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Published January 4, 2012, doi:10.4049/jimmunol.1102296 The Journal of

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

Dmitry V. Ostanin,*,† Elvira Kurmaeva,† Katie Furr,* Richard Bao,‡ Jason Hoffman,* Seth Berney,† and Matthew B. Grisham*

Active episodes of the inflammatory bowel diseases are associated with the infiltration of large numbers of myeloid cells including neutrophils, , and . 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 transfer model of chronic colitis, we found that induction of disease was associated with enhanced production of myelopoietic (IL-17 and G-CSF), increased production of neutrophils and monocytes, and infiltration of large numbers of myeloid cells into the mesenteric lymph nodes (MLNs) and colon. Detailed characterization of these myeloid cells revealed three major populations including Mac-1+ Ly6ChighGr-1low/neg cells (monocytes), Mac-1+Ly6CintGr-1+ cells (neutrophils), and Mac-1+Ly6Clow/negGr-1low/neg leukocytes (mac- rophages, dendritic cells, and ). In addition, we observed enhanced surface expression of MHC class II and CD86 on neutrophils isolated from the inflamed colon when compared with neutrophils obtained from the , the MLNs, and the of colitic mice. Furthermore, we found that colonic neutrophils had acquired APC function that enabled these to induce proliferation of OVA-specific CD4+ T cells in an Ag- and MHC class II-dependent manner. Finally, we observed a syner- gistic increase in proinflammatory and chemokine production following coculture 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 activation and proliferation as well as by enhancing production of proinflammatory mediators. The Journal of Immunology, 2012, 188: 000–000.

nflammatory bowel diseases (IBD; Crohn’s disease [CD] and Gr-1 in the (BM), circulation, mesenteric lymph ulcerative colitis) are multifactorial inflammatory disorders nodes (MLNs), the spleen, and colonic lamina propria (cLP). I of the gastrointestinal tract that result from a complex in- Although T cells undoubtedly play an important role in the in- teraction among genetic, environmental, and immune factors. duction and perpetuation of disease, the role of myeloid cells in Active episodes of the disease are associated with the infiltration disease pathogenesis has not been defined clearly. of large numbers of leukocytes including , gran- The accumulation of myeloid cells such as neutrophils, mono- ulocytes, monocytes, and macrophages (1–3). This enhanced in- cytes, and macrophages and others in the inflamed gut and their flammatory infiltrate is thought to contribute to the intestinal release of a variety of proinflammatory reactive oxygen and injury and dysfunction resulting in abdominal pain, rectal bleed- nitrogen species, cytokines, chemokines, and are thought ing, and diarrhea. Using mouse models of IBD, we and others to contribute to tissue injury and disruption of the epithelial barrier. (D.V. Ostanin, E. Kurmaeva, S.M. Berney, and M.B. Grisham, Indeed, -associatedmarkers are highlyexpressedin chronic unpublished observations; 4–7) have demonstrated that in addition active IBD (8), and the transepithelial migration of neutrophils to expansion of colitogenic T cells, induction of chronic intestinal into the bowel lumen to form crypt is a characteristic fea- inflammation is associated with a large and significant increase in ture of active disease (9, 10). Despite these observations, no clear the numbers of myeloid cells bearing markers CD11b (Mac-1) and consensus has been reached from several different studies that have attempted to define the role of myeloid cells in the pathogen- *Department of Molecular and Cellular Physiology, Louisiana State University esis of intestinal inflammation. Although some studies have sug- Health Sciences Center, Shreveport, LA 71130; †Department of Medicine, Louisiana ‡ gested that neutrophils or other myeloid cells promote inflammatory State University Health Sciences Center, Shreveport, LA 71130; and Department of Pathology, Louisiana State University Health Sciences Center, Shreveport, LA 71130 tissue injury (11, 12), other investigations were unable to confirm Received for publication August 8, 2011. Accepted for publication November 27, these findings (13, 14). Contributing even further to the confusion 2011. has been the observation that depletion of intestinal mononuclear This work was supported by a grant from the National Institutes of Health (PO1-DK phagocytes using clodronate–encapsulate liposomes worsened the 43785, Project 1, and Cores B and C) (to M.B.G.). D.V.O. is supported by funding from the Center of Excellence for Arthritis and Rheumatology (Louisiana State colonic inflammation induced by dextran sulfate sodium. The en- University Health Sciences Center, Shreveport, LA). hanced inflammation and injury observed in this study was found to Address correspondence and reprint requests to Dr. Matthew B. Grisham, Department be associated with an increased neutrophil infiltration into the co- of Molecular and Cellular Physiology, Louisiana State University Health Sciences lon, suggesting a pathogenic role for these cells (12). In contrast Center, 1501 Kings Highway, P.O. Box 33932, Shreveport, LA 71130-3932. E-mail address: [email protected] to these studies, recent reports have demonstrated that depletion The online version of this article contains supplemental material. of neutrophils actually exacerbates colonic inflammation and the systemic wasting syndrome in chronic (5, 6) and acute colitis Abbreviations used in this article: BM, bone marrow; cLP, colonic lamina propria; DC, ; DN, double-negative; IBD, inflammatory bowel diseases; LP, models (7), suggesting that neutrophils may function to suppress 2 2 lamina propria; MLN, mesenteric ; MPO, ; RAG / , intestinal inflammation. The depletion of neutrophils in these recombination activating gene-1–deficient; WT, wild-type. studies used the in vivo administration of the anti–Gr-1 Ab (clone Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 RB6-8C5) prior to or following induction of disease. Although this

