Eur. J. Biochem.251, 7342743 (1998) FEBS1998

Phenotypic difference between Bcgr and Bcgs macrophagesis related to differences in protein-kinase-C-dependent signalling

Martin OLIVIER 1, Paul COOK2, Juan DESANCTIS3, Zdenek HEL4, Wojciech WOJCIECHOWSKI4, Neil E. REINER5,Emil SKAMENE1 and Danuta RADZIOCH1

1 University of Laval, CHUL, Ste-Foy, Canada2 Department of Medicine, East Carolina University School of Medicine, Greenville, USA3 Central University of Venezuela, Faculty of Medicine, Institute of Immunology, Caracas, Venezuela4 Centre for the Study of Host Resistance, McGill University, Montreal General Hospital, Canada5 Departments of Medicine and Microbiology, University of British Columbia, Vancouver, Canada

(Received 3 November1997) 2 EJB 971552/1

Mice of diverse genetic backgrounds may be classified as being either resistant or susceptible toinfection withMycobacteria. These phenotypes appear to be determined by a single gene on chromosome1,theBcg gene, and are expressed at the level of the macrophagein vitro. When compared to macrophagesfrom mice of the susceptible phenotype (Bcgs), macrophages from mice of the resistant phenotype (Bcgr)show enhanced functional properties including increased expression of MHC class II molecules, increasednitric oxide production, and greater capacity to inhibit the growth of several intracellular pathogens. Thebacteriostatic activity of B10R and B10S macrophages correlated with the amount of nitric oxide pro-duced by the macrophages. Since protein kinase C (PKC) has been shown to be involved in the inductionof a range of macrophage functional activities, experiments were conducted to examine the possibilitythat phenotypic differences between Bcgr and Bcgs macrophages may be related to differences in PKC-dependent signalling. Macrophage cell lines were derived from mice congenic at theBcg locus that areeither resistant (B10R) or susceptible (B10S) to infection withMycobacteria. In the basal state, PKC-specific activity was significantly increased in the cytosolic fractions of B10R cells when compared toB10S cells. Following phorbol myristate acetate (PMA) treatment and following the stimulation withMycobacteria bovisBCG, PKC-specific activity increased significantly in membrane fractions of bothB10R and B10S cells, but the absolute level was significantly greater in particulate fractions from B10Rmacrophages. Furthermore, B10R cells had a superior ability to phosphorylate endogenous substratescompared to B10S macrophages. Scatchard analysis of phorbol ester receptors revealed no differencesbetween B10R and B10S cells. In contrast, the sensitivity of partially purified PKC from B10S cells toactivation in vitro by diacylglycerol was decreased by approximately 50% when compared to enzymefrom B10R cells. Western-blotting analysis using antibodies specific for PKC isoforms (A, β, δ, ε, ζ andη) showed similar levels of PKC isoforms present in B10R and B10S cells. To examine whether differ-ences in PKC activity of B10R and B10S cells had functional consequences, the induction ofc-fosgeneexpression was compared in the two cell lines. In response either to infection withM. bovisBCG or tostimulation with PMA,c-fosmRNA levels in B10R macrophages were increased 224-fold in comparisonto B10S macrophages. Since we have previously found that the bacteriostatic activity of B10R and B10Smacrophages correlated with the amount of nitric oxide produced by the macrophages, we have tested ifthe enhancement of PKC activity in these macrophages affects their ability to produce nitric oxide. Wehave found that interferon-γ-(IFNγ)-induced secretion of nitric oxide by B10R macrophages could beaugmented a few fold by the activation of PKC whereas, in B10S macrophages stimulated with IFNγ,nitric oxide release could be augmented by only about10220%. These results indicate that the differencesin PKC activity between B10R and B10S macrophages may contribute to altered responsiveness to IFNγthat results in different production of effector molecules crucial for bacteriostatic activity againstM. bovisBCG.

Keywords:macrophage;Mycobacterium bovisBCG; protein kinase C;Bcg gene; Nramp1.

The capacity of macrophages to become fully activated is influenced, at least in part, by a dominant autosomal gene,Bcg,important for the elimination of intracellular pathogens (i.e.My- that confers resistant (Bcgr) or susceptible (Bcgs) phenotypes tocobacteria, Leishmania, and others) that invade host cells. Theinfection with Mycobacteria bovisBCG, Leishmania donovaniextent of activation of murine macrophages is believed to beandSalmonella typhimurium(Bradley et al.,1979; Gros et al.,

1981 ; Plant and Glynn,1979; Skamene et al.,1982; Vidal etCorrespondence toD. Radzioch, Centre for the Study of Host Resis-al., 1995b).tance, Montreal General Hospital,1650 Cedar Ave., Room LH11-218,

Montreal, Quebec, Canada H3G1A4 It is now well established that theBcg gene is expressed byFax: 11 514 933 7146. mature macrophage (Lisner et al.,1983; Stach et al.,1984;Abbreviations. PMA, phorbol myristate acetate; PBt2, phorbol- Crocker et al.,1984; Goto et al.,1989; Olivier and Tanner,

12,13-dibutyrate ; Ac2Gro, diacylglycerol; CaI, calcium ionophore; PKC, 1987; Blackwell et al.,1988). Both primary macrophages cul-protein kinase C; H7,1-(5-isoquinolinylsulfonyl)-2-methylpiperazine ;tured in vitro and retrovirus-immortalized macrophage lines de-LAM, lipoarabinomannan; LPG, lipophosphoglycan; IFNγ, interferon-γ.rived from Bcgr and Bcgs mice display differential anti-myco-Enzyme.Protein kinase C (EC 2.7.1.37).

735Olivier et al. (Eur. J. Biochem. 251)

Cells. Macrophage cell lines were derived from the bonebacterial activity (Gros et al.,1988; Radzioch et al.,1991). TheNramp1gene, localized about130 kb distal toλ Mm1C165, and marrow of B10A and B10A. Bcgr-strain mice congenic at the

Bcg locus, as previously described (Radzioch et al.,1991). The50 kb proximal tovil, has been cloned by a positional approachand is considered to be theBcggene (Vidal et al.,1993; Govoni cells were cultured in Dulbecco’s Modified Minimal Essential

Medium (ICN Biomedicals) containing10% heat-inactivated fe-et al., 1996). It has been shown that this gene is exclusivelyexpressed in macrophages. TheNramp gene seems to encode tal bovine serum (Hyclone, Logan).

