JOURNAL OF INTERFERON RESEARCH 8:193-200 (1988)Mary Ann Liebert, Inc., Publishers

Early Steps in Interferon-7 Production:Possible Involvement of Ca2+-Calmodulin-

Dependent Enzymes

GUIDO ANTONELLI, VITO RUGGIERO, ALDO CEFARO, PAOLA Dì MARCO,PAOLA AMICUCCI, VITTORIO COLIZZI,1 and FERDINANDO DIANZANI

ABSTRACT

A23187 in combination with phorbol myristate acetate (PMA) strongly induces produc¬tion of interferon- (IFN- ) by human peripheral blood mononuclear cells (PBMC) andeven by murine PBMC, which respond poorly to A23187 alone. Macrophage depletionof PBMC strongly reduces IFN- production induced by several mitogens, but does notaffect IFN- production induced by A23187 and PMA. In addition the same stimuli areable in combination to induce strong amounts of IFN- , even in the Jurkat cell line.The protein kinase C inhibitor l-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7) andthe calmodulin antagonist 7V-(6-aminoehexyl)-5-chloro-l-naphthalenesulfonamide (W-7)were examined for their ability to inhibit IFN- production induced by PMA andA23187. At concentrations near the K\ for protein kinase C, H-7 failed to inhibit PMA-and A23187-induced IFN- production. In contrast, W-7 at low concentrations inhibitedIFN- production induced by the same stimuli. In addition OAG, which is known to di¬rectly activate protein kinase C, failed to act synergistically with A23187 in the inductionof IFN- . On the basis of these results we propose that A23187 and PMA may mimic theearly steps of lymphocyte activation, without the requirement of macrophage, bypassingantigen-, or lectin-induced signal. Our results suggest that Ca2*-calmodulin-dependentreactions other than protein kinase C activation may be essential for IFN- production,at least at level of the producing cells.

INTRODUCTION

Interferon^ (IFN- ), a lymphokine capable of modulating many cellular activities,'1·21 is pro¬duced by lymphocytes in response to mitogens or specific antigens (for review, see Ref. 3). Al¬

though the molecular events leading to IFN- production have not been fully unraveled, it isknown that accessory cells, such as macrophages bearing class II major histocompatibility antigens,are required for the phenomena.'4_6> The essential contribution(s) of macrophages to the induction

Institute of Virology, University of Rome "La Sapienza," Italy.'Department of Biology, University of Rome "Tor Vergata," Italy.

193

ANTONELLI ET AL.

of IFN- production is incompletely understood but it is thought to include physical presentationof soluble antigens and the elaboration of interleukin-1 (IL-1)'7·8' and other soluble factors.'9'

On the other hand, it has been proposed"0"13' that the activation of lymphocytes is mediatedby an early rise in cytosolic calcium concentration and we have shown that it is possible to induceIFN- by treatment of peripheral blood mononuclear cells (PBMC) with calcium ionophores."415'

There is now evidence from other systems that receptor-ligand interaction generates signals thattraverse the cell membrane and lead to the physiological response peculiar to the system."6"19'

2*Ca-calmodulin (CaM) and diacylglycerol (DAG) are general regulators of cellular activation;they are not species- or tissue-specific and they affect a number of cellular activities."2·18·20"22' Bio¬logical activities of CaM and DAG seem to occur through protein phosphorylation by CaM-depen¬dent protein kinases and calcium/phospholipid-dependent protein kinase (PK-C)."218) The preciserole of these kinases in the mechanism of lymphocyte activation still remains to be defined. Previ¬ously, we have shown that trifluoroperazine, a CaM antagonist, strongly reduces IFN- productioninduced by both mitogens'231 and antigens.'241 On the other hand, it has been suggested that IL-2production may involve PK-C activation.'25' Moreover, it has been reported that signal transduc¬tion, mediated by phospholipid turnover, can be mimicked by phorbol esters and calcium iono¬phores in combination, which activate PK-C and elevate intracellular calcium respectively."8'Thus, since phorbol myristate acetate (PMA) has been reported to superinduce IFN- productionstimulated by mitogens,'26' we first tested whether PMA exerts the same enhancing effect also incultures stimulated by calcium ionophores. Furthermore, to gain some insight into the mechanismof IFN- production and to evaluate better the roles of CaM-dependent intracellular enzymes andPK-C in cell activation, we used newly developed and highly specific pharmacologie inhibitors.Naphthalenesulfonamides (W-7) act as potent and specific inhibitors of CaM-dependent protein ki¬nases by inactivating CaM (CaM antagonists),'27' while isoquinolisulfonamides (H-7) are potentPK-C inhibitors.'28' These compounds have been proved to be useful tools for elucidating the bio¬logical significance of the reactions mediated by protein kinases.'27·29' This study aimed at examin¬ing the role of PK-C- and CaM-dependent enzymes in T-cell activation leading to IFN- productionby comparing the effects of both H-7 and W-7 on IFN- production stimulated by A23187 andPMA, which are able to trigger lymphocytes without a macrophage requirement.

