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The Journal of Immunology, 2005, 175: 5412-5422.
Copyright © 2005 by The American Association of Immunologists

Carboxylated Glycans Mediate Colitis through Activation of NF-{kappa}B1

Geetha Srikrishna2,*, Olga Turovskaya{dagger}, Raziya Shaikh{dagger}, Robbin Newlin*, Dirk Foell{ddagger}, Simon Murch3,§, Mitchell Kronenberg{dagger} and Hudson H. Freeze2,*

* The Burnham Institute, La Jolla, CA 92037; {dagger} La Jolla Institute for Allergy and Immunology, San Diego, CA 92121; {ddagger} Department of Pediatrics, University of Münster, Münster, Germany; and § Centre for Pediatric Gastroenterology, Royal Free and University College Medical School, London, United Kingdom


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The role of carbohydrate modifications of glycoproteins in leukocyte trafficking is well established, but less is known concerning how glycans influence pathogenesis of inflammation. We previously identified a carboxylate modification of N-linked glycans that is recognized by S100A8, S100A9, and S100A12. The glycans are expressed on macrophages and dendritic cells of normal colonic lamina propria, and in inflammatory infiltrates in colon tissues from Crohn’s disease patients. We assessed the contribution of these glycans to the development of colitis induced by CD4+CD45RBhigh T cell transfer to Rag1–/– mice. Administration of an anti-carboxylate glycan Ab markedly reduced clinical and histological disease in preventive and early therapeutic protocols. Ab treatment reduced accumulation of CD4+ T cells in colon. This was accompanied by reduction in inflammatory cells, reduced expression of proinflammatory cytokines and of S100A8, S100A9, and receptor for advanced glycation end products. In vitro, the Ab inhibited expression of LPS-elicited cytokines and induced apoptosis of activated macrophages. It specifically blocked activation of NF-{kappa}B p65 in lamina propria cells of colitic mice and in activated macrophages. These results indicate that carboxylate-glycan-dependent pathways contribute to the early onset of colitis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Crohn’s disease and ulcerative colitis are chronic, debilitating, and multifactorial inflammatory bowel diseases (IBD).4 The etiology of IBD is unknown but is thought to involve a dysregulated mucosal immune response to gut-derived bacterial Ags (reviewed in Refs.1 and 2). In IBD, the normal immune tolerance to bacterial Ags is lost, and in Crohn’s disease, and in several animal models, it is replaced by a Th1-skewed cytokine response (3). This response is characterized by the expression of proinflammatory cytokines such as TNF-{alpha}, IL-12, and IFN-{gamma}, recruitment of macrophages and neutrophils, and tissue damage. However, the initiating events and mechanisms that sustain inflammation in IBD are not well defined.

The transfer of CD4+CD45RBhigh T cells to immunodeficient mice results in a colitis with features in common with Crohn’s disease, including requirement for Th1 cytokine secretion (4, 5). Professional APCs of mucosal tissues are key factors in the induction of both effector and regulatory responses in this and other models of colitis. In the transfer model, expansion of CD4+ T cells in the colon of reconstituted mice, and subsequent colitis pathogenesis require normal intestinal bacterial flora (6) and expression of MHC class II molecules on APC of the host (7). In addition, GALT APC are believed to exert a major influence on the polarization of the T cell response (8, 9, 10). Activated macrophages also produce cytokines and are effector cells in the tissue-destruction phase of inflammation (11, 12). In fact, changes in phenotypically distinct macrophage populations in IBD have been suggested to promote development of chronic inflammation (13).

We earlier identified a new type of anionic modification on N-linked sugar chains (glycans) from macrophages and endothelial cells (14, 15). These glycans are distinct from selectin ligands. Their key structural component is a carboxylate residue other than sialic or uronic acids. We recently showed that some of the carboxylated N-glycans contain glutamic acid directly or indirectly linked to the outer regions of the sugar chain (16). The carboxylated glycans bind to annexin I, S100A8/A9, S100A12, and high mobility group box-1 protein (HMGB-1) (17, 18), which have been implicated in acute and chronic inflammation (19, 20, 21, 22, 23). S100A12 and HMGB-1 both bind to receptor for advanced glycation end products (RAGE), a cell surface signaling receptor implicated in the pathology of inflammation, cancer, diabetes, and Alzheimer’s disease (24, 25, 26, 27, 28). Structurally diverse ligands bind RAGE through its extracellular V-type domain, where two N-linked glycosylation sites are located (29). A subpopulation of RAGE molecules carries the carboxylated glycans, and deglycosylation of the receptor significantly decreases binding of S100A12 and HMGB-1, showing that ligand binding is glycan dependent (Ref.18 ; G. Srikrishna and H. H. Freeze, unpublished observations). This is significant because several studies show that blocking RAGE-ligand interactions alleviates progress of inflammatory pathologies (20, 30, 31, 32).

RAGE was believed to mediate colitis through binding to S100A12 (20). Although mice do not have a functional S100A12 gene (33), structure-function studies suggest that murine S100A8 is a functional homolog of human S100A12 (34). In addition, S100A12 is overexpressed in inflamed human colonic tissues, as is S100A9 (11, 35, 36, 37, 38).

