Acquisition of Antigen-Presenting Functions by Neutrophils Isolated from Mice with Chronic Colitis

Abstract

Active episodes of the inflammatory bowel diseases (IBD) are associated with the infiltration of large numbers of myeloid cells including neutrophils, monocytes and macrophages. The objective of this study was to systematically characterize and define the different populations of myeloid cells generated in a mouse model of chronic gut inflammation. Using the T cell transfer model of chronic colitis, we found that induction of disease was associated with enhanced production of myelopoietic cytokines (IL-17, G-CSF), increased production of neutrophils and monocytes and infiltration of large numbers of myeloid cells into the MLNs and colon. Detailed characterization of these myeloid cells revealed three major populations including Mac-1⁺Ly6C^highGr-1^low/neg cells (monocytes), Mac-1⁺Ly6C^intGr-1⁺ cells (neutrophils) and Mac-1⁺Ly6C^low/negGr-1^low/neg leukocytes (macrophages, dendritic cells, and eosinophils). In addition, we observed enhanced surface expression of MHC class II and CD86 on neutrophils isolated from the inflamed colon when compared to neutrophils obtained from the blood, MLNs and spleen of colitic mice. Furthermore, we found that colonic neutrophils had acquired antigen-presenting cell (APC) function that enabled these granulocytes to induce proliferation of ovalbumin-specific CD4⁺ T cells in an antigen- and MHC class II-dependent manner. Finally, we observed a synergistic increase in pro-inflammatory cytokine and chemokine production following co-culture of T cells with neutrophils in vitro. Taken together, our data suggest that extravasated neutrophils acquire APC function within the inflamed bowel where they may perpetuate chronic gut inflammation by inducing T cell activation and proliferation as well as by enhancing production of pro-inflammatory mediators.

Introduction

Inflammatory bowel diseases (IBD; Crohn’s disease, ulcerative colitis) are multifactorial inflammatory disorders of the gastrointestinal tract that result from a complex interaction among genetic, environmental and immune factors. Active episodes of the disease are associated with the infiltration of large numbers of leukocytes including lymphocytes, granulocytes, monocytes and macrophages (1-3). This enhanced inflammatory infiltrate is thought to contribute to the intestinal injury and dysfunction resulting in abdominal pain, rectal bleeding and diarrhea. Utilizing mouse models of IBD we and others have demonstrated that in addition to expansion of colitogenic T-cells, induction of chronic intestinal inflammation is associated with a large and significant increase in the numbers of myeloid cells bearing markers CD11b (Mac-1) and Gr-1 in the bone marrow, circulation, mesenteric lymph nodes (MLN), spleen and colonic lamina propria (4-8). Although T cells undoubtedly play an important role in the induction and perpetuation of disease, the role of myeloid cells in disease pathogenesis has not been clearly defined.

The accumulation of myeloid cells such as neutrophils, monocytes and macrophages and others in the inflamed gut and their release of a variety of pro-inflammatory reactive oxygen and nitrogen species, cytokines, chemokines and proteases are thought to contribute to tissue injury and disruption of the epithelial barrier. Indeed, neutrophil-associated markers are highly expressed in chronic active IBD (9) and the transepithelial migration of neutrophils into the bowel lumen to form crypt abscesses is a characteristic feature of active disease (10,11). Despite these observations, no clear consensus has been reached from several different studies that have attempted to define the role of myeloid cells in the pathogenesis of intestinal inflammation. While some studies have suggested that neutrophils or other myeloid cells promote inflammatory tissue injury (12,13) other investigations were unable to confirm these findings (14,15). Contributing even further to the confusion, has been the observation that depletion of intestinal mononuclear phagocytes using clodronate-encapsulate liposomes worsened the colonic inflammation induced by dextran sulfate sodium (DSS). The enhanced inflammation and injury observed in this study was found to be associated with an increased neutrophil infiltration into the colon suggesting a pathogenic role for these cells (13). In contrast to these studies, recent reports have demonstrated that depletion of neutrophils actually exacerbates colonic inflammation and the systemic wasting syndrome in chronic (6,7) and acute colitis models (8), suggesting that neutrophils may function to suppress intestinal inflammation. The depletion of neutrophils in these studies utilized the in vivo administration of the anti-Gr-1 antibody (clone RB6-8C5) prior to or following induction of disease. Although this strategy has previously proven effective in depleting neutrophils in healthy mice (16), administration of this antibody to mice with inflammation is known to induce profound and lethal respiratory and cardiovascular complications that may have devastating systemic effects including death of the animal (17,18). Another confounding variable that makes interpretation difficult is the fact that anti-Gr-1 antibody recognizes both Ly6G and Ly6C and thus its administration would deplete not only neutrophils (bearing Ly6G on their surface) but also Ly6C-positive myeloid cells with potent immunosuppressive properties (19,20). A recent study that analyzed the effects of in vivo anti-Ly6G (clone 1A8) and anti-Gr-1 (RB6-8C5) antibody administration on blood neutrophils and monocytes confirmed that the latter significantly depleted Gr-1^intF4/80⁺ monocytes (21).

Because of these experimental limitations and lack of a clear consensus as to the role of neutrophils and other myeloid cells in the pathogenesis of chronic gut inflammation, we undertook a systematic analysis and characterization of myeloid cell generation, phenotype and function in a mouse model of chronic gut inflammation.

Materials and Methods

Animals

Wild type (WT) mice, recombination activating gene-1 deficient (RAG^−/−; B6.129S7-Rag1^tm1Mom/J) mice and ovalbumin (OVA)-specific B6.Cg-Tg (TcraTcrb)425Cbn/J (OTII) mice (6-8 weeks of age) all on the C57Bl/6 background were purchased from The Jackson Laboratory (Bar Harbor, Maine). Animals were maintained on 12h/12h light/dark cycles in standard animal cages with filter tops under specific pathogen free (SPF) conditions in our animal care facility at LSU Health Sciences Center in Shreveport (LSUHSC-S) and given standard laboratory rodent chow and water ad libitum. All experimental procedures involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committee of LSUHSC-S and performed according to the criteria outlined by the National Institute of Health.

Antibodies and reagents

Antibodies were purchased either from BD Pharmingen (San Diego, CA) or eBioscience (San Diego, CA). Negative selection kits for isolation of mouse CD4⁺ T cells were purchased either from Invitrogen (Carlsbad, CA) or Stemcell Technologies (Vancouver, BC, Canada).

