MACROPHAGE ARGINASE REGULATION BY CCAAT/ENHANCER-BINDING... : Shock (original) (raw)
INTRODUCTION
Previous work characterized the expression of arginase activity in healing wounds and demonstrated that this enzyme is responsible for most local L-arginine metabolism during the resolution of the inflammatory phase of the tissue repair response (1). The temporal correlation between the expression of arginase activity in wounds and the initiation of the proliferative phase of repair supports the proposal that arginase activity is a marker for an “alternatively activated” macrophage phenotype endowed with tissue repair functions (2). Ornithine produced from L-arginine by arginase can be metabolized by ornithine decarboxylase to putrescine, thus initiating the synthesis of polyamines that can regulate cell proliferation in wounds and other inflammatory sites. Ornithine is, in addition, substrate for ornithine aminotransferase, the first enzyme in the synthesis of glutamate and proline, the latter used abundantly in the synthesis of collagen (3-6). The sustained expression of arginase in healing wounds is, therefore, likely to contribute essential metabolites to the repair process.
Macrophages can express both isoforms of arginase: arginase I, a cytosolic enzyme first described as a component of the urea cycle in the liver, and arginase II, which is localized in the mitochondrial matrix (7). Work using primary rat and murine macrophages and murine macrophage cell lines demonstrated that cAMP, Th2 cytokines (IL-4, IL-13, and IL-10), LPS, hypoxia, and other stimuli induce arginase expression, with variable specificity in the arginase isoform induced by different agonists in different cell types (4,7,8).
Relatively little is known about the identity of the transcription factors required for regulation of arginase expression in macrophages (9). The present study examined the role of C/EBPβ, a member of the CCAAT/enhancer-binding protein family of nuclear factors (for review, see Ref. 10). C/EBPβ has been shown to be required for the induction of rat liver arginase I by glucocorticoids (11), and by glucocorticoids and glucagon in isolated rat hepatocytes (12). Other members of the C/EBP family also participate in the regulation of nonmacrophage arginases, as evidenced by the decreased hepatic (13) and salivary gland arginase I expression (14) found in C/EBPα-null animals.
Reports demonstrating that cAMP stimulates C/EBPβ translocation and C/EBPβ-dependent gene transcription (15-17), STAT6, the canonical nuclear factor responsible for IL-4 responses, interacts with C/EBPβ in the regulation of gene expression by the cytokine (18-20), and LPS and hypoxia activate C/EBPβ (21-23), suggest the hypothesis that these diverse agonists could use C/EBPβ as a common regulatory factor in the induction of macrophage arginase. The hypothesis was tested in the experiments reported here.
MATERIALS AND METHODS
Cells and animals
C/EBPβ+/+ and −/− immortalized macrophage cell lines were a generous gift from Dr. Valeria Poli (University of Turin, Torino, Italy) (24). The macrophage phenotype of these cells was demonstrated by the originator (24). The C/EBPβ genotype of the cells was confirmed in the laboratory using polymerase chain reaction and immunoblotting using anti-C/EBPβ antibody as described below.
C/EBPβ−/− animals (B6.129 C/EBPβ) and wild-type controls (B6.129) were a kind gift from Dr. Peter Johnson (NCI-Frederick Cancer Center, Frederick, MD) (25). The animals' genotype was confirmed in the laboratory by tailing (DNeasy tissue kit; Qiagen, Valencia, CA) and polymerase chain reaction using primers: C/EBPβ: sense 5′-AGCCCCTACCTGGAGCCGCTCGCG-3′, antisense 5′-GCGCAGGGCGAACGGGAAACCG-3′; and Neo9: sense 5′-GTGCTCGACGTTGTCACTGAAGCGG-3′, antisense 5′-GATATTCGGCAAGCAGGCATCG-3′.
Animals were kept in barrier cages and were allowed food and water ad libitum. Brown University/Rhode Island Hospital veterinary personnel monitored animal welfare. All procedures involving animals were approved by the Lifespan Animal Welfare Committee. The experiments were performed in adherence to the National Institutes of Health Guidelines on the use of Laboratory Animals.
