![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 18, Issue 10, 3810-3819, October 2007
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Institutes of *Biochemistry I/Center for Drug Research, Development and Safety (ZAFES) and Clinical Pharmacology/ZAFES, Johann Wolfgang Goethe University, 60590 Frankfurt, Germany
Submitted December 11, 2006;
Revised June 11, 2007;
Accepted July 13, 2007
Monitoring Editor: J. Silvio Gutkind
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
and interleukin (IL) 12-p70 production, but increased formation of IL-8 and -10. Alternative macrophage activation required tumor cell death because a coculture with apoptosis-resistant colon carcinoma cells (RKO) or Bcl-2–overexpressing MCF-7 cells failed to induce phenotype alterations. Interestingly, phenotype alterations were achieved with conditioned media from apoptotic tumor cells, arguing for a soluble factor. Knockdown of sphingosine kinase (Sphk) 2, but not Sphk1, to attenuate S1P formation in MCF-7 cells, restored classical macrophage responses during coculture. Furthermore, macrophage polarization achieved by tumor cell apoptosis or substitution of authentic S1P suppressed nuclear factor (NF)-
B signaling. These findings suggest that tumor cell apoptosis-derived S1P contributes to macrophage polarization. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
triggers proinflammatory macrophage activation, characterized among other mediators, by the production of nitric oxide (NO), superoxide (O
), tumor necrosis factor (TNF)-, interleukin (IL)-1
and IL-6, thus resembling the M1 macrophage phenotype (Gordon, 2003
). In contrast, activation toward the M2 phenotype can be elicited by stimuli such as glucocorticoids, IL-4, -13, or -10 (Mantovani et al., 2002; Gordon, 2003).
In 1863 Virchow observed the presence of leukocytes in human tumors. Later on a link between cancer and chronic inflammation was suggested, and nowadays it seems accepted that macrophages are a major cell component infiltrating certain tumors (Mantovani et al., 2002). High numbers of tumor-associated macrophages (TAMs) often predict a poor survival prognosis for patients with solid human tumors, such as breast, prostate, ovarian, and cervical cancers (Bingle et al., 2002; Lewis and Pollard, 2006). Moreover, the presence of TAMs is often correlated with tumor cell survival. Tumor growth–promoting activities of TAMs are connected to alternative activation, because TAMs display a polarized M2 phenotype (Mantovani et al., 2002, 2004a). In contrast to M1-activated macrophages, TAMs show a reduced capacity to produce, e.g., TNF- or NO. TAMs not only support tumor survival and growth, but also contribute to metastasis, tumor angiogenesis, and immune evasion (Mantovani et al., 2004a; Pollard, 2004; Lewis and Pollard, 2006). Therefore, it is desirable to understand mechanisms of macrophage polarization toward the TAM phenotype because maneuvers to reprogram a M2 macrophage toward a M1 type may be beneficial (Sica et al., 2006). TAM polarization seems to be affected by the tumor microenvironment, with the likely contribution of tumor-derived molecules such as IL-4, IL-10, transforming growth factor (TGF)-, prostaglandin E2 (PGE2), and chemokines, as well as tumor hypoxia (Mantovani et al., 2002, 2004b; Lewis and Pollard, 2006).
Some years ago, another concept of tumor-induced macrophage polarization was introduced. Administration of apoptotic tumor cells reduced macrophage cytotoxicity against vital tumor cells (Reiter et al., 1999) and disrupting recognition of ACs by macrophages or dendritic cells in vivo induced tumor regression (Bondanza et al., 2004). Interactions of macrophages with apoptotic cells (ACs) provokes alternative activation profiles, which might be critical for termination of inflammation and/or repair of tissue during wound healing (Savill et al., 2002; Gregory and Devitt, 2004). ACs trigger the formation of IL-10, TGF-, or PGE2 from macrophages (Gregory and Devitt, 2004), but also release immunosuppressive molecules such as TGF- or IL-10 themselves (Tomimori et al., 2000; Chen et al., 2001). Recently, we noticed the release of sphingosine-1-phosphate (S1P) from ACs (Weigert et al., 2006), a sphingolipid known to be involved in tumor progression by promoting angiogenesis (LaMontagne et al., 2006).
Here we provide evidence for the release of S1P from apoptotic tumor cells as a modulator of macrophage polarization. In coculture, human monocyte-derived macrophages induced cell death of MCF-7 breast carcinoma cells, but not of apoptosis-deficient Bcl-2–overexpressing MCF-7 cells. Only apoptotic tumor cells decreased levels of TNF- and elevated those of IL-8 in macrophages after lipopolysaccharide (LPS) administration. The impact of apoptotic tumor cells on the macrophage phenotype was dependent on S1P production in apoptotic MCF-7 cells because knockdown of Sphk2 abrogated the phenotype shift as well as S1P production in cocultures. Interestingly, apoptotic MCF-7 cells and authentic S1P reduced nuclear factor (NF)-B activation in macrophages in response to LPS. These findings suggest a role for tumor cell apoptosis–derived S1P in macrophage polarization toward a TAM-like phenotype.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
was obtained from Roche (Indianapolis, IN).
