Protein SV-IV promotes nitric oxide production not associated with apoptosis in murine macrophages

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European Journal of Cell Biology 81, 185 ± 196 (2002, April) ¥ ¹ Urban & Fischer Verlag ¥ Jena http://www.urbanfischer.de/journals/ejcb

185

Protein SV-IV promotes nitric oxide production not associated with apoptosis in murine macrophages Carla Espositoa, Anna Cozzolinob, Raffaele Porta1)b, Loredana Mariniellob, Elisabetta Buomminocd, Francesco Morellicd, Vittoria Metaforacd, Salvatore Metaforacd a b c d

Department of Chemistry, University of Salerno, Salerno/Italy Department of Food Science, University of Naples ∫Federico II™, Naples/Italy CNR International Institute of Genetics and Biophysics, Naples/Italy Area di Ricerca del CNR, Servizio Tecnologie Biomolecolari, Naples/Italy

Received October 2, 2001 Received in revised version December 6, 2001 Accepted January 7, 2002

Protein SV-IV ± NO ± macrophages ± apoptosis ± bcl-2 ± c-myc SV-IV (seminal vesicle protein no. 4) is a potent immunomodulatory and anti-inflammatory secretory protein (Mr 9758) produced in large amounts by the rat seminal vesicle epithelium. Here we show that this protein possesses the ability to upregulate in J774 macrophages the expression of the gene coding for the inducible nitric oxide synthase (iNOS). The increase in NO production consequent on the marked enhancement of iNOS activity was not associated with apoptotic damage of the SV-IV-treated cells. In the same experimental model, however, LPS induced upregulation of iNOS coupled with an increase in NO production and marked apoptotic death. Differences in the ability of SV-IV and LPS to control the life/ death signal balance in target cells via trans-membrane activation of apoptotic (mediated by TNF-a and NO/iNOS system) and anti-apoptotic (mediated by bcl-2, c-myc, etc.) pathways are suggested to be the basis of the apoptotic fate of the experimentally treated cells. In addition, considering the important role played by NO in the process of mammalian reproduction, SV-IV may be involved in the fine tuning of NO concentration in the female genital tract mucosa via an SV-IVmediated control of iNOS gene expression in local macrophages.

Abbreviations. DXM Dexamethasone. ± eNOS Endothelial-type nitric oxide synthase. ± FAB Fast atom bombardment mass spectrometry. ± iNOS Inducible (or macrophage-type) nitric oxide synthase. ± L-NAME Nw-nitroL-arginine methyl ester. ± LPS Lipopolysaccharide. ± MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. ± NF-kB Nuclear factor-kB. ± nNOS Neuronal-type nitric oxide synthase. ± NO Nitric oxide. ± NOS Nitric oxide synthase. ± PAGE Polyacrylamide gel electrophoresis. ± 1)

Prof. Raffaele Porta, Department of Food Science, University of Naples ™Federico II∫, via Universita¡ 100, I-80055 Portici, Naples/Italy, e-mail: [email protected], Fax: ‡ 39 081 775 5116.

PCD Programmed cell death. ± ROMs Reactive oxygen metabolites. ± ROS Reactive oxygen species. ± RT-PCR Reverse transcription-polymerase chain reaction. ± SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ± SV-IV Seminal vesicle protein no. 4. ± TNF-a Tumor necrosis factor alpha. ± TUNEL Terminal deoxynucleotidyl transferasemediated dUTP nick end labeling.

Introduction Lipopolysaccharide (LPS or endotoxin, derived from the cell wall of gram-negative bacteria) is one of the most wellcharacterized macrophage-activating factors. It induces the production of various cytokines and pro- or anti-inflammatory mediators (Hasko¡ et al., 1996). Among the LPS-induced cytokines, TNF-a is a potent inducer of apoptosis and a proinflammatory protein that plays a pivotal role in the pathogenesis of endotoxic shock and other forms of inflammation (Beutler, 1995; Rath and Aggarwal, 1999). In addition, TNF-a was shown to be a key intermediate in the induction of NO synthesis in response to LPS (Szabo¡, 1995). In particular, it has been recently demonstrated that the LPS-induced apoptosis results from two independent mechanisms: first and predominantly, through the autocrine secretion of TNF-a (early apoptotic events), and second, through the production of NO (late phase of apoptosis) (Xaus et al., 2000). NO is a highly reactive, cytotoxic free radical that has been implicated in tissue damage in a variety of pathological conditions (Cartwright et al., 1997). The cytotoxicity resulting from a long-lasting NO generation produces programmed cell death (PCD) or apoptosis (Sarih et al., 1993; Hu and Van Eldik, 1996; Muhl et al., 1996; Sastry and Rao, 2000; Watanabe et al., 2000) of the target cells. NO-mediated PCD is characterized by marked morphological cell modifications, upregulation of p53, activation of caspases, chromatin condensation, DNA laddering, and is associated with alterations in the expression of

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186 C. Esposito, A. Cozzolino et al.

