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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
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:
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

Tissue samples were cut into small pieces capacity of these cells was compared with that of DC enriched from mononu- (1–2 mm3) and put in tissue-culture medium (RPMI 1640. [17]. were 1000 –5000 counts. frozen tissue sections (4 ␮m) were fixed in cold methanol:acetone (1:1) for 10 min. UK). Dutch modification. UK) background staining. and phycoerythrin-conjugated CD45. At least 100 cells were counted per sample. washed and stained with secondary biotinylated antibody for 1 h.12⫻105). Slides were air-dried. These conditions cyte antigen (HLA)-DR. Amersham Biosciences. “Controls” were patients under.97 correlations between scintillation counts and the counts obtained by imaging [20]. trol and Crohn’s omenta (56.5% w/v. availability of the alternative pathway precursor throughout the short pulse- conjugated CD1a. penicillin from the Ficoll-Paque-separated mononuclear cell population by first removing (100 IU ml–1). Of these patients. The for 1 h. High Wycombe. UK). a metrizamide gradient (14. and are potent stimulators of primary T cell Flow cytometry proliferation. We originally looked at collagenase-treated cells obtained using a population contained ⬍5% B and T cells from labeling with CD20 and CD3. age range 27– 62 years. UK). between the proportion of CD45⫹ve cells obtained from con- ratories) and were counterstained in hematoxylin. CD14 – amounts of fat. This 20-␮l culture technique produced earlier proliferative responses traction routine in Winlist. and CD86 (Serotec. UK) with 10% fetal calf serum. Sections washed in PBS were incu. washed in PBS. CD3.5%. 1). gold labeling was generally negative and always ⬍5 gold grains per cell. and omental biopsies were taken from patients undergoing surgery for 4⬘. Tissue was removed on a 100-␮m sieve.5 ⫻ 105 washed in phosphate-buffered saline (PBS). activity. washed. many with veiled projections. and FITC-conjugated CD83 (Alexis thymidine [19]. and streptomycin (100 ␮g ml–1) overnight at 37°C in 5% CO2. Samples were acquired immediately or stored trichloroacetic acid (5%). Dendritic cells in adipose tissue 547 . and any red cells were removed by low-density cells on a metrizamide gradient (14. The cell lysis. stained with ABC for 30 min at room marrow origin (Fig. CD40 (Serotec) for 4 h.MATERIALS AND METHODS bated with secondary antibody conjugated with tetramethyl rhodamine isothio- cyanate (TRITC) for 2 h and washed again in PBS. omental positive control. which cause problems in quantitating and handling cells. over three primary antibodies with normal serum. indicative of a bone antibodies (Dako Cytomation) overnight at 5°C. Macrophages and DC were identified from their charac- teristic morphology [16]. Cells were then blotted onto filters. transferred to a humidified (11. UK). Oxford. eight were not currently receiving treatment. a high proportion of PBS. Histogram analysis on the live cell gate used a tritium screen for 4 h and imaged in a phosphor imager (Amersham Winlist analysis software (Verity Software House. We 70% CD14 low (intensity of CD14 staining. sections were incubated with normal mouse serum for 1 h and Omental samples then incubated with FITC-conjugated primary antibody to CD86. The small number of cells obtained The large mononuclear cells were approximately 30% classical CD14 – DC and had a phenotype similar to those obtained with the walkout technique. -86. Percentage. cells. Significance was tested using two- cells were processed for electron microscopy.2⫻105 per mg. Poole. Biochemicals. and mounted under cover-slips using tained.6%⫾5. by virtue of the increased numbers of cells. In controls. Omentum from controls yielded a total of 2. the adherent macrophage population and after 24 h. which indicates the ratio of positive-to. and incubated with primary antibody to CD83 (Beckman majority of leukocytes from controls was large mononuclear Coulter. washed. and histological criteria. UK). freshly isolated.6). Control slides were prepared by replacing the ever. and developed with peroxidase substrate solution (Vector Labo. CD16. quantitated using ImageQuant software (Molecular Dynamics. added at a ratio of less than 1:200 allogeneic T cells. Some cells were surface-labeled way ANOVA of log transformed. and CD14 low populations have veiled morphology. Sigma) [17]. used routinely as a For electron microscopy between 2 and 10 ⫻ 105. CD80. ⬎10 grains were RESULTS considered positive. washed in PBS. and human peripheral blood DC were enriched Sigma.50⫾1. and incubated for 1 h at room temperature with 5% goat serum in patients with Crohn’s disease (Fig. chose to study only walkout cells more extensively to avoid release of excessive approximately ⬎40 times background for freshly isolated monocytes). and assessed for labeling for MHC Class II by counting gold particles per cell.36 ⫾ 0. 