- Email: [email protected]
Endotoxin and NO Induce MIP-1γ Gene Transcription in ANA-1 Murine Macrophages
Biochemical and Biophysical Research Communications 277, 617– 621 (2000) doi:10.1006/bbrc.2000.3718, available online at http://www.idealibrary.com on
Endotoxin and NO Induce MIP-1␥ Gene Transcription in ANA-1 Murine Macrophages Hong T. Guo, Charles Q. Cai, and Paul C. Kuo 1 Department of Surgery, Georgetown University Hospital, Washington, DC 20007
Received September 25, 2000
The host response to gram-negative endotoxin is characterized by an influx of inflammatory cells into host tissues, mediated in part by localized production of chemokines. In this study, using subtractive suppression hybridization analysis, we demonstrate that ANA-1 murine macrophages produce the CC chemokine, MIP-1␥, in response to LPS-mediated NO production. Gene transcription and protein translation are upregulated in the setting of LPS-induced NO synthesis. However, NO alone is a necessary but insufficient cofactor for induction of MIP-1␥ protein expression; an NO-independent LPS signalling pathway is also required. This study suggests a novel mechanism by which NO modulates the host inflammatory response to endotoxin. © 2000 Academic Press
In conditions of endotoxemia, inducible nitric oxide synthase (iNOS) production with subsequent synthesis of nitric oxide (NO) is associated with numerous cellular, biochemical and molecular regulatory functions which alter the host inflammatory response to sepsis. One approach to understanding regulatory mechanisms is the identification of patterns of gene expression associated with varying physiological states. Various methods to compare patterns of gene expression have been described, including differential hybridization screening, subtractive library construction, representational difference analysis (RDA), differential display, conventional cDNA array hybridization and serial analysis of gene expression (1). A technique called suppression subtractive hybridization (SSH) has recently been described which is based on technology similar to RDA but with modifications to normalize for mRNA abundance (1). SSH has previously been used to compare patterns of gene expression in breast cancer cell lines discordant for estrogen receptor expression (2). 1 To whom correspondence should be addressed at 4 PHC, 3800 Reservoir Road NW, Washington, DC 20007. Fax: 202-687-3004. E-mail: [email protected].
In a system of ANA-1 murine macrophages, we hypothesized that endotoxin (LPS)-mediated NO production induces a specific set of genetic programs which regulate downstream cellular functions. To identify genes differentially expressed in LPS-stimulated cells producing NO, RNA from LPS-treated cells was used as “tester” and RNA from LPS ⫹ N G-nitro-L-arginine methyl ester (L-NAME) treated cells was used as “driver.” In driver cells, L-NAME was added to LPS stimulated cells as a competitive substrate inhibitor of NO production. Individual cDNA clones generated by SSH were used as probes in Northern blot analysis to identify differentially expressed genes. Using SSH in ANA-1 murine macrophages, a number of the CC-class of chemokine genes were specifically induced in the presence of LPS stimulated NO synthesis. In this study, we demonstrate that gene transcription and protein expression of the murine CC chemokine, macrophage inflammatory protein-1␥ (MIP-1␥), are upregulated in the setting of LPS-induced NO synthesis. This study suggests a novel mechanism by which NO modulates the host inflammatory response to endotoxin. METHODS Cell culture and induction of NO synthesis in ANA-1 macrophages. ANA-1 macrophages (gift from Dr. George Cox, USUHS, Bethesda, MD) were maintained in DMEM with 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 g/ml streptomycin. LPS (100 ng/ml) was used in the absence of FCS (10%) to induce NO synthesis. In selected instances, the competitive substrate inhibitor of NO synthase, N G-nitro-L-arginine methyl ester (L-NAME 250 ng/ml), or the NO donor SNAP (100 M), or a combination of these compounds, was added. After incubation for 12 h at 37°C in 5% CO 2, the supernatants and cells were harvested for assays. Assay of NO production. NO released from cells in culture was quantified by measurement of the NO metabolite, nitrite. 50 l cell culture medium were removed from culture dish and centrifuged; the supernatants were mixed with 50 l sulfanilamide (1%) in 0.5 N HCl. After a 5-min incubation at room temperature, an equal volume of 0.02% N-(1-naphthyl) ethylenediamine was added. Following incubation for 10 min at room temperature, the absorbance of samples at 540 nm was compared with that of an NaNO 2 standard on a MAXLINE microplate reader.