Measurement of PKC activity. Macrophage cell lines B10Sfor a membrane protein that may play a role in membrane trans-port (Vidal et al.,1993,1995b). and B10R (23106 cells) were either untreated or treated with

PMA (10, 25, 50 or100 ng/ml,15 min at 37°C), and PKC-spe-Surface molecules isolated from mycobacteria such as thecell-wall-derived glycolipid lipoarabinomannan (LAM), or lipo- cific activities were determined in cytosolic and particulate frac-

tions after DE52 chromatography as previously described in de-phosphoglycan (LPG) fromLeishmania, have been shown to in-hibit certain macrophage functions including the oxidative burst tail (Olivier et al.,1992). Briefly, cells were homogenized using

20 strokes in a Dounce tissue grinder in 4 ml ice-cold 20 mMor interleukin-1 production (Chan et al.,1991 ; Frankenburg etal., 1990). The effects of LAM or LPG on macrophage func- Hepes, pH 7.4, 2 mM MgCl2, 10 mM EGTA, 2 mM EDTA,

2 mM dithiothreitol and the proteinase inhibitors pepstatin (2µg/tional properties have been attributed to inhibition of proteinkinase C (PKC) (Chan et al.,1991 ; McNeely and Turco,1987) ml), leupeptin (2µg/ml), benzamidine (400µg/ml), and aproti-

nin (1 µg/ml) (buffer A). Following ultracentrifugation atand, in the case of LPG, have been related to inhibition ofc-fosgene expression in response to extracellular stimuli (Descoteaux1000003g at 4°C for 1 h, supernatants were applied to DE52

columns equilibrated with 20 mM Hepes, pH 7.4, 2 mM EGTAet al., 1992). The cell-wall glycolipid LAM isolated from theavirulent H37Ra strain ofMycobacterium tuberculosis, rapidly 2 mM EDTA, and 2 mM dithiothreitol (buffer B). The corre-

sponding membrane pellets were solubilized (1 h at 4°C) ininduces early gene expression at the mRNA level (c-fos, JE,KC; Roach et al.,1993). In contrast, macrophages stimulated buffer A to which1% (mass/vol.) Nonidet P-40 was added and

then recentrifuged for1 h at1000003g at 4°C prior to the appli-with LAM (2500 ng/ml) isolated from the virulent Erdman donot show an induction of thec-fos, KC or JE mRNAs expres- cation of supernatants to DE52 columns. Unbound proteins were

washed from the columns with buffer B. Fractions containingsion. These results suggest that the level of induction of earlygene expression in macrophages, induced by various strains of PKC were eluted with buffer B containing 0.3 M NaCl. Eluates

were then assayed using a mixed micelle assay as described pre-Mycobacteria, may be correlated with their virulence (Roach etal., 1993). viously in detail (Olivier et al.,1992). The incorporation of ra-

dioactive phosphate from [γ-32P]ATP into the synthetic peptideThe induction of various macrophage functional responsessuch as the oxidative burst, major histocompatability class-II substrate [Ser25]PKC-(19231) (Peninsula Laboratories) was as-

sessed in 50µl final sample. Maximal activation of PKC wasprotein expression, interleukin1-β production, tumoricidal activ-ity and phagocytosis are thought to be regulated at least in part achieved in the presence of 2.25 mM Ca21, 60 µg/ml phosphati-

dylserine (Avanti Polar Lipids) and 6µg/ml 1,2-dioleoyl-rac-via PKC-dependent signalling (Myers et al.,1985; Kiyotaki andBloom, 1984; Celada and Schreiber,1986; Radzioch and Vare- glycerol (Ac2Gro; Sigma). Net PKC activity was determined by

subtracting the amount of radioactivity incorporated intosio, 1988; Olivier et al.,1992). Induction ofc-fosexpression inresponse to extracellular stimuli [phorbol myristate acetate [Ser25]PKC-(19231) in the absence of essential cofactors

(Ac2Gro, Phosphatidylserine and Ca21 from the maximal incor-(PMA), diacylglycerol (Ac2Gro), the calcium ionophore (CaI)]is also partially dependent on PKC (Radzioch et al.,1987). porated radioactivity.

The measurement of PKC activity following macrophage ac-Some of these functions (superoxide production, major histo-compatibility class II expression) are induced differently in tivation withM. bovisBCG was performed using a PKC assay

system (Gibco BRL Life Technologies) using a protocol de-macrophages of Bcgr and Bcgs background (Denis et al.,1988;Blackwell et al.,1988; Buschman et al.,1989; Radzioch et al., scribed by Thomas (Thomas et al.,1987), with some modifica-

tions made by Yasuda (Yasuda et al.,1990). Briefly, 231071991). M. bovis BCG-inducedc-fosexpression in these macro-phage cell lines has not yet been investigated. B10R macro- macrophages were stimulated withM. bovisBCG at10:1 bacte-

ria/macrophage for15, 30, 45, 60 and 90 min. Then, the cellsphages differ from B10S cells in their capacity to control theproliferation ofM. bovisBCG and differences in PKC activation were homogenized using 20 strokes in a Dounce tissue grinder

in a ice-cold lysing solution provided in the assay systembetween these cells may partially explain the numerous pleiotro-pic effects of theBcg gene reported previously (Denis et al., (20 mM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Tri-

ton X-100, 25µg/ml leupeptin and 25µg/ml aprotinin). The1988; Blackwell et al.,1988; Buschman et al.,1989; Radziochet al., 1991). In the present study, PKC cell signalling,c-fos membrane and cytosol fractions were separated by ultracentrifu-

gation at1000003g for 1 h at 4°C. The corresponding pelletsinduction and the influence of PKC on interferon-γ (IFNγ)-in-duced nitric oxide production were compared in B10R and B10S were solubilized in a buffer containing1% Nonidet P-40 and

then recentrifuged at1000003g for 1 h at 4°C. PKC-specificmacrophages.activities were determined in cytosolic and particulate fractionsafter DE52 column chromatography as previously described in

EXPERIMENTAL PROCEDURES detail (Olivier et al.,1992). The eluted fractions were analyzedfor PKC activity at the optimal conditions of the assay suggestedMaterials. [γ-32P]ATP and [20(n)-3H]phorbol-12,13-dibuty-

rate (TRK.826; specific activity, 46.4 Ci/g; I Ci5 37 GBq) in the instructions. Obtained samples were then spotted ontoDEAE-cellulose disks and subsequently acid washed and radio-were purchased from Amersham. The protein kinase inhibitor

1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7) was ob- activity determined in the beta counter. Net PKC activity wasdetermined by subtracting the amount of radioactivity incorpo-tained from Seikagaku America Inc. Phosphatidylserine was

purchased from Avanti Polar Lipids. Protein kinase C substrate rated into [Ser25]PKC-(19231) in the absence of essential co-factors (Ac2Gro, phosphatidylserine and Ca21) from the maximalpeptide [Ser25]-PKC(19231) was purchased from Gibco-BRL.

PMA and all other chemicals were purchased from Sigma.M. incorporated radioactivity.Analysis of phorbol ester receptors.Cytosolic fractionsbovis BCG (strain Montreal) was purchased from Armand

Frappier Institute. (1000003g) from B10S and B10R macrophage cell lines

736 Olivier et al. (Eur. J. Biochem. 251)

Fig. 1. Effect of PMA treatment on the specific activity of PKC in cytosolic (A) and particulate (B) B10R and B10S cell fractions.Macrophages(23106) were either untreated or treated with PMA (10, 25, 50 or100 ng/ml) for 15 min at 37°C and PKC activity was assayed after DE52chromatography by [Ser25]PKC substrate phosphorylation. Results are means of values obtained in four experiments. B10R (w), B10S (j). (*)PKC activity observed in the B10R cells was significantly different from that in the corresponding group of B10S cells, as calculated by pairedtwo-tailed t-test. Cytosolic fraction (basal),p 5 0.01; Membrane fraction (PMA, 50 ng/ml and100 ng/ml),p 5 0.04.

