Immunology 1990 70 332-337

L-arginine-dependent nitric oxide formation and nitrite releasein bone marrow-derived macrophages stimulated with bacterial

lipopeptide and lipopolysaccharide

S. HAUSCHILDT, E. BASSENGE,* W. BESSLER, R. BUSSE* & A. MULSCH* Institutfuir Immunobiologie and* Institut fir Angewandte Physiologie, Freiburg, FRG

Acceptedfor publication 21 March 1990

SUMMARY

This study shows that stimulating bone marrow-derived macrophages with either lipopolysaccharide(LPS) or the lipopeptide N-palmitoyl-S-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-(R)-cysteinyl-alanyl-glycine (Pam3Cys-Ala-Gly), a synthetic analogue of the N-terminal part of bacterial lipoprotein,leads to the formation of nitric oxide (NO) and nitrite (NO2r), a stable analogue of NO. NO was

detected by applying the chemiluminescence method and by measuring the activity of exogenouslyadded soluble guanylate cyclase (GC), which is strongly and selectively activated by NO. Synthesis ofNO and NO- occurs via activation of the L-arginine and NADPH-dependent enzyme(s) present inthe cytosol of bone marrow-derived macrophages. NO produced by this non-constitutive L-argininepathway is thought to be responsible for the cytostatic and killing properties ofmacrophages (Stuehr& Nathan, 1989). Macrophages stimulated either with LPS or Pam3Cys-Ala-Gly exhibited a 6-hr lagtime before engaging in nitrite synthesis, a time at which expression of the NO-forming enzyme hadalready reached its maximum. The regulation of NO and N02 synthesis during macrophagedevelopment seems to differ from that of cytokine synthesis. Whereas cytokine release varies during a

culture period up to 20 days, NO synthesis and expression of the NO-forming enzyme remainunaltered. These studies show that, similar to LPS, Pam3Cys-Ala-Gly is a potent activator of 'theoxidative L-arginine pathway' in bone marrow-derived macrophages. Whether both stimuli use thesame signal transfer mechanism to induce this pathway and whether NO synthesized by this pathwayis involved in the activation of the enzyme guanylate cyclase in macrophages requires clarification.

INTRODUCTION

Bacterial lipopeptides, analogues of the N-terminal part ofbacterial lipoprotein (Bessler et al., 1977), and lipopolysacchar-ides (LPS) are potent activators of macrophages (Adams &Hamilton, 1984; Hoffmann et al., 1989, 1988; Hauschildt et al.,1990). Once activated by these agents, macrophages producecytotoxic and cytostatic compounds such as cytokines (tumournecrosis factor-alpha, interleukin- 1) and reactive oxygen species(superoxide anion, hydrogen peroxide) (Hoffmann et al., 1989,1988; Hauschildt et al., 1990; Nathan, 1982; Urban et al., 1986;Onozaki et al., 1985). Only recently has nitric oxide (NO) beenidentified as a mediator of the L-arginine-dependent cytostasisand respiratory inhibition in tumour target cells (Stuehr &Nathan, 1989; Marietta et al., 1988; Hibbs et al., 1988). NO isformed from a guanido nitrogen of L-arginine by a NADPH-dependent enzyme system present in the cytosol of stimulatedmacrophages, the 'oxidative L-arginine pathway' (Marletta etal., 1988). So far, the factors regulating its expression and

Correspondence: Dr S. Hauschildt, Institut fur Immunobiologie,Stefan-Meier-Str. 8, D-7800 Freiburg, FRG.

activity are unknown. This study was carried out to analyse theeffect of the synthetic lipopeptide Pam3Cys-Ala-Gly and of LPSon the expression of the 'oxidative L-arginine pathway' in bonemarrow-derived macrophages. The formation of nitrite, onestable metabolite of nitric oxide, was measured in the culturesupernatant from macrophages grown for different periods oftime and stimulated with lipopeptide or LPS in differentconcentrations. Simultaneously, the expression of the enzyme(s)involved in NO formation in the cytosol of homogenizedmacrophages was measured. NO formation was detected bychemiluminescence and by determining the activity of anexogenously supplied purified soluble guanylate cyclase (GC)co-incubated with the cytosol. Soluble GC is a physiologicaltarget of NO (Murad, 1989) and is potently and selectivelyactivated by NO from biological or chemical sources. Thisapproach was used recently to detect NO formation in cytosolfrom endothelial cells and brain (Milsch et al., 1989).