apoptosis-associated proteins belonging to the bcl-2 family (Brune et al., 1998). NO is synthesized from L-arginine by the enzyme NO synthase (NOS). Three prototypical NOS isoforms, neuronal NOS (nNOS or NOS 1), inducible NOS (iNOS or NOS 2), and endothelial NOS (eNOS or NOS 3) have been isolated and extensively characterized (Hattori et al., 1994; Morris and Billiar, 1994). The iNOS is transcriptionally induced by immune activators such as cytokines and LPS in a wide variety of cells, including macrophages, neutrophils, endothelial cells, and vascular smooth muscle cells (Bredt and Snyder, 1994; Cartwright et al., 1997). In particular, the J774 murine macrophages are able to synthesize iNOS and produce NO in response to cytokines and LPS (Stuehr and Marletta, 1987). The protein SV-IV (seminal vesicle protein no. 4, according to its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)) is a basic (pI ˆ 8.9), thermostable, secretory molecule (Mr 9758) synthesized in large amounts by the rat seminal vesicle epithelium under strict androgen transcriptional control (Mansson et al., 1981; Abrescia et al., 1986). Proteins immunologically related to SV-IV are detectable in several rat tissues (uterus, lung, liver, brain, etc.) and in human seminal fluid (Abrescia et al., 1985; Metafora et al., 1987a). The protein SV-IV was purified to homogeneity and extensively characterized (Mansson et al., 1981; Pan and Li, 1982; McDonald et al., 1983; Abrescia et al., 1986; D×Ambrosio et al., 1993), and the gene coding for it was isolated, sequenced, and expressed in E. coli (D−Ambrosio et al., 1993). It has been clearly demonstrated that SV-IV possesses potent non-species-specific anti-inflammatory and immuno-modulatory properties (Galdiero et al., 1989; Metafora et al., 1989a, b; Camussi et al., 1990; Vuotto et al., 1993; Peluso et al., 1994; Romano-Carratelli et al., 1995; Mancuso et al., 1996; Tufano et al., 1996; Ialenti et al., 2001). Moreover, the binding of SV-IV to the plasma membrane of the rat epididymal spermatozoa markedly decreases their strong immunogenicity (Paonessa et al., 1984; Metafora et al., 1987b; Peluso et al., 1994). The antiinflammatory activity of SV-IVobserved either in carrageenantreated animals or in zymosan-induced air-pouch inflammation systems has been plausibly related to its ability to inhibit markedly the enzymatic activity of phospholipase A2 (PLA2), the first enzyme of the arachidonate cascade, and the synthesis and release of prostaglandin E2, both in vitro and in vivo (Metafora et al., 1989a; Camussi et al. 1990; Mancuso et al., 1996; Ialenti et al., 2001; unpublished data). The interference with the macrophage-T cell cooperation seems to be the mechanism at the basis of the SV-IV modulatory effects on the cell-mediated and humoral immune response (Metafora et al., 1989a; Camussi et al., 1990; Vuotto et al., 1993; Peluso et al., 1994; Romano-Carratelli et al., 1995; Tufano et al., 1996). Concerning these immuno-modulatory effects, we have shown in previous studies that SV-IV is both able to inhibit the production of IL-2, IL-1 and other pro-inflammatory cytokines (IFN-g, TNF-a, etc.) in human PBMC activated by either inflammatory stimuli or common mitogens, thus defining the immuno-suppressive effect of SV-IV (Metafora et al., 1989a; Vuotto et al., 1993; unpublished data), and to induce in vitro a marked release of a variety of cytokines (IFN-g, TNF-a, IL-6 and GM-CSF) from human non-activated PBMC as well as from isolated non-activated lymphocytes and monocytes, thus defining an immuno-stimulating effect of this protein (Tufano et al., 1996) in particular experimental conditions. On the basis of the above reported data and by taking in consideration the ability of SV-IV to interfere with the

biological activities of monocyte/macrophages (Galdiero et al., 1989; Metafora et al., 1989a; Camussi et al., 1990; Vuotto et al., 1993; Peluso et al., 1994; Romano-Carratelli et al., 1995; Tufano et al., 1996) and to induce the production of TNF-a (Tufano et al., 1996), a well known apoptosis- and iNOSexpression-inducing agent (Szabo¡, 1995; Kengatharan et al., 1996; Rath and Aggarwal, 1999), we were prompted to investigate the possibility that SV-IV could act on the J774 macrophage cell line as an inducer of apoptosis via TNF-a along the iNOS-expression-control pathway. The data reported in this paper show that the treatment of J774 cells with SV-IV induced, indeed, a marked increase in TNF-a and NO production together with a substantial upregulation of iNOS gene expression that were, surprisingly, not associated with apoptosis. In contrast, LPS induced in the same experimental model higher amounts of TNF-a associated to stronger stimulatory effects on the NO/iNOS system and, as expected, marked apoptosis. Interestingly, specific bioequivalent concentrations of LPS (1 ng/ml) and SV-IV (10 6 M), possessing the same ability to stimulate the NO/iNOS system, were found to be proapoptotic only in the case of LPS. This difference in the ability of SV-IV and LPS to induce PCD in target cells containing similar amounts of NO was ascribed to the ability of these two ligands to control the macrophage life/death signal balance via trans-membrane activation of different apoptotic (mediated by TNF-a and/or NO/iNOS system) or anti-apoptotic (mediated by bcl-2, c-myc, etc.) pathways. Possible implications in the process of mammalian reproduction of the SV-IV ability to promote NO production in macrophages are discussed.

Materials and methods Purification of the protein SV-IV The protein SV-IV was purified to homogeneity from the seminal vesicle secretion of adult rats (Fisher-Wistar strain) according to a previously published technique (Ostrowski et al., 1979). The purity of the protein was evaluated by 15% PAGE in denaturing or nondenaturing conditions, amino acid composition analysis, fingerprint technique, and fast atom bombardment (FAB) mass spectrometry (Ostrowski et al., 1979; Tufano et al., 1996). Neither lipopolysaccharide (LPS) nor tumor necrosis factor (TNF), as determined by specific, highly-sensitive colorimetric (Arvanitidou et al., 1999) and immunoenzymatic assays (Tufano et al., 1996), was detectable in the SV-IV preparations. The concentration of the purified SV-IV was measured by its molar absorption at 276 nm (4100/M cm), calculated on the basis of the tyrosine and phenylalanine residues present in the SV-IV polypeptide chain (Stiuso et al., 1999).

