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Keywords:

  • inflammatory bowel diseases;
  • Crohn's disease;
  • O-linked oligosaccharide;
  • IgA;
  • biomarker

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Background:

Ideal biomarkers are required to be developed for the diagnosis and prediction of the treatment of inflammatory bowel disease (IBD). We have reported that alteration of N-linked oligosaccharides of immunoglobulin (Ig) G is a novel diagnostic marker of IBD. Oligosaccharide alterations of IgA, however, have not been investigated in IBD patients.

Methods:

N- and O-linked oligosaccharides of serum IgA purified from 32 patients with Crohn's disease (CD), 30 patients with ulcerative colitis (UC), and 30 healthy volunteers (HV) were analyzed with high-performance liquid chromatography and mass spectrometry. Enzymes related to oligosaccharide attachment were investigated.

Results:

N-linked oligosaccharides of IgA were not different between IBD and HV. In contrast, the number of N-acetylgalactosamines per hinge glycopeptide (GalNAc/HP) in the O-linked oligosaccharides of IgA was significantly decreased in patients with CD compared with UC and HV. GalNAc/HP had high sensitivity and specificity for discriminating between CD and HV based on receiver operating characteristic analysis. Lower GalNAc/HP was associated with more severe disease activity of CD. Changes in GalNAc/HP levels in 6 weeks after treatment with infliximab were associated with the clinical activity of CD at 30 weeks. GalNAc transferase expression of naïve B cells and extent of GalNAc attachment in IgA were significantly decreased by interleukin-21 in vitro.

Conclusions:

The number of GalNAc attached in the IgA O-linked glycans of CD patients was significantly decreased, and strongly correlated with the clinical activity. Alterations of GalNAc attachment in IgA could be useful as a novel diagnostic and prognostic marker of CD. (Inflamm Bowel Dis 2012;)

Inflammatory bowel diseases (IBDs) are chronic intestinal disorders comprised of two major types: Crohn's disease (CD) and ulcerative colitis (UC). Although their pathophysiology remains unclear, recent studies have suggested that IBD is induced by a failure of the homeostatic mechanism of intestinal mucosal immune responses.1–3 To date, there are no serologic markers with sensitivity and specificity high enough to diagnose IBD.4 In addition, there are no ideal serologic markers to predict the clinical course of IBD. Thus, noninvasive serological markers of IBD must be developed in order to diagnose IBD and to predict responders to treatments. We have recently shown that oligosaccharide alterations in the N-glycan of serum immunoglobulin (Ig) G can be a novel diagnostic marker of IBD.5

In the mucosal immune system, IgA rather than IgG acts as the first line of defense against antigens or pathogens in the gut.6 Dysregulation of IgA-mediated homeostasis is suggested to trigger inflammatory disorders, such as IBD and celiac disease.7 IgA contains N-linked carbohydrates at position 263 in the constant region of heavy chain (CH) 2 and at position 459 in the tail-piece extension of CH3. IgA2 contains additional N-glycans in CH1 and CH2.8 In addition to the N-glycosylation sites, IgA1 has nine potential O-glycosylation sites in the 23-amino acid, proline-rich hinge region and usually four or five O-glycosylation sites are occupied by oligosaccharides.9 IgA2 lacks this hinge region and is not O-glycosylated. The mucin-type O-glycosylation is initiated by linking N-acetylgalactosamine (GalNAc) to the Ser or Thr of the protein backbone and this reaction is catalyzed by GalNAc transferases.10, 11 Oligosaccharide alterations of IgA related to disease have been studied in considerable detail in IgA nephropathy, in which IgA antibodies with agalactosyl O-linked oligosaccharides are precipitated in the nephron.12 Oligosaccharide alterations of IgA, however, have not been investigated in IBD.

Recently, mass spectrometry (MS) has been shown to be an effective tool for quantitation and measurement of site-occupancy of O-glycans.13 In this study we performed a detailed oligosaccharide analysis of N- and O-linked IgA glycans in IBD patients. We found that the number of N-acetylgalactosamines per hinge glycopeptide (GalNAc/HP) attached in the IgA O-linked glycopeptides of IBD patients was significantly decreased in patients with CD compared with healthy volunteers (HV). GalNAc/HP significantly negatively correlated with the Crohn's Disease Activity Index (CDAI) in CD. The enzymes related to the reduction of GalNAc/HP were further analyzed.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Subjects