MATERIALS AND METHODSMaterialsThe synthetic lipopeptide analogue N-palmitoyl-S-(2,3-bis(palmitoyloxy) - (2RS) - propyl) - (R) - cysteinyl - alanyl-

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Nitric oxide and nitrite

glycine(Pam3Cys-Ala-Gly) was prepared by chemical synthesis(Prass et al., 1987). NG-nitro-L-arginine, fl-NADPH and L-arginine were purchased from Serva (Heidelberg), and Escheri-chia coli lipopolysaccharide (LPS; E. coli serotype 055: B5),sulphanilamide and N- I -naphthyl-ethylendiamine were pur-chased from Sigma (Miinchen). NO/nitrogen mixture (1 p.p.m.V NO) and ultrapure nitrogen gas (99-99995 v%) was obtainedfrom Messer (Griesheim). All other chemicals were obtained asdescribed elsewhere (Miilsch et al., 1989; Milsch, Bohme &Busse, 1987).

Bone marrow-derived macrophage cultureCells obtained by flushing the femur of male BALB/c mice (bredin our institute) (age 8-12 weeks) were cultured in teflon bags inthe presence of L-cell-conditioned medium, as described in detailelsewhere (Hoffmann et al., 1989).

Preparation of culture supernatantBone marrow-derived macrophages taken from cultures on Day7 (unless otherwise indicated) were washed twice in RPMI- 1640(phenol red-free/5% fetal calf serum, FCS). The macrophageswere suspended in 2 ml RPMI-1640 at a density of 0 5 x 107/mlin 6-well culture plates (Falcon 3046, Becton-Dickinson, Heidel-berg) and incubated at 370 in 5% CO2 either in the presence orabsence of Pam3Cys-Ala-Gly or LPS. After 24 hr of incubation(unless otherwise indicated), the supernatants were collected,centrifuged and either analysed directly or stored at -70.

Preparation of cytosolMacrophages were scraped off the plastic dishes with a rubberpoliceman and kept suspended in ice-cold RPMI-1640 (Gibco,Eggenstein) or modified Tyrode's solution (composition: 10 mMHEPES, 137 mm NaCl, 2 7 mm KCI, 1-8 mm CaCl2 x 2 H20, 05mM MgCl2 x 6 H20, 0-36 mm NaH2PO4 x H20, 5 mm glucose;pH 7 5 adjusted with 0 1 N NaOH). Cells were washed twice(lOGOg, 5 min, 40) in 15 mm HEPES pH 7 5 (adjusted with 0 1 NNaOH) and homogenized by sonication (three times 10 seconds,100 Watt). The cytosolic fraction was then prepared bycentrifugation (1 hr 100,000 g supernatant). Aliquots werestored at - 30°. Protein was determined according to Bradford(1976).

Preparation of soluble guanylate cyclaseSoluble guanylate cyclase (GC) was purified to apparenthomogeneity from bovine lung, as described elsewhere (Miilsch& Gerzer, 1990). The purified enzyme was activated up to 30-fold by sodium nitroprusside (01 mM), which releases NOspontaneously (Feelish & Noack, 1987; Wright et al., 1989).