MATERIALS AND METHODS

Reagents: Phorbol myristate acetate (PMA), l-oleyl-2-acetylglycerol (OAG), A23187, galactoseoxidase, concanavalin A, staphylococcal enterotoxin (SEB), l-(5-isoquinolinylsulfonyl)-2-methyl-piperazine (H-7), and iV-(-6-aminohexyl)-5-chloro-l-naphtalene-sulfonamide (W-7) were obtainedfrom Sigma Chemical Company. PMA, OAG, A23187, H-7, and W-7 were dissolved in DMSO at0.5 mg/ml, 4.5 IO"2 M, 10 mM, 100 mM, 100 mM, respectively, and were diluted in culture me¬

dium just prior to use. SEB and concanavalin A were dissolved in PBS at 1 mg/ml and used at afinal concentration of 0.5 µg/ml (SEB) or 20 /¿g/ml (ConA). Galactose oxidase was dissolved inPBS (100 U/ml) and the treatment was performed as described previously.'9'

Cell Cultures and IFN Induction: PBMC were purified from human venous blood as describedpreviously.'15' Murine PBMC were obtained from spleens of C3H/N syngenic mice (Charles River).

Both murine and human PBMC (2.5 IO6 cells/ml) were cultured in 0.2 ml of RPMI 1640 me¬

dium supplemented with 10% fetal calf serum (Flow) and 50 /tg/ml gentamicin (Schering) in micro-titer plates (Falcon) in the presence of various inducers or inhibitors. After 24 h of culture at 37°C,the cells were removed and the supernatants were assayed for IFN activity. lymphocytes were prepared by passing PBMC through nylon wool columns after 24 h of previ¬

ous adherence on plastic dishes. The cells after depletion contained usually 90-95% OKT3-positivecells.'9' The Jurkat cell line was maintained in RPMI 1640 supplemented with 10% of FCS andpassaged by one-fifth dilution twice a week.

194

IFN- AND Ca2*-CALMODULIN ENZYMES

Viability Test: After each experiment cell viability was established by the dye uptake method andby the rate of incorporation of [3H]thymidine (Amersham, 1 mCi/mmole). Only experiments witha cell viability higher than 90% and a rate of [3H]thymidine incorporation comparable to controlvalues were considered.

IFN Assays and Characterization: Human IFN titer was determined by a previously describedassay130' and IFN activity is expressed as international units (IU). The IFN produced in these exper¬iments was characterized as IFN- by pH 2 sensitivity and neutralization by specific antibody(kindly provided by Dr. M.H. Langford, University of Texas, Galveston). Mouse IFN is expressedas IU and its titer was determined by a previously described assay.'6' Mouse IFN was characterizedas IFN- in that it was neutralized by antibodies to murine IFN- (kindly provided by Dr. G.J.Stanton, University of Texas, Galveston).

Control experiments showed that H-7 and W-7 added together with exogenous IFN did not af¬fect IFN activity.

RESULTS

Human PBMC were cultured in the presence of various concentrations of A23187 with or with¬out PMA. The cells were removed 24 h later and the supernates were collected for IFN assay.

The results of these experiments are shown in Table 1. Since no significant differences betweenvarious concentrations of PMA (10-100 ng/ml) were observed, we reported only one concentration(50 ng/ml) for PMA. It can be seen that PMA, although capable of inducing by itself only smallamounts of IFN, strongly enhanced IFN induction by A23187 (30- to 100-fold).

