Adipose tissue of human omentum is a major source of

dendritic cells, which lose MHC Class II and stimulatory

function in Crohn’s disease
Penelope A. Bedford,* Vesna Todorovic,* Edward D. A. Westcott,† Alistair C. J. Windsor,†
Nicholas R. English,* Hafid Omar Al-Hassi,* Kankipati S. Raju,‡ Sarah Mills,†
and Stella C. Knight*,1
*Antigen Presentation Research Group and †St. Mark’s Institute for Intestinal and Colorectal Disorders, Imperial
College London, Northwick Park and St. Mark’s Campus, Harrow, United Kingdom; and ‡Obstetrics & Gynaecology,
St. Thomas’ Hospital, London, United Kingdom

Abstract: Adipose tissue is reported to contain INTRODUCTION

monocyte-like pre-adipocytes, which may mature
into macrophages, contributing to local inflamma-
The omentum, like other adipose tissues, contains pre-adipo-
tion. Dendritic cells (DC) can be derived from
cytes, which share some properties with macrophages. These
monocytes and initiate and regulate primary im-
cells express pattern recognition receptors (PRR) such as
mune responses. We hypothesized, therefore, that
Toll-like receptors (TLR) [1] and chemokine receptors [2] and
adipose tissue may provide DC involved in local
secrete inflammatory mediators including tumor necrosis factor
immune activity. To test this, we studied cells from
␣ (TNF-␣) and interleukin-6 [3, 4]. Pre-adipocytes of the
human omental adipose tissue samples from 17
omentum are also reported to mature into macrophages, which
patients with benign gynecological disease. The hy-
contribute to local immune and inflammatory responses [5, 6].
pothesis that adipose tissue DC are involved in
Cells within adipose tissue are also now known to have stem
inflammatory disease was tested by comparing
cell activity [7–9]. However, accumulation of macrophages
these cells with those from 18 patients with Crohn’s
within adipose tissue in obesity and their involvement in
disease, where hypertrophy of adipose tissue sug-
inflammation have been highlighted recently [10, 11]. Den-
gests involvement in disease. A high proportion of
dritic antigen-presenting cells (DC; APC) initiate and regulate
the 1.33 ⴞ 0.12 ⴛ 105 CD45-positive cells/mg,
innate and adaptive immune responses. They can be derived
obtained from control omenta, expressed CD11c,
from stem cells shared with macrophages [12], and monocytes
CD1a, and CD83; costimulatory molecules CD40,
may give rise to DC [13]. We therefore hypothesized that DC,
CD80, and CD86; and major histocompatibility
like macrophages, might be obtained from adipose tissue, and
complex (MHC) Class II but little CD14, CD16, or
to test this hypothesis, we characterized myeloid cells migrat-
CD33. Omental cells showing morphological char-
ing from human omental tissue. We further proposed that DC
acteristics of DC were also observed. Metrizamide
derived from omentum could contribute to or reflect local
gradient-enriched DC from these populations were
inflammatory changes and so, looked for changes in omental
potent stimulators of primary proliferation of allo-
DC in the presence of inflammation in Crohn’s disease. Hy-
geneic T cells in mixed leukocyte reactions. In-
pertrophy of adipose tissue and fat-wrapping characterize
creased numbers of CD45ⴙ cells from omentum of
Crohn’s disease. This adipose tissue shows overexpression of
Crohn’s patients (4.50ⴞ1.08ⴛ105 CD45ⴙ cells/
peroxisome proliferator-activated receptor ␥ and increased
mg) contained higher percentages of CD11cⴙ and
production of TNF-␣ [14]. Increase in adipose tissue with its
CD40ⴙ cells (80.8ⴞ3.8% vs. 63.4ⴞ6, Pⴝ0.032;
inflammatory changes forms the basis for theories that these
77.9ⴞ4% vs. 58.8ⴞ6.5, Pⴝ0.029, respectively),
changes represent a fundamental part of the pathogenic pro-
but MHC Class II and stimulatory capacity were
cesses producing Crohn’s disease. We therefore used omental
almost completely lost (Pⴝ<0.001), suggesting in-
adipose tissue from these patients to identify whether changes
nate activation but lost capacity to stimulate adap-
in DC in adipose tissue might contribute to or reflect changes
tive immune responses. Granulocytes were also
in inflammatory disease.