617
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 277, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RNA preparation and Northern blot analysis. Total RNA was isolated from ANA-1 macrophages using Trizol reagent (GIBCO BRL, Rockville, MD). The RNA samples (10 g/lane) were fractionated by electrophoresis on a 1% agarose formaldehyde gel and transferred to Hybond-C nylon membrane (Amersham Pharmacia Inc.), Hybridization using 32P-dATP-labeled probes was performed at 42°C for 18 h in ULTRAhyb hybridization buffer (Ambion, Austin, TX). Following hybridization, filters were washed twice and subjected to autoradiography. cDNA probes were prepared by random primer labeling, followed by purification using a Sephadex G-50 minicolumn (BioMax Inc., Odenton, MD). Differential screening of the subtracted cDNA library. SSH was performed as previously described (2). To identify genes differentially expressed in LPS-stimulated cells producing NO, RNA from LPS-treated cells was used as tester and RNA from LPS ⫹ L-NAME treated cells was used as driver. Differentially expressed sequences in subtracted cDNA were amplified by PCR to amplify only cDNA with different adaptors at both ends. Further enrichment was performed by a second PCR amplification with nested primers. The differentially expressed sequences were inserted into a T/A vector, pT-Adv cloning vector (Clontech Inc.). After a blue/white visual assay, PCR was used to rapidly amplify cDNA inserts. PCR products were blotted on nylon membrane (Hybond N ⫹, Amersham). Following hybridization, positive clonies were sequenced with the ABI PRISM 377 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). Resulting sequences were compared to the Genbank database. Immunoblot analysis. The cells were lysed in buffer (0.8% NaCl, 0.02 KCl, 1% SDS, 10% Triton X-100, 0.5% sodium deoxycholic acid, 0.144 % Na 2HPO 4, and 0.024% KH 2PO 4, pH 7.4) and centrifuged at 12000g for 10 min at 4°C. Protein concentration was determined by absorbance at 650 nm using protein assay reagent (Bio-Rad). Protein was separated by 12% SDS–PAGE, and the products were electrotransferred to PVDF membrane (Amersham Pharmacia). The membrane was blocked with 5% skim milk PBS-0.05% Tween for 1 h at room temperature. After being washed three times, blocked membranes were incubated with the primary antibody for 1 h at room temperature, washed three times in PBS-0.05% Tween, and incubated with horseradish peroxidase conjugated second antibody for 1 h at room temperature. After an additional three times washing, bound peroxidase activity were detected by the ECL detection system (Amersham Pharmacia, Piscataway, NJ). Nuclear run-on assays. Macrophage nuclei were prepared in lysis buffer (10 mM Tris.Cl pH 7.4, 10 mM NaCl and 3 mM MgCl 2, 0.5% Nonident P-40) and pelletted at 500 g. The nuclei (2 ⫻ 10 7) were resuspended in 100 l glycerol buffer, then 150 Ci of ␣- 32P-UTP (800 Ci/mmol) in 100 l of 10 mM Tris.Cl (pH 8.0), 5mM DTT, 5 mM MgCl 2, 300 mM KCl, and 1 mM (each) ATP, CTP, and GTP for 30 min at 30°C was added. Labeled RNA was treated with 10 units RNasefree DNase I (GIBCO) for 5 min at 30°C and extracted with phenol: chloroform (24:1) and chloroform alone. Before ethanol precipitation, 10 g yeast tRNA was added, and labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution was neutralized by the addition of Hepes (acid free) to a final concentration of 0.24 M. After ethanol precipitation, the RNA pellet was resuspended in 10 mM N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (pH 7.4), 0.2% SDS, and 10 mM EDTA. MIP-1␥ cDNA was spotted onto nylon membranes with a slot blot apparatus (Bio-Rad); -Actin and pT-Adv vector served as positive and negative controls, respectively. Hybridization was performed at 42°C for 48 h with 5 ⫻ 10 6 cpm of labeled RNA in hybridization buffer [50% formamide, 4 ⫻ SSC, 0.1% SDS, 5⫻ Denhardt’s solution, 0.1 M sodium phosphate (pH 7.2), and 100 g/ml salmon sperm DNA]. After hybridization, the membranes were washed twice at room temperature in 2 ⫻ SSC and 0.1% SDS, and three times at 56°C in 0.1 ⫻ SSC and 0.1% SDS. The membranes were exposed to X-ray film with an intensifying screen. Statistical analysis. Data are expressed mean ⫾ standard of the mean of three or four assays. Statistical analysis was performed
FIG. 1. Effect of LPS stimulation on ANA-1 macrophage NO production. Semi-logarithmic plot of NO- LPS dose response relationships. Nitrite and nitrate production were determined in LPS stimulated ANA-1 macrophages, as described under Methods. (ANOVA for LPS alone, P ⫽ 0.0001; * P ⬍ 0.01 LPS vs LPS ⫹ L-NAME by Students t test).
using the Students t test or one way analysis of variance, as appropriate. P values less than 0.05 were considered significant.
