Introduction
Malignant tumors contain macrophages (Mps) as a major component of the host leukocytic infiltrate, and the role of Mps in tumor progression has generated contradictory evidence [
1]. It has been recognized that Mps can act either as negative regulators by achieving tumor cytotoxicity or as positive regulators by promoting tumor growth. Neovascularization, an essential step in tumor progression and metastasis development, can be modulated by the presence of Mps in the tumor microenvironment. Angiogenic stimuli can proceed from tumor cells and/or immune cells such as lymphocytes and Mps. We have previously demonstrated the ability Mps from tumor-bearing mice to exacerbate the angiogenic response elicited by LMM3 tumor cells (derived from a murine mammary adenocarcinoma), confirmed by CD31 positivity at the angiogenic site [
2]. There are several molecules, such as nitrogen metabolites, prostaglandins, vascular endothelial growth factor (VEGF), fibroblast growth factor and placental growth factor, that exert proangiogenic functions [
3]. Less knowledge is available about the autonomic regulation of tumor neovascularization. Here we investigate the role of the parasympathetic nervous system on the angiogenic activity exerted by peritoneal Mps from 7-day LMM3 mammary-tumor-bearing mice (TMps) by studying the expression and function of muscarinic acetylcholine receptors (mAchRs) in new blood vessel formation induced by TMps.
Detection of muscarinic acetylcholine receptor subtypes by Western blotting
Purified Mps (10
6 cells) were lysed at 4°C with 0.5 ml of 0.5% Nonidet P40, 10 mM Tris-HCl, pH 7.4, 5 mM MgCl
2, 50 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 0.1 mM orthovanadate and the following protease inhibitors: 10 μg/ml aprotinin, 10 μg/ml leupeptin, 5 mM PMSF and 50 μg/ml soybean trypsin inhibitor. Lysates were sonicated for 30 s and centrifuged at 3,000 r.p.m. for 10 min at 4°C. Supernatants were centrifuged at 10,000 r.p.m. for 20 min at 4°C. The resulting supernatants were stored at -80°C. Protein concentration was determined by the Lowry method [
6].
Samples were subjected to 7.5% SDS-PAGE minigel electrophoresis, with 30 μg of protein in each lane. Standards of known molecular masses were also seeded. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad) and washed with distilled water. The nitrocellulose strips were blocked in buffer (20 mM Tris-HCl, 500 mM NaCl, 0.05% Tween 20 (TBST) with 5% skimmed milk) for 1 hour at 20 to -25°C and subsequently incubated overnight with goat anti-M
1, anti-M
2 and anti-M
3 polyclonal antibodies (Santa Cruz Biotechnology) diluted 1:100 in TBST. After several rinses with TBST, strips were incubated with the second antibody (goat anti-mouse IgG conjugated with alkaline phosphatase, diluted 1:4,000 in TBST) at 37°C for 1 hour. Bands were revealed with a mixture of nitro blue tetrazolium chloride and 5-bromo-4-chloroindol-3-yl phosphate
p-toluidine salt (NBT/BCIP) [
7]. Quantification of the bands was performed with a computerized densitometer connected to an image analyzer (Bio-Rad GS700) and is expressed in optical density units per mm
2.
Arginase activity assay
Arginase activity was determined in cell lysates in accordance with methods described previously [
8]. In brief, 10
5 cells were treated or not with 100 nM carbachol in the absence or presence of 100 μM NOHA, 1 μM atropine, 1 μM pirenzepine, 1 μM methoctramine or 1 μM 4-DAMP. After being washed, cells were lysed with 0.5 ml of 0.1% Triton X-100, 25 mM Tris-HCl, pH 7.4, containing 5 mM MnCl
2. The enzyme was then activated by being heated at 56°C for 10 min. Arginine hydrolysis was performed by incubating 25 μl of the activated lysate with 25 μl of 0.5 M arginine, pH 9.7, at 37°C for 60 min. The reaction was stopped in acid medium. Urea concentration was measured at 540 nm with a microplate reader. Results are expressed as micromoles of urea per hour per million cells.
