©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Bacterial Lipopeptides Induce Nitric Oxide Synthase and Promote Apoptosis through Nitric Oxide-independent Pathways in Rat Macrophages (*)

(Received for publication, July 13, 1994; and in revised form, January 16, 1995)

Fulvia Terenzi (1) María J. M. Díaz-Guerra (1) Marta Casado (1) Sonsoles Hortelano (1) Silvia Leoni (2) Lisardo Boscá (1)(§)

From the  (1)Instituto de Bioquímica (CSIC-UCM), Facultad de Farmacia, Universidad Complutense, 28040 Madrid, Spain and the (2)Dipartimento di Biologia Cellulare e dello Sviluppo, Universitá ``La Sapienza,'' 00185 Roma, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Stimulation of resident peritoneal macrophages with S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysSerLys(4) or S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysAlaLys(4), two synthetic bacterial lipopeptides, promoted the expression of the inducible form of nitric oxide synthase, exhibiting a temporal pattern of nitric oxide release that was delayed with respect to the induction elicited by bacterial lipopolysaccharide. Treatment of macrophages with genistein blocked the nitric oxide synthesis triggered by the lipopeptides or lipopolysaccharide. Simultaneous incubation with lipopolysaccharide and lipopeptide resulted in an antagonistic effect on nitric oxide synthase mRNA levels and on nitrite plus nitrate release to the medium.

Triggering with bacterial lipopeptides induced macrophage programmed cell death. In macrophages activated with lipopeptide, apoptosis was observed even in the absence of nitric oxide synthesis, therefore indicating the existence of alternative pathways in the control of programmed cell death in these cells.


INTRODUCTION

The effect of lipopolysaccharide (LPS) (^1)by itself or in synergism with other cytokines on the induction of NOS in several tissues is now well documented(1, 2, 3, 4) . In M, IFN- and TNF-alpha synergize with LPS in the release of NO which is involved in the antimicrobial action of activated M(4) . However, in addition to LPS, other components of the bacterial cell wall such as membrane lipoproteins are able to modulate immunological responses in M and B and T lymphocytes, although their mechanism of action is less known(5, 6) . Regarding bacterial lipoproteins, previous work showed that their biological activity can also be reproduced by synthetic lipopeptides (TPP) that mimic the fatty acid esterification of these molecules(7) . Indeed, these lipopeptides have some technical advantages for the use in in vivo experiments since they do not produce necrosis upon injection and lack toxic and pyrogenic effects(7) . The activation elicited by these synthetic lipopeptides in M involves an early tyrosine phosphorylation of substrates and uses an upstream signaling pathway partially different from that triggered via LPS(8) .

Activation of M usually leads to programmed cell death and the release of NO by itself has been reported as one of the signals that mediates apoptosis at least in peritoneal M(9) . However, it is possible that in addition to this molecule other reactive intermediates or cytokines produced in the course of M activation may be involved in the apoptotic process characteristic of activated cells(9, 10) . Here we show that triggering of resident peritoneal rat M with TPP promotes NO synthesis with a kinetics different from that elicited by LPS, and the presence of both bacterial products results in a significant blockage of NOS expression and NO release. Moreover, in TPP-activated cells apoptosis may be obtained in the absence of NO synthesis, suggesting the existence of alternative pathways in cell death induction by TPP.


MATERIALS AND METHODS

Chemicals

Metabolites and biochemicals were from Sigma. Materials and chemicals for electrophoresis were from Bio-Rad. TPP and S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysAlaLys(4) (TPP-Ala) were from Boehringer (Mannheim, Germany) and were tested for endotoxin content that was below 0.1 ng/mg using the Limulus polyphemus test (Sigma). Serum and media were from Biowhittaker (Walkersville, MD).

