Eur. J. Immunol. 1991. 21: 495-500

Bernard Dugasa, Nathalie Paul-Euginea, Elisabeth G6noto, Jean Michel Mencia-HuertaA, Pierre Braqueta and Jean Pierre KolbO Institut Henri BeaufourA, Les Ulis and INSERM U 196, Institut Curieo, Pans

Mitogenic effect of the B subunit of cholera toxin

495

Effect of bacterial toxins on human B cell activation 11. Mitogenic activity of the B subunit of cholera toxin The B subunit of cholera toxin (CT) but not the entire CTwas found to induce the proliferation of resting human B lymphocytes. A significant mitogenic effect was observed for B subunit concentrations > 1 p,g/ml and reached a maximum of stimulation at 10 pg/ml. As already described for B lymphocytes preactivated with Staphylococcus aureus Cowan Strain I (SAC), B lymphocytes preactivated with the B subunit of CT,but not with the entire CT, were able to proliferate in response t o exogenous interleukin 2 (IL2) and to the low-molecular weight B cell growth factor (BCGF). To determine the transmembrane signaling system used by the B subunit of CT to mediate its biological effects, we compared the transmembrane signals used by the entire CT,its B subunit and SAC. In comparison to the entire CT,which directly activates adenylate cyclase and increases intracellular CAMP levels, neither the B subunit nor SAC modified the CAMPcontent. In contrast, although SAC induced inositol phosphate generation neither CTnor the separate subunits were able to induce such a production. Moreover, changes in the fluorescence of indo-1-loaded B lymphocytes revealed that mitogenic doses of either the B subunit or SAC induced a rapid and sustained increase in cytoplasmic free Ca2+ concentration ([Ca2+]i).The effect of the B subunit appeared to be largely dependent on the presence of extracellular Ca2+,because in Ca2+-freemedium no [Ca2+]iuptake was observed. In contrast, the SAC-induced [Ca2+]iuptake is substantially, but not totally, inhibited in Ca2+-freemedium, suggesting that part of the rise in [Ca2+]iwas due to the release from internal stores. Moreover, fluorimetric measurements on loaded cells with 2',7'-bis(carboxyethyl)-5(6')carboxyfluorescein revealed that SAC induced a rapid cytoplasmic alkalinization via activation of Na+/H+ exchange, whereas the entire CTand its B subunit had no effect on intracellular pH. Taken together, these data suggest that, in comparison to SAC, the mitogenic effect of the B subunit of CT was mediated through different intracellular biochemical pathways.

1 Introduction The toxin of Vibrio choferue(cholera toxin; CT)is a typical AD3 subunit toxin. The B subunit confers the binding specificity, whereas the A subunit possess the ADPribosyl-transferase activity [ 11.The pathogenic effect of CT is thought to result from its effect on a family of heterotrimeric regulatory proteins called guanine nucleotidebinding proteins ( G proteins) [2]. CTADP ribosylates the a chain of a stimulatory G protein (Gs) and thereby activates the adenylate cyclase [3], inducing an increase in intracytoplasmic cyclic AMP (CAMP)concentration.The effects of CT on the immune system are multiple and modulate various mononuclear cell functions [4-81. Recently, it has

been demonstrated that the B subunit of CT which binds exclusively to the GMI ganglioside on the cell surface [9, 101, could be mitogenic for rat thymocytes. Moreover, recent studies suggest that another bacterial toxin, pertussis toxin, could mediate its mitogenic effects on various cell types through the opening of Ca2+channels in the plasma membrane, and this via a biochemical pathway independent from the phosphatidylinositol metabolism [11].The purpose of this study was to investigate the biological effect of the B subunit of CT on purified B lymphocytes and also the mechanism whereby the binding of B subunit of CT to the membrane was transduced in B lymphocytes.

