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Institute of Biochemistry I, Faculty of Medicine, Johann Wolfgang Goethe-University Frankfurt, Frankfurt, Germany
1Correspondence: Johann Wolfgang Goethe-University, Faculty of Medicine, Institute of Biochemistry I, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. E-mail: bruene{at}zbc.kgu.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-stimulated murine RAW264.7 macrophages exposed to AC but not to either necrotic or viable human Jurkat cells was significantly reduced although iNOS expression remained high compared with controls. Analyzing iNOS activity in the cell extracts by using the radioactive L-arginine/citrulline conversion assay revealed increased ornithine production over citrulline in cells exposed to AC. RT-PCR, Western blot, and luciferase reporter analysis supported the idea of an arginase II increase in response to AC. The involvement of arginase in modulating NO formation in response to AC was substantiated by the arginase inhibitor N
-hydroxy-nor-L-arginine. Moreover, knockdown of arginase II by siRNA allowed recovery of NO production. Experiments with AC-conditioned medium demonstrated that a soluble lipid factor, rather than phagocytosis of AC, modulated NO production in macrophages. We conclude that AC release a lipid factor to modulate NO formation in macrophages via arginase II up-regulation, thereby contributing to innate immune regulation.—Johann, A. M., Barra, V., Kuhn, A-M., Weigert, A., von Knethen, A., Brüne, B. Apoptotic cells induce arginase II in macrophages, thereby attenuating NO production.
Key Words: phagocytosis • inflammation • alternative activation • inducible NO synthase
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. Engulfment and recognition of AC does not seem to be species specific, and major determinants for recognition are conserved between animals and humans (3
, 4)
. Moreover, it became clear that phagocytosis of AC regulates macrophage immune responses by shifting the cellular phenotype toward an anti-inflammatory one, also considered the M2 phenotype. Desensitization is characterized by the release of anti-inflammatory cytokines/prostanoids such as transforming growth factor (TGF)ß or prostaglandin (PG)E2 (2
, 5)
. It is also known that recognition of AC by macrophages attenuates the production of phorbolester-induced reactive oxygen species (6)
or NO production in response to lipopolysaccaride (LPS)/interferon- (IFN), as determined by reduced nitrite levels (7
, 8)
. However, molecular mechanisms underlying this behavior remain obscure.
There are three isoforms of NO-synthases (NOS): neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS). Only iNOS is strongly regulated at the expression level while eNOS and nNOS are usually considered to be constitutively expressed (9
, 10)
. In the presence of molecular oxygen and various cofactors, all isoenzymes convert L-arginine to citrulline and NO via the intermediate product N-hydroxy-L-arginine (11)
. Activation of the iNOS promoter, especially in macrophages, is an important step in regulating iNOS expression and thus NO formation in response to microbial compounds or cytokines (9)
. However, other mechanisms are known to affect NO production as well. For example, TGFß mediates iNOS protein degradation and reduces its mRNA stability (12)
. In addition, there are NOS inhibitory proteins such as NAP110 (13)
or kalirin (14)
, and substrate availability may become limiting via an increase in arginase activity (15
, 16)
.
Nowadays, two different arginase isoforms are recognized. Hepatic arginase (arginase I) is generally expressed in the liver and catalyzes the last step of urea synthesis (17)
but is also expressed in other cells (e.g., murine macrophages) (18)
, while arginase II is found in several extrahepatic tissues (17)
. Arginases degrade L-arginine to ornithine and urea, and therefore compete with iNOS for substrate (19)
. Indeed, murine RAW264.7 macrophages express arginase II to modulate NO production (15
, 20)
.
Considering the importance of AC in modulating immune responses in macrophages, it was our intention to characterize molecular mechanisms underlying reduced NO formation in macrophages on recognition of AC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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was from Roche Diagnostics (Mannheim, Germany). Culture supplements and FCS were ordered from PAA Laboratories (Cölbe, Germany). Oligonucleotides where bought from Metabion (Martinsried, Germany). Anti-actin antibody was purchased from Amersham Bioscience (Freiburg, Germany). The mouse anti-iNOS antibody was obtained from BD Biosciences (Heidelberg, Germany). Anti-arginase I and II antibodies were ordered from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TGFß1 and neutralizing TGFß1 antibody were purchased from R&D Systems (Wiesbaden-Nordenstadt, Germany). N-hydroxy-nor-L-arginine was ordered from Calbiochem (Bad Soden, Germany). [U-14C]-L-Arginine was delivered by MP Biochemicals (Eschwege, Germany). All chemicals were of the highest grade of purity and commercially available.
