Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2001;69:590-597.)
© 2001 by Society for Leukocyte Biology

Induction of soluble antitumoral mediators by synthetic analogues of bacterial lipoprotein in bone marrow-derived macrophages from LPS-responder and -nonresponder mice

Silke D. C. Pfannes*, Bernd Müller{dagger}, Stephan Körner{ddagger}, Wolfgang G. Bessler* and Petra Hoffmann*

* Institut für Molekulare Medizin und Zellforschung, AG Tumorimmunologie und Vakzineforschung, and
{dagger} AG Biophysik und Strahlenbiologie, Medizinische Fakultät der Universität Freiburg, 79104 Freiburg, and
{ddagger} Jomol Pharma GmbH, Regensburg, Germany

Correspondence: Silke Pfannes, AG Tumorimmunologie/Vakzine, Institut für Molekulare Medizin und Zellforschung, Stefan-Meier-Strasse 8, D-79104 Freiburg i.Br., Germany. E-mail: pfannes{at}uni-freiburg.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Macrophage-dependent antitumoral activity is partly mediated by soluble factors including cytokines, reactive-oxygen intermediates (ROIs), and reactive-nitrogen intermediates (RNIs). Activation of macrophages for tumor cytotoxicity can be achieved with various bacterial compounds, such as lipopolysaccharides (LPSs), muramyl-dipeptides, and lipopeptides. We studied the production and release of oxygen radicals, nitric oxide, and tumor necrosis factor {alpha} (TNF-{alpha}) by bone marrow-derived macrophages (BMDMs) of different mouse inbred strains after they were stimulated with the lipopeptide P3CSK4, a water-soluble synthetic analogue of the lipidated N terminus of bacterial lipoprotein. The lipopeptide was able to induce a strong, long lasting release of oxygen radicals in BALB/c mouse macrophages. Furthermore, it induced nitric oxide release from BMDMs of several mouse strains (BALB/c, C57Bl/6, C57Bl/10ScSn, Sv129, NMRI, and LPS-nonresponder C57Bl/10ScCr). Stimulation with P3CSK4 also resulted in comparable production of TNF-{alpha} in LPS-responder and nonresponder BMDMs from C57Bl/10ScSn mice and C57Bl/10ScCr mice, respectively. All three antitumoral mediators reached functional levels or concentrations as shown by the strong cytostatic/cytotoxic activity of lipopeptide-activated macrophages for the cell lines Abelson 8-1, M12.5/P815, and L929, which are sensitive to ROIs, nitric oxide, and TNF-{alpha}, respectively. We found that synthetic lipopeptides can induce the secretion of effective levels of soluble tumor-cytotoxic/cytostatic mediators in BMDMs of LPS-responsive and, of particular interest, also of LPS-unresponsive mice. This result could indicate that the highly effective bacterial-macrophage activators P3CSK4 and LPS use different receptors and/or different intracellular signal transduction pathways.

Key Words: nitric oxide • oxidative burst • tumor necrosis factor-{alpha} • lipopeptide


