Barab-Marszak, 2006

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Effect of tumor necrosis factor alpha and infliximab on apoptosisof B lymphocytes infected or not with EpsteineBarr virus

Fanny Baran-Marszak a,b,*, Christelle Laguillier a, Ibtissam Youlyouz c, Jean Feuillard c,Xavier Mariette a,d, Remi Fagard b, Martine Raphael a

a INSERM E109, CHU Bicetre, Assistance Publique-Hopit aux de Paris, Universite Paris 11 , 942 75 Le Kremlin Bicetre, Franceb Universite Paris13, EA3406, 93009 Bobigny, France

c UMR CNRS 6101, Facul te de Medecine, CHU Dupuytren, 87000 Limoges , Franced Service de Rhumatologie, CHU Bicetre, Assistance Publique-Hop itaux de Paris , Univers ite Paris 11, 94275 Le K remlin Bicetre, France

Received 1 June 2005; received in revised form 10 March 2006; accepted 19 March 2006

Abstract

Chronic inflammation and immunosuppressive therapies increase the risk of non-Hodgkin’s lymphoma associated or not with EpsteineBarr

virus (EBV) infection. A possible link between infliximab treatment and increased risk of lymphoma has been suggested. Indeed, infliximab

induces apoptosis of monocytes and activated T lymphocytes, but its effect on B lymphocytes infected or not with EBV is unknown. Secreted

tumor necrosis factor (TNF) a and the expression level of TNF receptor 1 (TNFR1) and TNFR2 were compared in EBV-positive and negative B-

cell lines. The impact of TNFa and infliximab on apoptosis of EBV-positive cells was analyzed regarding the activity of NF-kB. Increased ex-

pression of TNFa in EBV-positive cells suggested that infliximab could affect their survival. However, TNFa or infliximab incubation had no

effect on apoptosis of EBV-positive cells. Loss of NF-kB activity sensitized lymphoblastoid cell lines to TNFa-induced apoptosis, but no direct

effect of infliximab on apoptosis was detected. On the basis of our in vitro data, neither TNFa nor infliximab has a direct effect on apoptosis of B

lymphocytes and EBV-positive cell lines. Thus, if an increased incidence of lymphoma were induced by TNFa blockers, it would not involvea direct effect on B cells but rather an impaired immune surveillance by T cells.

Ó 2006 Elsevier Ltd. All rights reserved.

Keywords: Tumor necrosis factor a; Infliximab; B lymphocyte; Apoptosis; EpsteineBarr virus

1. Introduction

The incidence of lymphoma is increased in patients with

autoimmune diseases such as Sjogren’s syndrome, Hashimo-

to’s disease and rheumatoid arthritis (RA) [1e

6]. Most of

this increased risk is linked to the persistent activation of

autoimmune B lymphocytes, as in Sjogren’s syndrome [7], or

to the inflammatory activity of the disease, as in RA [8]. Inter-

estingly, the incidence of lymphoma is also increased in pa-

tients with non-autoimmune chronic inflammatory diseasessuch as pyothorax [9] or Crohn’s disease (CD) [1].

The increased risk of lymphoma in patients with autoim-

mune or inflammatory diseases may be also related to immu-

nosuppressive treatment. The two classical drugs that are

thought to increase this risk of lymphoma are methotrexate

(MTX) and azathioprine. In fact, a few cases of Epsteine

Barr virus (EBV)-associated lymphomas have been reported

with MTX treatment for autoimmune diseases [10,11], which

regressed after withdrawal of the drug, similar to what is ob-

served in EBV-associated lymphoproliferative disorders in

Abbreviations: RA, rheumatoid arthritis; CD, Crohn’s disease; MTX,

methotrexate; EBV, EpsteineBarr virus; TNF, tumor necrosis factor; NF-kB,

nuclear factor kB; LCL, lymphoblastoid cell line; LMP1, latent membrane

protein 1; PBMC, peripheral blood mononuclear cells; NGFR, nerve growth

factor receptor; I-kB, inhibitor of NF-kB; TNFR, TNF receptor; ELISA, en-

zyme-linked immunosorbent assay; PBS, phosphate-buffered saline.