[I 85071 2 Materials and methods Correspondence: Bernard Dugas, Laboratoire d'Immuno-allergologie, Institut Henri Beaufour, 1 Ave desTropiques, F-91952 Les Ulis, France

2.1 Reagents

pha'te

chem and prepared.as 2 m~ stock solution in DMSO.

0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991

0014-2980/91/0202-0495$3.50 + .25/0

496

Eur. J. Immunol. 1991. 21: 495-500

B. Dugas, N. Paul-Eughe, E. GCnot et al.

EGTA was obtained from Sigma Chemical Co. (St. Louis, MO), and was prepared as a 1 mM stock solution in water (pH 7.4). The following mAb (Immunotech, Marseille, France) were used to monitor the B lymphocyte populations by FCM: (a) the anti-CD20 (pan-B); (b) the anti-CD3 (pan-T); (c) the anti-CD14 (a monocyte marker). The low-molecular weight B cell growth factor (BCGF) was purchased from CTI (Cytokine Technology International, Buffalo, NY). Human rIL2 derived from E. cofi was purchased from Biotrans Co. (Los Angeles, CA) and had a sp. act. of 2 x lo7 U/mg. The PMA was purchased from Calbiochem and kept as a 1 mg/ml stock solution in DMSO.

stimuli. After lysing the cells a total of 200 p1 SN was assayed for cAMP content using the Amersham (Les Ulis, France) [3H]cAMP competitive assay system (TRK432) according to the manufacturers produce.

2.6 Measurement of [3H]inositol phosphates The conditions of incubation of the various stimuli with [3H]myoinositol-loadedB cells as well as general procedure of inositol phosphates on Dowex 1x8 columns have been described previously [13-151. It should be noticed that the inositol trisphosphate (InsP3) contains the two isomers of InsP3 (1, 4, 5 and 1, 3, 4), as well as InsP?.

2.2 B lymphocyte preparations PBMC were separated by centrifugation on Ficoll-Hypaque (Pharmacia, Uppsala, Sweden). Monocytic and NK cell depletion were done according toThiele et al. [12]. Briefly, PBMC were treated with 10 mM L-leucyl-methyl ester (Sigma) for 45 min at room temperature and washed three times. B lymphocytes were then isolated by two cycles of rosetting with aminoethylisothiouromium bromide hydrobromide-treated SRBC. These cells contained 98% of CD20+ cells, < O S % of CD3+ cells and C 1% of CD14+ cells. When these B lymphocytes were cultured in the presence of 10 pg/ml PHA-W (Wellcome Laboratories, Beckenham, GB) these preparations proved unable to proliferate. Moreover, after a 3-day culture in the presence of PHA, CT,the B subunit of CTor SAC, contained < 0.5% CD3+ cells. 2.3 B subunit- and SAC-activated B lymphocyte preparations

B lymphocytes were cultured at 2 x lo6 cells/ml concentration in a tissue culture flask (Becton Dickinson, Mountain View, CA) in complete RPMI 1640 medium (Flow Laboratories, Les Ulis, France) and the presence of optimal doses of either SAC (1/10OOO, v/v), the entire CTor of the A or B subunits of CT (3 pg/ml). After a 3-day culture in 5% C02 incubator, cells were recovered and extensively washed in RPMI 1640 medium. 2.4 Proliferation assay B lymphocytes were incubated at 2 x lo5 cells/well in flat-bottom microtest tissue culture plates (Becton Dickinson) in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 pg/ml streptomycin, 100 U/ml penicillin and 1% FBS (Flow). In the proliferation assay, B lymphocytes were cultured in the presence of various doses of CT or of its B subunit (1 ng to 10 pg/ml) and, as control, an optimal dose of SAC was used (l/lO OOO final concentration). The proliferative response was evaluated by dThd incorporation: 0.5 pCi = 18.5 kBq [3H]dThd (CEA, Saclay, France) was added for the last 10 h of a 3-day culture. 2.5 Determination of intracytoplasmic cAMP levels B lymphocytes (2 x loh celldml) were incubated 30 rnin in RPMI 1640 10% FBS in the presence of the indicated