40. CD40. Amersham. these cells. and -40. L-glutamine (2 mM). fixed in cold acetone (–20°C) for 5 min. but the results give Morphological studies ⬎0. FITC-conjugated time with low levels of radiation-induced damage in cells taking up the CD11c (Dako Cytomation.25–100 ⫻ 103 allogeneic peripheral blood mononuclear Cells were obtained by a walkout technique developed for enriching DC cells in triplicate. In samples from patients Bedford et al. than those obtained in conventional 200 ␮l cultures. Surgery was indicated from Large mononuclear cells of low density were enriched from omental cells over strictures/small bowel obstruction or fistulae. express costimulatory markers CD80. (500 –2000) with 6. age range 25– 69 years. UK).33⫾0. isolating nonadherent.2%⫾8. body followed by goat anti-biotin 10 nm gold prior to processing for electron microscopy. mean 53). incubated with normal mouse serum Crohn’s than in controls (4.2⫾0. normally distributed data for each of five with biotinylated anti-major histocompatibility complex (MHC) Class II anti.08 vs. Nottingham. 20 ␮l hanging drops in Terasaki plates.1 vs. Counts stimulated by concanavalin A. made using clinical parameters.001) were from omentum of chamber. Japan). 1. six were on Mixed leukocyte reactions (MLR) prednisolone with or without nonsteroidal. Biosciences). Counts were lower using imaging than those produced from scintillation counting. mean 37) with a diagnosis tories). The stimulatory populations from gut biopsies [15].6-diamidino-2-phenylindole-conjugated mounting medium (Vector Labora- Crohn’s disease (n⫽18. stimulate a MLR. allow accurate reflection of DNA synthesis in the cells. ⬍5 times isotype background vs. UK) overnight. 1). Slides were incubated with mouse anti-CD11c or HLA-DR primary these cells obtained expressed CD45. How- and mounted under cover-slips. and methanol. There was no significant difference temperature. technique for separating “pre-adipocytes”. omental samples studied in each patient group. To block nonspecific binding sites. washed with saline. The filter was exposed to at 4°C and acquired within 36 h. and significantly more cells hydrogen peroxide in methanol. CD19. Antibodies were 2 h with 3H thymidine to give a final concentration of 1 ␮g ml–1 (specific fluorescein isothiocyanate (FITC)-conjugated CD45 and CD80. radiographic studies. quenched for 30 min in 3% cells per mg tissue. and the remainder was receiving azathioprine or pentasa. clear cells of peripheral blood. Plates containing cell cultures of DC plus allogeneic T cells Cells were labeled with monoclonal antibodies to cell surface markers directly were inverted and cultured for 3 days over sterile saline at 37°C and pulsed for fluorochrome-conjugated and fixed in 1% paraformaldehyde. anti-inflammatory drugs. ME). Sigma) [18]. were determined using the super-enhanced D max sub. 2 Ci/mM. These cells were ⬎85% DC going abdominal surgery for benign gynecological conditions including uterine from electron microscopy and phenotyping and were used in varying numbers leiomas and cystadenomas (n⫽17. Amersham. as they ensure free CD14. Frozen sections were also prepared and stained with CD11c and MHC Class Cells migrating from omental biopsies II using the avidin-biotin complex (ABC) method (Vector Laboratories. Peter- borough. Some slides were double-labeled by times as many CD45⫹ cells were obtained from omentum in immunofluorescence microscopy. Negative controls were incubated with appropriate isotype controls. FITC. Ely. human leuko. cleared in xylene. and dried. CD34. and CD56 (Becton Dickinson. or Local Ethics Committee permission and informed patient consent were ob. P⫽0. Oxford. viewed using a JEOL JEM-1200 EX electron microscope (Osaka. Topsham. The accumulated counts on the storage phosphor screen were positive cells and the intensity ratio.