RESULTS LPS-Nitric Oxide Dose Response Relationship ANA-1 macrophage production of NO in response to a 12-h incubation with LPS (0 –10 g/ml) was determined in the presence and absence of the competitive substrate inhibitor, L-NAME (250 ng/ml) (Fig. 1). Nitrite levels in unstimulated Control cells were 23.1 ⫾ 3.3 nmol/mg. There was a significant concentrationdependent increase in media levels of nitrite, the NO metabolite, in response to LPS stimulation. (ANOVA P ⫽ 000.1) In the presence of [LPS] ⫽ 100 ng/ml, nitrite production was 74 ⫾ 5.6 nmol/mg. LPS ⫹ L-NAME treated cells exhibited levels of NO production that were not significantly from that of Controls for all concentrations of LPS utilized. Nitrite levels from cells treated with L-NAME alone did not differ from that of unstimulated Controls cells, 19.3 ⫾ 2.9 nmol/mg vs. 23.1 ⫾ 3.3 nmol/mg (p ⫽ NS). In subsequent assays, an LPS concentration of 100 ng/ml was utilized, unless otherwise stated. Differential Expression of the MIP-1␥ Gene Using SSH and Northern blot screening, genes exhibiting greater than a fivefold increase in expression in the presence of both LPS and NO were completely sequenced and queried against the NCBI GenBank
618
Vol. 277, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NO, SNAP (100 M), was then added with LPS ⫹ L-NAME, normalized MIP-1␥ mRNA expression was increased to a level not statistically different from that of LPS treated cells. In the setting of SNAP stimulation alone, MIP-1␥ mRNA expression was not different from that of Controls (data not shown). Serial Northern blot analyses were performed in the presence of actinimycin D (50 g/ml) to determine MIP-1␥ mRNA halflife. The calculated mRNA half-life was equivalent for all treatment groups, t 1/2 ⫽ 42 ⫾ 7.4 minutes. This indictaes that LPS induced NO synthesis does not increase steady state mRNA levels by alteration in MIP-1␥ mRNA half-life. These data indicate that LPSmediated NO production is associated with significantly increased MIP-1␥ mRNA expression in ANA-1 murine macrophages. In addition, NO is a necessary but insufficient cofactor. MIP-1␥ Gene Transcription Assays
FIG. 2. Northern blot analysis of MIP-1␥ and -actin mRNA in ANA-1 macrophages. (A) Northern blot analysis of MIP-1␥ and -actin mRNA, as described under Methods. Blot is representative of four experiments. (B) Histogram representation of densitometric analysis of normalized MIP-1␥ mRNA expression. (* P ⬍ 0.01 LPS vs Control, L-NAME, and LPS ⫹ L-NAME; ** P ⬍ 0.01 LPS ⫹ L-NAME ⫹ SNAP vs Control, L-NAME, and LPS ⫹ L-NAME by Students t test). Values are expressed as mean ⫾ SEM of four experiments.
database. One of these genes was found to be MIP-1␥. Northern blot analysis was performed to confirm that MIP-1␥ mRNA levels were upregulated in the setting of LPS-induced NO synthesis (Fig. 2). Baseline expression of MIP-1␥ mRNA was found in unstimulated Control cells. In the presence of LPS, MIP-1␥ mRNA expression, normalized to that of -actin, was increased approximately 10-fold. Inhibition of NO production by simultaneous addition of L-NAME to LPS treated cells significantly decreased OPN mRNA expression. In this setting, the level of normalized MIP-1␥ mRNA was ⬃75% that of Control cells. L-NAME alone did not alter MIP-1␥ mRNA levels. When an exogenous source of
Nuclear run-on assays were performed to determine whether LPS and NO increase MIP-1␥ gene transcription (Fig. 3). -actin cDNA served as the positive control, and pT-Adv plasmid as negative control. Ongoing MIP-1␥ gene transcription was noted in Control cells. The extent of transcription was noted to be increased by over fivefold in the cells stimulated with LPS (P ⬍ 0.01 vs Control). In the presence of LPS ⫹ L-NAME or L-NAME alone, the transcription of the MIP-1␥ gene was not different from that of unstimulated Control cells. Repletion of NO by addition of SNAP to LPS ⫹ L-NAME treated cells increased MIP-1␥ gene transcription by 100% when compared to Control cells (P ⬍ 0.05 vs Control). These data suggest that LPS induced NO production increases MIP-1␥ gene transcription. LPS-Mediated NO Synthesis and MIP-1␥ Protein Using a monoclonal MIP-1␥ antibody, immunoblot analysis was performed in supernatants collected from ANA-1 cells (Fig. 4). The supernatant from unstimulated Controls did not exhibit any immunoreactive MIP-1␥ protein. In contrast, MIP-1␥ protein was found in supernatant from LPS-treated cells. Inhibition of NO synthesis by addition of L-NAME with LPS resulted in ablation of MIP-1␥ protein expression. Cells treated with L-NAME alone did not express MIP-1␥ protein. Finally, addition of the exogenous NO source, SNAP, with LPS ⫹ L-NAME resulted in a level of MIP-1␥ protein expression that was not different from that of LPS treated cells. When cell lysates from the various treatment groups were assayed for immunoreactive MIP-1␥ protein, none was detected (data not shown). These data suggest that LPS induction of MIP-1␥ protein synthesis in ANA-1 murine macrophages is NO-dependent.