Endogenous substrate phosphorylation.B10R and B10S(107 cells/sample) were prepared as described above (includingDE52 chromatography) and equilibrium binding of [3H]phorbol- cells, untreated or treated with100 nM PMA for a 5 min period,

were pelleted by centrifugation, the supernatant was removed12,13-dibutyrate ([3H]PBt2) was analyzed. Briefly, 75µl par-tially purified cytosolic fractions in the presence or absence of and whole cell solubilized fractions were prepared by homoge-

nizing the cells in a buffer containing 20 mM Tris/HCl, pH 7.4,excess unlabelled PBt2 (32µM final concentration) were incu-bated with increasing concentration of [3H]PBt2 (02300 nM), 10 mM EDTA, 5 mM EGTA, 5 mM 2-mercaptoethanol,10 mM

benzamidine,1 mg/ml leupeptin, 50µg/ml phenylmethylsul-and fixed concentrations of phosphatidylserine (0.1mg/ml),CaCl2 (0.8 mM), and BSA (1 mg/ml). Following incubation for fonyl fluoride, 0.1 mg/ml ovalbumin, 0.1 unit/ml aprotinin and

0.1% Triton X-100. Cells were disrupted further by sonication30 min at 37°C, samples were treated with polyethylene glycol-8000 (PEG-8000;15%; mass/vol.) at 4°C for 30 min to precipi- at maximal power (Sonicator dismembranator model 300, Fisher

Scientific) for 10 s. Lysates were centrifuged at1000003g attate proteins. Precipitates were pelleted (145003g) and washedwith 15% PEG-8000. The incorporation of3H into proteins was 4°C for 20 min in a Beckman TL-100 ultracentrifuge. Protein

concentrations of high speed supernatants were determined bydetermined by liquid scintillation counting, and results subjectedto Scatchard analysis. the method of Bradford (Bradford,1976). Briefly, a 5-µl aliquot

of protein extract was added to 95µl of 0.5 M NaOH, then 252Western blot analysis. Equal concentrations of samples(40µg/lane) and a control whole mouse brain protein extract100µl mixture was added to1ml dye reagent concentrate (Bio-

Rad protein assay, Bio-Rad). After a10-min incubation at roomwere subjected to SDS/PAGE using a10% running gel. Proteinswere transferred to nitrocellulose filters (0.45µm pore size) at temperature, the absorbance was read at 595 nm and the protein

concentration was calculated using a standard curve prepared100 volts for 60 min using a Bio-Rad mini-transblot apparatus.Nonspecific sites on the filters were blocked with a150-min using the same protein assay.

Equal concentrations of whole cell solubilized fractionsincubation at room temperature in 2.7 mM sodium phosphate,1.5 mM potassium phosphate, 0.15 M NaCl, pH 7.4 (NaCl/Pi) (20µg) of B10R and B10S cells were incubated with 0.2 mM

EDTA, 0.05 mM EGTA,10 mM magnesium chloride, 0.75 mMcontaining 0.05% (by vol.) Tween 20 and 3% (mass/vol.) BSA,then incubated for16 h at 4°C with the appropriate antiserum. calcium chloride and 20 mM Tris/HCl, pH 7.4, in a total volume

of 50 µl. Protein kinase was activated by adding phosphatidyl-Rabbit antiserum directed against various isoforms (A, β, δ, ε, ζ)of PKC were kindly provided by Dr K. Ways (East Carolina serine and, diolein at concentrations of 5µg/100 µl and 0.6µg/

100µl, respectively. After warming the mixture to 27°C, theUniversity, Greenville, NC; Ways et al.,1992a, b).18 aminoacid C-terminal residue (INQDEFRNFSYVSPELQL) was used reaction was initiated by adding1 µM [γ-32P]ATP (5µCi/tube).

Incubations were terminated following a 60-s incubation by add-to prepare specific rabbit antisera against theη isoform of PKC.Filters were washed four times with NaCl/Pi containing 0.05% ing an equal volume of Laemmli stop solution and heating to

110°C for 3 min. Samples were then subjected to SDS/PAGE(by vol.) Tween 20. After a 90-min exposure at room temper-ature to125I-protein A, 0.03µCi/ml, in NaCl/Pi containing 3% using 3% and10% polyacrylamide stacking and running gels,

respectively. After staining, destaining and drying, the gels were(mass/vol.) BSA and 0.05% (by vol.) Tween 20, the filters wererinsed four times with NaCl/Pi containing 0.05% (by vol.) exposed to Kodak XAR film for 24248 h at270°C. Concurrent

electrophoresis of molecular-mass standards allowed appropriateTween 20. Dried filters were exposed to Kodak XAR film for5214 days. molecular-mass assignments to the phosphorylated substrates.

Induction of c-fosexpression byM. bovisBCG and phor-In vitro activation of PKC by Ac2Gro. The sensitivity ofpartially purified PKC to Ac2Gro in vitro was assayed as de-bol esters.The kinetics ofc-fosgene expression was examined

in B10R and B10S macrophages in response to exposure toscribed previously (Olivier et al.,1992). Cytosolic fractionsfrom both cell lines were applied to and eluted from DE52 as either phorbol ester (PMA) or BCG. Briefly,107 macrophages

cultured in 25-cm2 petri dishes were stimulated with PMApreviously described (Olivier et al.,1992). PKC activity in DE52eluates was assayed in the presence of increasing concentrations (10 ng or100 ng/ml) or viable BCG (20:1 ratio) for 15, 30, 60,

120 and 240 min. Macrophage monolayers were washed threeof Ac2Gro (0280 µg/ml). Phosphatidylserine and Ca21 concen-trations used in the assay were kept constant at 60µg/ml and times with warm Dulbecco’s Modified Eagle’s Minimal Essen-

tial Medium, and the cells were solubilized in guanidine2.5 mM, respectively.

737Olivier et al. (Eur. J. Biochem. 251)

Fig. 2. Effect of stimulation with M. bovis BCGon the specific activity of PKC in cytosolic (A) and particulate (B) B10R and B10S cellfractions. Macrophages (23107) were either unstimulated or stimulated withM. bovis BCG (bacteria/macrophage,10:1) for 15, 30, 45, 60 and90 min at 37°C and PKC activity was assayed after DE52 chromatography by [Ser25]PKC substrate phosphorylation. Results are means of valuesobtained in three experiments. B10R (w), B10S (j). (*) PKC activity observed in the B10R cells was significantly different from that in thecorresponding group of B10S cells, as calculated by paired two-tailedt-test. Cytosolic fractions (basal),p 5 0.01; Membrane fractions followingthe stimulation withM. bovisBCG (p,0.01).

isothiocyanate. Total RNA was purified by centrifugationthrough a cushion of CsCl (Chirgwin et al.,1979). 20µg totalRNA was separated on1.2% agarose gel containing 2.2 Mformaldehyde as previously described (Thomas,1980), and thenassessed by Northern-blot analysis. A1-kb PstI fragment ofthe v-fos gene was kindly provided by T. Curran (HoffmanLaRoche) and a glyceraldehyde-3-phosphate dehydrogenaseprobe was obtained from ATCC. The levels of mRNA werequantified by densitometry using a photodensitometer (Sciscan5000, USB) and normalized to glyceraldehyde-3-phosphate de-hydrogenase mRNA levels using several exposures of the sameNorthern blot, so the calculations were made based on a linearrange of X-ray film exposures.

Quantification of nitrite production by macrophages. Theestimation of nitrite in quadruplicate supernatant samples ofstimulated and nonstimulated macrophages was performed bycolorimetric spectrophotometry at 543 nm using equal volumesFig.3. Scatchard analysis of phorbol-ester-binding sites.Scatchard

analysis of the binding of [3H]PBt2 to PKC from B10R (w) and B10Sof the Griess reagent (1% sulfanilamide/0.1% N-(1-(j) cytosolic fractions (107 cells). Supernatants (100 0003g) were pre-naphthyl)ethylenediamine dihydrochloride/2.5% H3PO4) atpared from B10R and B10S cells and aliquots were incubated withroom temperature for10 min (Green et al.,1982). Results areincreasing concentrations of [3H]PBt2 in the absence or presence of ex-expressed as nitrite production (µmol · 24 h21 total proteincess unlabelled PBt2 (35 µM). Specific binding was taken as the differ-content macrophages21, estimated by the Bradford protein assayence between total binding (in the absence of unlabelled PBt2) and resid-(Bio-Rad). ual radioactivity detected in the presence of excess unlabelled ligand.The data are the mean values of four experiments,r 5 0.950 for B10Sandr 5 0.951 for B10R.

