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. 2002 Apr;105(4):509–514. doi: 10.1046/j.1365-2567.2002.01373.x

Lack of Shiga-like toxin binding sites in germinal centres of mouse lymphoid tissues

Yasuyuki Imai 1, Takashi Fukui 1, Asano Ikegaya 1, Tomoyuki Ishikawa 1, Yousuke Ono 1, Kohta Kurohane 1
PMCID: PMC1782679  PMID: 11985671

Abstract

B cells in germinal centres are known to express carbohydrate antigen CD77 in human lymphoid tissues. The CD77 antigen is specifically recognized by Shiga-like toxins (SLTs) that are produced by enterohaemorrhagic Escherichia coli O157:H7. To determine whether the binding subunits of Shiga-like toxin-1 (SLT-1B) could have adverse effects on the murine immune system when used as an immunogen, we investigated whether SLT-1B could bind to germinal centres of mouse lymphoid tissues. Frozen sections of peripheral lymph nodes and Peyer’s patches from immunized mice were tested for the presence of SLT-1B-binding sites by immunohistological methods. Germinal centres were not stained with SLT-1B, while they were intensely stained with peanut agglutinin (PNA), another marker of germinal centres. On the other hand, SLT-1B specifically bound to renal tubules and collecting ducts in frozen sections of mouse kidney. This is consistent with results from human tissues. We also demonstrated that B220/PNA double-positive populations in lymph nodes from immunized mice exhibited only marginal staining with SLT-1B. The present results suggest that SLTs would not impede germinal centre functions of the murine immune system.

Introduction

Infection with enterohaemorrhagic Escherichia coli(EHEC)(e.g. serotype O157:H7) can result in life-threatening complications such as haemolytic uremic syndrome (HUS).1 Shiga-like toxins (SLTs or verotoxins) are exotoxins and virulence factors of EHEC. The SLTs consist of a toxin component (A subunit), which inhibits protein synthesis, and cell-binding components (B subunit).2,3 The B subunits form a pentamer and recognize specific carbohydrate determinants, such as those displayed on glycolipid globotriaosylceramide (Gb3, Galα1-4Galβ1-4Glcβ1-1ceramide),4,5 also known as CD77.

CD77 is known to be a marker of Burkitt’s lymphoma cells and germinal centre B cells in human lymphoid organs.6 It has been suggested that infection with SLT-producing E. coli could compromise host defence mechanisms through inhibition of germinal centre functions. B cells from human tonsil that had been committed to immunoglobulin G (IgG) or immunoglobulin A (IgA) production were shown to be sensitive to SLT toxicity in vitro.7 These results indicate that SLTs may impair induction of immunological memory and class switch to the IgA isotype, which can neutralize toxin on the mucosal surface. Furthermore, B subunits alone have been reported to induce apoptosis in Burkitt’s lymphoma cells through ligation of CD77.8,9

We are currently studying mucosal immunity against SLTs, especially to challenge production of therapeutic antibodies. To produce monoclonal antibodies (mAbs) of IgA class against SLTs, toxicity against or induction of apoptosis in germinal centre B cells in mice may be a major problem. We previously prepared recombinant B subunits of SLT-1 (SLT-1B) as an immunogen and produced digoxigenin-labelled SLT-1B proteins (DIG-SLT-1B). The binding of DIG-SLT-1B to cell-surface CD77 on Burkitt’s lymphoma cell lines was demonstrated by flow cytometry.10 Furthermore, its binding to Gb3 glycolipids was shown by thin-layer overlay assay and by enzyme-linked immunosorbent assay (ELISA).10

To investigate possible damage to the murine immune system, we directly tested, by an immunohistological approach using DIG-SLT-1B, whether SLT-1B binds to the germinal centre of mouse lymphoid organs.