Preparation of Macrophages

Peritoneal resident M were prepared from male rats following a previous protocol(11) . Briefly, after light ether anesthesia the animals were killed by cervical dislocation and injected intraperitoneally with 50 ml of sterile RPMI 1640 medium (at 37 °C), and after 10 min of gentle distribution of the medium in the peritoneal cavity, the ascitic liquid was carefully aspirated to avoid hemorrhage and kept at 4 °C to prevent the adhesion of the macrophages to the plastic. After centrifugation at 200 times g for 10 min at 4 °C, the cell pellet was washed once with ice-cold PBS. Cells were seeded at 1 times 10^6/cm^2 in RPMI 1640 medium supplemented with 10% of heat inactivated fetal calf serum. After incubation for 1 h, the non-adherent cells were removed by extensive washing with ice-cold PBS. Except when otherwise stated, experiments were carried out in phenol-red free RPMI 1640 medium supplemented with 0.5 mM arginine and 10% of heat inactivated fetal calf serum. When arginine-free medium was used, the fetal calf serum was treated for 30 min at 37 °C with 1 unit/ml of arginase.

Determination of NO and H(2)O(2)

NO release was determined spectrophotometrically by the accumulation of nitrite and nitrate in the medium (phenol red-free) as follows: 250 µl of culture medium were transferred to 1.5-ml Eppendorf tubes, and the nitrate was reduced to nitrite with 0.5 units of nitrate reductase (Boehringer) in the presence of 50 µM NADPH, 5 µM FAD(12) . The excess of NADPH, which interferes with the chemical determination of nitrite, was oxidized in the presence of 0.2 mM pyruvate and 1 µg of lactate dehydrogenase. Nitrite was determined with Griess reagent (11) by adding 1 mM sulfanilic acid and 100 mM HCl (final concentration). After incubation for 5 min, the tubes were centrifuged and 200 µl of supernatant were transferred to a 96-well microtiter plate. After a first reading of the absorbance at 595 nm, 50 µl of naphthylenediamine (1 mM in the assay) were added. The reaction was completed after 15 min of incubation, and the absorbance at 595 nm was compared with a standard of NaNO(2). The H(2)O(2) secretion by cultured macrophages was measured after triggering with PDBu (13) as follows: cells attached to coverslips were incubated per triplicate with the indicated effectors (IFN-, TNF-alpha, and in the presence or absence of TPP) for 24 h. After two washes with PBS and one with 0.9% NaCl, the coverslips were placed in a fluorescence cuvette and triggered for 1 h with 100 ng/ml of PDBu. The release of H(2)O(2) was measured fluorometrically in the supernatant following the oxidation of scopoletin at 460 nm and in the presence of horseradish peroxidase that yields a non-fluorescent product when excited at 350 nm(14) .

RNA Extraction and Analysis

Total RNA (3-4 times 10^6 cells) was extracted following the guanidinium thiocyanate method(15) . After electrophoresis in a 0.9% agarose gel containing 2% formaldehyde, the RNA was transferred to a Nytran membrane (NY 13-N; Schleicher & Schuell, Germany) with 10 times SSC (10 times SSC is 1.5 M NaCl, 0.3 M sodium citrate, pH 7.4). The membrane was prehybridized, and the level of iNOS mRNA was determined using an EcoRI-HindII fragment from the iNOS cDNA(16) , labeled (45% of efficiency) with [alpha-P]dCTP using the Random Primed labeling kit (Boehringer). The membranes were washed with 0.1 times SSC and 0.1% SDS at room temperature for 10 min and twice at 50 °C for 30 min, followed by exposure to x-ray film (Kodak X-OMAT). Quantification of the films was performed by laser densitometry (Molecular Dynamics), using the hybridization with a beta-actin probe (0.6-kilobase EcoRI/HindIII fragment isolated from a VC 18 vector) as an internal standard.

Western Blot Analysis of iNOS

M cell layers (2 times 10^6) were rinsed twice with PBS and homogenized in 500 µl of boiling 250 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, and 2% beta-mercaptoethanol and after centrifugation in a microcentrifuge for 5 min, samples were briefly sonicated and centrifuged. Proteins were size separated in 8% SDS-polyacrylamide gel electrophoresis. The gels were processed as recommended by the iNOS mAb supplier (Transduction Laboratories), and after blotting onto a polyvinylidene difluoride membrane (Millipore) iNOS was revealed following the ECL technique (Amersham, Bucks, U.K.).