Induction of chronic gut inflammation

Chronic colitis was induced by the transfer of sorted WT CD4⁺CD45RB^high T-cells into RAG^−/− mice using our previously published method (22). Briefly, spleens were removed from donor WT mice and teased into single-cell suspensions in 1xPBS/4% fetal bovine serum (FACS buffer). CD4⁺ cells were obtained using negative selection kits according to the manufacturer’s protocol. Enriched CD4⁺ T cells were labeled with anti-CD4 and anti-CD45RB mAb and sorted into CD45RB^high (40% brightest) and CD45RB^low (15% dimmest) fractions using FACSAria (Becton-Dickinson, San Jose, CA) and were found to be >98% pure on post-sort analysis. Male RAG^−/− mice were injected (i.p.) with 5×10⁵ CD45RB^high, CD45RB^low or both T-cell subsets resuspended in 500 μl of PBS. For some experiments we adoptively transferred flow-purified CD4⁺CD25⁻ T cells into RAG^−/− recipients to induce colitis (5×10⁵; i.p. injected). In general, colitis induced by transfer of CD4⁺CD25⁻ T cells was comparable to that induced by CD45RB^high T cells, although we did see a slight reduction in the incidence of moderate/severe disease in the CD4⁺CD25⁻→RAG^−/− mice compared to the CD45RB^high→RAG^−/− mice. Clinical evidence of disease in mice (e.g. body weight loss and loose stool/diarrhea) was followed and recorded weekly from the time of the injection.

Histopathology

Eight weeks following T cell transfer or when the animals lost 15-20% of their original body weights, mice were euthanized, colons excised, measured and weighed. Gross colitic scores were assigned according to previously published criteria (22). Representative pieces of proximal and distal colon were fixed in 10% phosphate buffered formalin, sectioned and stained with hematoxylin & eosin (H&E). Blinded histopathological analysis of each colon was performed by a pathologist unaware the identity of each sample using our previously published scoring criteria (22-24).

Quantification of circulating leukocytes, and serum cytokines

Blood was withdrawn from anesthetized mice at the time of sacrifice via the inferior vena cava using a 25G needle attached to a 1ml heparinized syringe. After 500μl of heparinized blood was mixed with an equal volume of 3% Dextran (>400,000 MW) in PBS, the erythrocytes were allowed to settle for 30min at room temperature (RT), and leukocyte-rich supernatant was removed and stored on ice. Remaining erythrocytes in the supernatant were lyzed, leukocytes pelleted by centrifugation, resuspended in PBS, and mixed with crystal violet dissolved in 3% acetic acid for counting using a hemocytometer. Total blood leukocytes were expressed as cells/ml blood. For differential cell counts, 50,000-100,000 circulating or tissue-derived/flow purified leukocytes were re-suspended in 100μL of 1X PBS and dispensed into the cytospin chamber loaded with Colorfrost Plus Microscope Slides (Fisher Scientific) and cardboard filter (Thermo Electron Corporation) and centrifuged (600 rpm, 2 minutes) using Shandon Cytospin3 centrifuge. Following this samples were air dried for 1-2 minutes and stained using a Diff Quick kit (Siemens Healthcare Diagnostics) according to the manufacturer’s instructions. The slides were mounted under coverslip using Permount mounting agent (Fisher Scientific). At least 200 total cells were counted from at least 4-5 different fields and those with mononuclear or polymorphonuclear morphology were quantified and expressed as a percentage of the total cells counted.

For serum cytokine determinations, blood was withdrawn from anesthetized mice and allowed to clot for 30-60 min at room temperature. The blood was centrifuged (12,000g for 10min) in BD Microtainer serum separator tubes, the serum removed and stored at −80°C. Serum GM-CSF and IL-17 were quantified using ELISA kits (eBiosciences) according to the manufacturer’s instructions.

Myeloperoxidase assay

Representative pieces of colon were rinsed with ice-cold PBS, blotted dry, and immediately frozen with liquid nitrogen. The samples were stored at −80°C until thawed for MPO activity determination using the O-dianisidine method, as previously described (25,26). Briefly, samples were thawed, weighed, suspended (10% w/v) in 50 mM potassium phosphate buffer (KP_i), pH 6.0, containing 0.5% hexadecyltrimethylammonium bromide buffer (0.1 g/20 ml KP_i), and homogenized using Brinkmann Homogenizer. A 1ml sample of the homogenate was sonicated using a Labsonic 2000 generator and a Braun-Sonic 2000U transducer with a 40T needle probe tip (B. Braun Biotech International, Allentown, PA) two times for 15 seconds (approximate average power output: 15 W with continuous operation). The sample was then centrifuged at 12000 x g for 10 minutes at 4°C and the supernatant saved on ice. The reaction was initiated by adding a small aliquot of supernatant (30 μl) to a pre-warmed (to 37°C) reaction mixture containing 50 mM potassium phosphate (pH 6.0), O-dianisidine dihydrochloride (200μg/ml), and hydrogen peroxide (200μM). The reaction mixture was incubated for 5 minutes at 37°C, stopped by adding sodium azide (0.02% final concentration) and absorbance at 460 nm determined using a spectrophotometer. The amount of MPO in the colon was expressed as the units of MPO/g tissue. One unit is defined as the amount of enzyme necessary to produce a change in absorbance of 1.0 per minute.

Tissue lymphocyte isolation and flow cytometry analyses

Lymphocytes were obtained from the spleen, mesenteric lymph nodes (MLN) and colonic lamina propria and analyzed by flow cytometry as previously described (23,24,27). Briefly, spleens were removed from reconstituted RAG^−/−, teased into a single cell suspension using frosted ends of two glass slides. Red blood cells (RBCs) were removed by hypotonic lysis and the resulting leukocytes were washed passed through 70-micron cell strainer to remove large chunks and then resuspended in FACS buffer. Cells from MLNs were obtained by grinding the tissue through 70-micron nylon cell strainer (Falcon) in FACS buffer and further purified over 44/70% Percoll gradient. Lamina propria lymphocytes (LPLs) were prepared by digestion of finely minced intestinal pieces remaining after IEL isolation with RPMI-1640/4% FBS/2.5mM CaCl₂ and containing 150 units of collagenase type VIII (Sigma) twice for 40min each time in a 37°C orbital shaker. Lymphocytes were further enriched by centrifugation over a 44/70% Percoll gradient. For flow cytometry staining approximately 1×10⁶ cells were placed in individual wells of a 96-well plate, incubated first with FcR block (CD16/CD32) and then stained with appropriate antibody cocktails. After the staining, cells were fixed for 20 min. on ice in freshly prepared 2% ultra pure formaldehyde (Polysciences, Inc., Warrington, PA) and analyzed the next day on the FACSCalibur or BD LSR II (BD Biosciences). Flow cytometry data were analyzed using FlowJo software for PC (TreeStar, Ashland, OR, ver. 7.2.5).