Materials and methods

Animals

Specific pathogen-free female CD-1 (ICR) and BALB/c mice were purchased from SLC Japan (Shizuoka, Japan), and male C57BL/6 × DBA/2 F1 (BDF1) mice were purchased from CLEA Japan Inc. (Tokyo, Japan). All mice were used when 6–8 weeks of age. For parenteral immunization, mice were injected with 200 µg of ovalbumin (OVA) (Sigma, St. Louis, MO), subcutaneously, in complete Freund’s adjuvant (Difco, Detroit, MI), under ether anaesthesia. Brachial lymph nodes were collected 2–6 days after immunization. For oral immunization, mice were given 1 mg of OVA orally together with 5 µg of cholera toxin (List Biological Laboratory, Campbell, CA) as a mucosal adjuvant. Peyer’s patches were collected on days 3 and 4. Experiments were performed in accordance with ethical guidelines of the animal facilities of the University of Shizuoka.

Reagents

Preparation of SLT-1B was performed as described previously.10 Purified SLT-1B was labelled with digoxigenin as described previously.10 Fluorescein isothiocyanate (FITC)-labelled sheep anti-digoxigenin Fab fragments (FITC-anti-DIG) and horseradish peroxidase (HRP)-labelled sheep anti-digoxigenin Fab fragments (HRP-anti-DIG) were purchased from Boehringer Mannheim (Tokyo, Japan). Texas Red-avidin D, HRP-avidin D, biotin-conjugated peanut agglutinin (PNA) and 3-amino-9-ethylcarbazole (AEC) substrate kit were obtained from Vector (Burlingame, CA). Phycoerythrin (PE)-conjugated mAb rat anti-mouse B220 (clone RA3-6B2) and PC5-conjugated streptavidin were obtained from Beckman Coulter (Tokyo, Japan). RPMI-1640 was purchased from Gibco BRL (Grand Island, NY), fetal bovine serum (FBS) was obtained from HyClone Laboratory (Logan, UT), penicillin G from ICN Biomedicals (Costa Mesa, CA), streptomycin sulphate from Wako Pure Chemicals (Osaka, Japan), bovine serum albumin (BSA) fraction V and poly l-lysine from Sigma, paraformaldehyde from Nacalai Tesque (Kyoto, Japan), and Mayer’s haematoxylin and eosin Y (H & E) from Muto Pure Chemicals (Tokyo, Japan).

Immunostaining using SLT-1 binding subunits

Mouse kidneys, lymph nodes or small intestine fragments with Peyer’s patches were embedded in Tissue-Tek® O.C.T. compound (Miles, Elkhart, IN), and were frozen in a liquid nitrogen bath. Immunostaining was performed as described previously.11,12 In brief, cryostat sections (10-µm thick) were picked up on poly l-lysine-coated slides and air dried. The sections were fixed either in acetone for 30 seconds at 20° or in 2% (wt/vol) paraformaldehyde in 0·1 m phosphate buffer (pH 7·0) for 10 min at 20°. Non-specific binding sites were blocked by incubation for 10 min at 20° in Ca2+/Mg2+-free Dulbecco’s phosphate-buffered saline (PBS) containing 3% BSA. The sections were incubated with DIG-SLT-1B (2 µg/ml) in 3% BSA/PBS for 30 min at 20°. For detection of immunofluorescence, the sections were incubated with FITC-anti-DIG (0·2 µg/ml) in 3% BSA/PBS for 30 min at 20°. To detect PNA-binding sites, the sections were incubated with biotin-conjugated PNA (2 µg/ml) and then with Texas Red-avidin D (2·5 µg/ml). After each incubation and fixation, the sections were gently washed three times in PBS. After the final wash, the sections were mounted in Gel/mount (Biomeda, Foster City, CA) and analysed using a fluorescence microscope (BX60; Olympus, Tokyo, Japan). For detection of immunoperoxidase activity, the sections that had been reacted with DIG-SLT-1B were incubated with HRP-anti-DIG (1 : 500 dilution) for 30 min at 20°. Antibody binding was detected using the AEC substrate kit. The sections were counterstained in haematoxylin. The binding of biotin-PNA was revealed by incubation with HRP-avidin D (5 µg/ml) for 30 min at 20°.