Analysis of DNA Fragmentation

Internucleosomal DNA fragmentation was analyzed by agarose gel electrophoresis. The cell layers (1-2 times 10^6) were washed twice with ice-cold PBS and filled with 0.5 ml of 20 mM EDTA, 0.5% Triton X-100, 5 mM Tris-HCl, pH 8.0. After incubation for 15 min at 4 °C, the nuclei were removed by centrifugation at 500 times g for 10 min, and the resulting supernatant was centrifuged at 30,000 times g for 15 min. The fragmented DNA present in the soluble fraction was precipitated with 70% ethanol plus 2 mM MgSO(4), and aliquots were treated for 1 h at 55 °C with 0.3 mg/ml of proteinase K. After two extractions with phenol/chloroform, the DNA was resuspended and analyzed in a 2% agarose gel after staining with 0.5 µg/ml of ethidium bromide. Alternatively, samples were analyzed using an enzyme-linked immunosorbent assay cell death kit (Boehringer) in which the histone-associated DNA fragments of mono- and oligonucleosomes in the cytosol were detected using a sandwich-enzyme-immunoassay with anti-histone and anti-DNA-peroxidase antibodies. The quantification of the relative degree of apoptosis was determined by measuring the peroxidase activity at 405 nm and subtracting the contribution of cells at the initial time of culture (endogenous apoptosis). The average A of samples from control cells was 0.06 O.D. units.


RESULTS

TPP Promotes the Release of NO and H(2)O(2)

Two synthetic TPP derivatives (TPP and TPP-Ala) were used to determine the ability of cultured resident peritoneal M to release NO to the medium. As Fig. 1A shows, both lipopeptides promoted NO synthesis as reflected by the presence of nitrate and nitrite in the incubation medium. This M activation process exhibited a lag period of 6 h followed by a continuous release up to 48 h. The NO synthesis elicited by the lipopeptides was delayed when compared to that induced by LPS (Fig. 1A), suggesting that these bacterial products use partially different pathways in their mechanism of NOS induction. Additionally, the effect of LPS on NO synthesis resulted quantitatively higher (35% over the effect of TPP at 24 h) when compared to that elicited by the lipopeptides. Since both LPS and TPP have been reported to involve an increased tyrosine phosphorylation following cell triggering with these bacterial products, we determined the effect of genistein, a protein tyrosine kinase inhibitor, on the release of NO by M stimulated with LPS or TPP. As shown in Fig. 1A (bars) the release of nitrate and nitrites was completely blocked when cells were incubated in the presence of 100 µM of genistein, data that are in agreement with previous work(8) . To further characterize the inhibitory effect of genistein, M were stimulated either with TPP or LPS, followed by the addition of genistein at various times after priming. As Fig. 1B shows, when genistein was added during the 2 h following M priming, a complete blockage in the NO synthesis was observed. However, after this initial critical period, genistein resulted less effective in inhibiting TPP- than LPS-dependent NO release. The sensitivity to genistein was similar for TPP and LPS (Fig. 1B, inset). Fig. 2shows a very similar dose-dependent curve for both lipopeptides in nitrite and nitrate release, exhibiting a half-maximal effect at 0.8 µg/ml (nitrite concentration). In the following experiments only TPP was used.


Figure 1: Bacterial lipopeptides induce NO synthesis in M. Cultured resident peritoneal M (3 times 10^5) were incubated with 5 µg/ml of TPP (bullet), 5 µg/ml of TPP-Ala (), or 5 µg/ml of LPS (circle), and at the indicated times the concentration of nitrite plus nitrate released to the medium was measured. Alternatively, cells were incubated for 5 min with 100 µM of genistein prior to stimulation with 5 µg/ml of TPP (solid bars) or 5 µg/ml of LPS (open bars), and the nitrite and nitrate concentration were measured (panel A). When genistein was added at different times after stimulation with TPP (bullet) or LPS (circle) the NO synthesis was measured after 24 h of stimulation (panel B). The dose-dependent curve for the inhibition of NO release by genistein (added 5 min prior to M triggering) was measured after 24 h of stimulation with TPP or LPS (inset). Results show the mean ± S.E. of three experiments.