Cell culture In all the experiments reported in this paper we used murine macrophages in spite of the fact that SV-IV was a rat protein, because all the biological properties of this protein studied up to now are non-species specific. The murine monocyte/macrophage cell line J774 (American Type Culture Collection TIB 67) was grown as monolayers in tissue-culture flasks (75 cm2 growth area; Falcon) in Dulbecco×s modified Eagle×s medium supplemented with 10% (v/v) foetal calf serum (Euroclone, UK), 4 mM L-glutamine, 100 units/ml penicillin, and 100 mg/ml streptomycin (standard culture medium). No LPS was detected in foetal calf serum as determined by a specific biological assay (Arvanitidou et al., 1999). Mouse alveolar primary macrophages were prepared according to a previous published procedure (Brunelleschi et al., 1990). The culture conditions for these cells were essentially the same used for the J774 macrophage cell line. Cells were routinely tested for mycoplasma

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contamination. For use (standard protocol), cells were seeded into 12well plates or 10-cm dishes (Falcon) and allowed to adhere for 2 h at 37 8C. After the end of incubation, medium was replaced with fresh medium containing either SV-IV (10 9 ± 10 5 M), or various concentrations of LPS (0.25 ± 100 ng/ml; Difco Laboratories, Milano, Italy) or SVIV and LPS in combination, and the cells were incubated at 37 8C in a humidified atmosphere containing 5% CO2 and 95% air for a further 24 h in almost all the experiments. In the experiments in which nitrites and apoptosis were measured the cell incubation time was extended to 48 h. The cell viability was measured by both trypan blue exclusion test and MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Sigma Aldrich, Milano, Italy). In specific inhibition experiments dexamethasone (DXM; 10 6 M; Sigma) was added to macrophages treated with either LPS or SV-IV.

Nitric oxide measurement The NO produced by the iNOS-catalyzed reaction was evaluated by measuring with the Griess reagent the nitrite amounts released by the macrophages in their culture medium (Green et al., 1982). Following 24 ± 48 h incubation at 37 8C, 400 ml aliquots of culture medium were taken from the plates containing the cell monolayers, mixed with an equal volume of Griess reagent (0.5% sulfanilamide and 0.05% N1naphthylethylenediamine dihydrochloride in 2.5% phosphoric acid) and incubated at room temperature for 10 min. The absorbance of the reaction mixture was read at 570 nm with a spectrophotometer. The amount of nitrites released by the macrophages into the culture medium was expressed as nmol nitrites/5  106 cells/24 ± 48 h, using a sodium nitrite curve as a standard. Control experiments demonstrated that SVIV did not interfere with the Griess reaction.

Evaluation of iNOS activity iNOS activity was determined in crude homogenates of macrophages (J774 or rat alveolar cells). An appropriate number of cells was incubated for 24 h with either standard culture medium or various concentrations of LPS (0.25 ± 100 ng/ml) or SV-IV (10 9 ± 10 5 M) or LPS in combination with SV-IV. After the end of the incubation time, the cells were rinsed three times with ice-cold phosphate-buffered saline (PBS), removed from the culture plates with a cell scraper, collected, and transferred to microcentrifuge tubes. The sedimented cells were lysed by addition of 50 ml of ice-cold hypotonic homogenization buffer (1 mM EDTA, 1 mM EGTA, 25 mM Tris-HCl, pH 7.4). The iNOS activity present in 20 mg of homogenate proteins was evaluated by a NOS Detection Assay Kit (Stratagene, Milano, Italy) (Salter et al., 1991) according to the manufacturer×s instructions. In this assay, [3H]arginine (50 Ci/mmol; Amersham, IL, USA) was used as substrate and the reaction mixture was incubated for 30 min at 37 8C. Two blanks were included in the assay: one was prepared by omitting the homogenate, the other by adding the iNOS inhibitor Nw-nitro-Larginine methyl ester (L-NAME; 1 mM) to the reaction mixture before addition of the homogenate. The iNOS activity was expressed as citrulline pmol/mg protein/min. Control experiments demonstrated that SV-IV did not interfere with the iNOS detection assay.

Western blot analysis The expression of the iNOS protein was evaluated by Western blot. At the end of the various experiments, the treated (24 h treatment) or untreated cells were washed twice with ice-cold PBS, scraped off, collected by centrifugation (1000g, 5 min), resuspended in 50 mM TrisHCl, pH 7.5, and sonicated (30 s) on ice. Forty mg of sonicate proteins per lane were separated by SDS-7.5% PAGE and electroblotted on nitrocellulose. The blotted membrane was first soaked for 1 h in the blocking solution (150 mM NaCl, 5% skim milk, 10 mM Tris-HCl, pH 7.5) and then incubated overnight at 4 8C with the primary antibody to iNOS (rabbit polyclonal antibody to N-terminus of human iNOS; Santa Cruz, Germany). After the end of the incubation time, the blots were incubated for 1 h at room temperature with horseradish peroxidaseconjugated goat anti-rabbit IgG (Biorad) and finally developed by using diaminobenzamine as a substrate. All antibodies were used at a 1 : 1000 final dilution.