Flow cytometric detection using SLT-1 binding subunits

RAMOS cells, a cell line derived from Burkitt’s lymphoma, were cultured in RPMI-1640 supplemented with 10% FBS and penicillin (100 U/ml)/streptomycin (100 µg/ml), as previously described.10 Brachial lymph nodes were collected 6 days after immunization, and single-cell suspensions were prepared in PBS using 22-gauge needles. The lymph node cells (2 × 106) were incubated for 30 min at 37° in 0·2 ml of PBS containing 0·1% BSA and 0·1% NaN3, (PBS-BSA) with or without DIG-SLT-1B (10 µg/ml), with or without biotin-PNA (25 µg/ml), and with or without PE-anti-B220 (1 : 10 dilution). After washing in PBS-BSA, the cells were incubated in PBS-BSA containing FITC-anti-DIG (0·7 µg/ml) and PC5-streptavidin (1 : 10 dilution), for 30 min at 0°. The cells were analysed on a flow cytometer (EPICS XL; Beckman-Coulter) with an argon laser (488 nm) and with an appropriate combination of detection filter sets. In the case of RAMOS cells, immunostaining using only DIG-SLT-1B and FITC-anti-DIG was applied.

Results

Fixation conditions

Previous studies indicated that renal tubules and collecting ducts of human kidney are abundant in ligands for SLTs.13,14 As a result of this, we stained ICR mouse kidney frozen sections as a positive control. After fixation either with acetone or formaldehyde, kidney sections were incubated with DIG-SLT-1B. The binding sites were revealed by an immunoperoxidase method. In adult mice, renal tubules and collecting ducts were strongly stained with DIG-SLT-1B while glomeruli were not stained (Fig. 1a, b). The staining pattern showed no difference regardless of the method of fixation used (acetone or 2% paraformaldehyde). However, much stronger signals were obtained after acetone fixation than after formaldehyde fixation (data not shown). Furthermore, we have confirmed previously that cold acetone fixation (5 min at −20°), which has been used in an immunohistochemical detection of globo-series glycolipids by mAbs,15 produced the same results as acetone fixation at 20°. Therefore we decided to use acetone fixation for 30 seconds at 20° throughout our experiments.

Figure 1.

Figure 1

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Lack of Shiga-like toxin (SLT)-binding sites in germinal centres of the lymph nodes and Peyer’s patches from ICR mice. Acetone-fixed frozen sections of normal mouse kidneys [(a), (b) and (g)], mouse draining lymph nodes 6 days after subcutaneous immunization [(c), (d) and (e)], or mouse Peyer’s patches 4 days after oral immunization [(f) and (h),], were stained with digoxigenin-labelled SLT-1B proteins (DIG-SLT-1B) plus horseradish peroxidase (HRP)-anti-DIG [(a) and (b)]. The sections were then counterstained with haematoxylin. The other sections were stained with haematoxylin and eosin (c); biotin-conjugated peanut agglutinin (PNA) plus Texas Red avidin D (d) and (f); or DIG-SLT-1B plus fluorescein isothiocyanate (FITC)-anti-DIG (e), (g) and (h).(a): Cortical regions of the kidney. Renal tubules were strongly stained with DIG-SLT-1B (arrowheads) while glomeruli were not stained (single arrow).(b): Medullary regions of the kidney. Cross-sections of collecting ducts were strongly stained with DIG-SLT-1B.(d), (e), (f) and (h): Germinal centres (arrows) were strongly stained with PNA but not with DIG-SLT-1B. Arrowheads point towards the intestinal epithelium (f) and (h).(g): Collecting ducts in kidney medulla were strongly stained with DIG-SLT-1B, as revealed by immunofluorescence. Bars represent 50 µm [(a) and (b), × 230 magnification] or 200 µm [(c) to (h), × 70 magnification].