The subcutaneously implanted polyvinyl alcohol sponge (PVA Unlimited, Warsaw, IN) wound model has been described previously (1). The tissue response to the implanted sponges models precisely for the healing of soft tissue wounds. Sponges (five per animal) were implanted in C/EBPβ+/+ and −/− mice and were retrieved 10 days later as described in Albina et al. (1). Wound fluids were isolated from the sponges exactly as reported in Albina et al. and were assayed for arginase activity as indicated below. Total and differential counts of wound cells were performed using Hema-3-stained (Biochemical Sciences, Swedesboro, NJ) Cytospins (Shandon, Pittsburgh, PA). Resident peritoneal cells were harvested by lavage and were cultured as indicated below.
Cell culture
Cells were cultured overnight in RPMI 1640 (Life Technologies, Grand Island, NY) containing 1% fetal calf serum (HyClone, Logan, UT) at a density of 106 cells/mL, at a temperature of 37°C and 5% CO2 in humidified air. When indicated, cultures contained 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP, 0.5 mM; Sigma-Aldrich, St. Louis, MO), recombinant murine IL-4 (rmIL-4, 20 U/mL; BD PharMingen, San Diego, CA), or LPS (100 ng/mL, from Escherichia coli 055:B5; Difco Laboratories, Detroit, MI). The selected agonist concentrations were determined in preliminary experiments to induce maximal arginase activity in cultured murine peritoneal macrophages (not shown). Additional cells were cultured overnight in 1% O2 in gas-tight incubation chambers (Billups-Rothenberg, Del Mar, CA).
Immunoblotting
Postnuclear supernatants of cell lysates were size-fractionated in 12% SDS-polyacrylamide gels by electrophoresis, transferred to a nitrocellulose membrane, and blocked (5% nonfat dry milk in Tris buffer) overnight at 4°C. Arginase I was detected using a chicken anti-rat arginase I primary antibody (26) at a 1:50,000 dilution, horseradish peroxidase-conjugated rabbit anti-chicken immunoglobulin (1:5000; Jackson ImmunoResearch Laboratories, West Grove, PA), and enhanced chemiluminescence detection (Amersham, Piscataway, NJ). Arginase II was detected using an affinity-purified chicken antibody to purified human arginase II at a 1:500 dilution. Anti-C/EBPβ (C-19) rabbit polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and horseradish peroxidase-conjugated anti-rabbit immunoglobulin (Amersham) were used to blot for C/EBPβ. STAT6 was detected by immunoblotting after electrophoresis in 7.5% SDS-polyacrylamide using nuclear preparations (see below) from cultured cells and rabbit polyclonal anti-STAT6 (M20) or antiphospho-STAT6 antibodies (Santa Cruz Biotechnology).
Electrophoretic mobility shift analysis
Nuclear extract preparations and electrophoretic mobility shift analysis (EMSA) were performed as described (27) using oligonucleotides containing a STAT5/6 consensus sequence (Santa Cruz Biotechnology). Supershift analysis was carried out using rabbit polyclonal anti-STAT6 antibody (M20; Santa Cruz Biotechnology).
Arginase assay
Arginase activity was determined in wound fluids and cell lysates using the radiometric method of Russell and Ruegg (28) exactly as published (8). Results of the arginase assay are reported as nanomoles urea produced per minute per 106 cells.
Data presentation and statistical analysis
Experiments using macrophage cell lines were repeated at least three times each. Results from cell culture studies are means ± SD from three to five identical well replicates per culture condition in a representative experiment. Wounding and peritoneal cell harvesting experiments in C/EBPβ wild-type and −/− mice used six animals per group. Statistical analysis was performed using analysis of variance (ANOVA) or the Mann-Whitney U test, as appropriate to each individual experiment format. When not shown in bar graphs in the figures, the length of the error bars was smaller than the width of the border of the corresponding data bars.