The above studies, which suggest a role for RAGE, S100A12, and/or S100A8/A9 in colitis, do not indicate that carbohydrate modifications of RAGE or any other molecule are important for the recognition events that mediate inflammation. Because the aforementioned myeloid-related S100 proteins are carboxylate-glycan binding lectins (17), and glycans on RAGE are important for ligand binding, we hypothesized that carboxylated glycans expressed on RAGE, or other putative S100 protein receptors, may have an in vivo role in the development of colitis. We showed earlier that mAbGB3.1, a mAb raised against carboxylated glycans, blocks acute peritoneal inflammation in mice (15). In the present study, we evaluated the effect of this anti-glycan Ab on the development of colitis in Rag1–/– mice that were transplanted with CD4+CD45RBhigh T cells. We demonstrate a potent blocking effect of the Ab on colitis and provide evidence for an NF-{kappa}B-mediated mechanism for the downstream effects of glycan recognition.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Human tissues

Colorectal tissue samples from patients with chronic active colitis were obtained from the frozen tissue bank of Department of Gastroenterology at the University of Münster. Frozen normal colon tissues obtained at surgical resections were also provided by the Cooperative Human Tissue Network of National Cancer Institute. Five-micrometer frozen sections were cut with a cryostat, mounted, and stored at –80°C until analysis. Extra biopsy samples for isolation of lamina propria cells were obtained during endoscopy performed at the Royal Free and University College School of Medicine, London, after ethical approval and written informed consent from patients.

Mice

Donor (C57BL/6 x BALB/c)F1 (CB6F1) and Rag1–/– mice were purchased from The Jackson Laboratory and maintained under specific pathogen-free conditions. Donors were used between 6 and 12 wk of age. Recipients received adoptive transfers between 7 and 12 wk of age. All mice experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the La Jolla Institute of Allergy and Immunology.

Induction of colitis and Ab treatment

CD4+CD45RBhigh lymphocytes were isolated from spleens of donor mice as described (6). Recipients were each injected i.v. with 4–5 x 105 sorted CD4+CD45RBhigh lymphocytes in 100 µl of sterile PBS. In the "preventive protocol," mAbGB3.1 (10 µg/g) was administered i.v. in 100 µl of PBS once in 10 days, starting 1 wk before T cell transfer, over a period of 7 wk for the long-term study, and 3 wk for the short-term study. Control mice were administered an equivalent amount of isotype-matched control Ab (TIB-132; American Type Culture Collection; mAb against an idiotypic determinant on the P3X63Ag8 myeloma protein). The recipient mice were weighed once every 5–6 days. They were constantly monitored for clinical signs of illness, including general appearance, piloerection, diarrhea, and bloody stools. Diseased animals were sacrificed at 3 or 7 wk posttransfer, or when they had lost 20% of their initial weight. In the "therapeutic protocol," mice received the Abs weekly starting either 10 or 21 days after T cell transfer and sacrificed 7 wk posttransfer. Segments of colon and intestine were removed from all mice and fixed in PBS containing 4% formalin. Colon, spleen, and mesenteric lymph nodes (MLN) were also embedded in optimal cutting temperature compound, frozen in dry ice/methylbutane mixture (–65°C), and stored at –80°C until analysis.

Clinical scoring

Sections from formalin-fixed colon tissues were stained with H&E. Tissues were graded semiquantitatively according to an established scoring system (6) by investigators blinded to the conditions: inflammatory infiltrate in the lamina propria (score 0–3); mucin depletion (score 0–2); reactive epithelial hyperplasia/atypia (score 0–3); and number of inflammatory foci per 10 high power fields (score 1–3).

Immunochemical analysis of tissue sections

Cryosections (6 µm) were air-dried and fixed in cold acetone for 2 min at room temperature. Sections were rehydrated in PBS; endogenous peroxidases were neutralized with 1% hydrogen peroxide and blocked with avidin/biotin (Vector Laboratories). Samples were then incubated with the respective primary Abs, followed by biotin-conjugated secondary Abs. Binding was detected using streptavidin-peroxidase complex (Vectastain ABC (avidin/biotin complex) kit; Vector Laboratories) and diaminobenzidine (DakoCytomation). Sections were then counterstained with hematoxylin. Abs used were mAbGB3.1 (15), and Abs against mouse Ags CD4, CD11b, MAdCAM-1, CD86, TNF-{alpha}, IFN-{gamma}, F4/80, and CD11c (BD Pharmingen). mAbGB3.1 was biotinylated in situ using DakoCytomation Animal Research kit to reduce background due to anti-mouse Ig secondary reagent.

For colon sections from patients with Crohn’s disease, endogenous alkaline phosphatase was blocked with 20% acetic acid in PBS. After avidin-biotin blocking, samples were incubated with the respective primary Abs, followed by biotin-conjugated secondary Abs. Binding was detected using ABC-alkaline phosphatase and alkaline phosphatase substrate (Vector Laboratories). Abs used were mAbGB3.1, anti-human S100A12 (38), and anti-human S100A9 (BMA Biomedicals).

Isolation of cells from colonic lamina propria, spleen, and MLN

Colonic mucosal immune cells were isolated as described earlier (6, 7). Cell suspensions from spleen and MLN were obtained using standard protocols.

Flow cytometric analysis

Cells from spleen, MLN, or colonic lamina propria of mice were resuspended in HBSS staining buffer containing 1% BSA. After preincubation with a blocking anti-FcR Ab (BD Pharmingen), cells were stained with FITC or R-PE-conjugated Ab or with unlabeled Ab followed by labeled secondary reagent. Cells were washed and analyzed immediately on a FACScan flow cytometer (BD Biosciences). Abs used in this study include mAbGB3.1, rabbit anti-RAGE (a kind gift of Novartis Tsukuba Research Institute, Japan), and Abs to mouse CD4, CD11c, CD11b, {alpha}4{beta}7 integrin (BD Pharmingen), and CD205 (Serotec). Lamina propria cells from Crohn’s patients were isolated, and flow-cytometric analysis was performed as described previously (39).