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1102296 2 NEUTROPHILS AS APCs IN CHRONIC COLITIS strategy has previously proven effective in depleting neutrophils in Quantification of circulating leukocytes, and serum cytokines healthy mice (15), administration of this Ab to mice with inflam- Blood was withdrawn from anesthetized mice at the time of sacrifice via the mation is known to induce profound and lethal respiratory and inferior vena cava using a 25 g needle attached to a 1-ml heparinized syringe. cardiovascular complications that may have devastating systemic After 500 ml heparinized blood was mixed with an equal volume of 3% effects including death of the (16, 17). Another confounding dextran (.400,000 m.w.) in PBS, the erythrocytes were allowed to settle variable that makes interpretation difficult is the fact that anti–Gr-1 for 30 min at room temperature, and leukocyte-rich supernatant was re- moved and stored on ice. Remaining erythrocytes in the supernatant were Ab recognizes both Ly6G and Ly6C; thus, its administration would lysed, and leukocytes were pelleted by centrifugation, resuspended in PBS, deplete not only neutrophils (bearing Ly6G on their surface) but and mixed with crystal violet dissolved in 3% acetic acid for counting also Ly6C+ myeloid cells with potent immunosuppressive proper- using a hemocytometer. Total blood leukocytes were expressed as cells per ties (18, 19). A recent study that analyzed the effects of in vivo anti- milliliter of blood. For differential cell counts, 50,000–100,000 circulating or tissue-derived/flow-purified leukocytes were resuspended in 100 ml13 Ly6G (clone 1A8) and anti–Gr-1 (RB6-8C5) Ab administration on PBS and dispensed into the cytospin chamber loaded with Colorfrost Plus blood neutrophils and monocytes confirmed that the latter signifi- Microscope Slides (Fisher Scientific) and cardboard filter (Thermo Elec- cantly depleted Gr-1intF4/80+ monocytes (20). tron) and centrifuged (600 rpm, 2 min) using Shandon Cytospin3 centri- Because of these experimental limitations and lack of a clear fuge. Following this, samples were air-dried for 1–2 min and stained using consensus as to the role of neutrophils and other myeloid cells in the a Diff-Quick kit (Siemens Healthcare Diagnostics), according to the manufacturer’s instructions. The slides were mounted under coverslip pathogenesis of chronic gut inflammation, we undertook a sys- using Permount mounting agent (Fisher Scientific). At least 200 total cells tematic analysis and characterization of myeloid cell generation, were counted from at least four to five different fields, and those with , and function in a mouse model of chronic gut in- mononuclear or polymorphonuclear morphology were quantified and ex- flammation. pressed as a percentage of the total cells counted. For serum cytokine determinations, blood was withdrawn from anes- thetized mice and allowed to clot for 30–60 min at room temperature. The Materials and Methods blood was centrifuged (12,000 3 g for 10 min) in BD Microtainer serum separator tubes, and the serum was removed and stored at 280˚C. Serum GM-CSF and IL-17 were quantified using ELISA kits (eBioscience), Wild-type (WT) mice, recombination activating gene-1–deficient (RAG2/2; according to the manufacturer’s instructions. B6.129S7-Rag1tm1Mom/J) mice, and OVA-specific B6.Cg-Tg (TcraTcrb) 425Cbn/J (OTII) mice (6–8 wk of age) all on the C57BL/6 background Myeloperoxidase assay were purchased from The Jackson Laboratory (Bar Harbor, ME). Animals were maintained on 12-h/12-h light/dark cycles in standard animal cages Representative pieces of colon were rinsed with ice-cold PBS, blotted dry, with filter tops under specific pathogen-free conditions in our animal care and immediately frozen with liquid nitrogen. The samples were stored at 2 facility at Louisiana State University Health Sciences Center (Shreveport, 80˚C until thawed for myeloperoxidase (MPO) activity determination using LA) and given standard laboratory rodent chow and water ad libitum. All the O-dianisidine method, as described previously (24, 25). Briefly, sam- experimental procedures involving the use of animals were reviewed and ples were thawed, weighed, and suspended (10% w/v) in 50 mM potassium approved by the Institutional Animal Care and Use Committee of Loui- phosphate buffer (pH 6), containing 0.5% hexadecyltrimethylammonium siana State University Health Sciences Center and performed according to bromide buffer (0.1 g/20 ml potassium phosphate buffer), and homoge- the criteria outlined by the National Institutes of Health. nized using Brinkmann Homogenizer. A 1-ml sample of the homogenate was sonicated using a Labsonic 2000 generator and a Braun-Sonic 2000U Abs and reagents transducer with a 40T needle probe tip (B. Braun Biotech International, Allentown, PA) twice for 15 s (approximate average power output: 15 W Abs were purchased from either BD Pharmingen (San Diego, CA) or with continuous operation). The sample was then centrifuged at 12,000 3 eBioscience (San Diego, CA). Negative selection kits for isolation of mouse g for 10 min at 4˚C, and the supernatant was saved on ice. The reaction + CD4 T cells were purchased from either Invitrogen (Carlsbad, CA) or was initiated by adding a small aliquot of supernatant (30 ml) to a pre- StemCell Technologies (Vancouver, BC, Canada). warmed (to 37˚C) reaction mixture containing 50 mM potassium phos- phate (pH 6), O-dianisidine dihydrochloride (200 mg/ml), and hydrogen Induction of chronic gut inflammation peroxide (200 mM). The reaction mixture was incubated for 5 min at 37˚C, Chronic colitis was induced by the transfer of sorted WT CD4+CD45RBhigh stopped by adding sodium azide (0.02% final concentration), and absor- T cells into RAG2/2 mice using our previously published method (22). bance at 460 nm was determined using a spectrophotometer. The amount Briefly, were removed from donor WT mice and teased into a of MPO in the colon was expressed as the units of MPO per gram of tissue. single-cell suspension in 13 PBS/4% FBS (FACS buffer). CD4+ cells One unit is defined as the amount of enzyme necessary to produce a were obtained using negative selection kits, according to the manu- change in absorbance of 1.0/min. facturer’s protocol. Enriched CD4+ T cells were labeled with anti-CD4 and anti-CD45RB mAb and sorted into CD45RBhigh (40% brightest) and Tissue isolation and flow cytometry analyses low CD45RB (15% dimmest) fractions using FACSAria (BD Biosciences, Lymphocytes were obtained from the spleen, the MLNs, and the cLP and San Jose, CA) and were found to be .98% pure on postsort analysis. Male 2/2 3 5 high low analyzed by flow cytometry as described previously (22, 23, 26). Briefly, RAG mice were injected (i.p.) with 5 10 CD45RB , CD45RB , spleens were removed from reconstituted RAG2/2 and teased into a sin- or both T cell subsets resuspended in 500 ml PBS. For some experiments, 2 2 2 gle-cell suspension using frosted ends of two glass slides. RBCs were we adoptively transferred flow-purified CD4+CD25 T cells into RAG / removed by hypotonic lysis, and the resulting leukocytes were washed and recipients to induce colitis (5 3 105; i.p. injected). In general, colitis in- + 2 passed through 70-mm cell strainer to remove large chunks and then duced by transfer of CD4 CD25 T cells was comparable to that induced resuspended in FACS buffer. Cells from MLNs were obtained by grinding by CD45RBhigh T cells, although we did see a slight reduction in the in- 2 2 2 the tissue through a 70-mm nylon cell strainer (BD Falcon) in FACS buffer cidence of moderate/severe disease in the CD4+CD25 →RAG / mice 2 2 and further purified over 44/70% Percoll gradient. Lamina propria (LP) compared with the CD45RBhigh→RAG / mice. Clinical evidence of lymphocytes were prepared by digestion of finely minced intestinal pieces disease in mice (e.g., body weight loss and loose stool/diarrhea) was fol- remaining after intraepithelial lymphocyte isolation with RPMI 1640 lowed and recorded weekly from the time of the injection. medium/4% FBS/2.5 mM CaCl2 and containing 150 U collagenase type Histopathology VIII (Sigma-Aldrich) twice for 40 min each time in a 37˚C orbital shaker. Lymphocytes were further enriched by centrifugation over a 44/70% Eight weeks following T cell transfer or when the animals lost 15–20% Percoll gradient. For flow cytometry staining, ∼1 3 106 cells were placed of their original body weights, mice were euthanized, and colons were in individual wells of a 96-well plate, incubated first with FcR block excised, measured, and weighed. Gross colitic scores were assigned (CD16/CD32), and then stained with appropriate Ab mixtures. After the according to previously published criteria (21). Representative pieces of staining, cells were fixed for 20 min on ice in freshly prepared 2% ultra- proximal and distal colon were fixed in 10% phosphate-buffered formalin, pure formaldehyde (Polysciences, Warrington, PA) and analyzed the next sectioned, and stained with H&E. Blinded histopathological analysis of day on the FACSCalibur or BD LSR II (BD Biosciences). Flow cytometry each colon was performed by a pathologist unaware of the identity of each data were analyzed using FlowJo software for PC (version 7.2.5; Tree Star, sample using our previously published scoring criteria (21–23). Ashland, OR). The Journal of Immunology 3