Human Monocyte Isolation and Culture
Human monocytes were isolated as described (Von Knethen and Brune, 2001). In brief, monocytes were isolated from buffy coats (DRK-Blutspendedienst Baden-Württemberg-Hessen, Institut für Transfusionsmedizin und Immunhämatologie, Frankfurt am Main, Frankfurt, Germany) using Ficoll-Hypaque gradients (PAA Laboratories, Karlsruhe, Germany). Peripheral blood mononuclear cells were washed twice with phosphate-buffered saline (PBS) and were allowed to adhere to culture dishes (Primaria 3072, Becton Dickinson, Lincoln Park, NJ) for 1 h at 37°C. Nonadherent cells were removed. Monocytes were then differentiated into macrophages with RPMI 1640 containing 10% AB-positive human serum (PAA Laboratories) for 7 d.
Coculture Experiments
Primary human monocyte-derived macrophages were cultured at a density of 2 x 105 cells/ml. After differentiation, tumor cells were added at the same density, and cocultures were maintained for 5 d. Subsequently, residual tumor cells were removed from the plates by incubations with accutase (PAA Laboratories; Albee et al., 2007) for 5 min, which left adherence of macrophages unaltered. Remaining macrophages were treated with 1 µg/ml LPS and 100 U/ml IFN- or were exposed a second time to equal amounts of tumor cells. In some experiments higher ratios of tumor cells compared with macrophages were used, as indicated.
Quantification of Cell Death
Cocultures of human macrophages and tumor cells were harvested after 12, 24, or 48 h by incubation with accutase for 30 min, which removed both macrophages and tumor cells. Cell death was quantified by fluorescence-activated cell sorting (FACS) after incubations with a monoclonal human CD44-PE antibody (Ancell, Bayport, MN) to discriminate between macrophages and tumor cells, followed by the annexin V-fluorescein isothiocyanate (FITC) method (ImmunoTools, Friesoythe, Germany). In detail, quantification of macrophage versus tumor cell contents in cocultures was performed with FACS analysis using -CD44-PE. The macrophage population was identified by staining control macrophages, thereby gating this population, which was used to identify macrophages in cocultures. All remaining cell counts were attributed to tumor cells.
Production of Conditioned Media from Apoptotic Tumor Cells
Conditioned media from apoptotic MCF-7 or RKO cells were produced as described previously (Weigert et al., 2006). Briefly, MCF-7 cells, MCF-7 cells transfected with siRNA targeting SphK2, or RKO cells were exposed to 0.5 µg/ml staurosporine (Sigma) for 4 h in case of MCF-7 cells and 5 h in case of RKO cells. Subsequently, cells were washed twice with PBS, followed by an 2-h incubation in full medium. Thereafter, these conditioned media were harvested by centrifugation (13.000 x g, 5 min) and filtration through 0.2-µm pore filters, to remove apoptotic bodies.
Quantification of Cytokines
FACS analysis using BD Cytometric Bead Array Flex Sets following the instructions provided by the manufacturer allowed quantification of cytokines (TNF-, IL-10, IL-8, IL-12-p70) in the supernatant of cocultures. The samples were acquired with the FACSCanto (BD Biosciences, San Jose, CA) flow cytometer and analyzed with BD Biosciences' FCAP software. Performance matched the specifications of the manufacturer. Minimal detection limits were 1.9 pg/ml for TNF-, 3.8 pg/ml for IL-10, 4.9 pg/ml for IL-8, and 4.8 pg/ml for IL-12-p70. Routinely, medium from coculture setups were changed every 24 h. Thus, cytokine production reflects a sampling period corresponding to the last 24 h of the coculture, only. If not stated otherwise, cocultures lasted for 5 d with medium changes every 24 h.
Western Blot Analysis
Western blot analysis was performed as described (von Knethen et al., 2005). A mAb directed toward Bcl-2 (BD Transduction Laboratories, Lexington, KY) and polyclonal antibodies against Sphk1 or Sphk2 (Exalpha Biologicals, Watertown, MA) were used.
Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared as described (Von Knethen and Brune, 2001). An established electrophoretic mobility shift assay (EMSA) method, with slight modifications, was used (Camandola et al., 1996). Nuclear protein (20 µg) was incubated for 30 min at room temperature with 2 µg poly(dI-dC) from Amersham Biosciences (Freiburg, Germany), 2 µl buffer D (20 mM HEPES/KOH, 20% glycerol, 100 mM KCl, 0.5 mM EDTA, 0.25% Nonidet P-40, 2 mM DTT, 0.5 mM PMSF, pH 7.9), 4 µl buffer F (20% Ficoll-400, 100 mM HEPES/KOH, 300 mM KCl, 10 mM DTT, 0.5 mM PMSF, pH 7.9), and 250 fmol 5'-IRD700-labeled oligonucleotide (Metabion, Planegg-Martinsried, Germany) in a final volume of 20 µl. Specific p65 and p50 supershift antibodies (2 µg) were added as indicated. DNA–protein complexes were resolved at 200 V for 2 h using native 4% polyacrylamide gels and visualized with the Odyssey infrared imaging system (Li-Cor, Lincoln, Nebraska). Oligonucleotides with the consensus NF-B site (boldface letters) were used (Peng et al., 1995) : 5'-GCC AGT TGA GGG GAC TTT CCC AGG C-3'; 3'-C GGT CAA CTC CCC TGA AAG GGT CCG-5'. Supershift analysis was performed with -p65 and -p50 from Santa Cruz Biotechnology (Heidelberg, Germany). The latter antibody is known to attenuate the protein–DNA interaction rather than causing a classical supershift (He et al., 2004).