Immunoblots

Equivalent amounts of cell extract proteins from splenocytes or lamina propria cells were electrophoresed on denaturing and reducing 12% polyacrylamide gels, and transferred to nitrocellulose membranes. The blots were blocked with 10% dry skimmed milk, washed, and incubated with primary Abs followed by alkaline phosphatase-conjugated secondary Abs. Bound proteins were visualized using 5-bromo-4-chloro-3-indolyl phosphate/NBT (Sigma-Aldrich). Rabbit anti-mouse S100A8 and S100A9 were generated as described earlier (40).

Cell culture and LPS stimulation

RAW 264.7 cells obtained from American Type Culture Collection were maintained in DMEM (supplemented with glutamine, penicillin, and streptomycin and 10% FBS) at 37°C in a humidified incubator containing 5% CO2. For stimulation, cells were detached by vigorous pipetting and transferred to 12-well plates with fresh medium containing 2% serum at 1 x 106 cells/ml. Cells were stimulated with 1 µg/ml LPS from Escherichia coli serotype 0111:B4 (Sigma-Aldrich) in the presence or absence of 10 µg/ml mAbGB3.1 or isotype control Ab. Culture supernatants and cells were harvested at different time points after stimulation. Supernatants were stored at –80°C until analysis. RNA or nuclear extracts from cells were prepared immediately after harvest and stored at –80°C until analysis.

Measurement of cytokines and NO

TNF-{alpha}, IL-10, and IL-12 in culture supernatants or mice sera were measured using ELISA kits (BD Pharmingen). Total nitrite was measured using Griess reagent after reduction of NO3– to NO2– by nitrate reductase (R&D Systems).

RNA extraction and real-time PCR

PCR primers and TaqMan probes for TNF-{alpha} and IL-23 p19 were designed using PrimerSelect (DNAStar) and were obtained from BioSource International or PE Applied Biosystems. Primers and probes for GAPDH were obtained from PE Applied Biosystems. TaqMan probes contained a reporter dye (FAM) covalently attached to the 5' end and a quencher dye (BHQ) at the 3' end. Forward and reverse primers and probes were as follows: TNF-{alpha}: forward, 5'-CATCTTCTCAAAATTCGAGTGACAA-3', reverse, 5'-TGGGAGTAGACAAGGTACAACCC-3', and probe, 5'-FAM-CACGTCGTAGCAAACCACCAAGTGGA-BHQ1-3'; IL-23p19: forward, 5'-GTGCCCCGTATCCAGTGTGAAGA-3', reverse, 5'-GTGAAGTTGCTCCATGGGGCTATC, and probe, 5'-FAM-CCCACAAGGACTCAAGGACAACAG-BHQ1-3'. External standard for TNF-{alpha} was generated from total RNA extracted from mouse spleen by RT-PCR and cloned as described (41). Reference standard for IL-23 was obtained by various dilutions of RNA isolated from Con A-activated mouse spleen cells.

Total RNA was extracted from RAW264.7 or spleen cells using TRIzol reagent (Invitrogen Life Technologies). Reverse transcription and real-time PCR were performed in a single step, using the LightCycler-RNA Master Hybridization Probes kit (Roche Applied Science). Tth DNA polymerase, reaction buffer and dNTPs, forward and reverse primers, TaqMan probes, and purified RNA or standards at different dilutions in a total volume of 20 µl were directly added to LightCycler capillaries, which were inserted into a Roche LightCycler instrument. After incubating for 20 min at 61°C to allow mRNA reverse transcription, and an initial denaturation step at 95°C for 30 s, the cDNAs were amplified as described (41).

Measurement of apoptosis

Apoptosis of RAW264.7 cells at different time points after activation was measured by labeling with annexin V and propidium iodide (BD Pharmingen) followed by flow cytometry. Cell growth was measured by manual counting, and viability was assessed by trypan blue dye exclusion. Apoptosis was examined in colonic lamina propria of mice by a TUNEL assay using the ApopTag Fluorescein In Situ Apoptosis Detection kit (Chemicon International). Apoptotic macrophages were identified by double staining using rat anti-mouse F4/80 and Alexa Fluor 594-conjugated anti-rat IgG (Invitrogen Life Technologies).

Measurement of NF-{kappa}B binding activity in nuclear extracts

Nuclear extracts from RAW264.7 or lamina propria cells of mice were assayed for NF-{kappa}B binding activity using TransAM NF-{kappa}B assay kit (ActiveMotif) according to the manufacturer’s instructions. Mouse specific anti-p50 was obtained from Santa Cruz Biotechnology.

Statistical analysis

Statistical comparisons were performed using one-way ANOVA or Student’s t test. Differences were considered statistically significant when p < 0.05.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Endothelial cells and APC in human and mouse colon express carboxylated glycans

To begin to investigate a role of carboxylated glycans in the pathogenesis of IBD, we first examined tissues from patients with established IBD for glycan expression. In tissues from control subjects, anti-glycan Ab mAbGB3.1 stained endothelial cells and large mononucleated cells in the lamina propria (Fig. 1A). In tissues from two Crohn’s disease patients examined, the Ab also stained inflammatory infiltrates and serosal and submucosal aggregates of macrophages (Fig. 1B). The cells were identified as macrophages because they were large, mononucleated, and CD68 positive. Multiparameter analysis of lamina propria cells showed predominant expression of anti-glycan Ab epitope on CD11c+, CD80+, CD86+, and HLA-DR+ lamina propria cells from inflamed tissues (Table I). The infiltrating macrophage lesions in Crohn’s tissues also stained positive for S100A12 (Fig. 1, C and D) and S100A9 (not shown), whereas the S100 proteins were not detected in tissues from healthy controls, in agreement with other studies (38). Because S100A12 and S100A9 bind to carboxylated glycans recognized by mAbGB3.1 (henceforth referred to as anti-glycan Ab), this suggested up-regulation and colocalization of the ligand-receptor pair in inflamed regions of tissue in the patients.



