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*
Antigen Presentation Research Group, Imperial College School of Medicine, Northwick Park Institute for Medical Research, and
St. Marks Hospital, Harrow, Middlesex, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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0.6% of the mononuclear cells obtained from the lamina
propria, were endocytically active, and had the phenotype of immature
DC; they were CD40+ and expressed low levels of CD83 and
CD86, but little or no CD80 or CD25. Similar d0 DC populations were
isolated from the colonic mucosa of healthy controls and from both
inflamed and noninflamed tissue from patients with Crohn’s disease.
The lamina propria also contained a population of cells capable of
migrating out of biopsies during an overnight culture and
differentiating into mature DC with lower levels of endocytic activity
and high cell surface expression of CD40, CD80, CD86, CD83, and CD25.
This mature DC population was a potent stimulator of an allogeneic
mixed leukocyte (MLR). Overnight culture of cells isolated by enzymatic
digestion on d0 yielded DC with a phenotype intermediate between that
of the d0 cells and that of the cells migrating out overnight.
Overnight culture of colonic cells in which DC and
HLA-DR+lin+ cells were differentially labeled
with FITC-dextran suggested that some of the maturing DC might
differentiate from HLA-DR+lin+ progenitors.
This study presents the first analysis of the phenotype, maturational
status, and migratory activity of human gut DC. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DC may be involved in both responsiveness and nonresponsiveness to Ags
in the gut. Gut DC can acquire Ags such as OVA when these are fed to an
animal, and upon isolation these DC can activate OVA-specific T cells
(6). In contrast, treatment of mice with the growth factor
Flt3L, which boosts intestinal DC numbers, enhances the induction of
oral tolerance to soluble Ags (7). It is not yet clear to
what extent these outcomes are mediated by different DC populations or
by the same population responding to different signals in the local
environment. Heterogeneity of murine and rat intestinal DC has recently
been described (8, 9). DC from murine Peyer’s patches are
comprised of at least three populations: CD11b+
(myeloid) DC in the subepithelial dome, CD8
+
(lymphoid) DC in the interfollicular T cell regions, and a population
of CD11b-CD8- DC
present in both compartments (2, 10). Rat intestinal lymph
contains two discreet populations of DC (8).
CD4+/OX41+ lymph DC are
stronger stimulators of the allogeneic MLR than the
CD4-/OX41- DC population
and retain the ability to process complex Ags for longer when cultured
in vitro. Skin afferent lymph DC show similar heterogeneity in cattle
(9), suggesting that the presence of DC subpopulations in
lymph may not be a feature unique to the intestine. However, the
CD4-/OX41- rat intestinal
DC population appears to transport apoptotic epithelial cells to the T
cell areas of mesenteric lymph nodes and may be involved in the
maintenance of peripheral self tolerance (11).
Alterations in intestinal DC function could contribute to the poorly understood dysregulated immune responses that underlie the human inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis. However, little is known about DC in the human intestine. Cell populations with phenotypic properties consistent with DC have been identified in a number of immunocytochemical studies (12, 13, 14), and partially enriched populations of these cells have been shown to stimulate primary T cell responses in a primary allogeneic MLR (15). Detailed analysis of the phenotype and function of these cells is currently lacking.
In most nonlymphoid tissues DC are present as immature cells (reviewed
in Ref. 16). These immature DC express low levels of
costimulatory molecules and take up Ag very efficiently, but are poorly
stimulatory for T cells. In response to maturation signals, which
include microbial products and cytokines such as TNF-, these cells
change their pattern of expressed chemokine receptors and migrate to
the draining lymphoid tissue. During this process, DC down-regulate
their Ag acquisition machinery, up-regulate the cell surface expression
of MHC-peptide Ag complexes and costimulatory molecules, and acquire
their characteristic ability to stimulate naive T cells. It has
recently been suggested that an abnormal pattern of DC maturation, such
that mature cells fail to migrate but instead remain localized in the
tissue, could underlie chronic inflammatory processes
(17).
In the current study, we have used multicolor flow cytometry to characterize DC present in small amounts of biopsy tissue obtained from the human colon and for the first time have analyzed the phenotype and maturation state of DC present in tissue from inflammatory bowel disease patients and healthy controls.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Patients with active CD (n = 24), patients with
ulcerative colitis (UC) undergoing surveillance (n =
14), and those undergoing colonoscopy for other reasons (e.g., polyp
follow-up, family history of cancer, or investigation of rectal
bleeding) who were subsequently found to have normal histology
(controls; n = 22) were prospectively enrolled in the
study (Table I
). Six random colonic
biopsies were obtained from all UC and control patients and from 10 of
the patients with CD. Three rectal biopsies were studied from 14 CD
patients who were being assessed for anti-TNF- Ab treatment. The
characteristics of the patients are described in Table I. Disease
activity was assessed endoscopically with histological
confirmation.
