NF-KB ACTIVATION BY TUMOR NECROSIS FACTOR <y IN THE JURKAT T CELL LINE IS

INDEPENDENT OF PROTEIN KINASE A, PROTEIN KINASE C, AND Ca”-REGULATED KINASES

Jean Feuillard,’ HClkne Gouy,’ Georges Bismuth,’ Leo M. Lee,* Patrice Deb&’ Marie Kijrner’x”

NF-KB is a DNA-binding regulatory factor able to control transcription of a number of genes, including human immunodeficiency virus (HIV) genes. In T cells, NF-KB is activated upon cellular treatment by phorbol esters and the cytokine tumor necrosis factor (Y (TNFcK). In the present work, we investigated the molecular events leading to NF-KB activation by TNFcv in a human T cell line (Jurkat) and its subclone JCT6, which presents a deficiency in the PKA transduction pathway. We found that in both cell lines, both phorbol ester and TNFol were able to activate NF-KB. Phorbol activation was positively modulated by Ca” influx while TNFol activation was not. Furthermore, while PMA activation was inhibited by the PKC inhibitor staurosporin, the TNFa effect was unchanged. TNFcr did not activate CAMP production and its signal was not modulated by CAMP activators. Moreover, CAMP activators did not activate NF-KB in Jurkat cells. Thus, TNFol-induced NF-KB activation was found to be mediated by none of the major signal-mediating kinases such as protein kinase C (PKC), protein kinase A, or Ca*+ -regulated kinases. Furthermore, we found that cytoplasmic acidification facilitated NF-KB activation by both TNFa and PKC, by a mechanism that increases NF-KB/II& dissociation without affecting the NF-KB translocation step. Copyright o 1991 by W.B. Saunders Company

Tumor necrosis factor Q (TNFa) is a cytokine involved in inflammation and several immune re- sponses (for review see references 1 and 2). Further- more, TNFol was reported to activate the replication of human immunodeficiency virus (HIV) in latently in- fected T cells.3 It also induces preferential cytolysis of HIV-infected cells.4 Moreover, TNFol plasma concen- tration was reported to be significantly higher in AIDS patients as compared to healthy individuals.’ There- fore TNFa appears as a possible co-factor for HIV in development of AIDS.

TNFar activation of HIV replication and expres- sion of genes involved in the immune response, such that as for interleukin-2 (IL-2) receptor (Y, is triggered

‘Laboratoire d’Immunologie Cellulaire et Tissulaire, CNRS U625, Bat. CERVI, Hopital de la PitiC Salpetriere, 83, Bd. de l’Hopita1, 75013 Paris, France.

‘Nucleic Acid and Protein Synthesis Laboratory, NCI-FCRF, Pro- eram Resources Inc. Blde. 469. P.O. Box B. Frederick. MD 21701, USA.

.,

*To whom correspondence should be addressed. Copyright o 1991 by W.B. Saunders Company 1043-4666/91/0303-0009$05.00/O

KEY WORDS: NF-KBiprotein kinases/T cells/TNFa

CYTOKINE, Vol. 3, No. 3 (May), 1991: pp 257-265

by its interaction with specific cell surface receptors.6,7,8 Interaction of TNFa with its receptor results in a series of intracellular events including activation of the wide- spread transcription regulatory factor NF-KB. Both IL-2 receptor OL and HIV regulatory sequences harbor a transcription enhancer domain able to bind NF- KB. 9~10~11~12 NF-KB was shown to be involved in the regulation of a variety of genes including immunoglob- ulin K light chain in B cells, MHC class I, and several viral genomes.13~‘4~15~16

NF-KB is a 50-kD/65-kD heterodimer, interacting with a cytoplasmic inhibitor, 1~B.l~ Cloning of the murine ~50 subunit of NF-KB demonstrated its struc- tural identity with human KBFl,ls319 the MHC class I KB site-binding protein.” Moreover, p50 displays exten- sive amino acid sequence homology with the v-rel oncogene and the Drosophila maternal morphogene dorsal. l9 Recently ~85, which is the product of the c-rel gene, was shown to interact with the KB site.21

