Increased numbers of immature plasma cells in peripheral blood specifically overexpress chemokine receptor CXCR3 and CXCR4 in patients with ulcerative colitis

Authors


S. Hosomi, Department of Gastroenterology, Osaka City University Graduate School of Medicine, 1-4-3, Asahi-machi, Abeno-ku, Osaka 545-8585, Japan. E-mail: a96m062@med.osaka-cu.ac.jp

Summary

Ulcerative colitis (UC) is a chronic inflammatory bowel disease featuring infiltration by plasma cells producing immunoglobulins. We have reported previously the specific and significant proliferation of immature plasma cells in the inflamed colonic and pouch mucosa of UC patients. The aim of this study was to characterize peripheral blood immature plasma cells and the migration mechanisms of such immature plasma cells to inflamed sites in UC. The characteristics of peripheral blood immature plasma cells and chemokine receptor expression were examined by flow cytometry. Expression of mucosal chemokine was quantified using real-time reverse transcription–polymerase chain reaction and immunohistochemistry. The number of peripheral blood immature plasma cells was significantly higher in patients with active UC and active Crohn's disease (CD) than in healthy controls. The proportion of immature plasma cells was correlated positively with clinical activities of UC and CD. Many peripheral blood immature plasma cells were positive for CXCR3, CXCR4, CCR9 and CCR10. Expression of CXCR3 and CXCR4 in UC patients was significantly higher than in controls. CXCL9, CXCL10 and CXCL11 mRNA levels in colonic mucosa of inflamed IBD were higher than in controls. Immunofluorescence study also showed abundant CXCR3-positive immature plasma cells in the inflamed colonic mucosa of UC. Increased numbers of immature plasma cells may migrate towards inflammatory sites of UC via the CXCR3 axis, and may participate in UC pathogenesis.

Introduction

Ulcerative colitis (UC) and Crohn's disease (CD) are chronic, idiopathic and relapsing forms of inflammatory bowel disease (IBD) of unknown aetiology. A dysregulated inflammatory immune response has been suggested in the pathogenesis of IBD. Basal plasmacytosis, a dense infiltration of plasma cells in the lower one-third of the mucosa, is a classical pathological finding in UC [1]. Activation of humoral immunity based on T helper type 2 (Th2) cell-shifted immunity has been implicated in UC pathogenesis, whereas Th1-shifted activation of cellular immunity has an important role in CD pathogenesis [2]. In UC, lamina propria lymphocytes contain activated B cell subsets [3], and local immunoglobulin (Ig) G overproduction shifts to the highly complement-activating IgG1 subclass [4]. Hibi et al. [5,6] reported that lymphocytes isolated from the inflamed mucosa and peripheral blood of patients with UC secreted autoantibodies against colonic epithelial cells. Generation of autoantibodies to tropomyosin-5 [7,8], expressed by the intestinal epithelium, and perinuclear anti-neutrophil antibodies (p-ANCA) [9,10] are also a characteristic humoral immune response in patients with UC. However, little is known about the mechanisms and processes of B cell activation in the pathogenesis of inflammatory bowel disease.

We have reported the specific and significant proliferation of immature plasma cells in the inflamed colonic [11] and pouch [12] mucosa of patients with UC compared with healthy controls and patients with CD. Although these immature plasma cells may have important roles in abnormal humoral immune responses, including autoantibody production, the mechanisms of migration of immature plasma cells to inflamed sites of UC have not been clarified.

Chemoattractant cytokines (chemokines), together with tissue-specific adhesion molecules, have a central role in the migration of antibody-secreting cells to target tissue [13]. In particular, the chemokines/chemokine receptors axis is the most important regulator of leucocyte trafficking in infection or inflammation, by switching the expression of the chemokine receptor profile on these leucocytes during activation and differentiation [14,15]. The association between inappropriate activation of the chemokine network and several diseases has been investigated by many researchers [16]. In IBD, high expression of chemokine receptors on T cell lineages has been shown [17,18], however, little has been reported on the expression of chemokine receptors on B cell populations (including plasma cells).

The aim of this study was to characterize peripheral blood immature plasma cells and the migration mechanisms of such immature plasma cells to inflamed sites in IBD.