619
Vol. 277, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION
tion in Control cells is also expressed in ANA-1 macrophages exposed to LPS and the iNOS inhibitor, L-NAME. Repletion of NO to LPS ⫹ L-NAME cells in the form of the exogenous NO donor, SNAP, resulted in detection of increased levels of MIP-1␥ gene transcription, steady state mRNA levels and secreted protein. In all treatment groups, there was no immunoreactive MIP-1␥ protein noted in the cell lysates. These data suggest that LPS and NO not only increase MIP-1␥ gene transcription, but also induce MIP-1␥ protein translation. In addition, NO is necessary but sufficient to induce MIP-1␥ expression; an LPS-dependent signal transduction pathway is also required. MIP-1␥, also known as CCF-18 and MRP-2, is a member of the C-C family of murine chemokines. The human counterpart is, as yet unknown, but it has been hypothesized that CCL15/HCC2/leukotactin/MIP-1␦ may represent the human homologue (3). Little is known of MIP-1␥ function. However, in dendritic cells, Mohamafzadeh and coworkers found that MIP-1␥ functions to chemotactically recruits T cells, activated, nonactivated, CD4⫹, and CD8⫹ (4). Youn and colleagues also found that MIP-1␥ is a potent suppressor of growth factor stimulated colony formation by bone marrow myeloid progenitors (5). Poltorak et al. compared MIP-1␥ with MIP-1␣ and found that it acts as a pyrogen when administered intraventricularly (6). They both engage the same high affinity receptor on neutrophils and activate calcium release following cell contact. Additional data suggest that both MIP-1␥ and MIP-1␣ utilize a common signalling pathway. Of interest, they found that MIP-1␥ is expressed constitutively in a wide variety of tissues, including macrophages, and circulates at high concentrations, ⬃1 g/ml. The authors postulate that MIP-1␥ might regulate the “set point” for cell activation, diminishing responsiveness to itself and other CC chemokines. In stark contrast to our results, previous work by Mohamadzadeh failed to demonstrate LPS mediated increase in baseline XS52 dendritic cell expression of MIP-1␥ mRNA (4). However, in LPS-injected mice, Poltorak and coworkers found that net induction of MIP-1␥ mRNA expression was exhibited in spleen, heart and lung (6). Circulating macrophages or monocytes were not examined. In ad-
The host response to gram-negative endotoxin is characterized by an influx of inflammatory cells into host tissues, mediated in part by localized production of chemokines. In this study, we demonstrate that ANA-1 murine macrophages produce the chemokine, MIP-1␥, in response to LPS-mediated NO production. Transcription of the MIP-1␥ gene is upregulated in the presence of LPS and NO. Although secreted MIP-1␥ protein is only detected in the presence of LPS stimulation, MIP-1␥ gene transcription and steady state mRNA levels are both found in unstimulated Control macrophages. This steady state level of gene transcrip-
FIG. 4. Immunoblot analysis of secreted and cell lysate OPN protein in LPS stimulated ANA-1 macrophages, as described under Methods. Blot is representative of four experiments.
FIG. 3. Effect of LPS induced NO production on MIP-1␥ gene transcription in ANA-1 macrophages. (A) Nuclear run-on blot of MIP-1␥ gene transcription. Target DNA for MIP-1␥ was amplified by PCR based upon the published sequence. -actin and pT-Adv DNA served as positive and negative controls, respectively. Blot is representative of three experiments. (B) Histogram representation of LPS induced MIP-1␥ gene transcription normalized to -actin. (* P ⬍ 0.01 LPS vs Control, LPS ⫹ L-NAME, and L-NAME; ** P ⬍ 0.05 LPS ⫹ L-NAME ⫹ SNAP vs Control, LPS ⫹ L-NAME, and L-NAME by Students t test.) Values are expressed as mean ⫾ SEM of three experiments.