Figure 2: Nitrite and nitrate release in M activated with lipopeptides. M were incubated for 36 h with TPP (bullet) or TPP-Ala (), as described in Fig. 1. The release of nitrate (dotted line) and nitrite (continuous line) were measured. Results show the mean ± S.E. of three experiments.



Since the amount of nitrate released represented 27% of the nitrite concentration, these results suggested a moderate oxidation of nitrites in the course of TPP stimulation. Indeed, in addition to NO, TPP also promoted the release of H(2)O(2) when stimulated M cultures were triggered for 1 h with PDBu(13, 14) . This H(2)O(2) production was near additive to the effect elicited by TNF-alpha and IFN- (Fig. 3). These results prompted us to study the role of these cytokines on the TPP-dependent NO release. As Table 1shows, incubation of M with IFN-, TNF-alpha, or with both molecules inhibited the NO release induced by TPP. Opposite to this situation, a clear synergism between IFN- and TNF-alpha was observed in LPS stimulated M, suggesting that TPP and LPS use different pathways in their mechanism of NOS induction. However, an unexpected antagonism between TPP and LPS in NO release was observed.


Figure 3: TPP promotes PDBu-dependent H(2)O(2) release in M. Coverslip-attached cells were stimulated for 24 h with 2 ng/ml of IFN- or TNF-alpha in the absence (open bars) or presence (dashed bars) of 5 µg/ml of TPP. After this incubation period, the cell layers were extensively washed and triggered with 100 ng/ml of PDBu for 1 h to release H(2)O(2) that was determined fluorometrically by the oxidation of scopoletin in the presence of horseradish peroxidase. Results show the mean ± S.E. for triplicates from one of three independent experiments.





TPP and LPS Antagonize in iNOS Expression

The induction of iNOS mRNA in M stimulated with TPP and LPS was analyzed to establish the relative levels of message in each condition and to characterize the inhibitory effect of the simultaneous addition of both bacterial products at the messenger level. As Fig. 4shows, TPP and LPS increased the iNOS mRNA content (the 4.5 kilobase band) at 4 and 18 h, although to a different extent. However, when both TPP and LPS were simultaneously added, an important decrease in the mRNA levels (68% of the LPS-specific response at 18 h) was observed, in agreement with the inhibition of NO release in these conditions (Table 1).


Figure 4: TPP and LPS antagonize in promoting iNOS expression. M were incubated for the indicated periods of time in the absence (lane 1) or with 5 µg/ml of TPP (lanes 2 and 4) and 1 µg/ml of LPS (lanes 3 and 4), respectively. RNA was extracted and analyzed by Northern blot using a specific probe for iNOS. After normalization for beta-actin content, the relative amount of iNOS mRNA is shown in arbitrary units (a.u.). The figure shows 1 representative experiment out of three.



TPP Promotes Apoptosis Through NO-independent Mechanisms

NO by itself induces apoptosis in cultured M incubated with NO donors or cytokines that involve NO synthesis(9, 17) . Because TPP promoted both NO release and morphological changes associated with apoptosis, we tested to find out whether this cell death was exclusively due to NO or to other molecules released in the course of M activation. As Fig. 5A shows, TPP promoted DNA laddering even in the presence of NOS substrate analogue inhibitors, such as NMA. In this condition, the amount of nitrite and nitrate released to the medium was lower than in control unstimulated cells. To determine the effect of NO on the TPP-induced apoptosis, the nitrite plus nitrate concentration was measured at the time when the apoptotic process was quantified following the release to the cytosol of DNA containing nucleosomal complexes. As Fig. 5, B and C show, in the presence of NMA the NO synthesis remained unchanged (even lower than in unstimulated cells); however, a TPP-dependent apoptosis was observed. Addition of a chemical NO donor such as 3-morpholinosydnonimine (50 µM in the culture medium) to control M incubated with NMA triggered a rapid apoptotic process, indicating that NMA by itself did not interfere with the apoptotic response (Fig. 5C). To ascertain that TPP induces apoptosis in the complete absence of NO synthesis, M were incubated with arginine-free medium and with serum that was previously treated with arginase (1 unit/ml), and compared with the response in the presence of arginine. As Fig. 6shows, in this experimental model apoptosis prevailed in the absence of NO synthesis (lanes 1 and 2), confirming the existence of a NO-independent pathway involved in apoptosis induction in cultured rat M. To ensure that NOS was expressed in response to TPP regardless of the presence of arginine in the incubation medium, a Western blot of the cytosolic proteins of these cells was performed using an iNOS antibody. When cells were simultaneously stimulated with TPP and LPS, apoptosis prevailed and a decrease in iNOS protein (130 kDa) was evident (lane 5). Taking advantage of this arginine-free model we determined the degree of apoptosis induced by TPP in the complete absence of NO synthesis. As Fig. 7shows, in the above mentioned conditions TPP promoted M apoptosis exhibiting a half-maximal effect at 0.3 µg/ml, a lower concentration than required to produce the half-maximal NO synthesis (0.8 µg/ml; Fig. 2).