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Biopsies obtained during colonoscopy or sigmoidoscopy were collected in ice-cold Dutch modification of RPMI 1640 (Sigma, St. Louis, MO) supplemented with 10% FCS, 2 mM L-glutamine, gentamicin (25 µg/ml), and penicillin/streptomycin (100 U/ml) (complete medium). They were incubated with occasional agitation for 20 min at room temperature in calcium- and magnesium-free HBSS (Life Technologies, Paisley, U.K.) containing 1 mM DTT (Sigma). Biopsies were blotted, weighed, and transferred to HBSS containing 1 mM EDTA for removal of the epithelium. After 30-min incubation on a shaker at 37°C, they were washed in HBSS until the supernatant was macroscopically free of released epithelial cells. The EDTA and washing steps were repeated until no more epithelium was shed into the medium.
Isolation of lamina propria mononuclear cells (LPMC)
Two methods were used. In the first, fresh tissue was digested with 1 mg/ml collagenase D (Roche Molecular Products, Basel, Switzerland) in HEPES-buffered RPMI 1640 containing 20 µg/ml DNase I (Roche Molecular Products) and 2% FCS. Digestion at 37°C on a shaker was continued until visual inspection indicated that dissociation was complete. Typically this required an incubation of 90–180 min. Mononuclear cells were separated (650 x g, 20 min, room temperature) on Ficoll-Paque (Amersham Pharmacia, Aylesbury, U.K.) and washed in complete medium. In some experiments cells isolated by collagenase digestion were cultured overnight at 1.5 x 106/ml in complete medium; in some cases cells were labeled with 1 mg/ml FITC-dextran (Sigma; 30 min, 37°C) and washed three times before culture.
In the second method, cells migrating from cultured biopsies were recovered as described by Mahida and colleagues (18). Each biopsy was placed in 0.5 ml of complete medium in a well of a 24-well culture plate (Falcon) and incubated for 24 h at 37°C in 5% CO2. Migrating cells were recovered by centrifugation, and the biopsy was discarded or, in some experiments, cultured for one or more additional periods of 24 h.
Antibodies
Abs to HLA-DR (clone L243), CD3 (SK7), CD14 (M
P9), CD16
(B73.1), CD19 (4G7), CD34 (8G12), and CD62L (SK11) were purchased from
BD Biosciences (San Jose, CA). Those to CD40 (LOB7/6), CD80 (DAL-1 or
BB-1), CD86 (BU63), and were obtained from Serotec (Oxford, U.K.), and
Abs to CD25 (ACT-1) and CD11c (KB90) were purchased from Dako
(Carpenteria, CA). Anti-CD83 (HB15a) was purchased from Coulter
Immunotech (Hialeah, FL), and anti-CD1a (IK-B5) was obtained from
ImmunoKontact (Abingdon, U.K.). Anti-CD74 (M-B741) was purchased from
BD PharMingen (San Diego, CA). Isotype-matched controls were obtained
from the same manufacturers.
Flow cytometry
Ab labeling was performed in PBS supplemented with 1 mM EDTA and 0.02% sodium azide (FACS buffer). FCS was added at 15% during the labeling steps and at 2% during the washing stages. Data were acquired using a FACScan flow cytometer and were analyzed using CellQuest software (BD Biosciences). Dead cell and debris were excluded on the basis of light scatter. Using multicolor analysis, HLA-DR+ cells were divided into lin+ cells, which stained with a cocktail of Abs to T cells (CD3), B cells (CD19), NK cells (CD16), monocytes (CD14), and stem cells (CD34), and into lin- cells, which did not stain with this mixture. Absolute cell counts were obtained by simultaneous acquisition of Flow-Count fluorospheres (Coulter Immunotech), and levels of cell surface marker expression on gated populations were determined by geometric mean fluorescence intensity (MFI) with subtraction of values for isotyped-matched controls as appropriate.