present amongst the omental cells from Crohn’s
patients. Results indicated that omentum may pro-
vide DC, which could “police” local infections and
1
contribute to and/or reflect local inflammatory Correspondence: Antigen Presentation Research Group, Imperial College
activity. J. Leukoc. Biol. 80: 546 –554; 2006. London, Northwick Park and St. Mark’s Campus, 7W, Watford Road, Harrow
HA1 3UJ, UK. E-mail: s.knight@imperial.ac.uk
Received September 7, 2005; revised April 11, 2006; accepted May 19,
Key Words: mucosa 䡠 inflammatory bowel disease 䡠 fatty acids 2006; doi: 10.1189/jlb.0905501.

546 Journal of Leukocyte Biology Volume 80, September 2006 0741-5400/06/0080-546 © Society for Leukocyte Biology
MATERIALS AND METHODS
Omental samples
Local Ethics Committee permission and informed patient consent were ob-
tained, and omental biopsies were taken from patients undergoing surgery for
Crohn’s disease (n⫽18, age range 27– 62 years, mean 37) with a diagnosis
made using clinical parameters, radiographic studies, and histological criteria.
Of these patients, eight were not currently receiving treatment, six were on
prednisolone with or without nonsteroidal, anti-inflammatory drugs, and the
remainder was receiving azathioprine or pentasa. Surgery was indicated from
strictures/small bowel obstruction or fistulae. “Controls” were patients under-
going abdominal surgery for benign gynecological conditions including uterine
leiomas and cystadenomas (n⫽17, age range 25– 69 years, mean 53).
Cells were obtained by a walkout technique developed for enriching DC
populations from gut biopsies [15]. Tissue samples were cut into small pieces
(1–2 mm3) and put in tissue-culture medium (RPMI 1640, Dutch modification,
Sigma, Poole, UK) with 10% fetal calf serum, L-glutamine (2 mM), penicillin
(100 IU ml–1), and streptomycin (100 ␮g ml–1) overnight at 37°C in 5% CO2.
Tissue was removed on a 100-␮m sieve, and any red cells were removed by
lysis. We originally looked at collagenase-treated cells obtained using a
technique for separating “pre-adipocytes”. The small number of cells obtained
had a phenotype similar to those obtained with the walkout technique. We
chose to study only walkout cells more extensively to avoid release of excessive
amounts of fat, which cause problems in quantitating and handling cells.
Flow cytometry
Cells were labeled with monoclonal antibodies to cell surface markers directly
fluorochrome-conjugated and fixed in 1% paraformaldehyde. Antibodies were
fluorescein isothiocyanate (FITC)-conjugated CD45 and CD80, human leuko-
cyte antigen (HLA)-DR, and phycoerythrin-conjugated CD45, CD3, CD19,
CD14, CD16, CD34, and CD56 (Becton Dickinson, Oxford, UK); FITC-
conjugated CD1a, CD40, and CD86 (Serotec, Oxford, UK); FITC-conjugated
CD11c (Dako Cytomation, Ely, UK); and FITC-conjugated CD83 (Alexis
Biochemicals, Nottingham, UK). Samples were acquired immediately or stored
at 4°C and acquired within 36 h. Histogram analysis on the live cell gate used
Winlist analysis software (Verity Software House, Topsham, ME). Percentage-
positive cells and the intensity ratio, which indicates the ratio of positive-to-
background staining, were determined using the super-enhanced D max sub-
traction routine in Winlist.
Morphological studies
For electron microscopy between 2 and 10 ⫻ 105, freshly isolated, omental
cells were processed for electron microscopy. Some cells were surface-labeled
with biotinylated anti-major histocompatibility complex (MHC) Class II anti-
body followed by goat anti-biotin 10 nm gold prior to processing for electron
microscopy. At least 100 cells were counted per sample, viewed using a JEOL
JEM-1200 EX electron microscope (Osaka, Japan), and assessed for labeling
for MHC Class II by counting gold particles per cell. In controls, gold labeling
was generally negative and always ⬍5 gold grains per cell; ⬎10 grains were
considered positive. Macrophages and DC were identified from their charac-
teristic morphology [16].