Cell culture
The mouse monocyte/macrophage cell line RAW264.7, the human T cell line Jurkat, and the human monocytic cell line THP-1 were maintained in RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated FCS. Cells were checked for mycoplasma at regular intervals. For induction of apoptosis, medium without FCS was used. For isolation of mouse peritoneal macrophages, 8- to 10-wk-old female C57BL/6 mice were sacrificed and 5–10 ml Ringer solution (DeltaSelect, Dreieich, Germany) containing 5% FCS was injected into the peritoneum. Cells were recovered, pooled, seeded in full RPMI medium further supplemented with 1x nonessential amino acids, 10 µg/ml sodium pyruvate, and 10 mM HEPES buffer solution, and left to adhere on culture dishes (Primaria 3072, Becton Dickinson, Heidelberg, Germany) for 2 h. Then medium was changed and treatments were performed. THP-1 cells at a density of 3 x 105 cells/ml were differentiated into macrophages by treatment with 50 nM TPA for 24 h, washed and cultured for an additional 60 h without TPA, followed by individual stimulations.
Generation of apoptotic and necrotic cells
To generate AC, Jurkat cells were seeded in 10 cm dishes in medium without FCS, supplemented with 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were incubated for 3 h with 0.5 µg/ml staurosporine, then washed twice with medium. Necrotic cells were generated by heating an appropriate number of Jurkat cells for 30 min at 56°C. In all experiments, the ratio of apoptotic or necrotic cells to macrophages was kept at 5:1. Apoptotic vs. necrotic cell death was confirmed by flow cytometry using annexin V-FITC/PI staining (Immunotech, Marseille, France).
AC conditioned medium was obtained by incubating 2.5 x 106 AC/ml of medium. After 2 h, cells were centrifuged for 10 min at 1000 g. The supernatant was removed and filtered through a 0.2 µm cellulose syringe filter (Roth, Karlsruhe, Germany). The filtrate was taken as AC conditioned medium (CM). When using CM, full medium was removed and replaced with CM. To obtain CM devoid of S1P (CM/DMS), 20 µM dimethylsphingosine (DMS), an inhibitor of sphingosine kinases, was added to Jurkat cells. After the addition of DMS for 1 h, Jurkat cells were stimulated with 0.5 µg/ml staurosporine in FCS free medium for 3 h to initiate apoptosis. The formation of S1P, however, was inhibited. Afterward, CM was generated as described above. To obtain PGE2-free CM (CM/NS), the cyclooxygenase (COX) inhibitor NS398 was added to Jurkat cells for 1 h. Thereafter, CM was generated as before.
Griess assay
Nitrite, as a stable end product of NO metabolism, was determined in the supernatant of RAW264.7 cells or mouse peritoneal macrophages. Cells were seeded in 3 cm plates at a density of 1 x 105 cells/plate using 1 ml medium. After incubation for 24 h, cells were coincubated with AC or appropriate stimuli for 15 h. Thereafter, medium was changed and incubations were continued for 24 h with or without the further addition of 100 U/ml IFN. Nitrite formation was determined by the Griess assay according to the manufacturer's instructions (Promega, Heidelberg, Germany). Nitrite concentrations in the supernatant were calculated in comparison to standard concentrations of NaNO2 dissolved in culture medium or PBS. When the arginase inhibitor N-hydroxy-nor-L-arginine (nor-NOHA) was supplemented, medium was replaced with PBS after the 15 h preincubation period. Nor-NOHA was also present during stimulation with IFN. In the case of siRNA experiments, cells where cultured in 6 cm plates using 4 ml medium. Prior to the addition of IFN, cells where reseeded at a density of 5 x 105 cells/plate.
