INFECTION AND IMMUNITY, Jan. 1992, p. 231-236 Vol. 60, No. 10019-9567/92/010231-06$02.00/0Copyright © 1992, American Society for Microbiology

Roles of Protein Kinase C and G Proteins in Activation of MurineResting B Lymphocytes by Endotoxin-Associated Protein

JAYANT R. BANDEKAR, RAYMOND CASTAGNA, AND BARNET M. SULTZER*Department of Microbiology and Immunology, State University ofNew York,

Health Science Center at Brooklyn, Brooklyn, New York 11203

Received 22 May 1991/Accepted 21 October 1991

Endotoxin-associated protein (EP) from the outer membrane of gram-negative bacteria is a potentimmunomodulator. To examine the mechanism of EP stimulation, the protein kinase C inhibitors H7 andstaurosporine were used. Both DNA and RNA synthesis of EP-stimulated murine resting B cells werecompletely inhibited when inhibitors were added at 0 h, whereas 55 to 76% inhibition of DNA synthesis wasobserved when H7 was added after 12 h of stimulation. In contrast, HA 1004, which blocks protein kinase Aand protein kinase G activity, was relatively ineffective even at high concentrations, suggesting that the activityof protein kinase C is a primary mechanism of EP-induced murine B-cell proliferation. To examine the role ofG proteins in EP-induced DNA synthesis in B cells, the effects of pertussis toxin (PT), which inactivates certainG proteins, and the B oligomer of PT (PTB), which does not, were also examined. PT was found to inhibitEP-induced DNA synthesis in a dose-dependent manner. However, PIB also caused equivalent inhibition,suggesting that PTB may be responsible for most of the inhibitory effect seen with the holotoxin. These resultsserve to question whether G proteins are involved in the signal transduction that occurs during EP-inducedDNA synthesis in murine B cells.

Endotoxin-associated protein (EP) represents a selectnumber of outer membrane polypeptides of gram-negativebacteria which are coextracted with but separable from thelipopolysaccharide endotoxin (LPS) (36). EP is known to bea highly potent immunomodulator which stimulates prolifer-ation of B cells from LPS nonresponder mice as well ashuman peripheral blood lymphocytes (18, 19). EP alsoinduces nonspecific antibody production by mouse andhuman B lymphocytes, activates macrophages to produceinterferon and prostaglandin E2, and acts as a potent adju-vant for T-cell-dependent and T-cell-independent antigens(18, 19, 33-35). In addition, the EP extracted from Salmo-nella typhimurium can act as a protective antigen againstmouse typhoid (13, 14). Although the immunomodulatoryactivity of EP is well documented, its mechanism of activat-ing cells is not clear. On the other hand, some of the earlyevents in the activation of murine B lymphocytes to prolif-eration by agents that cross-link surface immunoglobulin(anti-slg) have been elucidated. Anti-slg induces phosphati-dylinositol (PI) turnover that leads to an increase in diacyl-glycerol (DAG) and inositol trisphosphate, which in turninduce the activation of protein kinase C (PKC) and therelease of intracellular ions of calcium (Ca2+), respectively(2, 6, 11, 27, 31). These events are believed to be importantfor inducing B-cell DNA synthesis. Supporting evidence forthis hypothesis has been provided by the finding that thecombination of phorbol esters that activate PKC and calciumionophores that increase Ca2+ also stimulates B-cell prolif-eration (28).LPS-induced murine B-cell proliferation, however, does

not involve either PI turnover or increase in Ca2+. Rather, ithas been proposed that LPS acts like a DAG analog, directlycausing the translocation and activation of PKC (3, 8, 21).Further studies have provided evidence that GTP-binding(G) regulatory proteins may also be involved in B-cell

* Corresponding author.