Granulocyte and myeloid cell isolation, T-cell proliferation assay and cytokine determinations

A single cell suspension was prepared from 3-4 pooled spleens and colonic LP cells and stained with anti-Mac-1 (CD11b), −Ly6C, and Gr-1 (Ly6G/C) Ab cocktail followed by sorting of Mac-1⁺ cells into Ly6C^intGr-1⁺ using FACSAria (BD). To obtain a highly enriched population of OTII CD4⁺ T cells, spleens from OTII mice were prepared as described above, stained with phycoerythrin (PE)-labeled antibodies to CD11c, Mac-1, CD8α and B220 and allophycocyanin (APC)-tagged anti-CD4 antibodies and sorted into CD4⁺[CD11c/Mac-1/CD8α/B220]^NEGATIVE. 50,000 OTII CD4⁺ T cells were co-cultured with the indicated number of sorted Neutrophils in the presence of 10μg/ml OVA peptide (323-339, AnaSpec Inc., San Jose, CA). Where indicated anti-CD1d (clone 1B1) or anti-MHC-II (M5/114.15.2) antibodies (1μg/ml final) were added according to previously published protocol (28). For fixation, sorted myeloid cells were fixed in culture medium containing 1% paraformaldehyde for 10 min. at 37°C followed by washing and additional 2×10minutes incubations in 120mM lysine as described by Brimnes et. al. (29). At the end of incubations, cells were washed additional 3 times in medium prior to culturing. Cells were allowed to proliferate for 72 hrs with the addition of 1μCi of ³H-thymidine for the last 8-18 hrs of culturing. Cells lysates were harvested onto fiberglass filters using semi-automated cell harvester (Brandel, Inc, Gaithersburg, MD) and radioactivity was quantified using a liquid scintillation counter (Wallac 1409).

For cytokine analysis negatively selected CD4⁺ T cells (50,000) isolated from spleens of WT mice were co-cultured in triplicate with sorted Neutrophils in a 96-well flat-bottom plates in the presence of plate-bound anti-CD3 and soluble CD28 (1μg/ml final) antibodies. At 48 hrs following the addition of the antibodies, supernatants from replicate wells were pooled, aliquoted and frozen at −80°C. Samples from two independent experiments were included for subsequent cytokine analysis. Multiplex cytokine analysis was carried out using Milliplex Mouse Cytokine/Chemokine Immunoassay (Millipore, Billerica, MA) according to the manufacturer’s instructions. Briefly, samples were thawed on ice and diluted using recommended buffer. Undiluted as well as 1:2 and 1:4 diluted samples were used for cytokine measurements and only those values that fell within the range of the standard curve were used in calculations. Samples were ran on Bio-Plex Luminex instrument (Bio-Rad laboratories, Hercules, CA). Data were analyzed using Miliplex Analyzer 3.1 xPONENT software (Millipore). Samples from repeat experiments were analyzed on the same run.

Neutrophils from spleens and colons of colitic mice were obtained by cell sorting. Ly6G⁺ cells from the bone marrow (BM) were obtained by positive selection using Ly6G-PE antibody and EasySep PE Positive Selection Kit (StemCell Technologies). Cells from peritoneal exudate were obtained 4-6 hrs after injection of 25mg of oyster glycogen type II re-suspended in 0.5ml of 1x PBS by peritoneal lavage. Ly6G⁺ cells from peritoneal exudate were isolated by positive selection. Cytospin was performed as described above.

RNA isolation and reverse transcription-PCR

Neutrophils from colitic spleens and colon LP were obtained by cell sorting. To obtain Ly6G⁺ cells from bone marrow, tibias from both legs of normal WT mice were flushed, and cells were stained with Ly6G-PE antibody at saturating concentrations, which was previously determined by titration. PE-expressing cells were isolated using EasySep® PE Positive Selection Kit (Stemcell Technologies, Vancouver, BC) according to the manufacturer’s instructions and were found to be >95% pure on post-analysis. Following positive selection, cells were processed for RNA isolation and reverse transcription PCR. Total RNA from cells was isolated using RNeasy Mini kit (Qiagen, Valencia, CA) according to the manufacturer’s instruction and treated with DNase I (Qiagen, Valencia, CA). RNA concentration and purity was assessed with NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Inc.) and 50ng was reversely transcribed with qScriptTM cDNA SuperMix (Quanta Biosciences, Inc.) according to the manufacturer’s protocol. PCR reactions were carried out in a 25 μL volume using 2720 Thermo Cycler (Applied Biosystems, Inc.) using primers specific for β2-microglobulin and histocompatibility class II antigen A, alpha chain (I-Aα). Primer pair sequences for I-Aα were obtained from Leibundgut-Landmann et. al. (30), for β2-microglobulin from RTPrimerDB database ((31) RTPrimerDB ID: 3584). Amplicons of expected length were resolved on a 1.5% agarose gel. For densitometry measurements, imageJ software (ver. 1.44p) (32) was used and the intensity of I-Aα band for each sample was normalized to that of β2-microglobulin.

Statistics

Data are presented as mean ± SEM (standard error of mean). Statistical significance between two groups was determined using two-sided Student’s t-test. Statistical significance between more than 2 groups was evaluated using a one way analysis of variance (ANOVA). Statistical significance between selected groups was evaluated using Dunnett post-hoc test. A probability (p value) of p < 0.05 was considered significant.

