Eur. J. Immunol. 1990.20: 969-976

Pertussis toxin is mitogenic for human B lymphocytes


Jean-Pierre Kolb., Elisabeth GenotoA, Elisabeth Petit-Koskas., Nathalie Paul-Eugeneo and Bernard DugasO

Effect of bacterial toxins on human B cell activation I. Mitogenic activity of pertussis toxin*

U 1% INSERM, Institut Curie., Paris and Institut Henri BeaufourO, Les Ulis

Pertussis toxin (PT) was found to elicit an increased thymidine uptake in resting B lymphocytes purified from human peripheral blood. A significant mitogenic effect was detected for toxin concentrations > 100 ng/ml (1 nM) and a plateau of stimulation was reached at 1000 ng/ml (10 nM). B cell blasts, activated by a first signal such as Staphylococcus uureus Cowan I or insolubilized anti-p chain antibody, were also stimulated to DNA synthesis by PT in the same range of concentrations. At lower sub-mitogenicconcentrations, the toxin potentiated the response to the low-molecular weight B cell growth factor (LMW-BCGF or 12-kDa BCGF), a progression factor for activated B cells. The “A” or catalytic subunit was devoid of any activity on B cells, suggesting the stimulatory effect of the toxin might be associated with the binding or “B” subunit, as it has been shown for T cells.This hypothesis was strengthened by the observation that, as in T cell, the whole toxin but not the “A” promoter,was able to induce calcium influx in these cells. In addition, the purified “B” oligomer alone was found to promote DNA synthesis in B cells. Finally, a fragment of the soluble cleaved form of the CD23 molecule (FceRII) could be involved in the process of PTmitogenicity for B cells.

1 Introduction Pertussis toxin (PT) is an exotoxin produced by Bordetellu pertussis, the bacterium responsible for whooping cough. This toxin, also called islet-activating protein [l], elicits stimulation of insulin secretion, sensitization to histamine [2], local inflammation and necrosis, lymphocytosis [3,4], lipolysis and hypoglycemia. As other AB toxins, PTconsists of a catalytic promoter, the A subunit of 28 kDa and a binding or B promoter [5]. The latter is a complex of four dissimilar chains of respective molecular mass of 23, 22, 11.7 and 9.3 kDa, assembled in a molar ratio of 1:1:2 : 1. While the A promoter displays the ADP ribosyltransferase catalytic activity [6], the B oligomer is involved in the binding of the toxin to cell membrane and promotes the passage of the A promoter through the cell surface. The effects of PT on the immune system are multiple and include both augmentation and suppression of antibody production, suppression of CTL response in mice [7], and enhancement of DTH by potentiation of the antigen reactivity of T cell populations of the L3T4 phenotype [8,9]. Most of the biological activities attributed to PT are believed to result from the catalytic ability of the A subunit to ADP-ribosylate some classes of GTP-binding or

[I 79621

* A

This work was supported by grant 6400 from ARC. Recipient of an ARC fellowship.

Correspondence: Jean-Pierre Kolb, U 196 “Recherche sur les Interferons”, Institut Curie, 26 rue d‘Ulm, F-75231 Paris, Cedex 05, France Abbreviations: SAC: Staphylococcus aureus Cowan I LMWBCGF Low-molecular weight B cell growth factor [Ca*+]i: Intracellular free calcium concentration IgE-BF IgE-binding factor PT: Pertussis toxin 0 VCH VerlagsgesellschaftmbH, D-6940 Weinheim, 1990

G proteins.The A subunit is able to transfer an ADP-ribose moiety from NAD to asparagine residues on certain G proteins. For instance, PT is able to ADP-ribosylate the ai subunit(s) of the Gi protein which mediates the receptorcoupled inhibition of adenylate cyclase, resulting in an inactivation of this Gi protein [lo]. PT is also able to ADP-ribosylate other G proteins such as the transducing (Gt), which is coupled to a cGMP selective phosphodiesterase and is involved in the stimulation of the rhodopsinretinal complex by light, and of some Gp proteins, which link ligand-activated receptor to the phosphoinositidase C responsible for the hydrolysis of polyphosphoinositides

WIThe toxin has been widely used as a tool to dissect the possible involvement of a G protein in the transduction pathway of various hormones and growth factors. We have previously reported that the B cell progression factor (low-molecular weight B cell growth factor; LMW-BCGF) elicited polyphosphoinositide hydrolysis in activated human B cells [12]. The possibility that a GTP-binding protein of the Gp type sensitive to PT might be involved in this process was thus tested. During the course of this study, we observed that purified human B cells were stimulated to DNA synthesis by the toxin. The present article describes the conditions of such mitogenic effect.The whole toxin and its B oligomer,but not the A promoter, are able to mobilize intracytoplasmic calcium and this activity parallels their ability to trigger B cells to enter the S phase of the cell cycle.