In mature B cells NF-KB is constitutively present in nuclei whereas in pre-B cell lines and T cells NF-KB is complexed with IKB in the cytoplasm. Activation of pre-B cells with lipopolysaccharide (LPS) or T cells with TNFa or phorbol esters and lectins (PHA) results in NF-KB dissociation from IKB and translocation into

257

258 I Feuillard et al.

the nucleus.” Although the molecular events leading to an active nuclear NF-KB are yet poorly understood, it is very likely that one of the essential steps involves protein phosphorylation.‘7~22~23 Furthermore, in vitro phosphorylation of purified IKB results in loss of its inhibitory activity.23 Recently, it has been demon- strated that at least two protein kinases, protein kinase C (PKC) and heme-regulated eIF-2 kinase, are able directly to phosphorylate IKB in vitro.23 However, it has been suggested that TNFo activation of NF-KB in T cells is independent of PKC,” although TNFo induces PKC translocation.z Furthermore the involvement of protein kinase A (PKA), a CAMP-dependent kinase, in NF-KB activation remains unclear. Indeed, a role for PKA in NF-KB activation remains highly controver- sial, 22X23,24,26 and no evidence of a direct phosphorylation of IKB by PKA has been reported so far.

In the present study we further investigated the mechanism of NF-KB activation by TNFol in T cells. In order to investigate the role of PKA, we developed a subclone of the human T cell line Jurkat, named JCT6, which does not produce CAMP in responce to cholera toxin treatment and prostaglandin E, (PGE,). We used JCT6 cells to study the involvement of PKA in NF-KB activation. We found that in both the Jurkat cell line and its JCT6 subclone, TNFa activation of NF-KB is independant of both PKC and PKA activators. Further- more, we found that TNFa activation of NF-KB was not mediated by Ca*‘-dependent protein kinases nor by intracellular pH (pH,) decrease, although pH, de- crease facilitated NF-KB activation by both TNFol and PKC.

RESULTS

Protein Kinase A and Protein Kinase C Involvement in NF-KB Activation by TNFa

We first examined the potential involvement of PKA in NF-KB activation. For this purpose, we devel- oped the cholera toxin (CT)-resistant subclone of Jurkat cells termed JCTB, which presents a defect at the G, protein level (see Materials and Methods and Fig. 1). Thus, we used JCT6 as a convenient cellular model for delineation of G, protein involvement in TNFa signal transduction pathway.

We examined the capacity of JCT6 clone to re- spond to TNFar and phorbol 1Zmyristate 13-acetate (PMA) plus phytohemagglutinin (PHA). The results, represented in Fig. 2A and 2B, show that both PMA plus PHA (PP) and TNFa activated NF-KB in JCT6 cells with equal efficiency as compared to Jurkat cells. These results suggest that G, protein is probably not involved in TNFor signal transduction. Besides, TNFcY did not induce a detectable increase in CAMP in either Jurkat cells or JCT6 cells in response to TNFo treat-

175-

150-

g 125-

5 (0

0 IOO- 7 1

; 75- V

3 50-

0

25-

CYTOKINE, Vol. 3, No. 3 (May 1991: 257-265)

Jurkat

JCT6

none PGE2 CT Forskolin TNF

Stimulation

Figure 1. CAMP production in Jurkat and JCT6 cells.

Cells were incubated for 10 min with PGE,, 4 h with cholera toxin (CT), 1 h with forskolin, and 2 h with TNFa. Reagent concentrations are indicated in the Materials and Methods.

ment (Fig. 1). We also examined, in both Jurkat and JCT6 cells, the effect of CAMP inducers on NF-KB activation. As shown in Fig. 3A and 3B, none of the CAMP inducers CT, PGE,, and forskolin induced NF-KB translocation into the nuclei. Finally, we investi- gated whether the actions of CAMP activators modu- late NF-KB activation by TNFa. We incubated Jurkat cells with CT, PGE,, or forskolin prior to TNFa treatment. No significant effect of CAMP activators on TNFa activation of NF-KB was seen (data not shown) suggesting that CAMP neither participated in NF-KB activation, nor modulated, either positively or nega- tively, the TNFcl activation pathway. These results indicate that in T cells, TNFol activation of NF-KB was not mediated by an increase of CAMP and therefore was probably independent of PKA.