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  		FIGURE 1. Expression of carboxylated glycans, RAGE, and S100 proteins. Immunohistochemical staining shows expression of anti-glycan Ab mAbGB3.1 epitope in normal human colon on endothelial cells (single arrow) and macrophages (double arrows) (A), and in inflamed colonic tissue of a patient with Crohn’s disease (B). Serial sections of the inflamed tissue (B and C) stained with anti-glycan Ab (B) and S100A12 (C) show that inflammatory infiltrates (indicated by arrows) express both the glycan epitope and the ligand. Enlarged image of an inflammatory infiltrate (D), stained with S100A12, shows the presence of large mononucleated macrophages. E-I represent analysis of non-reconstituted Rag1–/– mouse tissues and show anti-glycan Ab epitope (E and F), RAGE (G), their colocalization (H), and S100A8 and S100A9 (I) in colonic lamina propria (LP) and spleen. A and E, Sections stained with peroxidase-conjugated secondary reagent; B–D, sections stained with alkaline phosphatase-conjugated secondary reagent. Magnifications: A, x200; B and C, x100; D, x400; E, x400.

 

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  		Table I. mAbGB3.1 reactivity is expressed on APCs of colonic lamina propriaa

 
Using immunohistochemistry and flow-cytometric analysis, we also detected strong anti-glycan Ab reactivity in colonic lamina propria cells of non-reconstituted Rag–/– mice (Figs. 1, E and F). Soluble carboxylate-enriched glycopeptides decreased Ab binding to lamina propria cells, consistent with specific binding (Fig. 1F). Anti-glycan Ab reactivity was present on CD11c+ and CD205+ dendritic cells (DC) and CD11b+ macrophages (Table I). RAGE was also expressed in colonic lamina propria cells (Fig. 1G) and colocalized with anti-glycan Ab reactivity (Fig. 1H). Partial colocalization of anti-glycan Ab reactivity with RAGE suggested that other proteins besides RAGE also express the glycans. Immunoblotting showed the presence of S100A9 in cells of colonic lamina propria, and S100A8 and S100A9 in spleens of non-reconstituted Rag–/– mice (Fig. 1I). We also found HMGB-1 in spleen, MLN, and colonic lamina propria of these mice (not shown).

Anti-glycan Ab treatment blocks the onset of colitis in the adoptive cell transfer model

To further explore the role of the glycans in IBD, we tested the anti-carboxylate glycan Ab in the CD4+CD45RBhigh T cell transfer model of colitis. Animals were injected with a nonblocking control Ab or anti-glycan Ab once every 10 days and monitored for ~7 wk following cell transfer (half-life of the Abs in circulation was ~7–8 days). Reconstituted mice untreated or treated with the control Ab lost an average of 20–25% of their initial body weight (Fig. 2A) and suffered from severe diarrhea. Mice treated with the anti-glycan Ab showed minimal weight loss, did not have any diarrhea, and remained healthy throughout the treatment period. Histopathological examination of the colons revealed marked mucosal hyperplasia, goblet cell depletion, distortion of crypt architecture, and extensive inflammatory cell infiltration with occasional crypt abscesses in untreated or control Ab-treated mice, whereas in two-thirds of anti-glycan Ab-treated mice, colonic architecture was intact with minimal or no inflammatory changes (Figs. 2B and 3). Splenomegaly observed in reconstituted, untreated or control Ab-treated mice was also absent in all reconstituted mice treated with anti-glycan Ab (not shown).



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  		FIGURE 2. Anti-glycan Ab mAbGB3.1 treatment blocks colitis (preventive protocol). Recipient mice were administered with anti-glycan Ab mAbGB3.1 or a control Ab once every 10 days for 7 wk starting 1 wk before CD4+CD45RBhigh T cell transfer (arrow indicates cell transfer). A, Change in body weight over time is expressed as the percentage of starting weight (*, p < 0.05). B, Colonic inflammation was scored at the end of the experiment using an established scoring system (6 ). Each point represents mean ± SE of six mice in each group. *, p < 0.05 between reconstituted, untreated vs reconstituted, anti-glycan Ab treated.

 


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  		FIGURE 3. Minimal colonic inflammation in anti-glycan Ab-treated mice. H&E staining of representative proximal colon sections from Rag–/– mice 7 wk posttransfer: non-reconstituted (A), CD4+CD45RBhigh reconstituted (B), reconstituted mice treated with anti-glycan Ab (C), or reconstituted mice treated with control Ab (D). Extensive inflammatory cell infiltration, hyperplasia, mucin depletion, and crypt abscesses were observed in untreated and control Ab-treated mice, whereas in four of six anti-glycan Ab-treated mice, colonic architecture was intact with minimal or no inflammation. Images are at the same magnification (x200).

 
Anti-glycan Ab treatment initiated after cell transfer blocks progress of disease

In the transfer model, CD4+ T cell colonization in lamina propria is apparent 8–11 days after reconstitution, along with an early inflammatory infiltration (10). Severe colitis is established by 21 days posttransplantation. We therefore evaluated whether Ab administration after the initiation of colitis would block or reverse the disease. Mice received the Abs weekly starting either 10 or 21 days after T cell transfer and sacrificed 7 wk posttransfer. At 10 days posttransfer, we found evidence of inflammation in a parallel group of reconstituted, untreated mice (average inflammation scores of 2.5 in proximal and distal colons compared with 0.75 in non-reconstituted mice; n = 4). Anti-glycan Ab treatment started 10 days after cell transfer significantly impaired the progression of disease. This recovery was accompanied by reduced or no weight loss, good health, and reduced inflammation scores (Fig. 4). Splenomegaly observed in reconstituted, untreated or control Ab-treated mice was also absent in all reconstituted mice treated with anti-glycan Ab. However, administration of the anti-glycan Ab from 21 days after cell transfer did not prevent weight loss or symptoms of disease (not shown). This suggested that the Ab blocked an early step in colitis pathogenesis.