2 Materials and methods 2.1 B lymphocytes Peripheral blood leukocytes (PBL) were obtained from healthy donors as residues from platelet preparations and B 0014-2980/90/0505-0969$02.50/0


Eur. J. Immunol. 1990. 20: 969-976

J.-€? Kolb,E. Genot, E. Petit-Koskaset al.

cells were purified according to a technique previously BSA and 0.1% NaN3, 50 pl of a fluorescein (dichlorotridescribed [13]. Briefly, mononuclear cells (4 x lo9-6 X lo9), azinyl amino fluorescein)-conjugated affinity-purified isolated by centrifugation over Ficoll-Hypaque (Pharma- F(ab')2 fragment goat anti-mouse IgG ( H L) (Immunocia, Uppsala, Sweden), were applied to a column of nylon tech) was added and incubated for an additional 45 min on wool.The loosely adherent cells were recovered by mechan- ice. After washing, the cells were analyzed by FCM with an ical agitation, then rosetted with 2-aminoethylisothiouron- EPICS V (Coultronics) fluorescence-activated cell sorter, ium bromide, hydrobromide (AET)-treated SRBC. The gating on the viable B cell populations. Data analysis was non-rosetted cells were allowed to incubate overnight in performed on readings from 5000 cells per sample and plastic culture flasks to remove monocytes, then treated results are expressed as percent of positive cells after with a mixture of anti-Tcell mAb (anti-CD2, CD5, CD7; a subtraction of background fluorescence (cells incubated kind gift from Drs. J. Morizet and A. Bernard) in the with PBS containing 1% BSA followed by incubation with presence of rabbit C (Cedarlane, Hornby, Ontario, the fluoresceinated second antibody). Canada) to remove residual T cells.


The recovered population consisted of at least 96%-98% B cells, as estimated by immunofluorescence FCM with a goat anti-human Ig and with two pan-B mAb (CD19/B4 and CD20/B1, Coulter, Coultronics, Margency, France). After cell fractionation, 1x 108-3 x 108 purified B cells were usually recovered from one cytapheresis bag. T cells and monocytes contaminations were, respectively, below 2% and 0.1% , as estimated by immunofluorescence with the OKT3 and Mo-2 mAb. Resting B cells were further purified by centrifugation over a discontinuous gradient (70% -45%) of Percoll (Pharmacia) and were recovered in the fractions of high density. Activated B cells were obtained by a 3-day culture (1x 106-2 x 106 cells/ml in RPMI 1640 medium supplemented with 10% FCS) in the presence of either StaphyZococcus aurew Cowan I (SAC; 1/10000 v/v final dilution, a kind gift from C. Grillot-Courvalin) or insolubilized anti-p chain antibody (10 pg/ml; Bio-Rad, Richmond, CA). At the end of this activation step, B cell blasts were recovered by centrifugation over a discontinuous gradient of Percoll as the cells banding at the 50% and 45% interfaces. Confirmation of the nature and state of activation of these cells was assessed by FCM analysis with the pan-B markers and the presence of two activation markers, the transferrin receptor and the 4F2 antigen. 2.2 Antibodies

The following mAb were used to monitor the populations of resting and activated B cells by FCM: B1 (CD20, pan-B), B2 (CD21, anti-EBVC3d receptor) and B4 (CD19, pan-B) were purchased from Coulter; 2.7: anti-arsonate (IgGI), a gift from S. Amigorena (Institut Curie, Paris, France); OKT3 (CD3) was purchased from Ortho Diagnostic (Raritan, NJ); IOT9 (anti-transfemn receptor) was purchased from Immunotech (Marseille Luminy, France); 4F2, a kind gift f r o m y Richard, was available in ascites fluids obtained after injection of the hybridoma 4F2.Cl3 (-22) cells from American Type Culture Collection (Rockville, MD) in BALBlc mice.