620
Vol. 277, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dition, to date, no studies have been performed which examine protein expression in the presence and absence of LPS. Our data indicates that LPS mediated NO production significantly increase secreted MIP-1␥ protein levels by a mechanism of increased gene transcription and translation. The potential interaction between NO and MIP-1␥ metabolism has not been previously examined. However, Muhl and Dinarello examined production of a homologous CC chemokine, MIP-1␣, in human adherent peripheral blood mononuclear cells (7). They determined that the iNOS inhibitor, N-monomethyl-Larginine (L-NMMA), decreased LPS-induced MIP-1␣ protein release and steady state mRNA levels. In addition, IL-1 and TNF-␣ release were inhibited in this setting. In contrast, MCP-1 release was unaffected, while IL-10 release was enhanced. The authors did not examine MIP-1␥ mRNA half-life or MIP-1␥ gene transcription. They conclude that NO acts as a proinflammatory agent by inducing MIP-1␣ release in the setting of LPS stimulation. In our study, using LPSstimulated murine macrophages in culture, we demonstrate that both MIP-1␥ gene transcription and protein expression are NO-dependent. The paradigm of NO dependent gene transcription in the setting of LPS stimulation is not unique to the chemokine, MIP-1␥. Based upon our SSH data from murine macrophages, CC chemokines such as JE/ MCP-1 and C10/MRP-1, also exhibit increased mRNA and protein expression in the presence of LPS mediated NO production (unpublished observations). In a murine model of wound healing, Frank and coworkers recently found that NO is a negative regulator of RANTES chemokine expression (8). However, the effect of NO on RANTES gene transcription was not addressed. Nevertheless, expression of numerous proteins, such as gamma-glutamylcysteine synthetase and osteopontin, have been demonstrated to be influenced by NO and its effects upon gene transcription (9, 10). The underlying mechanisms are varied, but the majority center upon the participation of NO in redox chemistry. As an example, the formation of S-nitrosothiols underlies pathways of NO oxidation which lead to surrogate NO-like bioactivity and can result in allosteric receptor modification, inhibition of sulfhydryl-enzyme activities, and down-regulation of transcriptional activators or repressors (11). Certainly,
in the setting of LPS stimulation, our data suggest that NO is a necessary, but insufficient cofactor for upregulation of MIP-1␥ gene transcription and protein translation. MIP-1␥ chemokine expression requires both NO and LPS dependent signalling pathways. REFERENCES 1. Yang, G. P., Ross, D. T., Kuang, W. W., Brown, P. O., and Weigel, R. J. (1999) Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nuc. Acids Res. 27, 1517–1523. 2. Kuang, W. W., Thompson, D. A., Hoch, R. V., and Weigel, R. J. (1998) Differential screening and suppression subtractive hybridization indentified genes differentially expressed in an estrogen receptor-positive breast carcinoma cell line. Nuc. Acids Res. 26, 1116 –1123. 3. Rossi, D., and Zlotnick, A. (2000) The biology of chemokines and their receptors. Annu. Rev. Immunol. 18, 217–242. 4. Mohamadzadeh, M., Poltorak, A. N., Bergstresser, P. R., Beutler, B., and Takashima, A. (1996) Dendritic cells produce macrophage inflammatory protein-1gamma, a new member of the CC chemokine family. J. Immunol. 156, 3102–3106. 5. Youn, B. S., Jang, I. K., Broxmeyer, H. E., Cooper, S., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Elick, T. A., Fraser, M. J., and Kwon, B. S. (1995) A novel chemokine, macrophage inflammatory protein-related protein-2, inhibits colony formation of bone marrow myeloid progenitors. J. Immunol. 155, 2661–2667. 6. Poltorak, A. N., Bazzoni, F., Smirnova, I. I., Alejos, E., Thompson, P., Luheshi, G., Rothwell, N., and Bianchi, M. (1995) MIP1gamma: molecular cloning, expression and biological activities of a novel CC chemokine that is constitutively secreted in vivo. J. Inflammation 45, 207–219. 7. Muhl, H., and Dinarello, C. A. (1997) Macrophage inflammatory protein-1alpha production in lipopolysaccharide stimulated human adherent blood mononuclear cells is inhibited by the nitric oxide synthase inhibitor N-monomethyl-L-arginine. J. Immunol. 159, 5063–5069. 8. Frank, S., Kampfer, H., Wetzler, C., Stallmeyer, B., and Pfeilschifter, J. (2000) Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression. Biochem. J. 347, 265–273. 9. Kuo, P. C., Abe, K. Y., and Schroeder, R. A. (1996) Interleukin-1 induced nitric oxide production modulates glutathione synthesis in cultured rat hepatocytes. Am. J. Physiol. 271, C851–C862. 10. Takahashi, F., Takahashi, K., Maeda, K., Tominaga, S., and Fukuchi, Y. (2000) Osteopontin is induced by nitric oxide in RAW 264.7 cells. IUBMB Life 49, 217–221. 11. delaTorre, A., Schroeder, R. A., Punzalan, C., and Kuo, P. C. (1999) Endotoxin-mediated S-nitrosylation of p50 alters NFkappa B-dependent gene transcription in ANA-1 murine macrophages. J. Immunol. 162, 4101– 4108.
