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Cell density-enhanced expression of inducible nitric oxide synthase in murine macrophages mediated by interferon-β
NITRIC OXIDE
Biology and Chemistry
Nitric Oxide 8 (2003) 222–230 www.elsevier.com/locate/yniox
Cell density-enhanced expression of inducible nitric oxide synthase in murine macrophages mediated by interferon-b Aaron T. Jacobs and Louis J. Ignarro* Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA 90095, USA Received 13 January 2003; received in revised form 22 April 2003
Abstract Nitric oxide (NO) has an important cytotoxic role in host defense processes against invading microorganisms and neoplastic cells. Here we demonstrate the effect of culture density on the expression of NO synthase and NO production by lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. At high cell densities, the LPS-induced expression of iNOS message, protein, and activity is markedly enhanced. We demonstrate the effects to be mediated by a diffusible macrophage product. Increasing cell density correlates with activation of IFN-dependent signaling pathways. We observe enhanced phosphorylation of STAT-1 on tyrosine 701 and serine 727, and an increase in STAT-1 DNA binding. Expression of the IFN-stimulated transcription factor IRF-1 is also enhanced. The data are consistent with the reported involvement of IFN-b as an autocrine co-activator of iNOS expression. Considering the importance of NO as a cytotoxic mediator of host immunity, the data suggest that macrophage density is important in regulating the magnitude of NO production, and thus, the host response to infection. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Nitric oxide; Lipopolysaccharides; Cytokines; Interferons; Gene expression regulation; Signal transduction
Nitric oxide (NO)1 is an endogenous free radical species that is both a signaling agent and a cytotoxic molecule. It functions in such diverse processes as the regulation of blood flow, blood clotting, neurotransmission, and host defense (for review see [1–3]). NO is derived from oxidation of one of the equivalent guanidino nitrogens of L -arginine [4]. The generation of NO is catalyzed by NO synthase (NOS), a family of enzymes represented by three isoforms. NO synthesized by the endothelial and neuronal forms of NOS (eNOS and nNOS, respectively) is generated in moderate concentrations and primarily mediates physiological processes * Corresponding author. Fax: 1-310-206-0589. E-mail address: [email protected] (L.J. Ignarro). 1 Abbreviations used: NO, Nitric oxide; LPS, lipopolysaccharide; NOS, NO synthase; eNOS and nNOS, endothelial and neuronal forms of NOS; TLR4, toll-like receptor 4; NF-jB, nuclear factor kappa B; TNF-a, tumor necrosis factor-a; IL-1b, interleukin-1b; PBS, phosphate-buffered saline; RPA, Ribonuclease protection assay; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; GAS, c-activated sequence; [c-32 P]ATP, c-32 P-adenosine triphosphate; EMSAs, electrophoretic mobility shift assays; RPA, ribonuclease protection assay.
including vasodilatation, inhibition of platelet function, and synaptic neurotransmission. NO is also synthesized in the immune system by iNOS, where it facilitate the killing of invading microorganisms [5]. Whereas eNOS and nNOS activities are controlled primarily by intracellular calcium concentration, the activity of iNOS in macrophages is not. Instead, NO production in macrophages is related to the level of iNOS protein, which is regulated primarily at the level of gene transcription. The iNOS gene has elements within its promoter region that mediate expression in response to a number of factors including phorbol esters, cytokines, hypoxia, and the bacterial cell wall component, lipopolysaccharide (LPS) [6–9]. The induction of iNOS by LPS occurs through the activation of a toll-like receptor 4 (TLR4)dependent signaling cascade, resulting in the activation of nuclear factor kappa B (NF-jB) [10–12]. The stimulation of iNOS expression by LPS can be enhanced by the addition of specific cytokines, including tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and both type I (IFN-a=b) and type II (IFN-c) interferons [13,14]. The positive effects of these cytokines are
1089-8603/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1089-8603(03)00027-2
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reflected by the presence of corresponding enhancer elements in the iNOS promoter. IFN-b has been reported to function as an autocrine mediator in murine macrophages, where the LPS-stimulated synthesis and release of IFN-b facilitates iNOS induction in conjunction with LPS-dependent pathways [15,16]. Endogenous IFN-b augments LPS-dependent transcription of the iNOS gene by eliciting activation of JAK/STAT signaling pathways [17]. In murine macrophages, the activation of STAT-1 is regulated by the phosphorylation of both tyrosine (Y701) and serine (S727) residues. Interferon regulatory factors (IRFs) are an additional class of IFN-stimulated proteins that also mediate the synergy between IFN-b and LPS in macrophages. IRF-1 enhances the transcription of target genes, but is not expressed or is undetectable in unactivated macrophages. However, the expression of IRF-1 is strongly induced by activators of JAK/STAT signaling pathways, including IFN-b [18]. In the present report, we describe the enhancing effect of macrophage culture density on LPS-stimulated iNOS gene expression and the synthesis of NO. Furthermore, we provide evidence for the involvement of endogenous IFN-b and of IFN-stimulated signaling pathways in mediating these effects. We propose that the densitydependent regulation of NO production seen in macrophage culture relates to the ability of endogenous macrophage populations of different densities to generate nitrosative compounds and therefore to counteract pathogenic microbes at sites of infection.
Experimental procedures Cell culture and reagents RAW 264.7 macrophages were obtained from American Type Culture Collection and grown in 10 cm culture dishes containing DMEM (Mediatech) supplemented with 50 mM HEPES, 4 mM L -glutamine, 1.2 mM L -arginine, 10 IU/ml penicillin, 10 lg/ml streptomycin, 250 ng/ml amphotericin B, and 10% fetal bovine serum. Cells were grown to confluence, then split using 0.05% trypsin/0.53 mM ethylenediaminetetraacetic acid (EDTA), and plated at a density of 3 106 cells per 10 cm dish for propagation, or as indicated for experiments. Cells were counted using a hemacytometer, and viability was assayed using trypan blue dye exclusion, which was typically greater than 95%. Cultures were maintained until passage 20 and then discarded. LPS (Escherichia coli serotype 0128:B12, Sigma) was suspended in sterile water and added to obtain the desired concentration for experiments. For co-culture experiments, Costar Transwell inserts with a 7.5 cm diameter and 0.4 lm pore size were used. As much as 30 106 cells were allowed to adhere to the insert, which was immersed
