|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
Blood, 15 July 2006, Vol. 108, No. 2, pp. 525-535. Prepublished online as a Blood First Edition Paper on March 9, 2006; DOI 10.1182/blood-2005-09-3777.
IMMUNOBIOLOGY
Peroxisome proliferator-activated receptor
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
(PPAR) and inhibition of phospholipase A2 (PLA2) activity cooperate to alleviate CTL suppression. Of importance, purified tumor-associated macrophages display a similar M2 phenotype and are suppressive for antitumor CTLs, via a mechanism that can be almost completely reversed by PPAR ligands. Overall, our data identify PLA2 and especially PPAR as new potential therapeutic targets to subvert macrophage-mediated CTL suppression in cancer. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
expression and as such impair T-cell function.13-14 However, our own previous work demonstrates that arginase-1 activity is not necessarily implicated in the T-cell down-modulation by arginase-1+ myeloid suppressor cells (MSCs) from mice with progressing tumors, suggesting the existence of as-yet-unexplored suppressive mechanisms in these cells.15
Elevated arginase-1 activity is one of the hallmarks of IL-4– and/or IL-13–elicited alternatively activated myeloid cells (M2), as opposed to IFN- and/or TNF-induced classically activated myeloid cells (M1).16 Recently, the M2 concept has been broadened to myeloid cells developing in the presence of immune complexes and TLR ligands (M2b), or IL-10 (M2c),17 sharing overlapping functions: down-regulation of type I cytokine-driven inflammation and stimulation of angiogenesis and wound healing.18 This type of cell has been observed in different pathologies, including parasite infections19-21 and tumors.22 In a tumor context, M2 cells are, at least in part due to their immunosuppressive properties, associated with an exacerbation of the disease, whereas the induction of cytotoxic M1 contributes to tumor rejection.23-26 Therefore, a better understanding of the molecular and functional profile of cancer-associated M2 is warranted to use those cells as potential targets in cancer therapy.
In the present study, we aimed at obtaining a better insight into the origin, molecular profile, and cytotoxic T lymphocyte (CTL)–suppressive mechanism of in vitro–generated splenic MSCs from mice with progressing T-cell lymphoma tumors, and translating this information to the characteristics of in vivo–generated suppressive myeloid cells. We demonstrated that the monocytic fraction of splenic CD11b+Gr-1+ cells is sufficient for the generation of M2-oriented, CTL-suppressive macrophages. M2-associated genes, peroxisome proliferator-activated receptor (PPAR) and phospholipase A2 (PLA2), were identified as targets for suppression-lifting intervention, not only on in vitro–generated MSCs but, more importantly, also on M2-oriented tumor-associated macrophages.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Six- to 9-week-old female AKR mice (H-2k; Harlan, Horst, The Netherlands) were implanted subcutaneously in the flank with 2 x 106 BW-Sp3 cancer cells, and 5 to 7 weeks later mice were used for experiments.
Cell lines and media
The generation of BW-Sp3 and the BW-Sp3(B7-1) cells was described earlier.27-28 Cancer cells were maintained in RPMI 1640 medium, supplemented with 10% heat-inactivated FCS, 0.03% L-glutamine, 100 mg/mL streptomycin, and 100 mg/mL penicillin (Invitrogen, Frederick, MD). For splenocyte cultures, this medium was supplemented with 1 mM nonessential amino acids, 1 mM sodium pyruvate (Invitrogen), and 0.02 mM 2-ME (ME medium).
Fluorescence-activated cell sorter (FACS) staining
Cells were stained for 20 minutes at 4°C using conventional protocols: preincubation with anti-FcR Ab (2.4G2) before adding at 1 µg/106 cells anti-CD11b/PE or anti-CD11b/FITC (M1/70), anti–Gr-1/FITC (RB6-8C5), anti-Ly6G/FITC (1A8), anti-Ly6C/FITC (AL-21), anti-F4/80/PE (CI:A3-1), anti-CD31/PE (MEC13.3), anti-CD68/PE (FA-11), anti-CD11c/PE (HL3), or PE/FITC-conjugated isotype controls. The 7/4 antibody (gift of Dr Gordon Brown, Sir William Dunn School of Pathology, Oxford, United Kingdom) and anti–M-CSFR (AFS98; gift of Dr Pieter Leenen, Erasmus Medical Center, Rotterdam, the Netherlands) were used as purified Abs and hybridoma supernatant, respectively, detected by polyclonal anti–rat Ig/PE. All antibodies were from BD Biosciences (San Jose, CA), except anti-F4/80 and anti-CD68 (Serotec, Raleigh, NC).