621
Endotoxin and NO Induce MIP-1␥ Gene Transcription in ANA-1 Murine Macrophages Hong T. Guo, Charles Q. Cai, and Paul C. Kuo 1 Department of Surgery, Georgetown University Hospital, Washington, DC 20007
Received September 25, 2000
The host response to gram-negative endotoxin is characterized by an influx of inflammatory cells into host tissues, mediated in part by localized production of chemokines. In this study, using subtractive suppression hybridization analysis, we demonstrate that ANA-1 murine macrophages produce the CC chemokine, MIP-1␥, in response to LPS-mediated NO production. Gene transcription and protein translation are upregulated in the setting of LPS-induced NO synthesis. However, NO alone is a necessary but insufficient cofactor for induction of MIP-1␥ protein expression; an NO-independent LPS signalling pathway is also required. This study suggests a novel mechanism by which NO modulates the host inflammatory response to endotoxin. © 2000 Academic Press
In conditions of endotoxemia, inducible nitric oxide synthase (iNOS) production with subsequent synthesis of nitric oxide (NO) is associated with numerous cellular, biochemical and molecular regulatory functions which alter the host inflammatory response to sepsis. One approach to understanding regulatory mechanisms is the identification of patterns of gene expression associated with varying physiological states. Various methods to compare patterns of gene expression have been described, including differential hybridization screening, subtractive library construction, representational difference analysis (RDA), differential display, conventional cDNA array hybridization and serial analysis of gene expression (1). A technique called suppression subtractive hybridization (SSH) has recently been described which is based on technology similar to RDA but with modifications to normalize for mRNA abundance (1). SSH has previously been used to compare patterns of gene expression in breast cancer cell lines discordant for estrogen receptor expression (2). 1 To whom correspondence should be addressed at 4 PHC, 3800 Reservoir Road NW, Washington, DC 20007. Fax: 202-687-3004. E-mail: [email protected].
In a system of ANA-1 murine macrophages, we hypothesized that endotoxin (LPS)-mediated NO production induces a specific set of genetic programs which regulate downstream cellular functions. To identify genes differentially expressed in LPS-stimulated cells producing NO, RNA from LPS-treated cells was used as “tester” and RNA from LPS ⫹ N G-nitro-L-arginine methyl ester (L-NAME) treated cells was used as “driver.” In driver cells, L-NAME was added to LPS stimulated cells as a competitive substrate inhibitor of NO production. Individual cDNA clones generated by SSH were used as probes in Northern blot analysis to identify differentially expressed genes. Using SSH in ANA-1 murine macrophages, a number of the CC-class of chemokine genes were specifically induced in the presence of LPS stimulated NO synthesis. In this study, we demonstrate that gene transcription and protein expression of the murine CC chemokine, macrophage inflammatory protein-1␥ (MIP-1␥), are upregulated in the setting of LPS-induced NO synthesis. This study suggests a novel mechanism by which NO modulates the host inflammatory response to endotoxin. METHODS Cell culture and induction of NO synthesis in ANA-1 macrophages. ANA-1 macrophages (gift from Dr. George Cox, USUHS, Bethesda, MD) were maintained in DMEM with 10% heat-inactivated FCS, 100 units/ml penicillin, and 100 g/ml streptomycin. LPS (100 ng/ml) was used in the absence of FCS (10%) to induce NO synthesis. In selected instances, the competitive substrate inhibitor of NO synthase, N G-nitro-L-arginine methyl ester (L-NAME 250 ng/ml), or the NO donor SNAP (100 M), or a combination of these compounds, was added. After incubation for 12 h at 37°C in 5% CO 2, the supernatants and cells were harvested for assays. Assay of NO production. NO released from cells in culture was quantified by measurement of the NO metabolite, nitrite. 50 l cell culture medium were removed from culture dish and centrifuged; the supernatants were mixed with 50 l sulfanilamide (1%) in 0.5 N HCl. After a 5-min incubation at room temperature, an equal volume of 0.02% N-(1-naphthyl) ethylenediamine was added. Following incubation for 10 min at room temperature, the absorbance of samples at 540 nm was compared with that of an NaNO 2 standard on a MAXLINE microplate reader.
617
0006-291X/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.