2.3 Immunofluorescence study Resting or activated B cells (0.5 x 106cells) were incubated with an appropriate dilution of the various mAb for 30 min on ice. After washing with isotonic PBS containing 2%

2.4 Growth factors

LMW-BCGF (12-kDa BCGF) was purchased from CTI (Cytokine Technology International, Buffalo, NY). Before use, the various batches of growth factor were checked for the absence of IL2 activity on the murine CTLL-2 line and their B cell supporting activity was determined by a test of restimulation of human B cell blasts, preactivated by a 3-day culture in the presence of insolubilized anti-p antibody (10 p,g/ml) or of SAC (1/10000, v/v), as previously described [131. 2.5 PT

PT (islet-activating protein) was purchased from List Biological Laboratories (Campbell, CA: batches PT-28, 49A and 56A) or from Sigma (batch 118F-0861). The PT protomer (batch PTA-04) and B oligomer (batch FTB-07) were purchased from List.

2.6 Other chemicals The PKC inhibitor staurosporine (batch 901087) was purchased from Calbiochem (San Diego, CA). The adenylate cyclase activator [email protected][y-(N-methylpiperazino)-butyryl] forskolin or L858051, and the inactive 1,9-dideoxy forskolin were also purchased from Calbiochem. The calmodulin antagonist N-(6-aminohexyl) 5chloro-l-naphthalene-sulfonamideor W-7 was purchased from Sigma.

2.7 Proliferation studies Cells were incubated at 105 cells/well in the wells of microtitration trays in the presence of various concentrations of PT. Cultures were performed either in RPMI 1640 medium (Gibco, Saint-Ouen, France) supplemented with 10% FCS. After various times of incubation at 37 "C in an atmosphere of 5% C02 in air, cultures were pulsed for the last 18 h of incubation with 1 pCi/well (37 kBq/well) of [3H]dThd (TMM 79B; CEA, Gif-sur-Yvette, France; sp. act. 25 Cdmmol). At the end of the pulse, cells were harvested onto glass microfiber filter papers with a multiple automated sample harvester (Skatron, Flow Labs., Baar, Switzerland) and counted in a LKB (Bromma, Sweden) Rackbeta liquid scintillation spectrometer in the presence of 2 ml of scintillation mixture (Econofluor, NEN, Dreieich, FRG). Results are expressed as cpm k SEM of triplicate samples.

Eur. J. Immunol. 1990. 20: 969-976

Pertussis toxin is mitogenic for human B lymphocytes

2.8 Determination of the intracellular free calcium concentration ([Ca*+]i)

(A) l2




Indo-1 acetoxymethylester (indo-1 AM) was purchased from Calbiochem (La Jolla, CA) and prepared as a 2 mM stock solution in DMSO. Resting or activated B cells (5 x lo6) were suspended in RPMI 1640 medium with 2% FCS and loaded with 5 p~ of indo-1 AM during 30 min at 37 "C. In some experiments, the cells were previously loaded with 5 p~ BAPTA/AM (Calbiochem) for 30 min at 37 "C, prior to incubation with indo-1 AM. For fluorescence analysis, the cells were washed and resuspended in 2 ml of a pH 7.3 buffer maintained at 37 "C, containing 20 mM Hepes, 140 mM NaCl, 2 mM KCl, 10 m glucose, 1 mM MgC12, supplemented or not with 1m CaC12. The fluorescence signal was recorded using a Shimadzu (Kyoto, Japan) spectrofluorometer :excitation was at 331 nm and fluorescence emissions were measured at 400 nm. Baseline signal was obtained prior to the addition of the ligands. [Ca2+Ii in the cells is related to the fluorescence intensity (F) by the following equation:

n (C)

6 cells


[Ca2+]i= Kd X (F


Fmin)/(Fmax - F)

F,, was determined for each experiment by measuring fluorescence after the cells were lysed in TritonX-100 (Sigma). Fminwas determined by recording the fluorescence after addition of EGTA (2 mM) in the presence of Tris base to raise the pH of the sample above 7.3. The effective constant Kd of indo-1 has been reported to be 250 nM. 2.9 RIA for detection of sCD23DgE-BF

It was performed according to the technique previously described [14]. Briefly, 96-well microtiter plates were coated with 100 ml of mAb ER176, blocked with HBSS containing 10% BSA and finally washed with PBS.Test SN (75 pl) was added to the wells, and after 4 h at room temperature plates were washed and supplemented with 75 pl 1251-ER135 (2 x 105-3 x 105 c p d 7 5 pl, sp. act.: 2 x 104-3 x lo4 c p d n g ) . After overnight incubation, wells were washed and counted in a gamma counter. All samples were tested in duplicate, with interassay variation < 10%. The assay was calibrated by reference to a standard preparation of SN from RPMI 8866 cells given an arbitrary titer of lo5 U/ml (1 U = 150 pg).