Serum samples were collected from 32 patients with CD, 30 patients with UC, 30 HV, 17 patients with colonic inflammation including appendicitis, diverticulitis, and ischemic colitis (disease control; DC), and 10 patients with IgA nephropathy (Table 1). Intestinal tissues from patients with CD and HV were obtained during colonoscopy and surgery. All patients were Japanese and were recruited at the Department of Gastroenterology and Hepatology or Department of Geriatric Medicine and Nephrology, Osaka University Hospital (Suita, Osaka, Japan). Patients were diagnosed with CD or UC according to endoscopic, radiologic, histologic, and clinical criteria provided by the Council for International Organizations of Medical Sciences in the World Health Organization and the International Organization for the Study of Inflammatory Bowel Disease.14, 15 Disease behavior in CD (B1: nonstricturing and nonpenetrating; B2: stricturing; B3: penetrating) and disease severity in UC (S0: clinical remission; S1: mild; S2: moderate; S3: severe) were determined based on the Montreal Classification.16 Clinical activity was determined using the CDAI for CD17 or the Clinical Activity Index (CAI) for UC.18 IgA nephropathy was diagnosed histologically by renal biopsy. Detailed patient characteristics are presented in Table 1. Oligosaccharides of serum samples were measured before and after treatment with infliximab in 10 consecutive CD patients.

Table 1. Patient Characteristics
 HV (n = 30)DC (n = 17)UC (n = 30)CD (n = 32)IgAN (n = 10)
  1. IgAN, IgA nephropathy. The data are expressed as the mean ± SD in age, age at diagnosis, CRP, CAI, and CDAI. Other categories are shown as n (%).

Male/female13/179/815/1524/84/6
Age, y37 ± 939 ± 1641 ± 1436 ± 1232 ± 7
Age at diagnosis, y  35 ± 1327 ± 9 
Bowel surgery, n (%)  0 (0)19 (59) 
Extraintestinal manifestations, n (%)  0 (0)3 (9) 
Treatment     
 Salazosulfapyridine or mesalazine, n (%)  27 (90)29 (91) 
 Corticosteroids, n (%)  6 (20)3 (9) 
 Immunomodulators, n (%)  0 (0)3 (9) 
 Infliximab, n (%)  0 (0)0 (0) 
 Total parental nutrition or elemental diet, n (%)  2 (7)13 (41) 
Disease location     
 Small bowel/colon/small bowel and colon   11/5/16 
 Total /left colon/rectum and sigmoid  13/10/7  
Disease behavior of CD     
 Inflammatory/stricturing/penetrating/unknown   11/8/10/3 
Disease severity of UC     
 Clinical remission/mild/moderate/severe  4/15/9/2  
CRP, mg/dL7.7 ± 7.81.1 ± 3.31.1 ± 2.1 
CAI (UC) or CDAI (CD)  13 ± 7184 ± 89 

Analysis of N-linked Oligosaccharide of IgA by Reverse Phase High-performance Liquid Chromatography (HPLC)

IgA1 samples were isolated from 200 μL of serum using jacalin affinity chromatography (Vector Laboratories, Burlingame, CA).19 N-linked oligosaccharides were released from serum IgA1 and labeled with 2-aminopyridine, as described previously.20 Briefly, N-linked oligosaccharides were released from purified IgA1 samples by overnight incubation with 0.5 mU glycopeptidase F (Takara Bio, Shiga, Japan). Oligosaccharides were labeled with 2-aminopyridine by GlycoTag (Takara Bio) and incubated with 2 M acetic acid to remove sialic acids. Pyridylamino-oligosaccharides from IgA1 were analyzed on a reverse phase HPLC system (Waters, Milford, MA). Pyridylamino-oligosaccharides were detected using a fluorescence detector (Waters 2475) at wavelengths of 320 nm for excitation and 400 nm for emission.