although a lower percentage of the small cells generally expressed DC-associ- ated markers. which were CD45⫹. 2f) but was negligible in cells in Crohn’s (not shown).2⫾0.4% in controls and Crohn’s Crohn’s omentum were similar and showed two major cell patients. the macrophage marker CD16 (Fig. (a) A typical profile of forward. There was no evi- dence of CD56⫹ natural killer cells ( . as demonstrated in Figure 2. additionally. 2g). where positive-staining profiles and proportion of those cells positive after subtraction of the isotype control are shown. Langerhans cell-associated marker CD1a. control only shown).7⫾3. 2. The total numbers of cells The CD45⫹ cells from controls and Crohn’s patients were migrating out of fragments of omental adipose tissue after overnight incubation analyzed for a series of DC-associated markers. Many cells were bers of granulocytes. populations (Fig. b and c. shows representative histograms of the labeling for DC-associ- There was no significant difference in the proportion of CD45⫹ve cells ated markers in CD45⫹ve cells from a control and a Crohn’s obtained from control or Crohn’s omentum. Samples contained few cells positive for T and B cell markers CD3 and CD19. 2e) and myeloid marker CD33 (data not shown) were expressed on few or no cells.4 compared with 56.jleukbio. These putative T and B cells were confined to the smaller cells. Phenotypic markers found within large and small cells were similar. positive for DC-associated markers from 11 control and nine Crohn’s patients. similar results were observed in cells from Crohn’s omentum (data not shown). The profile for each antibody is typical of that obtained in at least five experiments. variable num. Figure 3 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. and those that are negative remain unshaded. A high proportion of cells expressed the with Crohn’s disease.5⫾5. These phenotypic characteristics suggested that conven- tional monocytes or macrophages were not present. Figure 4 shows the proportion of CD45⫹ve cells obtained from the Crohn’s than from control omentum (P⫽⬍0. was expressed on high numbers The light-scatter properties of CD45⫹ cells from control and of cells (78. September 2006 http://www. The presence of hematopoietic stem cells was indi- cated in controls from labeling with CD34 (Fig. 2a. There were significantly more cells patient. CD11c-positive (68. 1.and side-scatter of cells. and CD83. (b–f) The open histograms show the positive staining for the cell surface markers indicated. The costimulatory molecules CD40 and Fig. 2d) overall was low in comparison with that of monocytes from blood [positive/control intensity ratio of 2. Fig.9⫾3. there were. The isotype control (not shown) has been subtracted using the Winlist algorithm. Phenotypic profile of cells from control omentum by flow cytometric analysis.001).4⫾4. cells that are positive after subtraction of the isotype control are shown in the filled portions of the histograms. In addition.8 in Crohn’s disease).4 (n⫽3)]. a marker appearing during mat- Flow cytometry characterization uration of DC from monocytes.6 and 86. 548 Journal of Leukocyte Biology Volume 80. respectively). Expression of CD14 (Fig. Cells obtained from omental samples.4⫾6% in controls and 80.