Detection ofNO by activation ofguanylate cyclaseCytosol (0 03-1 mg protein/ml) was incubated for various times(10-30 min) at 370 with or without L-arginine (1 mM) andNADPH (0-1 mM) and the complete test mixture for formationof [32P]cGMP from [1-32P]GTP, in a total volume of 50 ,l. Themixture contained purified GC (1 yig/ml) and 0 1 mM [a-32P]GTP(0 2 ,uCi), 0 1 mm cGMP, 2 mm glutathione, 15 mm HEPES, pH7-5, 4 mM MgCl2, 1 mm 3-isobutyl-1-methylxanthine, 3.5 mMcreatine phosphate, 4 8 units creatine phosphokinase, and 0-1mg/ml bovine gamma-globulin. The enzymatic formation ofcGMP was stopped by the addition of 450 ,l zinc acetate (120mM) and 500 ,l sodium carbonate (120 mM). [32P]cGMP was

isolated by chromatography on acid alumina, and GC activity(nmol cGMP/min/mg purified GC) was calculated as describedelsewhere (Schultz & B6hme, 1984). The endogenous GCactivity present in the macrophage cytosol contributed less than1% to the total cGMP formation in the presence of purified GCand was therefore neglectable. The cytosol-enhanced GCactivity was calculated from the difference of the GC activity inthe presence or absence of cytosol.

Determination ofNO by chemiluminescenceNO was quantified in cytosolic incubates by chemiluminescence(Wright et al., 1989). Cytosol was incubated for 30 min in thebuffer used for the determination of GC activity (final volume0-2 ml), but without GC and [32P]GTP. Incubations wereperformed in a thermostated (370) plastic vial (1 ml volume)sealed with a rubber septum. After 30 min the gas-phase fromthe headspace above the septum was aspired with a gas-tightlockable syringe perforating the septum. The gas sample wasinjected through a septum directly into the gas line feeding achemiluminescence detector (Sievers, Boulder, CO), which wascontinuously flushed with ultrapure nitrogen (99 99995 v%).Two cooling traps chilled in a sodium chloride/ice mixture and adrying trap were placed between the injection port and thedetector. Calibration was performed by direct injection ofdefined volumes of NO/nitrogen gas mixture (1 p.p.m. V NO)into the carrier gas line.

Determination of nitriteNO- was quantified in the cell culture supernatant by diazo-tation (Green et al., 1982) and absorbance reading at 570 nm.The sensitivity of this method allowed the detection of + 01 JIMNO- in 0 28 ml sample volume on 96-well microtitre plates(Falcon 3072, Becton-Dickinson, Heidelberg) with a microplatereader (MR 600, Dynatech, Alexandria, VA). NO- values werecorrected for background levels of media determined in eachcase. Absence of interference by media and buffers was ascer-tained with aqueous standard NO- solutions.

RESULTS

L-arginine- and NADPH-dependent activation of purified solubleGC by macrophage-derived cytosol

Cytosolic fractions (0-5 mg/ml) isolated from macrophagesstimulated either with LPS (01 jIg/ml) or Pam3Cys-Ala-Gly(10 jyg/ml) enhanced basal GC activity 3 + 1 2-fold (n = 3 each)in the absence of L-arginine and NADPH. A 1-3+0 2-fold(n = 3) increase was observed when cytosol from unstimulatedmacrophages was used. Values of basal GC activity amountedto 20 + 10 nmol cGMP/min/mg GC. Addition of L-arginine andNADPH to cytosolic supernatants from stimulated macro-phages led to a marked increase up to 60-fold in guanylatecyclase activity; the EC50 values for L-arginine (0 1 mm NADPHpresent) and forNADPH (1 mm L-arginine present) being 30 yMand 1 ,UM, respectively (data not shown). Addition of L-arginineand NADPH to supernatants of unstimulated macrophagesresulted in a 2 6-fold increase in guanylate cyclase activity.