Transfections
To overexpress Bcl-2, MCF-7 cells were transfected with the pRc/CMVbcl2 plasmid (Messmer et al., 1996) using Nucleofector technology (Amaxa, Köln, Germany), according to the manufacturer's instructions. For knockdown of Sphk-isoforms, Sphk1-specific small interfering RNA (siRNA) or Sphk2 predesigned Hs_SPHK2_3 siRNA (Qiagen, Chatsworth, CA) were nucleofected into MCF-7 cells. Nucleofection efficiency was about 70%, as verified by flow cytometry after nucleofection of MCF-7 cells with pmaxGFP (Amaxa; data not shown). Overexpression of Bcl-2 and Sphk1 or Sphk2 knockdown was controlled by Western blot analysis 48 h subsequent to nucleofection. Knockdown of Sphk isoforms was controlled by siCONTROL nontargeting Duplex 1 (Dharmacon Research, Boulder, CO). After knockdown of Sphk isoforms, MCF-7 cells were cultured for another 24 h and subsequently were added to macrophage cultures.
S1P Quantification in Cell Culture Supernatants
Quantification of S1P from cell culture supernatants with liquid chromatography tandem mass-spectrometry (LC-MS/MS) was performed as described previously (Schmidt et al., 2006; Weigert et al., 2006). After 24 h, medium in cocultures of human macrophages and MCF-7 cells was replaced by medium free of FCS. Cocultures were maintained for another 24 h, and supernatants were harvested and analyzed. The lower limit of quantification was found to be 0.5 ng/ml. Variations in accuracy and intraday and interday precision (n = 6 for each concentration) were <10%.
Statistical Analysis
p values were calculated using the paired Student's t test combined with Bonferroni correction, as well as ANOVA. Differences were considered significant at p < 0.05, unless indicated otherwise.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, IL-10, and IL-8 in the coculture supernatants at different time points (Figure 1). The coculture provoked induction of TNF- and IL-10 at 24 h, followed by a decrease from 48 to 120 h, which was markedly stronger in case of TNF- (Figure 1A) compared with IL-10 (Figure 1B). In contrast, IL-8 production did not peak after 24 h, but increased steadily up to 120 h (Figure 1C). Cytokine production in either control macrophages or MCF-7 cells, cultured for 24 h was low. Interestingly, adding fresh MCF-7 cells for 24 h to the culture systems that already lasted for 120 h (second coculture) failed to induce TNF- (Figure 1A), but elicited an increase in IL-10 (Figure 1B), whereas IL-8 production remained high (Figure 1C). We concluded that MCF-7 cells, cocultured with macrophages elicited a transient proinflammatory response followed by an early production of TNF-, a more persistent formation of the anti-inflammatory cytokine IL-10 and a strong induction of IL-8.
|
at the end of the 5-d coculture with MCF-7 cells compared with control macrophages (Figure 2A). Residual MCF-7 cells were removed by incubations with accutase for 5 min, which left adherence of macrophages unaltered, thus proving that this setup exclusively determines cytokine production from macrophages (Figure 2B). Activation of naive macrophages with LPS/IFN- stimulated the production of TNF- and IL-10 but not of IL-8 compared with resting cells. Addition of LPS/IFN- to macrophages from the coculture setup produced significantly less TNF- and revealed markedly increased levels of IL-8, whereas IL-10 remained unchanged. In the past, decreased production of proinflammatory IL-12-p70 was connected to alternative macrophage activation and was attributed to increased IL-10 (Sica et al., 2000). Because IL-10 levels in macrophages from cocultures were enhanced compared with naive macrophages even without LPS/IFN- stimulation (Figure 1B), we investigated the amount of IL-12-p70 in our system. Administration of LPS/IFN- strongly induced IL-12-p70 in control macrophages, but remained significantly lower when stimulating macrophages from the coculture setup (Figure 2A). These results suggest that MCF-7 cells provoked a macrophage phenotype shift toward an alternative activation profile. Activation of macrophages from cocultures with LPS/IFN- substantiated this phenotype switch, when following the production of classical pro-versus anti-inflammatory mediators. Although principal observations pointing to alternative macrophage activation during coculture with MCF-7 cells were obvious (Figure 1), stimulation of naive versus coculture-primed macrophages with LPS/IFN- made the phenotype shift more definite. Therefore, we used LPS/IFN- stimulation in further experiments to demonstrate macrophage phenotype alterations. Infiltrating macrophages may comprise up to 50% of the tumor cell mass (Murdoch et al., 2004). Nevertheless, we asked whether or not higher cell numbers of tumor cells may change the phenotypic alterations that we observed. When we used ratios of 5:1 or 10:1 in cocultures, the decrease of TNF- became more pronounced, with more tumor cells added to macrophages, whereas the increase in IL-8 was unaffected (Figure 2C).