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Activated macrophages are of major importance for host defense against microbial infections and neoplasms [1 , 2 ]. Macrophage-dependent destruction of tumor cells is mediated by a number of different effector mechanisms, including soluble bioactive molecules with cytostatic/cytotoxic activity [3 ]. Among these are cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}) and interleukin (IL) 1ß [4 , 5 ], as well as reactive oxygen intermediates and reactive nitrogen intermediates (ROIs and RNIs, respectively), e.g., superoxide, hydrogen peroxide, and nitric oxide [6 ]. Bacterial compounds such as lipopolysaccharides (LPSs) and muramyl-dipeptides (MDPs) are potent activators of macrophages and have been investigated extensively as inducers of antitumoral activities [7 , 8 ]. We and others [9 10 11 ] recently have shown a similar immunostimulating potential for lipopeptides and lipoproteins from the outer cell walls of gram-negative bacteria. Thus, we could also show that synthetic lipopeptides, which represent analogues of the lipidated N-termini of native lipoproteins, stimulate macrophages for the enhanced production of cytokines such as TNF-{alpha}, IL-1, and IL-6 and the release of nitrogen radicals [12 , 13 ]. Furthermore, we could demonstrate that lipopeptide-stimulated macrophages are cytostatic/cytotoxic for tumor cells in vitro [14 ]. Mice with mutations in the LPS gene locus on chromosome 4 are highly resistant to LPS effects [15 , 16 ]. In contrast, no mouse nonresponder strain has been described so far for lipopeptides or lipoproteins [17 ]. Furthermore, lipoproteins and lipopeptides can induce similar mitogenic and immunogenic responses in cells from both LPS-responder and LPS-nonresponder mice [17 18 19 20 21 ]. Here we show that the water-soluble, synthetic lipopeptide N-palmitoyl-S-[2,3-bis(palmitoyloxy)-propyl]-(R)-cysteinyl-(lysyl)3-lysine (P3CSK4) induced the release of oxygen and nitrogen radicals as well as the production of TNF-{alpha} in bone marrow-derived macrophages from various mouse inbred strains. Macrophages from C57Bl/10ScSn (LPS-responder) and C57Bl/10ScCr (LPS-nonresponder) mice were activated to a comparable extent, and this stimulation could be enhanced by simultaneous addition of interferon {gamma} (IFN-{gamma}). Stimulation of ROI, RNI, and TNF-{alpha} production by P3CSK4 reached functional levels, as was shown by strong cytostasis/cytotoxicity of the lipopeptide-activated macrophages on Abelson 8-1, M12.5/P815, and L929 tumor cells, respectively.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
The synthetic lipopeptide P3CSK4 was prepared by chemical synthesis, as described by Metzger et al. [22 ], and was obtained from ECHAZ Microcollections, Tübingen, Germany. Recombinant mouse IFN-{gamma} was purchased from Pharmingen/Becton Dickinson, Hamburg, Germany. LPS from Salmonella abortus equi was a kind gift from C. Galanos, Max-Planck-Institut für Immunbiologie, Freiburg, Germany, and S. abortus equi S1301 kindly donated by B. Kleine, Institut für Molekulare Medizin und Zellforschung, AG Tumorimmunologie und Vakzineforschung, Freiburg, Germany. NG-Monomethyl-L-arginine (L-NMMA) and N-acetyl-L-cysteine (NAC) were purchased from Sigma (Deisenhofen, Germany).

Mice
Female and male strains BALB/c, C57Bl/6, C57Bl/10, and 129 SV mice, 6 to 10 weeks old, were obtained from the breeding facilities at the Max-Planck-Institut für Immunbiologie. For some experiments, strains C57Bl/6 and C57Bl/10 mice were purchased from Bomholtgård, Ry, Denmark.

Tumor cells
The murine fibroblast cell line L929, the murine mastocytoma cell line P815, and the murine B-cell lymphoma cell line M12.4 were kept in RPMI-1640 medium (Gibco BRL, Eggenstein, Germany), supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% nonessential amino acids, 2 mM L-glutamine, 100 U/mL of penicillin, and 100 µg/mL of streptomycin (cRPMI) (all from Seromed Biochrom KG, Berlin, Germany). The murine B-cell lymphoma cell line Abelson 8-1 was kept in Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L of glucose (Seromed Biochrom KG), supplemented with 10% FCS, 2 mM L-glutamine, 1% nonessential amino acids, 100 U/mL of penicillin, and 100 µg/mL of streptomycin (cDMEM).

Murine bone marrow-derived macrophages
Murine bone marrow-derived macrophages (BMDMs) were differentiated in vitro from bone marrow precursor cells as described in detail by Hoffmann et al. [14 ]. Briefly, bone marrow cells were flushed from femurs and tibias of 6- to 10-week-old mice, washed twice in RPMI 1640, and grown for 10 days in liquid cultures in Teflon film bags (SLG, Gauting, Germany) at 37°C and 5% CO2. The culture medium consisted of RPMI 1640 supplemented with 15% L-cell-conditioned medium as a source of macrophage colony-stimulating factor, 10% heat-inactivated FCS, 5% heat-inactivated horse serum, 1 mM sodium pyruvate (both from Seromed Biochrom KG), 50 U/mL of penicillin, 50 µg/mL of streptomycin, and 5 x 10-5 M 2-mercaptoethanol. Cultures were set up with 6 x 106 cells/50 mL. After harvesting, the macrophages were washed once, counted, and resuspended at 2 x 106 cells/mL in test medium. For the preparation of L-cell-conditioned medium, 105 L929 cells/mL were cultured in 100-mL batches in cell culture flasks (Falcon; Becton Dickinson, Heidelberg, Germany) in cRPMI 1640 at 37°C and 5% CO2. After 7 days, the culture supernatants were harvested, cleared from cell debris by centrifugation (1,500 x g, 4°C, 15 min), and stored at -20°C.