activation by LPS (12). Pertussis toxin (PT), which inacti-vates the alpha subunit of the Gi and other G proteins byADP ribosylation (4, 10, 16), has been shown to inhibitLPS-induced murine B-cell proliferation, as well as LPS-induced signalling in WEHI-231 B-lymphoma cells (12, 24).On the basis of these findings, we chose to examine the

role of PKC and G proteins in the activation of murine Blymphocytes by EP. Since C3H/HeJ B cells do not respondto LPS with either DNA synthesis or polyclonal antibodyproduction, they offered a model system that could becompared with previous results obtained with LPS re-sponder cells. By the use of specific PKC inhibitors we showthat PKC does play a vital role in the activation of C3H/HeJB cells by EP. In addition, the treatment of EP-activatedcells with PT resulted in partial inhibition of EP-stimulatedDNA synthesis; however, since the B oligomer of PT (PTB),which does not inactivate the Gi protein (38), is similarlyinhibitory, the role of G proteins in EP-activated B cells isprobably unlikely.

MATERIALS AND METHODS

Animals. C3H/HeJ mice were obtained from the NationalCancer Institute (Frederick, Md.). Mice of either sex wereused separately at 4 to 6 months of age and maintained onwater and Purina Chow ad lib.

Materials. EP from Salmonella typhi 0-901 was preparedas described previously (36). 1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride (H7), staurosporine, andphorbol myristate acetate (PMA) were obtained from Calbi-ochem (San Diego, Calif.). lonomycin was obtained fromSigma Chemical Co. (St. Louis, Mo.). HA 1004 [N-(2-guanidinoethyl)-5-isoquinolinesulfonamide hydrochloride]was obtained from Biomol Research Laboratories Inc. (Ply-mouth, Pa.). Purified PT and PTB were obtained from ListBiological Labs (Campbell, Calif.). PT (lot no. PT-74A) wasreported to have a residual adenylate cyclase activity of 2.5pM/min/,lg. PT (lot no. PT-76 AW) had a residual adenylate

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232 BANDEKAR ET AL.

cyclase activity of 0.5 pM/min/,ug. PTB (lot no. PTB-09A)contained 1% holotoxin as determined by the Chinese ham-ster ovary cell assay. All bioassays on PT and PTB werecarried out by List Labs.

Purification of B cells. Mice were sacrificed by cervicaldislocation, and single-cell suspensions were prepared fromthe spleens as previously described (36). The erythrocyteswere lysed by Tris-ammonium chloride treatment (5), and Tcells were depleted by treatment with anti-Thy-1.2 monoclo-nal antibodies (Sigma Chemical Co.) and rabbit Low ToxComplement (Cedarlane Laboratories, Hornby, Canada).Dense resting B cells were obtained by Percoll (Pharmacia,Uppsala, Sweden) density gradient separation. Cells in theband showing 1.076 to 1.08 density were selected andwashed twice with RPMI 1640 medium supplemented with5% fetal calf serum (Gibco Laboratories, Grand Island,N.Y.), 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), 100 U of penicillin per ml, and 100 ,ugof streptomycin per ml.DNA and RNA synthesis. Purified resting B cells were

cultured in the medium described above at either 2.5 x i05cells in 0.2 ml per well in 96-well U-bottom microtiter plates(Falcon Labware, Lincoln Park, N.J.) or 2.5 x 106 cells perml in tubes (12 by 75 mm; Falcon) depending on theexperiment. EP or other mitogens in the presence or absenceof the inhibitors, H7, staurosporine, HA 1004, PT, or PTBwere added at various times to the cultures as indicated inthe experiments. The cells were incubated at 37°C in 10%CO2 for 48 h, and DNA synthesis was measured by [3H]thy-midine incorporation by adding 1 ,uCi of [3H]thymidine (ICNBiochemicals, Costa Mesa, Calif.) in the last 18 h of incuba-tion. The effect of EP on RNA synthesis was studied byculturing purified resting B cells at 2.5 x 106/ml in tubes andby measuring [3H]uridine (ICN Biochemicals) incorporationby adding 1 ,uCi of [3H]uridine for various time periods asdescribed in the experiments. The cultures were harvestedonto glass fiber filters by using a cell harvester (Brandel,Gaithersburg, Md.), and [3H]thymidine and [3H]uridine in-corporation was measured by a Beckman liquid scintillationcounter, model 6000 IC (Beckman Instruments, Fullerton,Calif.).