* Corresponding author at: Service d’Hematologie biologique, Hopital Avic-

enne, 125 route de Stalingrad, 93000 Bobigny, France. Tel.: þ33 1 48 95 56

46; fax: þ33 1 48 95 56 28.

E-mail address: [email protected] (F. Baran-Marszak).

1043-4666/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.

doi:10.1016/j.cyto.2006.03.005

www.elsevier.com/locate/issn/10434666

Cytokine 33 (2006) 337e345

mailto:[email protected]


http://www.elsevier.com/locate/issn/10434666

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immunodeficient patients [12]. Thus, in RA with lymphoma,

MTX could be beneficial, resulting from efficient control of

the disease, or deleterious, leading to increased risk of EBV-

associated lymphoproliferations. This latter effect seems mar-

ginal, since in two large prospective studies, treatment with

MTX did not significantly increase the risk of non-Hodgkin’s

lymphoma with RA [6,13], although in one of these studies therisk of Hodgkin’s lymphoma (usually 10 times less frequent

than non-Hodgkin’s lymphoma) was increased in RA patients

treated with MTX.

Tumor necrosis factor a (TNFa) blockers are a new class of

therapeutic agents that are efficient in the treatment of refrac-

tory RA, spondylarthropathies and CD. TNFa has a dual role

and, depending on the cellular context, can induce cell survival

by activating nuclear factor kB (NF-kB) or trigger apoptosis

by activating caspases [14]. An increased secretion of TNFa

is observed in B lymphocytes upon EBV infection but lympho-

blastoid cell lines (LCLs) are resistant to TNFa-induced

apoptosis [15] because the viral latent membrane protein 1

(LMP1) protects against it.Infliximab is a chimeric monoclonal antibody that binds

specifically to human TNFa and neutralizes its biologic prop-

erties, but some effects of infliximab cannot be explained by

the neutralization of soluble TNFa alone [16]. For instance,

it induces the rapid suppression of mucosal inflammation in

CD and the specific increase of apoptosis in T lymphocytes

of the lamina propria in vivo and in vitro. Infliximab also in-

duces apoptosis in synovial monocytes/macrophages in RA

[17] and in CD through a caspase-dependent pathway [18].

Results of a few studies suggested an increased incidence

of lymphoma in RA patients treated with TNFa blockers

[19,20]. However, to date it has not been possible to discrim-inate between an increased incidence of lymphoma due to the

TNFa blockers themselves and an increased incidence because

most severe forms of RA are treated with TNFa blockers.

Such increased incidence, which is not established at the pres-

ent time, could be due either to a defect of immunosurveil-

lance of EBV-infected B cells by T cells or to a direct effect

of TNFa blockers on B cells infected or not by EBV. The pos-

sibility of an increased risk of lymphoma is one of the major

issues concerning the safety of long-term TNFa blocker use.

Only a few data are available concerning the action of

TNFa blockers on B cells infected or not with EBV. The pur-

pose of this work was to determine whether TNFa affects ap-

optosis in EBV-infected B lymphocytes, in Burkitt cell lines

infected or not with EBV and in B cells isolated from periph-

eral blood monoclonal cells (PBMCs), and whether infliximab

has a direct effect on apoptosis in these different types of B

cells in vitro.

2. Materials and methods

2.1. Cell culture

The LCL PRI and the Burkitt lymphoma cell line BL2 and

its EBV-infected counterpart BL2.B98.5 were grown at 37 C

in humidified 5% CO2 air in RPMI 1640 medium containing

GlutaMAX (GibcoBRL, Life Technologies, Cergy-Pontoise,

France) supplemented with 10% decomplemented fetal calf

serum (Dutscher, Brumath, France), 100 U/ml penicillin,

10 mg/ml streptomycin (GibcoBRL), 1 mM sodium pyruvate

(GibcoBRL), MEM vitamins 100Â (GibcoBRL) and 5 mg/ml

plasmocin (Cayla InvivoGen, Toulouse, France). Cells were

treated with TNFa (Invitrogen, Carlsbad, CA, USA), 10 ng/ ml for 30 min or 24 h, and infliximab (RemicadeÒ, Scher-

ing-Plough, Levallois-Perret, France), 5 mg/ml for 24 h.