Results

Chronic colitis is associated with increased myelopoiesis and elevations of serum myelopoietic cytokines

One of the hallmark features of chronic colitis induced in immunodeficient RAG^−/− mice injected with naïve (CD4⁺CD45RB^high) T cells is the large and significant accumulation of myeloid cells (neutrophils, monocytes, and macrophages) within the colonic LP when compared to non-colitic RAG^−/− mice injected with the CD45RB^low cells (CD45RB^low→RAG^−/−) (Supplementary Fig 1). In addition, this increased myeloid cell accumulation corresponds well with high levels of myeloperoxidase (MPO) activity in the colonic LP of colitic CD45RB^high→RAG^−/− mice compared to mice with little or no colitis (CD45RB^low→RAG^−/−) demonstrating the presence of neutrophils, monocytes and possibly eosinophils (Supplementary Fig 1D). Development of chronic colitis (Fig. 1A) is accompanied by a 5-6-fold increase in circulating leukocytes compared to unmanipulated RAG^−/− mice (Fig. 1B). Particularly striking was that the number of circulating neutrophils and PMN-ring cells increased approximately 10-fold in colitic mice when compared to unmanipulated RAG^−/− mice and 5-fold compared to WT mice (Fig 1C). Morphologic analysis of peripheral blood obtained from colitic mice showed that the vast majority of circulating leukocytes had a multi-lobed or donut-shaped nucleus, which are morphological features of mouse granulocytes such as neutrophils and PMN-“ring cells” (Fig 1E) (33,34). PMN-ring cells represent newly released and not fully matured “band” neutrophils, whereas maturation and segmentation of their nucleus leads to the appearance of “segmented” neutrophils (35). Both types of neutrophils constituted >95% of the total circulating leukocytes in colitic mice (Fig 1C and E).

Figure 1.

Development of chronic colitis is accompanied by enhanced myelopoiesis and increase in peripheral granulocyte numbers. A. Blinded histopathological scores of colons obtained from CD45RB^high→RAG^−/−, CD45RB^low→RAG^−/− and CD45RB^high+CD45RB^low→RAG^−/− mice for at least 11 animals per group from at least 2 independently conducted experiments. B. Total leukocyte numbers in peripheral blood of mice from the different groups. Blood leukocyte counts in mice were determined as described in the Materials and Methods section. C. Total numbers of PMNs/PMN-ring cells in peripheral blood in mice. Representative images of peripheral blood cytospins from wild-type (WT) (D) and colitic CD45RB^high→RAG^−/− mice (E) showing dramatic increases in peripheral blood granulocytes. Serum from 7-11 individual mice per group was analyzed for G-CSF (F) and IL-17 (G). Data in all graphs shown represent the mean±SEM. Significance between groups is indicated by asterisks: * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001.

Among the different cytokines responsible for stimulating myelopoiesis, we observed large and significant increases in serum granulocyte-colony stimulating factor (G-CSF) levels in colitic CD45RB^high→RAG^−/− mice when compared to either of the non-colitic CD45RB^low→RAG^−/− or CD45RB^high+CD45RB^low→RAG^−/− groups (Fig 1F). Serum level of interleukin-17 (IL-17) was significantly increased in colitic mice compared to the CD45RB^low→RAG^−/− group (Fig 1G), but was not significantly different from that in mice with attenuated disease, i.e. in CD45RB^high+ CD45RB^low→RAG^−/− group (Fig 1F and G).

Phenotypic analysis of leukocytes derived from the colon, MLNs and spleen of mice with chronic colitis

In order to more specifically define what different populations of leukocytes infiltrate animals with chronic disease, we analyzed leukocyte populations associated with colon, MLNs and spleen. Quantitatively, the numbers of CD4⁺ cells, Mac-1⁺Gr-1⁺ and Mac-1⁺Gr-1⁻ cells in the colons were significantly higher in the colitic group compared to the non-colitic CD45RB^low→RAG^−/− and CD45RB^high+CD45RB^low→RAG^−/− mice (Fig. 2A). We found that CD4⁺ T cells constituted a substantial portion (28%, range: from 4 to 43%) of the total cells obtained from the inflamed colon. In addition, we observed significant numbers of granulocytes expressing both Mac-1 and Gr-1 (Mac-1⁺Gr-1⁺) as well as those that expressed Mac-1, but not Gr-1 (Mac-1⁺Gr-1⁻). Both populations combined represented approximately 29% of total colonic LP cells (range: 15-42%) (Fig. 2B). We also observed large numbers of CD4⁺ T-cells within the spleen and MLNs in the colitic CD45RB^high→RAG^−/− mice. In addition to T cells, Mac-1-expressing cells were elevated in the MLNs and spleens in mice with active disease. We observed large and significant increases in the absolute numbers of Mac-1⁺Gr-1⁺ and Mac-1⁺Gr-1⁻ cells in the MLNs obtained from colitic when compared to their non-colitic controls (Fig. 2B). Although the numbers of Mac-1⁺Gr-1⁺ granulocytes were significantly increased in spleens from colitic mice compared to their non-colitic controls, the numbers of Mac-1⁺Gr-1⁻ cells were similar among all three groups.

Figure 2.

Enhanced accumulation of myeloid cells in mice with chronic colitis. A. Absolute numbers of CD4+ T cells, Mac-1⁺Gr-1⁺ and Mac-1⁺Gr-1⁻ were quantified in CD45RB^high→RAG^−/− (black bars), CD45RB^low→RAG^−/− (white bars) and CD45RB^high + CD45RB^low→RAG^−/− mice (hatched bars). At least 5 animals were analyzed per group and experiments were repeated at least twice. Data are expressed as mean±SEM. * indicates p<0.05, ** indicates p<0.01 and *** indicates p<0.001 compared to the RB^high control group. B. Representative dot plots showing percentages of Mac-1⁺Gr-1⁺ and Mac-1⁺Gr-1⁻ cells in the spleens, MLNs and colon LP. Numbers indicate percentages of cells within the gated leukocyte population (using forward and side scatter).

Identification of the different populations of murine myeloid cells by flow cytometry has been limited by the lack of specific markers. Mouse neutrophils and monocytes are commonly identified by flow cytometry using side scatter properties (granularity) and expression of Mac-1 and Gr-1 markers (36). Anti-Gr-1 antibody (clone RB6-8C5) recognizes both Ly6G, which is expressed primarily on mouse Neutrophils (17), and Ly6C, which is expressed on a variety of cells, including T cells and monocytes (37,38). Thus, Mac-1⁺Gr-1⁺ population can be further subdivided using anti-Gr-1 and anti-Ly6C antibodies (20). Therefore, we next characterized the tissue-associated myeloid cells and identified three major populations based on the expression of Mac-1 (CD11b), Gr-1(Ly6C/G) and Ly6C: Mac-1⁺Ly6C^highGr-1^low/neg (hereafter referred to as Ly6C^high), Mac-1⁺Ly6C^intGr-1⁺ (Gr-1⁺) and Mac-1⁺Ly6C^low/negGr-1^low/neg (double-negative, DN) (Fig 3A). Gr-1⁺ population contains mature and immature neutrophils and the Ly6C^high population contains recently recruited immature monocytes (20,39) that lose Ly6C expression following their maturation into tissue macrophages or DCs (40-43). The DN population consists of a heterogeneous mixture of mononuclear and granulocytic cells.