Lack of SLT binding to germinal centres in peripheral lymph nodes

Germinal centres were identified between days 3 and 6 in draining lymph nodes after subcutaneous immunization, as judged by H & E staining. Figure 1(c) demonstrates a representative result on day 6. Using immunofluorescence methods, germinal centres on day 6 were strongly stained with PNA (Fig. 1d), a marker for germinal centres.16 On the other hand, the germinal centre did not show any positive signals representing DIG-SLT-1B binding in consecutive serial sections (Fig. 1e). In positive control experiments, renal collecting ducts were strongly stained with DIG-SLT-1B under the same conditions (Fig. 1g). Germinal centres in lymph nodes were not stained with DIG-SLT-1B during days 3–5 (data not shown).

The binding of PNA and the lack of the binding of DIG-SLT-1B to the germinal centres were also confirmed by immunoperoxidase staining (data not shown). To further confirm the lack of DIG-SLT-1B binding to germinal centres of other mouse strains, lymph nodes from BALB/c and BDF1 mice were examined on day 6 after immunization by immunoperoxidase staining. DIG-SLT-1B did not bind to germinal centres in either strain, while the binding of PNA was readily detected (data not shown).

Lack of SLT binding to germinal centres in Peyer’s patches

Germinal centres were identified on days 3 and 4 after oral immunization. A representative result showing PNA staining of the germinal centre in Peyer’s patches on day 4 is shown (Fig. 1f). On the other hand, DIG-SLT-1B did not stain germinal centres in consecutive serial frozen sections (Fig. 1h). DIG-SLT-1B did not stain germinal centres on day 3 (data not shown).

Secondary reagents (HRP-anti-DIG, FITC-anti-DIG or Texas Red-avidin D) did not produce any positive signals when used alone.

Flow cytometric analyses on PNA-positive B cells from immunized mouse lymph nodes

Three-colour flow cytometric analyses of immunized lymph node cells were performed to examine the binding of DIG-SLT-1B to PNA-positive B cells at a single-cell level. B220+ (FL-2) and PNAbright (FL-4) populations were electronically gated, as described in the legend to Fig. 2(a) and Fig. 2(c). The gated populations represented 5·72% and 5·78% of total lymph node mononuclear cells in Fig. 2(a) and Fig. 2(c), respectively. When biotin-conjugated-PNA was not included as a control, cells in the gate represented only 0·35% of total lymph node mononuclear cells (data not shown). The binding of DIG-SLT-1B to the gated population was revealed by FITC-anti-DIG (FL-1). The gated population produced only a slight shift in the FL-1 signals in the presence of DIG-SLT-1B (Fig. 2b) as compared with those in the absence of DIG-SLT-1B (Fig. 2d). The mean fluorescence intensity (MFI) of the former and the latter samples were 0·90 and 0·50, respectively. In contrast, RAMOS cells were extensively stained with DIG-SLT-1B (Fig. 2f). The MFIs for RAMOS cells treated with or without DIG-SLT-1B were 11·4 and 0·49, respectively. These results confirmed that PNA-positive B cells from immunized mouse lymph nodes were essentially negative for the SLT-binding sites.

Figure 2.

Figure 2

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Lack of Shiga-like toxin (SLT)-binding sites on peanut agglutinin (PNA)-positive B cells from immunized ICR mouse lymph nodes. Draining lymph node cells obtained 6 days after subcutaneous immunization were stained with phycoerythrin (PE)-anti-B220 (FL-2) and biotin-PNA/PC5-streptavidin (FL-4). Cells were also treated with (a)and (b)or without(c) and (d)digoxigenin-labelled SLT-1B proteins (DIG-SLT-1B). The binding of DIG-SLT-1B was detected by fluorescein isothiocyanate (FITC)-anti-DIG (FL-1). The gated populations representing PNAbright/B220+ cells (the square in panel a) were analysed for the binding of DIG-SLT-1B (b). FL-1 signals from PNAbright/B220+-gated cells, without treatment of DIG-SLT-1B (the square in panel c), were shown as a background (d). The binding sites for SLT on RAMOS cells were demonstrated by treatment with (f) or without (e) DIG-SLT-1B, followed by incubation with FITC-anti-DIG (FL-1).