Induction and determination of nitric oxide release in murine BMDMs
Mature BMDMs were harvested, washed once, and resuspended in cRPMI medium. Cells (105/well) were seeded into the wells of 96-well flat-bottom microtiter plates (Falcon) and stimulated with various concentrations of P3CSK4 or LPS in the presence or absence of the costimulus IFN-{gamma} or the nitric oxide synthase (NOS) inhibitor L-NMMA in a total volume of 150 µL. Culture supernatants were harvested after 42 h (and for the kinetics studies also after 19 and 72 h). All assays were performed in triplicate. Production of nitric oxide was determined by measuring nitrite, a stable metabolite of nitric oxide, in the culture supernatants using the Griess reaction [6 ]: One hundred microliters of culture supernatant were mixed with 100 µL of Griess reagent [1% sulfanilamide and 0.1% N-(1-naphthyl)ethylendiamine in 2.5% phosphoric acid], and the absorbance at 550 nm was monitored with a Dynatech MRX enzyme-linked immunosorbent assay plate reader (Denkendorf, Germany). Nitrite concentrations were calculated by using sodium nitrite as a standard.

Macrophage-mediated growth inhibition of Abelson 8-1 tumor cells
Macrophage-mediated tumor-cytostatic/cytotoxic activity against Abelson 8-1 tumor cells was determined by applying the alkaline phosphatase assay, as described by Modolell et al. [23 ]. Briefly, cocultures of 5 x 103 Abelson 8-1 tumor cells together with 1 x 105 BMDMs were set up in cDMEM in flat-bottom microtiter plates in a total volume of 200 µL and incubated in the presence of various stimuli at 37°C and 10% CO2. After 3 days, the plates were centrifuged at 660 x g for 2 min, and the supernatants were decanted. To each well, 100 µL of buffer (pH 10.2) containing diethanolamine (200 mM), MgCl2 (2 mM), Triton X-100 (1%), and p-nitrophenylphosphate (10 mM) were added, and the plates were incubated for 60 min at room temperature in the dark on a horizontal shaker. The enzyme reaction was stopped by adding 100 µL/well of 0.5 M NaOH. Absorbance was measured at 405 and 490 nm in an automated enzyme-linked immunosorbent assay reader (MRX Dynatech, Denkendorf, Germany). Optical density values of cultures containing tumor cells and unstimulated effecter cells were set to 100%.

Macrophage-mediated cytostasis of P815 and M12.4 tumor cells
Target cells were seeded into flat-bottom microtiter plates (104/100 µL/well in cRPMI) and incubated at 37°C and 5% CO2 for 24 h. Mature BMDMs were harvested the following day, cell density was adjusted to 2 x 106/mL, and 50 µL were added to each well. The effecter cells were stimulated with either P3CSK4 or LPS from S. abortus equi S1301 in a final volume of 200 µL for 20 h. Then cells were pulsed for 4 h by the addition of 23.125 kBq [3H]TdR/well (925 kBq/mL; Amersham, Braunschweig, Germany). After freezing and thawing, labeled culture DNA was transferred to glass fiber filters with an automatic cell harvester (LKB1295-001; Pharmacia Upjohn, Freiburg, Germany). The incorporated radioactivity was measured in a liquid scintillation counter (LKB Betaplate 1205; Pharmacia Upjohn). Cytostasis was expressed as percentage of inhibition of [3H]TdR incorporation compared with control cultures (cocultures of unstimulated BMDMs and tumor cells).