Frozen sections were also prepared and stained with CD11c and MHC Class
II using the avidin-biotin complex (ABC) method (Vector Laboratories, Peter-
borough, UK). Slides were air-dried, fixed in cold acetone (–20°C) for 5 min,
washed in phosphate-buffered saline (PBS), quenched for 30 min in 3%
hydrogen peroxide in methanol, washed in PBS, transferred to a humidified
chamber, and incubated for 1 h at room temperature with 5% goat serum in
PBS. Slides were incubated with mouse anti-CD11c or HLA-DR primary
antibodies (Dako Cytomation) overnight at 5°C, washed and stained with
secondary biotinylated antibody for 1 h, stained with ABC for 30 min at room
temperature, and developed with peroxidase substrate solution (Vector Labo-
ratories) and were counterstained in hematoxylin, washed, cleared in xylene,
and mounted under cover-slips. Control slides were prepared by replacing the
primary antibodies with normal serum. Some slides were double-labeled by
immunofluorescence microscopy; frozen tissue sections (4 ␮m) were fixed in
cold methanol:acetone (1:1) for 10 min, incubated with normal mouse serum
for 1 h, washed, and incubated with primary antibody to CD83 (Beckman
Coulter, High Wycombe, UK) overnight. Sections washed in PBS were incu-
bated with secondary antibody conjugated with tetramethyl rhodamine isothio-
cyanate (TRITC) for 2 h and washed again in PBS. To block nonspecific
binding sites, sections were incubated with normal mouse serum for 1 h and
then incubated with FITC-conjugated primary antibody to CD86, CD80, or
CD40 (Serotec) for 4 h, washed in PBS, and mounted under cover-slips using
4⬘,6-diamidino-2-phenylindole-conjugated mounting medium (Vector Labora-
tories). Negative controls were incubated with appropriate isotype controls.
Mixed leukocyte reactions (MLR)
Large mononuclear cells of low density were enriched from omental cells over
a metrizamide gradient (14.5% w/v, Sigma) [17]. These cells were ⬎85% DC
from electron microscopy and phenotyping and were used in varying numbers
(500 –2000) with 6.25–100 ⫻ 103 allogeneic peripheral blood mononuclear
cells in triplicate, 20 ␮l hanging drops in Terasaki plates. The stimulatory
capacity of these cells was compared with that of DC enriched from mononu-
clear cells of peripheral blood, and human peripheral blood DC were enriched
from the Ficoll-Paque-separated mononuclear cell population by first removing
the adherent macrophage population and after 24 h, isolating nonadherent,
low-density cells on a metrizamide gradient (14.5%, Sigma) [18]. The cell
population contained ⬍5% B and T cells from labeling with CD20 and CD3.
The large mononuclear cells were approximately 30% classical CD14 – DC and
70% CD14 low (intensity of CD14 staining, ⬍5 times isotype background vs.
approximately ⬎40 times background for freshly isolated monocytes). CD14 –
and CD14 low populations have veiled morphology, express costimulatory
markers CD80, -86, and -40, and are potent stimulators of primary T cell
proliferation; these cells, added at a ratio of less than 1:200 allogeneic T cells,
stimulate a MLR. Plates containing cell cultures of DC plus allogeneic T cells
were inverted and cultured for 3 days over sterile saline at 37°C and pulsed for
2 h with 3H thymidine to give a final concentration of 1 ␮g ml–1 (specific
activity, 2 Ci/mM, Amersham Biosciences, Amersham, UK). These conditions
allow accurate reflection of DNA synthesis in the cells, as they ensure free
availability of the alternative pathway precursor throughout the short pulse-
time with low levels of radiation-induced damage in cells taking up the
thymidine [19]. Cells were then blotted onto filters, washed with saline,
trichloroacetic acid (5%), and methanol, and dried. The filter was exposed to
a tritium screen for 4 h and imaged in a phosphor imager (Amersham
Biosciences). The accumulated counts on the storage phosphor screen were
quantitated using ImageQuant software (Molecular Dynamics, Amersham, UK)
[17]. This 20-␮l culture technique produced earlier proliferative responses
than those obtained in conventional 200 ␮l cultures. Counts were lower using
imaging than those produced from scintillation counting, but the results give
⬎0.97 correlations between scintillation counts and the counts obtained by
imaging [20]. Counts stimulated by concanavalin A, used routinely as a
positive control, were 1000 –5000 counts. Significance was tested using two-
way ANOVA of log transformed, normally distributed data for each of five
omental samples studied in each patient group.