|
) in our system (Figure 2D). We noticed a continuous reduction of MCF-7 cells starting at 24 h after initiating the coculture system that resulted in a complete loss of tumor cells at 72 h (Figure 2D). To ensure that MCF-7 did not simply up-regulate CD44, we also monitored the morphology of cells in cocultures. Based on morphological criteria, MCF-7 cells were completely absent after 72 h (data not shown). Therefore, we suspected macrophage-induced AC death of cancer cells. To prove this assumption, cells from cocultures were harvested at 12, 24, and 48 h and stained with anti-CD44 to distinguish between macrophages and MCF-7 cells. In addition, annexin V staining quantified phosphatidylserine exposure as an early marker of cell death. Annexin V positive MCF-7 cells were noticed 24 h after coculture with macrophages. The amount of apoptotic MCF-7 cells increased to 
50% after 48 h, whereas no significant changes in annexin V binding were observed in macrophages from cocultures or control macrophages, i.e., cultured in the absence of MCF-7 cells (Figure 3A). Our working hypothesis predicted that only apoptotic tumor cells evoked phenotype changes in macrophages. In further experiments, we therefore used two cells lines that were resistant to cell death by cocultured macrophages. First, we used MCF-7 cells that overexpress Bcl-2 (Figure 3, B and D), and second, we used RKO cells, which turned out to be naturally resistant (Figure 3C). The reason for RKO cell survival in cocultures remained unknown. However, they expressed higher amounts of Bcl-2 than naive MCF-7 cells, which may account for apoptosis resistance (data not shown). A 48-h coculture of MCF-7-Bcl-2 or RKO cells with human macrophages showed no signs of cell death, either in tumor cells or in macrophages. Under these conditions, tumor cells and macrophages coexist, without initiation of cell death parameters. This is in contrast to the coculture of apoptosis sensitive MCF-7 cells exposed to macrophages.
|
–evoked cytokine responses after 5 d of coculture (Figure 4). Cytokine production of TNF-, IL-10, and IL-8 in naive macrophages after LPS/IFN- addition was significantly increased compared with unstimulated controls. Activation of macrophages coming from cocultures with MCF-7-Bcl-2 or RKO cells revealed a cytokine profile resembling that of naive macrophages after classic stimulation with LPS/IFN-. In contrast, cytokine formation of macrophages derived from cocultures with apoptotic sensitive MCF-7 cells was different. TNF- was lower, IL-8 was higher, and IL-10 remained unaltered. Apparently, MCF-7-Bcl-2 as well as RKO cells did not change the ability of macrophages to respond to LPS/INF- with the production of these cytokines compared with naive cells. Only the coculture with MCF-7 cells, which underwent AC death upon coculture with macrophages, altered cytokine production after LPS/IFN- stimulation. To corroborate that tumor cell death indeed was essential for alternative macrophage polarization, we induced apoptosis in RKO and MCF-7 cells with staurosporine and collected conditioned medium from these apoptotic tumor cells, which then was added to macrophages for 24 h. After changing the medium, macrophages were stimulated with LPS/IFN-. Supernatants from apoptotic tumor cells alone induced significant changes in the macrophage cytokine profile, independent of direct tumor cell–macrophage contacts. Secretion of IL-10 and IL-8 was enhanced, whereas IL-12-p70 production was decreased compared with naive macrophages (Figure 5A). Supernatants of apoptotic RKO or MCF-7 cells per se did not contain detectable amounts of cytokines (data not shown), except for IL-8 (about 200 pg/ml), which was nevertheless extremely low compared with the amounts that were produced by macrophages. Stimulation of macrophages exposed to tumor cell conditioned medium with LPS/IFN- elicited those phenotypic alterations, which we observed in direct cocultures, where tumor cell death occurred. TNF- and IL-12-p70 were decreased and IL-8 was enhanced, whereas IL-10 was high, but not significantly different from control macrophages (Figure 5A). We concluded that the macrophage phenotype shift was dependent on tumor cell apoptosis, rather than representing intrinsic features of different tumor cell lines. Moreover, because supernatants of apoptotic tumor cells evoked macrophage phenotype alterations, macrophage polarization demanded soluble, AC-derived factors. To further validate our data to be independent of a specific tumor cell line, we used four additional well-characterized tumor cell lines, such as the colon carcinoma cell lines Caco-2 and Colo201, the lung cancer cell line A549, and the hepatocellular carcinoma cell line Hep-G2 in our coculture system. After 5 d of coculture, Caco-2, Colo201, and A549 cells were absent from cocultures, whereas Hep-G2 cells were growing normally (data not shown). After removal of residual tumor cells and stimulation with LPS/IFN-, macrophages that had killed tumor cells displayed a cytokine profile comparable to, but even more pronounced than those from MCF-7 cocultures (Figure 5B). TNF- was significantly lower compared with control macrophages stimulated with LPS/IFN-, whereas IL-8 was significantly increased. Macrophages from cocultures with Hep-G2 cells displayed an activation profile similar to control macrophages, stimulated with LPS/IFN- (Figure 5B).