Materials and methods

This study was approved by the Ethics Committee of Osaka City University. Informed consent was obtained from all patients and healthy controls.

Patients and samples

The diagnosis of IBD was based on clinical, endoscopic and histopathological findings. The disease activity of UC patients was determined according to the Mayo Clinic score [19], whereas the disease activity of CD patients was determined according to the Crohn's Disease Activity Index (CDAI) [20]. Clinical remission was defined as a total Mayo Clinic score of 2 or fewer points in patients with UC, and a total CDAI <150 points in patients with CD.

Peripheral blood samples were obtained from 18 patients with UC of median age 39 years (25th–75th percentiles 32–53) and Mayo Clinic score 5 (2–7), 16 patients with CD aged 31 years (25–36·5) and CDAI 183·4 (129·2, 249·7) and 13 healthy controls aged 33 years (31–37). The location of CD involvement was ileal in two patients, ileocolonic in 13 patients and colonic in one patient. UC extent was pancolitis in 12 patients and left-sided colitis in six patients. For chemokine receptor expression assays, data were collected from 11 patients with UC, 13 patients with CD and 10 healthy controls.

Mucosal biopsy samples were obtained from inflamed and non-inflamed areas of the rectosigmoid colon from 35 patients with UC (18 inflamed and 17 non-inflamed specimens) and 28 patients with CD (13 inflamed and 15 non-inflamed specimens) during colonoscopy. The location of CD involvement was ileocolonic in 10 patients and colonic in 18 patients. UC extent was pancolitis in 19 patients, left-sided colitis in 12 patients and proctitis in four patients. For comparison, control biopsy specimens were taken from the normal rectosigmoid mucosa of 17 patients who underwent colonoscopy for various reasons (e.g. cancer screening). Endoscopic activity was determined using an endoscopic score of 0–2 (0, normal; 1, light erythema or granularity; 2, granularity, friability and bleeding, with or without ulcerations) according to Froslie's criteria [21], with a score of 0 or 1 regarded as non-inflamed.

Surgical samples obtained from 16 patients with UC (eight inflamed and eight non-inflamed specimens) and 15 patients with CD (eight inflamed and seven non-inflamed specimens). Macroscopically normal mucosa was obtained from seven patients who underwent surgery for colorectal cancer and was used as control samples.

Flow cytometric analysis

Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque Plus (Amersham Biosciences AB, Uppsala, Sweden) density centrifugation and washed in phosphate-buffered saline (PBS). To analyse the frequency of immature plasma cells and expression of the chemokine receptor, PBMCs were stained with fluorescein isothiocyanate (FITC)-conjugated mouse anti-human-CD20 (DakoCytomation, Glostrup, Denmark), allophycocyanin (APC)-conjugated mouse anti-human-CD19 (IOTest, Immunotech, Marseille, France), phycoerythrin (PE)-conjugated mouse anti-human-CD138 (DakoCytomation), PE-conjugated mouse anti-human-CD38 (DakoCytomation), PE-conjugated mouse anti-human-integrin α5/CD49e (R&D Systems, Incorporated, Minneapolis, MN, USA), PE-conjugated mouse anti-human-CXCR3 (R&D Systems, Incorporated), PE-conjugated mouse anti-human-CXCR4 (R&D Systems), PE-conjugated mouse anti-human-CXCR5 (R&D Systems), PE-conjugated mouse anti-human-CXCR6 (R&D Systems), PE-conjugated mouse anti-human-CCR9 (R&D Systems) and PE-conjugated rat anti-human-CCR10 (R&D Systems) in the dark at room temperature for 30 min. Isotype- and species- matched Ig was the negative control.

After washing in PBS, cells were analysed on a LSR II flow cytometer (BD Biosciences, San Jose CA, USA) using FACSDiva software.