Granulocyte and myeloid cell isolation, T cell proliferation Results assay, and cytokine determinations Chronic colitis is associated with increased and A single-cell suspension was prepared from three to four pooled spleens and elevations of serum myelopoietic cytokines cLP cells and stained with anti–Mac-1 (CD11b), -Ly6C, and –Gr-1 (Ly6G/ + int + One of the hallmark features of chronic colitis induced in immu- C) Ab mixture, followed by sorting of Mac-1 cells into Ly6C Gr-1 2/2 + high using FACSAria (BD). To obtain a highly enriched population of OTII nodeficient RAG mice injected with naive (CD4 CD45RB ) CD4+ T cells, the spleens from OTII mice were prepared as described T cells is the large and significant accumulation of myeloid cells above, stained with PE-labeled Abs to CD11c, Mac-1, CD8a, and B220 (neutrophils, monocytes, and macrophages) within the cLP when and allophycocyanin-tagged anti-CD4 Abs, and sorted into CD4+[CD11c/ 2/2 2 + compared with noncolitic RAG mice injected with the Mac-1/CD8a/B220] . A total of 50,000 OTII CD4 T cells were co- CD45RBlow cells (CD45RBlow→RAG2/2) (Supplemental Fig. 1). cultured with the indicated number of sorted neutrophils in the presence of 10 mg/ml OVA (323–339; AnaSpec, San Jose, CA). Where indi- In addition, this increased myeloid cell accumulation corresponds cated, anti-CD1d (clone 1B1) or anti-MHC class II (MHC-II) (M5/ well with high levels of MPO activity in the cLP of colitic 114.15.2) Abs (1 mg/ml final) were added according to previously pub- CD45RBhigh→RAG2/2 mice compared with mice with little or no lished protocol (27). For fixation, sorted myeloid cells were fixed in cul- colitis (CD45RBlow→RAG2/2) demonstrating the presence of ture medium containing 1% paraformaldehyde for 10 min at 37˚C, followed by washing and additional 10-min incubations twice in 120 mM neutrophils, monocytes, and possibly eosinophils (Supplemental lysine as described by Brimnes et al. (28). At the end of incubations, cells Fig. 1D). Development of chronic colitis (Fig. 1A) is accompanied were washed an additional three times in medium prior to culturing. Cells by a 5- to 6-fold increase in circulating leukocytes compared with 3 were allowed to proliferate for 72 h with the addition of 1 mCi [ H]thy- unmanipulated RAG2/2 mice (Fig. 1B). Particularly striking was midine for the last 8–18 h of culturing. Cells lysates were harvested onto that the number of circulating neutrophils and PMN-ring cells fiberglass filters using semiautomated cell harvester (Brandel, Gaithers- ∼ burg, MD), and radioactivity was quantified using a liquid scintillation increased 10-fold in colitic mice when compared with unma- 2/2 counter (1409; Wallac). nipulated RAG mice and 5-fold compared with WT mice (Fig. For cytokine analysis, negatively selected CD4+ T cells (50,000) isolated 1C). Morphologic analysis of peripheral blood obtained from from the spleens of WT mice were cocultured in triplicate with sorted colitic mice showed that the vast majority of circulating leukocytes neutrophils in a 96-well flat-bottom plates in the presence of plate-bound anti-CD3 and soluble CD28 (1 mg/ml final) Abs. At 48 h following the had a multilobed or donut-shaped nucleus, which are morpho- addition of the Abs, supernatants from replicate wells were pooled, ali- logical features of mouse granulocytes such as neutrophils and quoted, and frozen at 280˚C. Samples from two independent experiments PMN-“ring cells” (Fig. 1E) (32, 33). PMN-ring cells represent were included for subsequent cytokine analysis. Multiplex cytokine anal- newly released and not fully matured “band” neutrophils, whereas ysis was carried out using Milliplex Mouse Cytokine/Chemokine Im- maturation and segmentation of their nucleus leads to the ap- munoassay (Millipore, Billerica, MA), according to the manufacturer’s instructions. Briefly, samples were thawed on ice and diluted using the pearance of “segmented” neutrophils (34). Both types of neu- recommended buffer. Undiluted as well as 1:2 and 1:4 diluted samples trophils constituted .95% of the total circulating leukocytes in were used for cytokine measurements, and only those values that fell colitic mice (Fig. 1C,1E). within the range of the standard curve were used in calculations. Samples Among the different cytokines responsible for stimulating were run on Bio-Plex Luminex instrument (Bio-Rad Laboratories, Her- myelopoiesis, we observed large and significant increases in serum cules, CA). Data were analyzed using Miliplex Analyzer 3.1 xPONENT high 2/2 software (Millipore). Samples from repeat experiments were analyzed on G-CSF levels in colitic CD45RB →RAG mice when com- 2 2 the same run. pared with either of the noncolitic CD45RBlow→RAG / or Neutrophils from the spleens and colons of colitic mice were obtained high low→ 2/2 + CD45RB +CD45RB RAG groups (Fig. 1F). Serum by cell sorting. Ly6G cells from the BM were obtained by positive se- level of IL-17 was significantly increased in colitic mice compared lection using Ly6G-PE Ab and EasySep PE Positive Selection Kit low→ 2/2 (StemCell Technologies). Cells from peritoneal exudate were obtained 4– with the CD45RB RAG group (Fig. 1G) but was not 6 h after injection of 25 mg oyster glycogen type II resuspended in 0.5 ml significantly different from that in mice with attenuated disease 13 PBS by peritoneal lavage. Ly6G+ cells from peritoneal exudate were (i.e., in CD45RBhigh+CD45RBlow→RAG2/2 group) (Fig. 1F,1G). isolated by positive selection. Following positive selection, cells were processed for RNA isolation and RT-PCR. Cytospin was performed as Phenotypic analysis of leukocytes derived from the colon, the described above. MLNs, and the spleen of mice with chronic colitis RNA isolation and RT-PCR To more specifically define what different populations of leuko- cytes infiltrate animals with chronic disease, we analyzed leukocyte Total RNA from neutrophils obtained as described above was isolated using RNeasy Mini kit (Qiagen, Valencia, CA), according to the manufacturer’s populations associated with the colon, the MLNs, and the spleen. + + + instruction, and treated with DNase I (Qiagen). RNA concentration and Quantitatively, the numbers of CD4 cells and Mac-1 Gr-1 and 2 purity was assessed with NanoDrop ND-1000 spectrophotometer (Thermo Mac-1+Gr-1 cells in the colons were significantly higher in the Scientific), and 50 ng was reversely transcribed with qScriptTM cDNA colitic group compared with the noncolitic CD45RBlow→RAG2/2 SuperMix (Quanta Biosciences), according to the manufacturer’s protocol. and CD45RBhigh+CD45RBlow→RAG2/2 mice (Fig. 2A). We PCRs were carried out in a 25-ml volume using 2720 Thermo Cycler found that CD4+ T cells constituted a substantial portion (28%; (Applied Biosystems) using primers specific for b2-microglobulin and histocompatibility class II Ag A, a-chain (I-Aa). Primer pair sequences for range, 4–43%) of the total cells obtained from the inflamed colon. I-Aa were obtained from Leibundgut-Landmann et al. (30) and for b2- In addition, we observed significant numbers of granulocytes microglobulin from the RTPrimerDB database (RTPrimerDB ID: 3584) expressing both Mac-1 and Gr-1 (Mac-1+Gr-1+) as well as those (30). Amplicons of expected length were resolved on a 1.5% agarose gel. + 2 For densitometry measurements, ImageJ software (version 1.44p) (31) was that expressed Mac-1 but not Gr-1 (Mac-1 Gr-1 ). Both pop- used, and the intensity of I-Aa band for each sample was normalized to ulations combined represented ∼29% of total cLP cells (range, 15– + that of b2-microglobulin. 42%) (Fig. 2B). We also observed large numbers of CD4 T cells within the spleen and MLNs in the colitic CD45RBhigh→RAG2/2 Statistics mice. In addition to T cells, Mac-1–expressing cells were elevated Data are presented as mean 6 SEM (SE of mean). The statistical signif- in the MLNs and spleens in mice with active disease. We observed icance between two groups was determined using two-sided Student t test. large and significant increases in the absolute numbers of Mac-1+ The statistical significance between more than two groups was evaluated + + 2 using a one-way ANOVA. The statistical significance between selected Gr-1 and Mac-1 Gr-1 cells in the MLNs obtained from colitic groups was evaluated using the Dunnett post hoc test. A p value ,0.05 was when compared with their noncolitic controls (Fig. 2B). Although considered significant. the numbers of Mac-1+Gr-1+ granulocytes were significantly in- 4 NEUTROPHILS AS APCs IN CHRONIC COLITIS