We next investigated the involvement of PKC in signaling by TNFa. Treatment of JCT6 and Jurkat cells with staurosporine, a PKC inhibitor,*’ did not affect TNFol activation of NF-KB, whereas it strongly inhib- ited PMA plus PHA (PP) activation in both JCT6 and Jurkat cells (Fig. 2A and 2B). Thus, TNFol activation of NF-KB appears to be independent of PKC activation in Jurkat cells. This finding is in agreement with previous reports.24,28

E#ect of Ca” Ions on TNFa Activation of NF-uB

Ca*+ is an intracytoplasmic second messenger for a number of signals. Intracellular calcium increase stim-

NF-KB activation by TNFa / 259

A Jurkat cells

B JCT6 cells

I Stimulation 0 PPT 0 PPT’ Staurosporine - - - + + +

Stimulation ‘0 PPT 0 PPT’ Staurosporine - - - + + + 1_

1 2 3 4 5 6 123456

Figure 2. Gel retardation of “‘P-PRE with Jurkat and JCT6 cell line nuclear extracts after cell stimulation with TNFcl or PMA plus PHA.

Jurkat cell lines (A) and JCT6 cell line (B) were preincubated 30 min in the presence (lanes 4 to 6) or absence (lanes 1 to 3) of staurosporine. Stimulation with PMA plus PHA (PP) (1 anes 2, 5) and TNFu (lanes 3, 6) was carried out for 3 h. The EMSA was performed as described in Materials and Methods using 10 kg of nuclear protein per lane. NF-KB migration is indicated by the arrow.

A B Jurkat cells

Stimulation ‘0 TCTFPg’ Stimulation

JCT6 cells

‘0 TCT F Pg’

1 2 3 4 5 1 2 3 4 5

Figure 3. Gel retardation assay of “P-PRE with Jurkat and JCTQ nuclear extracts after cell stimulation with CAMP inducers.

Treatment of the Jurkat cell line (A) and JCT6 cell line (B) with CAMP activators cholera toxin (CT) (lane 3) forskolin (F) (lane 4), and prostaglandin E, (Pg) (lane 5) was carried out for 3 h as described in Materials and Methods. The amount of nuclear protein used for retardation of ‘*P-PRE was 10 up. All reagent concentrations are as indicated in the Materials and Methods section. NF-KB migration is indicated by the arrow.

260 I Feuillard et al.

I inn Stimulation 0 PP T 0 PP T 0 PP T

123456769

Figure 4. Effect of Ca’+ depletion on NF-KB activation.

Jurkat cells were preincubated for 10 min in Ca*+-containing (lanes 1 to 3) or Ca’+-free (lanes 4 to 6) medium prior to 1 h stimulation with PMA plus PHA (PP) (lanes 2,5) or TNFa (T) (lanes 3,6). Medium control conditions were performed in RPMI, 10% fetal calf serum (lanes 7 to 9). The amount of nuclear protein used for retardation of 32P-PRE was 15 ug. NF-KB migration is indicated by the arrow.

ulates several protein kinases, including PKC29 and calmodulin-regulated kinases (reviewed in reference 30). We investigated the role of Ca2+ in TNFol signal transduction and, therefore, the role of calmodulin- regulated protein kinases as mediators of the TNFo signal.