RESULTS

PKC activity in B10S and B10R cells.To examine PKC activa-tion, the subcellular distribution of enzymatic activity of PKC PMA, the levels of membrane-associated PKC in B10R cells

were significantly higher than that measured in B10S cellswas analyzed. In the basal state, PKC enzymatic activity in cyto-solic fractions was approximately 75% greater in B10R when (p,0.05; pairedt-test).

To examine PKC activation following the exposure of B10Rcompared to B10S (p,0.05 ; pairedt-test; Fig.1). In contrast,PKC activity associated with membrane fractions in the basal and B10S macrophages toM. bovisBCG, the subcellular distri-

bution of enzymic activity PKC was analyzed. In the basal state,state were similar in the two cell lines. In response to phorbolester, a dose-dependent translocation of PKC activity from the PKC enzymic activity in cytosolic fractions was approximately

75% greater in B10R when compared to B10S (p,0.05; pairedcytosol to the membrane was observed in both B10S and inB10R cells. In cells incubated with 50 ng/ml and100 ng/ml t-test; Fig. 2). In contrast, PKC activity associated with mem-

738 Olivier et al. (Eur. J. Biochem. 251)

Fig.6. Effect of PMA on endogenous substrate phosphorylation inFig. 4. Detection of PKC isoforms in B10R and B10S cell lines.PKC B10R and B10S cells.503106 cells were incubated either with vehicleisoforms (A, β, δ, ε, ζ and η) were detected using isoform-specific anti-or with 100 nM PMA. After a 5-min exposure, the cells were homoge-sera. Lanes were loaded with equal amounts of protein (40µg) from nized, and the whole cell solubilized fractions were prepared. TheB10R or B10S cells. The following dilutions of a specific antisera weresamples were incubated in the absence or presence of 0.75 mM Ca21,used:A (1:4000), β (1 :4000), δ (1 :2500), ε (1 :1000), ζ (1: 2000), η phosphatidylserine (5µg/100µl), and diolein (0.6µg/100µl). After(1 :1000). Rat brain total protein extract was used as positive control forSDS/PAGE, autoradiography was performed. Molecular-mass standardsthe PKC isoforms (CTL). are indicated to the left of the autoradiogram.

brane fractions in the basal state were similar in the two celllines. In response to stimulation withM. bovis BCG, a dose-dependent translocation of PKC activity from the cytosol to themembrane was observed in both B10S and in B10R cells. Incells stimulated withM. bovisBCG (10 :1 ratio of bacteria/mac-rophage), the levels of membrane-associated PKC in B10R cellswere significantly higher than that measured in B10S cells atevery time point post stimulation tested (p, 0.05; pairedt-test).

Analysis of phorbol ester receptors.Increased PKC-specificactivities in the basal state in cytosolic fractions and in mem-brane fractions following PMA treatment (100 ng/ml) in B10Rcells could be explained by increased enzyme protein levels,increased relative affinities of phorbol ester receptors, enhancedsensitivity of the enzyme to PMA or to a combination of thesefactors. To examine phorbol ester receptors, Scatchard analysisof [3H]PBt2 binding was carried out (Fig. 3). The results indicate

Fig. 5. In vitro activation of PKC by Ac2Gro. Cytosolic PKC was par- that 1000003g cytosolic fractions isolated from B10R andtially purified by DE52 chromatography from B10R (w) and B10S (j) B10S cells contained similar numbers of high-affinity phorbol-macrophages. Aliquots of 0.3 M NaCl column eluates were monitored

binding sites. The data shown represent one of four experimentsfor their protein content and assayed for PKC activity using [Ser25]PKCthat gave similar results. Mean values from the four experimentssubstrate. Data obtained at initial rates of reaction were linear with re-for Kd were 7.562.7 nM and11.666.2 nM (mean6SEM, notspect to time and protein concentration. The data were analyzed usingsignificantly different as determined by unpairedt-test), respec-Lineweaver Burke plots, by using expressiony 5 [V0 1 (Vmax2V0)3C]/tively, for B10S and B10R cells. Numbers of phorbol-ester-bind-(k 1 C), wherey is the rate of phosphorylation of specific [Ser25]PKC

substrate at any given concentration of Ac2Gro, C is the concentration ing sites were also similar for both cell lines (B10Sof Ac2Gro,V0 is the activity of PKC in the absence of Ac2Gro. Velocities 1300063200 sites/cells and B10R 10 70062700 sites/cells;were converted to the maximum [Ser25]PKC substrate phosphorylation.mean6SEM, not significantly different as determined by un-Results are means of values obtained in three experiments.Vmax values pairedt-test).calculated for B10R and B10S cells, were 4.3460.14 pmol · mg21 ·10 min21 and 2.7160.2 pmol · mg21 · 10 min21 (mean6SEM; n 5 3),

Western blot analysis.To examine whether the expression ofrespectively, and were significantly different (p 5 0.016) by unpairedspecific PKC isoforms was higher in B10R cells in comparisont-test. Km values for B10R (0.9460.058µg/ml, mean6SEM; n 5 3)to B10S cells, Western blot analysis was performed. Westernand B10S (2.4160.067µg/ml) were also significantly different (p 5

0.043) by two-tailedt-test. blotting using antibodies specific for PKC isoforms (A, β, δ, ε, ζ

739Olivier et al. (Eur. J. Biochem. 251)

Fig. 7. Effects of PMA on thec-fos mRNA expression.(A) B10R and B10S macrophages (107 cells) were treated with10 ng/ml PMA for 0, 30,60 or 120 min. RNA was extracted andc-fosmRNA expression was assessed by Northern-blot analysis. Expression ofc-fosmRNA was quantifiedby densitometric analysis of autoradiograms. (B) B10R and B10S macrophages (107 cells) were treated with100 ng/ml LPS,10 ng/ml or100 ng/ml PMA for 30 or 60 min. RNA was extracted andc-fosmRNA expression was assessed by Northern-blot analysis. Expression ofc-fos mRNAwas quantified by densitometric analysis of autoradiograms. MED, medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

andη) showed that B10S cells expressed similar levels of levels for Ac2Gro in comparison to the B10S cells, the capacity ofthese cells to phosphorylate endogenous substrates was com-of β, δ, ε, Z andη PKC isoforms (Fig. 4). Expression of PKCA

appeared to be higher in B10S cells. Therefore, the difference pared. The phosphorylation of endogenous substrates in theB10R extracts, both at basal state and following 5-min exposurein PKC activity observed between these cell types is not due to

higher levels of expression of any of the tested PKC isoforms to100 nM of PMA, was substantially higher when compared toextracts from B10S cells (Fig. 6).in B10R cells.