Mps were rinsed twice with ice-cold PBS and then scraped into 300 μl lysis buffer (50 mM Tris-HCl, pH 7.5, 0.1 mM EDTA, 0.1 mM EGTA, 1 μg/ml leupeptin, 1 μg/ml aprotinin and 0.1 mM PMSF). Lysis was completed by sonication. Samples (25 μg) were subjected to 10% SDS-PAGE as described previously [
9‐
11]. Nitrocellulose membranes were incubated overnight with a monoclonal anti-mouse arginase I antibody (BD Transduction Laboratories) or with a rabbit anti-arginase II antibody (a gift from Dr Masataka Mori). The secondary antibody anti-mouse or anti-rabbit IgG conjugated with alkaline phosphatase was added for 1 hour at 37°C. Proteins were revealed with NBT/BCIP and quantified by a densitometric analysis.
Prostaglandin E2assay
Prostaglandin E
2 (PGE
2) production by Mps was determined by RIA as described previously [
12]. Purified Mps (2 × 10
6 cells per sample) were incubated for 1 hour at 37°C in a Dubnoff bath with carbogen in 1 ml of MEM with or without 100 nM carbachol in the absence or presence of 1 μM atropine, 1 μM methoctramine or 1 μM 4-DAMP, 1 μM indomethacin or 10 μM NS-398. After incubation, cells were centrifuged for 10 min at 200
g and supernatants were frozen at -80°C until the assay was performed. For PGE
2 RIA, 100 μl samples or standards were incubated for 30 min with 500 μl of rabbit anti-PGE
2 antiserum (Sigma) at 4°C. Then 5 pg of [
3H]PGE
2 (specific radioactivity 154 Ci/mmol; New England Nuclear) was added to each tube. All dilutions were performed in 0.01 M PBS, pH 7.4, containing 0.1% BSA and 0.1% sodium azide. After incubation, a dextran-coated charcoal suspension was added to separate the bound and free fractions. The supernatants were removed from each tube and scintillation solution (Optiphase Hisafe 3; Wallac) was added to determine the amount of radioactivity present. Results are expressed in picograms per 10
5 cells.
Detection of cyclo-oxygenase (COX) isoforms by Western blotting
Purified Mps were washed twice in cold PBS and then resuspended in 300 μl of lysis buffer (20 mM Tris-HCl, 1 mM EDTA, 10 μg/ml leupeptin, 2 μg/ml aprotinin, 10 μg/ml dithiotreitol, 100 μg/ml soybean trypsin inhibitor, 1 mg/ml benzamidine). After 1 hour, lysates were centrifuged at 5,000 r.p.m. for 10 min. The resulting supernatants were stored at -80°C. Protein concentration was determined by the Lowry method [
6].
Samples were subjected to 7.5% SDS-PAGE minigel electrophoresis, with 30 μg of protein in each lane. Standards of known molecular masses were also seeded. After electrophoresis, proteins were transferred to a nitrocellulose membrane (Bio-Rad) at 4°C for 18 hours. Membranes were then washed with distilled water and incubated with blocking solution (5% skimmed milk in TBST) for 1 hour at 20 to -25°C. Membranes were incubated with rabbit polyclonal anti-COX-1 or anti-COX-2 antibodies (Cayman Chemical) in Tris-buffered saline for 90 min at room temperature. Then secondary anti-rabbit IgG antibody conjugated with alkaline phosphatase was added for 1 hour at 37°C. Proteins were revealed with NBT/BCIP and quantified by a densitometric analysis [
13].