Analysis of O-Linked Oligosaccharide of IgA Hinge Glycopeptides by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS)

IgA samples from 20 μL of serum were purified using affinity chromatography, which was manufactured by coupling anti-IgA antibody (Dako, Glostrup, Denmark) to a HiTrap NHS-activated HP column (GE Healthcare, Fairfield, CT). Isolated IgA was dissolved in a 0.5 mL solution of 6 M guanidine, 0.25 M Tris-HCl, pH 8.0, and then S-carbamidomethylated with 0.22 M iodoacetamide for 30 minutes at room temperature. After reaction, these chemicals were removed by gel filtration using an NAP5 column (GE Healthcare), and lysyl endopeptidase (Wako Pure Chemical Industries, Osaka, Japan) and trypsin (Promega, Fitchburg, WI) were added to the solution, which was then incubated at 37°C for 6 hours. Enrichment of glycopeptides from digests was carried out according to a method described previously.21 Briefly, a typical 100-μg digest was mixed with a 15-μL packed volume of Sepharose CL4B in butanol/ethanol/H2O (4:1:1, v/v). The gel was then incubated with an aqueous solvent of ethanol/H2O (1:1 v/v) for 30 minutes and the solution phase was recovered and dried using a vacuum concentrator. Desialylation of glycopeptides was carried out by incubation in 2 M acetic acid at 80°C for 2 hours. MALDI-TOF-MS was carried out on a Voyager DE Pro MALDI-TOF mass spectrometer with a nitrogen pulsed laser (Applied Biosystems, Foster City, CA). The peptide sample was mixed with 10 mg/mL of 2,5-dihydroxybenzoic acid dissolved in a 0.1% (v/v) trifluoroacetic acid and 50% (v/v) acetonitrile solution for measurement. The measurements were carried out in positive ion and linear TOF mode. The mass spectra acquired by at least 200 laser shots were accumulated and the measurement was repeated at least three times.22 The molar content of the component saccharides, GalNAc/HP and galactose per hinge glycopeptides (Gal/HP), was calculated by Equation 1 followed by Equation 2. Equation 1: (Glyco) peptide Peak % = [(Glyco) peptide Peak Intensity] / [Total (Glyco) peptide Intensity] × 10−2. Equation 2: GalNAc/HP, Gal/HP (mol/hinge glycopeptide) = Σ {(Glycopeptide Peak %) × (Number of GalNAc or Gal in the Glycopeptide)} × 10−2.13 The MALDI-TOF MS analysis was reproducible in repeated analysis of the same sample and we usually analyzed the oligosaccharides once per one sample.

Analysis of Anti-Saccharomyces cerevisiae Antibody (ASCA)

Serum ASCA concentrations were examined using the ASCA IgG enzyme-linked immunosorbent assay kit (Genesis Diagnostics, Cambridge, UK) according to the manufacturer's instructions. Values over 10 U/mL were defined as positive.

Isolation of B Cells and Naïve B Cells from Peripheral Blood Mononuclear Cells

Peripheral blood mononuclear cells were isolated from the heparinized venous blood of subjects by Ficoll–Hypaque density-gradient centrifugation. B cells and naïve B cells were separated by a B-cell isolation kit II and anti-IgD microbeads, respectively (Miltenyi Biotech, Bergisch Gladbach, Germany). Naïve B cells were cultured for 5 days in the presence of cytokines that induce IgA maturation; recombinant human CD40L (200 ng/mL; R&D Systems, Minneapolis, MN), interleukin (IL)-21 (50 ng/mL; BioSource International, Camarillo, CA), IL-10 (50 ng/mL; BioSource), or a proliferation-inducing ligand (APRIL, 500 ng/mL; R&D Systems).

Real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) for GALNT2

Total cellular RNA was isolated using Isogen-LS (Wako), and complementary DNA was synthesized from 0.1 to 0.5 μg of total RNA using the High Capacity RNA-to-cDNA Kit (Applied Biosystems). For TaqMan real-time RT-PCR, the reaction mixture was prepared by TaqMan Universal PCR Master Mix with predesigned and prelabeled TaqMan PCR primer and probe set for human polypeptide N-acetylgalactosaminyltransferase 2 (GALNT2), human IgA heavy chain, or human beta-actin endogenous control (Applied Biosystems). Real-time PCR was performed using an ABI PRISM 7900HT Sequence Detection System instrument and software (Applied Biosystems). Each sample was run in duplicate. The relative amount of GALNT2 RNA was calculated with the ΔΔCt method23 and normalized to IgA heavy chain.

Lectin Immunohistochemistry

Intestinal tissues from patients with CD and HV were embedded in paraffin. Tissue sections (5 μm thick) were deparaffinized, rehydrated, and subjected to antigen retrieval by incubation in a pressurized heating chamber (Dako). The sections were incubated in FITC-labeled mouse anti-human IgA1 or IgA2 (Southern Biotech, Birmingham, AL) and biotinylated jacalin (Vector Laboratories) followed by Texas red streptavidin (Vector Laboratories). The sections were mounted with Vectashield Hard Set Mounting Medium with DAPI (Vector Laboratories) and subjected to examination with a fluorescent microscope (Keyence BZ9000, Osaka, Japan).