|
|
). Considering existing evidence that associates S1P production with tumor vascularization and growth (LaMontagne et al., 2006; Visentin et al., 2006), we wanted to know whether S1P production in apoptotic MCF-7 cells contributed to macrophage polarization. This question was approached by using siRNA technology to knock down Sphk1 versus Sphk2 in MCF-7 cells. Knockdown of either Sphk isoforms in MCF-7 cells was successful (Figure 6D). siRNA supplied was isoform specific, and a control siRNA further proved specificity. As an additional control, we ruled out an interference of Sphk1 or Sphk2 knockdown on induction of MCF-7 cell death upon coculture with macrophages (Figure 6E). We then used naive macrophages, macrophages derived from a coculture with MCF-7 cells, or cells from a coculture of MCF-7 cells with either Sphk1 or Sphk2 being knocked down (Figure 6). After 5 d of coculture, tumor cells were removed, and macrophages were stimulated with LPS/IFN- to follow cytokine production of TNF- (Figure 6A), IL-10 (Figure 6B), or IL-8 (Figure 6C). As seen in previous experiments, macrophages from cocultures with MCF-7 cells displayed reduced TNF- and increased IL-8 but unaltered IL-10 production compared with naive macrophages exposed to LPS/IFN-. Knockdown of Sphk1 did not alter macrophage cytokine production compared with changes provoked by apoptotic MCF-7 cells. However, knockdown of Sphk2 affected the ability of MCF-7 cells to alter cytokine production in macrophages upon LPS/IFN- stimulation. With Sphk2 being suppressed in MCF-7 cells during the coculture, the macrophage response upon LPS/IFN- addition was similar to naive cells, producing high TNF- and low IL-8, with no changes in IL-10. A similar response in macrophages was noticed when exposed to conditioned media from apoptotic, i.e., staurosporine-treated MCF-7 cells, with SphK2 being knocked down by siRNA. Compared with conditioned media from apoptotic and SphK2-expressing MCF-7 cells, the release of IL-8 was attenuated and the production of IL-10 was lower in unstimulated macrophages (Figure 6F). Along that line, after stimulation with LPS/IFN, repression of TNF- seen with conditioned medium from apoptotic MCF-7 cells was reversed when exposed to conditioned medium derived from SphK2-kockdown cells (Figure 6F). To substantiate that tumor cell apoptosis and SphK2 knockdown in tumor cells affect the release of S1P, we used LC-MS/MS to quantify the lipid mediator, released into supernatants of cocultures of macrophages with MCF-7 cells or into the supernatant of MCF-7 cells treated with staurosporine (Table 1). Supernatants from cocultures of macrophages with MCF-7 cells, MCF-7 cells overexpressing Bcl-2, or MCF-7 cells with Sphk2 being knocked down, were collected at a time when apoptosis was high in MCF-7 cells, i.e., the 24–48-h period of coculture. Although S1P amounts in supernatants of control macrophages, cocultures of macrophages with Bcl-2–overexpressing- or Sphk2 knockdown MCF-7 cells were below the reliable limit of quantification (0.5 ng/ml), S1P levels in cocultures of macrophages with naive MCF-7 cells were significantly elevated (Table 1; p 
0.01). We obtained similar results when we quantified S1P in supernatants of viable or apoptotic, i.e., staurosporine-treated MCF-7 cells with or without SphK2 knockdown. Supernatants of viable MCF-7 lack S1P, whereas S1P was released from apoptotic MCF-7 cells upon staurosporine treatment. Knockdown of SphK2 abrogated this response significantly (Table 1; p 0.01). These results imply that S1P is generated by Sphk2 and is derived from dying MCF-7 cells.
|
|
B Activation in Macrophages). To validate our model with respect to this TAM marker, we looked for NF-B activation in macrophages exposed to apoptosis-sensitive versus -resistant MCF-7 cells. EMSA analysis showed NF-B activation in response to LPS/IFN- stimulation in macrophages from cocultures with MCF-7-Bcl-2 or RKO cells (Figure 7A, lanes 4–9). Supershift analysis was evident with an -p65 antibody, whereas -p50 reduced NF-B-DNA binding without shifting the complex to higher molecular mass. When macrophages had been cocultured with MCF-7 cells before stimulation with LPS/IFN-, activation of NF-B was impaired (Figure 7A, lanes 1–3). Negligible activation of NF-B was also seen when macrophages were pre-exposed for 2 d to authentic S1P (Figure 7B). Statistical quantification of relevant changes is shown in Figure 6C. Collectively, these data imply that S1P as well as apoptotic tumor cells suppress activation of NF-B in macrophages, whereas apoptotic resistant tumor cells do not share this behavior. Macrophage polarization by apoptotic tumor cells is evident at the level of cytokine production as well as NF-B activation.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Lewis and Pollard, 2006). Their protumor activities are associated with polarization toward an M2 phenotype. This alternative activation program is driven by the tumor microenvironment, which includes tumor-derived immunosuppressants as well as tumor hypoxia (Pollard, 2004). Our study points to tumor cell apoptosis as an additional process in affecting the tumor microenvironment and thus macrophage polarization (Kim et al., 2006). It has been shown previously that pretreatment of macrophages with apoptotic but not necrotic tumor cells reduced their cytotoxic behavior (Reiter et al., 1999). Considering the production of TNF- as a potentially harmful agent our work supports the notion that TNF- is produced in a coculture of macrophages with MCF-7 cells, with the latter ones being killed. However, once ACs had been generated in the coculture setup, the production of TNF- declined, and cocultures produced substantially smaller amounts of the cytotoxic agent upon LPS/IFN- stimulation compared with naive cell activation. Moreover, the second administration of fresh tumor cells to macrophages primed by apoptotic tumor cells did not elicit a new peak of TNF- formation and did not result in tumor cell killing (data not shown). Along that line, MCF-7 cells overexpressing Bcl-2 were not killed by macrophages, because Bcl-2–overexpressing cells, especially Bcl-2–expressing MCF-7 cells, are resistant to TNF-–induced apoptosis (Burow et al., 1998). Thus, the presence of ACs attenuates TNF- formation in macrophages, thereby affecting their cytotoxic potential toward tumor cells. Presumably the interaction with apoptotic tumor cells affects other mechanisms of macrophage cytotoxicity, which were investigated previously (Reiter et al., 1999). Nevertheless, tumor cell killing by macrophages that were primed with dying tumor cells beforehand was disabled. Apoptotic cells, besides reducing macrophage cytotoxicity, may further affect the tumor microenvironment because they release immune modulators such as TGF- and IL-10 (Tomimori et al., 2000; Chen et al., 2001). We did not observe the release of IL-10 from dying tumor cells in our system (data not shown). Nevertheless, conditioned medium from apoptotic tumor cells induced phenotypical alterations in macrophages.