Electron microscopy
Cells were fixed in 3% glutaraldehyde in 0.1 M PBS (pH7.4) at room temperature for 2 h and postfixed in 1% osmium tetroxide for 1 h at room temperature. The cells were washed overnight in distilled water. Block staining was then performed with 2% aqueous uranyl acetate in water for 4 h, followed by washing and dehydration in graded acetone. The samples were then infiltrated with araldite overnight, embedded, and cured at 65°C for 18 h. The sections were stained with lead citrate and examined under a JEOL 1200 EX transmission electron microscope (Peabody, MA).
Endocytosis assay
Endocytic activity was assessed by measuring uptake of the fluid phase marker FITC-dextran (Sigma). Thirty thousand to 50,000 LPMC were incubated at 37°C in complete medium containing 1 mg/ml FITC-dextran. Control incubations were performed at 4°C. At various time points incubation was stopped by transfer of the cells to ice-cold FACS buffer, and the cells were labeled and subjected to flow cytometry as described above.
Enrichment of DC
DC were enriched or depleted from the "walk-out" LPMC
population obtained from resection specimens (insufficient numbers were
obtained to perform separations on cells obtained from biopsies). For
negative selection of DC, LPMC were labeled with a cocktail of
FITC-conjugated Abs to CD3, CD14, CD16, CD19, and CD34; for negative
selection of an HLA-DR+ non-DC population, cells
were labeled with a cocktail of FITC-conjugated Abs to CD3, CD16, CD19,
CD34, and CD83. All labelings were labeled with immunomagnetic
anti-FITC microbeads (Miltenyi Biotec, Auburn, CA) and separated on
MiniMacs columns (Miltenyi Biotec) with the use of a flow restrictor.
Using these procedures DC were enriched 10-fold and depleted
4-fold, comprising >89% or <25% of the recovered
HLA-DR+ cells, respectively.
MLR
MLR were performed using a 20-µl hanging drop culture system (19). Irradiated (1,800 cGy) stimulator cells (LPMC or enriched populations) were added at between 625 and 5,000 cells to the wells of Terasaki plates containing 25,000 PBMC separated from the blood of a healthy control by centrifugation over Ficoll-Paque (described above). On the fifth day of culture, at 37°C in 5% CO2, wells were pulsed with [3H]thymidine (1 µg/ml; sp act., 2 Ci/mmol) and, after 2 h, harvested by blotting onto filter paper. Incorporation of [3H]thymidine was assayed by liquid scintillation counting.
Statistical analysis
Two-tailed t tests were used to compare cell proportions and cell surface marker expression. DC numbers were compared using the nonparametric Mann-Whitney-Wilcoxon test. Values of p < 0.05 were regarded as significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Leukocyte preparations were obtained from colonic biopsies by
immediate enzymatic digestion and by harvesting cells migrating out of
cultured tissue. These populations were termed day 0 (d0) collagenase
and d1 walk-out populations, respectively. Multicolor flow cytometry
permitted the identification of cell populations within this mixture
despite the presence of autofluorescent cells that characterize
tissue-derived populations (Figs. 1 and 2). Lin+ and
Lin- populations were identified among the
HLA-DR+ cells in both d1 walk-out and
d0 collagenase cells (Fig. 1). In all walk-out samples the
HLA-DR+Lin- cells were
exclusively CD11c+ (Fig. 1A). This was
also true for many of the d0 collagenase-digested samples (Fig. 1B), but in some samples a CD11c-
population was also present (Fig. 1C). In such cases, both
the CD11c+ and CD11c-
populations were large granular cells (data not shown), but the former
expressed higher levels of HLA-DR+ (Fig. 1),
allowing the two populations to be discriminated in subsequent analyses
(Fig. 1).
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illustrates further analysis of the staining obtained with d0
collagenase cells. The HLA-DR+
lin+ and HLA-DR+
lin- populations were located within the gate of
large cells, but were absent among cells with the light scatter
properties of lymphocytes (Fig. 2A). Isotype controls
confirmed the specificity of the labeling (Fig. 2B).
Labeling with individual components of the lineage cocktail indicated
that cocktail staining of the HLA-DR+
lin+ population could be attributed to
anti-CD14, because in the absence of this Ab the majority of cells
dropped out of the
HLA-DR+lin+ region (Fig. 2C; compare CD14 staining with that of the other lineage cocktail
components). CD3+ and CD19+
cells, lacking HLA-DR, could also be identified within the large cell
gate, as could a population of CD34+ cells
(Fig. 2C), which immunohistology suggested were vascular endothelial
cells (data not shown).