RESULTS
Cells migrating from omental biopsies
Omentum from controls yielded a total of 2.36 ⫾ 0.5 ⫻ 105
cells per mg tissue, and significantly more cells
(11.2⫾0.2⫻105 per mg, P⫽0.001) were from omentum of
patients with Crohn’s disease (Fig. 1); a high proportion of
these cells obtained expressed CD45, indicative of a bone
marrow origin (Fig. 1). There was no significant difference
between the proportion of CD45⫹ve cells obtained from con-
trol and Crohn’s omenta (56.6%⫾5.1 vs. 40.2%⫾8.6). How-
ever, by virtue of the increased numbers of cells, over three
times as many CD45⫹ cells were obtained from omentum in
Crohn’s than in controls (4.50⫾1.08 vs. 1.33⫾0.12⫻105). The
majority of leukocytes from controls was large mononuclear
cells, many with veiled projections. In samples from patients
Bedford et al. Dendritic cells in adipose tissue 547

Fig. 1. Cells obtained from omental samples. The total numbers of cells
migrating out of fragments of omental adipose tissue after overnight incubation
are shown (f) and the percentages of these cells expressing CD45 as measured
by flow cytometry (e) in samples from controls and from Crohn’s patients.
There was no significant difference in the proportion of CD45⫹ve cells
obtained from control or Crohn’s omentum. There were significantly more cells
obtained from the Crohn’s than from control omentum (P⫽⬍0.001).
with Crohn’s disease, there were, additionally, variable num-
bers of granulocytes.
Flow cytometry characterization
The light-scatter properties of CD45⫹ cells from control and
Crohn’s omentum were similar and showed two major cell
Fig. 2. Phenotypic profile of cells from control omentum by flow cytometric analysis. (a) A typical profile of forward- and side-scatter of cells, which were CD45⫹.
(b–f) The open histograms show the positive staining for the cell surface markers indicated. The isotype control (not shown) has been subtracted using the Winlist
algorithm; cells that are positive after subtraction of the isotype control are shown in the filled portions of the histograms, and those that are negative remain
unshaded. The profile for each antibody is typical of that obtained in at least five experiments.
548 Journal of Leukocyte Biology Volume 80, September 2006
populations (Fig. 2a, control only shown). Samples contained
few cells positive for T and B cell markers CD3 and CD19, as
demonstrated in Figure 2, b and c, where positive-staining
profiles and proportion of those cells positive after subtraction
of the isotype control are shown. These putative T and B cells
were confined to the smaller cells. Phenotypic markers found
within large and small cells were similar, although a lower
percentage of the small cells generally expressed DC-associ-
ated markers. Expression of CD14 (Fig. 2d) overall was low in
comparison with that of monocytes from blood [positive/control
intensity ratio of 2.2⫾0.4 compared with 56.5⫾5.4 (n⫽3)]. In
addition, the macrophage marker CD16 (Fig. 2e) and myeloid
marker CD33 (data not shown) were expressed on few or no
cells. These phenotypic characteristics suggested that conven-
tional monocytes or macrophages were not present; similar
results were observed in cells from Crohn’s omentum (data not
shown). The presence of hematopoietic stem cells was indi-
cated in controls from labeling with CD34 (Fig. 2f) but was
negligible in cells in Crohn’s (not shown). There was no evi-
dence of CD56⫹ natural killer cells (Fig. 2g).
The CD45⫹ cells from controls and Crohn’s patients were
analyzed for a series of DC-associated markers. Figure 3
shows representative histograms of the labeling for DC-associ-
ated markers in CD45⫹ve cells from a control and a Crohn’s
patient. Figure 4 shows the proportion of CD45⫹ve cells
positive for DC-associated markers from 11 control and nine
Crohn’s patients. A high proportion of cells expressed the
Langerhans cell-associated marker CD1a. Many cells were
CD11c-positive (68.4⫾6% in controls and 80.9⫾3.8 in
Crohn’s disease), and CD83, a marker appearing during mat-
uration of DC from monocytes, was expressed on high numbers
of cells (78.4⫾4.6 and 86.7⫾3.4% in controls and Crohn’s
patients, respectively). The costimulatory molecules CD40 and
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CD86 were expressed (Figs. 3 and 4), but the level of labeling
was low for CD80, suggesting that the cells were not fully
mature. Approximately half the cells in controls expressed
MHC Class II. The plasmacytoid DC marker CD123 was not
expressed (not shown). Gating on the population of larger cells
increased the proportion of cells labeling for markers associ-
ated with DC. In a typical example, over 90% of the large cells
were CD83-, CD11c-, and CD86-positive, and 78%, 59%, and
73% expressed CD40, CD80, and CD1a, respectively, indicat-
ing double-labeling of cells for DC lineage-associated markers
and costimulatory marker expression.