FIGURE 1. Development of chronic coli- tis is accompanied by enhanced myelopoi- esis and increase in peripheral numbers. A, Blinded histopathological scores of colons obtained from CD45RBhigh→ RAG2/2,CD45RBlow→RAG2/2,and CD45RBhigh+CD45RBlow→RAG2/2 mice for at least 11 animals/group from at least two independently conducted experiments. B, Total leukocyte numbers in peripheral blood of mice from the different groups. Blood leukocyte counts in mice were deter- mined as described in Materials and Meth- ods. C, Total numbers of PMNs/PMN-ring cells in peripheral blood in mice. Represen- tative images of peripheral blood cytospins from WT (D) and colitic CD45RBhigh→ RAG2/2 (E) mice showing dramatic in- creases in peripheral blood granulocytes. Serum from 7 to 11 individual mice/group was analyzed for G-CSF (F) and IL-17 (G). Data in all graphs shown represent the mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001.

creased in spleens from colitic mice compared with their non- major populations, based on the expression of Mac-1 (CD11b), colitic controls, the numbers of Mac-1+Gr-12 cells were similar Gr-1(Ly6C/G), and Ly6C: Mac-1+Ly6ChighGr-1low/neg (hereafter among all three groups. referred to as Ly6Chigh), Mac-1+Ly6CintGr-1+ (Gr-1+) and Mac-1+ Identification of the different populations of murine myeloid Ly6Clow/negGr-1low/neg (double-negative, DN) (Fig. 3A). Gr-1+ cells by flow cytometry has been limited by the lack of specific population contains mature and immature neutrophils, and the markers. Mouse neutrophils and monocytes are commonly iden- Ly6Chigh population contains recently recruited immature mono- tified by flow cytometry using side scatter properties (granularity) cytes (19, 38) that lose Ly6C expression following their matura- and expression of Mac-1 and Gr-1 markers (36). Anti–Gr-1 Ab tion into tissue macrophages or DCs (39–42). The DN population (clone RB6-8C5) recognizes both Ly6G, which is expressed pri- consists of a heterogeneous mixture of mononuclear and granu- marily on mouse neutrophils (16), and Ly6C, which is expressed locytic cells. on a variety of cells, including T cells and monocytes (36, 37). Ly6Chigh cells were identified in all tissues tested; however, they Thus, Mac-1+Gr-1+ population can be further subdivided using expressed varying levels of Gr-1: those isolated from the spleen anti–Gr-1 and anti-Ly6C Abs (19). Therefore, we next charac- had intermediate expression of Gr-1, whereas those from the colon terized the tissue-associated myeloid cells and identified three LP were Gr-1low. In addition, all Ly6Chigh cells were SSClow and The Journal of Immunology 5