Macrophage-mediated tumor cell cytotoxicity
The killing of labeled tumor cells was measured by a [3H]thymidine ([3H]TdR) release assay. Briefly, tumor cells were cultured in cRPMI and were labeled with 13.875 kBq of [3H]TdR/mL for 20 h at 37°C and 5% CO2. Four hours before use, the cells were washed twice with RPMI 1640 and maintained in cRPMI without radioactivity. The assays were performed in flat-bottom microtiter plates (Becton Dickinson) at an effector-to-target cell ratio of 10:1. BMDMs (105/100 µL) were added to the wells and stimulated with either P3CSK4 or LPS (from Escherichia coli strain O55:B5 [Sigma]) in a final volume of 200 µL for 2 h. Thereafter, the macrophages were washed twice, and 104 labeled tumor cells were added to each well in a final volume of 200 µL. The cocultures were incubated for 48 h at 37°C and 5% CO2. At the end of the coculture period, the radioactivity released into the supernatant was determined in an aliquot of 100 µL/well by liquid scintillation counting. Results are expressed as the percentages of specific lysis, as calculated by the following formula: percent specific lysis = ([cpmexp - cpmspont]/[cpmtotal - cpmspont]) x 100. Spontaneous release (cpmspont) was determined in microwells containing only labeled tumor cells. Total release (cpmtotal) was determined by lysis of the tumor cells with 0.5% sodium dodecyl sulfate.

Detection of ROIs by chemiluminescence
BMDMs were suspended in BM 86 medium (Boehringer Mannheim, Mannheim, Germany) supplemented with 2% FCS and placed into chemiluminescence (CL) tubes (5 x 105 BMDMs/100 µL). Four hundred microliters of Hanks’ balanced saline solution without phenol red (Seromed Biochrom KG) containing the stimulus were added, and the macrophages were stimulated for different periods at 37°C. The CL reaction was started by the addition of 10 µL of lucigenin (final concentration, 0.1 mM; Sigma), and the baseline was recorded for 5 min at 37°C. Thereafter, macrophages were incubated with a known inducer of CL (40 µL/tube of zymosan; final concentration, 500 µg/mL; Sigma), and the CL response was measured with an LB 9505 C luminometer (Berthold, Wildbad, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
P3CSK4 induces nitric oxide release in BMDMs from different mouse inbred strains
In a first set of experiments, we examined the release of nitric oxide from BMDMs after stimulation with either P3CSK4 or LPS. BMDMs from four different mouse inbred strains (BALB/c, C57Bl/6, C57Bl/10, and 129Sv) were differentiated in vitro from their bone marrow precursors under defined culture conditions. Mature macrophages were stimulated with 0.01–10 µg/mL of P3CSK4; LPS was added at 1 µg/mL, a concentration that proved to be optimal in earlier studies. Culture supernatants were examined for nitrite contents after 42 h. As shown in Figure 1 , P3CSK4 induced nitric oxide release in BALB/c BMDMs in a dose-dependent manner, resulting in a maximum nitrite concentration of 48 µM, as compared with 32 µM induced by LPS. Macrophages from C57Bl/6 and C57Bl/10ScSn mice reacted in a similar way, whereas the reactivity of BMDMs from 129Sv mice to P3CSK4 was less pronounced, yielding a maximum nitrite concentration of 19 µM, as compared with 24 µM after optimal LPS stimulation. The arginine analogue L-NMMA, a competitive substrate inducible-NOS (iNOS) activity, caused a dose-dependent inhibition of nitric oxide secretion in both BALB/c and C57Bl/10 macrophages (Fig. 2 ), indicating that the P3CSK4-induced nitric oxide release in BMDMs is dependent on NOS-mediated arginine metabolism.



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  		Figure 1. Dose-dependent induction of NO release by the lipopeptide P3CSK4. Murine BMDMs from the four different inbred mouse strains C57BI/6 (a), C57BI/10ScSn (b), BALB/c (c), and 129Sv (d) were seeded into the wells of 96-well microtiter plates and stimulated with P3CSK4 or LPS. NO release was determined by measuring the nitrite concentration in the culture supernatants using the Griess reaction. Values represent means ± SD of triplicate cultures.

 


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  		Figure 2. Inhibition of iNOS activity by L-NMMA. Macrophages from BALB/c, C57BI/10ScSn, or C57BI/10ScCr mice were stimulated with P3CSK4 without or with the iNOS inhibitor L-NMMA or in medium alone (control). Nitrite concentrations in the culture supernatants were determined using the Griess reaction. Values represent means ± SD of triplicate cultures.

