 colony-forming units (CFU)/ml, and E. coli, which was cultured in Luria broth (LB; Difco Labs, Detroit, MI) and used at 105 or 107 CFU/ml. DC were cultured overnight, in a 37°, 5% CO2 incubator, in RPMI-1640 containing stimulants and supplements. Following culture, supernatants were harvested and analysed for cytokines, and cell pellets were used to determine CCR9 phenotypic changes in response to the different treatments.

Determination of CCR9 levels on dendritic cells

DC surface antigen expression was investigated by dual monoclonal antibody (mAb) immunostaining. DC (2–5 × 105 cells) were aliquoted into tubes in 100 μl of staining buffer (RPMI-1640 + 10% FBS). Cells were blocked by incubation with the Fc receptor for immunoglobulin G (IgG) III/II (CD16/CD32; PharMingen, San Diego, CA) at 4°. DC were directly labeled with CD11c–phycoerythrin (PE) (HL3), and with CD40 (3/23), CD80 (B7-1; 16-10A1), CD86 (B7-2; GL-1) or major histocompatibility complex (MHC) class II I-Ad (AMS-32.1), all labeled with fluorescein isothiocyanate (FITC; PharMingen). To determine the changes in expression of CCR9 on DC, cells were labeled with CD11c–phycoerythrin (PE) and CCR9–FITC conjugates (R&D Systems). Gating was performed on live cells using species-specific IgG isotype controls in each experiment. At least 10 000 events were acquired on a fluorescence-activated cell sorter (FACScan; Becton Dickinson).

Fractionation of dendritic cell subsets

DC were labeled with CD11c–PE and CCR9–FITC conjugates for cell sorting. Isotype-matched hamster IgG–PE and rat IgG–FITC conjugates were used as negative controls. The sorted DC populations were collected in two subsets as CD11c+ CCR9high cells in the upper right-hand quadrant and as CD11c+ CCR9low DC in the upper left-hand quadrant of dot-plots. Collected DC were used in DC-T cell stimulation assays.

Dendritic cell stimulation of naive T cells

Spleens were harvested from C57BL6/J mice and T cells were purified using CD4 MACS beads and columns (Miltenyi Biotec). Recovered CD4 cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE)26,27 as previously described.22 Graded concentrations of DC subsets were added to T cells in 96-well flat-bottomed plates and cultured in a 37°, 5% CO2 incubator for 3 or 4 days. Harvested cells were stained with CD4 peridinin chlorophyll protein (PerCP, L3T4; RM4-5; PharMingen). More than 25 000 events were acquired on a FACScan and analysis was performed using fcs express v3 program software (De Novo Software, Ontario, Canada).

Determination of dendritic cell subsets in colons of mice

Induction of colitis in SCID mice represents a model of human inflammatory bowel disease (IBD). Colitis was induced by transfer of T cells into SCID mice, as described previously.22 Briefly, BALB/c spleen T cells were dual labeled with CD4 (PE; L3T4 GK.1.5; PharMingen) and CD45RB (FITC; C363.16A; PharMingen) mAbs, then sorted on a BD FACS Aria (Becton Dickinson, San Jose, CA), collecting the 35% most double-positive CD4 CD45RBhigh T cells. SCID mice were reconstituted with 0·5–1 × 106 T cells (injected in the lateral tail vein) for the development of colitis. On sacrifice of mice and harvest of colons, digestion of tissue was performed using collagenase and DNAse to obtain lamina propria mononuclear cells (LPMC), as described.28,29 LPMC were semipurified, using CD11c MACS beads and columns (Miltenyi Biotec), to obtain DC. Recovered CD11c+ cells were labeled with CD11c–PE and CCR9–FITC mAbs or relevant isotype controls and further purified on a FACS Aria (Becton Dickinson).

Cytokine quantification

Cell culture supernatants were centrifuged to remove the cells. Cytokine levels in the cell-free supernatant were measured using enzyme-linked immunosorbent assays (ELISAs) for bioactive interleukin (IL)-6, IL-10, IL-12p70, IL-17, IL-2 and interferon-γ (IFN-γ) using Duoset ELISA kits (R&D Systems).