For intracellular cytokine staining, 5 x 105 cells were stimulated for 6 hours with anti-CD3. GolgiPlug (BD Biosciences) was added during the last 4 hours. Following CD8/FITC (53-6.7) staining, cells were stained intracellularly using Cytofix/Cytoperm (BD Biosciences), according to the manufacturer's instructions. Antibodies were anti–IFN-/PE (XMG1.2) and rat IgG1 isotype/PE (R3-34) (BD Biosciences).
For detection of ROSs, cells were incubated with 2 µM dihydroethytium (DHE; Molecular Probes, Eugene, OR) for 1 hour, or 2 µM dichlorodihydrofluorescein diacetate (DCFDA; Molecular Probes) for 30 minutes at 37°C. Cells were washed, incubated at 4°C with anti-CD11b/PE, and analyzed on a FACSVantage SE flow cytometer (BD Biosciences).
In vivo depletion of granulocytes
The anti–Gr-1 Ab (RB6-8C5) was purified from hybridoma supernatant, using standard techniques, and tested for LPS contamination, using the LAL assay (BioWhittaker, Walkersville, MD). LPS-free Ab (100 µg) was injected intraperitoneally in 5- to 7-week tumor-bearing mice and 24 hours later spleens were taken.
Purification and depletion of cell populations
To isolate tumor-associated macrophages, tumors were dissected and chopped into small pieces before incubation with an enzyme mixture dissolved in RPMI 1640 for 30 minutes at 37°C: 400 U/mL collagenase type IV, 0.05 mg/mL collagenase type I, 0.025 mg/mL hyaluronidase (Sigma-Aldrich, St Louis, MO), 0.01 mg/mL Dnase I, 0.2 U/mL soybean trypsin inhibitor (Boehringer Mannheim, Mannheim, Germany). Single-cell suspensions were made in magnetic-activated cell sorter (MACS) buffer (degassed PBS, 0.5% BSA, 2 mM EDTA) and incubated (15 minutes, 8°C) with anti-CD11b microbeads (5 µL beads/107 cells; Miltenyi Biotec, Auburn, CA). CD11b+ cells were isolated on LS columns (Miltenyi Biotec), using the MidiMACS system (Miltenyi Biotec) according to the manufacturer's instructions.
To isolate peritoneal macrophages, peritoneal cells were collected in 0.34 M sucrose, resuspended in MACS buffer, incubated with anti-CD19 microbeads, and passed over LD columns (both Miltenyi Biotec), using the MidiMACS system (depletion of CD11b+CD19+ cells). Flow-through was collected, and remaining CD11b+ cells were isolated on LS columns. These cells were more than 90% CD11b+F4/80+ macrophages.
Depletion of splenic CD11b+ cells was performed using anti-CD11b microbeads and LD columns. To purify CD11b+Gr-1+ cells, splenocytes were first depleted of CD19+ and CD4+ cells, using anti-CD19 microbeads, anti-CD4 microbeads, and LD columns. Flow-through was collected, and remaining CD11b+ cells were isolated on LS columns as described in the first paragraph of this section. These cells were more than 90% CD11b+Gr-1+.
Evaluation of CTL activity
Splenocytes from naive AKR or BW-Sp3 progressors were restimulated at 2 x 107 splenocytes/well with 106 irradiated (110 Gray [11 000 rad]) BW-Sp3(B7-1) cells in 6-well plates (Falcon; Becton Dickinson, Lincoln Park, NJ) for 5 days in ME-medium, without addition of cytokines. When appropriate, the following compounds were added to the cultures at day 2: 200 U/mL superoxide dismutase (SOD, O2– scavenger; Sigma-Aldrich), 200 U/mL catalase (H2O2 scavenger; Sigma), 500 ng/mL anti–PD-L2 (eBioscience, San Diego, CA), 6 µM baicalein (12/15 LOX inhibitor; Sigma), 10 µM GW9662 (PPAR inhibitor; Sigma), 5 µM rosiglitazone (PPAR ligand; Cayman Chemical, Ann Arbor, MI), 1 µM 15-deoxy-
12,14-prostaglandin J2 (15d-PGJ2, PPAR ligand; Cayman Chemical), 3 µM aristolochic acid (PLA2 inhibitor; Sigma), 2.5 µM AACOCF3 (cPLA2 inhibitor; Biomol International, Plymouth Meeting, PA), 2.5 µM PACOCF3 (iPLA2 inhibitor; Calbiochem, San Diego, CA), and 0.5 µM ONO-RS-082 (sPLA2 inhibitor; Biomol International). Stock solutions of the latter 8 compounds were made in DMSO. Therefore, a corresponding volume of DMSO was added to cultures without these compounds.