 
Nitric oxide- and TNF-{alpha}-mediated tumor cytotoxicity of BMDMs stimulated with P3CSK4
We investigated the contribution of iNOS products to macrophage-mediated tumor cytotoxicity. The cytolytic activity of P3CSK4- or LPS-stimulated BALB/c BMDMs against the nitric oxide-sensitive tumor cell lines M12.4 and P815 was analyzed by a [3H]TdR release assay. Macrophages (105/well) were stimulated with either P3CSK4 or LPS from E. coli strain O55:B5 at a concentration of 50 µg/mL for 2 h. After repeated washing of the effector cells, the cocultures were started by the addition of 104 labeled target cells. Forty-eight hours later, [3H]TdR released into the culture supernatants was measured by liquid scintillation counting. As shown in Table 1 , stimulation of BALB/c BMDMs with P3CSK4 or LPS resulted in an increase of lysed M12.4 and P815 cells, as compared with control cultures. Cytotoxicity was mainly caused by RNIs, because it was markedly diminished in the presence of 1 mM L-NMMA.


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  		Table 1. Induction of Tumor Cytotoxicity in BALB/c BMDMs by P3CSK4

 
Apart from nitric oxide, there are other soluble mediators of tumor cytostasis and cytotoxicity released by activated macrophages, e.g., TNF-{alpha}. As shown in Table 1 , by means of lysis of the TNF-{alpha}-sensitive tumor cell line L929, P3CSK4 stimulation of BMDMs for only 2 h was sufficient to produce effective quantities of this cytokine, resulting in up to 85% lysis during the subsequent coculture. In contrast, stimulation of macrophages with LPS under the same conditions was less efficient, leading to the lysis of 47% of the tumor cells. Addition of L-NMMA to these cultures resulted in only partial inhibition of the cytotoxic effect, indicating that RNIs were most probably also involved in the process, however not as the main cause of tumor cell death.

Induction of the oxidative burst and of ROI-mediated antitumoral activity in BMDMs by P3CSK4
As demonstrated in the preceding experiments, lipopeptide-stimulated BMDMs are able to produce TNF-{alpha} and RNIs to attack different sensitive target cells. Other members of the family of cytostatic/cytotoxic factors released by activated macrophages are ROIs. We therefore investigated the induction of the oxidative burst in BMDMs by determination of the lucigenin-enhanced CL. For this approach, 5 x 105 BMDMs were stimulated with P3CSK4 or LPS from E. coli O111:B5 at various concentrations for 1 h at 37°C. Recording of the CL reaction was started after the addition of lucigenin and zymosan as the triggering substances. As can be seen in Figure 3a , both P3CSK4 and LPS primed BMDMs for the zymosan-mediated oxidative burst. In comparison to unstimulated BMDMs (i.e., CL activity set to 100%), P3CSK4- as well as LPS-stimulated macrophages showed maximum production of ROIs after an incubation period of 60 min. However, whereas the oxidative burst of LPS-primed BMDMs increased only twofold (200% CL), maximum CL of BMDMs stimulated with 10 and 50 µg/mL of P3CSK4 was over 400% and nearly 800% that of comparable control cells, respectively.



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  		Figure 3. Induction of oxidative burst in BMDMs. (a) Murine BMDMs were stimulated with P3CSK4 or LPS from E. coli O111:B4. Lucigenin was added to the samples, and a baseline of lucigenin-enhanced CL was recorded. The priming effect of P3CSK4 or LPS on the production of ROIs was determined after the addition of zymosan as triggering substance. (b) The time course of ROI production of murine P3CSK4 or LPS from E. coli O111:B4-primed BMDMs after restimulation with P815 tumor cells was recorded. (c) The CL response of cocultured BMDMs was integrated, and enhancement of ROI production was calculated by setting the CL response of cocultured, unstimulated BMDMs (Control) to 100%.

 
In further experiments, we demonstrated the release of ROIs by P3CSK4- and LPS-primed BMDMs after restimulation with P815 tumor cells (Fig. 3b and 3c) . Here again, the reaction of the P3CSK4-stimulated BMDMs was considerably stronger than that of cells stimulated with LPS.