Western blot analysis
Expression of iNOS, arginase I, arginase II, and actin was quantified by Western blot analysis. After individual incubations, cells were washed with ice-cold PBS, scraped off, lysed in 200 µl lysis buffer A (50 mM Tris, 150 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 1 mM PMSF, 1x protease inhibitor mix (Roche, Mannheim, Germany), pH 7.5), incubated on ice for 15 min, sonified, vortexed, and kept on ice for 20 min, followed by centrifugation (15,000 g, 15 min). Proteins (100 µg/sample) were resolved on 10% SDS-polyacrylamide gels and blotted onto nitrocellulose membranes with a semidry transfer cell. Anti-iNOS (1:1000), anti-arginase I (1:1000), anti-arginase II (1:1000), or anti-actin antibody (1:2000) was added and incubated overnight at 4°C. Afterward, nitrocellulose membranes were washed three times for 5 min each with TTBS. For protein detection, blots were incubated with a HRP-labeled goat anti-mouse secondary antibody (1:2000) or HRP-labeled goat anti-rabbit secondary antibody (1:2000), followed by ECL detection.
Citrulline assay
Arginase and iNOS activities were quantified using the citrulline assay basically as described (21)
. Briefly, after individual incubations, cells were washed with ice-cold PBS, scraped off, and lysed in 100 µl lysis buffer (25 mM Tris-HCl, 1 mM EDTA, and 1 mM EGTA) by three cycles of freeze-thawing using liquid nitrogen, vortexed, and kept on ice for 20 min, followed by centrifugation (15,000 g, 15 min). Proteins (50 µg) were added to a reaction mix (final concentration: 5 µM FAD, 5 µM FMN, 3 µM BH4, 1 mM NADPH, 10 µM [14C]-L-arginine, 100 µM cold arginine, 0.4 µM calmodulin, and 25 mM Tris/HCl pH 7.4), followed by incubations for 60 min at 37°C. Nor-NOHA was added at a final concentration of 1 mM. Reactions were terminated by adding 200 µl ice-cold methanol. Samples were dried in a SpeedVac vacuum dryer, resuspended in 10 µl H2O, and analyzed with TLC (CHCl3:CH3OH:NH4OH:H2O; 0.5:4.4:2:1) followed by autoradiography.
RNA extraction and quantitative real-time PCR
RNA from RAW264.7 or mouse peritoneal macrophages was extracted using peqGOLD RNAPure (Peqlab, Erlangen, Germany) according to the protocol supplied by the distributor. For RT reactions of mouse arginase I and II and ß2-microglobulin transcripts, we used the Advantage RT-for-PCR kit (Clontech, BD Biosciences, Heidelberg, Germany). Quantitative real-time PCR was performed using a MyiQ real-time PCR system (Bio-Rad, München, Germany) and the Absolute QPCR SYBR Green Mix (Abgene, Hamburg, Germany) according to the manufacturer's instructions. Sense and antisense primer (Metabion GmbH, Planegg, Germany) sequences and PCR product sizes were as follows: murine arginase I, TA = 60°C: 5'-AAG AAA AGG CCG ATT CAC CT-3'; 5'-CAC CTC CTC TGC TGT CTT CC-3', 201 bp (22)
, murine arginase II, TA = 60°C: 5'-ACA GGG TTG CTG TCA GCT CT-3'; 5'-TGA TCC AGA CAG CCA TTT CA-3', 298 bp (22)
; murine ß2-microglobulin, TA = 52°C: 5'-ACT GAC CGG CCT GTA TGC-3'; 5'-AGA CGG TCT TGG GCT CG-3' 298 bp. Annealing temperatures were calculated using the primer design program Oligo (MBI, Hanover, MD, USA). Controls of isolated RNA omitting reverse transcription during PCR were used to guarantee genomic DNA-free RNA preparations (data not shown). Quantification of real-time PCR results was performed using the Gene Expression Macro (V1.1) from Bio-Rad (München, Germany), taking ß2-microglobulin expression as the internal control.
Vector construction
To follow arginase II promoter activity, an arginase II luciferase reporter plasmid (mpArgII-pGL3luc) was generated. A 1894 bp fragment of the mouse arginase II promoter (GenBank #AF044680) was subcloned into a HindIII/NheI-digested pGL3-Basic vector (Promega, Mannheim, Germany) using the infusion kit (Clontech, BD Biosciences, Heidelberg, Germany). The arginase II promoter was obtained using the Expand High Fidelity PCR System (Roche) with murine genomic DNA. Sequences of the primers were as follows: TA = 62°C: 5'>3' 5'-GAG CTC TTA CGC GTG GAT ATC GAG CTC CCA GGA GGG AGA GAA TCT G-3', TA=62°C; 3'>5' 5'-CCG GAA TGC CAA GCT GCA GCC TCC CAC AGC TTC GAT G-3',1894 bp. Infusion sites are written in boldface letters. Correct orientation was verified by restriction analysis and sequencing.