All cultures in microtiter plate experiments were done insix replicates, while all cultures in tube experiments weredone in triplicate. Variation about the mean did not exceed± 10%. Unless otherwise noted, experiments were repeatedat least three times.

RESULTS

Effect of PKC inhibitors on EP-induced DNA synthesis inC3H/HeJ resting B cells. As previously reported, EP stimu-lates splenic B cells from LPS nonresponder C3H/HeJ miceto proliferate as well as to differentiate and produce poly-clonal antibodies (33, 34). However, for these inhibitionstudies it was necessary to use purified resting B cells tomore closely approximate cells in the Go phase of the cellcycle. As shown in Fig. 1, these lymphocytes likewiserespond to the induction of DNA synthesis induced by EP.When purified resting B cells were stimulated with EP in thepresence of various concentrations of H7 added at theinitiation of culture and DNA synthesis was measured after48 h, H7 inhibited the synthesis in a dose-dependent manner(Fig. 2). In addition, staurosporine was found to completelyblock DNA synthesis of EP-stimulated resting B cells at aconcentration of 0.1 puM (Table 1). Furthermore, HA 1004,which preferentially inhibits protein kinase A and protein

0~g) 500

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It 300/00 20-z

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(0,1) 10 20 50 100CONCENTRATION OF EP (ng /CULTURE)

FIG. 1. Stimulation of DNA synthesis in C3H/HeJ resting B cellsby EP. B cells were cultured in microtiter wells at 2.5 x 105 cells per0.2 ml and incubated for 48 h with various concentrations of EP, and[3H]thymidine (3H-TdR) uptake was measured as described inMaterials and Methods. The error bars represent the range of valuesabout the mean.

kinase G activity rather than PKC activity, was ineffective at25 to 50 ,uM and only a limited amount of inhibition of DNAsynthesis occurred at 100 ,uM (Table 2). These data suggestthat PKC activity is necessary for B-cell activation by EP.To examine the role of PKC in the later stages of activa-

tion, H7 at its optimal concentration (40 or 50 ,uM) wasadded 12 h after the culture was initiated, and DNA synthe-sis was measured at 48 h. As shown in Fig. 3, H7 almostcompletely inhibited DNA synthesis when added initiallyand when- added after 12 h of stimulation, 55 and 76.7%inhibition was observed with 40 p.M and 50 ,uM, respec-tively. These results indicate that EP-stimulated PKC activ-ity is needed later in the culture when most cells are in G1 sothat these B cells can progress into the S phase of DNA

zw0

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00 lo 20 30 40 50

CONCENTRATION OF H7 (LM)FIG. 2. Effect of various concentrations of H7 on EP-induced

DNA synthesis in C3H/HeJ resting B cells. B cells were cultured intubes at 2.5 x 106/ml for 48 h with EP (1 ,ug/ml) in the presence orabsence of various concentrations of H7. DNA synthesis wasmeasured by [3H]thymidine incorporation. Data are expressed asthe mean percent inhibition of [3H]thymidine incorporation. Back-ground control mean value = 2,103 cpm. EP stimulated mean value= 32,807 cpm.

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ENDOTOXIN PROTEIN-ACTIVATED PKC IN B CELLS 233

TABLE 1. Staurosporine inhibition of DNA synthesis by restingB lymphocytes activated by EP'

Staurosporine [3H]thymidine S(4M) incorporation (S.I.)b Inhibition

0 14.42.5 0.5 1000.1 0.8 1000.01 8.6 43.80.001 14.6 0

a Lymphocytes were activated by 10 jig of EP.b S.I. = stimulation index or the ratio of experimental to background

control counts per minute of [3H]thymidine incorporation. Background con-trol: mean counts per minute = 456 in microtiter plate cultures.

synthesis. In parallel experiments, B cells were stimulatedwith the PKC activator PMA (50 ng) and the calciumionophore ionomycin (1 ,uM). Treatment with 50 ,uM of H7inhibited DNA synthesis completely when H7 was added at0 h, whereas 83% inhibition was observed when H7 wasadded after 12 h of stimulation (Fig. 3). These results confirmthat PKC activity is needed later in the culture for mitogen-stimulated B cells to enter the S phase of DNA synthesis.