Statistical Analysis

Differences were tested with the Mann–Whitney U-test or the Kruskal–Wallis test followed by the Mann–Whitney U-test, where appropriate. Either the χ2 test, χ2 test with Yates' correction (when sample number was less than 10), or Fisher's exact test (when sample number was less than 4), where appropriate, was used for the comparison of frequencies. Sensitivity for each test result was defined as the probability of a positive test result in a patient with the disease under investigation. Specificity was defined as the probability of a negative test result in a patient without the disease under investigation. A receiver operating characteristic (ROC) curve was generated by plotting sensitivity versus 1-specificity.24, 25 Area under the curve (AUC) was calculated by JMP software (SAS Institute, Cary, NC). P < 0.05 was considered statistically significant.

Ethical Considerations

This study protocol was approved by the ethical committee of Osaka University Graduate School of Medicine and written informed consent was obtained from each participant.

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

N-linked Oligosaccharide Analysis of IgA in IBD Patients

We previously demonstrated that N-linked oligosaccharide structures of IBD patients are significantly different from those of healthy subjects.5 Terminal galactoses of N-linked oligosaccharides in serum IgG are highly depleted in patients with IBD. We first analyzed N-linked oligosaccharide structures of IgA1 using HPLC. The agalactosyl peaks of fucosylated N-linked oligosaccharides (G0F) of IgA1 were low and were similar between HV and CD (Fig. 1A). In addition, the galactosyl peaks of fucosylated N-linked oligosaccharides (G2F) of IgA1 were also similar between HV and CD (Fig. 1A). When the peak height ratios of G0F/G2F of N-linked oligosaccharides of IgA1 were calculated, they were not significantly different between CD, UC, and HV (data not shown), whereas the peak height ratios of G0F/G2F were increased in IgG samples of CD and UC compared with HV (Fig. 1B). These findings indicate that the levels of N-linked agalactosyl IgA oligosaccharides are not different between IBD and HV.

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Figure 1. Analysis of N-linked oligosaccharides of immunoglobulins purified from HV and IBD patients. (A) Representative profiles of N-linked IgA1 oligosaccharides of HV and CD patient. IgA was purified from the sera of HV and CD patients using jacalin affinity chromatography and the structures of IgA1 N-linked oligosaccharides were analyzed by HPLC. G0F, the agalactosyl peak of fucosylated N-linked oligosaccharides; G2F, the galactosyl peaks of fucosylated N-linked oligosaccharides. (B) The ratio of agalactosyl/galactosyl fraction of fucosylated oligosaccharides (G0F/G2F) in the N-linked oligosaccharides of IgG. Boxplots show 50% of the relevant patient population. The line inside the box represents median value. Whiskers indicate the 90th and 10th percentiles. †P < 0.05.

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Number of N-acetylgalactosamines in the O-linked Oligosaccharides of IgA Is Decreased in CD Patients

We next analyzed the O-linked oligosaccharides of IgA1. Because complex O-linked oligosaccharide structures are difficult to analyze by HPLC, we used MALDI-TOF MS for the detailed analysis of O-linked oligosaccharide structures of IgA1. Each peak of MALDI-TOF MS chart has been shown to correspond with each oligosaccharide attached in the IgA HP.13 The MALDI-TOF MS analysis revealed that the peaks corresponding to the five or six GalNAc sugar residues of CD patients were lower than those of HV (Fig. 2A). In contrast, the peaks corresponding to the three or four GalNAc sugar residues of CD patients were higher than those of HV (Fig. 2A). Similar results were obtained when we analyzed several serum samples obtained from CD patients and HV. To confirm that GalNAc attachment in the hinge region of IgA is decreased in CD compared with HV, we next calculated the number of GalNAc attached to the IgA HP in patients with CD, UC, DC, IgA nephropathy, and HV. The number of GalNAc per each HP (GalNAc/HP) was significantly lower in patients with CD and IgA nephropathy than in patients with UC, DC, and HV (Fig. 2B). In addition, the number of GalNAc/HP was significantly lower in patients with UC compared with HV (Fig. 2B). The number of GalNAc/HP in UC, however, was not different from that in DC (Fig. 2B). We next calculated the mean number of galactose residues per each HP (Gal/HP). The number of Gal/HP residues was significantly decreased in patients with IgA nephropathy compared with HV (Fig. 2C), which was consistent with a previous report.26 In contrast to the deficiency of GalNAc/HP in IBD patients, the Gal/HP levels were not significantly different between IBD patients and HV (Fig. 2C).