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Reverse transcription-polymerase chain reaction (RT-PCR) The messenger RNA, isolated by the mRNA Capture Kit (Roche Diagnostics S.p.A., Milano, Italy) from an appropriate number of macrophages incubated in the standard culture medium for 24 h (time at which PCD was undetectable) in the presence of either 10 6 M SV-IV, or 1 and 10 ng/ml LPS, or 10 6 M SV-IV in combination with either 1 or 10 ng/ml LPS, was transcribed by reverse transcriptase (superscript II, GIBCO-BRL) at 37 8C for 1.5 h according to the manufacturer×s protocol (final volume 20 ml). The cDNA contained in 2 ml of this reaction mixture was amplified in another reaction mixture containing, in a final volume of 25 ml, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 100 ng of both sense and antisense primers for either bcl-2 (sense, 5'-AACACCAGAATCAAGTGTTC-3'; antisense, 5'-TTCCCT TTGGCAGTAAATAG-3'), c-myc (sense, 5'-AACTTACAATCTGCGAGCCA-3'; antisense, 5'-AGCAGCTCGAATTTCTTCCAGATAT3'), p53 (sense, 5'-CCCTTCTCAAAAAACTTACC-3'; antisense, 5'TCATAACAAGCCCTAAAGTC-3') or iNOS (sense, 5'-GTTTCTTG TGGCAGCAGC-3'; antisense, 5'-CCTCGTGGCTTTGGGCTCCT3'), 100 mM deoxynucleoside triphosphate, and 1 U of Taq DNA polymerase (Roche Diagnostics). The reaction was carried out in a DNA thermal cycler (Promega). The PCRs were performed with 35 cycles in the exponential phase of amplification and always started with a 3-min denaturation step at 95 8C. The cycle for bcl-2 and p-53 was 95 8C, 30 s; 55 8C, 1 min; 72 8C, 1 min. The cycle for c-myc was 95 8C , 45 s; 50 8C, 45 s; 72 8C, 1 min. The cycle for iNOS was 95 8C, 45 s; 56 8C, 45 s; 72 8C, 45 s. A final 7 min at 72 8C was used in all cases. The PCR products were analyzed by electrophoresis on a 1.2% agarose gel in Tris-borate-EDTA (TBE) (Sambrook et al., 1989). The identities of the amplification products were confirmed by comparison of their sizes with the sizes expected from the known gene sequences. Coamplification of different cDNA sequences was performed by adding into the amplification reaction mixture the b-actin gene primers (10 ng of both sense and antisense; sense, 5'-CGTGGGCCGCCCTAGGCACCA-3'; antisense 5'-TTGGCCTTAGGGTTCAGGGGGG-3'). No products were detectable in control amplifications performed in the absence of cDNA or using as template non- reverse-transcribed mRNA.

TNF-a assay The amount of TNF-a occurring in the supernatants of J774 macrophages treated with the different stimuli for 24 h was evaluated by an immunoenzymatic method. The kit used for the cytokine determination was obtained from Genzyme Corporation (Cambridge, MA, USA) and used according to the manufacturer−s instructions. It is worth noting that in control experiments we found that the presence of SV-IV or LPS in the reaction mixture did not interfere neither with the immunological nor with the enzymatic reactions involved in the cytokine immunoenzymatic measurement.

Evaluation of apoptosis by DNA-flow cytometry At the end of the incubation time (48 h; apoptosis was clearly detectable only at this time) treated or untreated cells were centrifuged and directly stained in a propidium iodide (PI) solution (50 mg PI in 0.1% sodium citrate, 0.1% NP40, pH 7.4) overnight at 4 8C in the dark. Flow cytometric analysis was performed using a FACScan flow cytometer (Becton Dickinson, San Jose, CA, USA) interfaced with a Hewlett Packard computer (mod. 310) for data analysis. To evaluate cell apoptosis, PI fluorescence was measured as FL2 (Log scale) and the data analysis was performed by the CELL-FIT software (Becton Dickinson).

DNA fragmentation assay To evaluate by agarose gel electrophoresis the possible occurrence of internucleosomal hydrolysis of genomic DNA in treated macrophages, 2  106 cells were incubated in the standard culture medium for 48 h in the presence of either 106 M SV-IV, or 1 ng/ml LPS, or 106 M SV-IV in combination with 1 ng/ml LPS. After the end of the incubation time, the

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188 C. Esposito, A. Cozzolino et al.

cells were harvested by scraping, centrifuged at 500g, washed in PBS, and finally suspended in 100 ml of TNE (150 mM NaCl, 10 mM EDTA, 10 mM Tris-HCl, pH 8.0). The cell suspensions were lysed with 3 volumes of lysis buffer (0.2% SDS, 100 mg/ml RNase in TNE) and the lysates were incubated at 37 8C for 1 h. After incubation, 100 mg/ml proteinase K was added to the lysates and the mixtures were incubated for a further 2.5 h at 56 8C. The high-molecular-weight genomic DNA, extracted from the proteinase K-treated lysates according to a published procedure (Sambrook et al., 1989), was analyzed by electrophoresis (2 h, 80 V) in a 1% agarose gel containing ethidium bromide in TBE (0.045 M Tris-borate, 0.001 M EDTA, pH 8.0).

TUNEL DNA fragmentation in individual apoptotic cells was detected by the technique of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) of DNA strand breaks (Apoptosis Detection System, Fluorescein; Promega, Milan, Italy). Aliquots of 4  106 macrophages, treated for 48 h with either 10 6 M SV-IV, or 1 and 10 ng/ml LPS, or 10 6 M SV-IV in combination with either 1 or 10 ng/ml LPS, were harvested by scraping, centrifuged at 500g, washed in PBS, and fixed for 20 min in ice-cold 1% methanol. A reaction buffer containing terminal deoxynucleotidyl transferase and fluoresceindUTP was added to the fixed cells and the mixture was incubated (37 8C, 1 h) in a humidified chamber. The cells were stained with propidium iodide (1 mg/ml, 15 min) and observed by fluorescence microscopy with standard fluorescein excitation. Apoptotic cells were identified by the shrunken morphology and the yellow-orange-green fluorescence visible within the nucleus due to the fluorescein-12-dUTP incorporation at the 3'-OH ends of fragmented DNA. The negative control was represented by macrophages treated with the same TUNEL protocol but without the addition of terminal deoxynucleotidyl transferase.

Statistical analysis

The data have been reported as means  SEM (standard error of the mean) obtained from three separate experiments in which each point was performed in triplicate. The means were compared using analysis of variance (ANOVA) plus Bonferroni×s t-test, and a P value of less than 0.05 was considered significant.