In-vitro culture of CD19+ B cells

PBMCs isolated from healthy volunteers (as described above) were washed twice in PBS, and CD19+ B cells purified via positive selection on magnetic affinity cell sorting (MACS) columns (MACS CD19 MicroBeads human; Miltenyi Biotec, Auburn, CA, USA), using specific mouse anti-human CD19 specific microbeads. The purity of CD19+ B cells was tested by flow cytometry and found to be >95%. For the analysis of cell proliferation, CD19+ B cells were stained with 5(6)-carboxyfluorescein diacetate-N-hydroxysuccinimide ester (CFSE) (Dojin, Kumamoto, Japan). CD19+ B cells were washed twice in PBS and cells resuspended in PBS (1 × 106 cells/ml) containing CFSE at a final concentration of 2 µM. Cells were incubated at 37°C for 10 min. After washing in PBS, CFSE-treated cells were cultured in AIM-V (Gibco BRL Invitrogen, Grand Island, NY, USA) with recombinant interleukin-6 (IL-6) (20 ng/ml) (R&D Systems) for 7 days at 37°C in 5% CO2. Cultured cells were stained with APC-conjugated mouse anti-human-CD19 (IOTest, Immunotech) and PE-conjugated mouse anti-human-CD138 (DakoCytomation) in the dark at room temperature for 30 min, and analysed by flow cytometry. For studies of live cells, 7-aminoactinomycin D (7-AAD) (Immunotech) was added before flow cytometric analysis.

Intracellular staining for immunoglobulin

PBMCs isolated as described above were incubated with PE-conjugated mouse anti-human-CXCR3 (R&D Systems), PE-conjugated mouse anti-human-CXCR4 (R&D Systems), APC-conjugated mouse anti-human-CD19 (IOTest, Immunotech) and PE/Cy7-conjugated mouse anti-human-CD20 (BioLegend, San Diego, CA, USA). After washing twice with PBS, cells were fixed and permeabilized using BD Cytofix/Cytoperm Fixation/Permeabilization Kit (BD Biosciences), according to the manufacturer's instructions. Cells were stained subsequently with FITC-conjugated goat anti-human-IgM (KPL), FITC-conjugated anti-human-IgG (H + l) (KPL) and FITC-conjugated anti-human-IgA (α) (KPL, Gaithersburg, MD, USA) in the dark at room temperature for 30 min. Isotype- and species-matched Ig was the negative control. Cells were analysed by flow cytometry.

Quantification of levels of chemokine mRNA in colonic tissues by real-time reverse transcription–polymerase chain reaction (RT–PCR)

Total RNA was extracted and purified from rectosigmoid colon biopsy samples using the RNAqueous-Micro kit (Ambion Incorporated, Austin, Texas, USA), according to the manufacturer's instructions. The probes (Applied Biosystems Incorporated, Foster City, CA, USA) used for analysis were as follows: CXCL9 (Hs00171065_m1), CXCL10 (Hs00171042_m1), CXCL11 (Hs00171138_m1), CXCL12 (Hs00930455_m1), CCL27 (Hs00171157_m1) and CCL28 (Hs00219797_m). RT–PCR analysis was performed using an ABI prism 7700 sequence detection system instrument and software (Applied Biosystems). The reaction mixture was prepared according to the manufacturer's instructions using the SuperScript III Platinum One-Step quantitative RT–PCR (qRT–PCR) Kit (Invitrogen). Thermal cycling conditions were 50°C for 15 min and 95°C for 2 min, followed by 40 cycles of amplification at 95°C for 15 s and 60°C for 30 s. Total RNA was subjected to real-time RT–PCR for measurement of target genes, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal standard with TaqMan GAPDH control reagents (Applied Biosystems). Each resulting gene amount was divided by GAPDH gene amount to obtain a normalized value. The ratio of mRNA levels was normalized to the mean value for control samples.

Immunohistochemistry

Surgical samples were obtained from patients with CD and UC for immunohistological study. A segment (approximately 5 × 5 mm) was excised from surgically resected colon and immediately fixed in periodate/lysine/2% paraformaldehyde at 4°C for 6 h, and then incubated in a 10%–15%–20% sucrose gradient in PBS at 4°C for 6 h each. Specimens were embedded in octreotide (OCT) compound (Sakura Finetechnical Company, Tokyo, Japan) in dry ice/acetone, and stored at –80°C until use.