Effect of H7 on EP-induced RNA synthesis. When resting Bcells are stimulated by mitogens, most cells enter the G1phase of the cell cycle as indicated by increased synthesis ofRNA. Therefore, to determine the effect of a PKC inhibitoron the early stages of B-cell activation, RNA synthesis inEP-activated resting B cells in the presence or absence of H7was studied. As shown in Table 3, RNA synthesis measuredat various time intervals from 0 to 24 h was completelyblocked by PKC inhibition within the first 12 h of activationor even later in the culture period. These results support ourcontention that PKC is involved in both the early and laterstages of B-cell activation by EP.

Effect of PT and PTB on EP-induced DNA synthesis inresting B cells. To examine the role of G proteins in themitogenic stimulation of resting B cells, the effect of PT,which inactivates certain G proteins, was examined. Whenadded at the initiation of the culture of resting B cells or at 12h, PT inhibited EP-induced DNA synthesis in a dose-dependent manner (Fig. 4). However, differences were ob-tained depending on the lot of PT. As shown, PT lot no.PT-74A was more effective than PT lot no. PT-76 AW (Fig.4). This was also apparent with the inhibition of DNAsynthesis stimulated by PMA and ionomycin. It should benoted that the only measured difference between these PTlots was the level of residual adenylate cyclase activity (seeMaterials and Methods).PT is a heterodimer made up of an A and B subunit. ADP

ribosylation of the alpha subunit of the Gi protein is causedby the A subunit, whereas the B oligomer serves to bind the

TABLE 2. Effect of HA 1004 on EP-activated restingB lymphocytesa

[3H]thymidine tHA 1004 (p.M) SI.bInhibition

0 38.325.0 36.1 5.750.0 32.1 16.1

100.0 29.7 22.4

a Lymphocytes were activated by 100 ,ug of EP.b S.I. = stimulation index. Background control: mean counts per minute =

1,1% in microtiter plate cultures.

TABLE 3. Effect of inhibition of PKC on RNA synthesis inEP-stimulated B cellsa

EP H7 Pulse time (h) of [3HJuridine incorporation(1 pg) (50 FM) [3HJuridine (total mean cpm)

- - 0-12 8,043+ - 0-12 15,499+ + 0-12 3,083- - 14-18 2,246+ - 14-18 4,048+ + 14-18 1,573- - 12-24 8,637+ - 12-24 17,298+ + 12-24 8,003- - 20-24 1,270+ - 20-24 3,593+ + 20-24 898

a C3H/HeJ resting B cells (2.5 x 106/ml) were cultured in tubes with EP (1pLM) and with or without H7 (50 ,uM). [3H]uridine (1 ,Ci) was added at theindicated time, and the cells were harvested either 4 or 12 h later.

holotoxin to the cell surface receptor (38). As a control todetermine whether the effect of PT is due to its action on Gproteins or due to the effect of B-oligomer binding, PTB wasadded to the culture either at the initiation or 12 h after thestart of the culture and DNA synthesis was measured at 48 h.As shown in Fig. 5, PTB at concentrations equivalent to itsproportional composition of the holotoxin was able to inhibitDNA synthesis. In particular, the level of inhibition withPTB was strikingly similar to that obtained with PT lot no.PT-76 AW, which had a low adenylate cyclase activity.Furthermore, PMA and ionomycin stimulation of the B-cellDNA synthesis was comparatively resistant to PTB inhibi-tion (Fig. 5). Therefore, the inhibition observed due to thepertussis holotoxin appears to be due to the action of its Boligomer rather than the inactivation of the G protein by thePIT A subunit.