within the CD11chighF4/80+ population were SSClow and MHC- IIhigh, which is consistent with the profile for DCs. Cells bearing CD11clowF4/80high phenotype were also SSClow and MHC-IIhigh, which is consistent with a phenotype of macrophages. In contrast, CD11cnegF4/80low 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 (Supplemental Fig. 2A). Indeed, low-level expression of F4/80 in combination with Mac-1 expression is characteristic of eosinophils (43). Splenic CD11cnegF4/80neg cells were SSClow with intermediate expression of MHC-II. These cells had morphology typical of a , with a bean-shaped nucleus or a monocyte- ring cell with donut-shaped nucleus (Supplemental Fig. 2A) (33). In addition, all of these cells were SSClow (Supplemental Fig. 3). DN population in the MLN or cLP was more homogeneous than that in the spleen. In the MLN, this population contained mainly CD11cnegF4/80low cells. Side scatter and MHC-II expression revealed two populations that were SSChighMHC-IIlow (possibly eosinophils) and SSClowMHC-IIhigh, which appear to be monocytes and macrophages. In the cLP, the same two populations were found within the DN gate: eosinophils (SSChighMHC-IIlow) based on the cytospin analysis and -like (SSClowMHC-IIhigh) cells that, interestingly, did not express a classical marker of mature macrophages F4/80 (44). However, it is known that under certain inflammatory conditions F4/80 can be downregulated by mature macrophages, especially in the T cell-rich areas (45). Because Arnold et al. (42) demonstrated that Ly6C, which is expressed at high levels on circulating monocytes, is downregulated upon their maturation into tissue macrophages, the SSClowMHC-IIhigh pop- ulation most likely represents either mature macrophages or Ly6Clow-neg monocytes that can be found in the injured tissues (46).

+ FIGURE 2. Enhanced accumulation of myeloid cells in mice with Gr-1 neutrophils obtained from the inflamed colon express chronic colitis. A, Absolute numbers of CD4+ T cells, Mac-1+Gr-1+ and enhanced levels of MHC-II and CD86 + - high→ 2/2 Mac-1 Gr-1 were quantified in CD45RB RAG (black bars), Neutrophils isolated from patients with chronic inflammatory con- CD45RBlow→RAG2/2 (white bars), and CD45RBhigh + CD45RBlow→ 2 2 ditions have been documented to undergo “transdifferentiation” RAG / mice (hatched bars). At least five animals were analyzed per into DC-like cells, that is, they acquire the ability to act as APCs group and experiments were repeated at least twice. Data are expressed as mean 6 SEM. *p , 0.05, **p , 0.01, ***p , 0.001 compared with the and induce T cell activation (47–49). In addition, culturing human RBhigh control group. B, Representative dot plots showing percentages of or mouse neutrophils with proinflammatory cytokines induces Mac-1+Gr-1+ and Mac-1+Gr-12 cells in the spleens, MLNs, and colon LP. expression of MHC-II and costimulatory molecules (50–53) and Numbers indicate percentages of cells within the gated leukocyte pop- delays (54–56). Collectively, these reports suggest that ulation (using forward and side scatter). during chronic inflammation neutrophils undergo phenotypic and functional changes driven, in part, by the inflammatory cytokines had morphology of monocytes (data not shown). Last, these cells released by T cells. To assess whether neutrophils obtained from did not express macrophage marker F4/80 and dendritic cells tissues of colitic mice assume APC-like properties, we analyzed marker CD11c (Fig. 3B). Interestingly, MHC-II expression was surface expression of MHC-II and CD86 on these cells. We found very high on Ly6Chigh cells isolated from MLNs and colons but that Gr-1+ neutrophils isolated from the spleen, the MLNs, and the was low on cells isolated from spleens. cLP expressed low, moderate, and high levels of MHC-II, re- When we analyzed the Gr-1+ populations in all three tissues, we spectively (Fig. 3B). Because we established that Gr-1+ cells were found that these cells were granular (SSChigh) and negative for negative for CD11c and F4/80, MHC-II expression could not be both F4/80 and CD11c (Fig. 3B) and had the morphology char- due to contaminating DCs or macrophages. We also observed acteristics of PMNs and PMN-ring cells (Supplemental Fig. 2A). a modest upregulation of CD86 with the highest levels seen on We observed many fewer cells expressing a mature neutrophil cells isolated from the inflamed cLP (Fig. 3B). Comparison of morphology with a segmented nucleus within the Gr-1+ pop- median fluorescence intensity between neutrophils isolated from ulation isolated from the spleen when compared with MLNs and different tissues showed that cLP cells had a significantly elevated cLP. Furthermore, ∼75% of these cells displayed a ring-shaped expression of MHC-II and CD86 compared with those isolated morphology characteristic of immature “band” cells. In contrast, from the spleen (Fig. 3C), suggesting that cells that infiltrated Gr-1+ cells isolated from the cLP were mostly mature “seg- colons acquired phenotype of APC-like cells. We also analyzed mented” neutrophils (Supplemental Fig. 2B). expression of MHC-II and CD86 on DN and Ly6Chigh subsets of Analysis of the DN cells in the spleen revealed a heterogeneous myeloid cells from the different tissues and observed enhanced population of cells: CD11chighF4/80+,CD11cintF4/80high, CD11cneg surface expression of MHC-II on these cells (Fig. 3C). In general, F4/80low, and CD11cnegF4/80neg (Supplemental Fig. 3). All cells pattern of CD86 expression paralleled that of MHC-II for both the 6 NEUTROPHILS AS APCs IN CHRONIC COLITIS