Vol. 277, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
RNA preparation and Northern blot analysis. Total RNA was isolated from ANA-1 macrophages using Trizol reagent (GIBCO BRL, Rockville, MD). The RNA samples (10 g/lane) were fractionated by electrophoresis on a 1% agarose formaldehyde gel and transferred to Hybond-C nylon membrane (Amersham Pharmacia Inc.), Hybridization using 32P-dATP-labeled probes was performed at 42°C for 18 h in ULTRAhyb hybridization buffer (Ambion, Austin, TX). Following hybridization, filters were washed twice and subjected to autoradiography. cDNA probes were prepared by random primer labeling, followed by purification using a Sephadex G-50 minicolumn (BioMax Inc., Odenton, MD). Differential screening of the subtracted cDNA library. SSH was performed as previously described (2). To identify genes differentially expressed in LPS-stimulated cells producing NO, RNA from LPS-treated cells was used as tester and RNA from LPS ⫹ L-NAME treated cells was used as driver. Differentially expressed sequences in subtracted cDNA were amplified by PCR to amplify only cDNA with different adaptors at both ends. Further enrichment was performed by a second PCR amplification with nested primers. The differentially expressed sequences were inserted into a T/A vector, pT-Adv cloning vector (Clontech Inc.). After a blue/white visual assay, PCR was used to rapidly amplify cDNA inserts. PCR products were blotted on nylon membrane (Hybond N ⫹, Amersham). Following hybridization, positive clonies were sequenced with the ABI PRISM 377 Genetic Analyzer (PE Applied Biosystems, Foster City, CA). Resulting sequences were compared to the Genbank database. Immunoblot analysis. The cells were lysed in buffer (0.8% NaCl, 0.02 KCl, 1% SDS, 10% Triton X-100, 0.5% sodium deoxycholic acid, 0.144 % Na 2HPO 4, and 0.024% KH 2PO 4, pH 7.4) and centrifuged at 12000g for 10 min at 4°C. Protein concentration was determined by absorbance at 650 nm using protein assay reagent (Bio-Rad). Protein was separated by 12% SDS–PAGE, and the products were electrotransferred to PVDF membrane (Amersham Pharmacia). The membrane was blocked with 5% skim milk PBS-0.05% Tween for 1 h at room temperature. After being washed three times, blocked membranes were incubated with the primary antibody for 1 h at room temperature, washed three times in PBS-0.05% Tween, and incubated with horseradish peroxidase conjugated second antibody for 1 h at room temperature. After an additional three times washing, bound peroxidase activity were detected by the ECL detection system (Amersham Pharmacia, Piscataway, NJ). Nuclear run-on assays. Macrophage nuclei were prepared in lysis buffer (10 mM Tris.Cl pH 7.4, 10 mM NaCl and 3 mM MgCl 2, 0.5% Nonident P-40) and pelletted at 500 g. The nuclei (2 ⫻ 10 7) were resuspended in 100 l glycerol buffer, then 150 Ci of ␣- 32P-UTP (800 Ci/mmol) in 100 l of 10 mM Tris.Cl (pH 8.0), 5mM DTT, 5 mM MgCl 2, 300 mM KCl, and 1 mM (each) ATP, CTP, and GTP for 30 min at 30°C was added. Labeled RNA was treated with 10 units RNasefree DNase I (GIBCO) for 5 min at 30°C and extracted with phenol: chloroform (24:1) and chloroform alone. Before ethanol precipitation, 10 g yeast tRNA was added, and labeled RNA was treated with 0.2 M NaOH for 10 min on ice. The solution was neutralized by the addition of Hepes (acid free) to a final concentration of 0.24 M. After ethanol precipitation, the RNA pellet was resuspended in 10 mM N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (pH 7.4), 0.2% SDS, and 10 mM EDTA. MIP-1␥ cDNA was spotted onto nylon membranes with a slot blot apparatus (Bio-Rad); -Actin and pT-Adv vector served as positive and negative controls, respectively. Hybridization was performed at 42°C for 48 h with 5 ⫻ 10 6 cpm of labeled RNA in hybridization buffer [50% formamide, 4 ⫻ SSC, 0.1% SDS, 5⫻ Denhardt’s solution, 0.1 M sodium phosphate (pH 7.2), and 100 g/ml salmon sperm DNA]. After hybridization, the membranes were washed twice at room temperature in 2 ⫻ SSC and 0.1% SDS, and three times at 56°C in 0.1 ⫻ SSC and 0.1% SDS. The membranes were exposed to X-ray film with an intensifying screen. Statistical analysis. Data are expressed mean ⫾ standard of the mean of three or four assays. Statistical analysis was performed
FIG. 1. Effect of LPS stimulation on ANA-1 macrophage NO production. Semi-logarithmic plot of NO- LPS dose response relationships. Nitrite and nitrate production were determined in LPS stimulated ANA-1 macrophages, as described under Methods. (ANOVA for LPS alone, P ⫽ 0.0001; * P ⬍ 0.01 LPS vs LPS ⫹ L-NAME by Students t test).
using the Students t test or one way analysis of variance, as appropriate. P values less than 0.05 were considered significant.