2.2. Plasmid and transfection

The VINL Ik-Ba32/36a episomal vector was derived from

the previously described CKR 516 [21] vector by replacing the

enhanced green fluorescent protein-inducible marker with the

truncated version of the nerve growth factor receptor (NGFR)

lacking the cytoplasmic domain. This vector contains the

EBNA1 gene to ensure episomal replication, a bidirectional

tetracycline-inducible promoter driving the expression of two

independent cDNAs: Ik-Ba32/36a and NGFR, as a markerof induction. The expression of NGFR was determined by

flow cytometry and used to select cells with magnetic beads.

The induction of Ik-Ba32/36a was verified by Western

blotting.

A total of 1 mg vector/106 cells was transfected by double-

pulse electroporation (first pulse: 750 V, 25 mF and 201 U; sec-

ond pulse: 125 V, 3000 mF and 99 U). After 3 days of culture,

hygromycin-resistant cells (stable transfectants) were selected

(three weeks) by the use of 3 ml/ml hygromycin (Hygromycin B,

Sigma, Saint-Quentin Fallavier, France).

2.3. Selection of induced cells

Stably transfected cells were induced with 0.6 mg/ml doxy-

cyline for 24 h (Doxycycline, AP-HP, France) and labeled with

anti-low affinity NGFR (LNGFR) coupled with magnetic

micro beads for separation by the ‘‘MACSelectÔ LNGFR

System’’ procedure (Miltenyi Biotech, Paris, France). The in-

duced expression of LNGFR was assessed by flow cytometry.

CD19 quiescent B lymphocytes were separated from PBMCs

using the same method with anti-CD19.

2.4. Flow cytometry

Induced cells were washed in phosphate buffered saline

(PBS; BioMerieux, Marcy l’Etoile, France), incubated with

10 ml PE-labeled NGFR antibody (BD Pharmingen, Morangis,

France) and counted using a Coulter EPICS XL (Beckman

Coulter, Villepinte, France).

To measure the expression of TNFR1 and TNFR2, cells

were washed in PBS, incubated with 5 ml FITC-labeled

TNFR1 (FAB225F) and TNFR2 (FAB226F) antibodies

(R&D Systems Inc., Lille, France) and counted as described.

To measure the apoptosis rate, cells were washed in PBS,

resuspended in Annexin V binding buffer, incubated with

5 ml FITC-labeled Annexin V antibody (BD Pharmingen)

and 5 ml propidium iodide and counted as described.

338 F. Baran-Marszak et al. / Cytokine 33 (2006) 337 e 345



2.5. Western blotting

Viable cells were counted by the use of the trypan-blue

method. Total protein extracts were obtained as follows:

5 million cells were resuspended in lysis buffer containing

0.01% bromophenol blue (Sigma), 50 mM TriseHCl, pH 6.8, 2%

sodium dodecyl sulfate (Sigma), 2% glycerol (Sigma) and 2%2-mercaptoethanol (Sigma). The lysates were sonicated, boiled

and stored atÿ80 C. The extracts were separated on 10% poly-

acrylamide denaturing gel and transferred to nitrocellulose

membranes (Hybond-C Extra, Amersham Pharmacia Biotech,

Orsay, France). After transfer, the membranes were stained

with Ponceau-red (Sigma) to check that equal amounts of protein

were present in each lane. Membranes blocked for 2 h with 1%

non-fat milk (Regilait, Saint-Martin Belle Roche, France) in

Tris-buffered saline (TBS; 20 mM NaCl, 500 mM TriseHCl,

pH 8.0; Bio-Rad) were then incubated overnight with the first

antibody in TBS containing 1% non-fat milk. The antibodies

were anti-I-kBa (MAD 10B I-kBa, Dr R. Hay, University of

St Andrews, St Andrews, UK) at 1/50. After three washes inTBS and 1% non-fat milk, the corresponding horseradish