Arginase II reporter gene assay
To measure reporter activity, the mpArgII-pGL3luc plasmid that contains the arginase II promoter upstream of the luciferase gene was used. Macrophages were transfected using jetPEITM cationic polymer transfection reagent (Biomol, Hamburg, Germany) according to the directions of the manufacturer. 5 x 105 cells were seeded in 24 well plates for 7 h. After transfection, incubations continued for 24 h, followed by individual stimulation. Luciferase activity was determined 24 h later.
siRNA treatments
Arginase II-specific predesigned Mm_Arg2_3 siRNA (Qiagen, Hilden, Germany) was nucleofected into RAW264.7 cells using nucleofector technology (Amaxa, Köln, Germany) according to the manufacturer's instructions. After 2 h the medium was changed and cells were incubated for another 24 h in complete medium before use. Arginase II knockdown was controlled by Western blot analysis and compared with siCONTROLTM nontargeting Duplex #1 siRNA (Dharmacon, Chicago, IL, USA).
Statistical analysis
Each experiment was performed at least three times. Statistical analysis was applied using the 2-tailed Student's t test. Otherwise, representative data are shown.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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for 24 h increased nitrite values compared with controls or macrophages exposed to AC alone (Fig. 1
). When macrophages were preincubated with AC for 15 h prior to IFN stimulation, nitrite formation was significantly reduced. In contrast, neither necrotic nor viable Jurkat cells attenuated NO formation (data not shown). Parallel to nitrite determinations, we checked iNOS expression by Western blot analysis (Fig. 1)
. As anticipated, stimulation of macrophages with IFN induced a strong iNOS expression compared with controls or macrophages exposed only to AC. In cells pre-exposed to AC and stimulated with IFN, the amount of iNOS protein was comparable to cells stimulated with IFN alone, although nitrite values in these samples differed significantly. These experiments suggested that reduced NO formation is not caused by altered iNOS protein expression.
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Arginase modulates NO production in macrophages exposed to AC
After observing iNOS protein expression that was apparently inactive, the question arose as to whether iNOS was directly or indirectly blocked. Therefore, we followed enzyme activity in vitro by using the radioactive citrulline assay. In this assay, [14C]-L-arginine is converted to citrulline by iNOS, followed by separation of substrate vs. product by TLC chromatography. In controls, without the addition of protein we noticed residual citrulline and ornithine (Fig. 2
). As expected, lysates of RAW264.7 cells stimulated with 100 U/ml IFN for 24 h showed significantly increased iNOS activity as determined by increased levels of citrulline compared with untreated controls.
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When the assay was performed with macrophage extracts from cells exposed to AC beforehand, it showed citrulline levels compared with controls; interestingly enough, another spot appeared, which was identified as ornithine. This pointed to arginase induction by AC. The involvement of arginase activity was verified by supplying 1 mM of the arginase inhibitor nor-NOHA (23)
to lysates from AC-exposed macrophages, which blocked ornithine formation. When RAW264.7 cells were exposed to AC for 15 h and later stimulated for 24 h with IFN, an increase in both iNOS and arginase activity was noticed. This observation makes it less likely that AC modulate iNOS activity via an inhibitory protein, because then one would expect reduced citrulline production. Specificity of nor-NOHA was strengthened by the observation that IFN-induced iNOS activity was not affected by the presence of the inhibitor. As further controls, we showed that neither viable nor necrotic Jurkat cells affected arginase activity (i.e., provoked ornithine formation). Moreover, lysates from AC alone did not stimulate either citrulline or ornithine generation, excluding a potential impact of enzyme activities from AC themselves (data not shown).
To further strengthen the role of arginase in modulating iNOS activity in our system, we went on to analyze nitrite formation in macrophages under the influence of nor-NOHA. For these experiments, we changed medium for PBS after exposure to AC, thus excluding that arginine in the medium competes with nor-NOHA uptake and/or inhibition of arginase. As expected, nitrite production in RAW264.7 cells, incubated for 24 h with 100 U/ml IFN in PBS, was significantly higher compared with controls (Fig. 3
). Neither exposure to AC alone nor the addition of nor-NOHA provoked noteworthy nitrite formation. In addition, nor-NOHA did not affect nitrite formation in response to IFN stimulation, but significantly restored nitrite formation, which otherwise was abolished by AC.