As in cocultures with MCF-7 cells, IL-10 production in macrophages was enhanced with conditioned medium from dying tumor cells. Although mechanistically unexplained, an increase in IL-10 was correlated to defective NF-B binding in TAMs (Sica et al., 2000), and the inability to activate NF-B was also observed upon recognition of ACs by macrophages (Cvetanovic and Ucker, 2004). A short-term binding of ACs to macrophages did not affect NF-B DNA binding, but depleted p300 to impair transcriptional activation of NF-B (Cvetanovic and Ucker, 2004). Importantly, attenuated NF-B activity appeared critical in shaping the TAM phenotype (Biswas et al., 2006). Our experiments favored S1P, a soluble factor, in blocking NF-B, after long-term exposure of ACs to macrophages. This was unexpected, because NF-B activation rather than inhibition was shown for S1P in other cell systems (Siehler et al., 2001). Despite S1P might induce low NF-B activation, it completely abrogated TNF-–mediated stimulation of NF-B in THP-1 human monocytes (Kimura et al., 2006). Although mechanistically unclear at present, the impact of S1P on NF-B may help to understand macrophage polarization by apoptotic tumor cells. Activation versus inhibition may depend on the S1P receptor profile and/or duration toward S1P exposure, as well as the presence or absence of costimuli such as LPS/IFN-.
S1P from apoptotic MCF-7 cells changed the LPS/IFN- activation profile in macrophages. It suppressed TNF- formation but increased IL-8 production in macrophages upon LPS/IFN- activation and most likely provoked IL-10 liberation during phagocytosis of dying cells. Reduced TNF- production may result from a diminished NF-B activity, whereas alterations in interleukin production may be associated with activation of STAT1 and/or PI3K-signaling, because these pathways characterize M2 cells (Rauh et al., 2005; Biswas et al., 2006). Production of the immunosuppressive cytokine IL-10 is characteristic for TAMs. Although underlying molecular pathways remain unclear, it is known that S1P provoked secretion of IL-10 from T-cells and dendritic cells, which was correlated to a diminished proinflammatory activity in these cells (Idzko et al., 2002; Wang et al., 2005). Moreover, IL-10 formation may require PI3K signaling, a pathway which can be activated by S1P receptors (Taha et al., 2004). Furthermore, PI3K may negatively regulate TLR signaling during inflammation and has been proposed as a potential target to circumvent immunosuppression in cancer (Fukao and Koyasu, 2003).
As stated, IL-10 was previously related to defective IL-12 production in TAM, which was furthermore explained by defective NF-B signaling (Sica et al., 2000). IL-10 was produced in cocultures with MCF-7 cells, even after tumor cells were killed. Therefore, we expected that coculture-primed macrophages should produce less IL-12-p70, which indeed was the case. Because IL-12-p70 and TNF- production in macrophages from cocultures was equally affected, we did not follow the release of IL-12-p70 in further experiments. However, we showed that S1P reduced NF-B DNA-binding, which is likely to attenuate generation of IL-12-p70 as well.
Enhanced production of IL-8 in macrophages derived from MCF-7 cocultures is not immediately apparent considering the attenuated activity of NF-B, a known inducer of IL-8 (Hoffmann et al., 2002). Under conditions of S1P release, other transcriptional activators of IL-8 such as C/EBP, AP-1, or STAT1 may substitute for NF-B, and it will be interesting in the future to define how S1P enhances IL-8 production.
Despite uncertainties in IL-8 regulation, its production may explain how S1P contributes to tumor angiogenesis, because IL-8 expression was related to TAM-dependent angiogenesis and poor prognosis in uterine cancer (Fujimoto et al., 2002). Recent in vivo studies refer to a role of S1P in tumor vascularization (LaMontagne et al., 2006; Visentin et al., 2006). Forced S1P receptor desensitization with FTY720 abrogated S1P- and VEGF-induced angiogenesis and attenuated metastatic tumor growth (LaMontagne et al., 2006). In addition, the use of a monoclonal S1P antibody reduced tumor growth via inhibition of tumor angiogenesis and survival (Visentin et al., 2006). These observations support a direct connection between S1P production and tumorigenesis, although the source of S1P in human tumors is undefined.
Considering that high levels of Sphk1 in glioblastoma correlated with low patient survival (Van Brocklyn et al., 2005), it seems attractive to assume gain of expression regulation as an underlying mechanism. In our study, we identified Sphk2 activity in close association with tumor cell apoptosis by macrophages as another possible S1P origin. As shown by LC-MS/MS, the release of S1P from apoptotic, but not viable tumor cells induced macrophage polarization toward an alternatively activated phenotype, as characterized by a change in cytokine production, reduced tumor cytotoxicity and an attenuated NF-B response.