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above 2.5 106 cells in a 10 cm dish. Following the experiment, the insert was discarded and cells were collected from the bottom dish. Recombinant murine IFN-b and anti-mouse IFN-b antibody were obtained from Research Diagnostics. Ribonuclease protection assay Following treatment with experimental agents, cells were washed with cold phosphate-buffered saline (PBS), harvested using a cell scraper, and collected by centrifugation. Total RNA was extracted using the RNeasy Mini kit (Qiagen). RNA was quantified by measuring absorbance at 260 and 280 nm. Fifteen micrograms of RNA was dried using a Speed-Vac (Savant) and stored at )80 °C. Radiolabeled antisense RNA was synthesized using the Riboprobe transcription kit (Promega) and [a-32 P]cytosine triphosphate (CTP), 800 Ci/mmol (ICN). The template for antisense RNA for murine iNOS was purchased from Ambion and yields a protected fragment of 249 bases. The template for murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Pharmingen and yields a protected fragment of 97 bases. Ribonuclease protection assay (RPA) was performed using the RPA-III kit (Ambion), but substituting RNase ONE (Promega) for the supplied RNases. Protected fragments were resolved using a 5% polyacrylamide:bisacrylamide (19:1) TBU [25 mM Tris–borate; 2.5 mM EDTA; and 8 M urea] gel. Gels were dried and exposed to both autoradiographic film (Kodak BioMax Light) and a Molecular Dynamics PhosphorImager cassette (Amersham). The radiographic intensities of individual bands on the gels were determined using the program ImageQuant (Amersham). Protein extraction Following treatment with experimental agents, cells were washed with cold PBS, scraped into PBS, and collected by centrifugation. For total proteins, cell pellets were freeze-thawed three times in homogenization buffer [25 mM Tris, pH 7.4; 1 mM EDTA; 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA); 1 lg/ml aprotinin; 1 lg/ml leupeptin; 1 lg/ml pepstatin A; and 0.5 mM dithiothreitol (DTT)]. Samples were then centrifuged 5 min at 16,000g to remove debris and supernatant was stored at )80 °C. Protein concentrations were determined by the Bio-Rad assay and samples were diluted to 1 lg/ll. Nuclear extracts were prepared, as previously described [19]. Briefly, fresh cell pellets were resuspended in 800 ll icecold buffer A [10 mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 0.5 mM DTT; and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] and 50 ll of 10% Igepal CA-630 (NP-40) was added. Nuclei
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were pelleted by centrifugation at 1000g for 5 min at 4 °C and washed twice with buffer A. Nuclear proteins were then extracted by addition of buffer B [10 mM HEPES, pH 7.9; 420 mM NaCl; 1.5 mM MgCl2 ; 0.1 mM EDTA; 0.1 mM EGTA; 25% glycerol; 0.5 mM DTT; and 0.5 mM PMSF], and centrifuged at 16,000g for 10 min at 4 °C to remove debris. Protein concentrations were determined by the Bio-Rad assay and samples were diluted to 1 lg/ll. NOS activity assay Total protein extracts were examined for NOS activity using the NOSdetect assay kit (Stratagene) according to the manufacturerÕs protocol. This assay is based on measuring the NOS-catalyzed conversion of radiolabeled L -arginine to L -citrulline [20]. Briefly, 10 lg of protein extract is incubated with 1 lCi L -[2,3-3 H]arginine (42 Ci/mmol) in reaction buffer [25 mM Tris, pH 7.4; 3 lM tetrahydrobiopterin (BH4 ); 1 lM flavin adenine dinucleotide (FAD); 1 lM flavin mononucleotide (FMN); and 1 mM nicotinamide adenine dinucleotide phosphate (NADPH)] at 37 °C for 10 min. Reactions are stopped with excess stop buffer [50 mM HEPES, pH 5.5; 5 mM EDTA]. Unconverted L -[3 H]arginine is bound to a cation exchange resin in a spin column and the dpm of L -[3 H]citrulline in the eluate is determined using a scintillation counter. The NOSdetect protocol uses a final L -arginine concentration in enzyme reaction mixtures of 0.5 lM. Calculations revealed that no more than 2% conversion of total L -arginine was evident in any reaction mixture. Therefore, substrate concentration was not significantly compromised in any reaction. Protein immunoblots Five micrograms of protein per sample was resolved by SDS–PAGE and electroblotted onto a 0.2 lm nitrocellulose membrane using a Mini TransBlot apparatus (Bio-Rad). Membranes were blocked with blocking buffer [20 mM Tris, pH 7.6; 140 mM NaCl; 0.05% Tween 20; and 5% nonfat dried milk], prior to incubation with primary antibody diluted in blocking buffer. After washing three times in Trisbuffered saline (TBS) containing 0.1% Tween 20, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Chemiluminescence detection with Luminol Reagent was performed according to the manufacturerÕs protocol (Santa Cruz Biotech). Antibodies were obtained from the following sources: anti-phospho(Y701)-STAT-1, anti-IRF-1, and anti-iNOS, and all secondary antibodies were purchased from Santa Cruz Biotech; anti-phospho-(S727)-STAT-1 was purchased from Upstate.
Electrophoretic mobility shift assay Double stranded oligonucleotide encompassing the consensus c-activated sequence (GAS) 50 -TTCCCGT AA-30 (Geneka) was end-labeled using c-32 P-adenosine triphosphate ([c-32 P]ATP), 7000 Ci/mmol (ICN), and T4 polynucleotide kinase. Electrophoretic mobility shift assays (EMSAs) were performed according to an established method, with slight variation [21]. Briefly, 5 lg nuclear protein extract was incubated for 30 min at room temperature in binding buffer [25 mM HEPES, pH 7.9; 100 mM KCl; 5% Ficoll-400; 2% glycerol, 0.025% Igepal CA-630; 0.05 mM EDTA; 0.05 mM EGTA; 2 mM DTT; and 0.5 mM PMSF] containing 10 lg bovine serum albumin (BSA) and 1 lg poly(dIdC)–poly(dIdC) (Amersham–Pharmacia). For samples incubated with antibody, 2 lg anti-STAT-1 (Santa Cruz Biotech) was also included in the incubation mixture. About 100,000 cpm of radiolabeled consensus oligonucleotide (approximately 0.1 pmol) was then added to each sample, and incubated at room temperature for an additional 30 min. Samples labeled (C) indicate the addition of a 100-fold excess of unlabeled (cold) GAS oligonucleotide. Protein–DNA complexes were subsequently resolved in a 5% native Tris/taurine-buffered gel.
Results Cell density-enhanced iNOS transcription A positive correlation between the density at which RAW macrophages were plated and the induction of iNOS transcription by LPS was observed. Accordingly, the amount of iNOS mRNA induced in densely cultured cells was significantly greater in comparison to cells plated at low densities. RAW macrophages were plated at densities ranging from 2.5 106 to 30 106 cells per 10 cm dish and treated with LPS (100 ng/ml). For determination of iNOS message levels, total RNA collected 8 h after LPS addition was analyzed by ribonuclease protection assay (RPA). The mRNA for GAPDH was also quantified, and used as a control to normalize iNOS intensities. As anticipated, transcriptional expression of iNOS was strongly induced following treatment with LPS. We observed that the induction of iNOS mRNA by LPS was strongly enhanced when the cell density was increased (Fig. 1). Accordingly, in LPS-activated cells we observed an approximate 3-fold difference in the normalized levels of iNOS mRNA between cells at 2.5 106 and 30 106 cells per 10 cm dish. Data obtained using PhosphorImager analysis are represented numerically below the gel, reflecting the magnitude of iNOS expression relative to LPS-treated cells at the lowest density of 2.5 106 per dish (arbitrarily set at 1).
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Fig. 1. Increasing cell density enhances iNOS message expression induced by LPS. Macrophages were plated at increasing cell densities and activated with LPS (100 ng/ml) as indicated. Fifteen micrograms of total RNA collected 8 h following LPS induction was probed for iNOS and GAPDH (loading control) by RPA. Lane marked co-culture indicates RNA sampled from 2.5 106 cells, which was activated in the presence of 30 106 cells in a Transwell co-culture dish. Relative values for iNOS mRNA levels normalized to GAPDH are indicated below the gel and were obtained using PhosphorImager analysis. Results are representative of four independent experiments.