Figure 3.

Increased expression of MHC class II and CD86 on PMNs isolated from inflamed colons. A. Dot plot analysis of cells isolated from spleen, MLNs and colon LP showing expression of Ly6C vs. Gr-1. Representative dot plots were initially gated on Mac-1⁺ (CD11b) cells. B. Representative histograms showing expression of indicated markers (black line) on Gr-1^negLy6C^neg (DN), Gr-1⁺ PMNs, and Ly6C^high populations. C. Median fluorescent intensity (MFI) values show progressive increase in MHC-II and CD86 expression on PMNs and Ly6C^high cells isolated from colonic LP compared to those isolated from the spleen and MLN. ** indicates p<0.01 and *** indicates p<0.001. D. Expression of Ly6C and Gr-1 in peritoneal exudates cells. Dot plots in were initially gated on Mac-1⁺ cells. Exudate PMNs (ex. PMNs), Ly6C^high and DN populations are shown by indicated gates. E Histograms are gated on the corresponding populations identified in (D). For all graphs, staining with antibodies is indicated by black line whereas staining with isotype control antibody is indicated by shaded curves. F. PMNs isolated from colitic mice show increased expression of MHC-II mRNA compared to PMNs obtained from the bone marrow (BM) or peritoneal exudates (ex). RT-PCR of histocompatibility class II antigen A α chain and corresponding densitometry measurements. Gr-1⁺ PMNs from spleens and colons of colitic mice were obtains by cell sorting. Ly6G⁺ cells from the bone marrow (BM) or peritoneal exudates (ex) were obtained by positive selection. Ly6G negative fractions from BM (BM Ly6G-) and exudate (ex. Ly6G-) served as MHC-II positive control

Ly6C^high cells were identified in all tissues tested, however, they expressed varying levels of Gr-1: those isolated from the spleen had intermediate expression of Gr-1, while those from the colon LP were Gr-1^low. In addition, all Ly6C^high cells were SSC^low and had morphology of monocytes (data not shown). Lastly, these cells did not express macrophage marker F4/80 and dendritic cells marker CD11c (Fig 3B). Interestingly, MHC class II expression was very high on Ly6C^high cells isolated from MLNs and colons, but was low on cells isolated from spleens.

When we analyzed the Gr-1⁺ populations in all three tissues, we found that these cells were granular (SSC^high), negative for both F4/80 and CD11c (Fig. 3B) and had the morphology characteristics of PMNs and PMN-ring cells (supplementary figure 2A). We observed many fewer cells expressing a mature neutrophil morphology with a segmented nucleus within the Gr-1⁺ population isolated from spleen when compared to MLNs and colonic LP. Furthermore approximately 75% of these cells displayed a ring-shaped morphology characteristic of immature “band” cells. In contrast, Gr-1⁺ cells isolated from the colonic LP were mostly mature “segmented” neutrophils (supplementary figure 2B).

Analysis of the DN cells in the spleen revealed a heterogeneous population of cells: CD11c^highF4/80⁺, CD11c^intF4/80^high, CD11c^negF4/80^low and CD11c^negF4/80^neg (supplementary Fig. 3). All cells within the CD11c^highF4/80⁺ population were SSC^low and MHC-II^high, which is consistent with the profile for DCs. Cells bearing CD11c^lowF4/80^high phenotype were also SSC^low and MHC-II^high, which is consistent with a phenotype of macrophages. On the other hand, CD11c^negF4/80^low cells contained large granular cells based on their forward and side scatter profiles with intermediate expression of MHC-II. When DN cells were sorted and differentially stained (using Diff-Quick kit), some of these cells showed the cytoplasmic morphology and staining characteristics (red/pink granules) of eosinophils (supplementary figure 2A). Indeed, low level expression of F4/80 in combination with Mac-1 expression is characteristic of eosinophils (44). Splenic CD11c^negF4/80^neg cells were SSC^low with intermediate expression of MHC-II. These cells had morphology typical of a monocyte, with bean-shaped nucleus, or a monocytering cell with donut-shaped nucleus (supplementary figure 2A) (34). In addition, all of these cells were SSC^low (supplementary Fig 3). DN population in the MLN or colonic LP was more homogeneous than that in the spleen. In the MLN, this population contained mainly CD11c^negF4/80^low cells. Side scatter and MHC-II expression revealed two populations that were SSC^highMHC-II^low (possibly eosinophils) and SSC^lowMHC-II^high, which appears to be monocytes and macrophages. In the colonic LP the same two populations were found within the DN gate: eosinophils (SSC^highMHC-II^low) based on the cytospin analysis and macrophage-like (SSC^lowMHC-II^high) cells that, interestingly, did not express a classical marker of mature macrophages F4/80 (45). However, it is known that under certain inflammatory conditions F4/80 can be downregulated by mature macrophages especially in the T cell-rich areas (46). Since Arnold et.al. demonstrated that Ly6C, which is expressed at high levels on circulating monocytes is downregulated upon their maturation into tissue macrophages (43), the SSC^lowMHC-II^high population most likely represents either mature macrophages or Ly6C^low-neg monocytes that can be found in the injured tissues (47).

Gr-1⁺ neutrophils obtained from the inflamed colon express enhanced levels of MHC-II and CD86

Neutrophils isolated from patients with chronic inflammatory conditions have been documented to undergo “transdifferentiation” into DC-like cells, that is: they acquire the ability to act as antigen-presenting cells (APCs) and induce T cell activation (48-50). In addition, culturing human or mouse neutrophils with pro-inflammatory cytokines induces expression of MHC-II and co-stimulatory molecules (51-54) and delays apoptosis (55-57). Collectively, these reports suggest that during chronic inflammation neutrophils undergo phenotypic and functional changes driven, in part, by the inflammatory cytokines released by T cells. To assess whether neutrophils obtained from tissues of colitic mice assume APC-like properties, we analyzed surface expression of MHC-II and CD86 on these cells. We found that Gr-1⁺ neutrophils isolated from spleen, MLNs and colonic LP expressed low, moderate and high levels of MHC-II, respectively (Fig 5B). Since we established that Gr-1⁺ cells were negative for CD11c and F4/80, MHC-II expression could not be due to contaminating DCs or macrophages. We also observed a modest upregulation of CD86 with the highest levels seen on cells isolated from the inflamed colonic LP (Fig. 3B). Comparison of median fluorescence intensity (MFI) between neutrophils isolated from different tissues showed that colonic LP cells had significantly elevated expression of MHC-II and CD86 compared to those isolated from spleen (Fig 3C) suggesting that cells that infiltrated colons acquired phenotype of APC-like cells. We also analyzed expression of MHC-II and CD86 on DN and Ly6C^high subsets of myeloid cells from the different tissues and observed enhanced surface expression of MHC-II on these cells (Fig 3C). In general, pattern of CD86 expression paralleled that of MHC-II for both the DN and Ly6C^high populations. Next we wanted to determine whether MHC-II upregulation can be observed on neutrophils recruited in response to an acute inflammatory stimulus. To do this, we analyzed peritoneal exudate cells 4-6 hrs following the injection of oyster glycogen (Fig. 3D) (58). We found that these neutrophils had little surface expression of MHC-II or CD86 (Fig. 3E)

Figure 5.