Cryostat sections (thickness, 4 µm) were cut and pretreated with 5% normal donkey serum with PBS to inhibit non-specific protein binding. After washing in PBS, sections were first reacted with primary antibody at 4°C overnight. Rabbit anti-human-CXCR3 antibody (diluted 1:100 in PBS; GeneTex, Incorporated, San Antonio, TX, USA), mouse anti-human-CD3 antibody (diluted 1:200 in PBS; DakoCytomation), mouse anti-human-CD56 antibody (diluted 1:200 in PBS; DakoCytomation), mouse anti-human-CD68 antibody (diluted 1:200 in PBS; DakoCytomation), mouse anti-human-CD138 antibody (diluted 1:200 in PBS; DakoCytomation), goat anti-human-CD19 antibody (diluted 1:50 in PBS; C-20; Santa Cruz Biotechnology, Incorporated, Santa Cruz, CA, USA), rabbit anti-human-CD138 antibody (diluted 1:100 in PBS; Thermo Fisher Scientific Anatomical Pathology, CA, USA), mouse anti-human-CXCR3 antibody (diluted 1:50 in PBS; GeneTex, Incorporated), rabbit anti-human-CXCL9/MIG (diluted 1:200 in PBS; H-40; Santa Cruz Biotechnology), rabbit anti-human-CXCL10/IP-10 (diluted 1:200 in PBS; K-19; Santa Cruz Biotechnology) and rabbit anti-human-CXCL11/I-TAC (diluted 1:200 in PBS; FL-94; Santa Cruz Biotechnology) were the primary antibodies. Isotype- and species-matched Ig was the negative control. After washing three times in PBS containing 0·05% Tween-20 (PBS-T), sections were incubated for 1 h at room temperature with AlexaFluor 488 donkey anti-rabbit IgG antibody (diluted 1:200 in PBS-T; Molecular Probes/Invitrogen, Carlsbad, CA, USA), AlexaFluor 594 donkey anti-mouse IgG antibody (diluted 1:200 in PBS-T; Molecular Probes/Invitrogen) and AlexaFluor 350 donkey anti-goat IgG antibody (diluted 1:50 in PBS-T; Molecular Probes/Invitrogen). They were then mounted in fluorescent mounting medium (DakoCytomation). Images were taken under a fluorescence microscope (Olympus BX50, Olympus, Tokyo, Japan) equipped with a colour camera (Olympus Color Chilled 3CCD Camera, M-3204C). Results were obtained from serial sections.

To quantify the numbers of lamina propria CD19+ CD138+ CXCR3+ triple-positive cells, four random high-power fields in each specimen were counted and mean counts were calculated.

Statistical analysis

All values are expressed as medians (25th and 75th percentiles). Data were analysed non-parametrically using the Mann–Whitney U-test for unpaired samples. < 0·05 was considered significant. Correlations were analysed by Spearman's rank correlation analysis.

Results

Increase in immature plasma cells from peripheral blood in active UC and active CD

Three-colour flow cytometric analysis revealed that CD19+ CD20 cells in PBMC expressed CD138 and CD38 but no integrin α5/CD49e (VLA-5) (Fig. 1a). As reported previously [22], these cells were identified as peripheral blood immature plasma cells. This phenotype is the same among controls, UC and CD. As shown in Fig. 1b, the percentages of immature plasma cells among peripheral blood CD19+ cells were significantly higher in patients with active UC and active CD than in healthy controls (active UC versus controls, = 0·0047; and active CD versus controls, = 0·0218).

Figure 1.

(a) Representative flow cytometry plot of peripheral blood mononuclear cells (PBMCs) from healthy control (HC), ulcerative colitis (UC) and Crohn's disease (CD) stained for CD19 (allophycocyanin) and CD20 (fluorescein isothiocyanate) (upper). The phenotype of CD19+ CD20 cells was determined by three-colour flow cytometric analysis using CD138 [phycoerythrin (PE)], CD38 (PE) or CD49e (PE) (lower). (b) Percentages of immature plasma cells (CD19+ CD20 cells) among peripheral blood CD19+ cells from healthy controls (HC) and patients with remission UC (UC Rem), active UC (UC Act), remission CD (CD Rem), and active CD (CD Act), as determined by flow cytometry. Boxes show the median with 25th and 75th percentiles (lines at ends). Bars show the 10th and 90th percentiles. Circles show data points below the 10th percentile and above the 90th percentile. Numbers of patients are indicated. *< 0·05 versus control; **< 0·01 versus control.