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  		FIGURE 4. Anti-glycan Ab treatment after initiation of colitis blocks progress of disease (therapeutic protocol). Reconstituted mice were treated with anti-glycan Ab or a control Ab weekly for ~6 wk starting 10 days (indicated by arrow) after T cell transfer. A, Change in body weight over time is expressed as the percentage of starting weight (*, p < 0.05). B, Colonic inflammation was scored at the end of the experiment as indicated in Fig. 2. Each point represents mean ± SE of four mice per group. *, p < 0.05 between reconstituted, untreated vs reconstituted and anti-glycan Ab treated. Colons examined at 10 days posttransfer in a parallel group of reconstituted, untreated mice showed evidence of mild inflammation (see Results).

 
In the transfer model, lymphocyte reconstitution is associated with an initial homeostatic as well as Ag-driven expansion of CD4+ T cells in secondary lymphoid organs (7). This is followed by generation of organ-tropic T cells with a Th1 cytokine profile. Because APC play an important role in T cell maturation and activation, the anti-glycan Ab may block one or more of APC-mediated pathways early in T cell pathology. To understand the mechanisms underlying the protective effects of the anti-glycan Ab, we therefore conducted systematic examination of tissues of mice (7 or 3 wk after reconstitution) for cellular accumulation and expression of cytokines and other molecules.

Leukocyte accumulation is reduced in the colons of Ab-treated mice

We first examined accumulation of T cells in different compartments of reconstituted mice. At 7 wk posttransfer, CD4+ T cell infiltration was found throughout the intestine and colon in recipient mice, and this was unaffected by control Ab treatment. In mice treated with the anti-glycan Ab, there was a marked reduction in the accumulation of CD4+ T cells in the lamina propria of the colon (Fig. 5A). This effect was site specific, because accumulation of T cells in the small intestine (not shown) was unaffected by anti-glycan Ab treatment. This suggested that the colonic microenvironment, whose critical feature is the presence of a rich bacterial flora, contributes to the Ab-mediated effects.



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  		FIGURE 5. Anti-glycan Ab-treated mice show reduced accumulation of CD4+ T cells in colon. A, At 7 wk posttransfer, CD4+ T cell accumulation, assessed by immunochemical staining using anti-CD4, was reduced in colon (six mice per group). All images are at the same magnification (x200). B, At 3 wk posttransfer, the total number of CD4+ cells was quantitated by flow-cytometric analysis of cells from spleen, MLN, and lamina propria of control Ab- or anti-glycan Ab-treated mice. Each is the mean ± SE of three mice per group. *, p < 0.05.

 
We also examined CD4+ T cell accumulation 3 wk after T cell reconstitution. Compared with mice treated with the control Ab, the anti-glycan Ab significantly reduced accumulation of CD4+ T cells in the colonic lamina propria, but it did not affect accumulation in the spleen and MLN (Fig. 5B). This indicated that the anti-glycan Ab treatment affected early as well as late accumulation of T cells in the colon.

Next, we examined colon tissues of reconstituted mice for accumulation of inflammatory cells 7 wk posttransfer. In non-reconstituted mice, CD11b+ and F4/80+ myeloid cells were distributed throughout the lamina propria of both the proximal and distal colon (Fig. 6, shown for proximal colon). The expanded lamina propria of reconstituted mice untreated or treated with control Ab contained significant inflammatory infiltrates consisting predominantly of CD11b+ and F4/80+ macrophages and a smaller number of neutrophils in crypt abscesses (Fig. 6). Clusters of activated macrophages were confined to lamina propria, but in severe disease, they also extended to the submucosa. In reconstituted mice treated with anti-glycan Ab, accumulation of CD11b+ and F4/80+ cells was markedly reduced (Fig. 6).



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  		FIGURE 6. Anti-glycan Ab treatment blocks inflammatory cell infiltration and up-regulation of MAdCAM in the colon of reconstituted mice. CD11b+F4/80+ cellular infiltration and MAdCAM expression in mice colon 7 wk posttransfer was assessed by immunohistochemical staining using specific Abs (six mice per group). All images are at the same magnification (x200).

 
Anti-glycan Ab reduces expression of mucosal addressin cell adhesion molecule 1 (MAdCAM-1)

MAdCAM-1 and the gut-homing integrin {alpha}4{beta}7 are important for recruitment of T cells to the inflamed colonic tissue (42, 43). We therefore examined whether reduced T cell accumulation in the colons of anti-glycan Ab-treated mice was due to reduced frequency of gut tropic T cells or reduced expression of MAdCAM-1. Analysis of T cells from MLN of anti-glycan Ab-treated mice at 3 wk posttransfer showed no reduction in the frequency of {alpha}4{beta}7+ cells (11.3 ± 1.5%; n = 3), compared with the control Ab-treated mice (12.5 ± 0.7%; n = 3). However, markedly increased expression of MAdCAM-1 was seen in inflamed tissues from untreated or control Ab-treated mice, whereas the addressin expression on venules in colons of anti-glycan Ab-treated mice was similar to constitutive expression seen in non-reconstituted mice (Fig. 6). This suggested that the anti-glycan Ab could block the infiltration of gut tropic T cells to the colon by decreasing MAdCAM-1 expression.