Results Treatment of macrophages with SV-IV produces a marked increase in NO production Preliminary experiments showed that the kinetics of NO induction by LPS and SV-IV was linear in the first 24 h, the LPS-mediated kinetics being two times faster than that mediated by SV-IV. This finding suggested the optimal incubation time (24 h) to comparatively measure the NO2 production induced by either SV-IV or LPS treatment. After 48 h treatment, SV-IV- or LPS-mediated NO-induction plateau

levels were reached, the LPS plateau being two times higher than that of SV-IV. Treatment of J774 macrophages with various concentrations of LPS-free SV-IV (10 9 ± 10 5 M) for 24 h at 37 8C resulted in a marked dose-dependent increase in NO2 in the conditioned culture medium (Fig. 1A). The maximum increase observed (about 300%) was obtained with 10 6 M SV-IV. Higher doses of SV-IV produced a decrease in NO (Fig. 1A). This decrease was probably related to the biphasic dose-effect relationship of this protein, typical consequence of its concentration-dependent self-association equilibrium (shift at higher concentrations from the active monomeric form to the biologically inactive, non-toxic dimeric or trimeric configuration) (Stiuso et al., 1999). In the same experimental conditions, the treatment of the cells with 10 ng/ml LPS induced a response two times higher than that obtained with SV-IV (Fig. 1B); lower concentrations (0.5 ± 1 ng/ml) of LPS, however, produced the same increase in NO2 concentration in culture medium as 10 6 M SV-IV (Fig. 1B). The effect of these two different molecules (LPS and SV-IV) on NO synthesis was additive when they were administered in combination (Fig. 1C). The presence in the culture medium of 10 6 M dexamethasone (DXM), a potent inhibitor of the LPS ability to stimulate macrophage NO production, abrogated this specific metabolic response (Fig. 1A ± C). In the experiments described above, the cell viability, as monitored by both trypan blue exclusion test and MTT assay, was 90  5 % in all the SV-IV- or LPS-treated cells. The addition into macrophage culture medium of an appropriate concentration (50 U/ml) of TNF-a neutralizing antibodies (rabbit anti-mouse/rat TNF-a; Research Diagnostics Inc, Flanders, NJ) reduced by about 80% the NO production induced by 10 6 M SV-IVand by about 40% the NO production induced by 1 ± 10 ng/ml LPS (Table I). Most interestingly, at the NO concentrations induced by 10 6 M SV-IV and 1 ng/ml LPS apoptosis was detected (FACS analysis) in LPS- but not in SV-IV-treated cells (Table II), thus suggesting that, in addition to the TNF-a-mediated apoptotic pathway, some powerful anti-apoptotic pathway was also activated in the SV-IV-treated cells as a consequence of the functional interaction between this protein and its specific plasma-membrane receptors.

The increase in nitrite production in SV-IVtreated macrophages is associated with a substantial rise of iNOS enzymatic activity Kinetics of iNOS induction by LPS and SV-IV was found to be similar to that of NO. The ability of SV-IV to stimulate NO production in intact J774 cells was associated with a large increase in iNOS activity, as measured in vitro with macrophage

Tab. I. Effect of TNF-a neutralizing antibodiesa on the NO production induced by SV-IV or LPS in J774 macrophages. Cell treatment None (control) SV-IV (10 7 M ) SV-IV (10 6 M ) LPS (1 ng/ml) LPS (10 ng/ml)

NO2 produced (nmol/5  106 cells/24 h)

Inhibition, %

minus antibodies

plus antibodies

6  0.5 20  1 ** 26  0.4 ** 28  0.7 ** 42  0.8 **

6  0.5 4  0.8 ** 5  0.5 ** 17  0.6 ** 25  0.7 **

0 80 81 39 40

a Rabbit anti-mouse TNF-a ( Research Diagnostics Inc., Flanders, NJ ) neutralizing antibodies were added to the macrophage culture medium at a final concentration of 50 U/ml. The cell treatment was performed according to the standard protocol (see Materials and methods). Each value represents the mean  SEM of 4 independent determinations in which each point was performed in triplicate. ** ˆ P < 0.01 ( Bonferroni×s t-test) vs. the control value.

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Fig. 1. Effect of SV-IV on NO2 production in J774 and mouse primary alveolar macrophages. Cells were treated according to the standard protocol (see Materials and methods) with different concentrations of either 10 9 ± 10 5 M SV-IV (A and D: hatched bars) or 0.25 ± 100 ng/ml LPS (B and E: hatched bars) or 10 9 ± 10 5 M SV-IV in combination with 1 ng/ml LPS (C and F: criss-crossed bars); when the latter experiment was repeated by combining 10 9 ± 10 5 M SV-IV with

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10 ng/ml LPS similar results were obtained. Untreated cells: A ± F (white bars). Cells treated with 10 6 M DXM and 10 ng/ml LPS or 10 6 M SV-IV: A ± F (dotted bars). The values reported in the figure represent the means  SEM (error bars) of 4 independent determinations in which each point was performed in triplicate. Further experimental details are reported in Materials and methods.

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Tab. II. Evaluation of apoptosis by FACS analysis in J774 macrophages treated with SV-IV or LPS in the absence or in the presence of TNF-a neutralizing antibodiesa. Cell treatment None (control) SV-IV (10 7 M ) SV-IV (10 6 M ) LPS (1 ng/ml) LPS (10 ng/ml) SV-IV (10 6 M ) ‡ LPS (1ng/ml) LPS (1 ng/ml) ‡ DXM 10 6 M SV-IV (10 6 M ) ‡ LPS (1 ng/ml) ‡ DXM 10

Apoptosis, %

6

M

minus antibodies

plus antibodies

0 0 0 30.5  1.44 ** 33.48  1.73 ** 32.74  2.31 ** 0 0

0 0 0 15.14  1.75 ** 17.21  1.32 ** 16.00  2.58 ** 0 0

a

Rabbit anti-mouse TNF-a neutralizing antibodies were added to the macrophage culture medium at a final concentration of 50 U/ml. The cell treatment (48 h) was performed according to the standard protocol (see Materials and methods). Each value represents the mean  SEM of 3 independent determinations in which each point was performed in triplicate. ** ˆ P < 0.01 ( Bonferroni×s t-test) vs. the control value.