+

2.7 Determination of intracytoplasmic free Ca2+ concentration ([Ca2+]i) Resting B lymphocytes (5 x 106) were resuspended in RPMI 1640 with 2% FBS and loaded with 5 p~ of indo-1 during, at least, 30 rnin at 37°C [13]. For fluorescence analysis, cells were washed and resuspended in 2 ml of a pH 7.3 buffer (Na+ buffer) maintained at 37 "C, containing 20 mM Hepes, 140 mM NaCl, 2 mM KCI, 10 mM glucose, 1 mM MgC12, supplemented or not with 1 mM CaC12. Fluorescence signal was recorded using a Shimadzu (Kyoto, Japan) spectrofluorometer: excitation was at 331 nm and fluorescence emission was measured at 400 nm. Baseline signal was obtained prior the addition of the stimulus. [Ca2+]iin the cells is related to the fluorescence intensity (F) by the following equation: [Ca2+]i= Kd X (F - Fmin)/(Fmax - F) F,, was determined for each experiment by measuring the fluorescence after the cells were lysed in Triton X-100 (0.1%; Sigma). Fmin was determined by recording the fluorescence after addition of 2 mM EGTA in the presence of Tris base to raise the pH of the sample above 7.3.

2.8 Measurement of intracytoplasmic pH (pHi) pHi was determined fluorimetrically using BCECF/acetoxymethylester as intracellular probe. B lymphocyte suspensions (5 x lo7 cells/ml) in RPMI 1640 medium were loaded at 37°C for 30 rnin with 2 p~ BCECE Then, cells were sedimented and resuspended in the Na+ buffer at 3 x lo6 cells/ml. BCECF fluorescence was monitored with excitation at 495 nm (5-nm slit) and emission at 525 nm (10-nm slit). Calibration of fluorescence vs. pHi was obtained using the K+-nigericin method [16]. 2.9 Immunofluorescence The expression of CD3, CD20 and CD25 markers was determined by indirect immunofluorescence staining using anti-CD3, -CD20 and -CD25 mAb and F(ab')z of FITCconjugated anti-murine Ig. Briefly, cells (0.5 x 106/ml)were incubated at 4°C for 30 rnin in the presence of optimal doses of either anti-CD3, -CD20 or -CD25 mAb (5 pg/sample) and washed three times by centrifugation at loo00 rpm for 1 rnin in PBS containing 1% BSA.Then, the cells were

Mitogenic effect of the B subunit of cholera toxin

Eur. J. Immunol. 1991. 21: 495-500

incubated at 4°C in the presence of the F(ab')z FITCconjugated anti-murine Ig (5 pg/ml) and after three cycle of washing the fluorescence was measured with a cytofluorograph (FACStar+, Becton Dickinson, Grenoble, France).

3 Results

Table 1. Effect of the B subunit of CTand SAC on the expression of CD25-defined antigen on B lymphocytesa)

Challenge

Medium

3.1 Mitogenic effect of the B subunit of CT B lymphocytes were cultured with various doses of the B subunit of CT (10 ng to 10 pg/ml), or with an optimal dose of SAC (1/10 OOO, v/v) and [3H]dThd were evaluated after 3 days (Fig. l).The B subunit of CT,induced a dose-related increase in DNA synthesis and the magnitude of the response reached a maximum at 3 pg/ml, comparable in magnitude to that obtained with SAC stimulation. In control experiments neither the entire CTnor its A subunit were able to induce such a stimulation (data not shown).

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SAC B subunit Bsubunit B subunit Asubunit

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Effect of bacterial toxins on human B cell activation. II. Mitogenic activity of the B subunit of cholera toxin.

The B subunit of cholera toxin (CT) but not the entire CT was found to induce the proliferation of resting human B lymphocytes. A significant mitogeni...
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