PMNs synergize with T cells to enhance in cytokine production without an affect on proliferation. A-P and R. Co-culturing of WT CD4⁺ T cells with PMNs isolated from colonic LP of colitic mice results in increased production of cytokines and chemokines WT CD4⁺ T cells were cultured along (cross-hatched bars) or with the indicated number (shown on x-axis) of FACS-sorted PMNs (grey bars) in the presence of activating anti-CD3/CD28 antibodies for 48 hrs. Cytokine concentrations were measured using multiplex cytokine analysis. Data are expressed as the mean±SEM of serial dilutions (undiluted, 1:2 and 1:4) of each sample. Shown are results from a representative experiment of two independently conducted co-culture experiments with similar results. Proliferation of WT T cells in response to anti-CD3/CD28 antibodies in the absence or presence of PMNs (Q) was not substantially increased with the addition of PMNs.

Whale et. al. reported that neutrophils may passively acquire parts of plasma membranes from apoptotic or necrotic cells during phagocytosis (59). Thus, high expression of MHC-II may be due to trogocytosis by neutrophils of this molecule from the surface of neighboring APCs. To evaluate if Gr-1⁺ neutrophils actively transcribe MHC-II gene, we analyzed messenger RNA level of MHC class II in sorted neutrophils isolated from spleen and colon. We saw high levels of MHC-II antigen A α chain mRNA in neutrophils obtained from cLP (Fig 3F). In comparison, Ly6G⁺ neutrophils isolated from the bone marrow showed no MHC-II mRNA. Interestingly, Ly6G⁺ peritoneal exudates neutrophils showed some expression, but it was much less than Ly6G-negative cells, which consist of a mixed population of various hematopoietic cells. These data suggest that recruitment of circulating neutrophils into the site of inflammation may provide transcriptional signals for the upregulation of MHC-II mRNA expression. These results confirmed that surface MHC-II expression in neutrophils isolated from mice with colitis is not due to trogocytosis, but due to the transcription of mhc-II genes.

Neutrophils Induce T-Cell activation through an antigen-specific and MHC-II-dependent interaction

In order to determine whether neutrophils isolated from colitic mice could induce proliferation of T cells, we co-cultured these cells with flow-purified ovalbumin (OVA)-specific CD4⁺ T cells (OTII CD4⁺ cells) in the presence of OVA peptide. In our preliminary experiments we found that positively or negatively selected splenic OTII CD4⁺ cells extensively proliferated in the presence of OVA peptide without addition of accessory cells (data not shown), suggesting that these population contained APCs. Thus, to remove CD4⁺ DCs present in the spleen (60) as well as other contaminating APCs, we sorted splenic OTII cells into CD4⁺[CD11c/Mac-1/CD8α/B220]^neg cells. Only by sorting we were able to obtain a pure population of CD4⁺ T cells to use in our co-culture experiments that did not proliferate in the presence of peptide without the addition of accessory cells (Fig 4A). We found that neutrophils obtained from mice with active colitis in the presence of OVA peptide induced OTII T-cell proliferation in a cell number-dependent manner (Fig. 4A). Interestingly, at a 1:1 ratio of neutrophils to T cells those isolated from colon induced a 2-fold higher proliferation than those isolated from spleen, which correlated with their higher surface expression of MHC-II. Omission of antigen-presenting cells or addition of MHC-II blocking antibody completely inhibited antigen-induced proliferation of T cells (Fig 4B). The importance of antigen processing and presentation by cLP neutrophils was confirmed by formalin-fixation of these cells, which abolished their ability to trigger proliferation (Fig 4B). Taken together these experiments suggest that antigen-specific proliferation of T cells by neutrophils isolated from colitic mice is dependent upon their ability to internalize antigen and present it on their surface and is not due to non-specific binding of antigen to the surface of these cells.

Figure 4.

PMNs isolated from colitic mice induce proliferation of CD4⁺ T cells in an antigen-specific, MHC-II dependent manner. A. PMNs isolated from colitic mice can function as APC and trigger proliferation of antigen-specific T cells. FACS-sorted OTII CD4⁺ T cells were co-cultured with increasing numbers of sorted PMNs from spleens (white bars) or colonic LP (black bars) of colitic mice or WT iAccessory cells (hatched bars) in the presence of OVA peptide (10μg/ml). * indicates p<0.05, ** indicates p<0.01 compared to WT iAccessory cells. B. Antigen-specific T cell proliferation is abolished with the addition of MHC-II blocking antibody, but not in the presence of control anti-CD1d antibody. Fixation of PMNs or WT iAccessory cells also abolishes their ability to stimulate proliferation of T cells. Data are expressed as the mean counts per minute (cpm)±SEM from a representative experiment which was repeated 3-5 times.

Co-culture of Gr-1⁺ neutrophils with activated CD4⁺ T cells enhances the production of pro-inflammatory cytokines

We next wished to ascertain whether neutrophils were capable of modulating the production of cytokines by T cells. We found that addition of increasing numbers of neutrophils isolated from the colonic LP to activated WT CD4⁺ T-cells resulted in a cell number-dependent increases in the levels of pro-inflammatory mediators including IFN-γ, IL-17, TNF-α and IL-1β (Fig 5A-P). Increased production of cytokines that are thought to be important for myelopoiesis (e.g. IL-17, GM-CSF, G-CSF and IL-6) was also observed with the addition of more neutrophils. We also observed a modest increase in production of Th2 cytokines such as IL-4 and IL-5 when ratios of neutrophils:T cells approached 1:1. Concentrations of several chemokines, such as interferon gamma-induced protein 10 kDa (IP-10 or CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2) and regulated upon activation, normal T-cell expressed and secreted (RANTES or CCL5) and macrophage inflammatory protein (MIP-1α) also increased in a cell-dependent manner. It should be noted that there were no significant increase in T cell proliferation and even a slight decrease in IL-2 concentrations at a 1:1 ratio of neutrophils:T cells (Fig 5Q and R) suggesting that higher levels of cytokines in culture media cannot be due to the presence of more T cells. Slightly lower IL-2 levels with the addition of more neutrophils could be indicative of the inhibition of T cell proliferation despite the lack of any apparent effects on T cell proliferation as measured by the incorporation of ³H-thymidine (Fig 5R).