Proinflammatory cytokines are reduced in tissues and blood of anti-glycan Ab-treated mice

TNF-{alpha} and IFN-{gamma} are also important mediators of disease in the adoptive transfer model (4). Th1 cells produce IFN-{gamma}, whereas TNF-{alpha} is a product of both Th1 cells and macrophages that are activated by them. To investigate the role of the anti-glycan Ab in blocking activation of T cells and effector functions of Th1 cells, we analyzed expression of proinflammatory cytokines in tissues and sera of mice.

At 7 wk posttransfer, in reconstituted and untreated or control Ab-treated mice, increased IFN-{gamma} expression was associated with the presence of increased CD4+ lymphocytes in the lamina propria and submucosa, whereas in the colon of anti-glycan Ab-treated mice, IFN-{gamma} expression was minimal (Fig. 7). Anti-glycan Ab treatment also reduced the frequency of IFN-{gamma}-positive lymphocytes in spleen examined 3 wk posttransfer (Table II).



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  		FIGURE 7. IFN-{gamma} and TNF-{alpha} expression is reduced in tissues of anti-glycan Ab-treated mice. IFN-{gamma}- and TNF-{alpha}-positive cells in tissues of non-reconstituted and reconstituted mice at 7 wk posttransfer were detected using specific Abs (six mice per group). Arrows mark IFN-{gamma}+ cells. All images are the same magnification (x200).

 

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  		Table II. IFN-{gamma}-positive T cells in spleen

 
At 7 wk posttransfer, TNF-{alpha} was highly expressed in colonic lamina propria in reconstituted and untreated or control Ab-treated mice (Fig. 7). TNF-{alpha} expression showed a temporal correlation with macrophage (CD11b+F4/80+) infiltration, suggesting that macrophages are the principal source of TNF-{alpha}. Expression of TNF-{alpha} in colonic tissue was greatly reduced in anti-glycan Ab-treated mice. We examined the efficacy of Ab therapeutic modality on the production of TNF-{alpha} in colitis. TNF-{alpha} levels in blood were elevated as early as 10 days posttransfer and were maximum 7 wk posttransfer. When anti-glycan Ab treatment was initiated 10 days after T cell transfer, TNF-{alpha} measured 7 wk posttransfer was reduced to the levels seen in non-reconstituted mice (Table III).


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  		Table III. Levels of TNF-{alpha} in sera of reconstituted mice (therapeutic protocol)a

 
Anti-glycan Ab blocks proinflammatory cytokine production by macrophages and induces their apoptosis

To further confirm that anti-glycan Ab treatment down-regulates proinflammatory cytokine production in the colon, we analyzed the effects of the Ab on LPS-elicited production of cytokines by macrophages in vitro. LPS interacts with CD14 and the TLR4 complex on macrophages to activate multiple signaling pathways (44) and secretion of proinflammatory cytokines IL-12, TNF-{alpha}, and IL-1, as well as anti-inflammatory cytokines, including IL-10 (45). Anti-glycan Ab-reactive epitope is constitutively expressed on RAW264.7 macrophages (not shown). We therefore activated these macrophages in vitro with LPS in the presence or absence of anti-glycan Ab or the isotype control Ab. LPS increased TNF-{alpha} mRNA production in untreated cells by 3-fold (Fig. 8A). This was followed by an enhanced secretion of TNF-{alpha} into the culture medium. The anti-glycan Ab significantly reduced LPS-stimulated TNF-{alpha}. It also inhibited LPS-induced production of IL-23 mRNA, and secretion of IL-12, and NO, whereas it had no effect on the production of IL-10 (Fig. 8A). In addition, treatment of LPS-activated cells with the anti-glycan led to increased apoptosis of the macrophages, as determined by cellular growth (Fig. 8B) and annexin V staining (not shown). This was activation dependent, because the Ab had no effect on the growth of unstimulated cells.



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  		FIGURE 8. Anti-glycan Ab inhibits LPS-elicited cytokine expression and induces apoptosis of RAW264.7 macrophages. A, RAW264.7 macrophages in culture were activated with LPS in the presence or absence of anti-glycan Ab or an equivalent amount of isotype control Ab. Cellular cytokine mRNA (1 h after activation) was measured by real-time PCR. Secreted cytokines (in supernatants collected 20 h after activation), and NO were measured as described in Materials and Methods. Each point is the mean ± SE of two experiments, with duplicate measurements for each assay. *, p < 0.05 between activated, untreated vs activated, anti-glycan Ab treated. B, Treatment of LPS-activated cells with the anti-glycan Ab led to increased apoptosis, as determined by cellular growth (expressed as percentage of cells in unactivated cultures for each time point) and annexin V staining and flow cytometry (not shown). The Ab had no effect on the growth of unstimulated cells. Each point is the mean of duplicate measurements.

 
To determine whether the anti-glycan treatment induced apoptosis of activated (infiltrating) macrophages in vivo, we performed an immunochemical analysis of lamina propria macrophages 7 wk posttransfer in the therapeutic protocol. We identified apoptotic macrophages by F4/80 and TUNEL double staining. In untreated and treated mice, >90% of apoptotic cells were T cells. In the untreated mice and in the control Ab-treated mice, the TUNEL+ non-T cells did not show any overlap with F4/80-stained cells. In the anti-glycan Ab-treated mice, ~50% of TUNEL+ non-T cells were macrophages, because they were also positive for F4/80 (not shown). These results suggested that the anti-glycan Ab might specifically block proinflammatory but not anti-inflammatory cytokine production and may also promote apoptosis of infiltrating macrophages in colitis. Further confirmation would require more kinetic examination of apoptotic infiltrating macrophages at different time points after reconstitution in the untreated and treated mice.