3 Results 3.1 Mitogenic effect of PT on human PBL and purified T and B cells PBMC and purified Tand B cells were tested in parallel for their proliferative response to PT. T cells were purified from nylon wool-nonadherent cells by rosetting with AET-treated erythrocytes, followed by centrifugation over Ficoll-Hypaque. The rosetted cells were treated with an hypotonic shock of 6 s in distilled water to remove erythrocytes. This population consisted in >95% T cells as estimated by FCM fluorescence analysis with a pan-T (CD3) mAb. Resting B cells were prepared from the same PBL population, as described in the Sect. 2.1, and consisted in >97% CD20+ cells.


80 60 40

20 0










125 250 500 1000

P e r t u s s i s T o x i n (ng/ml)

Figure 1. Unseparated PBL (A) or purified T (B) or B (C) cells were cultured for 3 days at 105 cells/well in a microtiter plate in the presence or absence of various concentrations of PT. dThd uptake was estimated by a pulse of the radioactive precursor during t h e last 16 h of the culture. Control t3H]dThd incorporations (cpm) in the PBL, Tand B cell preparations were respectively of 22 270; 61 034 and 3625 in the presence of PHA (10 pglml) of 987; 793 and 15674 in the presence of anti-p (10 pg/ml), of 6383; 1861 and 3450 in the presence of LMW-BCGF (l/lO,v/v) and of 11088; 5807 and 66 733 in the presence of anti-p plus LMW-BCGE

PBL,Tand B cells were cultured for 3 days in the presence of various concentrations of PT ranging from 0.5 ng/ml to 1 mg/ml. As seen in Fig. 1, the unfractionated PBL as well as the purified B o r T cell populations responded to PT by a marked increase in dThd incorporation for concentrations of the toxin above 64ng/ml. The purity in the B cell preparation was further assessed by the lack of response to PHA and the expected stimulation by the combination of anti-p and LMW-BCGF. Conversely, T cells were strongly stimulated by PHA but only very poorly by the mixture of anti-p and LMW-BCGF. Taken together with the FCM analysis, the PT induced increase in dThd incorporation in the B cell preparation cannot be attributed to a few remaining contaminating T cells. 3.2 Mitogenic effect of PT on resting and activated B cells

Resting B cells were further purified from the bulk of B cells by centrifugation over a discontinuous gradient of


Eur. J. Imrnunol. 1990. 20: 969-976

J.-F! Kolb, E. Gtnot, E. Petit-Koskas et al.

Figurt-2. Purified resting B cells were cultured as describcd previously in the presence or absence of increased concentrations of VT. dThd uptake was cstirnated as reported in the legend of Fig. 1.

Figure4. Resting purified B cells were cultured as described previously. dThd uptakes were performed for 16 h after various timcs of incubation in the prescnce (m; loo0 ng/ml) or absence (M)

of PT.

PemU and were recovered in the fractions of high density. A typical experiment is represented in Fig. 2. A clear mitogenic effectwas evident for concentrations of the toxin above 100 nglml (about 1 nM). Maximal dThd incorporation was reached at loo0 ng/rnl with a plateau of stimulation for higher concentrations. Very similar results were obtained with various batches of PT, either from List or from Sigma origin (not shown).