RESULTS
Arginase expression in immortalized wild-type and C/EBPβ−/− macrophages
Studies were performed using C/EBPβ+/+ and −/− macrophage cell lines (24). Figure 1A shows arginase activity in cell lysates prepared 24 h after cell stimulation with 8-Br-cAMP (0.5 mM) or rmIL-4 (20 U/mL). Unstimulated C/EBPβ+/+ macrophages expressed more arginase activity than their C/EBPβ−/− counterparts. Arginase activity was enhanced in wild-type cells by 8-Br-cAMP and by rmIL-4. In contrast, 8-Br-cAMP failed to increase arginase activity in the C/EBPβ−/− cell line. The response of these cells to rmIL-4 was substantially blunted compared with that in wild-type controls. In agreement with results from the activity assay, immunoblots shown in Figure 1B demonstrate increased arginase I protein in lysates of wild-type cells stimulated with either agonist. Figure 1 also shows that there was no arginase I antigen induction by 8-Br-cAMP and a reduced response to rmIL-4 in C/EBPβ−/− cells. Arginase II protein was constitutively expressed in the C/EBPβ+/+ and −/− cell lines and was not modified by the addition of 8-Br-cAMP or rmIL-4 to the cell cultures (Fig. 1B).
Differential induction of arginase activity and arginase I protein by 8-Br-cAMP and rmIL-4 in C/EBPβ+/+ and −/− macrophage cell lines. (A) Immortalized C/EBPβ+/+ and −/− macrophages were cultured overnight as indicated in Materials and Methods with or without 8-Br-cAMP (0.5 mM) or rmIL-4 (20 U/mL). Cell lysates prepared at the end of culture were used in an arginase activity assay. Bars labeled with different characters are different (P < 0.05, two-way ANOVA with repeated measurements, Newman-Keuls). (B) Postnuclear supernatants from cells treated as just indicated were immunoblotted for arginase I or arginase II as indicated in Materials and Methods. Lanes contained 5 μg of protein in the case of macrophages assayed for arginase I, and 20 μg when blotted for arginase II. Liver was loaded at 3 μg of protein per lane, and kidney at 10 μg of protein per lane.
The lack of response of the C/EBPβ−/− macrophage cell line to the cAMP analog did not result merely from failure of 8-Br-cAMP to activate cAMP-dependent protein kinase (PKA) because 8-Br-cAMP stimulation of C/EBPβ+/+ and −/− cells resulted in identical PKA activity in both cell types when measured 15 min after stimulation (results not shown).
C/EBPβ is necessary for the induction of arginase by LPS and by hypoxia in immortalized macrophages
Figure 2 shows the effect of LPS (100 ng/mL; in the presence or absence of 0.5 mM 8-Br-cAMP), and of hypoxia (1% O2) on arginase activity in macrophages from C/EBPβ+/+ and −/− cell lines. As shown in the figure, LPS increased arginase activity in wild-type cells, an effect that was potentiated by 8-Br-cAMP. Neither response was found in C/EBPβ−/− cells. In a similar fashion, overnight exposure to 1% O2 determined an increase in arginase activity in C/EBPβ+/+, but not −/−, cells.
LPS and hypoxia induce arginase activity in C/EBPβ+/+ but not in −/− macrophage cell lines. Immortalized C/EBPβ+/+ and −/− macrophages were cultured overnight in media containing or not LPS (100 ng/mL) ± 8-Br-cAMP (0.5 mM), or in media equilibrated to 1% O2. Arginase activity of cell lysates generated at the end of culture was determined as indicated in Materials and Methods. Bars labeled with different characters are different (P < 0.05) for results with C/EBPβ+/+ mice. Results with C/EBPβ−/− cells are different from those in wild-type cells for all treatments (P < 0.05, two-way ANOVA with repeated measurements, Newman-Keuls).
Immortalized C/EBPβ−/− macrophages are not defective in expression or IL-4-stimulated activation of STAT6
STAT6 activation is required for the induction of arginase I by IL-4 (29,30). To determine whether the decreased arginase I response to rmIL-4 found in C/EBPβ−/− macrophages could result from alterations in STAT6 expression, translocation, or phosphorylation in the genetically modified cells, nuclear preparations from rmIL-4-treated C/EBPβ+/+ and−/− macrophage cell lines were subject to EMSA and immunoblotting. Figure 3A shows greater binding to an oligonucleotide containing the consensus sequence to STAT5/6 by nuclear extracts from rm-IL4-treated C/EBPβ−/− than +/+ cells. Supershifting with a specific antibody confirmed the identity of the induced band as STAT6. In full agreement with the EMSA findings, immunoblots shown in Figure 3B demonstrate that nuclear extracts from C/EBPβ−/− macrophages contain more basal STAT6 protein than the +/+ cells, and that the C/EBPβ null cells respond to rmIL-4 with greater accumulation of nuclear phospho-STAT6.