Alternatively, nonadherent cells were removed after 2 days of culture and restimulated for another 3 days in a new plate. To test cytotoxicity to CTLs, the compounds were added to these nonadherent cell cultures.
For each of these cultures, the nonadherent cells were recuperated after 5 days and loaded on Ficoll-Paque (Pharmacia Biotech, Piscataway, NJ) for gradient centrifugation. Viable cells were tested for their cytotoxicity toward 111In-labeled BW-Sp3(B7-1), as described earlier.15 Where appropriate, percent CTL recovery was calculated as follows: ([% specific lysis Total + inhibitor] – [% specific lysis Total])/([% specific lysis Nad] – [% specific lysis Total]) x 100.
For flow cytometry and functional assays, adherent cells were recovered at different time points, using Cell Dissociation Buffer (Gibco, Carlsbad, CA) and gentle scraping.
RNA extraction and quantitative reverse-transcriptase–polymerase chain reaction (RT-PCR) analysis
Total RNA (1 µg), prepared using TRIzol reagent (Invitrogen), was reverse-transcribed using oligo(dT) and SuperScript II RT (Invitrogen). Quantitative real-time PCR was performed in a Bio-Rad iCycler, with Bio-Rad iQ SYBR Green Supermix (Bio-Rad, Hercules, CA), using primers listed in Table 1. For all primers, each PCR cycle consisted of 1 minute at 94°C, 45 seconds at 55°C, and 1 minute at 72°C. Gene expression was normalized using ribosomal protein S12 as housekeeping gene.
|
All comparisons were tested for statistical significance (P < .05) via the unpaired t test, using GraphPad Prism 3.0 software (GraphPad Software, San Diego, CA).
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Although the heterogeneity of splenic immature CD11b+Gr-1+ MSCs in mice with progressing tumors has been recognized,8 this population has up to now been studied as one entity. In order to refine the composition of MSCs, we used the BW-Sp3 T-cell lymphoma model for which the expansion of CD11b+Gr-1+ cells in the spleens of AKR mice with progressing tumors (progressors) has been reported.15 FACS analysis on freshly isolated splenocytes from 5- to 7-week tumor-bearing mice revealed that, within the CD11b+Gr-1+ fraction, 2 populations exist, differing mainly in the Gr-1 and to a lesser extent also the CD11b expression level (Figure 1A). Of interest, cells staining intermediately for Gr-1 and high for CD11b (CD11bhiGr-1int; gate R1) had a low SSC profile (SSClo), suggestive of monocyte-oriented myeloid cells, while the Gr-1 high expressers that were also the highest CD11b expressers (CD11bhi+Gr-1hi; gate R2) had a higher SSC (SSChi), suggestive of a granulocytic cell type (Figure 1B). To further establish the nature of both MSC subpopulations, additional markers with a monocyte/granulocyte discriminative potential were evaluated. In the first instance, Ly6G and Ly6C seemed interesting candidates, since the anti–Gr-1 antibody (RB6-8C5) recognizes a shared epitope on these molecules.29 Ly6G is specifically expressed on granulocytic cells,29 while Ly6C expression is higher on immature monocytic than on granulocytic cells.30 It was demonstrated that the CD11bhi SSClo cells did not express the Ly6G marker (gate R3), but were the highest expressers of Ly6C (gate R5) (Figure 1A-B), corroborating their monocytic lineage. In contrast, the CD11bhi+ SSChi population was uniformly positive for Ly6G (gate R4) and Ly6C (gate R6), the latter being expressed at a somewhat lower level compared with the CD11bhi SSClo cells. Hence, the CD11bhi+ SSChi cells displayed typical characteristics of the granulocyte lineage.
Taylor et al31 used the 7/4 antigen to define myeloid cell heterogeneity in the spleen, with high expression levels on Gr-1hi neutrophils, but even higher levels on Gr-1int monocytes. Accordingly, splenic Gr-1int MSCs of BW-Sp3 progressors expressed higher 7/4 levels than the Gr-1hi MSCs, strengthening their identification as monocytic and granulocytic cells, respectively (Figure 1A).