To further substantiate these findings, BALB/c macrophages stimulated with various concentrations of P3CSK4 were cultured together with cells of the murine B-cell lymphoma Abelson 8-1 at an effector-to-target cell ratio of 20:1. Macrophage-mediated tumor cell growth inhibition (TCGI) was determined by measuring the alkaline phosphatase activity of the remaining viable tumor cells after 3 days. As can be seen in Figure 4 , P3CSK4-activated macrophages showed a strong, dose-dependent TCGI, resulting in up to 90% growth reduction at agent concentrations of 10, 1, and 0.1 µg/mL. Macrophages activated with 0.01 µg/mL of P3CSK4 were not able to bring about growth reduction. The activity of P3CSK4 could not be inhibited by the addition of L-NMMA at a concentration leading to complete blocking of nitric oxide release (1 mM) nor by addition of anti-TNF-{alpha} antibodies at appropriate concentrations (data not shown). In contrast, when preincubated for 1 h in medium containing the oxygen radical scavenger NAC, the macrophages showed a markedly reduced TCGI capacity (approximately 50% reduction at an NAC concentration of 83 µM and a P3CSK4 concentration of 0.1 µg/mL). This strongly suggests that P3CSK4 is capable not only of inducing effective quantities of nitric oxide but also of generating ROIs at concentrations sufficient for antitumoral effects.



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  		Figure 4. P3CSK4 induced macrophage-mediated TCGI against Abelson 8-1 tumor cells. For determination of tumor cell growth inhibition, murine BMDMs from BALB/c mice and Abelson 8-1 tumor cells were cocultured for 3 days in flat-bottom microtiter plates in the presence of P3CSK4 either alone or together with NAC. Tumor cell numbers at the end of the incubation time were determined by alkaline phosphatase assay as described in Materials and Methods. The growth of tumor cells in the presence of unstimulated macrophages served as controls (0% inhibition). Values represent means ± SD of triplicate determinations.

 
Induction of nitric oxide release and antitumoral activity in BMDMs from LPS-responder (C57Bl/10ScSn) and LPS-nonresponder (C57Bl/10ScCr) animals by P3CSK4
Cells from C57Bl/10ScSn mice showed a normal reaction to the bacterial stimulus LPS, as can be seen by the strongly enhanced nitric oxide release of their BMDMs after stimulation with 0.01–1 µg/mL of LPS for 42 h (Fig. 5a [insert]). The closely related C57Bl/10ScCr mice bear a mutation in the LPS gene located on chromosome 4. This renders their cells, as shown here for BMDMs, highly refractory to LPS stimulation (Fig. 5b [inset]). In contrast, BMDMs prepared from both C57Bl/10ScSn and the C57Bl/10ScCr animals released comparable quantities of nitric oxide after stimulation with the synthetic lipopeptide P3CSK4 for 42 h. When we determined the time course of this lipopeptide-induced nitric oxide production, we found very similar kinetics with cells from both LPS responder and nonresponder animals. Both showed a strong nitric oxide release during the first 24 h, which gradually declined thereafter until day 3 (Fig. 6 ).



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  		Figure 5. Comparison of NO release induced by P3CSK4 and LPS in BMDMs from LPS-responder C57Bl/10ScSn (A) and -nonresponder C57Bl/10ScCr (B) mice. Murine BMDMs were stimulated with P3CSK4 or LPS (inserts). Nitrite concentrations in the culture supernatants were determined using the Griess reaction. Values represent means ± SD of triplicate cultures.

 


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  		Figure 6. Time course of NO release induced by P3CSK4 and LPS. Murine BMDMs from LPS-responder C57Bl/10ScSn or LPS-nonresponder C57Bl/10ScCr mice were stimulated with P3CSK4 and LPS for the times indicated. Culture supernatants were harvested at the time points indicated and tested for nitrite contents using the Griess reaction. Values represent means ± SD of triplicate cultures.

 
IFN-{gamma} is known to enhance the release of nitric oxide from murine macrophages activated by other stimuli, e.g. LPS or TNF-{alpha}. In the next set of experiments, we therefore examined whether the P3CSK4-induced release of nitric oxide could also be enhanced by IFN-{gamma} in an additive or even synergistic way and, if so, whether LPS responder and nonresponder cells would respond in a similar way. BMDMs from C57Bl/10ScSn and C57Bl/10ScCr mice were stimulated with different concentrations of P3CSK4 (0.01–1 µg/mL) in the absence or presence of 0.5–12.5 U/mL of IFN-{gamma} for 42 h. As can be seen in Figure 7 , all IFN-{gamma} concentrations tested showed a marked synergistic activity especially with 0.1 and 1 µg/mL of P3CSK4. Similarly to LPS responder animals, LPS nonresponder macrophages produced nearly comparable amounts of NO2- after combined stimulation with the lipopeptide and IFN-{gamma}.