peroxidase-conjugated secondary anti-mouse antibodies (Bio-

Rad) at 1/5000, were added for 1 h. After being washed for 2 h

in TBS1% non-fat milk and0.1% Tween (Sigma), themembranes

underwent chemiluminescence (Lumiglo, Santa Cruz, CA, USA)

and autoradiography (Biomax, Kodak, Rochester, NY, USA).

2.6. Electrophoresis mobility shift assay (EMSA)

Nuclear and cytosolic proteins were extracted as described

previously [22]. Twenty micrograms of nuclear protein extract,

in 5 ml extraction buffer C (20 mM Hepes, 25% glycerol,

0.5 M NaCl, 1.5 mM MgCl2) were mixed with 1 ml of an-

nealed 32P-labeled oligonucleotide containing the kB site

(Genset, Paris, France) [23]. EMSA was performed as previ-

ously described [22]. The DNA-protein complexes were sepa-

rated on 6% non-denaturing polyacrylamide gel in 0.25ÂTriseBorateeEDTA buffer (Bio-Rad) by migration at 250 V.

The gel was dried and exposed to a phosphor-imaging screen

(Packard Instruments, Meriden, CT, USA). The radioactive

signal was visualized by the use of a phosphor system analyzer(Cyclone, Packard Instruments).

0

52

0

1

0,0

128

0100 101 102 103 104

128

0

10,0

20,0

30,0

40,0

50,0

60,0

medium PRI BL2 BL2B95.8

A

B

T N F α s

e c r e t i o n p g / m l

r e l a t i v e c e l l s n

u m b e r

log of fluorescence intensity

for TNFR1

100 101 102 103 104

log of fluorescence intensity

for TNFR2

BL2

BL2

B95.8PRI

BL2

BL2

B95.8PRI

Fig. 1. Secreted TNFa and expression of TNF receptors are increased in EBV-positive B lymphocytes. (A) Ten million cells of LCL (PRI), EBV-negative Burkitt

lymphoma (BL2) and their EBV-positive counterpart BL2.B95.8, were cultured in a new medium for 24 h at a concentration of 1 million cells/ml. After 24 h,

supernatants were concentrated on 10-kDa filters (Millipore) and the control (medium alone), and secreted TNFa was measured by ELISA. (B) Expression of

TNF receptors TNFR1 and TNFR2 was measured by flow cytometry in BL2, BL2.B95.8 and PRI. Histograms show the relative cells number and the log of fluo-

rescence intensity for TNR1 and TNFR2.




2.7. Quantification of cytokines in cell culture

supernatants by ELISA

Ten million cells were washed and grown in 15 ml com-

plete medium culture for 24 h. Supernatants were concentrated

to 500 ml with a centrifugal filter (cut-off:10 kDa; Amicon Ul-

tra-15 Centrifugal Filter Devices, Millipore, Bedford, MA,USA). Secreted TNFa was measured by ELISA (Quantikine

ELISA kit, R&D Systems). The optical density was deter-

mined by the use of a spectrophotometer (Multiskan EX,

Thermo, Cergy-Pontoise, France) set to 450 nm and was ana-

lyzed with use of the Ascent Software (Thermo). Duplicate

concentrations were averaged.

2.8. Quantitative RT e PCR

Total RNA was extracted from sorted NGFR-positive and

-negative cells by the use of the Qiagen kit following the rec-

ommendations of the manufacturer. We defined as referenceRNA a pool of RNAs extracted from different tonsils, lymph

nodes and spleens with benign reactive follicular hyperplasia.