c-fos mRNA expression in response to treatment with PMASensitivity of PKC to Ac2Gro. To examine whether increasedor infection with M. bovis BCG. To examine whetherc-foscytosolic PKC-specific activity in non-activated B10R cellsgene expression is modulated differently in B10S than in B10Rcould be accounted for by altered kinetic properties of the en-macrophages, total RNA was extracted from unstimulated cells,zyme, thein vitro responsiveness of cytosolic PKC from B10Rcells stimulated with PMA, or cells infected with BCG, and sub-and B10S macrophages to the allosteric activator Ac2Gro wasjected to the Northern blot analysis. To investigate the kineticsmeasuredin vitro in a mixed micelle assay. There was a signifi-of the effects of PMA onc-fosmRNA accumulation, B10R andcant difference in the Ac2Gro dose-response relationship of PKCB10S macrophages were treated with10 ng/ml PMA for 0, 30,isolated from B10S as compared with B10R cells (Fig. 5). PKC60 and120 min and the expression ofc-fos mRNA was mea-from B10S macrophages demonstrated a 50% lower sensitivitysured. Both cell lines constitutively expressed only very lowto Ac2Gro as measured by the concentration of activator requiredlevels ofc-fosmRNA (Fig. 7A). After stimulation with PMA, ato achieve half-maximum activation:Km(Ac2Gro) values for en-peak ofc-fosmRNA expression was observed after 30 min treat-zyme preparations from B10R and B10S cells, respectively,ment both in B10R and B10S cells and was followed by a grad-were 0.9460.058µg/ml and 2.4160.067µg/ml (mean6SEMual return to basal levels. In five individual experiments per-of values obtained in three independent experiments;p 5 0.043formed, a 223-fold increase inc-fos mRNA was consistentlyby unpairedt-test). Vmax values for the enzyme for the B10Rdetected in PMA-treated (10 ng/ml) B10S macrophages. In con-and B10S cell lines were also significantly different, and weretrast, a 6210-fold increase inc-fos mRNA was observed in4.3460.14 pmol ·µg21 · 10 min21 and 2.7160.2 pmol ·µg21

B10R macrophages. Even a10-fold increased dose of PMA· 10 min21 (mean6SEM, p 5 0.016).(100 ng/ml) was not able to induce thec-fos mRNA levels inB10S cells that were comparable to those observed in B10RPhosphorylation of endogenous substrates.Since B10R cells

were observed to have increased PKC activity and sensitivity cells in response to10 ng/ml of PMA (Fig. 7B). Interestingly,

740 Olivier et al. (Eur. J. Biochem. 251)

Fig. 8. Induction of c-fosmRNA in B10R and B10S macrophages inresponse to infection withM. bovis BCG. B10R and B10S cells (107)were stimulated with BCG (20:1 bacteria/macrophage) for1, 2 and 4 h,as indicated. Cells stimulated with PMA (100 ng/ml, 60 min) were usedas a positive control. Total RNA was extracted andc-fosmRNA expres-sion was assessed by Northern blot analysis. MED, medium. Expressionof c-fosmRNA was quantified by densitometric analysis of autoradio-grams. Integrated absorbance forc-fos mRNA expression was normal-ized to the levels of glyceraldehyde-3-phosphate dehydrogenase(GAPDH) mRNA in the same experimental sample.

no difference in thec-fos mRNA accumulation was observedbetween B10R and B10S cells stimulated with100 ng/ml LPS.A 223-fold increase inc-fosmRNA expression was consistentlyobserved in both B10R and B10S macrophages exposed to LPS.

The effects of infection of B10R and B10S macrophageswith M. bovisBCG was also examined. Significantc-fosmRNAaccumulation was observed, 2 h and 4 h following infection ofFig.9. Effect of PMA on IFNγ-induced NO secretion by B10R andeither B10R or B10S macrophages withM. bovisBCG (Fig. 8). B10S macrophages.(A) B10R (h) and B10S (j) macrophages wereIncreasedc-fos mRNA expression in response to BCG was,cultured in medium or10 U/ml IFNγ, for 24 h, or treated with10 U/mlhowever, consistently 224-fold higher in B10R macrophages IFNγ or medium alone for 6 h, then supplemented with10 ng/ml PMA

for additional18 h. NO release was measured using Griess reagent. Datacompared to B10S macrophages.represent one of the three independent experiments. Values presented aremeans6SD of quadruplicate samples used for each treatment. (B) B10REffect of PMA on IFN γ-induced NO secretion by B10R and(h) and B10S (j) macrophages were treated with10 U/ml IFNγ orB10S macrophages.We have previously shown that nitric oxidemedium alone for 6 h, then supplemented with102100 ng/ml PMA. NOcould curtail the proliferation ofM. bovisBCG and that morerelease was measured using Griess reagent. Data represent one of the

efficient control ofM. bovisBCG proliferation by B10R macro- three independent experiments. Values presented are means6SD of qua-phages compared to B10S macrophages correlates with their su-druplicate samples used for each treatment.perior ability to produce nitric oxide following IFNγ stimulation(Barrera et al.,1994). In the present study, we investigatedwhether B10R and B10S macrophages could be stimulated byPMA, either alone or in combination with IFNγ, to induce nitric by theBcg, Ity or Lsh genes, respectively (Bradley et al.,1979;oxide. The cooperative effects of PMA for NO release have been

Gros et al.,1981 ; Plant and Glynn,1979; Skamene et al.,1982).shown to be maximal at 6 h after IFNγ treatment (Jun et al.,These genes are either identical or closely linked on chromo-

1994). Therefore, both B10R and B10S macrophages were cul- some1. Recently, theNramp 1 gene was cloned and it is thetured for 6 h either in medium alone or in medium that contained

most theBcg gene (Vidal et al.,1993; 1995 a, b). Macrophage10 U/ml IFNγ, then supplemented with10 ng/ml PMA. PMAlines derived from the bone marrow of B10. A. Bcgs mice

alone did not induce NO secretion either in B10R or B10S mac- (B10S) and their congenic counterpart B10A. Bcgr mice (B10R)rophages, whereas10 ng/ml PMA in combination with10 U/ml

have been established and characterized in this laboratory (Rad-IFNγ synergistically increased NO production in B10Rzioch et al.,1991). These cells show different phenotypic prop-

(Fig. 9A). Barely detectable increase (about a10% increase) in erties. For example, relative to B10S cells, B10R cells showNO production was observed in B10S macrophages. The en-

increased bactericidal capacity and expression of I-Aβ mRNAhancing effect of PMA on IFNγ-induced nitric oxide release byin response to extracellular stimuli (Radzioch et al.,1991). Dif-

these macrophages was PMA-dose-dependent (102100 ng/ml ferences observed between these two macrophage lines are con-range tested ; Fig. 9B). At the highest dose of PMA tested

sistent with previous reports of the functional properties of pri-(100 ng/ml) we could detect 5-fold more nitrites released bymary bone-marrow-derived macrophages obtained from mice

IFNγ-stimulated B10R macrophages compared to B10S macro- that are either resistant or susceptible to infection withMyco-phages.

bacteria (Gros et al.,1981 ; Buschman et al.,1989; Denis etal., 1990). Thus, macrophages isolated from mice geneticallyresistant to mycobacterial infection show a greater capacity toDISCUSSIONcontrol the growth of various intracellular microorganisms (Nau-ciel et al.,1985; Van Dissel et al.,1987) when compared to cellsResistance in mice to infection with the intracellular patho-

gensMycobacterium, Salmonellaand Leishmaniais controlled isolated from genetically susceptible strain of mice (Denis et al.,

741Olivier et al. (Eur. J. Biochem. 251)