Final evidence for the distinction between both MSC populations came from the finding that the M-CSF receptor (M-CSFR), a prototypic monocyte/macrophage-lineage marker, was expressed only on the Gr-1int population (Figure 1C). In addition, F4/80 expression—another monocyte/macrophage lineage marker—was detected only on the Gr-1int cells, although not all of these cells scored positive (Figure 1C), a finding that was also reported for peripheral blood monocytes.32 Altogether, our data subdivide the CD11b+Gr-1+ splenic MSCs into CD11bhi Gr-1int SSClo Ly6Gneg Ly6Chi+ 7/4hi+ M-CSFRint monocytic cells and CD11bhi+Gr-1hiSSChiLy6GhiLy6Chi7/4hiM-CSFRneg granulocytic cells.
Finally, it should be remarked that these subpopulations already existed at low percentages in naive spleens (Figure 1D) and that both were equally expanded in function of tumor load. Subcutaneous BW-Sp3 inoculation can result in a dramatically different tumor outcome in different recipients, going from complete tumor rejection in some mice to tumor progression in others.15,27-28,33 At 6 weeks after tumor inoculation, no expansion of MSCs was seen in mice that had rejected their tumor (regressors); an intermediate expansion (2- to 3-fold) in mice whose tumors only recently started to progress after a stabilization phase (early progressors); and a strong expansion (6- to 8-fold) in mice with a longer history of progression and larger tumors (late progressors) (Figure 1D).
|
CD11b+Gr-1+ MSCs have been shown before to be responsible for the suppression of anti–BW-Sp3 CTLs upon in vitro culture.15 Having established the heterogeneity of splenic MSCs, we next wondered whether both subpopulations were strictly needed for this suppressive activity. Upon administration of anti–Gr-1 mAb to BW-Sp3 late progressors, the majority of the CD11b+Gr-1+ cells remaining in the spleen 24 hours after treatment were of the CD11bhiGr-1int phenotype (gate R1), lacking Ly6G (Figure 2A), with low SSC (Figure 2B) but expressing M-CSFR (Figure 2C), reminiscent of the monocytic MSC fraction. In quantitative terms, the percentage of splenic monocytic CD11b+Gr-1+ cells in the anti–Gr-1–treated mice (13.2% ± 3.1%) was on average in the same range as in untreated progressor mice (14.7% ± 5.0%).
In order to assess the impact of granulocyte depletion on the suppression of tumor-specific CTL activity, splenocytes from either naive, untreated progressor, or anti–Gr-1–treated progressor mice were restimulated in vitro with irradiated BW-Sp3(B7-1) cells for 5 days before testing the cytotoxic activity. In both anti–Gr-1–treated and untreated progressor spleens, CTL activity was as low as in naive spleens (Figure 3, Total). However, as opposed to naive spleens, strong anti–BW-Sp3 CTL activity was recovered by dissociating the plastic-nonadherent cells from the plastic-adherent splenocyte fraction in both types of progressors (Figure 3, Nad), reflecting the presence of CTL-suppressive cells in the progressor adherent population. Hence, the monocyte fraction of the CD11b+Gr-1+ splenocytes seemed to be sufficient for the generation of suppressive adherent cells in vitro.
|
|
Overall, our data firmly establish an association between suppression and the in vitro generation of mature macrophages but do not exclude a role for the CD11b+Gr-1+ precursors in CTL dysfunction. To directly compare the suppressive activity of both types of cells, a short-term assay was mandatory to preclude the possibility that CD11b+Gr-1+ cells would differentiate during the time course of the experiment. Therefore, purified CD11b+Gr-1+ cells or 5-day adherent splenocytes from either naive mice or late progressors were added at a 30% ratio to anti-CD3–stimulated regressor splenocytes (which contain activated antitumor CTLs, but only low amounts of CD11b+Gr-1+ cells, Figure 1D), and 6 hours later IFN- production by CD8+ cells was tested via intracellular FACS staining. Of interest, only late progressor—but not naive—adherent splenocytes significantly suppress the amount of IFN-–producing cells, while both type CD11b+Gr-1+ cells rather stimulate CD8+ IFN- production (Figure 5). Hence, under the given conditions, in vitro–differentiated macrophages from progressor spleens seem to be superior in suppressing T-cell functions compared with their naive counterparts or CD11b+Gr-1+ precursors.