Nature of the GC activating factor

Similar to the GC activating factor identified in the cytosol ofendothelial cells and of the cerebellum (Miilsch et al., 1989), the

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Figure 1. Time-course ofcGMP formation in the presence and absenceof cytosol. Cytosol (0-3 mg protein/ml) was isolated from 7-day-oldbone marrow-derived macrophages that had been incubated for 24 hr inthe presence of 0 1 pg/ml LPS. Purified soluble GC was incubated forvarious times in the presence (0) or absence (0) of cytosol (1 mM L-

arginine and 0-1 mm NADPH present). Formation of [32P]cGMP fromxa-32P]GTP was measured and expressed as pmol cGMP formed per mgsoluble GC. The experiment represents one of three determinationsperformed in triplicate. Values are the mean + SEM.

c 2000

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Figure 2. Effect of cytosolic protein concentration on activation of GC.Seven-day-old bone marrow-derived macrophages were incubated for24 hr in the presence of various concentrations of LPS [0-1 ng/ml (K), 1

ng/ml (0), 0 1 pg/ml (0), 10 jg/ml (A)], and 50 pg/ml Pam3Cys-Ala-Gly(-). Different concentrations of cytosolic preparations were assayed forGC activation (1 mM L-arginine, 0-1 mM NADPH present). GC activityis expressed as a percentage of SNP (100 pM)-stimulated activity in theabsence of cytosol. Values are the mean + SEM of two determinationsperformed in triplicate.

GC activating factor exhibited the biochemical features of NOgenerated by the oxidative L-arginine pathway. Activation was

stereospecific for L-arginine (data not shown). Activation was

strictly dependent on NADPH; replacement by NAD (0 1 mM)or NADH (01 mM) had no effect on GC activity. There was

some activation seen in the presence ofNADP (30 + 13% of thatofNADPH) (n = 3), which might have been due to reduction ofNADP to NADPH during the incubation. NO accumulated inthe headspace of a sealed incubation vial containing cytosol (0 2mg) from LPS (0 1 pg/ml)-stimulated macrophages and the GCassay mixture without GC and [(X-2P]GTP. After 30 min 10 + 2pmol NO were detected by the chemiluminescence method(n=3).

Time-course of GC activation

Figure 1 shows the progress ofcGMP formation by soluble GCin the absence or presence ofcytosol (0-3 mg protein/ml) isolatedfrom LPS (01 pg/ml)-stimulated macrophages. Compared tocGMP formation (nmol cGMP/mg GC) in the absence ofcytosol, addition of cytosol led to a 10-fold increase in activity.The increase occurred without any lag phase and was linear upto 30 min. Formation of cGMP was also found to be linear intime using different protein concentrations or cytosolic prep-

arations from macrophages stimulated with different concentra-tions of LPS (0 01-10 pg/ml) or Pam3Cys-Ala-Gly (10 pg/ml)(data not shown).

Concentration dependence of soluble GC activation by cytosolicprotein

As shown in Fig. 2, cytosol-induced GC activation (expressed as

a percentage of GC activity induced by 01 mm sodiumnitroprusside, SNP) was directly related to cytosolic proteinconcentrations. Comparing the dose-response curves obtainedwith cytosol from macrophages stimulated for 24 hr withdifferent concentrations of LPS (0-1 ng- I0 pg) and lipopeptide

_ 100

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100 Oj

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Figure 3. Dose-response curve of NO- release and GC activation.Seven-day-old bone marrow-derived macrophages were incubated for24 hr in the presence of various concentrations of Pam3Cys-Ala-Gly(circles) or LPS (triangles). Supernatants were assayed for NO2formation (closed symbols). GC activity (open symbols) was measuredin the presence of cytosol isolated from stimulated and unstimulatedcells. GC activity and NO- release are expressed as a percentage ofmaximum response obtained after stimulation with 0 1 pg LPS/ml or 10

pg Pam3Cys-Ala-Gly/ml. The results are means of duplicates from one

of three similar experiments. Standard errors between duplicates were

less than 2% of the mean.

(50 pg) showed that stimulating macrophages with higher LPSconcentrations (1 ng-10 pg) and lipopeptide enhanced thecytosol capacity to induce GC activity, i.e. less protein was

needed to obtain maximal GC activation. Interestingly, GCactivity measured in the presence of 100 gM SNP increased even

further upon addition of cytosol. In cytosol-free incubates, 100pM SNP was reported to induce maximal activation (Gerzer,Hofmann & Schultz, 1981). It is possible that NO formed by the'oxidative L-arginine pathway' in the cytosol reaches highersteady state concentrations than NO derived from the sponta-neous decay of SNP (Feelish & Noack, 1987).