Previously, other factors such as TGF- or phosphatidylserine were considered to provoke phenotype alterations in macrophages after the interaction with ACs (Chen et al., 2001; Savill et al., 2002). Because suppression of S1P release via SphK2 knockdown did not restore TNF- completely, these mediators might contribute in shaping the macrophage phenotype. However, S1P was shown to mimic TGF- responses by cross-activating the TGF-II receptor (Xin et al., 2004), which might support a prominent role of S1P. Taken together, our results suggest that induction of tumor cell apoptosis by invading macrophages, at least during early stages of tumor formation, may help to understand formation and action of TAMs.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
Address correspondence to: Bernhard Brüne (bruene{at}zbc.kgu.de)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bingle, L., Brown, N. J., and Lewis, C. E. (2002). The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J. Pathol 196, 254–265.[CrossRef][Medline]
Biswas, S. K. et al. (2006). A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation). Blood 107, 2112–2122.
Bondanza, A. et al. (2004). Inhibition of phosphatidylserine recognition heightens the immunogenicity of irradiated lymphoma cells in vivo. J. Exp Med 200, 1157–1165.
Burow, M. E., Weldon, C. B., Tang, Y., Navar, G. L., Krajewski, S., Reed, J. C., Hammond, T. G., Clejan, S., and Beckman, B. S. (1998). Differences in susceptibility to tumor necrosis factor alpha-induced apoptosis among MCF-7 breast cancer cell variants. Cancer Res 58, 4940–4946.
Camandola, S., Leonarduzzi, G., Musso, T., Varesio, L., Carini, R., Scavazza, A., Chiarpotto, E., Baeuerle, P. A., and Poli, G. (1996). Nuclear factor kB is activated by arachidonic acid but not by eicosapentaenoic acid. Biochem. Biophys. Res. Commun 229, 643–647.[CrossRef][Medline]
Chen, W., Frank, M. E., Jin, W., and Wahl, S. M. (2001). TGF-beta released by apoptotic T cells contributes to an immunosuppressive milieu. Immunity 14, 715–725.[CrossRef][Medline]
Cvetanovic, M., and Ucker, D. S. (2004). Innate immune discrimination of apoptotic cells: repression of proinflammatory macrophage transcription is coupled directly to specific recognition. J. Immunol 172, 880–889.
Draffin, J. E., McFarlane, S., Hill, A., Johnston, P. G., and Waugh, D. J. (2004). CD44 potentiates the adherence of metastatic prostate and breast cancer cells to bone marrow endothelial cells. Cancer Res 64, 5702–5711.
Fujimoto, J., Aoki, I., Khatun, S., Toyoki, H., and Tamaya, T. (2002). Clinical implications of expression of interleukin-8 related to myometrial invasion with angiogenesis in uterine endometrial cancers. Ann. Oncol 13, 430–434.
Fukao, T., and Koyasu, S. (2003). PI3K and negative regulation of TLR signaling. Trends Immunol 24, 358–363.[CrossRef][Medline]
Gordon, S. (2003). Alternative activation of macrophages. Nat. Rev. Immunol 3, 23–35.[CrossRef][Medline]
Gregory, C. D., and Devitt, A. (2004). The macrophage and the apoptotic cell: an innate immune interaction viewed simplistically? Immunology 113, 1–14.[CrossRef][Medline]
He, B., Chadburn, A., Jou, E., Schattner, E. J., Knowles, D. M., and Cerutti, A. (2004). Lymphoma B cells evade apoptosis through the TNF family members BAFF/BLyS and APRIL. J. Immunol 172, 3268–3279.
Hoffmann, E., Dittrich-Breiholz, O., Holtmann, H., and Kracht, M. (2002). Multiple control of interleukin-8 gene expression. J. Leukoc. Biol 72, 847–855.
Idzko, M. et al. (2002). Sphingosine 1-phosphate induces chemotaxis of immature and modulates cytokine-release in mature human dendritic cells for emergence of Th2 immune responses. FASEB J 16, 625–627.
Kim, R., Emi, M., Tanabe, K., and Arihiro, K. (2006). Tumor-driven evolution of immunosuppressive networks during malignant progression. Cancer Res 66, 5527–5536.
Kimura, T. et al. (2006). Sphingosine 1-phosphate receptors mediate stimulatory and inhibitory signalings for expression of adhesion molecules in endothelial cells. Cell Signal 18, 841–850.[CrossRef][Medline]
LaMontagne, K. et al. (2006). Antagonism of sphingosine-1-phosphate receptors by FTY720 inhibits angiogenesis and tumor vascularization. Cancer Res 66, 221–231.
Lewis, C. E., and Pollard, J. W. (2006). Distinct role of macrophages in different tumor microenvironments. Cancer Res 66, 605–612.
Mantovani, A., Allavena, P., and Sica, A. (2004a). Tumour-associated macrophages as a prototypic type II polarised phagocyte population: role in tumour progression. Eur. J. Cancer 40, 1660–1667.[CrossRef][Medline]
Mantovani, A., Sica, A., Sozzani, S., Allavena, P., Vecchi, A., and Locati, M. (2004b). The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 25, 677–686.[CrossRef][Medline]
Mantovani, A., Sozzani, S., Locati, M., Allavena, P., and Sica, A. (2002). Macrophage polarization: tumor-associated macrophages as a paradigm for polarized M2 mononuclear phagocytes. Trends Immunol 23, 549–555.[CrossRef][Medline]
Messmer, U. K., Reed, U. K., and Brune, B. (1996). Bcl-2 protects macrophages from nitric oxide-induced apoptosis. J. Biol. Chem 271, 20192–20197.
Murdoch, C., Giannoudis, A., and Lewis, C. E. (2004). Mechanisms regulating the recruitment of macrophages into hypoxic areas of tumors and other ischemic tissues. Blood 104, 2224–2234.