We hypothesized that the effect of cell density on iNOS induction might be due to the release of a soluble factor such as an interferon or other cytokine. To determine if a diffusible factor produced by the macrophages was contributing to the observed phenomenon, we designed a co-culture experiment. Accordingly, 2.5 106 macrophages were plated in a 10 cm dish, above which was inserted a cell culture well containing 30 106 macrophages. The two macrophage populations were separated by a 0.4 lm polycarbonate membrane, to allow exchange of secreted cell products. LPS (100 ng/ml) was added to the media of both cell populations, and after 8 h, total RNA was collected from the lower population of 2.5 106 cells. The results of the coculture experiment were consistent with the hypothesis that a diffusible substance contributed to the observed density effect (Fig. 1). The level of iNOS mRNA obtained from 2.5 106 cells was significantly enhanced (approximately 2.6-fold) when activated in proximity to 30 106 cells. These observations indicate that a macrophage-derived product was released from the macrophages at a density of 30 106 cells per dish and enhanced the induction of iNOS in the cells plated at 2.5 106 cells per dish. Cell density-enhanced iNOS protein expression Because the production of NO by macrophages is regulated primarily by iNOS transcript levels, we sought to determine if the positive effect of cell density on iNOS mRNA is also observed in increased levels of iNOS protein. Cells were therefore plated at densities, ranging
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from 2.5 106 to 30 106 cells per 10 cm dish and treated with LPS (100 ng/ml) for 10 h. Total protein was collected by freeze-thawing in detergent-free lysis buffer. Samples were subsequently assayed for the expression of iNOS protein by Western blot analysis and for iNOS enzymatic activity. Western blots were analyzed using the computer program NIH Image 1.62 to determine the relative intensities of individual bands. Data for each band were normalized to the level of actin as a loading control and expressed relative to LPS-treated cells at the lowest density of 2.5 106 per dish (arbitrarily set at 1). An enhanced expression of iNOS protein was observed in extracts obtained from cultures of increasing cell density (Fig. 2A). In addition, rates of NO production based on the conversion of 3 H-radiolabeled L -arginine to L -citrulline were measured in protein extracts. The data indicate that the enhanced level of iNOS protein expression observed in denser cell populations is reflected in a greater level of NOS catalytic activity (Fig. 2B). Accordingly, there was a greater than 2-fold increase in iNOS catalytic activity in samples collected from high-density (30 106 ) compared to low-density (2.5 106 ) cultures. Co-culture experiments were also performed for the measurement of iNOS protein levels and enzymatic activity. As much as 2.5 106 cells and 30 106 cells were plated in Transwell 10 cm dishes, the two populations being separated by a 0.4 lm polycarbonate membrane. Western blot analyses indicated that the level of iNOS protein for 2.5 106 macrophages was significantly enhanced by co-culture in comparison to 2.5 106 macrophages plated alone (Fig. 2A). Moreover, the catalytic rate of NO production for 2.5 106 macrophages in coculture was significantly enhanced, reflecting the higher levels of protein expression (Fig. 2B). These data suggest that a macrophage-derived soluble mediator participates in the enhancing effect of cell density on iNOS induction. Relationship between IFN-b and density-enhanced iNOS expression As described in the preceding experiments, we have established a positive correlation between cell culture density and iNOS induction. We have also demonstrated that this relationship is due, at least in part, to the effects of a soluble mediator produced by activated macrophages. IFN-b has been shown to function as an autocrine signal in murine macrophages, and is critical in the signaling events leading from LPS-challenge to expression of the iNOS gene [17]. The secreted cytokine augments iNOS transcription by stimulating the JAK/ STAT pathway, which acts in cooperation with the NFjB pathway in stimulating the iNOS promoter. Thus, it was hypothesized that the enhancing effect of IFN-b on iNOS transcription may account, in part, for the positive correlation between cell density and iNOS
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Fig. 2. Increasing cell density enhances iNOS protein expression induced by LPS. Macrophages were plated at increasing cell densities and activated with LPS (100 ng/ml) as indicated. Protein extracts were obtained by freeze-fracture method. (A) Five micrograms of protein extract was used per sample for Western blot analysis. Values for the relative expression of iNOS protein were determined using NIH Image 1.62 and normalized to the intensity of actin as a loading control. Results are representative of three independent experiments. (B) The rate of enzymatic conversion of 3 Hradiolabeled L -arginine to L -citrulline was measured. Values indicate means SEM for each condition ðn ¼ 3Þ. Data labeled ‘‘co-culture’’ was sampled from 2.5 106 cells, which was activated in the presence of 30 106 cells in a Transwell co-culture dish. *p < 0:005 as determined by unpaired StudentÕs t test.
expression. To characterize the relationship between IFN-b and LPS-induced iNOS transcription, a concentration–response relationship for IFN-b on iNOS mRNA was established. Accordingly, LPS-challenged macrophages at a low density of 2.5 106 cells per dish were simultaneously treated with LPS (100 ng/ml) and increasing amounts of recombinant murine IFN-b (10– 1500 U/ml). At 8 h following addition, total RNA was collected and subsequently analyzed by RPA for the message levels of iNOS and GAPDH (loading control). The data show that recombinant IFN-b significantly enhanced iNOS transcription by approximately 3.7-fold over LPS alone (Fig. 3). To determine the extent to which endogenous IFN-b from macrophages contributes to the density-dependent induction of iNOS transcription, specific antiserum was used to neutralize the effects of IFN-b in cell culture. RAW macrophages were plated at a density of either 2.5 106 or 30 106 cells per 10 cm dish. The highdensity cultures (30 106 ) were treated simultaneously with LPS (100 ng/ml) and anti-IFN-b neutralizing antibody, ranging from 5 to 1000 neutralization units (NU) per ml. Total RNA collected 8 h after treatment was then analyzed by RPA for the level of iNOS and GAPDH (loading control) transcripts. The data show that iNOS message levels in densely cultured cells were strongly attenuated with increasing antibody titer (Fig. 4), confirming a role for endogenous IFN-b in iNOS induction. Furthermore, incubation with a nonspecific anti-mouse IgG (5 lg/ml) was without effect (data not shown). Based on values obtained by PhosphorImager analysis, when treated with an antibody concentration of 1000 NU/ml, the induction of iNOS transcript in high-density culture was reduced to nearly
Fig. 3. Concentration-response relationship of IFN-b on LPS-induced iNOS mRNA expression. Macrophages were plated at a density of 2.5 106 cells per 10 cm dish and activated with LPS (100 ng/ml) plus increasing amounts of IFN-b as indicated. Fifteen micrograms of total RNA collected 8 h following LPS induction was probed for iNOS and GAPDH (loading control) by RPA. Relative values for iNOS mRNA levels normalized to GAPDH are indicated below the gel and were obtained using PhosphorImager analysis. Results are representative of three independent experiments.
equal the level of iNOS mRNA expressed in low-density culture. Because the density-dependent effect on iNOS transcription was almost completely attenuated by neutralizing antibody, these data suggest a principal role for IFN-b in mediating the level of iNOS message induction in LPS-treated macrophages. Cell density-enhanced activation of IFN-stimulated signaling pathways The effects of IFN-b on the transcription of target genes are communicated through activation of the JAK/STAT signaling pathway. Phosphorylated STAT
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Fig. 4. Neutralizing antibody against mouse IFN-b attenuates the density-enhanced expression of iNOS mRNA. Macrophages were plated at a density of 2.5 106 or 30 106 cells per 10 cm dish and activated with LPS (100 ng/ml). Cells plated at a density of 30 106 per dish were also treated simultaneously with an increasing titer of antimouse IFN-b antibody (a-IFN-b). One neutralization unit (NU) is defined as the quantity of antibody required to effectively neutralize one unit of mouse IFN-b (in a standardized antiviral assay). Fifteen micrograms of RNA collected 8 h following induction was probed for iNOS and GAPDH (loading control) by RPA. Relative values for iNOS mRNA levels normalized to GAPDH are indicated below the gel and obtained using PhosphorImager analysis. Results are representative of three independent experiments.
proteins mediate transcriptional enhancement by binding to conserved sequences in the promoters of IFNstimulated genes. Accordingly, the promoter region of the murine iNOS gene contains elements which facilitate the binding of STAT homo- and heterodimers, and confer sensitivity to the actions of IFN-b [22]. Maximal activation of STAT-1 transcriptional activity necessitates both serine (S727) and tyrosine (Y701) phosphorylation [23]. IFN-stimulated STAT-1 activation induces expression of a number of genes, including the transcription factor, IRF-1, which enhances iNOS transcription, owing to a critical IRF-element in the promoter region of the iNOS gene. Because IFN-b was observed to contribute to the density-dependent enhancement of iNOS induction, the effects of macrophage cell density on the phosphorylation of STAT-1 and on the induction of IRF-1 were examined. RAW cells were plated at densities ranging from 2.5 106 to 30 106 cells per 10 cm dish and treated with LPS (100 ng/ml). Nuclear proteins were extracted 3 h following LPS and diluted to equal protein concentrations. Western blots of nuclear proteins were probed with antibodies specific for phospho-(S727)STAT-1, phospho-(Y701)-STAT-1, IRF-1, and actin (loading control). A positive relationship was observed between cell density and both STAT-1 phosphorylation and IRF-1 expression (Fig. 5). In agreement with the hypothesis that IFN-b plays a significant role in the cell density-enhanced induction of iNOS, the data show that the levels of phospho-STAT-1 (both a and b) and IRF-1 were several-fold higher in high-density compared to low-density cell cultures. Moreover, experiments in
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Fig. 5. Increasing cell density enhances IFN-activated signaling pathways. Macrophages were plated at increasing cell densities and activated with LPS (100 ng/ml) as indicated. Nuclear proteins were sampled 3 h after LPS induction. Five micrograms of nuclear extract was used for immunoblot analysis. Data labeled ‘‘co-culture’’ was sampled from 2.5 106 cells, which was activated in the presence of 30 106 cells in a Transwell co-culture dish. Actin was used as a loading control. Results are representative of three independent experiments.