Expression of cell adhesion molecules on neutrophils obtained from tissues of colitic mice

The large majority of studies investigating the molecular determinants involved in leukocyte trafficking in chronic gut inflammation have focused on T and/or B cell-associated adhesion molecules. Given our data demonstrating that the inflamed MLNs and colon contain large numbers of neutrophils we wished to analyze surface expression of the major cell adhesion molecules on these cells found in blood and the different tissues in colitic mice. We found that expression of the α4 integrin was dramatically increased on neutrophils isolated from spleen and MLNs (Fig 6). Because we did not observe any detectable expression of α4β7 (LPAM-1) or β7 integrin, we concluded that expression of α4 on neutrophils from spleen and MLNs was largely due to the expression of α4β1. We did find that CD29 (β1 integrin) was highly expressed on circulating and tissue neutrophils obtained from mice with active colitis. Interestingly, the highest level of CD29 expression was seen on blood neutrophils where α4 expression was barely detectable. Since the β1 chain can pair with 7 different α subunits (α1-6 and αV) to form VLA(1-6) and αVβ1 integrins, respectively (61), our data would imply that β1 integrins other than VLA-4 may be playing a role in recruitment of neutrophils from the blood. Not surprising, we found that the β2 integrins CD11a (Fig 6) and CD11b (not shown, but was used for gating of CD11b+ cells in histograms shown in figure 6) were expressed on neutrophils isolated from blood, spleen, MLN and colonic LP. The expression of the α_E integrin (CD103) on neutrophils was relatively low which suggested that αEβ7 expression is minimal. In addition to the different integrins, we found that neutrophils obtained from the blood, spleen and MLNs, but not colonic LP were L-selectin-positive (Fig. 6). We also observed significant expression of ICAM-1 on neutrophils obtained from MLNs and colonic LP, whereas its expression on circulating and splenic neutrophils was somewhat reduced (Fig 6). ICAM-1 expression by APCs is critical for stabilization of immunological synapse and efficient priming of T cells (62). Thus, enhanced ICAM-1 expression by MLN and cLP neutrophils provides additional evidence for their acquisition of antigen-presenting capacity.

Figure 6.

Surface expression of different adhesion molecules on PMNs isolated from colitic mice. Representative histograms depicting expression of the indicated surface molecules (black line) were gated on PMNs (Mac-1⁺Ly6C^intSSC^highLy6G⁺) isolated from blood, spleen, MLNs or colonic LP. Staining with fluorescently-tagged isotype control antibody is depicted by shaded histogram plots.

Discussion

Histopathologic inspection of intestinal biopsies obtained from patients with active Crohn’s disease or ulcerative colitis reveal the presence of large numbers of myeloid cells such as neutrophils, monocytes, eosinophils and macrophages (1-3). In addition, several studies using clinical specimens obtained from patients with active IBD have demonstrated increased levels of intestinal IL-8, G-CSF and GM-CSF (63-65) that corresponded to a delay in neutrophil apoptosis (63). Because myeloid cells are capable of generating large amounts of pro-inflammatory mediators and reactive oxygen and nitrogen metabolites, investigators have proposed that these cells may play an important role in the inflammatory tissue injury observed in IBD (66-70). Indeed, transepithelial migration of neutrophils from the gut to the lumen to form crypt abscesses correlates with the worsening of disease (10,11). Unfortunately, no clear consensus has been reached from a variety of different experimental and clinical studies attempting to define the importance of myeloid cells in the pathogenesis of IBD. As a first step toward understanding the role that myeloid cells may play in the induction, perpetuation and/or resolution of chronic intestinal inflammation, we undertook a systematic analysis and detailed characterization of myeloid cell generation, phenotype and function in a mouse model of chronic gut inflammation.

Data obtained in the current study demonstrate that chronic gut inflammation is associated with enhanced plasma levels of granulo/myelopoietic cytokines, increased production of granulocytes and myeloid cells and infiltration of large numbers of neutrophils into the MLNs and colon. Our data confirm those of Monteiro et. al. and Nemoto and co-workers who demonstrated a robust myelopoietic response induced by adoptive transfer of CD4⁺ T cells into lymphopenic mice (7,71). In addition, the current study extends their findings by identifying, for the first time, three major populations of myeloid cells that infiltrate colon, MLNs and spleen of mice with chronic colitis. Our detailed analyses identified Mac-1⁺Ly6C^highGr-1^low/neg cells as monocytes, Mac-1⁺Ly6C^intGr-1⁺ cells as mature and immature neutrophils and Mac-1⁺Ly6C^low/negGr-1^low/neg (DN) as a heterogeneous mixture of DCs, macrophage-like cells and eosinophils. In all three tissues analyzed, we found that myeloid cells comprised a significant portion of infiltrating leukocytes in colitic mice. In the colon the combined number of Mac-1⁺Gr-1⁺ (which were mostly neutrophils) and Mac-1⁺Gr-1⁻ cells (which contained monocytes and DN cells) were approximately the same as the number of CD4⁺ T cells (Fig 2). In the MLNs, myeloid cells slightly outnumbered T cells and in the spleen we recovered approximately 10-fold more myeloid cells (primarily neutrophils) than T cells.

Based on cytospin and differential morphological analysis, neutrophils isolated from the colon were mostly “segmented” or mature, while a substantial number of those in the spleen had a donut-shaped nucleus characteristic of immature or “band” neutrophils (supplementary figure 3). Interestingly, analysis of the peripheral blood obtained from colitic mice showed that majority of circulating neutrophils had mature, segmented morphology (Fig 1E). This suggests that myelopoiesis observed during chronic inflammation, in addition to stimulating release of mature myeloid cells, also promotes egress of hematopoietic progenitors from the bone marrow with their subsequent accumulation in the spleen. Recently Swirski and colleagues showed that during ischemic myocardial injury the spleen can serve as a reservoir for undifferentiated monocytes that are capable of rapidly migrating to the injured site where they participate in wound healing (72). Thus, in the context of chronic inflammation, the spleen may function as a “depot” for accumulation and storage of myeloid cells and their precursors, presumably, for their subsequent utilization to fight infection or, as has been proposed for monocytes (72), in wound healing. Although we (73) and others (74) previously demonstrated in this T cell transfer model lack of spleen in recipient immunodeficient mice had no affect on the induction of colitis, in view of our new data it may be worthwhile to examine how splenectomy in mice with active colitis affects disease progression.