Statistical analysis

Data shown indicate the means and standard deviations, as determined against known concentrations, in cytokine standard curves using a Biotek synergy II ELISA reader and kc4 software (Winooski, VT). Significant differences in cytokine levels were determined using the Student’s t-test, with a P-value of < 0·05 as the limit.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

CCR9 is moderately expressed on bone marrow-derived dendritic cells

Bone marrow cells cultured in GM-CSF expressed CCR9 by day 1 of culture. Approximately 12% of leucocytes cultured in GM-CSF expressed CCR9 by day 1 of culture (Fig. 1a). For functional studies, DC were harvested on day 5 of culture because DC harvested at this time-point are more readily regulated by maturational stimuli than DC that are harvested at later time-points, which are often more mature and may be refractory to stimulation. To determine CCR9 expression on DC, cells harvested and purified on day 5 were stained with CD11c–PE and CCR9–FITC, or with matching isotypes. Alternatively, DC were treated with controls, including water, MRS, LB or ODN control, added in volumes of 10 μl into 1 ml of cells and then cultured overnight. In the dot-plot shown, CCR9 was expressed on 24% of CD11c+ cells (Fig. 1b). A similar trend of moderate CCR9 expression on freshly harvested or control-treated DC was consistently observed in experiments. There were no significant differences in the expression of CCR9 as a consequence of the addition of any of the control stimuli to DC. In other studies, the presence of CCR9 gene expression was confirmed by detection in RNA extracted from day 5-harvested DC using real-time polymerase chain reaction (PCR) (data not shown).

image

Figure 1.  CCR9 is moderately expressed on bone marrow (BM)-generated dendritic cells (DC). (a) BM cells (2 × 106) were cultured in 24-well plates for 1 day. Cells were removed from wells, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates, or with a control isotype (HIgG–PE and RIgG–FITC), and events were collected on a fluorescence-activated cell sorter (FACScan). (b) Day 5-harvested and purified BM-generated DC were subcultured at 1 × 106 cells/ml in the presence of control H2O (10 μl). DC were harvested the next day and labeled with monoclonal antibodies (mAbs), as shown. Dot-plots show basal levels of CCR9 expression on DC and are representative of more than six independent experiments. In experiments, the addition of control stimuli [H2O, MRS broth, Luria–Bertani broth or the cytosine–phosphate–guanosine (CpG) control] did not alter the expression of CCR9 on DC in comparison with CCR9 expressed on day 5-harvested DC.

Download figure to PowerPoint

Maturation of dendritic cells upon exposure to microbial stimuli

In the host, at sites such as the gastrointestinal tract and the liver, DC constantly encounter pathogen and pathogen-related molecules, which may trigger the expression of surface receptors, resulting in intracellular changes leading to DC maturation/activation. Studies have shown that CCR9 expression on cells may be implicated in gut pathogenesis.7,8,30 In the intestine, many bacteria are in contact with myeloid DC. Our goal here was to understand how bacteria or microbial products regulate DC maturation and then to determine whether there is a correlation between DC maturity and CCR9 changes. These investigations focused on how CpG, Gram-negative E. coli and a Gram-positive probiotic bacterium (L. plantarum) could alter DC costimulatory molecule expression, as a measure of DC maturation. Stimulation of DC with CpG resulted in the up-regulation of costimulatory molecules CD40 and CD86 by comparison with control stimuli (Fig. 2a,b). L. plantarum, at a concentration of 105 CFU/ml, did not significantly alter the expression of costimulatory molecules by comparison with control H2O or MRS broth. However, a non-pathogenic strain of E. coli, at a concentration of 105 CFU/ml, resulted in a significantly increased population of cells expressing CD40 and CD86 (Fig. 2a,b). With stimuli, CD80 expression was not significantly altered from its baseline level of about 55% expression on the surface of DC. Also, because of the constitutively high expression of MHC class II on these cells (about 75% expression) we did not use this marker as an indication of DC maturation.

image

Figure 2.  Costimulatory molecule regulation upon exposure of dendritic cells (DC) to microbial stimuli. DC were purified and stimulated with cytosine–phosphate–guanosine (CpG) (3 μm), Lactobacillus plantarum [105 colony-forming units (CFU)/ml] or E. coli (105 CFU/ml) in 1 ml of RPMI-1640/10% fetal bovine serum (FBS) and supplements, overnight in a 37°, 5% CO2 humidified incubator. Treatments were added in 10-μl quantities. Cells were collected the next day and labeled with CD11c–phycoerythrin (PE) and with CD40– or CD86–fluorescein isothiocyanate (FITC) conjugates. Histogram overlays show an increase in costimulatory molecule expression above that of H2O stimulation. The results are representative of two to three independent experiments.