which 2.5 106 macrophages were co-cultured in the presence of 30 106 macrophages demonstrate that the activation of these intracellular signals was related to the release of a soluble macrophage-derived interferon signal. To determine whether the observed increase in phospho-STAT-1 relates to nuclear activity, samples were tested by electrophoretic mobility shift assay (EMSA) for binding to a consensus GAS oligonucleotide. Again, there was a strong positive correlation
Fig. 6. Increasing cell density enhances STAT-1 binding to a consensus DNA sequence. Macrophages were plated at increasing cell densities and activated with LPS (100 ng/ml) as indicated. Nuclear proteins were sampled 3 h after LPS induction. Five micrograms of nuclear protein was assayed by EMSA for binding to a consensus GAS oligonucleotide. Preincubation with cold, unlabeled oligonucleotide (C) or with anti-STAT-1 antibody (a-STAT-1) significantly attenuated binding. Relative values for STAT-1 DNA binding are based on intensities of the bands determined by PhosphorImager. Results are representative of three independent experiments.
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between cell density and IFN-stimulated signaling, as the binding of STAT-1 to the radiolabeled GAS oligonucleotide was significantly enhanced in denser cell cultures (Fig. 6). Also, binding to the radiolabeled GAS oligonucleotide was attenuated by preincubation with either excess unlabeled (cold) GAS oligo, or with antiSTAT-1 antibody (a-STAT-1), confirming that the observed shift was due to STAT-1.
Discussion Production of NO by macrophages is regulated primarily through the tight control of iNOS gene expression, which is transcriptionally silent in the absence of activating stimuli. In the murine macrophage line RAW 264.7, iNOS is markedly induced when cells are incubated with the bacterial cell-wall product, LPS [24]. The transcriptional activation of the iNOS gene by LPS can be enhanced by the addition of cytokines, including TNF-a, IL-1b, and both type I (a=b) and II (c) IFNs. The expression of iNOS in RAW 264.7 macrophages involves the LPS-induced association of CD14 with TLR4 in an LPS-activation cluster. Intracellular signals initiated by the LPS activation cluster subsequently activate the transcription factor NF-jB by inducing the degradation of the inhibitory protein, IjB. The endogenous production of IFN-b triggered by NF-jB is critical in the processes leading to iNOS expression [25]. Accordingly, it was demonstrated that bone marrow macrophages derived from mice deficient in functional type I IFN receptor were unable to initiate iNOS transcription in response to the addition of LPS [26]. The mechanism by which IFN-b promotes iNOS expression is through activation of the JAK/STAT pathway. STAT-1 is phosphorylated in response to IFN-b and enhances the transcription of target genes through conserved GAS elements present in the promoter region of the iNOS gene. The transcription factor, IRF-1, induced by STAT-1 activation, is also critical in mediating iNOS transcription [27–29]. In studying the expression of iNOS in LPS-treated RAW macrophages, we demonstrate that density in cell culture strongly affects iNOS transcript levels. When normalized to the expression of GAPDH, comparatively more iNOS message is produced in high-density compared to low-density cell cultures. The positive effect of cell density on iNOS message expression was also reflected in the expression of iNOS protein by Western blot. This was confirmed by measuring NOS catalytic activity. We decided to examine the catalytic activity of iNOS in diluted cellular extracts, rather than sample the media for NO 2 and NO3 (end-products of NO). This was to avoid potential feedback-inhibition of iNOS by NO under the high concentrations of NO present in macrophage culture [30]. We propose that the
enhancement of iNOS expression caused by increasing cell density may have physiological importance in mediating the host response to infection, since larger macrophage populations could have greater capability in mounting an NO-based cytotoxic defense. To determine if the density-mediated effect was due to direct cell–cell contacts, or to a soluble macrophage product, co-culture experiments were conducted. The results of co-culture implied that a macrophage-derived soluble factor was involved in the enhancing effect of cell density on iNOS induction. Since IFN-b has been previously demonstrated to function as an autocrine signal in murine macrophages, we decided to investigate IFN-b as a potential mediator of the cell density effect. We observed that IFN-b enhanced the induction of iNOS message expression in low-density cultures; the magnitude of this effect was comparable to the enhancement observed in high-density cultures treated with LPS alone. Conversely, the addition of an IFN-b neutralizing antibody to cells stimulated with LPS alone nearly abolished the density-dependent enhancement of iNOS induction, suggesting that IFN-b plays a critical role in mediating this effect. Moreover, the absence of inducible IFN-a or IFN-c message expression is consistent with the view that the mediator is IFN-b (unpublished observations). The cytocidal or cytostatic actions of activated macrophages against pathogens and neoplastic cells are dependent on the production of NO [31,32]. When high concentrations of NO are achieved locally by activated macrophages, oxidation products such as nitrogen dioxide (NO2 ) and nitrous anhydride (N2 O3 ) may form [33]. This is because the chemical reactions leading to these species are multiple-order processes with respect to NO, and are thus highly dependent on the NO concentration [34,35]. These reactive nitrogen species are more potent oxidants than NO, are more reactive with cellular nucleophiles, and may contribute greatly to the observed cytotoxicity of macrophage-derived NO. The enhanced production of NO in dense macrophage populations may facilitate the formation of NO2 and N2 O3 , thereby increasing macrophage cytotoxicity. Accordingly, a 5-fold increase in cell density (assuming a proportional 5-fold increase in NO levels) was recently calculated to produce a 25-fold increase in the concentration of N2 O3 [36]. Since we observe that iNOS levels increase with cell density, we suggest that even greater increases in N2 O3 levels are possible. Therefore, a disproportional enhancement of cytotoxicity may occur where macrophage populations are dense. Interestingly, a study on the effect of culture density on iNOS induction in hepatocytes revealed a decrease in iNOS protein levels at 24 h after treatment [37]. The reduction in iNOS expression was reportedly caused by an NO-independent reduction in total protein synthesis following the treatment of hepatocytes with a cocktail of
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LPS, IL-1b, TNF-a, and IFN-c. This contrasts with our observation that iNOS protein levels were enhanced in densely cultured macrophages treated with LPS alone (Fig. 2). Differences between this earlier report and our investigation, including the use of a different cell type, a later time-point, and addition various cytokines make comparisons difficult, particularly since the cause of the reduction in protein synthesis was undetermined. The effect of cell density on the cytotoxic role of macrophages is seldom addressed. However, in a recent study, the ability of human monocytes to suppress infection with Mycobacterium bovis was shown to be highly dependent on cell density [38]. Accordingly, mycobacterial growth was successfully inhibited by a cell density of 2 105 , but not by 5 104 macrophages per culture dish. A number of factors were evaluated as possible reasons why dense cultures were more effective at controlling infection. Interestingly, growth of M. bovis was not enhanced by the addition of the iNOSinhibitor L -N-iminoethyllysine, suggesting that NO had an insignificant role. However, other investigators have reported that NO derived from human macrophages is indeed critical in suppressing M. bovis infections [39]. The discrepancy in findings suggests that further study is needed in this area. In this respect, we propose that the enhancement of iNOS induction in dense macrophage populations may contribute a hostÕs ability to control infections when NO can play a cytotoxic role.
Acknowledgments This work was supported by grants from the National Institutes of Health HL35014 and HL40922. We gratefully acknowledge Dr. Jon M. Fukuto for valuable scientific guidance.