Figures 3 and 4 also record differences between phenotypes
of cells from control samples and those from Crohn’s disease
patients. A significantly higher proportion of cells from Crohn’s
omenta expressed CD11c, suggesting an increase in putative
myeloid DC. The costimulatory molecules CD40, CD80, and
CD86 were more highly expressed in cells from Crohn’s pa-
tients, and this elevation was significant for CD40. The inten-
sity of staining for CD40 and CD86 was also greater in cells
from Crohn’s patients (e.g., Fig. 3). In cells from some patients,
staining with many antibodies was approximately 90%, even in
total cell populations (e.g., for CD11c, CD40, and CD86 in Fig.
3), and it was again clear that there was multiple labeling of
cells using these antibodies. The most striking change was the
almost-complete loss of MHC Class II in cells from Crohn’s
patients, which was not up-regulated even after exposure of the
cells to lipopolysaccharide for 2 days in culture. This loss of
MHC contrasted with normal levels of MHC Class II on parallel
blood samples (n⫽3) of metrizamide gradient-separated blood
DC after 24 h of incubation.
Morphological identification of DC
In cytospins of omental walkout cells, we confirmed that there
were only occasional lymphocytes and that large mononuclear
cells formed the major cell population, some containing obvi-
ous lipid droplets; the latter cells appear to be developing
adipocytes and may account for the presence of CD45-negative
cells within the samples. In control samples, granulocytes were
not identified, but in samples from Crohn’s patients, granulo-
cytes formed 20 ⫾ 8% of the cells (range 5– 45%). As DC are
difficult to identify securely using light microscopy, we sought
to confirm the presence of DC using electron microscopy. DC
enriched using metrizamide gradients were studied in two
control samples and two from Crohn’s patients. The majority
(⬎90%) of cells from the controls appeared myeloid cells, of
which ⬎70% were identified as DC (Fig. 5, a and c); the
remainder was not clearly identifiable or was macrophages
(with many round vacuoles of different sizes, heterochromatic
nuclei with a rounded or horseshoe shape, and multiple or-
ganelles of different types present in a cytoplasm, which often
appeared darker than that of the DC). Cells of three morpho-
logical types of DC previously identified in DC separated from
4
Fig. 3. Profiles of staining for different markers of the CD45⫹ cells from
omentum. Cells migrating from omental fragments were double-labeled for
CD45 and for the markers indicated. The profile of the CD45⫹ cells positively
stained for different markers is shown. The portion positive after subtraction of
the isotype control is shown in the filled portions of the histograms, and those
negative remain unfilled. The percentage positive and the positive/control
intensity ratio denoting the level of staining are shown on each histogram. The
labeling of omental cells from a control and a Crohn’s is shown. These plots are
representative of the labeling obtained from 11 controls and nine Crohn’s
patients.
Bedford et al. Dendritic cells in adipose tissue 549
Fig. 4. Numbers of CD45⫹ omental cells positive
for different markers. Cells migrating from omental
fragments were double-labeled for CD45 and for the
markers indicated and the proportions positive
shown after subtraction of the isotype control using
the Winlist algorithm. The percentage-positive val-
ues for cells from controls and Crohn’s patients
were compared, and significant differences are in-
dicated.
human peripheral blood were identified in each sample [16].