Analysis of the kinetics of walk-out cultures demonstrated that the
majority (
80%) of DC were recovered during the first 24 h of
culture and that leukocytes obtained at this time point were relatively
enriched for DC (2.2% of recovered cells at 0–24 h, 1.1% at 24–48
h, and 0.3% at 48–72 h), suggesting that DC migration is more rapid
than that of other cell populations. Electron microscopy of walk-out
cells confirmed the presence of DC (Fig. 3). DC are heterogeneous by electron
microscopy and have been classified into types 1, 2, and 3
(20). Type 1 DC are smaller, irregularly shaped cells with
small cell surface projections and heterochromatic nuclei in which the
chromatin is present in a thick band around the margin and in small
condensed areas. Type 2 DC are larger with few projections and have a
euchromatic nucleus with chromatin disaggregated and present as a thin
band at the nuclear margin. Type 3 DC have a veiled appearance
and euchromatic nuclei. The significance of these different
morphological forms is not yet clear, but all three were present within
the walk-out population. We did not observe Birbeck granules in
the walk-out colonic DC.
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),
supporting the identification of the Lin- and
Lin+ subpopulations as DC and
monocyte/macrophages, respectively.
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The expression of additional markers on the surface of d0
and d1 colonic HLA-DR+lin-
cells was assessed using three-color flow cytometry. Where necessary,
CD11c+ and CD11c-
subpopulations were separated on the basis of level of HLA-DR
expression as shown in Fig. 1. Fig. 5
shows representative data for this type of FACS experiment. The DC
obtained after the culture of biopsies for 24 h had the phenotype
of mature cells consistent with their potent ability to stimulate an
allo-MLR. They were CD83 positive and expressed high levels of CD80,
CD86, CD40, and CD25. In contrast, the CD11c+ d0
population had a phenotype consistent with immature DC, i.e.,
CD40+ but expressing low levels of CD83 and CD86
with little or no CD80 and CD25.
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To rule out the possibility that exposure to collagenase/DNase may remove markers from the surface of DC, mature DC were generated by culture of peripheral blood mononuclear cells, incubated with the enzyme mixture, and subsequently labeled. Expression of CD86 and CD40 was unaffected by exposure to the collagenase mix (CD86, MFI of 115 without collagenase treatment vs 115 for treated cells; CD40, MFI of 47 without collagenase vs 43 for treated cells). CD11c was expressed at high levels after enzymatic treatment (MFI = 149), although levels were reduced compared with those in untreated cells (MFI = 244). CD83 was also slightly affected by collagenase treatment (MFI = 143 for untreated cells; MFI = 71 for treated cells), but levels were still more typical of d1 walk-out colonic DC than of d0 colonic DC. These experiments also failed to show any evidence of a toxic affect of enzyme treatment on mature DC.
Endocytic activity of colonic DC
That d0 and d1
CD11c+HLA-DR+lin-
are immature and mature DC populations, respectively, is supported by
their endocytic activities (Fig. 6). The
d0 cells were endocytically active, with a level of FITC-dextran uptake
intermediate between that of gut lymphocytes and that of
HLA-DR+lin+
monocyte/macrophages. Endocytosis by d1 walk-out
CD11c+HLA-DR+lin-
cells was less than that by their d0 counterparts (Fig. 6), consistent
with increased maturity. Those DC remaining in the tissue also
maintained their endocytic activity, suggesting that maturation and
migration go hand-in-hand.
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On the basis that cell recoveries by the d0 collagenase technique
were likely to best reflect the numbers present in the tissue, we used
this method to measure DC numbers. Overall,
CD11c+ DC comprised 0.59 ± 0.35% of the
colonic cell population (n = 52). This was equivalent
to a median of 114 CD11c+ DC/mg of tissue
(n = 51; interquartile range, 78–220). Proportions of
DC did not differ significantly between controls and CD patients or
between inflamed and noninflamed CD tissue (the means were 0.62, 0.57,
and 0.61% for control, noninflamed CD tissue, and inflamed CD tissue,
respectively). The median number of DC isolated was higher from
inflamed tissue (146/mg of tissue compared with 115 for both control
and noninflamed CD tissue; Fig. 7A), but this difference did
not reach statistical significance.