Download figure to PowerPoint

Release of cytokines in stimulated dendritic cell cultures

Changes in DC costimulatory molecules are often followed by intracellular changes that lead to changes in cytokine release. Therefore, having initially observed increases in DC costimulatory molecule expression as a result of exposure to CpG and to E. coli, we examined the ability of these microbial stimuli to stimulate cytokines in DC. Because CCR9 may be important in intestinal disorders,7,8,30 we examined the release of Th1, Th2 and pro-inflammatory cytokines involved in the cure or in the pathogenesis of gut inflammation.31–35 The levels of IL-10, IL-6, IL-17, IL-12 p70, IL-2 and interferon-γ (IFN-γ) were measured in DC cultures after microbial stimulation. The most potent inducer of cytokines was CpG, which stimulated the release of a high level of IL-6 in DC cultures, moderate levels of IL-10, low levels of IL-12 p70, and no IL-17, IL-2 or IFN-γ. Like CpG, E. coli (105 CFU/ml) was a potent stimulator of IL-6 release, showing lower levels of IL-10, but not to the same extent as with CpG treatment (Fig. 3a). L. plantarum, at a dose of 105 CFU/ml, gave little stimulation of DC regarding either changes in costimulatory molecules or in cytokine release, indicating little activation of cells with this treatment.

image

Figure 3.  Early release of cytokines upon dendritic cell (DC) stimulation. (a) Purified DC (1 × 106 cells/ml) were cultured in 24-well plates, as described above, with the addition of cytosine–phosphate–guanosine (CpG) (3 μm), Lactobacillus plantarum [105 colony-forming units (CFU)/ml] or Escherichia coli (105 CFU/ml). Supernatants were collected after 24 hr and cytokine release was investigated. The results are representative of three independent experiments. (b) Purified DC (1 × 105 cells/well, 200 μl) were cultured in 96-well flat-bottom plates. Cells were treated with CpG (3 μm), L. plantarum (107 CFU/ml) or E. coli (107 CFU/ml). Supernatants were harvested at the time-points indicated and the amounts of cytokines released were determined using enzyme-linked immunosorbent assay (ELISA). Means and standard deviations of duplicate assay wells are shown. P-values indicate levels of significance for cytokine release above that of the control (water). IL, interleukin.

Download figure to PowerPoint

The kinetics of cytokines released revealed that CpG and E. coli stimulated the production of significantly high levels of the pro-inflammatory cytokine IL-6 by day 1 of culture. Upon stimulation of DC with a high dose (107 CFU/ml) of L. plantarum, IL-6 and comparatively moderate levels of IL-10 were released. This stimulus was much less efficient at activating pro-inflammatory IL-6 in DC than either CpG or E. coli (Fig. 3b).

CCR9 expression is reduced in mature dendritic cells

With the finding that DC stimulated with bacterial agents expressed different amounts of surface CD40 and CD86, and released different concentrations of cytokines in culture, both of which are associated with DC maturation/activation, we then focused on how CCR9 expression varied in these populations of DC. Cells were stimulated with microbial agents overnight, as described in a previous section, and CCR9-expressing cells were investigated by dual mAb staining. Treatment with CpG and E. coli resulted in cell populations with drastically reduced (threefold lower) numbers of CCR9-expressing DC, whereas cells treated with L. plantarum, MRS, LB or water, which did not alter costimulatory molecule expression, also did not induce a significant change in the population of DC expressing this receptor (Fig. 4). Additionally, treatment of DC with GM-CSF and IL-4 day 5–6 led to an up-regulation of the DC costimulatory molecule CD86 (18–31%) and fewer DC expressing CCR9 (Fig. 4). Taken together, our observations show that stimuli which matured DC (CpG, E. coli, GM-CSF and IL-4), also led to DC populations with low CCR9 expression, to the extent that the percentage of DC expressing CCR9 was negligible after treatment with activation stimuli (Fig. 4). It is noteworthy that even though 105 CFU/ml of L. plantarum did not alter CCR9 expression, in other experiments we found that increasing the dose to 107 CFU/ml was sufficient to induce a significant reduction in CCR9 (data not shown).

image

Figure 4.  CCR9 on dendritic cells (DC) is up-regulated in response to maturation stimuli. Purified DC (1 × 106 cells/ml, 24-well plates) were stimulated with cytosine–phosphate–guanosine (CpG) (3 μm), Lactobacillus plantarum (105 CFU/ml), Escherichia  coli (105 CFU/ml) or granulocyte–macrophage colony-stimulating factor (GM-CSF) (7 ng/ml) plus interleukin-4 (IL-4) (20 ng/ml), overnight in a 37°, 5% CO2 incubator. Cells were stained with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates or with relevant isotypes (HIgG–PE and RIgG–FITC). The isotype staining shown is representative of background fluorescence obtained with cells from each treatment. Other dot-plot panels show CCR9 expression when DC were treated with different stimuli or with the control (water). The results are representative of three to five independent experiments.