Dendritic cells in adipose tissue 549 . granulo- cytes formed 20 ⫾ 8% of the cells (range 5– 45%). Cells migrating from omental fragments were double-labeled for CD45 and for the markers indicated. CD80. 3). The percentage positive and the positive/control intensity ratio denoting the level of staining are shown on each histogram. The plasmacytoid DC marker CD123 was not were CD83-. The most striking change was the almost-complete loss of MHC Class II in cells from Crohn’s patients. even in total cell populations (e.. 5. the remainder was not clearly identifiable or was macrophages (with many round vacuoles of different sizes. a and c). DC enriched using metrizamide gradients were studied in two control samples and two from Crohn’s patients. and multiple or- ganelles of different types present in a cytoplasm.g. and 73% expressed CD40. Fig. staining with many antibodies was approximately 90%. of which ⬎70% were identified as DC (Fig. The majority (⬎90%) of cells from the controls appeared myeloid cells. over 90% of the large cells MHC Class II. and 78%. and those negative remain unfilled. which was not up-regulated even after exposure of the cells to lipopolysaccharide for 2 days in culture. the latter cells appear to be developing adipocytes and may account for the presence of CD45-negative cells within the samples.g. heterochromatic nuclei with a rounded or horseshoe shape. Bedford et al. The labeling of omental cells from a control and a Crohn’s is shown. some containing obvi- ous lipid droplets. Gating on the population of larger cells was low for CD80. 3 and 4). suggesting that the cells were not fully increased the proportion of cells labeling for markers associ- mature. A significantly higher proportion of cells from Crohn’s omenta expressed CD11c. 3. Profiles of staining for different markers of the CD45⫹ cells from omentum. indicat- ing double-labeling of cells for DC lineage-associated markers and costimulatory marker expression. we sought to confirm the presence of DC using electron microscopy. Cells of three morpho- logical types of DC previously identified in DC separated from 4 Fig. we confirmed that there were only occasional lymphocytes and that large mononuclear cells formed the major cell population. In a typical example. but in samples from Crohn’s patients. granulocytes were not identified. respectively. The inten- sity of staining for CD40 and CD86 was also greater in cells from Crohn’s patients (e. and CD86-positive. CD11c-. suggesting an increase in putative myeloid DC. and this elevation was significant for CD40. In cells from some patients. The portion positive after subtraction of the isotype control is shown in the filled portions of the histograms.. CD80. As DC are difficult to identify securely using light microscopy. and CD86 in Fig. and CD1a. Morphological identification of DC In cytospins of omental walkout cells. and it was again clear that there was multiple labeling of cells using these antibodies. and CD86 were more highly expressed in cells from Crohn’s pa- tients. 59%. CD40. The profile of the CD45⫹ cells positively stained for different markers is shown. These plots are representative of the labeling obtained from 11 controls and nine Crohn’s patients.CD86 were expressed (Figs. The costimulatory molecules CD40. 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. 3). Approximately half the cells in controls expressed ated with DC. but the level of labeling expressed (not shown). Figures 3 and 4 also record differences between phenotypes of cells from control samples and those from Crohn’s disease patients. which often appeared darker than that of the DC). for CD11c. In control samples.

proliferative activity in allogeneic T cells. DC with to or higher than those found in the normal MLR using metri- a smoother cell boundary and fewer processes. double-staining of cells DISCUSSION was confirmed using fluorescently labeled antibodies to CD83. stimu- confirming the loss of MHC Class II observed by flow cytom. there was again a high prepon. omental cells stimulated higher levels of proliferation than derance of veiled cells clearly identified as DC by electron peripheral blood DC. and there was iment shown. as exemplified by costimulatory molecules (CD40. lation was still increasing with a dose of stimulator cells.jleukbio. identified shown in Figure 5. allogeneic T cell proliferation. Stimulation Langerhans cells and cells with longer processes. Small numbers of these sep- They included cells with electron dense nuclei. omentum. ogy were seen in the omentum in interstices between the The lack of primary T cell stimulation with cells from Crohn’s adipocytes. Large mononuclear ing by light and electron microscopy and function in stimulat- cells were separated from omental cells using a metrizamide ing. as exem. In frozen sections. Cells from all veiled cells in the afferent lymph. and each of the This paper identifies human adipose tissue. In the Crohn’s omentum. major repository of myeloid DC by plified for CD86 in Figure 5. (P⫽⬍0. The percentage-positive val- ues for cells from controls and Crohn’s patients were compared. three of five samples from Crohn’s nogold labeling (Fig. and immunostain- primary. as indicated in the representative exper- microscopy (65% and 80% in the two studied). The higher stimulation could light microscopy. lower counts when DC from blood were used. and many had high levels of patients induced no significant MLR (Fig. allogeneic omental DC at less electron-dense nuclear morphology. September 2006 http://www. and the other gold staining (Fig. which had a was observed when adding putative. 