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Biology and Chemistry
Nitric Oxide 8 (2003) 222–230 www.elsevier.com/locate/yniox
Cell density-enhanced expression of inducible nitric oxide synthase in murine macrophages mediated by interferon-b Aaron T. Jacobs and Louis J. Ignarro* Department of Molecular and Medical Pharmacology, UCLA, Los Angeles, CA 90095, USA Received 13 January 2003; received in revised form 22 April 2003
Abstract Nitric oxide (NO) has an important cytotoxic role in host defense processes against invading microorganisms and neoplastic cells. Here we demonstrate the effect of culture density on the expression of NO synthase and NO production by lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. At high cell densities, the LPS-induced expression of iNOS message, protein, and activity is markedly enhanced. We demonstrate the effects to be mediated by a diffusible macrophage product. Increasing cell density correlates with activation of IFN-dependent signaling pathways. We observe enhanced phosphorylation of STAT-1 on tyrosine 701 and serine 727, and an increase in STAT-1 DNA binding. Expression of the IFN-stimulated transcription factor IRF-1 is also enhanced. The data are consistent with the reported involvement of IFN-b as an autocrine co-activator of iNOS expression. Considering the importance of NO as a cytotoxic mediator of host immunity, the data suggest that macrophage density is important in regulating the magnitude of NO production, and thus, the host response to infection. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Nitric oxide; Lipopolysaccharides; Cytokines; Interferons; Gene expression regulation; Signal transduction
Nitric oxide (NO)1 is an endogenous free radical species that is both a signaling agent and a cytotoxic molecule. It functions in such diverse processes as the regulation of blood flow, blood clotting, neurotransmission, and host defense (for review see [1–3]). NO is derived from oxidation of one of the equivalent guanidino nitrogens of L -arginine [4]. The generation of NO is catalyzed by NO synthase (NOS), a family of enzymes represented by three isoforms. NO synthesized by the endothelial and neuronal forms of NOS (eNOS and nNOS, respectively) is generated in moderate concentrations and primarily mediates physiological processes * Corresponding author. Fax: 1-310-206-0589. E-mail address: [email protected] (L.J. Ignarro). 1 Abbreviations used: NO, Nitric oxide; LPS, lipopolysaccharide; NOS, NO synthase; eNOS and nNOS, endothelial and neuronal forms of NOS; TLR4, toll-like receptor 4; NF-jB, nuclear factor kappa B; TNF-a, tumor necrosis factor-a; IL-1b, interleukin-1b; PBS, phosphate-buffered saline; RPA, Ribonuclease protection assay; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride; GAS, c-activated sequence; [c-32 P]ATP, c-32 P-adenosine triphosphate; EMSAs, electrophoretic mobility shift assays; RPA, ribonuclease protection assay.
including vasodilatation, inhibition of platelet function, and synaptic neurotransmission. NO is also synthesized in the immune system by iNOS, where it facilitate the killing of invading microorganisms [5]. Whereas eNOS and nNOS activities are controlled primarily by intracellular calcium concentration, the activity of iNOS in macrophages is not. Instead, NO production in macrophages is related to the level of iNOS protein, which is regulated primarily at the level of gene transcription. The iNOS gene has elements within its promoter region that mediate expression in response to a number of factors including phorbol esters, cytokines, hypoxia, and the bacterial cell wall component, lipopolysaccharide (LPS) [6–9]. The induction of iNOS by LPS occurs through the activation of a toll-like receptor 4 (TLR4)dependent signaling cascade, resulting in the activation of nuclear factor kappa B (NF-jB) [10–12]. The stimulation of iNOS expression by LPS can be enhanced by the addition of specific cytokines, including tumor necrosis factor-a (TNF-a), interleukin-1b (IL-1b), and both type I (IFN-a=b) and type II (IFN-c) interferons [13,14]. The positive effects of these cytokines are
1089-8603/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S1089-8603(03)00027-2
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reflected by the presence of corresponding enhancer elements in the iNOS promoter. IFN-b has been reported to function as an autocrine mediator in murine macrophages, where the LPS-stimulated synthesis and release of IFN-b facilitates iNOS induction in conjunction with LPS-dependent pathways [15,16]. Endogenous IFN-b augments LPS-dependent transcription of the iNOS gene by eliciting activation of JAK/STAT signaling pathways [17]. In murine macrophages, the activation of STAT-1 is regulated by the phosphorylation of both tyrosine (Y701) and serine (S727) residues. Interferon regulatory factors (IRFs) are an additional class of IFN-stimulated proteins that also mediate the synergy between IFN-b and LPS in macrophages. IRF-1 enhances the transcription of target genes, but is not expressed or is undetectable in unactivated macrophages. However, the expression of IRF-1 is strongly induced by activators of JAK/STAT signaling pathways, including IFN-b [18]. In the present report, we describe the enhancing effect of macrophage culture density on LPS-stimulated iNOS gene expression and the synthesis of NO. Furthermore, we provide evidence for the involvement of endogenous IFN-b and of IFN-stimulated signaling pathways in mediating these effects. We propose that the densitydependent regulation of NO production seen in macrophage culture relates to the ability of endogenous macrophage populations of different densities to generate nitrosative compounds and therefore to counteract pathogenic microbes at sites of infection.
Experimental procedures Cell culture and reagents RAW 264.7 macrophages were obtained from American Type Culture Collection and grown in 10 cm culture dishes containing DMEM (Mediatech) supplemented with 50 mM HEPES, 4 mM L -glutamine, 1.2 mM L -arginine, 10 IU/ml penicillin, 10 lg/ml streptomycin, 250 ng/ml amphotericin B, and 10% fetal bovine serum. Cells were grown to confluence, then split using 0.05% trypsin/0.53 mM ethylenediaminetetraacetic acid (EDTA), and plated at a density of 3 106 cells per 10 cm dish for propagation, or as indicated for experiments. Cells were counted using a hemacytometer, and viability was assayed using trypan blue dye exclusion, which was typically greater than 95%. Cultures were maintained until passage 20 and then discarded. LPS (Escherichia coli serotype 0128:B12, Sigma) was suspended in sterile water and added to obtain the desired concentration for experiments. For co-culture experiments, Costar Transwell inserts with a 7.5 cm diameter and 0.4 lm pore size were used. As much as 30 106 cells were allowed to adhere to the insert, which was immersed