RAGE and carboxylated glycan binding proteins S100A8, S100A9, and HMGB-1 are up-regulated in colitis

Both IFN-{gamma} and TNF-{alpha} stimulate macrophage expression of S100A8 (46), and S100A9 expression is up-regulated in activated spleen cells (47). S100A8 is chemotactic in mice (48, 49) and S100A9 promotes integrin-mediated adhesion of phagocytes (50). Together, they may provide a strong stimulus for early infiltration of neutrophils and monocytes. We therefore examined whether S100A8 and S100A9 are up-regulated in inflamed tissues of reconstituted mice. Immunoblot analysis showed that S100A8 and S100A9 are strongly up-regulated in spleen and colonic lamina propria of reconstituted and untreated or control Ab-treated mice examined 3 wk posttransfer, but not in anti-glycan Ab-treated mice (Fig. 9). We also found up-regulation of RAGE in colonic lamina propria and spleens of untreated and control Ab-treated mice, but not in anti-glycan Ab-treated mice (Fig. 9). The two forms of RAGE seen in spleen may represent proteins encoded by alternatively spliced mRNAs (51). In addition, we also detected HMGB-1 in the sera of mice with inflammation, whereas it was undetectable in unreconstituted mice (not shown).



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  		FIGURE 9. Myeloid-related proteins S100A8 and S100A9 and RAGE are up-regulated in tissues from colitic mice. Twenty micrograms of extract proteins were prepared from splenocytes or colonic lamina propria cells of mice 3 wk posttransfer, separated on SDS-PAGE gels, and probed using specific Abs after transfer. Lanes: 1, non-reconstituted; 2, reconstituted, untreated; 3, reconstituted, anti-glycan Ab treated; 4, reconstituted, control Ab treated. The two forms of RAGE at 45 and 32 kDa in spleen may represent splice variants.

 
Because anti-glycan Ab treatment started 10 days after cell transfer significantly impaired progression of disease, we next examined whether S100A8, S100A9, and RAGE are up-regulated in tissues at 10 days posttransfer. ELISA quantitation showed that S100A9 was moderately up-regulated in spleen (~2-fold; p < 0.005), but not in colonic lamina propria, compared with non-reconstituted mice (n = 4 each group; data not shown). Expressions of RAGE and S100A8 in spleen and colonic lamina propria were unaffected 10 days posttransfer in both spleen and lamina propria. This suggests that S100A9 up-regulation in spleen may be an early event during initiation of colitis, and may play an important role in the anti-glycan Ab-mediated effects.

Anti-glycan Ab reduces activation of NF-{kappa}B p65

Because HMGB-1, S100A8, S100A9, and S100A12 are all strong inducers of NF-{kappa}B (20, 52), the above results suggested that NF-{kappa}B activation could play a role in carboxylated glycan-mediated effects. RAGE promoter has two NF-{kappa}B binding sites and activation of NF-{kappa}B by RAGE ligation results in up-regulation of the receptor, thus amplifying the signal and initiating a pathological cycle of cellular perturbation.

At 3 wk posttransfer, NF-{kappa}B p65 was highly up-regulated in lamina propria cells of recipients untreated or treated with control Ab, but was significantly reduced in cells from anti-glycan Ab-treated mice (Fig. 10). In vitro, LPS-stimulated RAW264.7 macrophages showed significant activation of NF-{kappa}B, which was blocked by anti-glycan Ab treatment. We observed inhibition of activation of NF-{kappa}B p65, but not that of other NF-{kappa}B/Rel family members (Fig. 10). This finding is interesting because NF-{kappa}B p65 is increased in colonic lamina propria of Crohn’s disease patients (53, 54, 55), and administration of p65 antisense oligonucleotides reduces clinical and histological signs of trinitrobenzene sulfonic acid- or dextran sulfate sodium-induced colitis (56, 57, 58). We also found a mild but statistically significant up-regulation of NF-{kappa}B p65 in the spleen (~1.5-fold; p < 0.05; not shown), but not in colonic lamina propria of mice 10 days posttransfer, compared with non-reconstituted mice (n = 4 each group), concomitant with an increase in S100A9 in spleen, and elevated TNF-{alpha} in serum (Table III). Because S100A9 binds carboxylated glycans (17), and S100A9 can induce NF-{kappa}B activation (52), inhibition of NF-{kappa}B activation could be a crucial factor in the disease-blocking effects of the anti-glycan Ab.



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  		FIGURE 10. Anti-glycan Ab treatment inhibits activation of NF-{kappa}Bp65 in RAW264.7 macrophages. Nuclear extracts were prepared from lamina propria cells from reconstituted mice 3 wk posttransfer (A) and RAW264.7 macrophages treated with 1 µg/ml LPS (with and without priming with IFN-{gamma}) for 60 min in the presence of anti-glycan Ab or a control Ab (B). Twenty micrograms of nuclear lysate protein was incubated in wells coated with consensus NF-{kappa}B binding oligonucleotide sequences, and bound protein was measured using anti-p65 or anti-RelB. *, p < 0.05 between reconstituted, untreated vs reconstituted, anti-glycan Ab-treated mice (n = 3) (A) or LPS-activated, untreated vs LPS-activated, anti-glycan Ab-treated macrophages (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Oligosaccharides are increasingly being recognized as important mediators of signaling in innate and adaptive immune responses (59, 60, 61, 62, 63). Considerable diversity of oligosaccharide structures provides enormous potential for information display on cell surfaces and specific recognition by different lectins. Glycans that have the same structure can also have different functions depending upon the proteins and cell types that carry them. Examples are the selectin ligands that mediate both inflammation-initiated leukocyte rolling as well as physiological lymphocyte homing and recirculation. However, not all glycan structures in mammals have been proven, much less functionally characterized. We earlier identified a family of novel carboxylated glycans on endothelial cells and macrophages that mediate inflammation. Here, we show that interfering with the interaction between these glycans and their putative lectin partners using a monoclonal anti-glycan Ab prevents the pathogenic process in a mouse model of IBD.