To study the effect of Ca*’ on TNFa activation of

9 e 1 e e Costimulation 0 0 ,O 0 P ,O 0 f -0

Stimulation OOOPPPTTT

CYTOKJNE, Vol. 3, No. 3 (May 1991: 257-265)

NF-KB, we incubated Jurkat cells in a Ca*+-free me- dium and stimulated the cells by TNFo and PP after addition of either 1 mM Ca*’ or 0.1 mM EGTA (Fig. 4). In the presence of extracellular Ca*‘, both PP and TNFa efficiently activated NF-KB as compared to their effects in control experiments performed in complete culture medium (Fig. 4, lanes 1 to 3 and 7 to 9). However, when extracellular medium was depleted of Ca*+ by EGTA, PP treatment of Jurkat cells failed to activate NF-KB (Fig. 4, compare lane 2 with lane 5). In contrast, TNFol-induced activation was unaffected by extracellular Ca2+ depletion (Fig. 4, compare lane 3 with lane 6) suggesting that TNFa activation of NF-KB is independent of Ca*’ influx. In order to test this hypothesis, we next examined the effect of three Ca2’ influx activators, the Ca2+-ionophore ionomycin, the lectin PHA, and the CD3 monoclonal antibody UCHTl. These three Ca2’ influx activators systematically poten- tiated NF-KB activation by PMA (Fig. 5, compare lane 4 with lanes 5 and 6, and compare lane 11 with lane 13). In contrast, when cells were treated with TNFa in the presence of either ionomycin or PI-IA, NF-KB activa- tion remained unaffected (Fig. 5, compare lane 7 with lanes 8 and 9). Furthermore, TNFo treatment of Jurkat cells did not increase Ca*+ intracellular concen- tration (not shown). These results indicated that intra- cellular free Ca*’ is not involved in the TNFa activa- tion pathway leading to NF-KB translocation. Involvement of a calmodulin-regulated kinase as medi- ator of the TNFo signal seems therefore to be ex- cluded.

Eflect of pH on NF- KB Activation

It has been reported that treatment of peripheral blood lymphocytes (PBL), with PI-IA or ionomycin, induces calcium-dependant cytoplasmic acidification.3’

S8 0 0:: 0 POP

123456789 10 11 12 13

Figure 5. Effect of Ca”-influx inducers on NF-KB stimulation.

The effects of Ca*‘-influx inducers alone (lanes 2, 3, 12) or in the presence of PMA (lanes 5, 6, 13) or TNFa (lanes 8, 9) were tested in conditions de- scribed in Materials and Methods. Treatment of Jurkat cells with PHA and ionomycin (Iono.) was performed in flasks whereas stimulation with UCHTl monoclonal anti-CD3 antibodies (aCD3) was per- formed in 6-well plates coated with the aCD3. The amount of nuclear protein used for retardation of “P-PRE was 10 ug. NF-KB migration is indicated by the arrow.

NF-KB activation by TNFcl / 261

We investigated the effect of acidification on NF-KB activation. We first studied the effect of pH in vitro by incubating cytosolic extracts from untreated Jurkat cells with 32P-labeled PRE oligonucleotide at various pH ranging from 8.0 to 5.5. Overlapping pH between the three buffers used (HEPES, MOPS, and MES) permitted us to assess that the effect was not due to the chemical nature of buffers. In cytosols, maximum activation of NF-KB was achieved at pH 6.5 (Fig. 6A). The specificity of the pH-activated NF-KB was assessed by competition experiments in HEPES buffer pH 7.0 (not shown). The activation of NF-KB by acidification of protein-DNA incubation media was not due to an increased affinity of NF-KB for the oligonucleotide, since the binding of NF-KB extracted from PP-treated Jurkat cell nuclei was partially inhibited when the pH was equal to or less than 6.5 (Fig. 6B). This result indicated that although binding of NF-KB to its specific DNA sequence was favored at pH 7 to 8, acidic pH induced dissociation of NF-KB from IKB in the cyto- plasma in a manner comparable to the previously described detergent dissociation.32,33