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Nitric oxide and nitrite

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Figure 4. Time-course of NOj release and expression of GC activationby macrophage cytosol. Seven-day-old bone marrow-derived macro-

phages (7 x l07/14 ml) were incubated for different periods of time in theabsence (squares) or presence of 10 pug/ml Pam3Cys-Ala-Gly (circles)and of 01 I ug/ml LPS (triangles). Supernatants were assayed for NO2formation (closed symbols), and GC activity (open symbols) was

measured in the presence of cytosol induced from stimulated andunstimulated cells. The results are means ofduplicates from one of threesimilar experiments. Standard errors between triplicates were less than2% of the mean.

Dose-response curve of LPS- and Pam3Cys-Ala-Gly-inducedexpression of the oxidative L-arginine pathway

Seven-day-old bone marrow-derived macrophages were incu-bated for 24 hr in the absence or presence of graded concentra-tions of Pam3Cys-Ala-Gly and LPS (Fig. 3). Addition ofcytosolic supernatants (0-2 mg/ml) isolated from these cells topurified GC caused an increase in activity which depended on

the dose of stimulus added. Maximum activity was reachedwhen stimulation was carried out with 01 HIg LPS/ml (335 + 53 8nmol cGMP/min/mg GC) or 10 jag Pam3Cys-Ala-Gly/ml(310 + 78 nmol cGMP/min/mg GC). Control values (cytosolfrom unstimulated cells) were 33 6+7-9 nmol cGMP/min/mgGC. Similar to GC activation, the release of NO- into theculture supernatant was dependent on the concentration of thestimulus added. Values rose from 1-7 + 0-6 jM (controls) up to175 + 2-8 jiM (LPS; 0 1 jIg/ml) and 25 5 +1 jIM (Pam3Cys-Ala-Gly; 10 jig/ml). The standardized dose-response curves ofNO2release and GC activation (indicating the cytosolic NO synthe-sis) were nearly identical for each particular stimulus, but variedbetween the two stimuli tested.

Time-course of expression of the oxidative L-arginine pathway

Seven-day-old bone marrow-derived macrophages were incu-bated for 0, 2, 6, 10, 16 and 24 hr in the presence or absence ofLPS (0 1 jIg/ml) or Pam3Cys-Ala-Gly (10 jig/ml), before culturesupernatants and cytosolic preparations were assayed for NO2formation and GC activation, respectively. As shown in Fig. 4,both stimuli led to a considerable increase in expression ofNOsynthesis (indicated by cytosol induced GC activity) after 6 hr ofincubation, and after 10 hr the maximum was reached. Incontrast, NO- release did not reach its maximum until 24 hr ofincubation, and at incubation times shorter than 10 hr there washardly any NO- detectable.

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07 9 13 20

Days of cultureFigure 5. Influence of macrophage development on expression of theoxidative L-arginine pathway and NO- release. Bone marrow-derivedmacrophages cultured for different periods of time were harvested andincubated for an additional 24 hr in the absence (control) (0) or presence

of 1O pg/ml Pam3Cys-Ala-Gly (-) or 01 I jg/ml LPS (U). Supernatantswere assayed for NOy formation and GC activity was measured in thepresence of cytosol. The results are the mean + SEM from one of threeexperiments performed in triplicate.

Dependency of the expression of the oxidative L-arginine pathwayon developmental stages of the macrophages

Bone marrow-derived macrophages were cultured for 7, 9, 13and 20 days. Subsequently, they were incubated for 24 hr in thepresence or absence of LPS (0 1 jIg/ml) or Pam3Cys-Ala-Gly (10jug/ml). Culture supernatants were tested for NO- formation,and expression of NO synthesis (indicated by cytosol-inducedGC activation) was assayed in the cytosol. Stimulation witheither stimulus led to a significant increase in NO- release andcytosol-stimulated GC activation on all days tested (Fig. 5). Theduration of the culture period affected neither the extent ofNO2release nor the extent of GC activation significantly. However,after 20 days of culture, there was a slight decrease in cytosol-induced GC activation, which was not reflected by a decrease inNO- release.