CTL-suppressive macrophages are M2 oriented
Depending on the environment they encounter, macrophages can evolve into a continuum of distinct subsets, of which M1 and M2 are the 2 extremes. Of note, we previously reported a high arginase activity in the suppressor cells of the BW-Sp3 tumor model,15 suggesting an M2 orientation. To further expand this finding, we quantified—using real-time PCR—the expression of selected M2-associated genes, comparing the 5-day plastic-adherent cells from restimulated late progressor and naive spleens. The latter population also contained about 85% F4/80hiCD68hi macrophages (data not shown), making its composition comparable with the progressor cells. The tested genes were selected from a collection of M2-associated transcripts, obtained in our lab from a subtracted cDNA library between M1 and M2 elicited in a Trypanosoma brucei brucei infection model.35
Table 2 ("P/N at least 2") demonstrates that many of these M2-associated genes were at least 2-fold up-regulated in the progressor adherent splenocytes compared with the naive. Namely, the high induction of well-documented murine M2 markers, such as arginase-1,16 fibronectin-1,36 and especially Ym35 and FIZZ1,35 strengthens the notion that these cells were strongly M2 polarized. The presence of other IL-4– and/or IL-13–inducible genes (CCL24,37 E-cadherin,38 MGL-1,39 MGL-2,39 coagulation factor XIIIa,40 PD-L2,41 macrophage mannose receptor,42 cysteinyl leukotriene R1,43 and PPAR40,44) further contributed to the M2 nature of the MSC-derived macrophages and probably reflected the enhanced production of these cytokines in the progressor versus the naive splenocyte cultures (data not shown). Other genes listed in Table 2 ("P/N 
2") had not been linked to M2 cells before, although functionally most of them fit within some recurrent themes, which might partially overlap and represent known functions of M2: genes involved in anti-inflammation (selenoprotein P,45 coagulation factor XIIIa,46 PLA2GVII/PAF-AH,47 PPAR48), wound healing (fibronectin-1,36 arginase-1,16 coagulation factor XIIIa49), lipid metabolism (FIZZ1,50 prosaposin,51 PPAR52) and the related arachidonic acid metabolism (PLA2GIVa,53 PLA2GVII/PAF-AH,47 cysteinyl leukotriene R1,43 PPAR48), Th2-associated chemotaxis (CCL8,54 CCL2437), and pattern recognition receptors of the C-type lectin family (MGL-1,55 MGL-2,55 macrophage mannose receptor42).
|
|
|
ligands and PLA2 inhibitors cooperate to diminish M2-associated CTL suppressionNext we wondered whether any of the M2-associated gene products, listed in Table 2 ("P/N at least 2"), had an impact on the CTL-suppressive mechanism of these macrophages. Hereto, splenocytes from 5- to 7-week tumor-bearing mice were restimulated in vitro for 5 days, with or without specific inhibitors to block M2 gene function added at day 2.
|
PD-L2 is another M2-associated molecule with a known function in inhibiting T-cell activation, through direct interaction with the inhibitory receptor PD-1.56 PD-L2 mRNA is up-regulated in the splenic suppressor macrophages (Table 2 "P/N at least 2"), resulting in a readily detectable surface expression of the protein (data not shown). However, addition of neutralizing anti–PD-L2 antibodies to the cultures did not recover CTL activity, excluding PD-L2 as a candidate suppressor molecule in this model (Figure 6A).
Another possible pathway of T-cell inactivation is initiated by the IL-4–induced macrophage 12/15-lipoxygenase (LOX) activity, thereby generating potential ligands for PPAR, a member of the ligand-dependent nuclear receptor family.57 The mRNA for both 12/15-LOX and PPAR was detected in the progressor adherent splenocytes, though only PPAR expression was up-regulated compared with naive adherent splenocytes (Table 2 "P/N at least 2"). To assess this putative suppressive mechanism, CTL activity was investigated in the presence of baicalein, a selective 12/15-LOX inhibitor, or of the PPAR antagonist GW9662. Of interest, as shown in Figure 6C, antitumor CTL activity was actually further decreased by both compounds. This effect could not be attributed to a direct toxicity of the compounds toward CTLs, since addition of baicalein or GW9662 to nonadherent splenocyte cultures did not alter the observed cytotoxicity (Figure 6C). This finding suggested that PPAR stimulation in macrophages could be beneficial for CTL function. Therefore, potent synthetic and endogenous PPAR-specific ligands—rosiglitazone and 15d-PGJ2, respectively—were tested for their impact on CTL activity. Of importance, the selected doses of the ligands did not affect the viability of the suppressor population (data not shown), nor did they have an impact on the CTL activity of nonadherent splenocyte cultures (Figure 6C). In the total splenocyte cultures, both ligands could partially restore CTL activity, but the effect remained limited to about 25% recovery (Figure 6C).