FIGURE 3. Increased expression of MHC-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 versus Gr-1. Representative dot plots were initially gated on Mac-1+ (CD11b) cells. B, Representative histo- grams showing expression of indicated markers (black line) on Gr-1negLy6Cneg (DN), Gr-1+ PMNs, and Ly6Chigh populations. C, Median fluorescent intensity (MFI) values show progressive increase in MHC-II and CD86 expression on PMNs and Ly6Chigh cells isolated from cLP compared with those isolated from the spleen and MLN. **p , 0.01, ***p , 0.001. D, Expression of Ly6C and Gr-1 in peritoneal exudates cells. The dot plot was initially gated on Mac-1+ cells. Exudate PMNs (ex. PMNs) and Ly6Chigh and DN populations are shown by indicated gates. E, Histograms are gated on the corresponding populations identified in D. For all graphs, staining with Abs is indicated by black line, whereas staining with control Ab is indicated by shaded curves. F, PMNs isolated from colitic mice show increased expression of MHC-II mRNA compared with PMNs obtained from the BM or peritoneal exudates. RT-PCR of histocompatibility class II Ag A a-chain and corresponding densitometry measurements. Gr-1+ PMNs from spleens and colons of colitic mice were obtained by cell sorting. Ly6G+ cells from the BM or peritoneal exudates were obtained by positive selection. Ly6G2 fractions from BM (BM Ly6G2) and exudate (ex. Ly6G2) served as MHC-II+ control.

DN and Ly6Chigh populations. Next, we wanted to determine MHC class II in sorted neutrophils isolated from the spleen and whether MHC-II upregulation can be observed on neutrophils the colon. We saw high levels of MHC-II Ag A a-chain mRNA in recruited in response to an acute inflammatory stimulus. To do neutrophils obtained from cLP (Fig. 3F). In comparison, Ly6G+ this, we analyzed peritoneal exudate cells 4–6 h following the neutrophils isolated from the BM showed no MHC-II mRNA. injection of oyster glycogen (Fig. 3D) (57). We found that these Interestingly, Ly6G+ peritoneal exudates neutrophils showed some neutrophils had little surface expression of MHC-II or CD86 (Fig. expression, but it was much less than Ly6G2 cells, which consist 3E). of a mixed population of various hematopoietic cells. These data Whale et al. (58) reported that neutrophils may passively ac- suggest that recruitment of circulating neutrophils into the site of quire parts of plasma membranes from apoptotic or necrotic cells inflammation may provide transcriptional signals for the upregu- during . Thus, high expression of MHC-II may be lation of MHC-II mRNA expression. These results confirmed that due to trogocytosis by neutrophils of this molecule from the sur- surface MHC-II expression in neutrophils isolated from mice with face of neighboring APCs. To evaluate whether Gr-1+ neutrophils colitis is not due to trogocytosis but due to the transcription of actively transcribe MHC-II gene, we analyzed mRNA level of mhc-II genes. The Journal of Immunology 7

Neutrophils induce T cell activation through an Ag-specific and crease in production of Th2 cytokines such as IL-4 and IL-5 when MHC-II–dependent interaction ratios of neutrophils:T cells approached 1:1. Concentrations of To determine whether neutrophils isolated from colitic mice could several chemokines, such as IFN-g–induced protein 10 (IP-10 or induce proliferation of T cells, we cocultured these cells with flow- CXCL10), monocyte chemotactic protein-1 (MCP-1 or CCL2), purified OVA-specific CD4+ T cells (OTII CD4+ cells) in the regulated upon activation, normal T-cell expressed, and secreted presence of OVA peptide. In our preliminary experiments, we (RANTES or CCL5), macrophage inflammatory protein-1a (MIP- found that positively or negatively selected splenic OTII CD4+ 1a or CCL3), and the chemokine KC (GRO-a or CXCL1) also cells extensively proliferated in the presence of OVA peptide increased in a cell-dependent manner. It should be noted that there without addition of accessory cells (data not shown), suggesting were no significant increases in T cell proliferation and even that these population contained APCs. Thus, to remove CD4+ DCs a slight decrease in IL-2 concentrations at a 1:1 ratio of neu- present in the spleen (59) as well as other contaminating APCs, trophils:T cells (Figs. 5Q,5R), suggesting that higher levels of we sorted splenic OTII cells into CD4+[CD11c/Mac-1/CD8a/ cytokines in culture media cannot be due to the presence of more B220]neg cells. Only by sorting we were able to obtain a pure T cells. Slightly lower IL-2 levels with the addition of more neu- population of CD4+ T cells to use in our coculture experiments trophils could be indicative of the inhibition of T cell proliferation despite the lack of any apparent effects on T cell proliferation as that did not proliferate in the presence of peptide without the 3 addition of accessory cells (Fig. 4A). We found that neutrophils measured by the incorporation of [ H]thymidine (Fig. 5R). obtained from mice with active colitis in the presence of OVA Expression of cell adhesion molecules on neutrophils obtained peptide induced OTII T cell proliferation in a cell number- from tissues of colitic mice dependent manner (Fig. 4A). Interestingly, at a 1:1 ratio of neu- trophils to T cells, those isolated from colon induced a 2-fold The large majority of studies investigating the molecular deter- higher proliferation than those isolated from the spleen, which minants involved in leukocyte trafficking in chronic gut inflam- correlated with their higher surface expression of MHC-II. mation have focused on T and/or -associated adhesion Omission of APCs or addition of MHC-II blocking Ab com- molecules. Given our data demonstrating that the inflamed MLNs pletely inhibited Ag-induced proliferation of T cells (Fig. 4B). The and colon contain large numbers of neutrophils, we wished to importance of Ag processing and presentation by cLP neutrophils analyze surface expression of the major cell adhesion molecules on was confirmed by formalin fixation of these cells, which abolished these cells found in blood and the different tissues in colitic mice. their ability to trigger proliferation (Fig. 4B). Taken together, these We found that expression of the a4 integrin was dramatically in- experiments suggest that Ag-specific proliferation of T cells by creased on neutrophils isolated from the spleen and the MLNs neutrophils isolated from colitic mice is dependent on their ability (Fig. 6). Because we did not observe any detectable expression of to internalize Ag and present it on their surface and is not due to a4b7 (lymphocyte Peyer’s patch adhesion molecules-1) or b7 nonspecific binding of Ag to the surface of these cells. integrin, we concluded that expression of a4 on neutrophils from the spleen and the MLNs was largely due to the expression of Coculture of Gr-1+ neutrophils with activated CD4+ T cells a4b1. We did find that CD29 (b1 integrin) was highly expressed on enhances the production of proinflammatory cytokines circulating and tissue neutrophils obtained from mice with active We next wished to ascertain whether neutrophils were capable of colitis. Interestingly, the highest level of CD29 expression was modulating the production of cytokines by T cells. We found that seen on blood neutrophils where a4 expression was barely de- addition of increasing numbers of neutrophils isolated from the cLP tectable. Because the b1 chain can pair with seven different a + to activated WT CD4 T cells resulted in a cell number-dependent subunits (a1–6 and aV) to form VLA(1–6) and aVb1 integrins, increases in the levels of proinflammatory mediators including respectively (60), our data would imply that b1 integrins other IFN-g, IL-17, TNF-a, and IL-1b (Fig. 5A–P). Increased produc- than VLA-4 may be playing a role in recruitment of neutrophils tion of cytokines that are thought to be important for myelopoiesis from the blood. Not surprising, we found that the b2 integrins (e.g., IL-17, GM-CSF, G-CSF, and IL-6) was also observed with CD11a (Fig. 6) and CD11b (data not shown but was used for the addition of more neutrophils. We also observed a modest in- gating of CD11b+ cells in histograms shown in Fig. 6) were