Figure 5: TPP promotes apoptosis in the absence of NO synthesis. M (2 times 10^6) were incubated for 36 h in the absence (lanes 1 and 3) or presence of 5 µg/ml of TPP (lanes 2 and 4) and 1 mM NMA (lanes 3 and 4). The DNA laddering and nitrite plus nitrate concentration in the medium were assayed (panel A). The time course of NO release in the absence (circle, bullet) or presence of TPP (box, ) (panel B), and the relative content in apoptotic cells (panel C) were determined. Filled symbols correspond to assays in the presence of 1 mM NMA. To have a positive control of NO-dependent apoptosis, cells were incubated with 50 µM of 3-morpholinosydnonimine (), a NO donor (panel C). An A value of 0.06 O.D. units corresponding to control cells was subtracted from each sample. Results show 1 representative experiment out of three.




Figure 6: TPP induces apoptosis in arginine-free medium and promotes iNOS expression. M were incubated for 36 h in arginine-free medium (lanes 1 and 2) or in its presence (lanes 3-5) and were stimulated with 5 µg/ml of TPP (lanes 2 and 4), or TPP plus 1 µg/ml of LPS (lane 5). The extent of apoptosis (open bars) or NO release (dashed bars) were measured. An A value of 0.05 O.D. units corresponding to control cells was subtracted from each sample. At the time of sampling, cells were homogenized and analyzed by Western blot using an iNOS mAb.




Figure 7: Dose dependence curve of apoptosis in M stimulated with TPP in arginine-free medium. M were incubated for 36 h with the indicated concentrations of TPP and the extent of apoptosis (circle) and NO release (bullet) were measured. Results show a representative experiment out of three cell preparations.




DISCUSSION

Several bacterial cell wall products, among which are LPS, lipoproteins, murein, and membrane proteins, share in common the ability to stimulate various types of immune cells(18) . However, since the chemical structure of these molecules exhibits great differences between them, it is conceivable that the signaling pathways activated after cell triggering with bacterial products might show a certain degree of specificity. For example, lipopeptides specifically activate G(i) proteins in human neutrophils(19) . Additionally, tyrosine phosphorylation of MAP kinases 1 and 2 has been reported after TPP activation of murine M from LPS-responsive and -nonresponsive strains; however, for LPS MAP activation is observed only in LPS-responsive strains, and the lack of activation in the LPS-nonresponsive counterparts has been situated at a post-receptor step but prior to MAP kinase activation(8) . As a result of M activation with cytokines and bacterial products, these cells release various secretory molecules (10) exhibiting a high chemical activity (NO, H(2)O(2), and O(2)). Regarding NO release by TPP-activated M, it has been shown that tyrosine kinase inhibitors, such as genistein, effectively cancelled NO production, revealing the necessity of tyrosine phosphorylation in the pathway that involves NOS expression, and a similar conclusion applies for LPS(20) . However, except for a prolonged sensitivity to genistein when added at various times after LPS triggering, no significant differences have been observed between LPS and TPP regarding the involvement of protein tyrosine kinase activation following M activation.