Endogenous PPAR ligands are lipid mediators, generated through the arachidonic acid metabolism. The initiating regulatory enzymes of this pathway are the phospholipases A2, which are broadly classified into secretory (sPLA2), cytosolic Ca2+-dependent (cPLA2), and cytosolic Ca2+-independent (iPLA2) PLA2s.58 Of interest, though only the cPLA2 group IVa mRNA was up-regulated more than 2-fold in the suppressor macrophages (Table 2 "P/N at least 2"), other sPLA2 or iPLA2 members also tended to have a slightly higher expression in these cells (Table 2 "P/N less than 2"). Therefore, we wondered whether modulating PLA2 activity, either alone or in combination with PPAR stimulation, influenced CTL activity. Supplementing the cultures with a nontoxic dose of a general PLA2 inhibitor, aristolochic acid, recovered CTL activity on average by 19.53% ± 5.79% (Figure 6D). However, in combination with rosiglitazone or 15d-PGJ2, the increase in cytotoxicity reached 58.19% ± 10.38% and 53.04% ± 11.10%, respectively (Figure 6D), demonstrating that PPAR stimulation and PLA2 inhibition can cooperate in diminishing suppression by M2 cells.
Subsequently, we investigated whether the suppression-reversing effects of aristolochic acid could be attributed to the inhibition of a particular subfamily of PLA2. To this end, combinations of sPLA2-specific (ONO-RS-082), cPLA2-specific (AA-COCF3), and iPLA2-specific (PACOCF3) inhibitors were added to the CTL cultures together with 15d-PGJ2, and antitumor cytotoxicity was monitored. As shown in Figure 6E, a combination of ONO-RS-082, AACOCF3, and 15d-PGJ2 was minimally needed for the CTL activity to rise above the level seen with 15d-PGJ2 alone. Yet, the best recovery of CTLs was repeatedly noticed when combining all inhibitors with 15d-PGJ2 (Figure 6E), suggestingthat sPLA2, cPLA2, and iPLA2 activities contributed to PLA2-mediated suppression.
PPAR ligands subvert CTL suppression by M2-oriented tumor-associated macrophages
We next asked whether similar CTL-suppressive mechanisms would apply to freshly isolated tumor-associated macrophages (TAMs), since at least part of these cells was reported to be derived from the in vivo differentiation of CD11b+Gr-1+ splenocytes.12 To isolate TAMs, tumor single-cell suspensions were made and the CD11b+ fraction was collected via magnetic sorting. About 95% of the viable CD11b+ tumor-associated cells were F4/80hiCD68hi, identifying them as mature macrophages (data not shown). In order to discern specific characteristics of TAMs, these cells were compared with tumor-distal F4/80hi peritoneal macrophages from the same tumor-bearing mice.
In the first instance, we determined the activation state of TAMs. Therefore, the expression of a selected group of genes was assessed in both types of macrophages via real-time PCR. Clearly, the most prominent M2 markers—FIZZ1, Ym, fibronectin-1, and arginase-1—were all several fold higher in tumor-associated macrophages (Table 3). This finding was corroborated by an elevated arginase enzymatic activity in TAM versus peritoneal macrophages (Figure 7), altogether suggesting that the tumor-infiltrating macrophages had a more pronounced M2 phenotype than their tumordistal counterparts.
|
ligands and PLA2 inhibitors. In this context, it is important to note that the PPAR gene is more prominently expressed in TAMs compared with tumor-distal macrophages (Table 3). Hence, splenocytes from progressors were first depleted of CD11b+ cells via MACS, replenished with 15% freshly purified TAMs, and restimulated in vitro for 5 days, with or without PPAR ligands and/or PLA2 inhibitors. Figure 8 demonstrates that depletion of CD11b+ cells resulted in a greatly enhanced CTL activity in progressor spleens, but addition of 15% TAMs reduced the cytotoxicity almost back to the level seen in the total splenocyte culture. Reminiscent of the suppressive mechanism by in vitro–generated M2 macrophages, L-NMMA/norNOHA supplementation had only a modest effect on CTL activity and aristolochic acid only partially reversed TAM-mediated suppression (Figure 8). However, addition of rosiglitazone or 15d-PGJ2, whether or not combined with aristolochic acid, almost completely restored CTL activity, suggesting that PPAR stimulation could be an efficient tool to subvert TAM-mediated T-cell suppression. |
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
|
|
Antitumor CTL activity in these cultures is generally low but can be rescued when separating the CTLs from the plastic-adherent cells after 2 days of culture, suggesting an association between the presence of macrophages from day 2 onward and CTL suppression. Indeed, in granulocyte-depleted cultures, both macrophage formation and CTL suppression are intact. Direct proof of late progressor adherent splenocyte-mediated T-cell suppression came from the observation that these cells restrict the number of IFN-–producing CD8+ T cells upon short-term (6 hour) anti-CD3 stimulation. Remarkably, CD11b+Gr-1+ cells rather stimulate IFN- production in this setup, suggesting that freshly isolated, nondifferentiated CD11b+Gr-1+ cells are either not suppressive at all or at least unable to suppress anti-CD3–mediated T-cell activation.