lysates. The data reported in Figure 2A demonstrate, in fact, that the highest iNOS-activating effect was observed when 10 6 M SV-IV was used in the assay. Higher doses of SV-IV resulted in a decrease in iNOS activity probably related to the biphasic dose-effect relationship of this protein (Stiuso et al., 1999). In the same experimental conditions the treatment of the cells with 10 ng/ml LPS produced a response about 65% higher in comparison with that observed after cell treatment with SVIV, whereas 0.5 ± 1 ng/ml LPS produced the same increase in iNOS activity as 10 6 M SV-IV (Fig. 2B). The stimulating effect of LPS and SV-IV on iNOS activity was found to be additive when the two molecules were given in combination (Fig. 2C). When appropriate concentrations of L-NAME, a structural analogue of arginine acting as competitive inhibitor of iNOS, were included in the assays, the enzymatic activity was practically abrogated (Fig. 2A ± C). In these experimental conditions, the cell viability, monitored by both trypan blue exclusion test and MTT assay, was 90 5 % in all the SV-IV- or LPS-treated cells. It is well known that LPS induces in rodent macrophages not only iNOS activity but also arginase activity (Wang et al., 1995). By evaluating the arginase activity of untreated or SV-IVtreated macrophages, as measured by quantitating the [14C]urea derived from [14C]L-arginine (Wang et al., 1995), we found that SV-IV did not possess the ability to stimulate this enzymatic activity.

The iNOS gene expression is upregulated in SVIV-treated macrophages To verify whether the SV-IV-dependent increase in iNOS activity was due to an upregulation of iNOS expression, we evaluated by semiquantitative RT-PCR the amount of iNOS mRNA occurring in the total RNA prepared from macrophages treated or not for 24 h at 37 8C with SV-IV or LPS alone or in combination. In these experimental conditions, the cell viability, monitored by both trypan blue exclusion test and MTT assay, was 90  5 % in all SV-IV- or LPS-treated cells. The data reported in Figure 3A show that iNOS mRNA was barely detectable in the control untreated cells, whereas it was definitely upregulated in the cells stimulated with either SV-IV (10 6 M) or LPS (1 ng/ml) alone, and with both in combination. It is worth noting that in the latter case the stimulating effect of the two molecules was rather additive than synergistic. The semi-quantitative evaluation of each PCR product was

achieved by integrating the peak area obtained by densitometry of the ethidium bromide-stained agarose gels (iNOS (600 bp): 655  65, 3615  400, 16333  1400, 23701  2700; b-actin (300 bp): 19321  1500, 19848  1650, 19526  1600, 19638  1550; software used: NIH image V.16). The ratio between the yield of each amplified product and co-amplified b-actin (iNOS/b-actin mRNA ratio: 0.0339, 0.1851, 0.8364, 1.2068) allows a relative estimate of mRNA levels in the samples analyzed. Similar results were obtained when the iNOS protein amount was assessed by Western blot after SV-IV (10 6 M) and LPS (1 ng/ml) treatment of the cells (Fig. 3B). The results of densitometric analysis of the iNOS protein (130 kDa), following interaction of the electrophoretically separated protein bands with the specific anti-iNOS antibody and immunostaining with horseradish peroxidase-conjugated goat anti-rabbit IgG and diaminobenzamine, are here reported: 9092  850, 12513  1380, 17731  1660, 20539  2350; background: 495  65 (Fig. 3B; software used: NIH image V.16).

Macrophage treatment with SV-IV does not induce apoptosis To test whether the increase in NO induced apoptosis in J774 cells following their treatment with SV-IVand LPS for 48 h, we measured some morphological and biochemical parameters typical of this particular form of cell death. In these experiments the cell treatment was protracted from the standard 24 h to 48 h because apoptosis was detectable only at this time. When the cells were treated with SV-IV (10 6 M) no signs of apoptosis were observed. In particular, we found: a) absence of morphological signs of apoptosis, as defined by phase-contrast light microscopy (data not shown); b) absence of apoptotic cells (TUNEL analysis: Fig. 4B; FACS analysis: Table II) as in control untreated cells (TUNEL analysis: Fig. 4A; FACS analysis: Table II); c) absence of DNA fragmentation (DNA laddering; Fig. 4E, lane 2); d) no changes of bcl-2 and c-myc expression (RT-PCR analysis: Fig. 4F, G, lane 2; Western blot with mouse anti-bcl-2 (C-2) and anti-c-myc (C-33) monoclonal antibodies (Santa Cruz Biotechnology, Milano, Italy): data not shown) in comparison with untreated cells (RT-PCR analysis: Fig. 4F, G, lane 1; Western blot with mouse anti-bcl-2 (C-2) and anti-c-myc (C-33) monoclonal antibodies: data not shown); e) absence of p53 expression both in untreated and SV-IV-treated cells (RT-PCR analysis: data not shown).

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Fig. 2. Effect of SV-IV on NO synthase activity in J774 and mouse primary alveolar macrophages. Cells were treated according to the standard protocol (see Materials and methods) with different concentrations of either 10 9 ± 10 5 M SV-IV (A and D: hatched bars) or 0.25 ± 100 ng/ml LPS (B and E: hatched bars) or 10 9 ± 10 5 M SV-IV in combination with 1 ng/ml LPS (C and F: criss-crossed bars); when the latter experiment was repeated by combining 10 9 ± 10 5 M SV-IV with

Protein SV-IV and nitric oxide 191

10 ng/ml LPS similar results were obtained. Untreated cells: A ± F (white bars). Cells treated with 10 ng/ml LPS or 10 6 M SV-IV with 1 mM L-NAME added into the enzymatic assay: A ± F (black bars). The values reported in the figure represent the means  SEM (error bars) of 4 independent determinations in which each point was performed in triplicate. Further experimental details are reported in Materials and methods.

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192 C. Esposito, A. Cozzolino et al.