In order to investigate whether acidification partic- ipates in NF-KB activation in vivo, we studied the effect of intracellular pH (pH,) variations on NF-KB in situ. Without stimulation, the intracytoplasmic pH of the Jurkat cell line ranged from 7.4 to 7.5 (not shown). We incubated the cells in the “K’ medium” at pH 7.0, 7.3, and 7.5 as described in Materials and Methods, in the

presence of the K’/H’ ionophore nigericin to equili- brate pH, with the pH of the extracellular K’ medium (pHe).34 It has been shown that in the K’ medium, calcium influx is strongly reduced.35 Therefore, the incubation conditions used in our experiment allow the study of the pH effect independently of calcium influx. Once equilibrated at the right pH, cells were treated with PP or TNFcx and their nuclear and cytosolic extracts were tested for NF-KB by electrophoretic mobility shift assay (EMSA). In contrast to in vitro experiments, no activated NF-KB was found in either cytoplasmic fractions (not shown) or nuclear extracts of cells incubated in nigericin pH 7.3 or 7.0 media (Fig. 7, lanes 4,7). Thus, a drop in pH, from 7.5 to 7.0 alone was not sufficient to dissociate NF-KB from its cytoplas- mic inhibitor in situ. Control experiments were per- formed in K’ medium, pH 7.3, in the absence of nigericin and in RPMI. TNFa activation of NF-KB was identical in RPM1 and control K’ medium (Fig. 7, lanes 3 and 6). This result is in agreement with the previous finding that TNFa activates NF-KB indepen- dently of calcium influx (Figs. 4 and 5). The Jurkat cell line treatment with PP in control K’ medium failed to activate NF-KB as compared to control medium, RPM1 (Fig. 7, lanes 2 and 5), pointing out the requirement for calcium influx for NF-KB activation by PP treatment. However, when treated with PP or TNF~L, NF-KB activation was enhanced by lowered pH, (Fig. 7, lanes 11,12 and 14, 15) as compared to the “resting” pH 7.5

B

Buffer MES MOPS HEPES Buffer MES MOPS HEPES

nnn nnn PH 515 6 6,5 6,5 7 7,5 7 715 8 PH 5,5 6 6,5 6,5 7 7.5 7 7.5 a

1,. 7.i

123456789 123456789

Figure 6. Effect of pH on NF-KB binding in vitro.

Proteins were extracted from cytosol (A) or nuclei (B) of untreated cells (A) or PMA plus PHA (PP)-treated Jurkat cells (B). Equal amounts of extracted proteins were incubated with 10 kg SS-DNA and 1 ng of ‘*P-PRE in medium buffered at pH 5.5 to 8.0 using 20 mM MES, MOPS, and HEPES as buffers. The time of incubation and the gel retardation assays were performed in standard conditions described in Materials and Methods. NF-KB migration is indicated by the arrow.

262 1 Feuillard et al. CYTOKJNE, Vol. 3, No. 3 (May 1991: 257-265)

Nigericin

Medium Conditions

Stimulation

I I RPM1 PH 7,3 PH 795 PH 793 PH 790

nnnnn 0 PP T 0 PP T 0 PP T 0 PP T 0 PP T

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

(Fig. 7, lanes 8 and 9) Since nigericin did not induce [Ca”+], change (not shown), these results indicated that cytoplasmic acidification facilitated NF-KB activation during both TNFa and PP treatment and this effect was independant of calcium influx.

When Jurkat cells were treated with ionomycin or PHA in the presence of Ca’*, pH, dropped from 7.45 to 7.25 (data not shown). A comparable pH, drop did not occur in Jurkat cells after TNFol treatment. Thus, acidification of the cytoplasm facilitated NF-KB activa- tion by TNFa, even though a drop in pH, was not an event mediating the TNFa activation signal.

DISCUSSION

In this study we have investigated various possible transduction pathways of the TNFa signal, leading to NF-KB activation in T cells. Our results showed that neither PKC nor PKA mediate the TNFcx activation of NF-KB. Furthermore, probably none of the Ca*+- regulated kinases participates in TNFar signal transduc- tion since TNFol did not modulate calcium influx and calcium influx did not alter TNFLx activation of NF-KB (Figs. 4 and 5). The fact that PKC was shown not to be involved in NF-KB activation during the TNFcx treat- ment is somehow paradoxical. Indeed, PKC stimula- tion by PMA activates NF-KB, and it was reported that TNFIx induces PKC translocation to the cell membrane in Jurkat cellsz5 A possible explanation might reside in the kinetics and the degree of stimulation of PKC activity by TNFol versus PMA. It was shown that TNFa-induced PKC translocation reaches its maxi- mum at 6 min in Jurkat cells.25 After 10 min of TNFa stimulation, translocated PKC drops to 50% of the maximum. Control experiments showed that after 10 min of PMA treatment, the PKC translocation sur- passes the maximum TNFa-induced translocation.