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. gradient designed to enrich DC. were also present. whereas the response had reached a plateau and was lower with dritic morphology was also confirmed by immunostaining in the higher dose of omental cells. Amongst mononuclear cells in the two stimulated only low levels of proliferation (P⫽⬍0. g and h. as a potential. showing that significant numbers of these cells migrated from human omental tissue fragments in culture with more than Allogeneic T cell stimulation fivefold more DC per mg tissue than are found per ml blood. possibly be a result of a greater maturity of the omental DC. and CD86).org . etry. DC The functional hallmark of DC is their potency in stimulating were identified from phenotype. 6a). there was staining for CD11c but not for MHC Class II. there were also scattered granulocytes vis- ible (not shown). similar to that of 1–2% of responding mononuclear cells (Fig. but also. An 550 Journal of Leukocyte Biology Volume 80. not only were there no evidence of staining of these cells for MHC Class II. which gave the highest level of labeling. some vacuoles. b and c). CD80.05). The counts were similar and numerous short pseudopods as shown in Figure 5. Numbers of CD45⫹ omental cells positive for different markers. The presence in omentum of mononuclear cells of den. By contrast. 4. morphology. from flow cytometry and electron microscopy. human peripheral blood were identified in each sample [16].001). primary MLR of myeloid cells labeled positively for MHC Class II by immu. In the experiment shown. The samples from Crohn’s patients. arated cells stimulated MLR (Fig. Over 70% five control omenta stimulated significant. 6b). resembling zamide-enriched blood DC as a stimulus (Fig.Fig. Clusters of cells of veiled/dendritic morphol. and significant differences are in- dicated. In the Crohn’s disease samples. 5. 5d). e and f. 6). and specific staining of these cells for CD11c is patients was consistent with loss in MHC Class II. 6c). primary.

Dendritic cells in adipose tissue 551 . (d) The gold grains per cell for the surface DR-labeling in the two experiments performed are shown. (c) The types of myeloid cells identified in the omental cells from two control patients. Cellular as well as bio. which we obtained from gut. the proportion positive for HLA-DR staining is shown in the solid portion of the histograms. Macrophage. In controls. with decreased MHC Class II and stimulatory function enteric lymph nodes are embedded in the human omentum. Morphology of omental DC. 5. (h) The same section as g with label specific for CD86 (FITC-labeled). which was absent from control slides. probably related to inflammation in described here and similar cells. We adapted this method to obtain integral to Crohn’s disease. (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. Cell nuclei. particularly in perinodal adipose tissue. (a) A typical DC from a control omentum is shown. There were few adipose tissue. and those negative are hatched. The presence of putative DC in enriched DC from human omental samples. (g) Frozen section of control omentum. M␾. indicates crease in innate activation of DC but loss of ability to stimulate that DC are indeed present in adipose tissue of the omentum. ⬎10 grains was considered positive. which is close to the spleen. as these cells floated and were removed. so increases during local stimulation with endotoxin. bore witness that Our identification of DC migrating from human adipose tissue immunological changes. although no higher expression of costimulatory molecules. mes- CD40. which adipocytes present. DC can be obtained as they mature and migrate out of skin chemical changes within omental adipose tissue may thus be or gut tissue fragments [15]. were distinguishable. An in. in cells from omentum in Crohn’s patients. showing that the veils were associated with mononuclear cells in the section. was sug. No evidence of DR-labeling was seen in either of the two Crohn’s patients studied. positive-peroxidase staining. The original bars indicate 1 ␮m. (b) An example of immunogold staining of the veils of a DC. increased proportion of putative myeloid (CD11c⫹) DC and gested in studies of adipose tissue from rats. showing two cells specifically labeled for CD83 (TRITC-labeled). adaptive immunity may be indicated. avoiding problems of having large quantities of fat present. (f) Control for e using serum instead of specific antibody and with only hematoxylin staining visible. gold labeling was always ⬍5 gold grains per cell.Fig. mouse omentum [24]. particularly characterization of these cells was undertaken [21–23]. which were stained with hematoxylin. occurred in cells within omental adipose tissue. Bedford et al.