RNA levels for the TNFa gene were quantified in parallel in

the different RNA extracts and in the RNA pool on an ABI

PRISM 7000 automat by the use of the TaqManR ‘‘Assay on

demandÔ’’ gene expression reference system (Applied Bio-

system, website: http//www.appliedbiosystem.com) (product

reference: Hs00174128-m1). The Abl1 gene was used as a ref-

erence gene for the control of amplification (product refer-

ence: Hs00245443-m1). Reverse transcription of 2 mg of

total RNA involved the use of the Archive kit RT from Ap-

plied Biosystems in a final volume of 50 ml. From this, a vol-

ume of 1.25 ml of cDNA was used for gene amplification. All

these steps were performed following the recommendations of

the manufacturer. The relative expression levels of the genes

were calculated as previously reported [24], with Abl1

mRNA expression used for normalization.

3. Results

3.1. TNFa, TNFR1 (p55) and TNFR2 (p75) are

differentially expressed in EBV-positive B-cell lines

The expressions of TNFa and its receptors were measured

in the LCL PRI and BL cell lines infected (BL2.B95.8) or not

(BL2) with EBV. The secretion of TNFa was measured by

ELISA in cell culture supernatants, and the expression of

TNFR1 and TNFR2 was determined by flow cytometry.TNFa secretion was elevated in PRI LCL cells (52 pg/ml)

(Fig. 1A). In contrast with BL2 cells, which did not release

any detectable amount of TNFa, BL2 B95.8 cells secreted

1 pg/ml of TNFa after 24 h of culture in a new medium. Al-

though low, this amount of TNFa was sufficient to induce

IkBa degradation in BL2 cells (see Fig. 5A, lane 3). The ex-

pression of the TNF receptors TNFR1 and TNFR2 was high

in all EBV-positive cells (Fig. 1B), but in our experiments it

was higher in BL2B95.8 than in PRI cells. PRI cells that se-

creted the highest levels of TNFa were subsequently used to

A

I-kBα

Ponceau red

15

7.8

0,0

5,0

10,0

15,0

B

I-kBm- +

- +

I-kBm - + - +

NGFR

FSC

0

0,5

1

1,5

2

2,5

C

I-kBm

2.3

1.3

T N F α

r e

l a t i v

e

e x p r e

s s i

o n

l e v

e l

T N F α s

e c r e t i o n p g / m l

Fig. 2. Specific inhibition of NF-kB reduces TNFa secretion. (A) LCL PRI cells were stably transfected with a tetracycline-inducible vector construct with the

cDNAs coding for I-kBa mutated on serine 32 and 36 (I-kBm) and NGFR as a marker of induction. Expression of NGFR revealed by flow cytometry and

I-kBa by Western blotting was induced after 0.6 mg/ml doxycycline treatment for 24 h. (B) After 24 h of induction, NGFR-positive cells were magnetically selected.

NGFR-positive cells and NGFR-negative cells were cultured for 24 h, supernatants were then concentrated, and secreted TNF a was measured by ELISA. (C)

mRNA expression levels of IkBm selected positive and negative cells were analyzed by quantitative RTePCR. The relative expression level of TNFa mRNA

was calculated with Abl1 mRNA expression used for normalization.


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analyze the regulation of the expression of the TNFa gene and

the effects of TNFa and infliximab on apoptosis.

3.2. The expression of TNFa is under the control of

NF-k B in LCLs

To specifically inhibit the transcriptional factor NF-kB, themutated form of I-kBa (mutated on serines 32 and 36) was

over-expressed in PRI cells. Cells were stably transfected

with the NGFR/mutated I-kBa-inducible vector. Inducibility

by doxycycline was verified by flow cytometry and Western

blotting (Fig. 2A). The number of NGFR-positive cells varied

from 30% to 60% after 24 h of induction by doxycycline; cells

were therefore selected in order to analyze homogeneous

populations of NGFR-positive cells. Secretion of TNFa was

measured by ELISA in the supernatants of the selected NGFR-

positive and NGFR-negative cells. The secretion of TNFa was

reduced from 15 to 7.8 pg/ml in NGFR-positive selected cells

(Fig. 2B) and the relative TNFa mRNA expression level was

twofold lower after inhibition of NF-kB (Fig. 2C).