Download figure to PowerPoint

Dendritic cell populations lacking CCR9 induce high levels of cell division in naïve T cells

To understand in more detail the changes in DC expression of CCR9 and DC function we investigated the ability of CCR9high-expressing and CCR9low-expressing DC populations to induce cell division in naïve CD4+ T cells. Few CCR9-expressing DC remained in culture after treatment with activation stimuli (Fig. 4). Thus, to study the relationship between DC phenotypic expression of CCR9 and DC function we adopted the following approach in which DC subsets were fractionated on the basis of CCR9 expression, without further manipulation. Day 5-generated BM-derived DC were purified and dually labeled with CCR9–FITC and CD11c–PE mAbs. Gating against the negative isotype controls in dot-plots, DC were sorted by flow cytometry and collected in two separate fractions: CD11c+ CCR9 (CCR9low cells) in the upper left quadrant; and CD11c+ CCR9+ (CCR9high) cells in the upper right quadrant (as shown in Fig. 1b). Allogeneic T cells typically give higher immune responses than syngeneic T cells in mixed leucocyte responses, so we studied the ability of sorted CCR9high and CCR9low DC to stimulate naïve allogeneic T cells. After 4 days in co-culture, cells were harvested and labeled with CD4 PerCP mAb. The amount of CFSE incorporated by T cells as they divided during culture was determined by the percentage of cells in the histogram peaks.

CCR9low DC had a greater capacity to induce division in T cells than CCR9high DC. Thus, CCR9low DC induced cell division in 22% of cells, whereas only 7% of T cells were in division after stimulation with CCR9high DC (Fig. 5a). T-cell division was less with fewer DC in co-cultures (data not shown). Further investigations showed that CCR9low DC could stimulate T cells to divide to levels similar to unfractionated DC and hence this CCR9low DC subset contributes to the bulk of cell division observed in T cells (Fig. 5b).

image

Figure 5.  CCR9low dendritic cells (DC) are potent stimulators of naïve T cells. (a) DC purified by magnetic antibody cell sorting (MACS) were sorted by flow cytometry into CCR9low and CCR9high fractions. Allogeneic C57BL/6J-purified spleen T cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE) and added to 96-well flat-bottomed plates at a fixed number (2 × 105 cells/well). Sorted DC fractions were added to T cells at 1 × 105 cells /well or at 5 × 104 cells/well. Cultures were harvested 4 days later and labeled with CD4 peridinin chlorophyll protein (PerCP) (FL3). Dead cells were gated out and > 25 000 events were collected on a flow cytometer. Histograms show CD4-stained T cells that were labeled for CFSE at a DC : T-cell ratio of 1:2, and are representative of three separate experiments. (b) Unfractionated DC and CCR9low DC were used to stimulate T cells, as described above, at a DC : T-cell ratio of 1:2. CCR9low DC were almost as potent stimulators of T cells as unfractionated DC.

Download figure to PowerPoint

CCR9low dendritic cells stimulated the release of interleukin-2 in allogeneic dendritic cell–T-cell cultures

To investigate the cytokines involved in CCR9high and CCR9low DC subset stimulation of T cells, we examined the resulting culture supernatants for the presence of cytokines in co-cultures. At the time of harvest of co-cultures we examined co-culture supernatants for the presence of cytokines.