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above 2.5 106 cells in a 10 cm dish. Following the experiment, the insert was discarded and cells were collected from the bottom dish. Recombinant murine IFN-b and anti-mouse IFN-b antibody were obtained from Research Diagnostics. Ribonuclease protection assay Following treatment with experimental agents, cells were washed with cold phosphate-buffered saline (PBS), harvested using a cell scraper, and collected by centrifugation. Total RNA was extracted using the RNeasy Mini kit (Qiagen). RNA was quantified by measuring absorbance at 260 and 280 nm. Fifteen micrograms of RNA was dried using a Speed-Vac (Savant) and stored at )80 °C. Radiolabeled antisense RNA was synthesized using the Riboprobe transcription kit (Promega) and [a-32 P]cytosine triphosphate (CTP), 800 Ci/mmol (ICN). The template for antisense RNA for murine iNOS was purchased from Ambion and yields a protected fragment of 249 bases. The template for murine glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was purchased from Pharmingen and yields a protected fragment of 97 bases. Ribonuclease protection assay (RPA) was performed using the RPA-III kit (Ambion), but substituting RNase ONE (Promega) for the supplied RNases. Protected fragments were resolved using a 5% polyacrylamide:bisacrylamide (19:1) TBU [25 mM Tris–borate; 2.5 mM EDTA; and 8 M urea] gel. Gels were dried and exposed to both autoradiographic film (Kodak BioMax Light) and a Molecular Dynamics PhosphorImager cassette (Amersham). The radiographic intensities of individual bands on the gels were determined using the program ImageQuant (Amersham). Protein extraction Following treatment with experimental agents, cells were washed with cold PBS, scraped into PBS, and collected by centrifugation. For total proteins, cell pellets were freeze-thawed three times in homogenization buffer [25 mM Tris, pH 7.4; 1 mM EDTA; 1 mM ethylene glycol-bis (b-aminoethyl ether)-N,N,N0 ,N0 -tetraacetic acid (EGTA); 1 lg/ml aprotinin; 1 lg/ml leupeptin; 1 lg/ml pepstatin A; and 0.5 mM dithiothreitol (DTT)]. Samples were then centrifuged 5 min at 16,000g to remove debris and supernatant was stored at )80 °C. Protein concentrations were determined by the Bio-Rad assay and samples were diluted to 1 lg/ll. Nuclear extracts were prepared, as previously described [19]. Briefly, fresh cell pellets were resuspended in 800 ll icecold buffer A [10 mM HEPES, pH 7.9; 10 mM KCl; 0.1 mM EDTA; 0.1 mM EGTA; 0.5 mM DTT; and 0.5 mM phenylmethylsulfonyl fluoride (PMSF)] and 50 ll of 10% Igepal CA-630 (NP-40) was added. Nuclei
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were pelleted by centrifugation at 1000g for 5 min at 4 °C and washed twice with buffer A. Nuclear proteins were then extracted by addition of buffer B [10 mM HEPES, pH 7.9; 420 mM NaCl; 1.5 mM MgCl2 ; 0.1 mM EDTA; 0.1 mM EGTA; 25% glycerol; 0.5 mM DTT; and 0.5 mM PMSF], and centrifuged at 16,000g for 10 min at 4 °C to remove debris. Protein concentrations were determined by the Bio-Rad assay and samples were diluted to 1 lg/ll. NOS activity assay Total protein extracts were examined for NOS activity using the NOSdetect assay kit (Stratagene) according to the manufacturerÕs protocol. This assay is based on measuring the NOS-catalyzed conversion of radiolabeled L -arginine to L -citrulline [20]. Briefly, 10 lg of protein extract is incubated with 1 lCi L -[2,3-3 H]arginine (42 Ci/mmol) in reaction buffer [25 mM Tris, pH 7.4; 3 lM tetrahydrobiopterin (BH4 ); 1 lM flavin adenine dinucleotide (FAD); 1 lM flavin mononucleotide (FMN); and 1 mM nicotinamide adenine dinucleotide phosphate (NADPH)] at 37 °C for 10 min. Reactions are stopped with excess stop buffer [50 mM HEPES, pH 5.5; 5 mM EDTA]. Unconverted L -[3 H]arginine is bound to a cation exchange resin in a spin column and the dpm of L -[3 H]citrulline in the eluate is determined using a scintillation counter. The NOSdetect protocol uses a final L -arginine concentration in enzyme reaction mixtures of 0.5 lM. Calculations revealed that no more than 2% conversion of total L -arginine was evident in any reaction mixture. Therefore, substrate concentration was not significantly compromised in any reaction. Protein immunoblots Five micrograms of protein per sample was resolved by SDS–PAGE and electroblotted onto a 0.2 lm nitrocellulose membrane using a Mini TransBlot apparatus (Bio-Rad). Membranes were blocked with blocking buffer [20 mM Tris, pH 7.6; 140 mM NaCl; 0.05% Tween 20; and 5% nonfat dried milk], prior to incubation with primary antibody diluted in blocking buffer. After washing three times in Trisbuffered saline (TBS) containing 0.1% Tween 20, membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody. Chemiluminescence detection with Luminol Reagent was performed according to the manufacturerÕs protocol (Santa Cruz Biotech). Antibodies were obtained from the following sources: anti-phospho(Y701)-STAT-1, anti-IRF-1, and anti-iNOS, and all secondary antibodies were purchased from Santa Cruz Biotech; anti-phospho-(S727)-STAT-1 was purchased from Upstate.
Electrophoretic mobility shift assay Double stranded oligonucleotide encompassing the consensus c-activated sequence (GAS) 50 -TTCCCGT AA-30 (Geneka) was end-labeled using c-32 P-adenosine triphosphate ([c-32 P]ATP), 7000 Ci/mmol (ICN), and T4 polynucleotide kinase. Electrophoretic mobility shift assays (EMSAs) were performed according to an established method, with slight variation [21]. Briefly, 5 lg nuclear protein extract was incubated for 30 min at room temperature in binding buffer [25 mM HEPES, pH 7.9; 100 mM KCl; 5% Ficoll-400; 2% glycerol, 0.025% Igepal CA-630; 0.05 mM EDTA; 0.05 mM EGTA; 2 mM DTT; and 0.5 mM PMSF] containing 10 lg bovine serum albumin (BSA) and 1 lg poly(dIdC)–poly(dIdC) (Amersham–Pharmacia). For samples incubated with antibody, 2 lg anti-STAT-1 (Santa Cruz Biotech) was also included in the incubation mixture. About 100,000 cpm of radiolabeled consensus oligonucleotide (approximately 0.1 pmol) was then added to each sample, and incubated at room temperature for an additional 30 min. Samples labeled (C) indicate the addition of a 100-fold excess of unlabeled (cold) GAS oligonucleotide. Protein–DNA complexes were subsequently resolved in a 5% native Tris/taurine-buffered gel.
Results Cell density-enhanced iNOS transcription A positive correlation between the density at which RAW macrophages were plated and the induction of iNOS transcription by LPS was observed. Accordingly, the amount of iNOS mRNA induced in densely cultured cells was significantly greater in comparison to cells plated at low densities. RAW macrophages were plated at densities ranging from 2.5 106 to 30 106 cells per 10 cm dish and treated with LPS (100 ng/ml). For determination of iNOS message levels, total RNA collected 8 h after LPS addition was analyzed by ribonuclease protection assay (RPA). The mRNA for GAPDH was also quantified, and used as a control to normalize iNOS intensities. As anticipated, transcriptional expression of iNOS was strongly induced following treatment with LPS. We observed that the induction of iNOS mRNA by LPS was strongly enhanced when the cell density was increased (Fig. 1). Accordingly, in LPS-activated cells we observed an approximate 3-fold difference in the normalized levels of iNOS mRNA between cells at 2.5 106 and 30 106 cells per 10 cm dish. Data obtained using PhosphorImager analysis are represented numerically below the gel, reflecting the magnitude of iNOS expression relative to LPS-treated cells at the lowest density of 2.5 106 per dish (arbitrarily set at 1).
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Fig. 1. Increasing cell density enhances iNOS message expression induced by LPS. Macrophages were plated at increasing cell densities and activated with LPS (100 ng/ml) as indicated. Fifteen micrograms of total RNA collected 8 h following LPS induction was probed for iNOS and GAPDH (loading control) by RPA. Lane marked co-culture indicates RNA sampled from 2.5 106 cells, which was activated in the presence of 30 106 cells in a Transwell co-culture dish. Relative values for iNOS mRNA levels normalized to GAPDH are indicated below the gel and were obtained using PhosphorImager analysis. Results are representative of four independent experiments.