Of a total of 18 experiments performed with B cells from different normal blood donors, 14 responded in this typical way, while 2 donors displayed a limited, although significant, response and in the 2 remaining cases no obvious mitogenic effect of the toxin could be detected. The toxin was also clearly mitogenic for B cell blasts activated by a primary 3-day culture in the presence of SAC The doseresponse curve was comparable to that obtained with resting B cells and similar results were obtained when anti-F chain was used instead of SAC as the first signal of activation (not shown). As for quiescent B cells, the A protomer of the toxin was devoid of mitogenic activity.

mitogenicactivity,evenwhen testedat high concentrations. In contrast, the B oligomer was able to induce dThd incorporation in these celh and a plateau of stimulation was reached for concentrations similar to those obtained with the whole toxin. Since the B protomercontains < 1% of the intact toxin, it is unlikely that the effect may be due to this residual contamination. (A) R e s t i n g 6 c e l l s


3.3 Mitogenic efleci of the PT B promoter

The A and B subunits of PT were tested in paralle on B cells. As Seen in Fig. 3, the A protomer was devoid of

‘“1 2

( 8 ) SAC-activated 6








Figure 3. Purified B cells were cultured as previously with various concentrations of PT (W) or B (.) protomer. dThd uptake was estimated as before.

Figure 5. Resting B cells (A) or SAC-preactivated B cells (B)were seeded at 5 x 10511 ml in the wells ofa 24well culture tray in the presence loo0 ng/ml) or absence (8)of PT. The numhr of viable cells recovered after various timcs of culturc werc then recorded and are presented as the mean2SEM of triplicatc samplcs.


Eur. J. Immunol. 1990.20: 969-976

Pertussis toxin is mitogenic for human B lymphocytes


3.4 Kinetics of PT-dependent DNA synthesis in B cells Resting B cells were cultured for various times in the presence of a mitogenic concentration of PT (1000 ng/ml). As seen in Fig. 4,the stimulation reached a peak between days 3 and 4 of incubation, then declined but was still significant at day 6.The same profile was observed for 3-day SAC-activated B cells (not shown). 3.5 Effect of PBL on B cell multiplication Resting B cells (A) or SAC-preactivated B cells (B) were cultured together with PT (1000 ng/ml) or control medium and cell recovery was recorded after various times of incubation. As seen in Fig. 5 , PTwas unable to evoke a true cell multiplication, although small but significantly higher numbers of cells were recovered around day 4, when compared to the control cultures. Besides, this effect was dependent on the concentration of PTand there was a strict correlation between the amount of [3H]dThdincorporated into the cells and the number of living cells recovered (Fig. 6).

Anti-p-activated B cell blasts were cultured with an optimum concentration of a commercial preparation of natural LMW-BCGF (lo%, v/v) and with sub-mitogenic concentrations of PT. In these conditions, a potentiating effect of the toxin could be observed (Fig. 7). To test the sensitivity of a growth factor-induced signaling pathway to this toxin, via the ADP ribosylation of a GTP-binding protein, lower concentrations of PT are generally used. No inhibition of the LMW-BCGF driven proliferation could be observed with concentrations of the toxin in the range 0.1 t o 10 ng/ml, suggesting the signaling pathway of LMW-BCGF,

- 40

700 -30

+ .f

When B cells were stimulated by PT (500 ng/ml) in a buffer with an external calcium concentration of 1mM, a slow rise in [Ca2+Iicould be detected which peaked at 3 min and remained in plateau for at least 30 min (Table l).When the cells were loaded with BAPTA, an intracellular calcium chelator, this slow rise was unaffected, suggesting its independency from intracellular pools. In the same experiments, the A subunit was unable to induce such an increase in [Ca2+Ii,When B cells were stimulated by PT in a calcium-free medium, no rise in [Ca2+Ii was observed. Taken together, these results indicate that the observed rise in [Ca2+]ielicited by PT was mostly due to the entry of extracellular calcium. Table 1. Effect of PTon [Ca2+Iimobilization in human B lymphocytesa)




E, 0


which involves polyphosphoinositide hydrolysis [121, is not sensitive to the toxin (not shown). 3.7 Effect of intact PT and of its A protomer on calcium mobilization

3.6 Effects of PT and LMW-BCGF on B cell blasts


Figure 7. B cells were activated for 3 days by a primary culture in the presence of 10 pg/ml of insolubilized anti-p antibody.The blast cells were set in culture as previously described, in the presence of various submitogenic concentrations of PTor not (m) and together with an optimum concentration of LMW-BCGF ( dThd incorporation was determined as before.


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2 %



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Pertussis Toxin

Effect of bacterial toxins on human B cell activation. I. Mitogenic activity of pertussis toxin.

Pertussis toxin (PT) was found to elicit an increased thymidine uptake in resting B lymphocytes purified from human peripheral blood. A significant mi...
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