FIGURE 4. PMNs isolated from colitic mice induce proliferation of CD4+ T cells in an Ag-specific, MHC-II–dependent manner. A, PMNs isolated from colitic mice can function as APC and trigger proliferation of Ag-specific T cells. FACS-sorted OTII CD4+ T cells were cocultured with increasing numbers of sorted PMNs from spleens (white bars) or cLP (black bars) of colitic mice or WT iAccessory cells (hatched bars) in the presence of OVA peptide (10 mg/ ml). *p , 0.05, **p , 0.01 compared with WT iAccessory cells. B, Ag-specific T cell proliferation is abolished with the addition of MHC-II blocking Ab but not in the presence of control anti-CD1d Ab. Fixation of PMNs or WT iAccessory cells also abolishes their ability to stimulate proliferation of T cells. Data are expressed as the mean cpm 6 SEM from a representative experiment, which was repeated three to five times. 8 NEUTROPHILS AS APCs IN CHRONIC COLITIS

FIGURE 5. PMNs synergize with T cells to enhance in cytokine production without an effect on proliferation. A–P and R, Coculturing of WT CD4+ T cells with PMNs isolated from cLP 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 (gray bars) in the presence of activating anti-CD3/CD28 Abs for 48 h. Cytokine concentrations were measured using multiplex cytokine analysis. Data are expressed as the mean 6 SEM of serial dilutions (undiluted, 1:2 and 1:4) of each sample. Shown are results from a representative experiment of two independently conducted coculture experiments with similar results. Proliferation of WT T cells in response to anti-CD3/CD28 Abs in the absence or presence of PMNs (Q) was not substantially increased with the addition of PMNs. expressed on neutrophils isolated from blood, spleen, MLN, and by APCs is critical for stabilization of immunological synapse cLP. The expression of the aE integrin (CD103) on neutrophils and efficient priming of T cells (61). Thus, enhanced ICAM-1 was relatively low, which suggested that aEb7 expression is expression by MLN and cLP neutrophils provides additional minimal. In addition to the different integrins, we found that evidence for their acquisition of Ag-presenting capacity. neutrophils obtained from the blood, the spleen, and the MLNs but not cLP were L- positive (Fig. 6). We also observed sig- Discussion nificant expression of ICAM-1 on neutrophils obtained from Histopathologic inspection of intestinal biopsies obtained from MLNs and cLP, whereas its expression on circulating and splenic patients with active Crohn’s disease or ulcerative colitis reveal the neutrophils was somewhat reduced (Fig. 6). ICAM-1 expression presence of large numbers of myeloid cells such as neutrophils, The Journal of Immunology 9

FIGURE 6. Surface expression of different adhesion molecules on PMNs isolated from colitic mice. Representative histograms depict- ing expression of the indicated surface mole- cules (black line) were gated on PMNs (Mac-1+ Ly6CintSSChighLy6G+) isolated from blood, spleen, MLNs, or cLP. Staining with fluo- rescently-tagged isotype control Ab is depicted by shaded histogram plots.