Another novel finding obtained in the current study is that neutrophils isolated from the chronically inflamed colon have acquired APC phenotype and function based upon their enhanced expression of MHC-II and CD86 on mRNA and protein levels as well as their ability to induce T cell activation in an antigen- and MHC-II-dependent manner. Although it can be argued that a small number of contaminating DCs and/or monocytes/macrophages could account for the APC phenotype and function, our in-depth multi-color flow cytometric analyses confirmed that these cells did not contain CD11c⁺ (DCs) or F4/80⁺ (macrophages) cells (Fig 3B). We also show that induction of T cell proliferation is contingent upon MHC-II expression since blocking antibody completely abrogated proliferation of T cells. Lastly, we demonstrate that proliferation of T cells does not occur when antigen binds non-specifically to the surface of fixed cells, but is dependent upon the ability of live neutrophils to internalize, process and present antigen on their surface in association with MHC-II. These data are in agreement with several other reports demonstrating that neutrophils isolated from patients with chronic inflammatory conditions may undergo “transdifferentiation” into APCs and acquire the ability to induce T-cell activation in vitro (48-50). Liu et.al. reported that peripheral monocytes isolated from CD patients showed increased expression of co-stimulatory molecules (75). “Transdifferentiation” refers to a process whereby a non-stem cell with a specific function transforms into a different type of cell and has been used frequently to describe acquisition of antigen-presenting characteristics by neutrophils (49,50). This term, however, may not accurately reflect this phenomenon, because these cells retain morphology of neutrophils.

The specific mechanisms by which neutrophils acquire their APC function within the colon have not been identified, however, it has been demonstrated that culturing of circulating neutrophils with GM-CSF, IFN-γ and/or IL-3 induces the expression of both MHC-II and co-stimulatory molecules (51-53). In addition to triggering conversion into APC-like cells, GM-CSF and IFN-γ can also delay apoptosis and increase lifespan of granulocytes (55-57,76-78). Indeed, acquisition of APC-like properties by neutrophils appears to be dependent upon inflammatory cytokine-driven upregulation of MHC-II/CD86 as cells obtained after an acute inflammatory response (i.e. 4-6 hrs peritoneal exudates cells) do not show substantial surface expression of MHC-II or CD86 (Fig. 3E)

We also show that these extravasated neutrophils highly express accessory molecules that are important in T cell/APC interactions. For example, interaction between LFA-1 on T-cells with ICAM-1 on APCs has been shown to be critical for optimal T cell activation (79,80) and expression of ICAM-1 by antigen-presenting cells is thought to be important for the proper functioning of the immunological synapse during T cell activation (62,81). Thus, high level of ICAM-1 on cLP neutrophils is consistent with their more potent T-cell stimulatory ability. In support of this contention, while we demonstrate that both spleen and cLP neutrophils are capable of triggering antigen-specific T-cell proliferation, those isolated from the colon are more potent as APCs (on a cell-to-cell basis) (Fig. 4A).

In addition to promoting T cell proliferation, we report that co-culturing of neutrophils with T cells increased the production of different chemokines known to be chemo-attractive for T cells (IP-10, RANTES), neutrophils (KC) and monocytes (MCP-1) (Fig 5). These data suggest that neutrophils may play an important role in promoting the recruitment of monocytes and effector T cells into the inflamed gut. Data presented in the current study also demonstrate that neutrophils, but not T cells, are the primary producers of the key granulopoietic and pro-inflammatory cytokines, such as IL-6 and G-CSF (82-85).

The molecular determinants required by neutrophils to migrate from the blood to the different tissues during the development of chronic gut inflammation have not been well-defined. Under physiological conditions L-selectin is known to promote neutrophil recruitment into the inflammatory sites (86). Here we found that circulating neutrophils in colitic mice express high levels of L-selectin (Fig 6). Analysis of integrin expression revealed that circulating neutrophils also express high levels of the β2 (e.g. LFA-1 and Mac-1) and β1 integrins, but no LPAM-1, VLA-4 or α_Eβ₇, suggesting that other integrins of the β1 family may be important mediators for promoting recruitment of neutrophils from the blood into the lymphoid (MLNs) and non-lymphoid (gut) tissues. With respect to adhesion molecules it is noteworthy that we observed the highest level of β1 integrin expression on the surface of circulating neutrophils (Fig 6). Analysis also revealed some interesting tissue-specific differences in surface molecule expression. For example, neutrophils isolated from spleen and MLNs of colitic mice expressed significant amounts of α4 integrin on their surface, while those in the blood or colon did not. Although it may reflect organ-specific induction, which is necessary for cellular localization within the specific tissue, we think that expression of α4 on the surface of neutrophils is an indicator of their immature phenotype (our unpublished observations). Haile et.al. suggested that loss of α4 expression correlates with maturation of CD11b⁺Gr-1^dull/int cells (87).

Taken together, our data obtained demonstrate that chronic colitis is associated with a dramatic myelopoiesis, which correlates with the infiltration of large numbers of neutrophils into the colon and the MLNs. These innate immune cells acquire the phenotype and function of APCs that may provide additional stimulatory cues to colonic effector T cells, thereby perpetuating chronic gut inflammation. Because neutrophils represent a significant portion of cells infiltrating the intestine of patients with IBD, strategies aimed at reducing their numbers and/or function may prove useful as a therapeutic strategy for treating patients with IBD. The selective removal of granulocytes and monocytes through apheresis is one of the available treatment options for IBD patients in Japan and Europe. However, it should be remembered that while selective removal of granulocytes (e.g. neutrophils and eosinophils) may be beneficial, depletion of circulating monocytes, which function to promote tissue healing (43,47), may counteract these positive effects. Indeed, it was recently reported that leukapheresis to remove both monocytes and neutrophils (88), failed to demonstrate clinical efficacy in a large-scale placebo-controlled clinical trial (89). Nevertheless, a better understanding of the roles that the different myeloid cells play in immune function and their contribution to the pathogenesis of chronic gut inflammation may ultimately reveal new therapeutic strategies for the treatment of patients with IBD.