Inhibition of the oxidative L-arginine pathway by NG-nitro-L-arginine

NG-nitro-L-arginine is a competitive inhibitor of L-arginine atthe site of NO synthesis in several mammalian cells expressingthe oxidative L-arginine pathway, whereas its D-stereoisomer isinactive (Milsch et al., 1989; Mulsch & Busse, 1989). Experi-ments were carried out to determine whether this compound was

able to inhibit NO- formation induced by LPS and Pam3Cys-Ala-Gly. NG-nitro-L-arginine (1 mM) or NG-nitro-D-arginine (1mM) were added to macrophages (1 x 107/2 ml) which were

stimulated for 24 hr with LPS (0-1 jIg/ml) or Pam3Cys-Ala-Gly(10 jig/ml). In the presence of NG-nitro-L-arginine LPS- andPam3Cys-Ala-Gly- induced NOj release was reduced by about80% (LPS: 32-1 + 4 jiM; LPS+ NG-nitro-L-arginine: 4-1 + 0 5 jiM;

Pam3Cys-Ala-Gly: 21-3 +23 ,uM; Pam3Cys-Ala-Gly + NG-nitro-L-arginine: 6-0+0 6 jiM). NG-nitro-D-arginine had no effect(data not shown).

335

336 S. Hauschildt et al.

DISCUSSION

The data presented here show that the synthetic lipopeptidePam3Cys-Ala-Gly and LPS induce the formation of NO andNO- in bone marrow-derived macrophages. These studiesconfirm the finding of Stuehr & Marletta (1985) who showedthat mouse peritoneal macrophages produce NO- and NO- inresponse to LPS. Through experiments with cell cultures andinbred strains of mice that had defects in T and B cells, theyidentified macrophages as being the main source of NO3-formation. When these authors observed an increase in NOj3formation in mice that had been treated with bacillus Calmette-Guerin they concluded that endogenous lymphokines were alsopowerful modulators of macrophage NO-/NO- synthesis(Stuehr & Marletta, 1987). Subsequent work revealed thatIFN-y and TNF-i// or IFN-a/f/y and LPS can interactsynergistically to induce NO- release (Ding, Nathan & Stuehr,1988).

As reported by Marletta et al. (1988), NO is an intermediatein the pathway of L-arginine to NO- and NO- and, inaccordance with their data, the present study found that NOformation is catalysed by a cytosolic enzyme which shows anabsolute requirement for L-arginine and NADPH. The methodused to detect NO formation was based on the fact that NO is apotent activator of soluble GC (Murad, 1989). Having a shorthalf-life in the range of seconds (Palmer, Ferrige & Moncada,1987), NO does not accumulate in incubates. This is reflected inthe linear time-course ofcGMP formation by guanylate cyclasein the presence of cytosol. NO is rapidly scavenged bysuperoxide anions (Blough & Zafiriou, 1985) and protected bysuperoxide dismutase (Palmer et al., 1987). The fact thatcytosolic GC activation is inhibited by the generator ofsuperoxide, LY 83583 (Mulsch et al., 1988), and is enhanced bysuperoxide dismutase (Mulsch et al., 1989) indicates thatactivation is highly specific for NO. Furthermore, directevidence was obtained for the synthesis of NO in macrophagecytosol by the chemiluminescence method (Marletta et al., 1988;Wright et al., 1989; Palmer et al., 1987). According to thefollowing criteria the activator was identified as a product of theoxidative L-arginine pathway: activation required the presenceof the L-form of arginine and depended solely on NADPH as co-factor. The L-arginine analogue NG-nitro-L-arginine, a potentcompetitive inhibitor ofcytosol-induced GC activation (Miulsch& Busse, 1989), but not the D-analogue, inhibited NO- releasefrom intact cells, indicating the stereospecificity of inhibition.Furthermore, NO- release showed a similar dependency on theconcentration of the stimulus used as cytosol-enhanced GCactivation.