A significant positive correlation (ρ = 0·796, = 0·0010) was found between the percentage of immature plasma cells among peripheral blood CD19+ cells and clinical activity of patients with UC (Mayo Clinic score) (Fig. 2a). In CD, a significant positive correlation (ρ = 0·609, = 0·0184) was also found between the percentage of immature plasma cells among peripheral blood CD19+ cells and clinical activity of CD (CDAI) (Fig. 2b). A significant positive correlation was observed between the percentage of immature plasma cells among peripheral blood CD19+ cells and serum C-reactive protein (CRP) level of patients with UC (ρ = 0·618, = 0·0109) and CD (ρ = 0·711, = 0·0059) (Fig. 2c,d).

Figure 2.

(a) Correlation between percentages of immature plasma cells among peripheral blood CD19+ cells and disease activity determined by Mayo Clinic score in ulcerative colitis (UC) (ρ = 0·796, = 0·0010). (b) Correlation between percentages of immature plasma cells among peripheral blood CD19+ cells and disease activity determined by Crohn's Disease Activity Index (CDAI) in Crohn's disease (CD) (ρ = 0·609, = 0·0184). (c) Correlation between percentages of immature plasma cells among peripheral blood CD19+ cells and serum C-reactive protein (CRP) level of patients with UC (ρ = 0·618, = 0·0109). (d) Correlation between percentages of immature plasma cells among peripheral blood CD19+ cells and serum CRP level of patients with CD (ρ = 0·711, = 0·0059). Correlations were analysed by Spearman's rank correlation analysis.

Proliferation of peripheral blood immature plasma cells in vitro

Analysis of CD19+ cells cultured for 7 days demonstrated a higher expression of CD138 on CD19+ proliferating (CFSElo) cells compared with non-proliferating CD19+ cells (CFSEhi) (Fig. 3b). This result revealed that these CD19+ CD138+ cells (peripheral blood immature plasma cells) were more proliferative than the other CD19+ cells.

Figure 3.

Proliferation of peripheral blood immature plasma cells. Peripheral blood CD19+ cells were stained with 5(6)-carboxyfluorescein diacetate-N-hydroxysuccinimide ester (CFSE) and then cultured with recombinant interleukin (IL)-6 for 7 days. CD19+ proliferating (CFSElo) and CD19+ non-proliferating (CFSEhi) cells are shown (upper). The percentage of CD138+ cells per CD19+ cells in each population were determined by flow cytometry (lower). These in-vitro data are gated on 7-AAD-negative cells. These data are representative of three independent experiments.

Expression of chemokine receptors on peripheral blood immature plasma cells

Expression of CXCR3 on peripheral blood CD19+ CD20 immature plasma cells was significantly (= 0·0102) higher in patients with UC 68·9% (66·9–85·4%) than in healthy controls 58·25% (52·7–63·8%), although it did not differ significantly between patients with CD 63·5% (50·4–79·3%) and healthy controls (Fig. 4). Expression of CXCR4 on peripheral blood immature plasma cells was also significantly (= 0·0371) higher in patients with UC 57·0% (42·5–67·6%) than in healthy controls 46·7% (32·1–50·7%), although it did not differ significantly between patients with CD 44·5% (39·9–48·1%) and healthy controls (Fig. 4). However, there is no correlation among these CXCR expression and clinical activity of UC.

Figure 4.

Percentage of chemokine receptor expression (CXCR3, CXCR4, CXCR5, CXCR6, CCR9 and CCR10) on peripheral blood immature plasma cells from healthy controls (HC) and patients with ulcerative colitis (UC) and Crohn's disease (CD), as determined by three-colour flow cytometry. Analysis was gated on CD19+ CD20 cells. Numbers of patients are indicated. *< 0·05 versus control.

The population of CXCR5- and CXCR6-positive cells in peripheral blood immature plasma cells was 6·6% and 6·0% in healthy controls, respectively, and there was no difference between patients with IBD and healthy controls (Fig. 4). The population of CCR9-positive cells in peripheral blood immature plasma cells was 16·7% in healthy controls, and there was no difference between patients with IBD and healthy controls (Fig. 4). The population of CCR10-positive cells in peripheral blood immature plasma cells was 60·7% in controls, and there was no difference between patients with IBD and healthy controls (Fig. 4).