They included cells with electron dense nuclei, some vacuoles,
and numerous short pseudopods as shown in Figure 5. DC with
a smoother cell boundary and fewer processes, resembling
Langerhans cells and cells with longer processes, which had a
less electron-dense nuclear morphology, similar to that of
veiled cells in the afferent lymph, were also present. Over 70%
of myeloid cells labeled positively for MHC Class II by immu-
nogold labeling (Fig. 5, b and c), and many had high levels of
gold staining (Fig. 5d). Amongst mononuclear cells in the
samples from Crohn’s patients, there was again a high prepon-
derance of veiled cells clearly identified as DC by electron
microscopy (65% and 80% in the two studied), and there was
no evidence of staining of these cells for MHC Class II,
confirming the loss of MHC Class II observed by flow cytom-
etry. The presence in omentum of mononuclear cells of den-
dritic morphology was also confirmed by immunostaining in
light microscopy. Clusters of cells of veiled/dendritic morphol-
ogy were seen in the omentum in interstices between the
adipocytes, and specific staining of these cells for CD11c is
shown in Figure 5, e and f. In the Crohn’s disease samples,
there was staining for CD11c but not for MHC Class II. In the
Crohn’s omentum, there were also scattered granulocytes vis-
ible (not shown). In frozen sections, double-staining of cells
was confirmed using fluorescently labeled antibodies to CD83,
which gave the highest level of labeling, and each of the
costimulatory molecules (CD40, CD80, and CD86), as exem-
plified for CD86 in Figure 5, g and h.
Allogeneic T cell stimulation
The functional hallmark of DC is their potency in stimulating
primary, allogeneic T cell proliferation. Large mononuclear
cells were separated from omental cells using a metrizamide
550 Journal of Leukocyte Biology Volume 80, September 2006
gradient designed to enrich DC. Small numbers of these sep-
arated cells stimulated MLR (Fig. 6). The counts were similar
to or higher than those found in the normal MLR using metri-
zamide-enriched blood DC as a stimulus (Fig. 6a). Stimulation
was observed when adding putative, allogeneic omental DC at
1–2% of responding mononuclear cells (Fig. 6b). Cells from all
five control omenta stimulated significant, primary MLR
(P⫽⬍0.001). By contrast, three of five samples from Crohn’s
patients induced no significant MLR (Fig. 6c), and the other
two stimulated only low levels of proliferation (P⫽⬍0.05). The
omental cells stimulated higher levels of proliferation than
peripheral blood DC, as indicated in the representative exper-
iment shown. In the experiment shown, not only were there
lower counts when DC from blood were used, but also, stimu-
lation was still increasing with a dose of stimulator cells,
whereas the response had reached a plateau and was lower with
the higher dose of omental cells. The higher stimulation could
possibly be a result of a greater maturity of the omental DC.
The lack of primary T cell stimulation with cells from Crohn’s
patients was consistent with loss in MHC Class II, identified
from flow cytometry and electron microscopy.
DISCUSSION
This paper identifies human adipose tissue, as exemplified by
omentum, as a potential, major repository of myeloid DC by
showing that significant numbers of these cells migrated from
human omental tissue fragments in culture with more than
fivefold more DC per mg tissue than are found per ml blood; DC
were identified from phenotype, morphology, and immunostain-
ing by light and electron microscopy and function in stimulat-
ing, primary, proliferative activity in allogeneic T cells. An
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Fig. 5. Morphology of omental DC. (a) A typical DC from a control omentum is shown. (b) An example of immunogold staining of the veils of a DC. The original
bars indicate 1 ␮m. In controls, gold labeling was always ⬍5 gold grains per cell; ⬎10 grains was considered positive. (c) The types of myeloid cells identified
in the omental cells from two control patients; the proportion positive for HLA-DR staining is shown in the solid portion of the histograms, and those negative are
hatched. M␾, Macrophage. (d) The gold grains per cell for the surface DR-labeling in the two experiments performed are shown. No evidence of DR-labeling was
seen in either of the two Crohn’s patients studied. (e) A light microscopy picture of a cluster of putative DC immunostained with CD11c on a frozen section of
omentum from a Crohn’s patient showing the dark, positive-peroxidase staining, which was absent from control slides. Cell nuclei, which were stained with
hematoxylin, were distinguishable, showing that the veils were associated with mononuclear cells in the section. (f) Control for e using serum instead of specific
antibody and with only hematoxylin staining visible. (g) Frozen section of control omentum, showing two cells specifically labeled for CD83 (TRITC-labeled). (h)
The same section as g with label specific for CD86 (FITC-labeled).