This experimental approach was also applied to the murine system, where other investigatorswere unable to show lymphocyte activation induced by calcium ionophores alone.'25' Specif¬ically, murine PBMC were cultured for 24 h in the presence of A23187 and/or PMA at variousconcentrations. The supernatants were collected and assayed for IFN activity. It can be seen (Table1) that A23187, which alone is a poor IFN inducer in this system, did induce substantial productionof IFN- when used in combination with PMA (50 ng/ml).

To test whether the aforementioned stimuli affected lymphocytes only, we followed the same

experimental approach on isolated cells and in a T-cell line. The results, shown in Table 2, indi¬cate that A23187 and PMA are able to induce large amounts of IFN also in purified cells, thussuggesting that these agents do bypass the macrophage requirement while other inducers (ConA,SEB, and galactose oxidase) do not.

Table 1. Production of IFN- in Human and Murine PBMC afterStimulation by A23187 with or without PMA

Treatment IFN-y (IU/ml) production in

A23187 (µ ) PMA (ng/ml) Human PBMC Murine PBMC

-

<3 <30.1

-

32 31

-

316 1050 10 3

0.1 50 1,000 101 50 31,623 316

The cells were cultured with A23187 (0.1 jtMor 1 µ ) in the presence or absence ofPMA (50 ng/ml). After 24 h the supernates were collected for IFN assay. Values aremeans of several experiments (where the SD did not exceed 3 IU).

195

ANTONELLI ET AL.

Table 2. IFN- Production by Macrophage-Depleted Human PBMC andJurkat Cell Line3

IFN-y (lU/ml)* in

Macrophage-depletedcIFN inducer Control PBMC PBMC Jurkat

None <3 <3 <3

Staphylococcal enterotoxin 1,000 32 < 3Concanavalin A 100 10 < 3Galactose oxidase 1,000 10 <3A23187 316 316 3A23187 + PMA 31,623 31,623 316

aData represent means of three experiments in which the SD did not exceed 0.5 log. Threefold dif¬ference in IFN titer is significant at < 0.05.

bControl macrophage-depleted human PBMC or Jurkat cells were treated with various agents asdescribed in Materials and Methods. After 24 h incubation at 37°C, supernatant fluids were assayedfor IFN activity.

cMacrophage depletion was performed by sequential adherence to plastic followed by adherence tonylon fiber columns.

To analyze the effect of PMA, a reported PK-C activator, and to test whether the aforemen¬tioned PMA-induced superproduction of IFN- occurs through the activation of PK-C, we com¬

pared the activity of two inhibitors, namely W-7 and H-7. W-7 inhibits CaM-dependent phosphor¬ylation with a A'j (constant of inhibition) value of 12 µ , while inhibiting PK-C with a /f¡ value of110 µ .<3" In contrast H-7 inhibits PK-C with K¡ value of 6.0 µ , while inhibiting CaM-dependentphosphorylation with a A"¡ value of 97 µ .'28'

The effects of H-7 and W-7 on IFN- induction by 1 µ A23187-in the presence of PMA (50ng/ml) are shown in Fig. 1. At low concentrations, H-7 failed to inhibit IFN- production afterstimulation with A23187 and PMA, being effective only at the highest concentration used (96 µ ).In contrast, W-7 inhibited IFN- production also when the inhibitor was added at a concentration '

of 12 µ , which is reported to be inhibitory only on CaM-dependent phosphorylation.In sum, A23187 and PMA strongly induce T-lymphocyte activation leading to IFN- production,

but the effect of PMA is not completely attributable to the activation of PK-C, since inhibition ofPK-C by H-7 failed to reduce IFN- production while inhibition of CaM by W-7 did.

To test this hypothesis further we carried out experiments with OAG, a synthetic analog of DAGknown to be a direct activator of PK-C."8' Specifically, human PBMC were treated with a combi¬nation of A23187 and OAG at various concentrations reported to be active in other systems.'32"34'The results of two representative experiments are shown in Table 3. Only a slight increase in IFN- production was observed when PBMC were treated with both agents.