STAT6 regulation in C/EBPβ+/+ and −/− macrophage cell lines. Immortalized C/EBPβ−/− and wild-type macrophages were cultured for 1 h with or without rmIL-4 (20 U/mL). EMSA and nuclear preparation immunoblots were performed as described in Materials and Methods. (A) EMSA of nuclear preparations against consensus-binding sequence for STAT5/6 and supershift with antiSTAT6 antibody. (B) Immunoblot of nuclear preparations using antiphosphoSTAT6 and antiSTAT6 antibodies. Relative densitometry results are shown.
Cellularity and arginase activity of wounds in wild-type and C/EBPβ−/− mice
Sterile wounds were inflicted upon wild-type and C/EBPβ−/− mice, and wound cells and extracellular wound fluids were harvested 10 days after injury. Preliminary studies using wild-type mice established that maximal arginase activity in extracellular wound fluids is found 10 to 14 days after wounding (data not shown).
Wounds in wild-type and C/EBPβ−/− animals did not differ in total cell count or macrophage number (Table 1). Wounds in C/EBPβ−/− animals, however, contained more polymorphonuclear leukocytes than those in wild-type mice (Table 1). Table 1 also shows that arginase activity in fluids harvested from wild-type mice was almost twice as high as that in fluids from C/EBPβ−/− mice.
Characterization of the cellular infiltrate and of arginase activity in wound in C/EBPβ +/+ and −/− mice.
Resident peritoneal macrophages from C/EBPβ−/− mice express less basal and inducible arginase activity than those from wild-type animals
Resident peritoneal cells were harvested from wild-type and C/EBPβ−/− mice, enriched for macrophages by adherence, and incubated overnight in culture media containing or not 8-Br-cAMP (0.5 mM) or rmIL-4 (20 U/mL). Results in Figure 4 show that lysates from unstimulated macrophages from wild-type animals contained more arginase activity than those from C/EBPβ−/− mice. Cells from wild-type animals responded to the cAMP analog and to rmIL-4 with increased arginase activity, whereas those from C/EBPβ−/− animals did not augment their arginase activity when exposed to 8-Br-cAMP and had only a modest, albeit significant, response to rmIL-4. Arginase activity correlated with arginase I protein abundance (not shown).
Differential induction of arginase activity by 8-Br-cAMP and rmIL-4 in peritoneal macrophages isolated from wild-type and C/EBPβ−/− mice. Peritoneal cells were harvested by lavage from C/EBPβ+/+ and −/− mice (n = 6 per group). Macrophages were isolated by adherence to plastic and were cultured overnight in media containing or not 8-Br-cAMP (0.5 mM) or rmIL-4 (20 U/mL). Arginase activity was measured in cell lysates generated at the end of culture. Bars labeled with different characters are different (P < 0.05, two-way ANOVA with repeated measurements, Newman-Keuls).
DISCUSSION
Recent reports indicating that elevated arginase expression and activity are involved in many physiologic and pathophysiologic processes [including wound healing (1), asthma (30,31), and trauma (32)] have focused attention on the identification of the agents and mechanisms responsible for regulating the expression of the arginases, especially arginase I, in inflammatory cells. Although macrophage arginases have been shown to be induced by cell-permeable analogs of cAMP, Th2 cytokines, LPS, and hypoxia (7,8,26), there is relatively little information on the specific regulatory molecules that modulate enzyme expression in these cells. Based on evidence reviewed in the Introduction, this study tested the hypothesis that C/EBP proteins, and more specifically, C/EBPβ, may be a common required factor for the induction of arginase expression in macrophages by disparate agonists.