Discussion

In human lymphoid tissue, CD77 is known to be a marker of germinal centres. Together with chemokine BLC, which recruits B cells into follicles,17 recent reports have also suggested functional roles of certain molecules with the CD77 determinants on B cells.6 Ligation of CD40 on human extrafollicular B cells by CD40 ligand (CD40L) resulted in the expression of cell-surface CD77 and the subsequent sensitivity to toxicity of SLT-1. Having sequence homology with B subunits of SLTs, CD19 has been proposed as a possible receptor for CD77.18 As cell-to-cell adhesion based on CD19 and CD77 interaction has been observed,18 CD77 may participate in B-cell adhesion to follicular dendritic cells that express CD19. These results suggest that CD77 is not just a marker of germinal centre B cells but plays some functional roles in formation of the germinal centre in human lymphoid tissues.

Binding specificity of SLTs4,5 indicates that CD77-positive germinal centre B cells are one of the cellular targets for SLTs. This means that immunological memory, affinity maturation and immunoglobulin isotype switch could be impeded by the effects of SLT. From this standpoint, production of high-affinity antibodies against SLTs has been believed to be a difficult task. Although recent reports indicate that SLT-1 also blocks lymphocyte activation in the bovine system,19 it is not well documented whether the effects of SLT-1 on germinal centre B cells could be generalized to other species. Our results demonstrated, by an immunohistological approach, that SLT-1B did not bind to germinal centres in mouse lymphoid tissues. Consistent with these results, we recently reported that IgG blocking antibodies were produced in BALB/c mice after parenteral immunization with SLT-1B.20 As CD77 appeared to be functionally involved in the human system, the types of molecule involved in germinal centre formation, instead of CD77, remain to be elucidated in the mouse.

In contrast to CD77, which is the Gal α1–4 Gal determinant, PNA-binding sites are expressed both in mouse16,21 and in human22 germinal centres. In this study, we confirmed the expression of PNA-binding sites in mouse germinal centres. The results indicated that the expression of carbohydrate determinants recognized by PNA (Gal β1–3 GalNAc) was conserved between human and mouse germinal centres.

We also demonstrated (by three-colour flow cytometric analysis) that PNA-positive lymph node B cells from immunized mice were essentially negative for the SLT-1B-binding sites. We have previously reported that Burkitt’s lymphoma cells (RAMOS, Raji and Daudi) were extensively stained with DIG-SLT-1B.10 In fact, RAMOS cells were shown to display a number of binding sites for DIG-SLT-1B under the present conditions.

It is less probable that germinal centres of ICR mice did not display binding sites for SLT-1B as a result of allelic variation in the CD77 synthase itself, because SLT-1B binding was readily demonstrated using kidney sections from ICR mice. It is still theoretically possible that failure of SLT-1B binding was based on a difference in the regulation of CD77 synthase because of allelic variation. To rule out such possibility, different strains of mice were examined. Neither BALB/c mice nor BDF1 mice were found to produce binding sites for SLT-1B in lymph node germinal centres.

In conclusion, germinal centres in mouse peripheral lymph nodes and Peyer’s patches did not display binding sites for SLT-1B.

Acknowledgments

This work was supported by Grants-in-aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan (12033213, 12680638) and TERUMO Life Science Foundation. We thank Ms Aya Takatsuki in our department for technical assistance.

Abbreviations

BSA

bovine serum albumin

DIG

digoxigenin

Gal

galactose

GalNAc

N-acetyl-galactosamine

HRP

horseradish peroxidase

MFI

mean fluorescence intensity

PNA

peanut agglutinin

SLT

Shiga-like toxin

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