Very low levels of IFN-γ, IL-17, IL-10 or IL-12 p70 were observed in these co-cultures. Low levels of IL-6 were released in these supernatants. These levels of IL-6 were higher in co-cultures with CCR9low DC than in co-cultures with CCR9high DC, which were usually below the level of detection. However, the predominant cytokine released was IL-2, which was present at much higher levels in T-cell cultures stimulated with CCR9low DC than in T-cell cultures stimulated with CCR9high DC (Fig. 6). Thus, CCR9low DC were superior in their ability to stimulate T-cell division in comparison with CCR9high DC, and this process of active cell division resulted in the production of a significant amount of IL-2.

image

Figure 6.  CCR9low dendritic cells (DC) induce the release of interleukin-2 (IL-2) in co-cultures. Co-cultures were set up as described above. Supernatants were harvested on day 4 and the cytokine levels were determined. Means and standard deviations of duplicate wells are shown. P-values indicate significantly higher levels of cytokine release in CCR9low DC–T-cell co-cultures compared with cytokine release in CCR9high DC–T-cell co-cultures at corresponding DC : T-cell ratios (1:2 or 1:4). Changes in IL-2 release are representative of three independent experiments.

Download figure to PowerPoint

Expression of CCR9 on colon lamina propria dendritic cells

DC are present in very low numbers in the colons of normal mice. Thus, most of our studies of the intestine have focused on DC from the intestines of mice with colitis where a high percentage of these cells are found. Here, these initial studies on lamina propria DC from SCID mice with colitis revealed that CCR9 is expressed by about one-third (32%) of CD11c+ cells (Fig. 7). As freshly isolated LPMC from the colon of mice with colitis comprise approximately 15% of CD11c+ cells (DC),22 about 5% of LPMC are DC-expressing CCR9.

image

Figure 7.  CCR9 is moderately expressed on lamina propria dendritic cells (DC). Lamina propria mononuclear cells (LPMC) were recovered from colons of mice with colitis by collagenase digestion. Cells were purified using CD11c magnetic antibody cell sorting (MACS) beads, labeled with CD11c–phycoerythrin (PE) and CCR9–fluorescein isothiocyanate (FITC) conjugates and further purified on a Becton Dickinson FACS Aria. CCR9+ (upper right) and CCR9 (upper left) DC are shown in the dot-plot quadrants.

Download figure to PowerPoint

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

CCR9 has recently come to the forefront of investigations as a molecule that may be of importance in the regulation of intestinal disorders. The function of this molecule on cells in disease is not well understood. In the case of IBD, an orally active inhibitor of CCR9 [Traficet-EN® (CCX282-B); ChemoCentryx, Mountain View, CA] is undergoing trials for the treatment of Crohn’s disease. However in celiac disease a reduction in the number of CCR9-expressing CD3+ T cells appears to be associated with active disease.7 These and other studies,8,30,36 taken together, suggest that the regulation of CCR9-bearing T-cell immune responses appears to be a function of disease and possibly the anatomical location of these cells.

Few studies have addressed the possible function of CCR9 on DC. Wendland et al.13 reported CCR9 as a gut-homing receptor for plasmacytoid DC. A possible role for CCR9 on myeloid-derived DC has not yet been reported. The plasticity of DC37 encompasses the idea that DC are immature in most tissues, but upon encountering antigens or pathogen-associated molecules, several genes in these cells are altered as surface and intracellular changes occur in these cells. Apart from their well-known role as antigen-presenting cells,16–20 DC regulate immune responses in health and mediate immune tolerance, whereas in disease they stimulate and perpetuate immune responses.22–24 At sites such as the gut and liver, myeloid DC encounter a multiplicity of microbial antigens, and the immune response elicited depends upon the activation state induced in the DC, which is largely reflected in the phenotype of these cells. Thus, antigen will stimulate DC to exhibit a surface phenotype consistent with activation/maturation or tolerance.

The goal of this study was to determine the relationship between CCR9 expression and activation state of DC. DC were generated from the BM of mice, a source frequently used to study immune responses of these leucocytes. Potent DC maturation was observed following stimulation of DC with LPS, which was similar to the effect observed upon stimulation of DC with CpG (i.e. upregulation of CD40 and of CD86; M.L. Drakes, personal observations), so we decided to focus our investigations on only one of these stimulatory agents.

L. plantarum (105 CFU/ml) did not alter DC costimulatory molecule expression; however, a 105 CFU/ml dose of E. coli resulted in a significant maturation of cells, with observed increases in costimulatory molecule expression. The inability of L. plantarum to upregulate DC surface markers, as observed for E. coli, may be a result of the potential of Lactobacillus spp. to have in vivo probiotic properties and because of its tendency to downregulate immune responses and maintain immunological balance.38,39 Interestingly, even though high doses of L. plantarum could stimulate the release of pro-inflammatory IL-6, the levels of IL-6 produced were significantly lower by comparison with those produced following E. coli stimulation, and the proportion of IL-10 to IL-6 release was higher for L. plantarum than for E. coli or CpG, suggesting that L. plantarum activates only low levels of pro-inflammatory cytokines.