We identified these CTL-suppressive macrophages as M2-oriented cells, based on the up-regulated expression (compared with naive adherent splenocytes) of well-established M2 markers, such as FIZZ1,35 Ym,35 fibronectin-1,36 arginase-1,16 and the MGLs,39 or other IL-4/IL-13–regulated genes ("Results"). This M2 skewing correlates with the detection of IL-4, IL-13, and IL-10 in the culture supernatant, while the M1-driving cytokine IFN is absent (J.A.V.G., unpublished data, July 2001). In addition, a number of novel M2 markers, not regulated by Th2 cytokines, but consistently found in in vivo–elicited M2 in different pathologies (PLA2 GVII/PAF-AH, prosaposin, TREM-2b)72 were also present. Of note, real-time PCR was performed on plastic-adherent populations enriched in macrophages (85%) but not on entirely pure macrophages. Therefore, we cannot a priori exclude that some of the genes listed in Table 2 ("P/N at least 2") are also expressed in other cells besides myeloid cells or macrophages. However, we favor the notion that the observed differences in gene expression reflect mainly the differences between macrophage populations, since (1) all the listed genes are known to be expressed in myeloid cells, and, even more importantly, many of them (including Ym, PD-L2, TREM-2b, Cathepsin L, Macrophage mannose receptor, MGL-1, MGL-2) are predominantly expressed in myeloid cells such as macrophages and DCs, the latter of which are not present in our cultures;, and (2) all genes listed in Table 2 ("P/N at least 2") have been, based on literature data and our own studies on peritoneal macrophages from a Trypanosoma brucei brucei infection model (G.H.G. and G.R., unpublished data), associated with M2-oriented macrophages. In addition, the most prominent genes were also up-regulated in purified TAMs.
Kusmartsev and Gabrilovich12 reported that splenic Gr-1+ cells are precursors of F4/80+ TAMs in vivo. Therefore, in order to correlate the characteristics of in vitro–generated CD11b+Gr-1+–derived M2 macrophages with the in vivo situation, we purified TAMs from mice with progressing BW-Sp3 tumors. In the first instance, it was important to find that TAMs were also M2 oriented, making a comparison between the in vitro and in vivo situation not far fetched. However, it should be noted that in a side-by-side comparison, most M2 markers have a significantly higher expression in the in vitro–generated macrophages compared with TAMs, suggesting that the 5-day in vitro culture strongly polarizes these cells.
Another point of comparison between both types of macrophages is their suppressive activity toward antitumor CTLs. Since macrophages make up a significant portion of BW-Sp3 tumors (J.A.V.G., unpublished data, June 2005), these cells could therefore have an important impact on intratumoral T-cell activity. Recently, the combined activity of arginase-1 and iNOS was shown, in a number of murine tumor models, to be important for the suppressive activity of tumor-infiltrating CD11b+ myeloid cells12 and splenic CD11b+Gr-1+ cells.11 By lowering L-arginine availability, arginase-1 can switch on the reductase domain of iNOS, thereby generating ROSs.11 Remarkably, neither the in vitro–generated splenic macrophages15 nor the TAMs from the BW-Sp3 model use this suppressive mechanism, despite high arginase enzyme activity in these cells. The reason for the discrepancy with other models is not clear, though it could be linked to the fact that the mouse AKR strain used in this study is strongly type-2 oriented and, hence, biased to the development of M2 cells with possibly different characteristics compared with C57Bl/6 or Balb/c M2.64 For example, enhanced arginase-1 activity and lowered L-arginine concentrations were shown to impede iNOS mRNA translation and protein stability in some macrophages.65
We describe the stimulation of PPAR by synthetic (rosiglitazone) and endogenous (15d-PGJ2) ligands as a novel strategy to diminish CTL suppression by both in vitro–generated splenic M2 and TAMs. Of interest, the efficiency of this treatment on TAMs, representing true in vivo–differentiated macrophages, is much higher. In macrophages, stimulation of this nuclear receptor has profound anti-inflammatory effects,66-67 suggesting that inflammation-linked molecules could be responsible for TAM-mediated suppression. Several of such molecules, including STAT-1,12 IFN,12 TNF,12,68 and prostaglandins,68 have been implicated in TAM suppressive activity before, so further research will be needed to clarify this issue in this model.