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  		Figure 7. Enhancement of the P3CSK4-induced NO release from murine BMDMs by IFN-{gamma}. Murine BMDMs from C57Bl/10ScSn and C57Bl/10ScCr mice were stimulated with P3CSK4 either alone or together with IFN-{gamma}. Nitrite concentrations in supernatants were determined using the Griess reaction. Values represent means ± SD of triplicate cultures.

 
Finally, we compared the P3CSK4-induced tumor cytostatic activity of BMDMs from LPS-responder (C57Bl/6) and LPS-nonresponder (C57Bl/10ScCr) mice (Fig. 8 ). Both types of macrophages were cocultured with L929 or P815 tumor cells in the presence of either 50 µg/mL of P3CSK4 or 50 µg/mL of LPS (from E. coli O55:B5) for 24 h. Proliferating tumor cells were labeled with [3H]TdR during the final 4 h. P3CSK4 induced a strong cytostatic activity against the TNF-{alpha}-sensitive cell line L929 as well as against the nitric oxide-sensitive cell line P815 in BMDMs from both LPS-responder and -nonresponder animals. As expected, only BMDMs from LPS-responder mice showed a comparable cytostatic activity against the two tumor cell lines after stimulation with LPS.



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  		Figure 8. Cytostatic activity of BMDMs against L929 or P815 tumor cells. Tumor cells were incubated in microtiter plates. BMDMs were added to each well and stimulated with P3CSK4 or LPS from S. abortus equi S1301. Proliferating tumor cells were pulsed with [3H]TdR. Cytostasis was expressed as percentage of inhibition of [3H]TdR incorporation into target cell DNA compared to control cultures.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several constituents of the bacterial cell wall including LPS, MDP, and lipoprotein are potent activators of cells of the innate immune system, especially of monocytes and macrophages. As a result, these cells release an array of cytokines and other low-molecular-weight bioactive molecules known to be involved in antimicrobial and antitumoral defense reactions. One potent mediator of tumor cell lysis is the nitrogen radical nitric oxide [24 ]. In macrophages, nitric oxide is produced by the cytosolic enzyme iNOS (type II NOS) during the oxidative conversion of L-arginine to L-citrulline [25 ]. We have previously shown that the lipopeptides P3CAG and P3CSK4, two synthetic lipopeptide analogs of the N-terminal part of bacterial lipoprotein, induce nitric oxide formation in BALB/c macrophages in a time- and dose-dependent manner [13 , 26 ]. Here we extended our earlier data by showing that nanomolar concentrations of the lipopeptide P3CSK4 could induce iNOS enzyme activity and the release of nitric oxide not only in BMDMs from BALB/c mice but also to a comparable extent in macrophages from four other mouse inbred strains, including the LPS-responder strain C57Bl/10ScSn and the congenic LPS-nonresponder strain C57Bl/10ScCr.

Animals of the C57Bl/10ScCr strain, like those of the LPS-nonresponder strain C3H/HeJ, bear a mutation in the LPS gene on chromosome 4 [15 ]. However, whereas the C3H/HeJ allele (Lpsd) is codominant, that of C57Bl/10ScCr animals is strictly recessive [15 ]. In addition, the two strains differ in their IFN-{gamma} response to microbial stimuli; whereas C3H/HeJ (as well as C57Bl10/ScSn) splenocytes readily produce IFN-{gamma} after stimulation with various bacteria, C57Bl/10ScCr mice are unable to exhibit such an IFN-{gamma} response [27 ]. Yaegashi et al. [28 ] showed that this is due to the inability of the respective macrophages to produce IFN-ß after microbial stimulation and thereby provide the necessary help for the IFN-{gamma}-producing T and natural killer cells. On the other hand, Munder et al. [29 ] reported that murine BMDMs themselves are able to produce large amounts of IFN-{gamma} if stimulated properly (in their case with a combination of IL-12 and IL-18). Especially for the induction of nitric oxide release, IFN-{gamma} is known to be an extremely potent costimulus [30 ], either directly or by inducing TNF-{alpha} [31 ].