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Figure 2. Analysis of O-linked oligosaccharides in hinge glycopeptides of IgA. Hinge glycopeptides that contain O-linked oligosaccharides were isolated and analyzed by MALDI-TOF mass spectrometry. Mass spectra of 30 HV, 17 patients with DC, 30 patients with UC, 32 patients with CD, and 10 patients with IgAN were evaluated. (A) Representative charts of MALDI-TOF mass spectra of O-linked oligosaccharides in IgA1 hinge glycopeptides of HV and CD. IgA was purified from the sera using IgA affinity chromatography and hinge glycopeptides that contain O-linked oligosaccharides were isolated. □, GalNAc; •, Gal; and □4•3 represents 4 GalNAc and 3 Gal attached to the hinge glycopeptide of IgA. The peaks corresponding to the five or six GalNAc sugar residues of CD patients (closed arrows) were lower than those of HV. In contrast, the peaks corresponding to the three or four GalNAc sugar residues of CD patients (open arrows) were higher than those of HV. (B) The number of GalNAc per hinge glycopeptide (GalNAc/HP) was analyzed using MALDI-TOF mass spectra. (C) The number of galactose residues per hinge glycopeptide (Gal/HP) was analyzed. DC, disease control; IgAN, IgA nephropathy. Boxplots show 50% of the relevant patient population. The line inside the box represents median value. Whiskers indicate the 90th and 10th percentiles. †P < 0.05 to HC, DC, UC, and CD; ††P < 0.05 to HV, DC, and UC; †††P < 0.05 to HV.

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Decrease of GalNAc in IgA O-linked Oligosaccharides Is Associated with an Increase in CDAI

We then investigated the relationship between GalNAc/HP and the clinical activity of CD and UC. GalNAc/HP showed a significantly negative correlation with CDAI in CD (Fig. 3A). When the CD patients were divided into those with active disease (CDAI ≥150) and those in remission (CDAI <150), GalNAc/HP was significantly lower in the active patients than in the remissive patients (Fig. 3B). GalNAc/HP levels were significantly lower in patients with stricture or penetration (Montreal category: B2+B3) than those with no evidence of stricture or penetration (Montreal category: B1, Fig. 3C). GalNAc/HP also showed a significantly negative correlation with CAI in UC (Fig. 3D). GalNAc/HP in active UC patients (CAI ≥6), however, did not significantly differ from that in patients in remission (CAI <6, Fig. 3E). In addition, GalNAc/HP levels were not significantly different among the Montreal categories (Fig. 3F). These findings indicate that the decreased GalNAc levels in IgA O-linked oligosaccharides are associated with active disease status of CD.

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Figure 3. Relationship between GalNAc/HP and disease activity. (A) Relationship between GalNAc/HP and CDAI in CD. (B) GalNAc/HP of CD patients in remission (CDAI <150) and in an active stage (CDAI ≥150). (C) GalNAc/HP of CD patients with category B1 and B2+B3 in the Montreal Classification. (D) Relationship between GalNAc/HP and CAI in UC. (E) GalNAc/HP of UC patients in CAI <6 and in CAI ≥6. (F) GalNAc/HP of UC patients with category S0, S1, S2, S3, and S4 in the Montreal Classification. †P < 0.05.

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GalNAc/HP as a New Serologic Marker for CD

We then investigated the effectiveness of the IgA O-linked oligosaccharide analysis as the serologic marker of IBD. In order to define the optimal cutoff value with the highest diagnostic accuracy, we performed multiple ROC curve analyses for distinct GalNAc/HP. The highest AUC was calculated for a GalNAc/HP of 4.50 with a sensitivity of 0.81 and specificity of 1.00. We defined GalNAc/HP <4.50 as “GalNAc deficient.” The GalNAc deficient rate in CD, UC, HV, and DC was 81%, 30%, 0%, and 11%, respectively (Fig. 4A). We next compared the sensitivity and specificity of GalNAc/HP with those of ASCA for the discrimination of IBD using an ROC curve. Both the sensitivity and specificity of GalNAc/HP were higher than those of ASCA for differentiation of CD and HV (AUC of GalNAc/HP vs. ASCA; 0.94 vs. 0.75, Fig. 4B). Moreover, both the sensitivity and specificity of GalNAc/HP were higher than those of ASCA for the differentiation of CD and UC (AUC of GalNAc/HP vs. ASCA; 0.71 vs. 0.59, Fig. 4C).