increased proportion of putative myeloid (CD11c⫹) DC and
higher expression of costimulatory molecules, particularly
CD40, with decreased MHC Class II and stimulatory function
in cells from omentum in Crohn’s patients, bore witness that
immunological changes, probably related to inflammation in
gut, occurred in cells within omental adipose tissue. An in-
crease in innate activation of DC but loss of ability to stimulate
adaptive immunity may be indicated. Cellular as well as bio-
chemical changes within omental adipose tissue may thus be
integral to Crohn’s disease. The presence of putative DC in
adipose tissue, particularly in perinodal adipose tissue, which
increases during local stimulation with endotoxin, was sug-
gested in studies of adipose tissue from rats, although no
characterization of these cells was undertaken [21–23]; mes-
enteric lymph nodes are embedded in the human omentum.
Our identification of DC migrating from human adipose tissue
described here and similar cells, which we obtained from
mouse omentum [24], which is close to the spleen, indicates
that DC are indeed present in adipose tissue of the omentum.
DC can be obtained as they mature and migrate out of skin
or gut tissue fragments [15]. We adapted this method to obtain
enriched DC from human omental samples. There were few
adipocytes present, as these cells floated and were removed, so
avoiding problems of having large quantities of fat present.

Bedford et al. Dendritic cells in adipose tissue 551

Fig. 6. MLR stimulated by omental DC. Peripheral blood DC were enriched from 24 h nonadherent Ficoll-separated mononuclear cells on metrizamide gradients.
Omental DC were also enriched over metrizamide. Small numbers, 1000 (䡬) or 2000 (‚), of these cells were added to different numbers of responder cells in 20
␮l hanging drop cultures, and the stimulation was measured by uptake of tritiated thymidine into the cells in a 2-h pulse of thymidine on Day 4 of culture after
blotting them onto a filter and imaging the filter in a phosphor imager. (a) The uptake in a normal MLR in this microculture system using peripheral blood DC.
(b) Stimulation with control omental DC and (c) effect of adding DC from Crohn’s omentum. The background proliferation of unstimulated responder cells (f) is
shown in each graph. The control and omental DC each caused a significant stimulation (P⫽⬍0.001), but the Crohn’s omental DC caused little (P⫽0.05, n⫽2)
or no (n⫽6) significant stimulation.

There was some blood contamination within omental samples,
and some cells could possibly derive from this contamination.
However, the preponderance of myeloid cells and the presence
of higher numbers of DC/mg tissue than there are DC/ml blood
ruled out blood as the source of the majority of cells obtained.
Occasional omental samples showed pale areas, which may
have been “milky spots”, but these areas proved impossible to
dissect out, as they were fluid rather than structured. The lack
of lymphocytes also suggested that we were not obtaining
immune cells from structured lymphoid tissue within the fat.
All control, omental samples were from females, whereas the
Crohn’s patients were evenly split between male and female.
However, analysis of the numbers of cells from male and
female Crohn’s patients showed no significant differences in
numbers of omental cells. We also saw no evidence that the
treatment of some patients was significantly altering the cells
that we obtained or their functions.
Expression of MHC Class II is generally considered a de-
fining characteristic of DC and was well represented on a high
proportion of the cells from control omentum. The lower MHC
Class II found on cells in Crohn’s could indicate a loss of DC
from omentum in Crohn’s. However, the similarity of the phe-
notype of cells from controls and Crohn’s patients argues that
these are related to the DC and have lost or lack MHC Class II.
The information that control omentum contains stem cells,
including hematopoietic stem cells, as indicated from the
CD34 labeling, however, could mean that these cells represent
cells developing down an alternative maturation pathway. The
presence of granulocytes in omentum in Crohn’s could indicate
that some omental stem cells are maturing down a granulocytic
pathway, although recruitment of additional cells from the
circulation is an alternative possibility. There is evidence of a
close relationship between granulocytes and DC. Thus, gran-
ulocytes can be driven to acquire DC characteristics [25], and
an imbalance between granulocytes and DC, favoring produc-
tion of the former, may contribute to production of autoimmune
disease [26].