The downregulation observed of CCR9 by maturation stimuli such as E. coli, CpG and IL-4, is consistent with the results of other studies in which T cells activated in vivo and ex vivo showed significantly decreased CCR9 expression.7 Having determined that DC CCR9 expression could be regulated by maturation stimuli, we conducted studies to understand whether cells expressing different levels of CCR9 could induce different immune responses. The finding that DC populations low in CCR9 expression are capable of potently stimulating T cells, while populations expressing high levels of CCR9 are modest stimulators of T cells, raises different possibilities. First, it must be considered that there may be insufficient costimulatory molecule expression on CCR9-expressing DC for efficient stimulation of T cells. Second, CCR9high-expressing DC may be able to induce suppressor function in T cells. Finally, CCR9high DC may be more readily subject to apoptosis than CCR9low DC and may not survive in culture as long as CCR9low DC to enable continued stimulation of T cells over the culture period. However, the observation that CCR9low DC are more potent stimulators of T cells, combined with the fact that DC maturation stimuli reduces CCR9 on DC, argues in favor of the fact that a low level of CCR9 on DC is consistent with the maturity of these cells.

IL-2 was the major cytokine released in co-cultures, and release of this cytokine was highly skewed towards cultures of CCR9low DC T-cell stimulation, with significantly lower amounts released from CCR9high–DC co-cultures. Whether this IL-2 is produced by DC or by T cells in these co-cultures remains to be determined, but the presence of this cytokine in culture is consistent with the ability of DC to stimulate T cells in several phases, resulting in IL-2 production over the first 12 hr, as reported previously.40 Addition of harvested IL-2-rich supernatants from DC–T-cell day 1 co-cultures, to parallel day-1-cultured T cells was not sufficient to consistently cause robust T-cell proliferation (M.L. Drakes, personal observations). Thus, IL-2 release, together with the presence of DC costimulatory molecules interacting with T-cell molecules (including CD40 ligand and CD28), are probably responsible for driving T-cell proliferation in CCR9low DC co-cultures.

The significance of IL-2 production and T-cell proliferation by the interaction of CCR9low DC with T cells may be an important phenomenon in disease conditions and lends support to the hypothesis that T cells can distinguish between DC subsets (in this case CCR9low and CCR9high DC), such that the nature of the T-cell adaptive response in vivo may be fine-tuned by the presence of different DC subsets.

This is a study of CCR9 expression and the functional significance of this receptor on dendritic cells. Stimuli that downregulated CCR9 also upregulated costimulatory molecule expression on DC and induced the release of high levels of pro-inflammatory cytokines. Because CCR9low DC are potent stimulators of naïve CD4 T cells and were the major contributors to T-cell division in co-cultures, we suggest that there is an inverse relationship between CCR9 expression and the activation/maturation state of DC. The ability of CCR9low DC to direct the magnitude of T-cell division suggests that this DC fraction may have the ability to be of a more pathogenic nature and may not be beneficial at sites of immune reactivity in vivo. The contribution of CCR9high and CCR9low DC to Th1 and Th2 responses is presently under investigation. Even with the possible differences between in vivo and ex vivo generated DC, studies on BM-propagated DC provide a useful model for the understanding of DC-subset immune responses and suggest that the level of DC CCR9 expression may be a crucial regulator of immune responses. Additionally, our initial studies showing the presence of CCR9 on lamina propria DC underscores the potential of these DC subsets to act as regulators of tolerance or immunity at sites of inflammation. Future studies with DC from CCR9-deficient (−/−) mice and with lamina propria DC from animals with intestinal inflammation will further unravel the importance of DC CCR9 in health and disease.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References