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Figure 4. Effectiveness of GalNAc/HP-positive rate as a serologic marker for IBD. (A) GalNAc/HP-positive rates were analyzed in CD, UC, HV, and DC. The cutoff point of GalNAc deficient was determined “GalNAc/HP = 4.50” by ROC curve. Fisher's exact test was used for the comparison between CD and UC, HV, or DC and between UC and HV. *1P < 0.05 to UC, HV, and DC. *2P < 0.05 to HV. (B) The ROC curves for GalNAc/HP (solid line) and ASCA (dotted line) levels for the discrimination between CD and HV, or (C) between CD and UC. Sensitivity is represented on the y-axis and 1-specificity on the x-axis.

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Alteration of O-linked Oligosaccharides Is Detectable in Inflamed Mucosa of CD Patients

To investigate where GalNAc-deficient IgA is produced in IBD patients, we performed lectin immunohistochemistry by using jacalin (galactose-GalNAc binding lectin) of intestinal tissues obtained from the patients with CD and HV. The number of the cells costained with anti-IgA1 antibody and jacalin in the lamina propria was lower in CD patients than in HV (Fig. 5). In contrast, lamina propria cells costained with anti-IgA2 antibody and jacalin were not observed in either CD patients or HV (Supporting Fig. 1). These findings show GalNAc in O-linked IgA oligosaccharides is deficient in the mucosal IgA1-producing cells of CD patients.

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Figure 5. Alterations of IgA1 O-linked oligosaccharides in the inflamed intestine of CD patients. Immunohistochemistry of the small intestine costained with anti-IgA1 antibody and jacalin, a galactose-GalNAc-specific lectin. Tissue sections of the small intestine of HV and inflamed small intestine of CD were stained with anti-IgA1-FITC antibody (green) and jacalin-Texas red (red). Double-positive cells for IgA1 and jacalin were observed in the mucosa of HV (arrowhead), but were decreased in the inflamed mucosa of CD. Representative pictures are shown.

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Expression of GalNAc Transferase mRNA Is Decreased in the Inflammatory Condition

In patients with IgA nephropathy, decreased activity of galactose transferase (C1GALT1; core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase, 1) in B cells is responsible for the reduction of galactose in the IgA O-linked oligosaccharides.27 We therefore investigated GalNAc transferase (GALNT2; polypeptide N-acetylgalactosaminyltransferase 2) expression, which may be responsible for the attachment of GalNAc in the peripheral blood B cells of the patients with CD. GALNT2 mRNA expression in the peripheral blood B cells and immortalized B cells by infection with Epstein–Barr virus isolated from IBD patients, however, was not decreased when compared with HV (data not shown). Because oligosaccharide alterations in IgA are suggested to take place in the inflamed mucosa, we next stimulated naïve B cells with CD40L, IL-21, and IL-10, all of which have been shown to provide signals for the maturation of IgA producing cells in the mucosa.28, 29 CD40L is essential for T-cell-dependent isotype switching of naïve B cells to IgA-producing cells, and both IL-10 and IL-21 can switch activated naïve B cells to IgA-producing plasma cells in the presence of CD40L. IL-21 is increased in the inflamed mucosa of the patients with CD.30 When the naïve B cells were stimulated with CD40L and IL-21 in vitro, GALNT2 was significantly decreased when compared with CD40L alone or CD40L and IL-10 (P < 0.05; Fig. 6A). Consistent with the decrease in GALNT2, GalNAc/HP levels in the IgA O-linked oligosaccharides were lower in the presence of CD40L and IL-21 (GalNAc/HP, 4.37) than the presence of CD40L alone (GalNAc/HP, 4.47; Fig. 6B).

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Figure 6. Reduction of GALNT2 expression and GalNAc of IgA O-linked oligosaccharides in B cells stimulated by IL-21. (A) The levels of polypeptide GalNAc transferase 2 (GALNT2) mRNA expression in B cells stimulated by cytokines that develop IgA-producing cells (CD40L, CD40L/IL-10, and CD40L/IL-21 [n = 4-9]). Results are shown as mean ± SE. †P < 0.05. (B) A representative chart of MALDI-TOF mass spectra of IgA O-linked oligosaccharides purified from B cells stimulated with CD40L (solid black spectrum) and CD40L/IL-21 (dotted red spectrum).