Figure 7. Effect of cytoplasmic acidification on NF-KB activation.

Proteins were extracted from cell nuclei after the following treatments: lanes 1 to 3, stimulation of Jurkat cells in RPM1 as control conditions of cell treatment; lanes 4 to 6, stimulation in K’ medium (pH 7.3) in absence of nigericin; lanes 7 to 15, stimulation in the presence of nigericin at pH 7.5 (lanes 7 to 9) pH 7.3 (lanes 10 to 12), and 7.0 (lanes 13 to 15). Lanes 2, 5, 8, 11, 14: 1 h stimulation by PMA plus PHA (PP). Lanes 3, 6, 9, 12, 15: 1 h stimulation by TNFa (T). NF-KB migration is indi- cated by the arrow.

Therefore, the time and the extent of PKC activation by TNFa might not be sufficient for NF-KB activation.

In contrast with Jurkat cells, JCI6 cells are not able to produce CAMP in responce to CT and PGE, treatment (Fig. 1). CT induces ADP-ribosylation of the (Y subunit of the G, protein (see reference 36 for review). This modification of the G, protein stimulates adenylate cyclase to produce CAMP. PGE,, by interact- ing with a specific receptor, also activates CAMP production via G, protein.37 The lack of responsiveness of JCT6 cells to CT and PGE, is probably associated with the incapacity of the G, protein OL subunit to be ADP-ribosylated (our current investigations). The fact that TNFcx induced NF-KB with equal efficiency in both Jurkat cells and JCT6 cells suggests that TNFa uses a second messenger system that does not involve G, protein. Logically, this finding was further supported by the fact that TNFa did not induce CAMP production in normal Jurkat cells. Furthermore, agents which were able to activate CAMP production in Jurkat cells, such as CT, PGE,, and forskolin, failed to activate NF-KB, suggesting that in those cells the PKA pathway is not involved in NF-KB activation. The fact that CAMP was reported to be involved in activation of NF-KB in YT natural killer and 7OZ/3 pre-B cell lines” could be explained by a tissue specificity for NF-KB activation pathways. Recently, IKB was isolated from the cytosol of human placenta. In that particular tissue two forms of IKB exist, the smaller 37-kD form being predomi- nant.39 Possibly, in some tissues, such as the pre-B cell line 702/3, a “PKA-responsive” IKB is the predomi- nant form of IKB, while in other cells, such as the Jurkat cell line, the dominant form of IKB is “PKA unresponsive.” Furthermore, ~50 could be also a target for PKA signaling since a potential PKA phosphoryla- tion site was reported to be located close to the C terminus of ~50.‘~

NF-KB activation by TNFa / 263

We also showed that acidification of the cytoplasm facilitated NF-KB activation. The pH facilitation did not discriminate between PP and TNFcx treatment. This could be explained by a conformational effect of pH on the NF-KB/IKB complex rather than a specific effect of the pH on a enzymatic system. Although a drop of pH, from 7.5 to 7.0 induced an in vitro dissociation of the IKB/NF-KB complex, alone it was not sufficient to induce NF-KB activation in situ (or to irreversibly dissociate NF-KB from IKB). A possible explanation for these data is that cytoplasmic acidifica- tion alone dissociated NF-KB from IKB (as suggested by the in vitro data), but it was not able to induce translocation into the nuclei. In contrast, TNFa or PMA plus PHA treatment induced both the cytoplas- mic NF-KB/IKB dissociation and the translocation of NF-KB into the nuclei. These results argue for the existence of at least two steps of NF-KB activation. The first step would consist in NF-KB/IKB dissociation, and the second step would be translocation into the nuclei by an active mechanism which remains to be deter- mined. Our results indicate that the intracellular pH drop facilitated NF-KB activation by TNFa by increas- ing the amount of cytosolic “free” NF-KB.