This study was supported by National Institutes of Health grants DK-65859 and AI-055710. 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.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosures
  9. References
  • 1
    Hosoe N, Miura S, Watanabe C et al. Demonstration of functional role of TECK/CCL25 in T lymphocyte–endothelium interaction in inflamed and uninflamed intestinal mucosa. Am J Physiol Gastrointest Liver Physiol 2004; 286:G45866.
  • 2
    Kunkel EJ, Campbell DJ, Butcher EC. Chemokines in lymphocyte trafficking and intestinal immunity. Microcirculation 2003; 10:31323.
  • 3
    Norment AM, Bogatzki LY, Gantner BN, Bevan MJ. Murine CCR9, a chemokine receptor for thymus-expressed chemokine that is up-regulated following pre-TCR signaling. J Immunol 2000; 164:63948.
  • 4
    Papadakis KA, Landers C, Prehn J, Kouroumalis EA, Moreno ST, Gutierrez-Ramos JC, Hodge MR, Targan SR. CC chemokine receptor 9 expression defines a subset of peripheral blood lymphocytes with mucosal T cell phenotype and Th1 or T-regulatory 1 cytokine profile. J Immunol 2003; 171:15965.
  • 5
    Zaballos A, Gutierrez J, Varona R, Ardavin C, Marquez G. Cutting edge: identification of the orphan chemokine receptor GPR-9-6 as CCR9, the receptor for the chemokine TECK. J Immunol 1999; 162:56715.
  • 6
    Zabel BA, Agace WW, Campbell JJ et al. Human G protein-coupled receptor GPR-9-6/CC chemokine receptor 9 is selectively expressed on intestinal homing T lymphocytes, mucosal lymphocytes, and thymocytes and is required for thymus-expressed chemokine-mediated chemotaxis. J Exp Med 1999; 190:124156.
  • 7
    Olaussen RW, Karlsson MR, Lundin KE, Jahnsen J, Brandtzaeg P, Farstad IN. Reduced chemokine receptor 9 on intraepithelial lymphocytes in celiac disease suggests persistent epithelial activation. Gastroenterology 2007; 132:237182.
  • 8
    Papadakis KA, Prehn J, Moreno ST et al. CCR9-positive lymphocytes and thymus-expressed chemokine distinguish small bowel from colonic Crohn’s disease. Gastroenterology 2001; 121:24654.
  • 9
    Wurbel MA, Philippe JM, Nguyen C et al. The chemokine TECK is expressed by thymic and intestinal epithelial cells and attracts double- and single-positive thymocytes expressing the TECK receptor CCR9. Eur J Immunol 2000; 30:26271.
  • 10
    Eksteen B, Grant AJ, Miles A et al. Hepatic endothelial CCL25 mediates the recruitment of CCR9+ gut-homing lymphocytes to the liver in primary sclerosing cholangitis. J Exp Med 2004; 200:15117.
  • 11
    Feng N, Jaimes MC, Lazarus NH, Monak D, Zhang C, Butcher EC, Greenberg HB. Redundant role of chemokines CCL25/TECK and CCL28/MEC in IgA+ plasmablast recruitment to the intestinal lamina propria after rotavirus infection. J Immunol 2006; 176:574959.
  • 12
    Pabst O, Ohl L, Wendland M, Wurbel MA, Kremmer E, Malissen B, Forster R. Chemokine receptor CCR9 contributes to the localization of plasma cells to the small intestine. J Exp Med 2004; 199:4116.
  • 13
    Wendland M, Czeloth N, Mach N, Malissen B, Kremmer E, Pabst O, Forster R. CCR9 is a homing receptor for plasmacytoid dendritic cells to the small intestine. Proc Natl Acad Sci USA 2007; 104:634752.
  • 14
    Iwata M, Hirakiyama A, Eshima Y, Kagechika H, Kato C, Song SY. Retinoic acid imprints gut-homing specificity on T cells. Immunity 2004; 21:52738.
  • 15
    Mora JR, Bono MR, Manjunath N, Weninger W, Cavanagh LL, Rosemblatt M, Von Andrian UH. Selective imprinting of gut-homing T cells by Peyer’s patch dendritic cells. Nature 2003; 424:8893.
  • 16
    Banchereau J, Steinman RM. Dendritic cells and the control of immunity. Nature 1998; 392:24552.
  • 17
    Hart DN. Dendritic cells: unique leukocyte populations which control the primary immune response. Blood 1997; 90:324587.
  • 18
    Lanzavecchia A, Sallusto F. Regulation of T cell immunity by dendritic cells. Cell 2001; 106:2636.
  • 19