Expression of intracellular Ig in peripheral blood immature plasma cells

Flow cytometric analysis of intracellular Ig demonstrated that peripheral blood immature plasma cells were positive for IgG or IgA, but negative for IgM (Fig. 5). Populations of IgG-positive cells in CXCR3+ and CXCR4+ immature plasma cells were 80·8% and 76%, respectively. Populations of IgA-positive cells in CXCR3+ and CXCR4+ immature plasma cells were 13·1% and 8%, respectively (Fig. 5).

Figure 5.

Immunoglobulin (Ig) type of peripheral blood immature plasma cells. Intracellular staining for Ig (IgM, IgG and IgA) was performed in CD19+ CD20 cells by three-colour flow cytometry. Intracellular staining for Ig (IgG and IgA) was performed in CD19+ CD20 CXCR3+ cells and CD19+ CD20 CXCR4+ cells by four-colour flow cytometry. These data are representative of three independent experiments.

Quantification of levels of chemokine mRNA in colonic tissues

CCL27 mRNA was not detected in mucosal samples (data not shown). Mucosal mRNA level of CXCL9 was significantly higher in the inflamed colonic mucosa of patients with UC than in healthy controls (inflamed UC versus controls, = 0·0105) (Fig. 6a). Similarly, mRNA level of CXCL9 was significantly higher in the inflamed colonic mucosa of patients with CD than in controls (inflamed CD versus controls, = 0·0047) (Fig. 6a). CXCL10 mRNA level was significantly higher in the colonic mucosa of patients with UC (inflamed and non-inflamed) than in healthy controls (inflamed UC versus controls, = 0·0146; and non-inflamed UC versus controls, = 0·0220) (Fig. 6b). CXCL10 mRNA was significantly higher in the inflamed colonic mucosa of patients with CD than in healthy controls (inflamed CD versus controls, = 0·0152) (Fig. 6b). CXCL11 mRNA level was significantly higher in the inflamed colonic mucosa of patients with UC than in controls (inflamed UC versus controls, = 0·0248) (Fig. 6c). CXCL11 mRNA was significantly higher in the inflamed colonic mucosa of patients with CD than in controls (inflamed CD versus controls, = 0·0113) (Fig. 6c). No difference in CXCL12 and CXCL28 mRNA expression between patients with IBD and controls was found (Fig. 6d,e).

Figure 6.

Chemokine mRNA levels in colonic tissues of healthy controls (HC) and patients with non-inflamed ulcerative colitis (UC) (UC-NI), inflamed UC (UC-I), non-inflamed Crohn's disease (CD) (CD-NI) and inflamed CD (CD-I). Real-time reverse transcription–polymerase chain reaction (RT–PCR) analysis was performed, and mRNA levels were normalized to the mean value for control samples. (a) CXCL9 mRNA level. (b) CXCL10 mRNA level. (c) CXCL11 mRNA level. (d) CXCL12 mRNA level. (e) CCL28 mRNA level. Numbers of patients are indicated. *< 0·05 versus control; **< 0·01 versus control.

Localization of CX chemokine receptor 3-positive cells and CX chemokine ligands in the colonic mucosa

To identify the type and distribution of immune cells that express the chemokine receptor CXCR3, we conducted immunofluorescence studies on surgically resected colonic tissue of controls and IBD patients. Double- or triple-immunofluorescence experiments on colonic mucosa revealed that CD3+ T cells, CD56+ natural killer (NK) cells, CD19+ cells and CD138+ cells all expressed CXCR3, while CD68+ macrophages did not. In the ulcer base of UC, most CXCR3 expression was found on immature plasma cells double-positive for CD19 and CD138 (Fig. 7). In accord with our previous data [11], numerous CD19+ CD138+ immature plasma cells were observed in the inflamed UC mucosa (Fig. 8a–e). A triple-immunofluorescence study revealed that the number of immature plasma cells expressing CXCR3 was significantly higher in the inflamed colonic mucosa of UC patients than in the colonic mucosa of controls (inflamed UC versus controls, = 0·0026) (Fig. 8c,d,f).

Figure 7.