Detection of VEGF by Western blotting
Production of VEGF was measured in lysates from untreated TMps or TMps treated with 100 nM carbachol for 1 hour in the absence or presence of 1 μM atropine, 1 μM pirenzepine, 1 μM methoctramine or 1 μM 4-DAMP or the enzyme inhibitors 100 μM NOHA, 1 μM indomethacin or 10 μM NS-398. Cells were then cultured without FCS at 37°C for 24 hours in 100 mm Petri dishes. After being washed twice with cold PBS, TMps were lysed in 10 mM Tris-HCl, pH 8, 1% Triton X-100, 100 mM NaCl, 10 mM EGTA, 10 mM EDTA, with protease inhibitors (1 mM PMSF, 10 μg/ml leupeptin, 10 μg/ml aprotinin). After 1 hour in an ice bath, lysates were centrifuged at 10,000 r.p.m. for 10 min at 4°C. Samples were subjected to 10% SDS-PAGE electrophoresis. Proteins were transferred to nitrocellulose membranes and, after several rinses with doubly distilled water, were blocked with 5% skimmed fat milk in TBST buffer. The primary antibody (goat polyclonal anti-VEGF; Santa Cruz Biotechnology) was added for 18 hours, and the secondary antibody anti-goat IgG conjugated with alkaline phosphatase was added for 1 hour at 37°C. Proteins were detected with NBT/BCIP and quantified by densitometric analysis [
13,
14].
Drugs
All drugs were purchased from Sigma-Aldrich unless otherwise stated. Solutions were prepared fresh daily.
Statistics
Results are given as means ± SEM for at least three independent experiments. The statistical significance of differences between groups was analyzed by analysis of variance, Tukey's modified t-test or the Mann–Whitney test, using the STAT PRIMER program; P < 0.05 was considered to be statistically significant.
Discussion
Mps perform multiple functions that are essential in tissue remodeling, wound healing, inflammation and immunity. These cells form the major component of the mononuclear leukocyte population of some solid tumors [
1,
15]. In the 1980s, Polverini and Leibovich demonstrated that tumor-associated Mps isolated from 3-methycholanthrene-induced rat fibrosarcoma were potent stimulators of
in vivo neovascularization and bovine endothelial cell proliferation; depletion of Mps from tumor cell suspensions significantly decreased their angiogenic potential, suggesting that neovascularization was mediated in part by Mps [
16].
Taking into account the fact that murine mammary adenocarcinomas arising spontaneously in BABL/c mice in our laboratory are poorly infiltrated by Mps, we showed that peritoneal Mps from 7-day tumor-bearing mice, when present at low concentrations, contribute to the enhancement of LMM3 angiogenesis by providing polyamine precursors to tumor cells [
2]. Although the origin of tumor-infiltrating Mps has been discussed extensively, evidence supports both recruitment from the circulating pool of monocytes and the proliferation of the local Mps population, and it has recently been discussed that Mps could become angiogenic in the presence of diverse stimuli such as growth factors or low oxygen tension as well as soluble tumor antigens [
17,
18]. Here we show that peritoneal TMps, when inoculated in a number equal to that of LMM3 tumor cells, themselves elicit a potent angiogenic response. In contrast, 'unstimulated' NMps did not promote angiogenesis in our model. Further investigation is required to determine whether TMps activation occurs in the host-tumor interface or can be triggered at distance by soluble cytokines and/or tumor antigens.
Other authors have shown that the levels and functions of lymphocytes, granulocytes, Mps and natural killer cells are under the regulation of the autonomic nervous system [
19]. We showed that the activation of mAchR in TMps by the cholinergic agonist carbachol increases their angiogenic ability. The participation of muscarinic receptors was demonstrated by preincubating cells with the non-selective muscarinic antagonist atropine. Angiogenesis is now considered an important step during inflammation and cancer, and it might be necessary as a local, protective response against invasion by pathogens and the proliferation of transformed cells. It is also important in tumor growth and metastasis. The nervous system reflexively regulates the inflammatory response and it has been recently documented that acetylcholine, the principal vagal neurotransmitter, significantly attenuates the release of cytokines (tumor necrosis factor, IL-1, IL-6 and IL-18, but not the anti-inflammatory cytokine IL-10) in lipopolysaccharide-stimulated human macrophage cultures [
20]. These anti-inflammatory actions are generally related to nicotinic receptor stimulation [
21]. In our model, mAchR stimulation seems to be promoting pro-inflammatory actions by stimulating angiogenesis induced by TMps. It remains to be tested whether the activation of nicotinic receptors in TMps might be exerting anti-inflammatory actions.