1990; Stach et al.,1984; Stokes et al.,1988). Although presum- zyme from B10R cells (Fig. 5). Furthermore, the phosphoryla-ably influenced by theBcg gene, the molecular bases for the tion of endogenous substrates in the B10R extracts, both in thefunctional difference between macrophages from Bcgr and Bcgs basal state and following exposure to PMA, was substantiallymice are not well understood. Several macrophage functionalhigher compared to B10S extracts (Fig. 6). The exact basis forproperties have been shown to be induced via biochemical path- different kinetic properties of the enzyme from B10S and B10Rways that apparently involve PKC (Myers et al.,1985; Kiyotaki cells remains to be determined, but this could be related to al-and Bloom,1984; Radzioch and Varesio,1988; Olivier et al., tered activation in intact cells bearing BCG-susceptible back-1992). For example, Politis and Vogel (1990) demonstrated that ground. The results (Fig. 6) may also be accounted for by differ-the phagocytosis of SRBC mediated by the macrophage FcγR ence in the expression or availability of the PKC substrates, suchwas nearly completely inhibited in the presence of the potentas F-actin (Blobe et al.,1996) or MARCS proteins (TaniguchiPKC inhibitor-H7. A PKC-dependent signalling pathway hadet al.,1994; Brawn et al.,1995) in the B10R and B10S macro-also been shown to be involved in the induction of major histo-phages.compatibility class-II molecule expression by IFN-γ (Politis and To examine whether these observed differences in PKC acti-Vogel, 1990), as well as in the induction ofc-fosmRNA accu- vation between B10R and B10S cells had functional conse-mulation in both fibroblasts and macrophages (Radzioch et al.,quences, the induction ofc-fosgene expression was compared1987). in the two cell lines. In response to stimulation with either PMA

These findings suggest that difference in cell signalling in-(Fig. 7A and B) or to infection withM. bovis BCG (Fig. 8)volving PKC is, a potential basis to explain divergent functionalc-fos mRNA levels in B10R cells were increased 225 fold inproperties between Bcgr and Bcgs macrophages. To investigatecomparison to cells from B10S mice. Thus, enhanced activationthis possibility, PKC expression and activation in macrophagesof PKC in B10R macrophages was shown to correlate with thederived from mice either resistant (B10R) or susceptible (B10S) higher level ofc-fosmRNA expression in these two cell types.to infection with Mycobacteriawere examined. In the basal To examine whether these observed differences in PKC acti-state, PKC-specific activity was significantly increased in cyto-vation between B10R and B10S cells had also other functionalsolic fractions of B10R cells when compared to B10S cells. consequences, we have analyzed the effect of PKC activation onFollowing PMA treatment and stimulation withM. bovisBCG, secretory functions of B10R compared to B10S macrophages. ItPKC-specific activity increased in membrane fractions of bothhas been previously shown that nitric oxide could curtail theB10R and B10S cells, but the absolute level was significantlyproliferation ofMycobacteria(Barrera et al.,1994; Green et al.,greater in particulate fractions from B10R macrophages (Fig.1 1994; Brown et al.,1995). Furthermore, more efficient controland Fig. 2). Scatchard analysis of PBt2 binding revealed no dif- of M. bovisBCG proliferation by B10R macrophages comparedferences in the number of phorbol ester receptors between B10R to B10S macrophages correlated with their superior ability toand B10S cells (Fig. 3). Relatively increased PKC-specific ac-produce nitric oxide following IFNγ stimulation (Barrera et al.,tivity in cytosolic extracts of B10R macrophages could be re-1994). Since B10R and B10S macrophages differ in their PKClated either to differences in PKC abundance or to differentialactivity following PMA stimulation, we examined whether thisstates of the enzyme between B10R and B10S cells, or to a difference correlated with the ability of these macrophages tocombination of these factors. For example, Hopper et al. (1989) secrete NO. Therefore, we determined whether B10R and B10Sdemonstrated that diminished expression of one isoform of PKCmacrophages could be stimulated by PMA, either alone or in(PKCβ) had a prominent impact on the extent of PKC activationcombination with IFNγ, to release nitric oxide. We found thatin intact cells. They observed that the KG-1a cell line, a sponta- the augmentation of IFNγ-induced nitric oxide release by PMAneous variant of the myeloid leukemia cell line KG-1, showed was 5-fold more prominent in B10R macrophages compared toa reduced capacity to differentiate into macrophages in response

B10S macrophages. Overall, these data implicate the PKC acti-to phorbol ester. This correlated with diminished levels of PKCβvation in the pathway of macrophage activation leading to the

mRNA and PKC translocation in KG-1a cells.NO release. The difference in PKC activity between B10R andWestern-blot analysis to examine expression of PKC iso-B10S macrophages may be responsible for the difference in theirforms using antibodies specific for PKCA, β, δ, ε, ζ andη iso-bacteriostatic function againstM. bovisBCG.forms showed that B10S cells expressed similar levels ofβ, δ,

Brown and co-workers (1994) demonstrated that treatmentε, ζ andη PKC isoforms and a higher a PKC expression com-of IFNγ-activated macrophages that transiently expressed majorpared to B10R cells (Fig. 4). Therefore, the higher specific activ-histocompatibility class-II gene (I-A) with PMA resulted in theity of PKC observed in B10R cells is not explained by higherinduction of I-A expression persistence. They have also shownlevels of mRNA expression of any of the PKC isoforms tested.that addition of a high dose of rIFNγ to macrophages previouslyThe relative increase in the PKC-specific activity in B10Rtreated with a low dose of rIFNγ resulted in the synergistic acti-cells might be due either to different levels of expression of yetvation of PKC. Furthermore, they have shown that the additionunknown PKC isoform, or related to divergent properties of theof the high dose of rIFNγ to macrophages from BALB/c. Bcgsenzyme, such as the extent of enzyme phosphorylation in themice, previously treated with the low dose of rIFNγ, failed tobasal state. For example, Borner et al. (1989) observed that theactivate high levels of PKC activity observed after similar treat-level of PKC phosphorylation in resting breast cancer cells de-ment of macrophages from BALB/c. Bcgr mice. Previously, wetermined the extent of enzyme activation in response to phorbolhave also found that IFNγ-stimulated B10R and B10S macro-ester. In these cells, PKC was found to be synthesized as anphages differ in their ability to express major histocompatibilityinactive, nonphosphorylated 74-kDa protein that was sequen-class-II protein (Radzioch et al.,1991). Our results and the re-tially converted into 77-kDa and 80-kDa active forms, followingsults of Brown and co-workers are consistent with the possibilitytwo phosphorylation steps. The 80-kDa form of PKC was shownthat theBcg gene (Nramp1gene) is involved in the regulationto be more responsive to further activation in comparison to theof PKC activation. The differences at the level of PKC activity77-kDa species (Borner et al.,1989).between macrophages derived from mice resistant and suscepti-Examination of enzymic properties of partially purified PKCble to infection withMycobacteriareported in this manuscript,from the B10R and B10S cells revealed that the sensitivity ofmay, at least in part, be responsible for some of the phenotypicPKC from B10S cells to activationin vitro by diacylglycerol

was decreased by approximately 50% when compared to en- differences observed between these macrophages.