The results reported in this work add new information regarding cell activation and apoptotic death induction of M by bacterial lipopeptides, which seem to exhibit some specific characteristics when compared with the effects elicited by LPS. The release of NO after TPP stimulation is delayed and quantitatively lower with respect to the response elicited after LPS challenge. This is opposite to the temporal pattern of MAP kinase phosphorylation that, at least in murine M, is more rapidly activated by lipopeptides(8) . Moreover, simultaneous treatment of the cells with both TPP and LPS results in a blockage of the response, as reflected by the decrease in iNOS mRNA levels and protein, and NO release to the medium, which suggests that some signals are released in the course of the dual stimulation resulting in a partially antagonistic response. This result was unexpected since cooperation would be observed in view of the use of some common transduction pathways (i.e. early protein tyrosine kinase stimulation and MAP kinase activation).

In rat M TPP in addition to NO synthesis promotes the release of H(2)O(2), clearly observed after triggering with phorbol esters, and therefore contributing to cell activation with various oxygen reactive intermediates; however, the study of the modulation by IFN- and TNF-alpha of the TPP response revealed that these cytokines antagonize the production of NO (a behavior opposite to their cooperative action in LPS stimulated macrophages, refs. 13, 14), but result additive (and therefore independent) with TPP in the production of H(2)O(2).

An additional difference between TPP and LPS in M activation concerns the commitment for apoptosis after stimulation. In the absence of NO synthesis LPS fails to trigger an effective apoptosis(9, 17) , whereas under these conditions TPP retains the ability to induce cell death of activated M, which suggests that an alternative NO-independent apoptotic pathway is operative in TPP-stimulated cells. Our results show that when NO is produced, the relative amount of DNA fragmentation is moderately increased (30%), suggesting that NO is not the main apoptotic inducer and that a cooperation exists between pathways that trigger apoptosis. However, it remains to be determined whether NO may influence the fraction of the cell population exhibiting DNA cleavage.

Regarding the mechanism of action of TPP in promoting apoptosis, several possibilities could be envisaged. The contribution of NO has been attributed either to a blockage in the energetic metabolism (via aconitase inhibition) or to a direct alteration in the DNA structure as result of nucleotide deamination directly due to NO, and both cases have been discussed previously(9, 17) . In addition to these mechanisms, there is a possible occurrence in TPP-activated M of an enhanced susceptibility to H(2)O(2) or other oxidant species released in the course of the activation. An example of this situation has been reported in retrovirus infected T cells that exhibit an extreme sensitivity to H(2)O(2) for apoptosis(21) . Indeed, the occurrence of physiological pathways leading to apoptosis of antigen-presenting M following CD4 T cell activation has been proposed to be genetically programmed into the repertoire of M functions(22, 23) .

Finally, and in keeping with the view that M are programmed for cell death upon activation(23) , it is possible that the apoptosis observed in TPP-activated M lies on the release of several reactive oxygen intermediates (NO, H(2)O(2), O(2)) which may trigger the expression of genes or modulate the activity of transcriptional factors such as NF-kB or AP-1 that would be responsible for this process(24) .


FOOTNOTES

*
This work was supported by Grant PM92-070 from CICYT, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Instituto de Bioquímica, Facultad de Farmacia, 28040 Madrid, Spain.

(^1)
The abbreviations used are: LPS, lipopolysaccharide; TPP, S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysSerLys(4); TPP-Ala, S-[2,3-bis(pamitoyloxy)-(2R,2S)-propyl]-N-palmytoyl-(R)-CysAlaLys(4); NMA, N^G-methyl-L-arginine; M, macrophage; NOS, nitric oxide synthase; IFN-, interferon ; TNF-alpha, tumor necrosis factor alpha; PBS, phosphate-buffered saline; PDBu, phorbol 12,13-dibutyrate; MAP, mitogen-activated protein.


ACKNOWLEDGEMENTS

We thank Dr. Q.-W Xie and Dr. C. F. Nathan for the iNOS cDNA probe, O. G. Bodelón for technical assistance and E. Lundin for help in the preparation of the manuscript.


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