3.3. B lymphocytes resist TNFa-triggered apoptosis

Peripheral blood CD19-positive cells, LCL PRI, Jurkat (T-

cell lines used as a control) and EBV-positive and -negative

BL cell lines were incubated with TNFa for 24 h, and the

number of Annexin V-positive cells was determined. The

rate of apoptosis (Annexin V-positive cells) was slightly

higher after TNFa treatment in the Jurkat T-cell line (20e

28% Annexin V-positive cells) but was unchanged in all B-

cells studied (Fig. 3A). The DNA binding activity of NF-kB

(p50/p65), which is known to be maximal in LCL and EBV-infected BL cells, was not stimulated by TNFa (Fig. 4, compare

lane 2 to lane 1 and lane 8 to lane 7) whereas it was stimulated

in Jurkat (Fig. 4, lane 11 compared to lane 10). The sensitivity

of LCLs to TNFa was therefore studied after specific inhibi-

tion of NF-kB. NGFR/mutated I-kBa-transfected cells were

induced and NGFR-positive (IkBm positive) and NGFR-negative

cells (IkBm negative) were selected. As expected, NGFR-

positive cells expressing IkBm showed an increased of sponta-

neous apoptosis from 43% to 56% of Annexin V-positive cells

(Fig. 3B, compare panel b to panel a), and NGFR-negative

cells (IkBm negative) were insensitive to TNFa from 43%

to 38% Annexin V-positive cells after TNFa treatment

(Fig. 3B, compare panel c to panel a). On the other hand,

NGFR-positive cells (IkBm-positive cells) were clearly sensi-

tized to apoptosis and this was enhanced by TNFa treatment

from 56% before TNFa to 66% Annexin V-positive cells after

TNFa treatment (Fig. 3B, panel d).

3.4. Infliximab has no effect on apoptosis of B

lymphocytes even after NF-k B inhibition

Supernatants from LCL PRI or EBV-positive BL2 were

used as a source of TNFa and transferred to BL2. Supernatants

from LCL PRI or EBV-positive BL2 transferred to BL2 cells

induced IkBa degradation in 30 min (Fig. 5A, lanes 1e

3).