Peng, H. B., Libby, P., and Liao, J. K. (1995). Induction and stabilization of I kappa B alpha by nitric oxide mediates inhibition of NF-kappa B. J. Biol. Chem 270, 14214–14219.
Pollard, J. W. (2004). Tumour-educated macrophages promote tumour progression and metastasis. Nat. Rev. Cancer 4, 71–78.[CrossRef][Medline]
Rauh, M. J., Ho, V., Pereira, C., Sham, A., Sly, L. M., Lam, V., Huxham, L., Minchinton, A. I., Mui, A., and Krystal, G. (2005). SHIP represses the generation of alternatively activated macrophages. Immunity 23, 361–374.[CrossRef][Medline]
Reiter, I., Krammer, B., and Schwamberger, G. (1999). Cutting edge: differential effect of apoptotic versus necrotic tumor cells on macrophage antitumor activities. J. Immunol 163, 1730–1732.
Savill, J., Dransfield, I., Gregory, C., and Haslett, C. (2002). A blast from the past: clearance of apoptotic cells regulates immune responses. Nat. Rev. Immunol 2, 965–975.[CrossRef][Medline]
Schmidt, H., Schmidt, R., and Geisslinger, G. (2006). LC-MS/MS-analysis of sphingosine-1-phosphate and related compounds in plasma samples. Prostaglandins Other Lipid Mediat 81, 162–170.[CrossRef][Medline]
Sica, A., Saccani, A., Bottazzi, B., Polentarutti, N., Vecchi, A., van Damme, J., and Mantovani, A. (2000). Autocrine production of IL-10 mediates defective IL-12 production and NF-kappa B activation in tumor-associated macrophages. J. Immunol 164, 762–767.
Sica, A., Schioppa, T., Mantovani, A., and Allavena, P. (2006). Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur. J. Cancer 42, 717–727.[CrossRef][Medline]
Siehler, S., Wang, Y., Fan, X., Windh, R. T., and Manning, D. R. (2001). Sphingosine 1-phosphate activates nuclear factor-kappa B through Edg receptors. Activation through Edg-3 and Edg-5, but not Edg-1, in human embryonic kidney 293 cells. J. Biol. Chem 276, 48733–48739.
Taha, T. A., Argraves, K. M., and Obeid, L. M. (2004). Sphingosine-1-phosphate receptors: receptor specificity versus functional redundancy. Biochim. Biophys. Acta 1682, 48–55.[Medline]
Tomimori, Y., Ikawa, Y., and Oyaizu, N. (2000). Ultraviolet-irradiated apoptotic lymphocytes produce interleukin-10 by themselves. Immunol. Lett 71, 49–54.[CrossRef][Medline]
Van Brocklyn, J. R., Jackson, C. A., Pearl, D. K., Kotur, M. S., Snyder, P. J., and Prior, T. W. (2005). Sphingosine kinase-1 expression correlates with poor survival of patients with glioblastoma multiforme: roles of sphingosine kinase isoforms in growth of glioblastoma cell lines. J. Neuropathol. Exp. Neurol 64, 695–705.[Medline]
Visentin, B. et al. (2006). Validation of an anti-sphingosine-1-phosphate antibody as a potential therapeutic in reducing growth, invasion, and angiogenesis in multiple tumor lineages. Cancer Cell 9, 225–238.[CrossRef][Medline]
von Knethen, A., Tautenhahn, A., Link, H., Lindemann, D., and Brune, B. (2005). Activation-induced depletion of protein kinase C alpha provokes desensitization of monocytes/macrophages in sepsis. J. Immunol 174, 4960–4965.
Von Knethen, A. A., and Brune, B. (2001). Delayed activation of PPARgamma by LPS and IFN-gamma attenuates the oxidative burst in macrophages. FASEB J 15, 535–544.
Wang, W., Graeler, M. H., and Goetzl, E. J. (2005). Type 4 sphingosine 1-phosphate G protein-coupled receptor (S1P4) transduces S1P effects on T cell proliferation and cytokine secretion without signaling migration. FASEB J 19, 1731–1733.
Weigert, A., Johann, A. M., von Knethen, A., Schmidt, H., Geisslinger, G., and Brune, B. (2006). Apoptotic cells promote macrophage survival by releasing the antiapoptotic mediator sphingosine-1-phosphate. Blood 108, 1635–1642.
Xin, C., Ren, S., Kleuser, B., Shabahang, S., Eberhardt, W., Radeke, H., Schafer-Korting, M., Pfeilschifter, J., and Huwiler, A. (2004). Sphingosine 1-phosphate cross-activates the Smad signaling cascade and mimics transforming growth factor-beta-induced cell responses. J. Biol. Chem 279, 35255–35262.
This article has been cited by other articles:
|
|
 |
|
  N. Weis, A. Weigert, A. von Knethen, and B. Brune Heme Oxygenase-1 Contributes to an Alternative Macrophage Activation Profile Induced by Apoptotic Cell Supernatants Mol. Biol. Cell, March 1, 2009; 20(5): 1280 - 1288. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. M. Johann, A. Weigert, W. Eberhardt, A.-M. Kuhn, V. Barra, A. von Knethen, J. M. Pfeilschifter, and B. Brune Apoptotic Cell-Derived Sphingosine-1-Phosphate Promotes HuR-Dependent Cyclooxygenase-2 mRNA Stabilization and Protein Expression J. Immunol., January 15, 2008; 180(2): 1239 - 1248. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