Our results showed that TNFa activation of NF-KB is not mediated by PKC, although PKC is able to activate NF-KB when stimulated with phorbol esters. This argues in favor of multiple sites as targets for NF-KB activation. Positive cooperation between these sites might account for the synergistic activation of NF-KB by TNFa and PMA stimulation.”

The fact that TNFa activation of NF-KB is not mediated by PKC, PKA, or calmodulin-dependant protein kinases reinforces the hypothesis of a specific kinase as mediator of the TNFa signal. Indeed, TNFa was shown to induce protein phosphorylation,40~4’ even though no known Ser/Thr kinases were shown to mediate TNFa signaling. Furthermore, no tyrosine kinase was shown to be activated by TNFa. Moreover, phosphorylation of IKB as a consequence of TNFo stimulation was not reported. Therefore the possibility that there is a TNFa-induced post-translational modifi- cation different from phosphorylation of IKB remains to be investigated.

MATERIALS AND METHODS

Cells Lines and Monoclonal Antibodies The Jurkat cell line, a CD4’ human malignant T cell line

(clone E6-l), was kindly provided by Dr. A. Alcover (Institut Pasteur, Paris, France) and was cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and stan- dard concentrations of L-glutamine and antibiotics. The Jurkat T cell subclone, named JCT6, was obtained by a long-term culture of E6-1 cells in complete culture medium in the presence of CT holotoxin. During the selection phase,

cells were progressively adapted to grow with continuous exposure to increasing concentrations of CT (5 to 100 @ml). Toxin additions were renewed twice a week and the cell density always maintained between 5 x lo5 and 5 x lo6 per ml. After one month of selection in these culture conditions the resulting cells were cloned twice by limiting dilutions and the clones expanded in complete culture medium. Second cloning and subsequent cultures were performed without any addition of CT. Resistance of JCT6 to the CT anti- proliferative effect coincides with its loss of CAMP produc- tion after both PGE, and CT treatment (Fig. 1). However, when JCT6 cells were treated with forskolin, a diterpene substance capable of interacting directly with adenylate cyclase,42 CAMP production was comparable that in the Jurkat cell line (Fig. 1).

UCHTl, a CD3 specific monoclonal antibody, was a kind gift of Dr. P.C.L. Beverlay (Imperial Cancer Research, London, UK).

Reagents

Phorbol 12-myristate 13-acetate (PMA) and forskolin were obtained from Sigma (St. Louis, MO, USA). Phytohe- magglutinin (PHA) was from Welcome (Paris, France). Cholera toxin (CT) and prostaglandin E, (PGE2) were from Campbell (CA, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively. Ionomycin, 2’,7’bis-(carboxyethyl) 5,6- carboxy-fluorescein (BCECF) acetoxymethylester (BCECF/ AM), staurosporine, and nigericin were purchased from Calbiochem (Meudon, France). TNFo was a kind gift of Ch. Damais (CNRS UPR405, Paris, France). Ro-20-1724 was a kind gift of Hoffmann-La Roche Laboratories (Base& Switzer- land). 2-(N-morpholino)ethane sulfonic acid (MES), 3-(N- morpholino)propane sulfonic acid (MOPS), and N-(ZHy- droxyethyl)piperazine-N’-(2-ethane sulfonic acid) (HEPES) were from Sigma (St. Louis, MO, USA). Ethylene glycol- O,O’-bis(2-aminoethyl)-N,N,N’,N’-tetraacetic acid (EGTA) was from Fluka Chemie AG (Buchs, Switzerland).