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Phenotype of DC from healthy and IBD colonic tissue
When levels of CD80, CD86, CD25, and CD83 were compared,
CD11c+ DC from IBD patients and controls appeared
to be at a similar stage of maturation. For all four markers, levels
were significantly (p < 0.001) higher on d1
walk-out cells than on the d0 population (Fig. 7B). CD80 and
CD25 were almost completely absent on d0 cells, whereas CD86 and CD83
were expressed at low and variable levels. A similar pattern of results
was obtained with two different CD80-reactive Abs, DAL-1 and BB-1,
although staining of the d1 cells with BB-1 tended to be brighter (data
not shown). This was not due to BB-1’s reported cross-reactivity with
invariant chain (CD74) (22) as the d1 walk-out population
did not stain with anti-CD74 (data not shown).
Maturation of colonic DC
To address the question of whether freshly isolate DC have the
potential to undergo maturation, we examined the phenotype of cells
prepared on d0 by collagenase digestion and then cultured overnight.
Cells cultured overnight in this way up-regulated CD40, CD80, CD86, and
CD25 compared with the starting d0 population, confirming a potential
to undergo maturation. However, all markers were expressed at higher
levels on DC migrating out of biopsies in parallel cultures (Fig. 8). Furthermore, nonmigrating DC obtained
on d1 by collagenase digestion of the cultured biopsy expressed lower
levels of these cell surface markers than DC that had migrated out of
the tissue (data not shown).
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In four of five experiments in which cells were compared on d0 and
after overnight incubation, an increase (30–70%) in the recovered
number of CD11c+ DC was noted, raising the
possibility that some cells initially outside the
HLA-DR+lin- gate acquire
this phenotype during culture. A likely candidate for the source of
such cells is the
HLA-DR+lin+ population, as
the differentiation of CD14+ cells into DC is
well established. To address this issue we took advantage of the
differential uptake of FITC-dextran by
HLA-DR+lin- and
HLA-DR+lin+ cells (Figs. 6 and 9). Lamina propria cells were
isolated by collagenase digestion, labeled with FITC-dextran, washed,
and analyzed immediately or following overnight culture. As previously
described, the HLA-DR+lin+
population labeled strongly with FITC-dextran, and this was maintained
during the overnight culture (Fig. 9). The DC population on d0 took up
a smaller amount of FITC-dextran, and this was clearly distinguishable
from the amount present in either fresh or cultured
HLA-DR+lin+ populations.
After culture a population of DC displayed a level of FITC labeling
characteristic of the
HLA-DR+lin+ population
(Fig. 9). Lymphocytes displayed little or no labeling before or after
culture (data not shown). These results are consistent with a
differentiation of FITC-dextran-bearing
HLA-DR+lin+ cells into DC
during overnight culture and provide further support for the importance
of a pathway of DC maturation that proceeds via a monocyte
intermediate.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The relationship between the freshly isolated and mature DC populations remains to be determined, but the simplest view is that the population identified on d0 matures into the population identified on d1. This would parallel the situation that occurs with Langerhans’ cells in skin explants; immature epidermal Langerhans cells migrate out of the skin via the dermis and in doing so begin to mature (23, 24). However, cells differentiating into mature DC from other populations, such as the HLA-DR+Lin+ population, may also contribute. Following labeling of this latter population with FITC-dextran and overnight culture, cells appear within the DC (HLA-DR+lin-) gate with a level of FITC labeling characteristic of the HLA-DR+lin+ population. These findings are suggestive of differentiation of HLA-DR+lin+ cells into DC, an interpretation favored by the increase in DC numbers during culture. However, the possibility that high levels of FITC-dextran are transferred to a subpopulation of DC or that maturation somehow influences FITC fluorescence cannot currently be excluded. Nonetheless, the HLA-DR+Lin+ cells are CD14+, and there is abundant evidence that CD14+ cells can differentiate into DC in vitro and in vivo, particularly under conditions of inflammation (25, 26, 27, 28). Thirdly, the CD11c+ DC population could potentially differentiate from the CD11c-HLA-DR+lin- population, although there is no evidence for interconversion of CD11c+ and CD11c- DC populations in peripheral blood (21). The CD11c- HLA-DR+lin- population labeled with few of the other Abs tested and its nature remain to be determined. It may correspond to the CD11c- HLA-DRlow CD14- macrophage population recently isolated from normal colonic mucosa (29) or to the population with DC-like properties that was enriched from lamina propria by Pavli and colleagues and expressed little or no CD11c (15). If they constitute a subpopulation of DC, they differ from CD11c- peripheral blood DC in their lack of CD123 and CD62L expression (A. J. Stagg, S. J. Bell, R. Rigby, M. A. Kamm, and S. C. Knight, unpublished observations) (21). Importantly, the lack of these markers on HLA-DR+lin- cells in the preparations from colonic mucosa suggests that there is minimal contribution by contaminating peripheral blood.