742 Olivier et al. (Eur. J. Biochem. 251)

The authors wish to thank Ms Marie Boule for excellent technical Descoteaux, A. & Matlashewski, G. (1989) c-fos and tumor necrosisfactor gene expression inLeishmania donovani-infected macro-assistance. This work was supported by the Medical Research Council

of Canada grant MI-10707 (D. R.). It was also partially supported by phages,Mol. Cell. Biol. 9, 522325227.Descoteaux, A., Matlashewski, G. & Turco, S. J. (1992) Inhibition ofthe Medical Research Council of Canada grants MT-12671 (M. O.) and

MA8633 (N. R.), Juttings Scholarship (W. W) and by the FRSQ Scholar- macrophage protein kinase C-mediated protein phosphorylation byLeishmania donovanilipophosphoglycan,J. Immunol. 149, 30082ships : Senior (D. R.) and Junior I (M. O.). The costs of publication of

this article were defrayed in part by the payment of page charges. This 3015.Fan, X.-D., Goldberg, M. & Bloom, B. R. (1988) Interferon-gamma-article must be therefore be hereby marked ‘advertisement’ in accor-

dance with18 U.S.C. Section1734 solely to indicate this fact. induced transcriptional activation is mediated by protein kinase C,Proc. Natl Acad. Sci. USA 85, 512225125.

Frankenburg, S., Leibovici, V., Mansboch, N., Turco, S. J. & Rosen,G. (1990) Effect of glycolipids ofLeishmania parasiteson humanmonocyte activity,J. Immunol. 145, 428424289.

REFERENCES Goto, Y., Buschman, E. & Skamene, E. (1989) Regulation of host resis-tance to Mycobacterium intracellularein vivo andin vitro by the BcgBlackwell, J. M., Toole, S., King, M., Dawda, P., Roach, T. I. A. &gene,Immunogenetics 30, 2182221.Cooper, A. (1988) Analysis of Lsh gene expression in congenic B10.

Govoni, G., Vidal, S., Gauthier, S., Skamene, E., Malo, D. & Gros, P.L-Lshr mice,Curr. Top. Microbiol. Immunol. 137, 3012309.(1996) The Bcg/Ity/Lsh locus : genetic transfer of resistance to infec-Blobe, G. C., Stribling, D. S., Fabbro, D., Stabel, S. & Hannum, Y. A.tions in C57Bl/6J mice transgenic for the Nramp1 Gly169 allele,(1996) Protein kinase beta II specifically binds to and is activatedInfec. Immun. 64, 292322929.by F-actin.J. Biol. Chem. 271, 15823215830.

Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Whisnok, J.Borner, C., Filipuzzi, I., Wartmann, M., Eppenberger, V. & Fabbro, D.S. & Tannenbaum, S. R. (1982) Analysis of nitrite, nitrite, and15n-(1989) Biosynthesis and posttranscriptional modifications of proteinnitrate in biological fluids,Anal. Biochem. 126, 1312138.kinase C in human breast cancer cells,J. Biol. Chem. 264, 139022

Green, S. J., Scheller, L. F., Marletta, M. A., Seguin, M. C., Klotz, F.13909.W., Slater, M., Nelson, B. J. & Nacy, C. A. (1994) Nitric oxide:Bradford, M. M. (1976) A rapid and sensitive method for the quantifica-cytokine-regulation of nitric oxide in host resistance to intracellulartion of microgram quantities of protein utilizing the principle of pro-pathogens,Immunol. Lett. 43, 87294.tein-dye binding,Anal. Biochem. 72, 2482254.

Gros, P., Skamene, E. & Forget, A. (1981) Genetic control of naturalBradley, D. J., Taylor, B. A., Blackwell, J. M., Evans, E. P. & Freeman,resistance toMycobacterium bovis(BCG) in mice,J. Immunol. 127,J. (1979) Regulation ofLeishmaniapopulation within the host. III.241722421.Mapping of the locus controlling susceptibility to visceral Leishma-

Hamilton, T. A., Becton, D. L., Somers, S. D., Gray, P. W. & Adams, D.niasis in the mouse,Clin. Exp. Immunol. 37, 7214.O. (1985) Interferon-γ modulates protein kinase C activity in murineBrawn, M. K., Chiou, W. J. & Leach, K. L. (1995) Oxidant-inducedmacrophages,J. Biol. Chem. 260, 137821381.activation of protein kinase C in UC11MG cells,Free Radical Res.

Horiguchi, J., Spriggs, D., Imamura, K., Stone, R., Leubbers, R. & Kufe,22, 23237.D. (1989) Role of arachidonic acid metabolism in transcriptional in-Brown, D., Faris, M., Hilburger, M. & Zwilling, B. S. (1994) The induc-duction of tumor necrosis factor gene expression by phorbol ester,tion of persistence of I-A expression by macrophages from BcgrMol. Cell. Biol. 9, 2522258.mice occurs via a protein kinase C-dependent pathway,J. Immunol.

Hooper, W. C., Abraham, R. T., Ashendel, C. L. & Woloschak, G. E.152, 132321331.(1989) Differential responsiveness to phorbol esters correlates withBrown, D. H., LaFuse, W. & Zwilling, B. S. (1995) Cytokine-mediateddifferential expression of protein kinase C in KG-1 and KG-1a hu-activation of macrophages fromMycobacterium bovisBCG-resistantman myeloid leukemia cells,Biochem. Biophys. Acta 1013, 47254.and -susceptible mice: differential effects of corticosterone on anti-

Ina, Y., Koide, Y., Nezu, N. & Yoshida, T. O. (1987) Regulation ofmycobacterial activity and expression of Bcg gene (CandidateHLA class II antigen expression: intracellular signalling moleculesNramp),Infect. Immun. 63, 298322888.responsible for regulation by IFN-gamma and cross-linking of FcBuschman, E., Taniyama, T., Nakamura, R. & Skamene, E. (1989) Func-receptors in HL-60 cells,J. Immunol. 139, 171121717.tional expression of Bcg gene in macrophages,Immunol. Res. 140,

Jun, C.-D., Choi, B.-M., Hoon-Ryu, Um, J.-Y., Kwak, H.-J., Lee, B.-S.,7932797.Paik, S.-G., Kim, H.-M. & Chung, H.-T. (1994) Generation of nitricCelada, A. & Schreiber, R. D. (1986) Role of protein kinase C andoxide inhibits formation of superoxide in macrophages during activa-intracellular calcium mobilization in the induction of macrophagetion, J. Immunol. 153, 368423690.tumoricidal activity by interferon-γ, J. Immunol. 137, 237322379.

Kiyotaki, C. & Bloom, B. R. (1984) Activation of murine macrophageChan, J., Fan, X., Hunter, S. W., Brennan, P. J. & Bloom, B. R. (1991)cell lines. Possible involvement of protein kinase in stimulation ofLipoarabinomannan, a possible virulence factor involved in persis-superoxide production,J. Immunol. 133, 9232931.tence ofMycobacterium tuberculosiswithin macrophages,Infect. Im-

Laemmli, U. K. (1970) Cleavage of structural proteins during the assem-mun. 59, 175521761.bly of the head of bacteriophage T4,Nature 227, 6802685.Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, W. J.

Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. L. (1951)(1979) Isolation of biologically active ribonucleic acids from sourcesProtein measurement with Folin phenol reagent,J. Biol. Chem. 193,enriched in ribonucleases,Biochemistry 18, 529425306.2652275.Christiansen, N. O. & Borregaard, N. (1989) Translocation of protein

Lissner, C. R., Swanson, R. N. & O’Brien, A. D. (1983) Genetic controlkinase C to subcellular fractions of human neutrophils,Scand. J.of innate resistance of mice toSalmonella typhimurium: expressionImmunol. 29, 4092414.of the Ity gene in peritoneal and splenic macrophages isolatedinChung, I. Y., Kwon, J. & Benveniste, E. N. (1992) Role of protein kinasevitro, J. Immunol. 131, 300623013.C activity in tumor necrosis factor alpha gene expression. Involve-

McNeely, T. B. & Turco, S. J. (1987) Inhibition of protein kinase Cment at the transcriptional levels,J. Immunol. 149, 389423902.activity by the Leishmania donovanilipophosphoglycan,Biochem.Crocker, P. R., Blackwell, J. M. & Bradley, D. J. (1984) Expression ofBiophys. Res. Commun. 148, 6532657.the natural resistance gene Lsh in resident liver macrophage,Infect.