The ability of infliximab to neutralize TNFa induced-degrada-

tion of I-kBa was showed by Western-blotting (Fig. 5B). Su-

pernatants from LCL PRI or EBV-positive BL2 pre-treated

with infliximab were transferred to BL2 cells (Fig. 5A, lanes

4e6). In BL2, I-kBa degradation was prevented by the treat-

ment with infliximab indicating that TNFa was neutralized. In

LCLs, there was no change in the I-kBa levels following in-

fliximab treatment (Fig. 5B), indicating that infliximab treat-

ment had no direct effect on IkBa degradation. In addition,

infliximab treatment had no effect on the DNA binding activ-

ity of NF-kB in LCL PRI and EBV-positive BL2 cells (Fig. 4,

lanes 3, 6, 9, 12). Finally, infliximab treatment had no effect on

the rate of apoptosis (number of Annexin V-positive cells was

unchanged) (Fig. 6A). We then tested the effect of infliximab

treatment following inhibition of NF-kB, which sensitized

cells to apoptosis. Although the inhibition of NF-kB enhanced

apoptosis of selected IkBm-positive cells (from 12% to 73% of

Annexin V-positive cells), infliximab had no additional effect

on apoptosis of IkBm-negative or -positive cells (11% to 12%

B

0

10

20

30

CD19 PRI BL2 BL2B95.8 JURKAT

0

TNF

A

% o

f a n n e x i n V - p o

s i t i v e c e l l s

I-kBm

- TNFα

+ TNFα

b

d

- +a

c

Annexin V

Fig. 3. In B lymphocytes, including LCLs, TNFa has no effect on apoptosis,

inhibition of NF-kB sensitizes LCLs to TNFa-induced-apoptosis. (A) LCL

PRI, BL2, EBV-positive BL2 (BL2.B95.8), CD19-positive selected quiescent

B lymphocytes from PBMCs and a control T-cell line (Jurkat) were treated

with 10 ng/ml TNFa (TNF) for 24 h. Apoptosis rate was measured by flow cy-

tometry with Annexin V. (B) Stably transfected LCL PRI cells were induced

with doxycycline for 24 h, the selected NGFR-positive cells (I-kBmþ) (panels

b and d) and NGFR-negative (I-kBmÿ) (panels a and c) cells were then treated

(panels c and d) or not (panels a and b) with 10 ng/ml TNFa for 24 h. Cell

apoptosis was measured by flow cytometry after Annexin V binding.




for IkBm-negative cells treated with infliximab, and 75%

to 73% for IkBm-positive cells treated with infliximab)

(Fig. 6B).

4. Discussion

In this study, we demonstrated that neither TNFa nor inflix-

imab had an effect on apoptosis in B cells infected or not with

EBV. The increased production of TNFa by EBV-positive

cells, particularly LCLs, suggested a possible effect of inflix-

imab on these cells. However, our observation that infliximab

had no effect on apoptosis of LCLs, even when cells were

sensitized to apoptosis by inhibition of NF-kB suggests that

TNFa had no autocrine action in these cells and that inflixi-

mab had no TNFa-independent effect.Previous studies have demonstrated that exogenous TNFa

increases DNA synthesis and immunoglobulin production by

mitogen-activated human B cells [25]. In addition, TNFa is

an autocrine growth factor in B-cell chronic lymphocytic leu-

kemia [25,26]. Finally, TNFa is one of the earliest genes tran-

scribed after antigen stimulation of B cells and is not sufficient

for but augments anti-Ig or anti-CD40þ interleukin-4-induced

B-cell proliferation [27]. In EBV-infected B cells, TNFa had

no effect on apoptosis, although it could compete with

p50/p65

p50/p50

000 Inf

6 7 8 9 10 11 12

tnf 0Inf Inf Inf tnf tnf tnf

PRI BL2 BL2B95.8 Jurkat

21 3 4 5

Fig. 4. TNFa or infliximab have no effect on the DNA binding activity of NF-kB in B lymphocytes. LCL PRI (lanes 1, 2 and 3), BL2 (lanes 4, 5 and 6), BL2.B95.8

(lanes 7, 8 and 9) and Jurkat cells (lanes 10, 11 and 12) were treated with TNFa (tnf) (lanes 2, 5, 8, 11) or infliximab (inf) (lanes 3, 6, 9, 12) for 24 h, and NF-kB

activity (p50/p65) was analyzed by EMSA.

A

B

1-kBα

Ponceau red

I-kBα

Ponceau red

Infliximab

1 2 3 4 5 6

BL2 BL2

0 P R I s u

p

B L 2 B 9 5

. 8 s u p

0 P R I i n f l i x

i m a b

p r e t r e

a t e

d s

u p

B L 2 B 9 5

. 8 i n

f l i x i m

a b

p r e t r e

a t e

d s

u p

0

Fig. 5. Infliximab neutralizes the secreted TNFa but has no effect on the TNF-induced I-kBa degradation in EBV-infected B cells. (A) Supernatants from EBV-

positive cells (PRI sup or BL2.B95.8 sup) were transferred to EBV-negative BL cells (BL2) for 30 min. The total protein extracts were separated on gel and I-kBa

degradation was analyzed by Western blotting. BL2 incubated with: LCL PRI supernatant (lane 2), BL2.B95.8 supernatant (lane 3), infliximab-pretreated LCL PRI

supernatant (lane 5), infliximab-pretreated BL2.B95.8 supernatant (lane 6). (B) Absence of a direct effect of infliximab. LCL PRI cells were treated with infliximab

for 24 h and expression of I-kBa was analyzed by Western blotting on total protein extracts.




lymphotoxin b (TNFb), which was identified as an autocrine

growth factor [28]. The constitutive activation of NF-kB by

LMP1 in LCLs [29,30] leads to the induction of target genes

such as Bcl2, Bfl1, Bcl-xl and cIAP1e2 involved in the inhi-

bition of apoptosis [31e33], the mechanism by which EBV-

infected B cells were shown to be resistant to TNFa-induced

apoptosis [15].