We hypothesized that the effect of cell density on iNOS induction might be due to the release of a soluble factor such as an interferon or other cytokine. To determine if a diffusible factor produced by the macrophages was contributing to the observed phenomenon, we designed a co-culture experiment. Accordingly, 2.5 106 macrophages were plated in a 10 cm dish, above which was inserted a cell culture well containing 30 106 macrophages. The two macrophage populations were separated by a 0.4 lm polycarbonate membrane, to allow exchange of secreted cell products. LPS (100 ng/ml) was added to the media of both cell populations, and after 8 h, total RNA was collected from the lower population of 2.5 106 cells. The results of the coculture experiment were consistent with the hypothesis that a diffusible substance contributed to the observed density effect (Fig. 1). The level of iNOS mRNA obtained from 2.5 106 cells was significantly enhanced (approximately 2.6-fold) when activated in proximity to 30 106 cells. These observations indicate that a macrophage-derived product was released from the macrophages at a density of 30 106 cells per dish and enhanced the induction of iNOS in the cells plated at 2.5 106 cells per dish. Cell density-enhanced iNOS protein expression Because the production of NO by macrophages is regulated primarily by iNOS transcript levels, we sought to determine if the positive effect of cell density on iNOS mRNA is also observed in increased levels of iNOS protein. Cells were therefore plated at densities, ranging
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from 2.5 106 to 30 106 cells per 10 cm dish and treated with LPS (100 ng/ml) for 10 h. Total protein was collected by freeze-thawing in detergent-free lysis buffer. Samples were subsequently assayed for the expression of iNOS protein by Western blot analysis and for iNOS enzymatic activity. Western blots were analyzed using the computer program NIH Image 1.62 to determine the relative intensities of individual bands. Data for each band were normalized to the level of actin as a loading control and expressed relative to LPS-treated cells at the lowest density of 2.5 106 per dish (arbitrarily set at 1). An enhanced expression of iNOS protein was observed in extracts obtained from cultures of increasing cell density (Fig. 2A). In addition, rates of NO production based on the conversion of 3 H-radiolabeled L -arginine to L -citrulline were measured in protein extracts. The data indicate that the enhanced level of iNOS protein expression observed in denser cell populations is reflected in a greater level of NOS catalytic activity (Fig. 2B). Accordingly, there was a greater than 2-fold increase in iNOS catalytic activity in samples collected from high-density (30 106 ) compared to low-density (2.5 106 ) cultures. Co-culture experiments were also performed for the measurement of iNOS protein levels and enzymatic activity. As much as 2.5 106 cells and 30 106 cells were plated in Transwell 10 cm dishes, the two populations being separated by a 0.4 lm polycarbonate membrane. Western blot analyses indicated that the level of iNOS protein for 2.5 106 macrophages was significantly enhanced by co-culture in comparison to 2.5 106 macrophages plated alone (Fig. 2A). Moreover, the catalytic rate of NO production for 2.5 106 macrophages in coculture was significantly enhanced, reflecting the higher levels of protein expression (Fig. 2B). These data suggest that a macrophage-derived soluble mediator participates in the enhancing effect of cell density on iNOS induction. Relationship between IFN-b and density-enhanced iNOS expression As described in the preceding experiments, we have established a positive correlation between cell culture density and iNOS induction. We have also demonstrated that this relationship is due, at least in part, to the effects of a soluble mediator produced by activated macrophages. IFN-b has been shown to function as an autocrine signal in murine macrophages, and is critical in the signaling events leading from LPS-challenge to expression of the iNOS gene [17]. The secreted cytokine augments iNOS transcription by stimulating the JAK/ STAT pathway, which acts in cooperation with the NFjB pathway in stimulating the iNOS promoter. Thus, it was hypothesized that the enhancing effect of IFN-b on iNOS transcription may account, in part, for the positive correlation between cell density and iNOS
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Fig. 2. Increasing cell density enhances iNOS protein expression induced by LPS. Macrophages were plated at increasing cell densities and activated with LPS (100 ng/ml) as indicated. Protein extracts were obtained by freeze-fracture method. (A) Five micrograms of protein extract was used per sample for Western blot analysis. Values for the relative expression of iNOS protein were determined using NIH Image 1.62 and normalized to the intensity of actin as a loading control. Results are representative of three independent experiments. (B) The rate of enzymatic conversion of 3 Hradiolabeled L -arginine to L -citrulline was measured. Values indicate means SEM for each condition ðn ¼ 3Þ. Data labeled ‘‘co-culture’’ was sampled from 2.5 106 cells, which was activated in the presence of 30 106 cells in a Transwell co-culture dish. *p < 0:005 as determined by unpaired StudentÕs t test.
expression. To characterize the relationship between IFN-b and LPS-induced iNOS transcription, a concentration–response relationship for IFN-b on iNOS mRNA was established. Accordingly, LPS-challenged macrophages at a low density of 2.5 106 cells per dish were simultaneously treated with LPS (100 ng/ml) and increasing amounts of recombinant murine IFN-b (10– 1500 U/ml). At 8 h following addition, total RNA was collected and subsequently analyzed by RPA for the message levels of iNOS and GAPDH (loading control). The data show that recombinant IFN-b significantly enhanced iNOS transcription by approximately 3.7-fold over LPS alone (Fig. 3). To determine the extent to which endogenous IFN-b from macrophages contributes to the density-dependent induction of iNOS transcription, specific antiserum was used to neutralize the effects of IFN-b in cell culture. RAW macrophages were plated at a density of either 2.5 106 or 30 106 cells per 10 cm dish. The highdensity cultures (30 106 ) were treated simultaneously with LPS (100 ng/ml) and anti-IFN-b neutralizing antibody, ranging from 5 to 1000 neutralization units (NU) per ml. Total RNA collected 8 h after treatment was then analyzed by RPA for the level of iNOS and GAPDH (loading control) transcripts. The data show that iNOS message levels in densely cultured cells were strongly attenuated with increasing antibody titer (Fig. 4), confirming a role for endogenous IFN-b in iNOS induction. Furthermore, incubation with a nonspecific anti-mouse IgG (5 lg/ml) was without effect (data not shown). Based on values obtained by PhosphorImager analysis, when treated with an antibody concentration of 1000 NU/ml, the induction of iNOS transcript in high-density culture was reduced to nearly
Fig. 3. Concentration-response relationship of IFN-b on LPS-induced iNOS mRNA expression. Macrophages were plated at a density of 2.5 106 cells per 10 cm dish and activated with LPS (100 ng/ml) plus increasing amounts of IFN-b as indicated. Fifteen micrograms of total RNA collected 8 h following LPS induction was probed for iNOS and GAPDH (loading control) by RPA. Relative values for iNOS mRNA levels normalized to GAPDH are indicated below the gel and were obtained using PhosphorImager analysis. Results are representative of three independent experiments.
equal the level of iNOS mRNA expressed in low-density culture. Because the density-dependent effect on iNOS transcription was almost completely attenuated by neutralizing antibody, these data suggest a principal role for IFN-b in mediating the level of iNOS message induction in LPS-treated macrophages. Cell density-enhanced activation of IFN-stimulated signaling pathways The effects of IFN-b on the transcription of target genes are communicated through activation of the JAK/STAT signaling pathway. Phosphorylated STAT
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Fig. 4. Neutralizing antibody against mouse IFN-b attenuates the density-enhanced expression of iNOS mRNA. Macrophages were plated at a density of 2.5 106 or 30 106 cells per 10 cm dish and activated with LPS (100 ng/ml). Cells plated at a density of 30 106 per dish were also treated simultaneously with an increasing titer of antimouse IFN-b antibody (a-IFN-b). One neutralization unit (NU) is defined as the quantity of antibody required to effectively neutralize one unit of mouse IFN-b (in a standardized antiviral assay). Fifteen micrograms of RNA collected 8 h following induction was probed for iNOS and GAPDH (loading control) by RPA. Relative values for iNOS mRNA levels normalized to GAPDH are indicated below the gel and obtained using PhosphorImager analysis. Results are representative of three independent experiments.
proteins mediate transcriptional enhancement by binding to conserved sequences in the promoters of IFNstimulated genes. Accordingly, the promoter region of the murine iNOS gene contains elements which facilitate the binding of STAT homo- and heterodimers, and confer sensitivity to the actions of IFN-b [22]. Maximal activation of STAT-1 transcriptional activity necessitates both serine (S727) and tyrosine (Y701) phosphorylation [23]. IFN-stimulated STAT-1 activation induces expression of a number of genes, including the transcription factor, IRF-1, which enhances iNOS transcription, owing to a critical IRF-element in the promoter region of the iNOS gene. Because IFN-b was observed to contribute to the density-dependent enhancement of iNOS induction, the effects of macrophage cell density on the phosphorylation of STAT-1 and on the induction of IRF-1 were examined. RAW cells were plated at densities ranging from 2.5 106 to 30 106 cells per 10 cm dish and treated with LPS (100 ng/ml). Nuclear proteins were extracted 3 h following LPS and diluted to equal protein concentrations. Western blots of nuclear proteins were probed with antibodies specific for phospho-(S727)STAT-1, phospho-(Y701)-STAT-1, IRF-1, and actin (loading control). A positive relationship was observed between cell density and both STAT-1 phosphorylation and IRF-1 expression (Fig. 5). In agreement with the hypothesis that IFN-b plays a significant role in the cell density-enhanced induction of iNOS, the data show that the levels of phospho-STAT-1 (both a and b) and IRF-1 were several-fold higher in high-density compared to low-density cell cultures. Moreover, experiments in
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Fig. 5. Increasing cell density enhances IFN-activated signaling pathways. Macrophages were plated at increasing cell densities and activated with LPS (100 ng/ml) as indicated. Nuclear proteins were sampled 3 h after LPS induction. Five micrograms of nuclear extract was used for immunoblot analysis. Data labeled ‘‘co-culture’’ was sampled from 2.5 106 cells, which was activated in the presence of 30 106 cells in a Transwell co-culture dish. Actin was used as a loading control. Results are representative of three independent experiments.