In addition, when PPAR stimulation alone has a limited effect—as is the case for the in vitro–generated M2—results could be improved by a combination treatment with PLA2 inhibitors. Note that the best results were obtained with a combination of cPLA2, sPLA2, and iPLA2 inhibitors. All 3 PLA2 types collaborate to maximize the mobilization of arachidonic acid,58 which is the precursor of the eicosanoids, in cells. Inhibition of PLA2 could therefore diminish the production of potentially T-cell–suppressive eicosanoids,69 but could also lead to an up-regulation of PPAR transcripts,70 possibly explaining the cooperative action of both treatments.
Current data provide a rationale for the use of PPAR ligands and/or PLA2 inhibitors to improve antitumor immunity, for example in immunotherapeutic settings. Of note, PPAR ligands such as the synthetic thiazolidinediones (eg, rosiglitazone) are currently used to treat diabetes but have also demonstrated antitumor effects in preclinical models.71
|
Acknowledgements |
|---|
|
Footnotes |
|---|
Prepublished online as Blood First Edition Paper, March 9, 2006; DOI 10.1182/blood-2005-09-3777.
Supported by a "Prospective Research for Brussels" postdoctoral grant from the Brussels government (J.A.V.G.); by a doctoral grant from the Vrije Universiteit Brussel (S.M.); by a postdoctoral fellowship from the "Institute for Promotion and Innovation by Science and Technology in Flanders" (IWT-Vlaanderen) (G.R.); by a grant from IWT-Vlaanderen for "Generisch Basisonderzoek aan de Universiteiten" (IWT-GBOU); and by the "Fund for Scientific Research Flanders" (FWO-Vlaanderen).
J.A.V.G. designed and performed research, analyzed data, and wrote the paper; S.M., Y.L., L.B., and K.D.G. performed research; G.H.G. and G.R. designed and performed research; and P.D.B. designed research.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Jo A. Van Ginderachter, Lab of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Building E, Level 8, Pleinlaan 2, B-1050 Brussels, Belgium; e-mail: jvangind{at}vub.ac.be.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
IG-H3. Scand J Immunol. 2001;53: 386-392.[CrossRef][Medline]
[Order article via Infotrieve] system by IL-13. J Immunol. 2005;174: 834-845.
: biochemical characterization of its oligomeric nature. J Biol Chem. 2002;277: 42011-42016. activated by macrophage-derived 12/15 lipoxygenase ligands. J Biol Chem. 2002;277: 3973-3978. is a negative regulator of macrophage activation. Nature. 1998;391: 79-82.[CrossRef][Medline]
[Order article via Infotrieve] agonists inhibit production of monocyte inflammatory cytokines. Nature. 1998;391: 82-86.[CrossRef][Medline]
[Order article via Infotrieve] activation inhibits tumor progression in non-small-cell lung cancer. Oncogene. 2004;23: 100-108.[CrossRef][Medline]
[Order article via Infotrieve]
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. P. Lepique, K. R. P. Daghastanli, I. M. Cuccovia, and L. L. Villa HPV16 Tumor Associated Macrophages Suppress Antitumor T Cell Responses Clin. Cancer Res., July 1, 2009; 15(13): 4391 - 4400. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. Eruslanov, S. Kaliberov, I. Daurkin, L. Kaliberova, D. Buchsbaum, J. Vieweg, and S. Kusmartsev Altered Expression of 15-Hydroxyprostaglandin Dehydrogenase in Tumor-Infiltrated CD11b Myeloid Cells: A Mechanism for Immune Evasion in Cancer J. Immunol., June 15, 2009; 182(12): 7548 - 7557. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Movahedi, M. Guilliams, J. Van den Bossche, R. Van den Bergh, C. Gysemans, A. Beschin, P. De Baetselier, and J. A. Van Ginderachter Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity Blood, April 15, 2008; 111(8): 4233 - 4244. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Z. Duan, M. G. Usher, and R. M. Mortensen Peroxisome Proliferator-Activated Receptor-{gamma}-Mediated Effects in the Vasculature Circ. Res., February 15, 2008; 102(3): 283 - 294. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Caljon, J. Van Den Abbeele, B. Stijlemans, M. Coosemans, P. De Baetselier, and S. Magez Tsetse Fly Saliva Accelerates the Onset of Trypanosoma brucei Infection in a Mouse Model Associated with a Reduced Host Inflammatory Response Infect. Immun., November 1, 2006; 74(11): 6324 - 6330. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2006 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
(PPAR