It is generally admitted that EBV infection promotes TNFa

production, in BL cells this secretion is variable and may be

very low [34]; we found that intracytoplasmic TNFa was

high in BLs (not shown) contrasting with the low secretion.

The increased secretion of TNFa in EBV-infected cells is con-

sistent with the presence of kB sites in the promoter region of

the TNFa gene [35]. Our finding of increased expression of

both TNFR1 and TNFR2 in EBV-infected cell lines confirmed

the results of previous studies [36,37] but we did not find any

correlation between the receptor number, receptor affinity and

the cytotoxic effect of TNFa [38]. Interestingly, although

TNFR1 triggering activates both caspases and NF-kB [39],

TNFa and infliximab had no effect on the activity of NF-kB

and the rate of apoptosis in the EBV-positive B cells. There-

fore, these cells appear to be insensitive to TNFa. Neverthe-

less, in agreement with earlier observations, the cells

underwent spontaneous apoptosis following inhibition of

NF-kB [31], became sensitive to the apoptosis induced by

TNFa [15], but remained insensitive to infliximab even after

0

10

20

30

40

CD19 PRI BL2 BL2B95.8

% o

f a n n e x i n V - p o s i t i v e c e l l s

Annexin V

I-kBm

Without

Infliximab

A

B

With

Infliximab

0

Infliximab

11 %

12 %

75 %

73 %

Fig. 6. Treatment with infliximab has no effect on apoptosis of B lymphocytes infected or not with EBV even after NF-kB inhibition. (A) LCL PRI, BL2,

BL2.B95.8 and CD19-positive selected quiescent B lymphocytes from PBMCs were treated with 5 mg/ml infliximab for 24 h. Apoptosis rate was measured by

flow cytometry with Annexin V. (B) Stably transfected LCL PRI cells were induced with doxycycline for 24 h, and selected, the NGFR-positive ((I-kBmþ) (panels

b and d) and the NGFR-negative (I-kBmÿ) (panels a and c) cells were treated (panels c and d) or not (panels a and b) with infliximab for 24 h. Cell apoptosis was

measured by flow cytometry after Annexin V binding.




the inhibition of NF-kB. Thus confirming the absence of a di-

rect effect of infliximab on LCLs. In addition, we ruled out the

possibility of increased apoptosis following a direct interaction

of infliximab with transmembrane TNFa as suggested in T

lymphocytes [16]. After neutralization of TNFa with inflixi-

mab no effect on apoptosis of LCLs was observed confirming

that TNFa had no autocrine role in EBV-infected cells. Thus,in comparison with its ability to induce apoptosis in T cells of

the lamina propria in vivo and in vitro in CD, of blood mono-

cytes in CD and RA and of synovial monocytes/macrophages

in RA, infliximab is unable to increase apoptosis in B cells,

which demonstrates that the action of TNFa blockers may

vary in different cellular types.

The possible increased risk of lymphoma with TNF antag-

onists is one of the major issues concerning the long-term

safety of TNFa blockers. Even if a few studies suggest that

the incidence of lymphoma is increased in RA patients treated

with TNFa blockers [20,21], it is impossible to determine if

this incidence is linked with anti-TNFa-treated RA being

more active or corresponds to excessive risk induced by theTNFa blocker itself. If an increased incidence of lymphoma

was induced by TNF blockers, it would not involve, on the ba-

sis of our in vitro data, a direct effect of the treatment of B

cells infected or not with EBV but rather an impaired immune

surveillance by T cells.

Acknowledgments

This work was supported in part by a grant from Schering-

Plough. C.L. was supported by the Fondation pour la re-

cherche medicale (FRM).

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