Tab. III. Effect of SV-IV or LPS on TNF-a release from J774 macrophages. Cell treatment a

TNF-a release (pg/ml)

None (control) SV-IV (10 7 M ) SV-IV (10 6 M ) LPS (1 ng/ml) LPS (10 ng/ml)

50  10 80  8 * 110  25 ** 1065  42 ** 4500  120 **

a

The cell treatment was performed according to the standard protocol (see Materials and methods). Each value represents the mean  SEM of 3 independent determinations in which each point was performed in triplicate. * ˆ P < 0.05, ** ˆ P < 0.01 ( Bonferroni×s t-test) vs. the control value.

Fig. 3. Expression of iNOS in J774 macrophages following 10 6 M SVIV and 1 ng/ml LPS treatments. The expression of iNOS in untreated or SV-IV/LPS-treated cells was evaluated by RT-PCR (A), and Western blot analysis (B). The data reported in the figure are the results of a typical single experiment that was repeated three times with practically identical results. Quantitation of each electrophoretic band by scanning densitometry showed that the variability of the data was about  10% (see Results). Further experimental details are reported in Materials and methods.

In contrast, apoptosis was easily induced in these cells following their exposure to LPS (1 ng/ml). Typical apoptotic morphological phenotype (TUNEL analysis: see Fig. 4C), clear DNA laddering (Fig. 4E, lane 3), inhibition of both bcl-2 and cmyc expression (RT-PCR analysis: Fig. 4F, G, lane 3; Western blot with mouse anti-bcl-2 (C-2) and anti-c-myc (C-33) monoclonal antibodies: data not shown), and absence of p53 expression both in untreated and LPS-treated cells (RT-PCR analysis: data not shown), were observed. When the macrophages were treated with 10 6 M SV-IV in combination with 1 ng/ml LPS the results did not differ from those obtained with LPS alone (Fig. 4D; Fig. 4E ± G, lane 4; Western blot with mouse anti-bcl-2 (C-2) and anti-c-myc (C-33) monoclonal antibodies: data not shown). The addition into macrophage culture medium of an appropriate concentration (50 U/ml) of anti-TNF-a neutralizing antibody (rabbit anti-mouse/rat TNF-a; Research Diagnostics Inc, Flanders, NJ) produced an about 50% decrease in the apoptotic damage of LPS-treated cells without any apparent effect on SV-IV-treated cells (Table II).

Effect of SV-IV and LPS on TNF-a production by J774 macrophages The amount of TNF-a occurring in the supernatants of J774 macrophages treated with either SV-IV or LPS was evaluated by an immunoenzymatic method. The data reported in Table III indicate that doses of LPS (1 ng/ml) or SV-IV (10 6 M), inducing equivalent NO production in the target cells, are able to induce in LPS-treated cells amounts of TNF-a that are about 10 times higher than those induced in SV-IV-stimulated cells. This result suggests that the higher release of TNF-a from

LPS-treated cells could be responsible for apoptosis observed in these cells but not in SV-IV-treated cells. To verify this hypothesis, we added to the medium of cells treated with 10 6 M SV-IV an amount of TNF-a (950 pg/ml; Research Diagnostic Inc.) equivalent to the difference between the amount of TNF-a induced by 1 ng/ml of LPS and that induced by 10 6 M SV-IV (see Table III). Unexpectedly, the addition of these amounts of TNF-a did not trigger apoptosis in these cells.

The activity of SV-IV is confirmed using primary mouse macrophages To verify if mouse macrophage-like cell lines are faithful predictors of responses of non-transformed macrophages derived from normal tissues, we repeated all the experiments using primary mouse alveolar macrophages. The results obtained confirmed not only the NO/iNOS-stimulating activity of SV-IV (Figs. 1D ± F and 2D ± F), but also its differences from LPS as apoptosis-inducing agent (data not shown).

Discussion In this paper data are reported showing that the treatment of J774 macrophages with SV-IV increases markedly their ability to produce NO. This increase is associated with a significant enhancement of iNOS activity caused, in turn, by an upregulation of iNOS gene expression at both protein (immunoblot) and mRNA level (semiquantitative RT-PCR). The increase in iNOS gene expression might be related to the binding of SV-IV to SV-IV-specific plasma membrane tyrosine-kinase-associated receptors (Kd ˆ 10 8 M) (Paonessa et al., 1984; Porta et al., 1990, 1991; Romano-Carratelli et al., 1995; Tufano et al., 1996; Stiuso et al., 1999; unpublished data) followed by an intracytoplasmic protein phosphorylation cascade. The upregulation of iNOS gene expression by SV-IV could be either direct or indirect via TNF-a autocrine production. Different data are in line with the latter hypothesis: 1) SV-IV has been clearly demonstrated to possess the ability to induce the production of substantial amounts of TNF-a from non-activated monocytes (Tufano et al., 1996); 2) TNF-a is a potent inducer of iNOS in different cell types (Szabo¡, 1995; Rath and Aggarwal, 1999); 3) anti-TNF-a antibodies are capable of neutralizing the SV-IV-dependent NO production in J774 macrophages (this paper). The additive biochemical effect observed when SV-IV was administered in combination with LPS, was probably related to

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Fig. 4. Apoptotic effect induced by SV-IV and LPS treatments in J774 macrophages. A ± D: TUNEL assay. A, untreated cells; B, cells treated with 10 6 M SV-IV; C, cells treated with 1 ng/ml LPS; D, cells treated with 10 6 M SV-IV in combination with 1 ng/ml LPS. Normal cells are red-stained throughout the cytoplasm and not reduced in volume. Apoptotic damage of experimentally treated cells is shown by marked shrinkage in cell volume, occurrence of small red-stained apoptotic bodies, and green-yellow fluorescence localized within the nuclei of shrunken apoptotic cells. Magnification is the same in (A ± D). E: internucleosomal DNA fragmentation assay. Lane 1, untreated cells; lane 2, cells treated with 10 6 M SV-IV; lane 3, cells treated with 1 ng/ml LPS; lane 4, cells treated with 1 ng/ml LPS and 10 6 M SV-IV; lane M, 100 bp ladder used as a molecular weight marker. F: evaluation of bcl-2