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To elucidate whether the impact of AC on RAW264.7 cells required phagocytosis, we treated macrophages with cytochalasin D to block phagocytosis by inhibiting actin polymerization. To further inquire whether cell-cell contacts between AC and macrophages were required, we used AC conditioned media (CM) supplied to macrophages (Fig. 4
). As a readout, we followed nitrite formation and iNOS expression. Adding cytochalasin D to IFN-stimulated RAW264.7 cells in the presence of AC did not impair the ability of AC to attenuate NO formation, nor did we notice attenuated iNOS protein expression. To our surprise, the addition of AC-conditioned media reproduced inhibition of nitrite formation to a similar extent as AC themselves. Moreover, iNOS expression was not altered as seen with AC. CM gained from apoptotic MCF-7 cells provoked inhibition of NO production similar to that of Jurkat-derived CM (data not shown). These experiments suggest that neither binding nor phagocytosis of AC is required to modulate arginase expression. Rather, a soluble factor released from AC attenuated NO formation in IFN-stimulated macrophages.
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To obtain supporting information on arginase induction and to further specify whether arginase I or arginase II was involved, we performed real-time PCR analysis. RAW264.7 cells were treated with CM and mRNA was analyzed as described in Materials and Methods (Fig. 5
A). CM provoked a marked increase in arginase II mRNA compared with controls, while the level of arginase I mRNA remained unaltered. The strikingly higher levels of arginase II mRNA in CM-treated cells compared with unstimulated controls resulted from very low arginase II mRNA levels in the latter ones.
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After considering earlier reports that TGFß might inhibit NO formation (7)
, we analyzed mRNA content of arginase I/II under the influence of TGFß as well. Incubating RAW264.7 cells with 5 ng/ml TGFß for 15 h affected neither arginase I nor arginase II mRNA amounts (Fig. 5A
). However, in the Griess assay, TGFß (5 ng/ml) reduced IFN-induced NO production from 30 µM to roughly 10 µM, which proved its action (data not shown). To further specify that CM provoked arginase II expression, we performed Western blot and reporter analysis as described in Materials and Methods. Treatment of RAW264.7 cells with CM increased protein levels of arginase II (Fig. 5C
) whereas arginase I expression remained absent (Fig. 5B
). Experiments with AC produced similar results (data not shown). Treatment of cells with 1 mM cAMP, known to cause arginase I expression (24)
, indeed increased arginase I and II protein amounts, proving that our test system was capable of expressing both arginase isoforms. To follow arginase II promoter activity, we cloned a 1894 bp promoter fragment in front of the luciferase gene as outlined under Materials and Methods. Arginase II promoter reporter analysis showed an 
35-fold increase in luciferase activity in macrophages treated with CM, whereas cAMP led to only a 5-fold increase (Fig. 5D
).
To validate modulation of arginase expression and NO production, we performed arginase II real-time and Western blot analysis in primary mouse peritoneal macrophages (Fig. 6
A, B). As expected, treatment of primary macrophages with CM for 24 h significantly increased arginase II mRNA expression as well as arginase II protein expression compared with controls. The minor mRNA induction in primary mouse cells compared with RAW264.7 macrophages, as seen in Fig. 5A
, may result from higher basal arginase II mRNA levels in primary cells. Furthermore, nitrite determinations were performed in primary mouse peritoneal macrophages (Fig. 6C
). Peritoneal macrophages produced 20 µM nitrite in response to 1 µg/ml LPS and 50 µM nitrite in response to 100 U/ml INF- when stimulated for 24 h. Nitrite formation in response to either LPS or IFN was significantly reduced when exposed to CM.
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In addition, we used differentiated THP-1 cells treated with CM for 24 h and performed Western blot analysis to verify arginase II expression in human cells (Fig. 6D
). Indeed, an increased arginase II expression was observed compared with untreated controls. Griess assay was not performed with THP-1 cells, since it is accepted that human macrophages do not produce measurable amounts of NO in vitro (25)
.
To prove the relevance of arginase II in attenuating NO production, we knocked down arginase II by siRNA and measured nitrite production after treatment with CM. In siRNA-treated cells, nitrite formation in response to IFN, which was normally suppressed by CM, was significantly recovered (Fig. 7
). To control the efficiency of siRNA knockdown, we performed arginase II Western blot analysis. While CM increased the arginase II protein amount, its expression was largely impaired in the siRNA approach. Experiments with nontargeting control siRNA left nitrite production unaltered. We conclude that treatment of RAW264.7 macrophages with AC up-regulated arginase II, thereby attenuating NO production.