DISCUSSION

Synergism between calcium ionophores and phorbol esters has been shown to occur in a numberof different cell types.125·32·35"37' Here we show, in agreement with others,'38' that such synergismoccurs also in terms of IFN- production. We demonstrate also that A23187 alone is a poor activa¬tor of murine lymphocytes, but in combination with PMA it induces a substantial production ofIFN- in lymphoid cell populations. Furthermore, this activation does not require the presence of

196

IFN- AND Ca"-CALMODULIN ENZYMES

ToINHIBITOR

ISO-CONCENTRATION (f»M)

FIG. 1. Effect of the inhibitors H-7 and W-7 on IFN- induction stimulated in human PBMC byA23187 (1 µ ) and PMA (50 ng/ml). The cells were cultured with: A23187, PMA, and H-7 (O) atvarious concentrations, or A23187, PMA, and W-7 ( ) at various concentrations.

Table 3. IFN- Production by PBMC Stimulated byA23187 and/or OAG*

Inducers

A23187 OAG

IFN-y (IU/ml)

Exp. 1 Exp. 2

1 µ

1 µ 1 µ

4.5 "5 M4.5 "6 M4.5 "5 M4.5 "6 M

100<10<10316158

178<10<10316562

aHuman PBMC were cultured in the presence of A23187 and/orOAG at various concentrations. The supernatants of cultures werecollected for IFN assay after 24 h.

197

ANTONELLI ET AL.

macrophages, since we obtained the same results also by using Jurkat T-cell line or purified cellsinstead of PBMC. Thus, in the murine system, as well as in the human system, the treatment withboth agents bypasses the requirement for antigen- or lectin-induced signal and may mimic the earlysteps of lymphocyte activation. PMA has been used as an activator of PK-C in several studies."8·25'In fact PMA has been shown to activate PK-C directly and is believed to mimic the action of DAG,which is produced as a result of the breakdown of inositol phospholipids."8' However, many otheractivities have been attributed to PMA, such as effects on protein alkylation139' or phospholipaseA2.'40' We show here, however, that the synergism between A23187 and PMA leading to IFN- production is not completely attributable to an activation of PK-C. In fact when we used an inhibi¬tor of PK-C, namely H-7, we did not observe a reduction of IFN- production stimulated byA23187 and PMA. We obtained only a partial reduction by H-7 when we used it at a concentrationthat has been reported to inhibit also CaM-dependent enzymes. On the other hand W-7, a specificcalmodulin antagonist which at low doses completely blocks CaM-activity and, potentially, alsoCaM-dependent protein kinases, substantially inhibits IFN- production both at high and at lowconcentrations.

These data suggest that inhibition of PK-C is not sufficient by itself to block IFN- productionand that other CaM-dependent steps are involved. Moreover, the inhibition of cellular responses toPMA by W-7 suggests that the site of its action is not only PK-C but also other intracellular en¬

zymes. This hypothesis is supported by the finding that OAG alone, which activates PK-C in situ,neither activates production of IFN- nor strongly enhances IFN induction by A23187. In this re¬

spect, OAG failed to mimic the action of phorbol esters also in other systems,'35 41' thus suggestingthat these agents have other pleiotropic effects.

On the other hand, studies that made use of these inhibitors have indicated that W-7 or other cal¬modulin antagonists inhibit cell proliferation127·42' or neutrophil activation,'29' and H-7 inhibitsPMA-induced release of histamine from human basophils,'29' expression of viral antigens in Ep-stein-Barr virus (EBV)-transformed cell line,'43' and transcription of the c-fos oncogene in U937'44'over a concentration range used in this study.

We did not address the question of whether the inhibitory effect of W-7 is due to either general¬ized CaM antagonism or specific inhibition of CaM-dependent protein kinases activity. Thus, thedata do not help to distinguish between the possibilities that cellular activation is specifically due to

Ca2*-CaM-dependent protein kinases or to other CaM-dependent processes. Studies to establishwhich CaM-dependent enzyme(s) is induced in the process are in progress.

ACKNOWLEDGMENTS

We wish to thank Miss Enrica Di Francesco for excellent secretarial help. This work was sup¬ported by a grant from CNR (P.F. Controllo Malattie da Infezione), by the Italian Ministry ofEducation (project 40%), Cenci Bolognetti, and Associazione Italiana per la Ricerca sul Cancro.

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Address reprint requests to:Dr. Guido AntonelliInstitute of VirologyUniversity of Rome

Viale di Porta Tiburtina, 2800180 Rome, Italy

Received 23 June 1987/Accepted 15 September 1987

200