monocytes, eosinophils, and macrophages (1–3). In addition, of the peripheral blood obtained from colitic mice showed that several studies using clinical specimens obtained from patients majority of circulating neutrophils had mature, segmented mor- with active IBD have demonstrated increased levels of intestinal phology (Fig. 1E). This suggests that myelopoiesis observed dur- IL-8, G-CSF, and GM-CSF (62–64) that corresponded to a delay ing chronic inflammation, in addition to stimulating release of in neutrophil apoptosis (62). Because myeloid cells are capable of mature myeloid cells, also promotes egress of hematopoietic generating large amounts of proinflammatory mediators and re- progenitors from the BM with their subsequent accumulation in active oxygen and nitrogen metabolites, investigators have pro- the spleen. Recently, Swirski et al. (71) showed that during is- posed that these cells may play an important role in the chemic myocardial injury the spleen can serve as a reservoir for inflammatory tissue injury observed in IBD (65–69). Indeed, undifferentiated monocytes that are capable of rapidly migrating to transepithelial migration of neutrophils from the gut to the lumen the injured site where they participate in . Thus, in to form crypt abscesses correlates with the worsening of disease the context of chronic inflammation, the spleen may function as (9, 10). Unfortunately, no clear consensus has been reached from a “depot” for accumulation and storage of myeloid cells and their a variety of different experimental and clinical studies attempting precursors, presumably, for their subsequent utilization to fight to define the importance of myeloid cells in the pathogenesis of infection or, as has been proposed for monocytes (71), in wound IBD. As a first step toward understanding the role that myeloid healing. Although we (72) and others (73) previously demon- cells may play in the induction, perpetuation, and/or resolution strated in this T cell transfer model lack of spleen in recipient of chronic intestinal inflammation, we undertook a systematic immunodeficient mice had no affect on the induction of colitis, analysis and detailed characterization of myeloid cell generation, in view of our new data, it may be worthwhile to examine how phenotype, and function in a mouse model of chronic gut in- splenectomy in mice with active colitis affects disease progression. flammation. Another novel finding obtained in the current study is that Data obtained in the current study demonstrate that chronic gut neutrophils isolated from the chronically inflamed colon have inflammation is associated with enhanced plasma levels of granulo/ acquired APC phenotype and function, based on their enhanced myelopoietic cytokines, increased production of granulocytes and expression of MHC-II and CD86 on mRNA and protein levels myeloid cells, and infiltration of large numbers of neutrophils into as well as their ability to induce T cell activation in an Ag- and the MLNs and colon. Our data confirm those of Monteiro et al. (70) MHC-II–dependent manner. Although it can be argued that a and Nemoto et al. (6) who demonstrated a robust myelopoietic small number of contaminating DCs and/or monocytes/macro- response induced by adoptive transfer of CD4+ T cells into lym- phages could account for the APC phenotype and function, our in- phopenic mice. In addition, the current study extends their find- depth multicolor flow cytometric analyses confirmed that these ings by identifying, for the first time, to our knowledge, three cells did not contain CD11c+ (DCs) or F4/80+ (macrophages) cells major populations of myeloid cells that infiltrate the colon, the (Fig. 3B). We also show that induction of T cell proliferation is MLNs, and the spleen of mice with chronic colitis. Our detailed contingent upon MHC-II expression because blocking Ab com- analyses identified Mac-1+Ly6ChighGr-1low/neg cells as monocytes, pletely abrogated proliferation of T cells. Last, we demonstrate Mac-1+Ly6CintGr-1+ cells as mature and immature neutrophils, that proliferation of T cells does not occur when Ag binds non- and Mac-1+Ly6Clow/negGr-1low/neg (DN) as a heterogeneous mix- specifically to the surface of fixed cells but is dependent on the ture of DCs, macrophage-like cells, and eosinophils. In all three ability of live neutrophils to internalize, process, and present Ag tissues analyzed, we found that myeloid cells made up a signifi- on their surface in association with MHC-II. These data are in cant portion of infiltrating leukocytes in colitic mice. In the colon, agreement with several other reports demonstrating that neu- the combined number of Mac-1+Gr-1+ (which were mostly neu- trophils isolated from patients with chronic inflammatory con- trophils) and Mac-1+Gr-12 cells (which contained monocytes and ditions may undergo “transdifferentiation” into APCs and acquire DN cells) were approximately the same as the number of CD4+ the ability to induce T cell activation in vitro (47–49). Liu et al. T cells (Fig. 2). In the MLNs, myeloid cells slightly outnumbered (74) reported that peripheral monocytes isolated from CD patients T cells, and in the spleen, we recovered ∼10-fold more myeloid showed increased expression of costimulatory molecules. “Trans- cells (primarily neutrophils) than T cells. differentiation” refers to a process whereby a non-stem cell with a On the basis of the cytospin and differential morphological specific function transforms into a different type of cell and has analysis, neutrophils isolated from the colon were mostly “seg- been used frequently to describe acquisition of Ag-presenting mented” or mature, whereas a substantial number of those in the characteristics by neutrophils (48, 49). This term, however, may spleen had a donut-shaped nucleus characteristic of immature or not accurately reflect this phenomenon, because these cells retain “band” neutrophils (Supplemental Fig. 3). Interestingly, analysis morphology of neutrophils. 10 NEUTROPHILS AS APCs IN CHRONIC COLITIS

The specific mechanisms by which neutrophils acquire their MLNs. These innate immune cells acquire the phenotype and APC function within the colon have not been identified; however, function of APCs that may provide additional stimulatory cues to it has been demonstrated that culturing of circulating neutrophils colonic effector T cells, thereby perpetuating chronic gut inflam- with GM-CSF, IFN-g, and/or IL-3 induces the expression of both mation. Because neutrophils represent a significant portion of cells MHC-II and costimulatory molecules (50–52). In addition to infiltrating the intestine of patients with IBD, strategies aimed at triggering conversion into APC-like cells, GM-CSF and IFN-g can reducing their numbers and/or function may prove useful as a ther- also delay apoptosis and increase the lifespan of granulocytes (54– apeutic strategy for treating patients with IBD. The selective re- 56, 75–77). Indeed, acquisition of APC-like properties by neu- moval of granulocytes and monocytes through apheresis is one of the trophils appears to be dependent on inflammatory cytokine-driven available treatment options for IBD patients in Japan and Europe. upregulation of MHC-II/CD86 because cells obtained after an However, it should be remembered that although selective removal acute inflammatory response (i.e., 4–6 h peritoneal exudates cells) of granulocytes (e.g., neutrophils and eosinophils) may be beneficial, do not show substantial surface expression of MHC-II or CD86 depletion of circulating monocytes, which function to promote tissue (Fig. 3E). healing (42, 46), may counteract these positive effects. Indeed, it We also show that these extravasated neutrophils highly express was recently reported that leukapheresis to remove both monocytes accessory molecules that are important in T cell/APC interactions. and neutrophils (87) failed to demonstrate clinical efficacy in a For example, interaction between LFA-1 on T cells with ICAM-1 large-scale placebo-controlled clinical trial (88). Nevertheless, a on APCs has been shown to be critical for optimal T cell activation better understanding of the roles that the different myeloid cells (78, 79), and expression of ICAM-1 by APCs is thought to be play in immune function and their contribution to the pathogenesis important for the proper functioning of the immunological synapse of chronic gut inflammation may ultimately reveal new therapeutic during T cell activation (61, 80). Thus, a high level of ICAM-1 on strategies for the treatment of patients with IBD. cLP neutrophils is consistent with their more potent T cell stim- ulatory ability. In support of this contention, although we dem- Disclosures onstrate that both the spleen and cLP neutrophils are capable of The authors have no financial conflicts of interest. triggering Ag-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 References coculturing of neutrophils with T cells increased the production 1. Korzenik, J. R., and D. K. Podolsky. 2006. Evolving knowledge and therapy of of different chemokines known to be chemoattractive for T cells inflammatory bowel disease. Nat. Rev. Drug Discov. 5: 197–209. 2. Le Berre, N., D. Heresbach, M. Kerbaol, S. Caulet, J. F. Bretagne, J. Chaperon, (IP-10 and RANTES), neutrophils (KC), and monocytes (MCP-1) M. Gosselin, and M. P. Rame´e. 1995. Histological discrimination of idiopathic (Fig. 5). 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