Protein SV-IV and nitric oxide 193

expression by RT-PCR assay. Lane 1, untreated cells; lane 2, cells treated with 10 6 M SV-IV; lane 3, cells treated with 1 ng/ml LPS; lane 4, cells treated with 1 ng/ml LPS and 10 6 M SV-IV; lane M, 100 bp ladder used as a molecular weight marker. G: evaluation of c-myc expression by RT-PCR assay. Lane 1, untreated cells; lane 2, cells treated with 10 6 M SV-IV; lane 3, cells treated with 1 ng/ml LPS; lane 4, cells treated with 1 ng/ml LPS and 10 6 M SV-IV; lane M, 100 bp ladder used as a molecular weight marker. In (F and G) the lower bands refer to the housekeeping b-actin mRNA used as internal control in the semiquantitative RT-PCR. When all the experiments reported in this figure were repeated with 10 ng/ml LPS, similar results were obtained. Further experimental details are reported in Materials and methods.

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194 C. Esposito, A. Cozzolino et al.

the interaction of these two ligands with structurally and functionally different receptors. A recent report indicates, in fact, that the receptor for LPS is composed of three membraneassociated proteins (CD14 (a specific tyrosine-kinase-associated receptor), CD11b/CD18, and Toll-like receptor (TLR) 4) the coordinate engagement of which with LPS seems to be a requirement for an optimal signaling delivery to the macrophage (Perera et al., 2001). In addition, the possibility of some direct interaction between LPS and SV-IV is ruled out by previously published data showing that SV-IV does not react in vitro with LPS (Vuotto et al., 1993). We have found that the LPS-mediated stimulation of NO/ iNOS production in J774 macrophages was markedly higher than that induced by SV-IV (see Results). The higher level of TNF-a inducible by LPS in monocytes/macrophages in comparison with that inducible by SV-IV in the same cell types (Tufano et al., 1996 ; Table 3 of this paper) may reasonably be at the basis of this finding. Stimulation of macrophage arginase by SV-IV and consequent limitation of the availability of L-arginine (common substrate for both arginase and iNOS) for iNOS was ruled out as a factor contributing to the low efficiency of SV-IV-mediated NO production because SV-IV was found unable to interfere in vitro with the activity of this enzyme (see Results). In contrast, the difference between LPS and SV-IV in the ability to stimulate NO production was clearly related to the observed difference in their NO/iNOS induction kinetics (see Results). The finding that the NO/iNOS induction kinetics of LPS is faster than that of SV-IV suggests that the differential fate of the LPS- and SV-IV-treated cells could be simply related to the higher amount of TNF-a and NO induced by LPS in comparison with that induced by SV-IV. The different amounts of TNFa induced by these two bioactive molecules could also trigger or not apoptosis by acting via a mechanism other than that based on NO/iNOS stimulation, namely through activation of caspase 8 upon TNF-a receptor engagement, the apoptotic effect being dose-dependent and requiring a threshold level of active caspase 8 before PCD initiation (Hu et al., 2000). Comparison of dose-effect curves obtained with either LPS or SV-IV demonstrates, however, that bioequivalent concentrations of these molecules (1 ng/ml LPS and 10 6 M SV-IV), inducing in the target cells similar levels of NO (see Results), produced different biochemical and biological effects, namely expression of bcl-2 and c-myc and no apoptosis in SV-IVtreated cells as in control cells, suppression of bcl-2 and c-myc associated with substantial apoptotic death in LPS-stimulated cells. These results, together with the data showing the different ability of SV-IV and LPS to induce NO/iNOS and TNF-a and the insensitivity of SV-IV-treated cells to doses of TNF-a clearly apoptogenic for LPS-treated cells (see Results), suggest that in our experimental model LPS and SV-IV regulate the macrophage PCD by modulating the life/death balance of these cells through a differential trans-membrane activation of specific apoptotic and anti-apoptotic pathways. The molecular mechanisms underlying these biochemical and biological events are, most probably, related to a different trans-membrane signaling function of structurally different membrane receptors. One more question concerns the possible physiological significance of the ability of SV-IV to upregulate iNOS in macrophage cells. Evidence has been reported in the literature suggesting the possibility that appropriate concentrations of NO can play a pivotal role in the process of mammalian

reproduction by regulating important biological events such as the sperm capacitation and hyperactivation, and the binding of spermatozoa to the zona pellucida of mature oocytes (Manco and Abrescia, 1988; Herrero et al., 1997; Rosselli et al., 1998; Sengoku et al., 1998; Yeoman et al., 1998; Herrero et al., 1999). It has been demonstrated that, following ejaculation, part of the protein SV-IV is adsorbed on the sperm cell plasma membrane and part of it is free in the seminal plasma (Paonessa et al., 1984; Manco and Abrescia, 1988). In addition to spermatozoa, the main target cells of SV-IV are lymphocytes and macrophages which are known to be largely represented in the mucosa of the female genital tract (Paonessa et al., 1984; Galdiero et al., 1989; Metafora et al., 1989a, b; Camussi et al., 1990; Vuotto et al., 1993; Peluso et al., 1994; Romano-Carratelli et al., 1995; Tufano et al., 1996). The interaction of SV-IV with the male gametes and with the cell population (immunocytes, endothelial cells, epithelial cells, etc.) occurring in this anatomical region might actively contribute, via the upregulation of the iNOS gene expression appropriately coupled to the endocellular activation of powerful bcl-2- and c-myc-mediated anti-apoptotic pathways, to the fine tuning of the local biochemical milieu required for a successful egg fertilization process. In contrast with this hypothesis, data have been reported in the literature showing that fertility is quite normal in iNOS knockout mice (Wei et al., 1995). Possible redundant biochemical pathways controlling the same biological event, however, can still make our hypothesis standing. Acknowledgements. We are very grateful to Mr. Salvatore Baiano and Mr. Francesco Moscatiello for their skilful, excellent technical assistance.

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