NF-{kappa}B/Rel family of plieotropic transcription factors play a pivotal role in host immune and inflammatory responses (64, 65, 66) and are believed to be important in the pathophysiology of IBD. They are activated in response to a range of stimuli, including pathogens, proinflammatory cytokines, and stress. They regulate expression of proinflammatory cytokines, adhesion molecules, and inducible NO synthase, and play an antiapoptotic role in many systems (67). Our study shows that carboxylated glycans may play a critical role in early events of colitis pathology by blocking NF-{kappa}B activation. However, the specific events and mediators remain unknown. The Ab may block either a single step or multiple steps during the initiation or recurrence of disease. Likely early targets include DC maturation, DC-T cell interactions, proliferation and polarization of T cells, homing, and infiltration and survival of inflammatory cells into the colon. The sugar chains may mediate multiple interactions involving different cell types and ligand-receptor pairs, such as RAGE and its ligands, and a network, rather than a linear sequence in the activation of NF-{kappa}B.

Although the effects of the Ab are predominantly localized to the colon, peripheral effects such as blocking initial Ag-driven expansion and activation of T cells in spleen cannot be ruled out. CD4+ T cells in colon and spleen have an activated phenotype at early time points following cell transfer (7). DCs from colon migrate to draining lymph nodes and peripheral sites providing a mechanism for systemic activation of T cells to mucosal Ags (68). The anti-glycan Ab may have an initial inhibitory effect on systemic expansion and activation of T cells. Reduced IFN-{gamma}-positive T cells in spleen of anti-glycan Ab-treated mice, associated with reduced expression of S100A8 and S100A9 and RAGE in spleen 3 wk posttransfer support this view. In addition, we also found up-regulation of S100A9 and NF-{kappa}B in the spleen of mice 10 days posttransfer. These findings underscore the importance of NF-{kappa}B-mediated initial events in secondary lymphoid organs in the onset of disease.

Possible roles of S100 proteins and RAGE in the early onset of colitis and in mediating the protective effects of the anti-glycan Ab need further exploration. We showed earlier that RAGE from bovine and mouse lung express the glycans (18), and the glycans mediate RAGE-ligand binding (18). RAGE ligation leads to long-term activation of NF-{kappa}B and inflammation (20, 69). S100 proteins and HMGB-1 released from activated inflammatory cells provide a potent positive feedback for sustained pathology by activating RAGE-mediated signaling pathways, activation of NF-{kappa}B and up-regulation of RAGE expression (25). Sustained RAGE activation combined with an exaggerated host response in the absence of regulatory mechanisms such as seen in IBD and experimental colitis could lead to irreversible complications of disease. The anti-glycan Ab could block this cycle by interfering with RAGE-ligand interaction. However, RAGE knockout mice have normal adaptive immune responses (70). In preliminary studies, we found that CD4CD45RBhigh cells from RAGE–/– mice were as effective as wild-type donor T cells in eliciting disease (not shown). Also, the presence of RAGE–/–GB3.1+/+ immune cells in wild-type mice (Fig. 1; G. Srikrishna and H. H. Freeze, unpublished observations) brings up the question whether the glycans mediate their effects through RAGE, or independent of RAGE, or both. In addition, the cellular receptors for S100A8/A9 have not been identified. Our future studies will address these questions.

In summary, our findings indicate that carboxylated glycans expressed on APC and other cells may play critical roles in the initiation and progression of colitis. Because colon tissues from Crohn’s disease patients express increased levels of the glycan epitope, further characterizing and targeting of the carboxylated glycan-dependent pathway(s) may be a promising new approach to the treatment of human IBD.


   Acknowledgments
 
We are grateful to Dr. Nissi Varki (University of California, San Diego, La Jolla, CA) for expert histology advice and for critical reading of the manuscript. We thank Charles DeRossi (The Burnham Institute), Dr. Patrick Stordeur (Erasme University Hospital, Brussels, Belgium), and David Boyle and Dr. Sanna Rosengren (University of California, San Diego) for help and advice with cytokine real-time PCR assays. We also thank Dr. Paul Ashwood (Royal Free and University College Medical School, London) for flow-cytometric analysis of human Crohn’s samples, Jonamani Nayak (The Burnham Institute) for excellent technical help, and Douglas Haynes for help with illustrations. Dr. Angelika Bierhaus (University of Heidelberg, Heidelberg, Germany) kindly provided the RAGE knockout mice used in initial studies mentioned in Discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the Broad Medical Research Program of the Eli and Edythe L. Broad Foundation and by National Institutes of Health Grants R01-CA92608 (to H.H.F. and G.S.) and PO1 DK46763 (to M.K.). Back

2 Address correspondence and reprint requests to Dr. Geetha Srikrishna or Dr. Hudson H. Freeze, Glycobiology Program, The Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. E-mail addresses: gsrikrishna{at}burnham.org and hudson{at}burnham.org Back

3 Current address: Clinical Sciences, Warwick Medical School, Coventry CV2 2DX, U.K. Back

4 Abbreviations used in this paper: IBD, inflammatory bowel disease; HMGB-1, high mobility group box-1 protein; RAGE, receptor for advanced glycation end products; MLN, mesenteric lymph node; DC, dendritic cell; MAdCAM-1, mucosal addressin cell adhesion molecule 1. Back

Received for publication May 17, 2005. Accepted for publication August 9, 2005.


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