Expression of CXCR3 (green) on the lamina propria T cells (CD3, red), natural killer (NK) cells (CD56, red), macrophages (CD68, red) and immature plasma cells (CD19, blue; CD138, red) in active ulcerative colitis (UC). The specificity of each antibody was confirmed by negative reactivity of isotype- and species-matched immunoglobulin.

Figure 8.

Triple immunofluorescent staining for CD19 (blue), CD138 (green) and CXCR3 (red) in the lamina propria of control (a), non-inflamed ulcerative colitis (UC) (b), inflamed UC (c,d) and inflamed Crohn's disease (CD) (e). (d) High-magnification images of the boxed areas in (c). The specificity of each antibody was confirmed by negative reactivity of isotype- and species-matched immunoglobulin. Adjacent sections were stained with haematoxylin and eosin. This figure is representative of staining performed in four controls and six patients with UC or CD. Scale bar represents 200 µm. (f) Numbers of CD19, CD138 and CXCR3 triple-positive cells in the lamina propria of healthy controls (HC) and patients with non-inflamed UC (UC-NI), inflamed UC (UC-I), non-inflamed CD (CD-NI) and inflamed CD (CD-I). Numbers of patients are indicated. **< 0·01 versus control.

Immunofluorescence study also showed increased expression of CXCL9, 10 and 11 in the inflamed colonic mucosa of UC and CD patients compared with control colonic mucosa. As reported elsewhere [23][24], CXCL9, 10 and 11 expression was detected on neutrophils and lamina propria mononuclear cells (Fig. 9a–d).

Figure 9.

Immunofluorescent staining for CXCL9, CXCL10 and CXCL11 in the lamina propria of control (a), non-inflamed ulcerative colitis (UC) (b), inflamed UC (c) and inflamed Crohn's disease (CD) (d). The specificity of each antibody was confirmed by negative reactivity of isotype- and species-matched immunoglobulin. Scale bar represents 200 µm.

Discussion

Memory B cells activated by antigen in secondary lymphoid tissues leave the lymphoid tissues and migrate through the vessels to target tissues [13]. Activated B cells differentiate into plasma blasts that express CD19+ CD20 CD138+ integrin α5/CD49e phenotype, and then differentiate into CD138+ cells within a few days [25]. These CD19+ CD20 CD138+ CD49e cells in the peripheral blood are defined as early (immature) plasma cells because they lack the CD49e expression characteristic of bone marrow plasma cells [22,26]. These immature plasma cells represent 0·1–0·2% of the total PBMCs in healthy donors, but these peripheral blood immature plasma cells increase in the patients with active systemic lupus erythematosus (SLE), bacterial septicaemia and liver cirrhosis [22]. In the present study, because three-colour flow cytometric analysis of PBMC revealed that peripheral blood CD19+ CD20 cells expressed CD138 and CD38 but not CD49e, these peripheral blood CD19+ CD20 cells could be identified as immature (early) plasma cells. The population of immature plasma cells among peripheral blood CD19+ cells in patients with active UC and active CD were significantly higher than healthy controls. This is the first study to describe increased peripheral blood immature plasma cells in active UC and CD. There was a significant positive correlation between the percentage of immature plasma cells among peripheral blood CD19+ cells and the clinical activity of UC and CD. Alteration in peripheral blood immature plasma cells is therefore considered to be a good parameter to predict clinical activity. The increased numbers of immature plasma cells in active SLE and bacterial septicaemia decrease after treatment [22], therefore an increased number of peripheral blood immature plasma cells may reflect mobilization of B cells into mature plasma cells.

Earlier, we showed that the immature plasma cells that infiltrated into the inflamed colonic mucosa, vermiform appendix and pouchitis mucosa of patients with UC exhibited high degrees of labelling for Ki-67 [11,12,27]. The present study revealed that immature plasma cells in peripheral blood proliferated in vitro in response to IL-6.

Despite an increase in peripheral blood immature plasma cells among patients with UC and CD, infiltration of immature plasma cells in inflamed mucosa is specific for UC [11]. These observations may indicate that the processes of cells ‘homing’ to the inflamed mucosa differ in UC and CD. To elucidate the mechanisms of peripheral blood immature plasma cells homing via chemokine receptors, we analysed chemokine receptor expression on peripheral blood immature plasma cells.