Cell Stimulation Cells were stimulated with TNFo (100 U/ml) or 1 l&ml

phytohemagglutinin (PHA) or 50 rig/ml phorbol 1Zmyristate 13-acetate (PMA) or with both PMA plus PHA (PP) for 1 to 3 h at 37°C. Forskolin, cholera toxin, and PGE, were used at 0.1 mM, 100 rig/ml, and 1 FM, respectively. Ionomycin was used at 340 nM. Stimulations by UCHTl were carried out with immobilized antibodies. Inhibition assays were per- formed by preincubating cells with 50 @ml staurosporine for 30 min prior to cell stimulation. Cell stimulations were performed in RPM1 medium except for the extracellular calcium ([Ca”],) depletion assay (Fig. 4) and cytoplasmic pH assays (Fig. 7). For the [Caz+le depletion assay cells were incubated in “Na+ medium” (140 mM NaCl, 5 mM KCI, 10 mM glucose, and 20 mM HEPES, pH 7.4). Either 1 mM CaCl, or 100 uM EGTA were added to Na’ medium when indicated. The effect of intracellular pH (pH,) on the activa- tion of NF-KB was determined by incubating Jurkat cells in “K’ medium” (130 mM KCI, 10 mM NaCI, 1 mM CaCl,, 10 mM glucose, 20 mM Tris-HEPES) buffered at three pH’s: 7.5, 7.3, and 7.0. Nigericin (700 @ml) was added to K+ medium 10 min prior to TNFcl or PP stimulation of the cells.

264 I Feuillard et al. CYTOKINE, Vol. 3, No. 3 (May 1991: 257-265)

Nigericin was removed with 5 mgiml bovine serum albumin.34 Cells were washed twice in phosphate-buffered saline (1 x PBS, pH 7.4) prior to protein extraction.

Protein Preparation Nuclear proteins were extracted according to Osborn et

a1.28 except that both cytoplasmic and nuclear fractions were ultracentrifuged and kept in their extraction buffers (buffer A and C, respectively) instead of being dialyzed against buffer D. Protein concentration was determined according to Bradford.43 Typically it ranged from 5 to 12 mg/ml. Proteins were diluted in buffer D (pH 7.9) for gel retardation assays.

Oligonucleotides The probe for NF-KE% in gel retardation assays was a

31-mer synthetic double-stranded oligonucleotide containing a direct repeat of the KB site, the sequence of which was determined by the positive regulatory element (PRE) of the HIV LTR. Its sequence and characteristics of binding to proteins extracted from Jurkat cells were described previ- ously.” For EMSA, PRE was end-labeled with 32P with phage T4 kinase.

Electrophoretic Mobility Shift Assay (EMSA) Nuclear protein extracts (10 to 15 pg) were preincu-

bated for 15 min on ice with 10 p,g of salmon sperm DNA (SS-DNA). Both protein and SS-DNA were diluted in buffer D (pH 7.9). Protein extracts were diluted in buffer D (pH 7.9) in order to normalize protein concentration in each lane. 32P-labeled PRE oligonucleotide (100,000 to 500,000 cpm/ng) was added and the reaction was allowed to proceed at room temperature for an additional 15 min. Bound and free oligonucleotides were then separated as described.” To determine the effect of pH on NF-KB in vitro the incubation with the oligonucleotide was carried out in HEPES, MOPS, or MES buffers, so that the resulting pH of the reaction mixtures ranged from 5.5 to 8.0 as indicated in Fig. 6A and 6B.

Determination of CAMP Level in Jurkat Cells CAMP levels in Jurkat cells were determined by double

radio-immunoassay as described.4s The cell stimulations were performed in the presence of 20 mM Ro-20-1724, a CAMP phosphodiesterase inhibitor. CAMP levels were measured using the Arnersham CAMP assay kit (Amersham, Les Ulis, France).

Measurement of pHi Calibration of pH, was performed using nigericin in “K+

medium” as described.34 Briefly, after loading the cells with the fluorescent probe BCECF/AM, the cells were resus- pended in K’ medium. The fluorescence of BCECF was measured using 500 and 530 nm as excitation and emission wavelengths, respectively. The K+/H’ ionophore nigericin was added in the medium after establishment of a stable baseline. The pH of K’ medium was varied from 6.8 to 7.6 by adding either KOH or HCl to the cell suspension.

Acknowledgments We are grateful1 to Mr. Daniel Cefai’ for helpful

advice during in situ pH and calcium experiments and to Mrs. Nadine Tarantino for skillful technical assis- tance. We thank Dr. Annick Harel-Bellan for helpful discussion and reading of the manuscript.

REFERENCES

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