Myers, M. A., McPhail, L. C. & Snyderman, R. (1985) RedistributionImmunol. 43, 103321040.of protein kinase C activity in human monocytes: correlation withCurran, T. & Franza, B. R. Jr (1988) Fos and Jun : the AP-1 connection,the activation of the respiratory burst,J. Immunol. 135, 341123416.Cell 55, 3952397.

Nauciel, C., Vilde, F. & Ronco, E. (1985) Host response to infectionDenis, M., Forget, A., Pelletier, M. & Skamene, E. (1988) Respiratorywith the temperature-sensitive mutant ofSalmonella typhimuriuminburst in congenic Bcgr and Bcgs macrophages,Clin. Exp. Immunol.a susceptible and resistant strains of mice,Infect. Immun. 49, 523273, 3702375.527.Denis, M., Forget, A., Pelletier, M., Gervais, F. & Skamene, E. (1990)

Nishizuka, Y. (1986) Studies and perspectives of protein kinase C,Sci-Killing of Mycobacteria smegmatisby macrophages from geneticallysusceptible and resistant mice,J. Leuk. Biol. 47, 25230. ence 233, 3052312.

743Olivier et al. (Eur. J. Biochem. 251)

Nishizuka, Y. (1992) Intracellular signalling by hydrolysis of phospho- Taniguchi, H., Manenti, S., Suzuki, M. & Titani, K. (1994) Myristoy-lipids and activation of protein kinase C,Science 258, 6072614. lated alanine-rich C kinase substrate (MARCKS), a major protein

Olivier, M., Brownsey, R. W. & Reiner, N. E. (1992) Defective stimulus- kinase substrate, is andin vivo substrate of proline-directed proteinresponse coupling in human monocytes infected withLeishmania kinase(s). A mass spectroscopic analysis of the posttranscriptionaldonovaniis associated with altered activation and translocation of modifications,J. Biol. Chem. 269, 18299218302.protein kinase C,Proc. Natl Acad. Sci. USA 89, 748127485. Thomas, P. (1980) Hybridization of denatured RNA and small DNA

Olivier, M. & Tanner, C. E. (1987) Susceptibilities of macrophage pop- fragments transferred to nitrocellulose,Proc. Natl Acad. Sci. USAulations to infectionin vitro by Leishmania donovani, Infect. Immun. 77, 520125205.55, 4672471.

Thomas, T. P., Gopalakrishna, R. & Anderson, W. B. (1987). HormonePlant, T. & Glynn, A. A. (1979) LocatingSalmonellaresistance gene onand tumor promoter-induced activation or membrane association ofmouse chromosome1, Clin. Exp. Immunol. 37, 126.protein kinase C in intact cell,Methods Enzymol. 141, 3992411.Politis, A. D. & Vogel, S. (1990) Pharmacologic evidence for the

Van Dissel, J. T., Stikkelbrock, J. J., Michel, J. B., Leijh, P. C. & Vanrequirement of protein kinase C in IFN-induced macrophage FcFurth, R. (1987) Salmonella typhimurium-specific difference in rategamma receptor and Ia antigen expression,J. Immunol. 145, 37882of intracellular killing by resident peritoneal macrophages fromSal-3795.monella-resistant CBA and Salmonella-susceptible C57Bl/10 mice,Roach, T. I. A., Barton, C. H., Chatterjee, D. & Blackwell, J. M. (1993)

Macrophage activation: lipoarabinomannan from avirulent and viru- J. Immunol. 138, 442824434.lent strains ofMycobacterium tuberculosisdifferentially induces the Vidal, S. M., Malo, D., Vogan, K., Skamene, E. & Gros, P. (1993) Natu-early genes c-fos, KC, JE and tumor necrosis factor-alpha,J. Immu- ral resistance to infection with intracellular parasites: isolation of anol. 150, 188621896. candidate for Bcg,Cell 73, 4692485.

Radzioch, D., Bottazzi, B. & Varesio, L. (1987) Augmentation ofc-fos Vidal, S., Gros, P. & Skamene, E. (1995a) Natural resistance to infectionmRNA expression by activators of protein kinase C in fresh, termi- with intracellular parasites : molecular genetics identifies Nramp1 asnally differentiated resting macrophages,Mol. Cell. Biol. 7, 5952 the Bcg/Ity/Lsh locus,J. Leuk. Biol. 58, 3822390.599. Vidal, S., Tremblay, M. L., Govoni, G., Gauthier, S., Sebastiani, G.,Radzioch, D., Hudson, T., Boule, M., Barrera, L., Urbance, J. W., Vare-

Malo, D., Skamene, E., Olivier, M., Jothy, S. & Gros, P. (1995b) Thesio, L. & Skamene, E. (1991) Genetic resistance/susceptibility toIty/Lsh/Bcg locus : natural resistance to infection with intracellularMycobacteria: Phenotypic expression in bone marrow derived mac-parasites is abrogated by disruption of Nramp1 gene,J. Exp. Med.rophage lines,J. Leuk. Biol. 50, 2632272.182, 6552666.Radzioch, D. & Varesio, L. (1988) Protein kinase C inhibitors block the

Ways, D. K., Riddle, R., Ways, M. & Cook, P. (1991) Effect of phorbolactivation of macrophages by IFNβ but not by IFNγ, J. Immunol.esters on cytosolic protein kinase C content and activity in the human140, 125921263.

Radzioch, D. & Varesio, L. (1991) c-fos mRNA expression in macro- monoblastoid U937 cell,J. Biol. Chem. 206, 125821264.phages is downregulated by interferon-γ at the posttranscriptional Ways, D. K., Cook, P., Webster, C. & Parker, P. (1992a) Effect of phor-level,Mol. Cell. Biol. 11, 271822722. bol esters on protein kinase C-zeta,J. Biol. Chem. 267, 479924805.

Skamene, E., Gros, P., Forget, A., Kongshavn, P., St-Charles, C. & Tay-Ways, D. K., Messer, B. R., Garris, T. O., Qin, W., Cook, P. P. & Parker,lor, B. A. (1982) Genetic regulation of resistance to intracellular pa- P. (1992b) Modulation of protein kinase C-epsilon by phorbol estersthogens,Nature 297, 5062509. in the monoblastoid U937 cell,Cancer Res. 52, 560425609.

Stach, J. L., Gros, P., Forget, A. & Skamene, E. (1984) Phenotypic ex- Yasuda, I., Kishimoto, A., Tanaka, S., Tominaga, M., Sakurai, A. &pression of genetically controlled natural resistance byMycobacte- Nishizuka, Y. (1990) A synthetic peptide substrate for selective assayrium bovis(BCG), J. Immunol. 132, 8882892.

of protein kinase C,Biochim. Biophys. Res. Commun. 166, 12202Stokes, R. W., Orme, I. M. & Collins, F. M. (1986) Role of mononuclear1227.phagocytes in expression of resistance and susceptibility toMyco-

bacterium aviuminfections in mice,Infect. Immun. 54, 8112819.