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Alteration of Oligosaccharides in IgA Recovers and Predicts Clinical Course After Treatment with Infliximab

We next investigated whether GalNAc/HP in IgA is affected by the treatment with infliximab, an antibody against tumor necrosis factor-alpha. When the levels of GalNAc/HP were compared between 0 and 6 weeks after the treatment with infliximab in 10 CD patients, GalNAc/HP was significantly higher at 6 weeks than 0 weeks (P < 0.05, Fig. 7A). In seven patients whose GalNAc/HP was increased at 6 weeks compared with that at 2 weeks, CDAI scores were decreased at 30 weeks compared with 0 weeks (Fig. 7B). In contrast, in three patients whose GalNAc/HP was decreased at 6 weeks compared with that at 2 weeks, CDAI scores were elevated after the treatment, including one patient who required small bowel resection (Fig. 7B).

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Figure 7. Alterations of O-linked oligosaccharides after treatment of infliximab. (A) GalNAc/HP levels before and after the treatment of infliximab. Ten patients with CD were treated with infliximab and serum samples were collected at 0 (before infliximab), 2, and 6 weeks. GalNAc/HP levels at 6 weeks were significantly higher than those at 0 weeks (†P < 0.05). (B) CDAI scores of CD patients from 0 and 30 weeks after treatment with infliximab. Three patients with decreased GalNAc/HP levels at 6 weeks compared with that at 2 weeks are indicated as dotted lines in (A,B). Cross mark shows that the patient received bowel surgery at 9 weeks.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

We recently reported that terminal galactoses are highly depleted in the N-linked oligosaccharides of IgG in IBD patients when compared with healthy subjects.5 IgA, rather than IgG, functions as a first line of defense in the mucosal immune system and functional abnormalities of IgA have been shown to cause mucosal immune disorders, such as IBD; selective IgA deficiency is shown to be associated with development of IBD.31, 32 An association of linear IgA dermatosis and IBD has also been reported.33 The association of IgA oligosaccharide alterations and IBD, however, has not yet been reported. We demonstrate for the first time that O-linked, but not N-linked, oligosaccharide structures of serum IgA1 are significantly altered in patients with CD compared with healthy subjects. Alterations of O-linked oligosaccharides were not due to galactose-deficiency, but rather to GalNAc-deficiency. Galactose-deficiency of N-linked oligosaccharides in IgG and GalNAc-deficiency of O-linked oligosaccharides in IgA were also reported in patients with rheumatoid arthritis, which is a similar immunologic disorder as CD.34 Our results suggest that CD has a biochemical disorder similar to rheumatoid arthritis for the formation of oligosaccharides of IgA and IgG. Association of O-linked oligosaccharides in IgA and disease development is best studied in detail in IgA nephropathy. Galactose-deficiency of O-linked oligosaccharides in IgA is commonly observed in patients with IgA nephropathy and can be a cause of nephron damage.35, 36 Indeed, IgA nephropathy is sometimes associated both with UC37 and CD,38 and IgA nephropathy followed after IBD is called secondary IgA-nephropathy.39 The O-linked oligosaccharide structures of IBD patients, however, were different from those of IgA nephropathy in terms of galactose deficiency and none of the IBD patients in our cohort was suffering from IgA nephropathy. Thus, the mechanism and consequence of the alteration of O-linked oligosaccharides in IgA of IBD patients are different from those in patients with IgA nephropathy.

Among the several serologic markers reported to have diagnostic value for IBD, ASCA and perinuclear antineutrophil cytoplasmic antibody have been extensively investigated and shown to be effective for discriminating CD and UC, respectively.40, 41 More recently, additional serum antibodies have been reported in CD patients, including those against the Pseudomonas-associated sequence I2, outer membrane porin C of Escherichia coli, and against the bacterial flagellin cBir1.42–44 Most recently, novel anti-glycan antibodies, such as against mannan (IgG anti-covalently attached anti-Saccharomyces cerevisiae antibodies) and anti-mannobioside (Man [a1,3]) carbohydrate IgG antibodies, were reported to link to CD.45 These serologic markers carry high specificities comparable to ASCA, ranging from 80% to 90%.46 Moreover, elevated levels of novel anti-glycan antibodies are associated with early disease onset, complicated disease behavior, and IBD-related surgery. On the other hand, the sensitivities of these markers, ranging from 20% to 55%, are not high enough for clinical use.46 We demonstrated that GalNAc/HP, which has relatively high sensitivity and specificity for IBD, is a potential diagnostic marker for IBD. In addition, GalNAc/HP reflects the clinical activity of CD and can have a predictive value for treatment. Further studies are required regarding the efficacy of GalNAc/HP for the diagnosis and prediction in IBD patients using larger samples and a high-throughput screening system needs to be established.