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We next aimed to characterize the AC-derived soluble factor that decreased NO production. To check for a protein, we heated CM for 2 h at 100°C (CM/100°C) and supplied the heat-inactivated fraction in combination with 100 U/ml IFN to RAW264.7 cells, followed by nitrite determination. Despite heat treatment, CM still attenuated nitrite formation (Fig. 8
A). Next, we performed chloroform methanol extractions (chloroform: methanol; 2:1) of CM (CM/CHCL3). The organic phase was evaporated, reconstituted in PBS containing 1 mg/ml BSA, and added back to macrophages in combination with IFN. The Griess assay showed that the lipid phase of CM suppressed nitrite formation. These results indicated the release of a lipid-soluble component into CM, which blocked NO formation in macrophages. Moreover, data of Fig. 8A
indicate that CM also reduced nitrite formation in response to LPS, which was provided as an alternative stimulus to IFN to provoke NO formation. The most obvious candidates known to be released from apoptotic cells that might be involved in blocking NO formation are S1P, PGE2, and TGFß (Fig. 8B
). To determine the involvement of S1P, we treated AC with DMS, a known inhibitor of sphingosine kinase and thus an inhibitor of S1P production in AC. DMS allowed us to generate S1P-free CM (CM/DMS). Blocking cyclooxygenases in AC, we used NS398 to generate PGE2-free CM (CM/NS). Alternatively, NS398 (100 µM) was directly added to macrophages, blocking cyclooxygenases, thus attenuating formation of prostanoids, which may act via a feedback loop. Moreover, a neutralizing antibody was used to scavenge TGFß in the CM. Experimentally, IFN-stimulated nitrite formation in macrophages was suppressed by CM, as seen before. The ability to suppress NO production was also seen with CM/DMS, CM/NS, NS, or in the presence of 1 µg/ml neutralizing TGFß antibody. Thus, S1P, cyclooxygenase-derived lipid messengers, and TGFß were not involved in suppressing NO formation in IFN-stimulated macrophages. The inability of S1P to convey arginase expression was further excluded when S1P failed to induce the arginase II reporter (data not shown). Thus, initial characterization of the arginase II-inducing factor ruled out a protein component and made the involvement of a lipid factor likely.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The altered macrophage phenotype is associated, among other alterations, with decreased proinflammatory mediator production of, for example, TNF-
or NO (5
, 7)
and/or increased anti-inflammatory mediator release such as PGE2 or TGFß (26)
. Formation of NO can be considered a prototypical marker of classic macrophage activation resembling a fundamental component of their phagocytic, bactericidal, and tumoricidal activities. In our study we aimed to understand molecular mechanisms whereby AC attenuate NO formation in classical (i.e., IFN-stimulated macrophages). We corroborated earlier findings on the ability of AC to block NO formation in macrophages (7
, 8
, 27)
and went on to demonstrate that AC did so by enhancing arginase II expression/activity without altering iNOS expression. Unexpectedly, a soluble lipid factor released from AC transmitted the inhibitory response.