The concentration of lipopeptide required to induce maxi-mal NO and NO- formation exceeds that of LPS (Fig. 3). Thisdifference in concentration requirement can vary greatly,depending on the biological response examined. Measuringcytotoxicity against L929 cells, for example, shows that lessPam3Cys-Ala-Gly than LPS is needed to produce the sameresponse (Hoffmann et al., 1989). The dose-response curves ofNO formation and NO- release obtained after immunostimula-tion for 24 hr are almost identical for each stimulus tested. Thiscorrelation between NO- and NO production suggests that theyare co-products of the same reaction. As has been shown byMarletta et al. (1988), NO- and NO- were derived from theoxidation of one of the two chemically equivalent guanido

nitrogens. The NO3 formed was not derived from NO- butboth end-products were partitioned from a common interme-diate, NO.

The progress of NO- release and the expression of theenzyme catalysing the formation of NO from L-arginine differsignificantly (Fig. 4). Therefore, the extent of the nitriteformation at a given time cannot serve as a measure of theexpression of NO forming enzyme(s). The actual expression ofthis pathway can be assessed either by determining NO directlyor by measuring cytosol-induced GC activation in the presenceof saturating concentrations of L-arginine and NADPH. Weshowed that 6 hr after stimulation maximal expression of the L-arginine pathway was achieved. In accordance with the presentdata, Stuehr & Marletta (1987) reported that the onset of NO-/NO- release started with a lag phase of about 4-12 hr afterimmunostimulation. Unlike in endothelial and neuronal cells(Palmer et al., 1987; Garthwaite, Charles & Chess-Williams,1988), a short-term modulation of NO synthesis has not beenobserved in macrophages. The present data and reports fromother groups suggest that the L-arginine pathway must beinduced in macrophages and is not constitutive.

The duration of the culture period does not influence thecapacity of the macrophages to produce NO/NOR to anysignificant extent (Fig. 5). This is in contrast to the finding thatsecretion of cytokines strongly depends on the developmentalstage of the macrophages. It is shown that IL-6 and TNF-asecretion increased from Day 6 to Day 20 whereas IL- I secretionincreased until Day 13 and decreased on Day 20 (Hauschildt etal., 1990). Thus, it seems that expression of cytokines isregulated separately from the production of NO/NOR duringdistinct differentiation phases of the culture period examined.NO has been proposed to be the final effector molecule

causing specific patterns of metabolic inhibition in tumourtarget cells (Hibbs et al., 1988; Stuehr & Nathan, 1989). Themetabolic changes include inhibition of mitochondrial respi-ration, inhibition of the citric acid cycle enzyme aconitase andinhibition ofDNA replication. Not only in macrophages, whichare specialized in host defence, but also in endothelial and braincells the L-arginine/NO pathway is expressed (Garthwaite et al.,1988; Knowles et al., 1989; Palmer et al., 1988). NO formed byendothelial cells acts as a vasodilator via stimulation of solubleGC and consequent elevation of cGMP (Murad, 1989). Thestimulation of GC by NO has also been shown to occur in thecentral nervous system (Knowles et al., 1989). It is very likelythat NO synthesized in macrophages not only displays cellkilling properties. Since it is known that cyclic nucleotides areinvolved in several macrophage functions, and that macro-phages treated with NO generating agents show a large increasein cGMP levels (Bromberg & Pick, 1980), NO might also serveas a paracrine and/or autocrine signalling agent by stimulatingsoluble GC. This potential signal transduction pathway may beof importance in the regulation of the immune system.

ACKNOWLEDGMENTSWe gratefully acknowledge the skilful technical assistance of MrsChristel Herzog and Mrs Philippi-Schulz. This work was supported bythe Deutsche Forschungsgemeinschaft (Bu 436/4-1, Be 859/6-1).

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