which 2.5 106 macrophages were co-cultured in the presence of 30 106 macrophages demonstrate that the activation of these intracellular signals was related to the release of a soluble macrophage-derived interferon signal. To determine whether the observed increase in phospho-STAT-1 relates to nuclear activity, samples were tested by electrophoretic mobility shift assay (EMSA) for binding to a consensus GAS oligonucleotide. Again, there was a strong positive correlation
Fig. 6. Increasing cell density enhances STAT-1 binding to a consensus DNA sequence. Macrophages were plated at increasing cell densities and activated with LPS (100 ng/ml) as indicated. Nuclear proteins were sampled 3 h after LPS induction. Five micrograms of nuclear protein was assayed by EMSA for binding to a consensus GAS oligonucleotide. Preincubation with cold, unlabeled oligonucleotide (C) or with anti-STAT-1 antibody (a-STAT-1) significantly attenuated binding. Relative values for STAT-1 DNA binding are based on intensities of the bands determined by PhosphorImager. Results are representative of three independent experiments.
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between cell density and IFN-stimulated signaling, as the binding of STAT-1 to the radiolabeled GAS oligonucleotide was significantly enhanced in denser cell cultures (Fig. 6). Also, binding to the radiolabeled GAS oligonucleotide was attenuated by preincubation with either excess unlabeled (cold) GAS oligo, or with antiSTAT-1 antibody (a-STAT-1), confirming that the observed shift was due to STAT-1.
Discussion Production of NO by macrophages is regulated primarily through the tight control of iNOS gene expression, which is transcriptionally silent in the absence of activating stimuli. In the murine macrophage line RAW 264.7, iNOS is markedly induced when cells are incubated with the bacterial cell-wall product, LPS [24]. The transcriptional activation of the iNOS gene by LPS can be enhanced by the addition of cytokines, including TNF-a, IL-1b, and both type I (a=b) and II (c) IFNs. The expression of iNOS in RAW 264.7 macrophages involves the LPS-induced association of CD14 with TLR4 in an LPS-activation cluster. Intracellular signals initiated by the LPS activation cluster subsequently activate the transcription factor NF-jB by inducing the degradation of the inhibitory protein, IjB. The endogenous production of IFN-b triggered by NF-jB is critical in the processes leading to iNOS expression [25]. Accordingly, it was demonstrated that bone marrow macrophages derived from mice deficient in functional type I IFN receptor were unable to initiate iNOS transcription in response to the addition of LPS [26]. The mechanism by which IFN-b promotes iNOS expression is through activation of the JAK/STAT pathway. STAT-1 is phosphorylated in response to IFN-b and enhances the transcription of target genes through conserved GAS elements present in the promoter region of the iNOS gene. The transcription factor, IRF-1, induced by STAT-1 activation, is also critical in mediating iNOS transcription [27–29]. In studying the expression of iNOS in LPS-treated RAW macrophages, we demonstrate that density in cell culture strongly affects iNOS transcript levels. When normalized to the expression of GAPDH, comparatively more iNOS message is produced in high-density compared to low-density cell cultures. The positive effect of cell density on iNOS message expression was also reflected in the expression of iNOS protein by Western blot. This was confirmed by measuring NOS catalytic activity. We decided to examine the catalytic activity of iNOS in diluted cellular extracts, rather than sample the media for NO 2 and NO3 (end-products of NO). This was to avoid potential feedback-inhibition of iNOS by NO under the high concentrations of NO present in macrophage culture [30]. We propose that the
enhancement of iNOS expression caused by increasing cell density may have physiological importance in mediating the host response to infection, since larger macrophage populations could have greater capability in mounting an NO-based cytotoxic defense. To determine if the density-mediated effect was due to direct cell–cell contacts, or to a soluble macrophage product, co-culture experiments were conducted. The results of co-culture implied that a macrophage-derived soluble factor was involved in the enhancing effect of cell density on iNOS induction. Since IFN-b has been previously demonstrated to function as an autocrine signal in murine macrophages, we decided to investigate IFN-b as a potential mediator of the cell density effect. We observed that IFN-b enhanced the induction of iNOS message expression in low-density cultures; the magnitude of this effect was comparable to the enhancement observed in high-density cultures treated with LPS alone. Conversely, the addition of an IFN-b neutralizing antibody to cells stimulated with LPS alone nearly abolished the density-dependent enhancement of iNOS induction, suggesting that IFN-b plays a critical role in mediating this effect. Moreover, the absence of inducible IFN-a or IFN-c message expression is consistent with the view that the mediator is IFN-b (unpublished observations). The cytocidal or cytostatic actions of activated macrophages against pathogens and neoplastic cells are dependent on the production of NO [31,32]. When high concentrations of NO are achieved locally by activated macrophages, oxidation products such as nitrogen dioxide (NO2 ) and nitrous anhydride (N2 O3 ) may form [33]. This is because the chemical reactions leading to these species are multiple-order processes with respect to NO, and are thus highly dependent on the NO concentration [34,35]. These reactive nitrogen species are more potent oxidants than NO, are more reactive with cellular nucleophiles, and may contribute greatly to the observed cytotoxicity of macrophage-derived NO. The enhanced production of NO in dense macrophage populations may facilitate the formation of NO2 and N2 O3 , thereby increasing macrophage cytotoxicity. Accordingly, a 5-fold increase in cell density (assuming a proportional 5-fold increase in NO levels) was recently calculated to produce a 25-fold increase in the concentration of N2 O3 [36]. Since we observe that iNOS levels increase with cell density, we suggest that even greater increases in N2 O3 levels are possible. Therefore, a disproportional enhancement of cytotoxicity may occur where macrophage populations are dense. Interestingly, a study on the effect of culture density on iNOS induction in hepatocytes revealed a decrease in iNOS protein levels at 24 h after treatment [37]. The reduction in iNOS expression was reportedly caused by an NO-independent reduction in total protein synthesis following the treatment of hepatocytes with a cocktail of
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LPS, IL-1b, TNF-a, and IFN-c. This contrasts with our observation that iNOS protein levels were enhanced in densely cultured macrophages treated with LPS alone (Fig. 2). Differences between this earlier report and our investigation, including the use of a different cell type, a later time-point, and addition various cytokines make comparisons difficult, particularly since the cause of the reduction in protein synthesis was undetermined. The effect of cell density on the cytotoxic role of macrophages is seldom addressed. However, in a recent study, the ability of human monocytes to suppress infection with Mycobacterium bovis was shown to be highly dependent on cell density [38]. Accordingly, mycobacterial growth was successfully inhibited by a cell density of 2 105 , but not by 5 104 macrophages per culture dish. A number of factors were evaluated as possible reasons why dense cultures were more effective at controlling infection. Interestingly, growth of M. bovis was not enhanced by the addition of the iNOSinhibitor L -N-iminoethyllysine, suggesting that NO had an insignificant role. However, other investigators have reported that NO derived from human macrophages is indeed critical in suppressing M. bovis infections [39]. The discrepancy in findings suggests that further study is needed in this area. In this respect, we propose that the enhancement of iNOS induction in dense macrophage populations may contribute a hostÕs ability to control infections when NO can play a cytotoxic role.
Acknowledgments This work was supported by grants from the National Institutes of Health HL35014 and HL40922. We gratefully acknowledge Dr. Jon M. Fukuto for valuable scientific guidance.
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