Experiments using C/EBPβ−/− and wild-type immortalized macrophage cell lines demonstrated that the absence of C/EBPβ resulted in reduced basal expression of arginase I and abolished the response to 8-Br-cAMP. Although rmIL-4-induced arginase I in C/EBPβ−/− cells, enzyme protein and activity did not reach levels found in identically stimulated wild-type cells (Fig. 1). Similar results were obtained with primary macrophages from wild-type and C/EBPβ−/− mice (Fig. 4). Arginase II was constitutively expressed in the cells regardless of their C/EBPβ genotype, and was not responsive to stimulation by 8-Br-cAMP or rmIL-4.
Previous evidence confirmed that STAT 6 activation is required for the induction of arginase by IL-4 (29,30). The ability of cells from the C/EBPβ−/− line, which exhibited a blunted arginase response to IL-4, to recruit STAT6 to the nucleus after cytokine stimulation and the phosphorylation status of nuclear STAT6 ruled out a defect in the STAT6 pathway in these cells (Fig. 3). The modest arginase I induction despite increased STAT6 activation by IL-4 in C/EBPβ−/− cells demonstrates a requirement for both nuclear factors for maximal transcriptional induction of the enzyme by the cytokine. This conclusion is supported by reports indicating the IL-4 response elements in the promoters of the immunoglobulin germline ε (18,19) and “found in inflammatory zone-1” (FIZZ1) (20) require binding of STAT6 and C/EBPβ, and by the observation that the IL-4 response element in the arginase I promoter requires binding of STAT6 and C/EBPβ (M. Gray, M. Poljakovic, and S. Morris, unpublished results, and Ref. 33). Current findings, then, confirm and extend molecular findings indicating that both nuclear factors STAT6 and C/EBPβ are required for maximal arginase I induction by IL-4 in macrophages.
Additional experiments demonstrated a requirement for C/EBPβ in the induction of arginase activity by LPS and hypoxia in the macrophage cell lines (Fig. 2). In the case of LPS, evidence has been published demonstrating increased nuclear localization of C/EBPβ in macrophages and splenocytes after LPS treatment (21,22). In regard to the induction of arginase by hypoxia, it was shown that hypoxia upregulates C/EBPβ in rat lungs in vivo and that it increases nuclear localization of the transcription factor in cultured pulmonary microvascular smooth muscle cells (23). Although the promoter elements required for arginase induction by LPS or hypoxia have not yet been identified, present results demonstrate a role for C/EBPβ in the transcriptional regulation of arginase by these stimuli.
The biological relevance of findings in immortalized macrophage cell lines was confirmed by studies employing wild-type and C/EBPβ−/− mice. Analysis of wound cellularity in the C/EBPβ−/− animals demonstrated a delay in the resolution of the inflammatory phase of repair that was evidenced by the persistence of polymorphonuclear leukocytes in the cellular infiltrate of the wound (Table 1). Most likely explaining these findings, C/EBP-binding sites have been identified in the promoters of multiple cytokines and chemokines participating in inflammatory reactions (for review, see Ref. 34), and the functional relevance of these sites to mediator production demonstrated in C/EBPβ−/− macrophages (24). An altered cytokine/chemokine milieu may have thus delayed the normal evolution of the inflammatory response from the early polymorphonuclear leukocyte-rich infiltrate toward a macrophage-predominant infiltrate in wounds of C/EBPβ−/− mice. Wound fluids and unstimulated peritoneal macrophages from C/EBPβ−/− mice contained significantly less arginase activity than those from C/EBPβ+/+ animals, thus confirming a role for C/EBPβ in the regulation of arginase activity in macrophages in vivo.
Present findings demonstrate that C/EBPβ is necessary for the induction of arginase I in macrophages by cAMP, IL-4, LPS, and hypoxia. They do not, however, define whether C/EBPβ directly regulates transcription of the arginase I gene or does so through indirect mechanisms. In this connection, a recent communication indicated that macrophages have constitutively activated C/EBPβ and proposed that cooperation with other nuclear factors is required for the transcriptional activation of certain C/EBPβ-dependent genes (22). Although further work will be required to fully define the molecular mechanisms for the regulation of arginase I by C/EBPβ, findings reported here identify a key role this nuclear factor in the differentiation of macrophages along the alternatively activated phenotypic pathway (2). In doing so, they extend our current understanding of the participation of C/EBP proteins in the regulation of innate immune responses.
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Keywords:
Inflammation; cAMP; IL-4; LPS; hypoxia
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