Peripheral blood DC were enriched from 24 h nonadherent Ficoll-separated mononuclear cells on metrizamide gradients. The background proliferation of unstimulated responder cells (f) is shown in each graph. whereas the interest in future experiments to examine in more detail DC Crohn’s patients were evenly split between male and female. the preponderance of myeloid cells and the presence immune activity in the gut [27]. There is evidence of a been considered. The involve- from omentum in Crohn’s. were unexpected. However. as they were fluid rather than structured. dietary fats can be beneficial in its treatment [30]. Thus. taken to- presence of granulocytes in omentum in Crohn’s could indicate gether with the increased TNF-␣ production in adipose tissue that some omental stem cells are maturing down a granulocytic in Crohn’s. may contribute to production of autoimmune tribution is altered in Crohn’s disease and that manipulation of disease [26]. The that we obtained or their . The changes in putative DC in However. 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. although questions of cause and effect have close relationship between granulocytes and DC. Significant changes in cellular cells developing down an alternative maturation pathway. The control and omental DC each caused a significant stimulation (P⫽⬍0. A reduction in the capacity of of higher numbers of DC/mg tissue than there are DC/ml blood cells to express MHC and to initiate adaptive immune re- ruled out blood as the source of the majority of cells obtained. and Finally. but the Crohn’s omental DC caused little (P⫽0. from these other compartments. as well as DC changes in the gut [29]. Systemic changes in numbers and of lymphocytes also suggested that we were not obtaining functions of DC in Crohn’s disease have recently been reported immune cells from structured lymphoid tissue within the fat. gran. omental samples were from females. 6. lend weight to the idea of greater involvement of pathway.Fig. The components of adipose tissue in Crohn’s disease. [28]. and it will be of All control. MLR stimulated by omental DC. and some evidence suggests that fatty acid dis- tion of the former. We also saw no evidence that the patients with Crohn’s. There was some blood contamination within omental samples. although recruitment of additional cells from the adipose tissue in disease pathogenesis than has previously circulation is an alternative possibility. September 2006 http://www. Innate immune mechanisms to bacteria me- and some cells could possibly derive from this contamination. n⫽2) or no (n⫽6) significant stimulation. perhaps via effects on TLR [31]. could mean that these cells represent be a source of local APC. providing a link between the activity of treatment of some patients was significantly altering the cells cells in the colon in Crohn’s and those in the omentum. as indicated from the migrating from the omentum suggests that adipose tissue may CD34 labeling. which may ure of DC to mature and elicit appropriate adaptive immune have been “milky spots”. sponses could perhaps represent a mechanism indicating fail- Occasional omental samples showed pale areas. merely a consequence of the disease process has been postu- The information that control omentum contains stem cells. The elevated production of TNF-␣ in fining characteristic of DC and was well represented on a high the adipose tissue in Crohn’s thus provides a further link proportion of the cells from control omentum. One possible The lack of Class II and loss of T cell stimulatory function of mechanism of action of fatty acids is via modulation of mac- cells with the phenotype and morphology of DC in omentum rophages. CD40 levels on DC in the colon normalize after treatment with Expression of MHC Class II is generally considered a de. The lack an increased permeability. differentially. Omental DC were also enriched over metrizamide. The lower MHC between the aberrant activity in the colon and expression of Class II found on cells in Crohn’s could indicate a loss of DC costimulatory markers and the function of DC. yet to be elucidated. (a) The uptake in a normal MLR in this microculture system using peripheral blood DC. diated by PRR such as TLR on DC might then underlie the However. lated [14]. favoring produc. but these areas proved impossible to activity to deal with infecting organisms entering via a gut with dissect out. the similarity of the phe. which has female Crohn’s patients showed no significant differences in also been observed in DC isolated from the lamina propria of numbers of omental cells.05. ment of adipose tissue in the development of Crohn’s disease notype of cells from controls and Crohn’s patients argues that rather than the concept that the changed fat distribution is these are related to the DC and have lost or lack MHC Class II. anti-TNF antibody [29]. however.jleukbio. of these cells were added to different numbers of responder cells in 20 ␮l hanging drop cultures. ulocytes can be driven to acquire DC characteristics [25]. 1000 (䡬) or 2000 (‚). The presence of primary antigen-presenting DC including hematopoietic stem cells.001). (b) Stimulation with control omental DC and (c) effect of adding DC from Crohn’s omentum. analysis of the numbers of cells from male and Crohn’s include an increase in CD40 expression. Small numbers. Fatty acids may also 552 Journal of Leukocyte Biology Volume 80. different fatty acids can modulate immune functions an imbalance between granulocytes and DC.

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