There are several possibilities to attenuate NO formation. Besides modulation of iNOS protein amount and mRNA content (12)
, inhibitory proteins (13
, 14)
or reduced substrate availability via expression of arginase (15
, 16)
have been suggested. In our experiments, expression modulation of arginase II mRNA and protein, arginase activity determinations, use of the arginase inhibitor nor-NOHA, siRNA knockdown experiments, as well as arginase promoter activity strongly suggest that AC use arginase II induction to halt NO formation in IFN-stimulated macrophages. The substantial formation of nitrite in the nor-NOHA experiments performed in PBS can be explained by the existence of not freely exchangeable intracellular L-arginine pools (28
, 29)
, which may be accessible for arginase and iNOS under the given experimental conditions. Complete reconstitution of NO production during the arginase II siRNA experiments proved that the effect was indeed arginase II dependent. In the context of the citrulline assay, it is important to note that arginase II has been shown to be 50% active even in the absence of Mn2+, although addition of Mn2+ will be required to obtain full arginase activity (30)
. When we performed the 14C-arginine/citrulline conversion assay, altered arginase activity could not be foreseen and thus was performed in the absence of Mn2+, which certainly underestimates arginase activity. Nevertheless, the ornithine signal was indicative for arginase activity. Thus, limited substrate supply to iNOS by arginase activity is important to regulate NO production under conditions when iNOS is expressed in macrophages. It is somehow unexpected to note that signals derived from AC turned out to be potent regulators of arginase expression/activity. Previous reports proposed that the ability of AC to attenuate NO formation in murine resident peritoneal macrophages was TGFß mediated (7)
. The authors suggested that TGFß shifted arginine metabolism toward polyamine biosynthesis, thus impairing NO production. Suggestions were based on the study of Boutard et al., who proposed TGFß-mediated up-regulation of arginase or induction of factors regulating the affinity of arginase for its substrate (31)
. However, the authors observed that inhibition of NO production in rat peritoneal macrophages occurred much earlier than arginase activity, which required 24 h of TGFß exposure, and therefore suggested an early and late mechanism, with only the latter one being arginase mediated. As we recognized arginase induction after 4 h of incubation with CM (data not shown), a contribution of TGFß is unlikely. Along that line, the group of Barksdale et al. have already shown that TGFß alone does not increase arginase expression in RAW264.7 macrophages (32)
. Our results showing that a TGFß-neutralizing antibody left CM-mediated NO inhibition unaltered strengthen these findings. However, a recent study in TGFß receptor-deficient cells pointed to arginase I up-regulation after phagocytosis of AC (33)
. Although RAW264.7 macrophages were used as in our system, we noticed up-regulation of arginase II rather than arginase I as well as high iNOS expression. Furthermore, we proved an increase in arginase II expression in the human monocyte/macrophage cell line THP-1 as well as primary mouse peritoneal macrophages in combination with attenuated NO production. One might speculate whether the apoptotic stimulus employed to evoke cell death, the time used to generated AC, as well as potential mediator release from AC and/or the time exposing macrophages to AC will affect distinct signaling pathways in macrophage to modulate NO formation. These variables require further attention.
Our experiments with CM demonstrated that cell-cell contacts were not necessary to attenuate IFN-mediated NO production. In fact, a soluble lipid factor released from AC seemed to be responsible for the up-regulation of arginase II expression. The release of soluble factors by AC, such as the established anti-inflammatory cytokine IL-10 (2)
or the caspase-3-mediated release of lipid attraction signals (34)
alluring macrophages to apoptotic cells, has been described. Recently our group identified sphingosine-1-phospate (S1P) as another soluble factor released by AC, which influences the macrophage phenotype (35)
. S1P was a potential candidate in modulating arginase II in our system, but S1P-free CM (CM/DMS) failed to restore NO production, thus making an involvement of this lipid unlikely. It has also been suggested that PGE2 might induce arginase activity in murine macrophages, although the isoform was not specified (36)
. Using the cyclooxygenase inhibitor NS398, which attenuated AC-induced PGE2 production in our system (data not shown), we were unable to alter CM-induced arginase II expression, thus ruling out an influence of PGE2 and/or other COX-derived prostanoids on NO inhibition. Furthermore CM/NS and the addition of NS398 to macrophages did not restore NO formation. In addition, an influence of the protein factors IL-10 or TGFß can be excluded based on the use of heat-inactivated CM. Additional studies will have to clarify the identity and function of the lipid factor (or factors) present in CM that cause increased arginase II activity. In contrast to the classical proinflammatory macrophage activation (M1 phenotype) mediated by LPS or IFN, apoptotic cells seem to activate macrophages alternatively, provoking an anti-inflammatory M2 phenotype that has been described in response to TH2 cytokines (37
, 38)
. In this respect, the TH2 cytokine IL-13 has been reported to induce arginase I activity in mouse inflammatory peritoneal macrophages and/or to suppress iNOS protein translation, depending on the time and intensity of the applied stimulus (39)
. This may point to regulatory features of both arginase I and arginase II under different patho-physiological settings.
In summary, our study adds to observations that immune regulatory pathways are activated in macrophages in response to AC. The release of a so far unknown lipid factor by AC reduced NO production in response to IFN by inducing arginase II expression, thus lowering L-arginine availability but leaving iNOS protein expression unaltered. We propose that arginase II up-regulation participates in reprogramming macrophages subsequent to recognition of a soluble lipid factor released by AC.
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ACKNOWLEDGMENTS |
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Received for publication November 30, 2006. Accepted for publication March 29, 2007.
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REFERENCES |
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0.05 vs. IFN