M
1, M
2 and M
3 antagonists decreased the carbachol stimulation of neovascularization induced by TMps, showing a collaborative activation of different mAchR subtypes in the neovascular response. It has recently been documented that different mAchR activation controls different functions in distinct systems simultaneously. The activation of M
1 and M
3 receptors by carbachol induces pigment granule dispersion in isolated retinal pigment epithelium from bluegill. Carbachol-induced pigment granule dispersion is blocked by the muscarinic antagonist atropine, by the M
1 antagonist pirenzepine and by the M
3 antagonist 4-DAMP [
22]. We also showed that the activation of M
1, M
2 and M
3 receptors by carbachol is involved in the proliferation of two different murine mammary adenocarcinoma cell lines, LM3 and LM2 [
23].
The carbachol stimulation of angiogenesis induced by TMps occurs by the signaling of M
1 to arginase, because pirenzepine totally blocked the carbachol stimulation of urea production. Arginase I and II are overexpressed in TMps in comparison with NMps and are involved in the positive modulation by TMps of angiogenesis induced by LMM3 mammary tumor cells [
2]. We were the first to report that carbachol was able to stimulate the proliferation of tumor cells by arginine metabolism through arginase linked to M
1 receptors in LM2 cells, derived from M2 murine mammary adenocarcinoma [
23].
We also observed that methoctramine partly blunted carbachol-stimulated vascularization and urea formation, indicating that M
2 receptors are also involved in this effect. We have previously documented a collaborative action of M
2 and M
3 receptor activation by carbachol, which increases amylase secretion in lipopolysaccharide-inflamed salivary glands by stimulating PGE
2 liberation [
7]. We are therefore reporting that the expression and function of M
1 and M
2 receptors are involved in the control of angiogenesis induced by TMps, by stimulating polyamine synthesis in these cells.
The tumor microenvironment is rich in inflammatory cytokines, growth factors and chemokines, but generally poor in cytokines associated with a sustained immune antitumor response. It is now accepted that tumor-associated Mps produce soluble mediators that contribute to tumor progression. Our results indicate that the parasympathetic nervous system positively modulates neovascularization induced by TMps by stimulating M
3 receptors and PGE
2 liberation. Because indomethacin and NS-398 blunted the carbachol action on PGE
2 synthesis, the COX-1 and COX-2 isoenzymes are involved in angiogenesis induced by TMps. In particular, COX-2 protein expression is highly upregulated in TMps in comparison with NMps (data not shown). Several authors have stated that there is a role of COX-2 expression and function not only in tumors but also in immune cells from the host [
14,
18,
25]. The overproduction of this prostanoid could be responsible for an autocrine loop that also promotes immunosuppression of the host.
Previous results indicate that activation of G-protein-coupled receptors encoded by Kaposi's sarcoma-associated herpesvirus could be increasing VEGF expression and promoting an angiogenic response that characterizes Kaposi's sarcoma lesions [
26]. In support of this view, we observed that stimulation of mAchR in TMps by carbachol increased the 45 kDa isoform of VEGF. This effect is linked to activation of the M
2 and M
1 receptors, which in turn promote the arginine metabolic pathway through arginase. We have previously observed that the arginase pathway is involved in the angiogenic response induced by LMM3 cells derived from a murine mammary adenocarcinoma. These cells, which exert a potent angiogenic response quantitatively similar to that induced by TMps, also produce significant amounts of VEGF [
13]. Our results show that VEGF production by TMps depends partly on arginase metabolism because NOHA decreases VEGF production. Pretreatment of cells with COX inhibitors also diminished VEGF derived from TMps. In this way, the expression of COX-1 and COX-2 and their product PGE
2 has been shown to be promoters of angiogenesis by modulating the synthesis of various factors, including VEGF [
13,
27]. It must be taken into account that the stimulation of VEGF expression by COX-derived PGE
2 in TMps is independent of M
3 receptor activation.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
E de la T performed the Western blot assays, LD performed the in vivo angiogenesis experiments and the statistical analysis, MAJ made helpful criticism in discussion, TG developed the anti-arginase II antibody, ESL participated in the study design and coordination, and MES performed RIAs and conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.