We have shown that GalNAc attachment in IgA hinge region was decreased in CD. Since galactose-deficiency has been reported to be caused by systemic deficiency of galactosyltransferase in IgA nephropathy,47 we speculated that GalNAc deficiency was due to the defect of GalNAc transferase activity in IBD patients. When freshly isolated peripheral blood B cells and IgA-producing cell lines obtained from IBD patients were analyzed, GalNAc transferase activity of IBD patients, however, was not decreased compared with that of HV. Lectin immunohistochemistry of the small intestine revealed that GalNAc-positive cells were rarely detectable in IgA1-producing cells in the lamina propria of CD patients. Thus, a great difference was observed in the affinity to jacalin of IgA1-producing cells between the inflamed mucosa of CD and normal mucosa of HV. The difference in the amount of GalNAc attached to serum IgA1 between CD and HV, however, was relatively small. The affinity to jacalin on IgA1-producing cells in noninflamed tissue of CD was high similarly to HV (data not shown). It is speculated that small changes in the serum IgA1 oligosaccharides in CD may be due to the dilution of highly GalNAc-deficient oligosaccharides produced in inflammatory tissue with oligosaccharides in noninflamed tissue, which are similar to those of HV. To investigate the mechanisms of GalNAc-deficiency in the inflamed lesion of CD, we evaluated GALNT2 expression in IgA-producing cells stimulated with inflammatory cytokines related to the maturation of IgA-producing cells. We showed that GALNT2 expression was significantly lower in B cells stimulated by CD40L and IL-21 than by CD40L alone or CD40L and IL-10. In addition, GalNAc attachment was less in B cells stimulated with CD40L and IL-21 than CD40L alone. It has been reported that isotype switching from naïve B cells to IgA plasma cells can be induced by CD40L together with IL-10 and/or IL-21, and it is called the “T-cell-dependent pathway”.29 Likewise, APRIL and BAFF (B-cell-activating factor belonging to the tumor necrosis factor family) have also been reported to induce isotype switching to IgA in a T-cell-independent manner.28 IL-21 is a cytokine that strongly induces IgA production,29 and elevated IL-21 levels in the mucosa and elevated IL-21 receptor expression are observed in IBD patients.30 Although IL-10 induces IgA production similarly to IL-21, production of IL-10 is decreased in CD patients.48, 49 When we cultured naïve B cells with APRIL, APRIL did not affect GALNT2 expression in vitro (data not shown). These results suggested that exposure to IL-21 in the mucosa of IBD can be one of the mechanisms for the defective GalNAc attachment in IgA and GalNAc/HP might be recovered after the introduction of infliximab by the restoration of the mucosal cytokine network.

To date, it is unknown if the function of IgA with GalNAc deficiency is altered in IBD patients. It has been reported that O-linked oligosaccharides strengthen the attachment of IgA and bacteria as well as the binding of IgA and mannan binding lectin, which is associated with complement activation.9 It has also been reported that T cells express receptors that have binding affinity with IgA O-linked oligosaccharides.50 These reports suggest that oligosaccharide alterations of IgA possibly serve to modify the immune function of CD. Further studies are needed to investigate the function of IgA with altered O-linked oligosaccharides and these projects are ongoing in our laboratory.

In summary, GalNAc/HP in O-linked oligosaccharides of IgA is significantly decreased in CD patients and reflects disease activity and clinical course. These findings suggest that GalNAc/HP can be a novel serologic marker for CD, and thus be of predictive value of CD treatments.

Acknowledgements

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

We thank Dr. Jan Novak, Department of Microbiology, University of Alabama at Birmingham for providing the polymeric and monomeric IgA1 myeloma protein that is deficient in galactose in O-linked glycans.

REFERENCES

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  8. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
IBD_22876_sm_SuppFig1.tif1876KSupporting Information Figure 1. Immunohistochemistry of the small intestine co-stained with anti-IgA2 antibody and jacalin. Tissue sections of the small intestine of HV and CD were stained with anti-IgA2-FITC antibody (green) and jacalin-Texas red (red). Representative pictures are shown.

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