It is currently unclear whether the DC (or precursors of DC) migrate out of the biopsy through pores in the basement membrane, as occurs for other leukocytes in similar culture systems (18), or whether they exit in the opposite direction as if migrating to draining lymphoid tissue (30). After 24 h in culture these walk-out DC express uniformly high levels of maturation-dependent markers. This maturation appears more rapid than that reported for Langerhans cells migrating out of murine skin tissue (23) or for human Langerhans’ cells in culture (31, 32). This discrepancy may reflect a history of increased Ag exposure that is characteristic of the mucosal population. The walk-out culture system may provide a useful model in which the roles of cytokines, chemokines, and other molecules in the maturation of gut DC can be dissected. The supernatant of cultured colonic biopsies is rich in proinflammatory cytokines (R. Rigby, S. J. Bell, M. A. Kamm, S. C. Knight, and A. J. Stagg, unpublished observations). Although CD80, CD86, CD25, and CD40 are up-regulated during overnight culture of d0 collagenase DC, they do not reach the levels expressed on walk-out DC. This incomplete maturation may result from rather nonspecific effects of tissue dissociation, but it could also indicate an absence of important signals provided during migration through the tissue and across the basement membrane. There is evidence that DC can receive maturational signals during migration across endothelial tissue (27).
Immunohistological studies have described disease-associated changes in APC populations in IBD (12, 13, 33, 34, 35, 36), including evidence for activation in early aphthoid lesion in CD. However, the contributions of these cells in general and of DC in particular to the immune dysregulation underlying these conditions (37) are not known. Abnormality of DC function has been reported in one transgenic model of colitis (38). In experimental models of autoimmune disease, injection of Ag-bearing DC can induce inflammation (39, 40, 41) and the neogenesis of organized lymphoid structures (41). Sallusto and Lanzavecchia have recently suggested that the failure of mature DC to leave peripheral tissue and migrate to lymph nodes could underlie such processes in some inflammatory diseases (17). Mature DC remaining in the tissue could recruit recently activated T cells and participate in rounds of mutual stimulation with these cells with the accompanying production of growth and differentiation factors. In postchlamydial reactive arthritis we found DC bearing chlamydial Ags localized within inflamed joints (42). If the processes proposed by Sallusto and Lanzavecchia occur in CD, mature DC should be identifiable within inflamed lamina propria. We found no evidence for such mature DC, expressing high levels of costimulatory and other maturation-dependent markers, among the cells extracted immediately from colonic tissue. This was the case regardless of whether the tissue was inflamed. Recently, DC identified in an immunohistochemical study as mature on the basis of CD86 expression have been identified in the gastric mucosa in autoimmune gastritis (43), suggesting that nonmigrating mature DC might contribute to other forms of intestinal inflammation.
Although fully mature DC were not found in cells from IBD colon, it is possible that subtle alterations in functionally important DC cell surface molecules would be revealed in a larger study or that other activities of DC, such as the production of soluble mediators, contribute to local inflammatory reactions. It could be argued that mature DC are present in the inflamed lamina propria, but are not extracted by the methods used, are lost during the separation procedure, or have their phenotype altered by exposure to the digestion enzymes. In other contexts collagenase treatment has proved effective in extracting tightly bound DC subpopulations (44). Control experiments on mature DC prepared from peripheral blood suggested that the low expression of DC maturation markers on d0 colonic DC is unlikely to be due to a deleterious effect of enzymatic treatment. Furthermore, positive control markers, including CD45 and MHC class I, are expressed at high level on gut DC obtained by enzymatic digestion.
We found no statistically significant evidence for an increase in DC numbers in inflamed colonic tissue, although median values were higher. This may simply reflect the imprecise nature of quantifying enzyme-extracted cells. Alternatively, this initially surprising finding may be explained if the DC that are extracted from the tissue represent the net balance between recruitment of cells and migration to the draining lymph nodes. Animal studies suggest that inflammatory stimuli may increase both the recruitment and the rate of exit of mucosal DC (1, 45, 46). Thus, the numbers of DC resident in the tissue at a given time need not be obviously increased. It follows that gut DC may mediate their immunoregulatory effects in the draining lymph nodes, and it is here that their influence in IBD may be exerted. We hope to address these questions in future studies.