Whether murine BMDMs respond to lipopeptide stimulation by releasing IFN-{gamma} is presently under investigation. However, our results in which C57Bl/10ScCr cells showed a slightly lower secretion of nitric oxide after lipopeptide stimulation as compared with macrophages of the congenic C57Bl/10ScSn animals could reflect a less pronounced reaction of the former and in consequence a lower endogenous production of IFN-{gamma} after lipopeptide stimulation. In agreement with this assumption are the results we obtained after adding exogenous IFN-{gamma} to the two cell populations: In C57Bl/10ScCr macrophages, the synergistic effect was much more pronounced and showed a more stringent dose dependency than in C57Bl/10ScSn cells, resulting finally in the release of comparable quantities of nitric oxide from both cell populations.

Cox et al. [32 ] showed that NO must reach a threshold level to be active in tumor cell destruction. The same is likely to be true for other soluble mediators of tumor cytotoxicity, e.g., TNF-{alpha} and oxygen radicals. We previously showed that production of TNF-{alpha} is induced in murine BMDMs after stimulation with synthetic lipopeptides [12 ]. Here we demonstrated for the first time a release of ROIs by lipopeptide-stimulated BMDMs that exceeded by far the oxidative burst induced in these cells by LPS. Furthermore, we showed that all three of the soluble mediators reached functional, i.e., tumoricidal, levels after stimulation of BMDMs with P3CSK4, because we obtained substantial killing or growth arrest of tumor cells sensitive to either TNF-{alpha} (L929), RNIs (P815 and M12.4), or ROIs (Abelson 8-1) after cocultivation with lipopeptide-activated macrophages.

Well-known targets for regulation by ROIs within the cells are several proteins known to be involved in LPS signaling, namely the mitogen-activated protein kinases (MAPKs) extracellular regulated kinases 1 (ERK1) and 2 (ERK2), as well as the nuclear transcription factor NF-{kappa}B [33 , 34 ]. After stimulation of murine macrophages (BMDMs as well as cells of the macrophage cell line RAW 264.7) with the lipopeptide P3CSK4, we could detect both a dose- and time-dependent activation of the MAPKs ERK1 and ERK2, as well as a translocation of the activated NF-{kappa}B [M. R. Müller, S. D. C. Pfannes, M. Ayoub, P. Hoffmann, W. G. Bessler, and K. Mittenbühler, unpublished results]. Hence, intracellular signaling by LPS and lipopeptides seems to follow similar routes as far as these two steps are concerned. Earlier within the signal transduction chain, however, there seem to be differences between LPS- and lipopeptide-driven stimuli: Both molecules bind to cells via CD14, but whereas the presence of this molecule is essential for LPS signaling, it is not for lipopeptides, but only enhances its effect [35, 36; M. R. Müller et al., unpublished results]. Several recent publications identify different members of the Toll receptor family as being responsible for the transmembrane signaling step after binding of LPS to CD14. Kirschning et al. [37 ] showed transfer of LPS responsiveness to otherwise LPS-unresponsive cells by transfection with human Toll-like receptor (TLR)-2. In contrast, Poltorak et al. [38 ] demonstrated that the LPS gene (see above) corresponds to the gene of TLR-4 and that the LPS-nonresponder mice used here (C57Bl/10ScCr) bear a null mutation in this gene. Because we could show a comparable reactivity to lipopeptides for macrophages from both C57Bl/10ScSn LPS-responder and C57Bl/10ScCr LPS-nonresponder animals, which was reflected in a similar release of nitric oxide as well as similar cytostatic and cytotoxic activity against various tumor target cell lines, participation of TLR-4 in lipopeptide signaling seems highly unlikely. Preliminary results indicated that the release of ROIs is also similar in these LPS-responder and -nonresponder macrophages after lipopeptide stimulation [P. Hoffmann et al., unpublished results]. With the same line of evidence, Takeuchi et al. [39 ] demonstrated that macrophage-activating lipopeptide-2-induced cytokine and NO synthesis was mediated by TLR-2. Whether other members of the TLR family also play a role in lipopeptide signaling must be elucidated in future studies.


   ACKNOWLEDGEMENTS
 
This work was supported by a grant from Deutsche Forschungsgemeinschaft and by Fonds der Chemischen Industrie.

The authors appreciate the excellent technical assistance of Angelika Haber and Marianne Eckert.

Received May 30, 2000; revised November 27, 2000; accepted November 29, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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