    Liu YJ. Dendritic cell subsets and lineages, and their functions in innate and adaptive immunity. Cell 2001; 106:25962.
  • 20
    Mellman I, Steinman RM. Dendritic cells: specialized and regulated antigen processing machines. Cell 2001; 106:2558.
  • 21
    Stagg AJ, Hart AL, Knight SC, Kamm MA. The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria. Gut 2003; 52:15229.
  • 22
    Drakes ML, Blanchard TG, Czinn SJ. Colon lamina propria dendritic cells induce a proinflammatory cytokine response in lamina propria T cells in the SCID mouse model of colitis. J Leukoc Biol 2005; 78:1291300.
  • 23
    Leithauser F, Krajina T, Trobonjaca Z, Reimann J. Early events in the pathogenesis of a murine transfer colitis. Pathobiology 2002; 70:15663.
  • 24
    Ninomiya T, Matsui H, Akbar SM, Murakami H, Onji M. Localization and characterization of antigen-presenting dendritic cells in the gastric mucosa of murine and human autoimmune gastritis. Eur J Clin Invest 2000; 30:3508.
  • 25
    Drakes ML, Czinn SJ, Blanchard TG. Regulation of murine dendritic cell immune responses by Helicobacter felis antigen. Infect Immun 2006; 74:462433.
  • 26
    Lyons AB. Analysing cell division in vivo and in vitro using flow cytometric measurement of CFSE dye dilution. J Immunol Methods 2000; 243:14754.
  • 27
    Suchin EJ, Langmuir PB, Palmer E, Sayegh MH, Wells AD, Turka LA. Quantifying the frequency of alloreactive T cells in vivo: new answers to an old question. J Immunol 2001; 166:97381.
  • 28
    Drakes ML, Czinn SJ, Blanchard TJ. Isolation and purification of colonic lamina propria dendritic cells. Cytotechnology 2004b; 46:15161.
  • 29
    Van Der Heijden PJ, Stok W. Improved procedure for the isolation of functionally active lymphoid cells from the murine intestine. J Immunol Methods 1987; 103:1617.
  • 30
    Rivera-Nieves J, Ho J, Bamias G, Ivashkina N, Ley K, Oppermann M, Cominelli F. Antibody blockade of CCL25/CCR9 ameliorates early but not late chronic murine ileitis. Gastroenterology 2006; 131:151829.
  • 31
    Asseman C, Mauze S, Leach MW, Coffman RL, Powrie F. An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation. J Exp Med 1999; 190:9951004.
  • 32
    Ogawa A, Andoh A, Araki Y, Bamba T, Fujiyama Y. Neutralization of interleukin-17 aggravates dextran sulfate sodium-induced colitis in mice. Clin Immunol 2004; 110:5562.
  • 33
    Powrie F, Leach MW, Mauze S, Menon S, Caddle LB, Coffman RL. Inhibition of Th1 responses prevents inflammatory bowel disease in scid mice reconstituted with CD45RBhi CD4+ T cells. Immunity 1994; 1:55362.
  • 34
    Simpson SJ, Shah S, Comiskey M, De Jong YP, Wang B, Mizoguchi E, Bhan AK, Terhorst C. 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 1998; 187:122534.
  • 35
    Yamamoto M, Yoshizaki K, Kishimoto T, Ito H. IL-6 is required for the development of Th1 cell-mediated murine colitis. J Immunol 2000; 164:487882.
  • 36
    Onai N, Kitabatake M, Zhang YY, Ishikawa H, Ishikawa S, Matsushima K. Pivotal role of CCL25 (TECK)-CCR9 in the formation of gut cryptopatches and consequent appearance of intestinal intraepithelial T lymphocytes. Int Immunol 2002; 14:68794.
  • 37
    Huang Q, Liu D, Majewski P, Schulte LC, Korn JM, Young RA, Lander ES, Hacohen N. The plasticity of dendritic cell responses to pathogens and their components. Science 2001; 294:8705.
  • 38
    Madsen KL, Doyle JS, Jewell LD, Tavernini MM, Fedorak RN. Lactobacillus species prevents colitis in interleukin 10 gene-deficient mice. Gastroenterology 1999; 116:110714.
  • 39
    Pena JA, Rogers AB, Ge Z, Ng V, Li SY, Fox JG, Versalovic J. Probiotic Lactobacillus spp. diminish Helicobacter hepaticus-induced inflammatory bowel disease in interleukin-10-deficient mice. Infect Immun 2005; 73:91220.
  • 40
    Mempel TR, Henrickson SE, Von Andrian UH. T-cell priming by dendritic cells in lymph nodes occurs in three distinct phases. Nature 2004; 427:1549.