There is direct evidence for recruitment of CD14+ monocytes into mucosal tissue in CD (47), and as discussed above, these cells may represent an important pool of DC precursors. Our finding of a reduced proportion of HLA-DR+lin+ (almost all of which are CD14+) in CD tissue compared with control tissue could reflect an increase in in vivo differentiation into DC in the disease state.
Collectively, these data illustrate the feasibility of using flow cytometry to identify and characterize DC in cell populations isolated from small amounts of human gut tissue. For the first time the phenotype and maturational and migratory activities of DC from both healthy and IBD gut tissue have been determined.
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Footnotes |
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2 Abbreviations used in this paper: DC, dendritic cell; CD, Crohn’s disease; IBD, inflammatory bowel disease; LPMC, lamina propria mononuclear cell; MFI, mean fluorescence intensity; UC, ulcerative colitis; d0, day 0; d1, day 1.
Received for publication September 6, 2000. Accepted for publication February 6, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, MIP-3
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J. Exp. Med. 179.
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 J O Lindsay, K Whelan, A J Stagg, P Gobin, H O Al-Hassi, N Rayment, M A Kamm, S C Knight, and A Forbes Clinical, microbiological, and immunological effects of fructo-oligosaccharide in patients with Crohn's disease Gut, March 1, 2006; 55(3): 348 - 355. [Abstract] [Full Text] [PDF]
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P Middel, D Raddatz, B Gunawan, F Haller, and H-J Radzun Increased number of mature dendritic cells in Crohn's disease: evidence for a chemokine mediated retention mechanism Gut, February 1, 2006; 55(2): 220 - 227. [Abstract] [Full Text] [PDF]
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 K. B. Gurney, J. Elliott, H. Nassanian, C. Song, E. Soilleux, I. McGowan, P. A. Anton, and B. Lee Binding and Transfer of Human Immunodeficiency Virus by DC-SIGN+ Cells in Human Rectal Mucosa J. Virol., May 1, 2005; 79(9): 5762 - 5773. [Abstract] [Full Text] [PDF]
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B Eksteen, L S K Walker, and D H Adams Immune regulation and colitis: suppression of acute inflammation allows the development of chronic inflammatory bowel disease Gut, January 1, 2005; 54(1): 4 - 6. [Full Text] [PDF]
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A L Hart, K Lammers, P Brigidi, B Vitali, F Rizzello, P Gionchetti, M Campieri, M A Kamm, S C Knight, and A J Stagg Modulation of human dendritic cell phenotype and function by probiotic bacteria Gut, November 1, 2004; 53(11): 1602 - 1609. [Abstract] [Full Text] [PDF]
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J.-F. Fonteneau, M. Larsson, A.-S. Beignon, K. McKenna, I. Dasilva, A. Amara, Y.-J. Liu, J. D. Lifson, D. R. Littman, and N. Bhardwaj Human Immunodeficiency Virus Type 1 Activates Plasmacytoid Dendritic Cells and Concomitantly Induces the Bystander Maturation of Myeloid Dendritic Cells J. Virol., May 15, 2004; 78(10): 5223 - 5232. [Abstract] [Full Text] [PDF]
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 H. Karlsson, P. Larsson, A. E. Wold, and A. Rudin Pattern of Cytokine Responses to Gram-Positive and Gram-Negative Commensal Bacteria Is Profoundly Changed when Monocytes Differentiate into Dendritic Cells Infect. Immun., May 1, 2004; 72(5): 2671 - 2678. [Abstract] [Full Text] [PDF]
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S. Goddard, J. Youster, E. Morgan, and D. H. Adams Interleukin-10 Secretion Differentiates Dendritic Cells from Human Liver and Skin Am. J. Pathol., February 1, 2004; 164(2): 511 - 519. [Abstract] [Full Text] [PDF]
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A J Stagg, A L Hart, S C Knight, and M A Kamm The dendritic cell: its role in intestinal inflammation and relationship with gut bacteria Gut, October 1, 2003; 52(10): 1522 - 1529. [Abstract] [Full Text] [PDF]
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 L. Gardner and A. Moffett Dendritic Cells in the Human Decidua Biol Reprod, October 1, 2003; 69(4): 1438 - 1446. [Abstract] [Full Text] [PDF]
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