The high expression of CXCR3 on peripheral blood memory B cells in rheumatoid arthritis and on B cells in cerebrospinal fluid in multiple sclerosis have been reported [28,29]. Several studies have shown that CXCR3 ligand chemokines (CXCL9/MIG, CXCL10/IP10 and CXCL11/ITAC) are up-regulated in inflammatory sites of UC and CD [30–33]. In the present study, flow cytometry experiments revealed that many peripheral blood immature plasma cells were positive for CXCR3, and its expression on peripheral blood immature plasma cells was up-regulated selectively in patients with UC. Real-time RT–PCR data showed increased mRNA expression of CXCR3 ligand chemokines (CXCL9, 10 and 11) in the inflamed colonic mucosa of patients with UC and CD compared to that in controls. These findings were corroborated by immunohistochemistry studies that demonstrated the following results: (1) increased CXCL9, 10 and 11 expression on the inflamed colonic mucosa of UC and CD patients, especially on inflamed UC mucosa, compared to control mucosa; and (2) an abundance of CXCR3 expressing CD19+ CD138+ immature plasma cells on the inflamed colonic mucosa of UC patients compared to controls. However, in CD patients, CXCR3 expression on immature plasma cells was not high. Specific to UC patients, the up-regulation of both CXCR3 and its ligands may trigger immature plasma cell migration into inflamed colonic mucosa. These CXCR3-positive peripheral blood immature plasma cells contained mainly IgG. Our previous study showed that immature plasma cells in inflamed colonic mucosa of UC contained IgG [11]. This result explains the local IgG overproduction and generation of autoantibodies in patients with UC.

CXCR4 expression on peripheral blood immature plasma cells was also up-regulated selectively in patients with UC. IgA and IgG antibody-secreting cells (ASCs) with CXCR4 expression migrate to the bone marrow and mucosal sites in response to CXCL12/SDF1α[13]. However, mRNA expression of the CXCR4 ligand CXCL12/SDF1α in colonic mucosa was not significantly different between IBD and controls. These findings indicate that CXCR4 axis may play a less important part in the migration of immature plasma cells to inflamed colonic mucosa in UC than the CXCR3 axis.

Most peripheral blood immature plasma cells were negative for CXCR5 and CXCR6. CXCR5 expression on B cells is decreased on plasma cells during plasmacytic differentiation [34]. Human bone marrow plasma cells express CXCR6, and the CXCR6–CXCL16 axis contributes to migration and tissue localization of human plasma cells in bone marrow [35]. These findings indicate that peripheral blood immature plasma cells are intermediate between B cells and plasma cells.

The CCR9–CCL25/TECK and CCR10–CCL28/MEC axes have homeostatic roles in mucosal immunity by homing of antigen-specific IgA ASCs to small intestinal mucosa [36] and epithelial cells in various mucosal tissues [37], respectively. Many peripheral blood immature plasma cells were negative for CCR9 (16·7%), and no difference was found in the expression of CCR9 on peripheral blood immature plasma cells between IBD and controls. Many peripheral blood immature plasma cells were positive for CCR10 (60·7% positive), but no differences in CCR10 expression were found on peripheral blood immature plasma cells or in CCL28 expression in colonic mucosa between patients with IBD and controls. These results suggest that CCR9 and CCR10 may not explain the migration of peripheral blood immature plasma cells to inflamed sites in UC specifically.

Hardi et al. [38] reported positive safety data from Phase 1 trials of anti-CXCL10 monoclonal antibody (mAb) (MDX-1100) for the treatment of UC and rheumatoid arthritis. Inhibition of the CXCL10/CXCR3 axis may become a novel therapeutic approach for the treatment of UC due to the reduction of the migration of immature plasma cells. The pathogenic effects of immature plasma cells have not been studied in detail. Further studies are needed to clarify the functions of immature plasma cells in the pathogenesis of UC.

In conclusion, our study demonstrates that peripheral blood immature plasma cells in patients with IBD are elevated with a positive correlation to clinical activity. CXCR3 expression on peripheral blood immature plasma cells is selectively up-regulated in patients with UC. These results indicate that immature plasma cells may migrate towards inflammatory sites of UC via the CXCR3 axis and participate in the pathogenesis of UC.

Disclosure

The authors declare no conflicts of interest.

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