0

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00 0-

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FIG. 3. Inhibition of DNA synthesis in EP- or PMA-ionomycin-stimulated B cells by H7. B cells in microtiter well culture werestimulated with (A) EP (100 ng) or (B) PMA (50 ng) and ionomycin(1 FLM) for 48 h. H7 (40 or 50 ,uM) was added at 0 or 12 h after theinitiation of the culture. DNA synthesis was measured by [3H]thy-midine incorporation. Stimulation Index, ratio of experimental tocontrol counts per minute of [3H]thymidine incorporation. Panel A:control mean value = 800 cpm; EP-stimulated mean value = 23,813cpm. Panel B: control mean value = 564 cpm; PMA-ionomycin-stimulated mean value = 68,704 cpm.

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234 BANDEKAR ET AL.

0o

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z

0Loti0000 UN 00 NW

-000 -0000 00 00cOOOO Cv O OOC3O O CUU-280 20 2°O gg gg

FIG. 4. Effect of PT on EP-stimulated B cells. B cells in micro-titer well culture were incubated with various mitogens for 48 h, andPT in variouts concentrations was added at 0 or 12 h after theinitiation of the culture. (A) 100 ng of EP. (B) 1 ,ug of EP. (C) 100 ngof PMA + 1 ,uM ionomycin. DNA synthesis was measured by[3H]thymidine incorporation. Data are expressed as mean percentinhibition of [3H]thymidine incorporation. Control mean value =

1,139 cpm. EP (100 ,ug)-stimulated mean value = 44,446 cpm. EP (1,ug)-stimulated mean value = 105,047 cpm. PMA-ionomycin-stimu-lated mean value = 92,043 cpm. 0, PT (adenylate cyclase activity =0.5 pmol/min/,ug); 0, PT (adenylate cyclase activity = 2.5 pmol/min/ikg)-

DISCUSSION

Quiescent, noncycling (GO) B cells can be modulated by a

variety of physiological mediators or bacterial products, andregulation of this activation process is very complex. Multi-ple pathways of signal transduction may be involved. Exten-sive studies by stimulating murine B cells with anti-sIg haveshown that cross-linking of sIg leads to the activation ofPLCcausing rapid turnover of PI (6, 11, 25, 27, 30). The hydro-lysis products inositol triphosphate and DAG cause the

00

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50 F0FIG. 5. Effect of the B subunit of PT on EP-stimulated B cells. B

cells in microtiter well culture were incubated with various mitogensfor 48 h and PTB in various concentrations was added at 0 or 12 hafter the initiation of the culture. (A) 100 ng of EP. (B) 1 ,ug of EP.(C) 100 ng of PMA + 1 ,uM ionomycin. DNA synthesis wasmeasured by [3H]thymidine incorporation. Data are expressed asmean percent inhibition of [3H]thymidine incorporation. On aweight basis, PTB represents 75% of the pertussis holotoxin.

release of Ca2" from the intracellular compartment andactivation of PKC, respectively (11, 29). Both Ca2+ andPKC are important in inducing cell proliferation. PMA,which activates PKC, and ionomycin, which is a calciumionophore, do not stimulate cell proliferation alone, butcombined treatment induces cell proliferation (28). On theother hand, LPS does not cause PI hydrolysis or release ofCa2" from internal sources, but it does act as a DAG analogactivating PKC (3, 21). Indeed, the PKC inhibitor H7 (23)inhibits LPS-induced murine B-cell activation as well asanti-slg-induced human B-cell activation (1, 22, 26). Stauro-sporine, another inhibitor of PKC (37), also blocks theactivation of human tonsillar B cells (39). In our experi-ments, the inhibition by H7 was dose dependent and, fur-thermore, partial inhibition was observed when H7 wasadded 12 h after stimulation with EP, indicating that a degreeof continuous PKC activity during the culture period isneeded. Furthermore, our results with staurosporine, whichhas a greater binding affinity for PKC than H7, showedhighly effective inhibition of DNA synthesis stimulated byEP at much lower concentrations than H7.Although these results indicate an important role for PKC

in EP-induced mitogenic stimulation of B cells, H7 andstaurosporine have been reported to inhibit other proteinkinases (9, 23). Because HA 1004, among the isoquinoline-sulfonamide derivatives, has the weakest inhibitory activityagainst PKC and is active against cyclic GMP- and cyclicAMP-dependent protein kinases, it is useful as a control forH7. Our results showed that HA 1004 is not effective as aninhibitor of EP-induced DNA synthesis for resting B cells,further supporting the need for PKC activity. Nevertheless,it is essential that direct measurements of changes in proteinkinase levels in activated and inhibited B lymphocytes alsobe made.