The lack of Class II and loss of T cell stimulatory function of
cells with the phenotype and morphology of DC in omentum
were unexpected. Innate immune mechanisms to bacteria me-
diated by PRR such as TLR on DC might then underlie the
immune activity in the gut [27]. A reduction in the capacity of
cells to express MHC and to initiate adaptive immune re-
sponses could perhaps represent a mechanism indicating fail-
ure of DC to mature and elicit appropriate adaptive immune
activity to deal with infecting organisms entering via a gut with
an increased permeability. Systemic changes in numbers and
functions of DC in Crohn’s disease have recently been reported
[28], as well as DC changes in the gut [29], and it will be of
interest in future experiments to examine in more detail DC
from these other compartments. The changes in putative DC in
Crohn’s include an increase in CD40 expression, which has
also been observed in DC isolated from the lamina propria of
patients with Crohn’s, providing a link between the activity of
cells in the colon in Crohn’s and those in the omentum. The
CD40 levels on DC in the colon normalize after treatment with
anti-TNF antibody [29]. The elevated production of TNF-␣ in
the adipose tissue in Crohn’s thus provides a further link
between the aberrant activity in the colon and expression of
costimulatory markers and the function of DC. The involve-
ment of adipose tissue in the development of Crohn’s disease
rather than the concept that the changed fat distribution is
merely a consequence of the disease process has been postu-
lated [14]. The presence of primary antigen-presenting DC
migrating from the omentum suggests that adipose tissue may
be a source of local APC. Significant changes in cellular
components of adipose tissue in Crohn’s disease, taken to-
gether with the increased TNF-␣ production in adipose tissue
in Crohn’s, lend weight to the idea of greater involvement of
adipose tissue in disease pathogenesis than has previously
been considered, although questions of cause and effect have
yet to be elucidated.
Finally, different fatty acids can modulate immune functions
differentially, and some evidence suggests that fatty acid dis-
tribution is altered in Crohn’s disease and that manipulation of
dietary fats can be beneficial in its treatment [30]. One possible
mechanism of action of fatty acids is via modulation of mac-
rophages, perhaps via effects on TLR [31]. Fatty acids may also

552 Journal of Leukocyte Biology Volume 80, September 2006 http://www.jleukbio.org

block DC function, an effect that is independent of nuclear
factor-␬B activation [32]. Fats are taken up into developing
DC, and this uptake is modulated by the effects of cytokine,
antigen, or location [24]. The presence of ␻-3 fatty acids
particularly can down-regulate immune activity, possibly via
the effects of downstream molecules such as prostaglandins
[33]. ␻-3 fatty acids fed to rats down-regulate the stimulatory
functions of DC [34], and prostaglandins modulate DC function
[35], suggesting that DC are affected by environmental fatty
acids and their downstream products. Perinodal adipose tissue
is high in unsaturated fatty acids, which can down-regulate the
activity of stimulated lymphocytes [36]. The DC derived from
adipose tissue itself may therefore provide a conduit for effects
of fatty acids on the immune system. There is loss of ␻-3 fatty
acids in the plasma in Crohn’s disease [37, 38] and consequent
changes in downstream molecules such as prostaglandins with
their effects on DC function [35], providing possible mecha-
nisms via which fatty acids within the adipose tissue may
influence APC and produce changes in immune activity in
Crohn’s disease. We showed that the usual preponderance of
unsaturated fatty acids in perinodal adipose tissue was absent
in tissue from patients with Crohn’s disease with a major loss
in ␻-3 fatty acids [39]. However, changes in environmental
fatty acids may not be mirrored in lymphoid cells, and the
interactions between environmental fatty acids and the cells of
the immune system may be critical in determining the meta-
bolic and immune activity [40]. We observed that the fatty
acids in lymph node cells from patients with Crohn’s disease,
unlike those in plasma and adipose tissue, showed a dispro-
portionate loss in ␻-6 fatty acids, leading to a significantly
lower-than-normal ␻-6/␻-3 ratio [39]. Such an increase in the
relative proportion of anti-inflammatory ␻-3 fatty acids in
immune cells from Crohn’s patients may provide a mechanism
for the loss in immune activation observed.
In conclusion, these novel studies of DC showing their
presence in omental adipose tissue and changes in their num-
ber, phenotype, and function in Crohn’s disease identify a
number of mechanisms by which these omental DC may be
involved in local immune responses and altered in inflamma-
tory disease. These data, establishing that DC are present in
adipose tissue, also pave the way for evaluation of their source,
development pathways, lineage relationships, and a more de-
tailed examination of the way in which adipose tissue DC may
be involved in local immune responses in health and in dis-
ease.
ACKNOWLEDGMENT
This work was supported by a grant from the Medical Research
Council UK.
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