Recently, the role of G proteins in the activation oflymphocytes also has received increased attention. SinceADP ribosylation irreversibly modifies and uncouples Gproteins from the receptor, an agent such as PT, whichcatalyzes ADP ribosylation of the alpha subunit ofG protein,has been used extensively to investigate G protein involve-ment in lymphocyte activation. In this manner Dziarskistudied the mitogenic activation of mouse B lymphocytes byLPS and other bacterial mitogens, concluding that G pro-teins are involved in B-cell activation since ribosylation ofPT substrates correlated with the inhibition of LPS-inducedDNA synthesis (12). PT has also been reported to blockLPS-stimulated interleukin-1 production in P388D1 macro-phage cells at doses consistent with the modification of a Gprotein (24).

In the experiments reported here, PT can block EP-induced DNA synthesis in murine B cells. It was alsoobserved that the lot of PT which had five times higherresidual adenylate cyclase activity (2.5 pmol/min/,ug) thanthe second lot (0.5 pmol/min/,ug) was more effective inblocking DNA synthesis. Dziarski has reported that B-cellstimulation by PMA and ionomycin is relatively insensitiveto PT (12) and in our experiments when PT with low residualadenylate cyclase activity was used, activation by PMA andionomycin was not blocked. In contrast, the PT with higheradenylate cyclase activity did inhibit DNA synthesis stimu-lated by PMA and ionomycin from 50 to 60%, suggesting thatthe contaminating enzyme contributed to the inhibitionobserved with the PT. Of greater significance is the fact thatthe membrane binding subunit or the B oligomer of PT,which does not cause ADP ribosylation of G proteins, alsoblocked DNA synthesis in resting B cells stimulated by EP.

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ENDOTOXIN PROTEIN-ACTIVATED PKC IN B CELLS 235

PTB, which represents 75% of the pertussis holotoxin on aweight basis, was as effective as the pertussis holotoxin thathad low adenylate cyclase activity. Therefore, most, if notall, of the inhibition of the EP-stimulated DNA synthesis weobserved was probably due to the B oligomer. Similarfindings have been made with T-cell activation by using theB oligomer (20). Additionally, Sommermeyer and Resch (32)found that PT produced more effects on Jurkat humanlymphoma cells than an ADP-ribosylation reaction of Gproteins and cautioned that if PT is used to discern theinvolvement of G proteins in signal transduction, one mustmake sure that the observed effects are not due to thebinding of the toxin. Consequently, our results support thenotion that the G proteins are most likely not a majorcomponent in the activation of B lymphocytes by EP, atleast as defined by PT.

Finally, it has been recently reported that stimulation withanti-slg also caused activation of protein tyrosine kinase,which may be important in the mitogenic stimulation of Bcells, as is the case in the activation of T lymphocytes (7, 15,17, 40). In addition, we have preliminary data on the specificprotein tyrosine kinase inhibitor Genisteine indicating thatprotein tyrosine kinase activity is also necessary for EP andLPS stimulation of DNA synthesis in mouse resting Blymphocytes. Therefore, it seems likely that various signaltransduction pathways are involved in mitogenic stimulationof lymphocytes which result in the activation of eithercalcium-phospholipid-dependent PKC, cyclic AMP-depen-dent protein kinase A, or protein tyrosine kinase. Clearly,the precise role of each of these kinases and other secondarymessengers induced by EP and LPS needs to be evaluatedfurther.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant Al 28526from the National Institute of Allergy and Infectious Diseases.We thank Janice Howard for her excellent assistance in the

preparation of this manuscript.

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