RESULTS LPS-Nitric Oxide Dose Response Relationship ANA-1 macrophage production of NO in response to a 12-h incubation with LPS (0 –10 g/ml) was determined in the presence and absence of the competitive substrate inhibitor, L-NAME (250 ng/ml) (Fig. 1). Nitrite levels in unstimulated Control cells were 23.1 ⫾ 3.3 nmol/mg. There was a significant concentrationdependent increase in media levels of nitrite, the NO metabolite, in response to LPS stimulation. (ANOVA P ⫽ 000.1) In the presence of [LPS] ⫽ 100 ng/ml, nitrite production was 74 ⫾ 5.6 nmol/mg. LPS ⫹ L-NAME treated cells exhibited levels of NO production that were not significantly from that of Controls for all concentrations of LPS utilized. Nitrite levels from cells treated with L-NAME alone did not differ from that of unstimulated Controls cells, 19.3 ⫾ 2.9 nmol/mg vs. 23.1 ⫾ 3.3 nmol/mg (p ⫽ NS). In subsequent assays, an LPS concentration of 100 ng/ml was utilized, unless otherwise stated. Differential Expression of the MIP-1␥ Gene Using SSH and Northern blot screening, genes exhibiting greater than a fivefold increase in expression in the presence of both LPS and NO were completely sequenced and queried against the NCBI GenBank
618
Vol. 277, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
NO, SNAP (100 M), was then added with LPS ⫹ L-NAME, normalized MIP-1␥ mRNA expression was increased to a level not statistically different from that of LPS treated cells. In the setting of SNAP stimulation alone, MIP-1␥ mRNA expression was not different from that of Controls (data not shown). Serial Northern blot analyses were performed in the presence of actinimycin D (50 g/ml) to determine MIP-1␥ mRNA halflife. The calculated mRNA half-life was equivalent for all treatment groups, t 1/2 ⫽ 42 ⫾ 7.4 minutes. This indictaes that LPS induced NO synthesis does not increase steady state mRNA levels by alteration in MIP-1␥ mRNA half-life. These data indicate that LPSmediated NO production is associated with significantly increased MIP-1␥ mRNA expression in ANA-1 murine macrophages. In addition, NO is a necessary but insufficient cofactor. MIP-1␥ Gene Transcription Assays
FIG. 2. Northern blot analysis of MIP-1␥ and -actin mRNA in ANA-1 macrophages. (A) Northern blot analysis of MIP-1␥ and -actin mRNA, as described under Methods. Blot is representative of four experiments. (B) Histogram representation of densitometric analysis of normalized MIP-1␥ mRNA expression. (* P ⬍ 0.01 LPS vs Control, L-NAME, and LPS ⫹ L-NAME; ** P ⬍ 0.01 LPS ⫹ L-NAME ⫹ SNAP vs Control, L-NAME, and LPS ⫹ L-NAME by Students t test). Values are expressed as mean ⫾ SEM of four experiments.
database. One of these genes was found to be MIP-1␥. Northern blot analysis was performed to confirm that MIP-1␥ mRNA levels were upregulated in the setting of LPS-induced NO synthesis (Fig. 2). Baseline expression of MIP-1␥ mRNA was found in unstimulated Control cells. In the presence of LPS, MIP-1␥ mRNA expression, normalized to that of -actin, was increased approximately 10-fold. Inhibition of NO production by simultaneous addition of L-NAME to LPS treated cells significantly decreased OPN mRNA expression. In this setting, the level of normalized MIP-1␥ mRNA was ⬃75% that of Control cells. L-NAME alone did not alter MIP-1␥ mRNA levels. When an exogenous source of
Nuclear run-on assays were performed to determine whether LPS and NO increase MIP-1␥ gene transcription (Fig. 3). -actin cDNA served as the positive control, and pT-Adv plasmid as negative control. Ongoing MIP-1␥ gene transcription was noted in Control cells. The extent of transcription was noted to be increased by over fivefold in the cells stimulated with LPS (P ⬍ 0.01 vs Control). In the presence of LPS ⫹ L-NAME or L-NAME alone, the transcription of the MIP-1␥ gene was not different from that of unstimulated Control cells. Repletion of NO by addition of SNAP to LPS ⫹ L-NAME treated cells increased MIP-1␥ gene transcription by 100% when compared to Control cells (P ⬍ 0.05 vs Control). These data suggest that LPS induced NO production increases MIP-1␥ gene transcription. LPS-Mediated NO Synthesis and MIP-1␥ Protein Using a monoclonal MIP-1␥ antibody, immunoblot analysis was performed in supernatants collected from ANA-1 cells (Fig. 4). The supernatant from unstimulated Controls did not exhibit any immunoreactive MIP-1␥ protein. In contrast, MIP-1␥ protein was found in supernatant from LPS-treated cells. Inhibition of NO synthesis by addition of L-NAME with LPS resulted in ablation of MIP-1␥ protein expression. Cells treated with L-NAME alone did not express MIP-1␥ protein. Finally, addition of the exogenous NO source, SNAP, with LPS ⫹ L-NAME resulted in a level of MIP-1␥ protein expression that was not different from that of LPS treated cells. When cell lysates from the various treatment groups were assayed for immunoreactive MIP-1␥ protein, none was detected (data not shown). These data suggest that LPS induction of MIP-1␥ protein synthesis in ANA-1 murine macrophages is NO-dependent.
619
Vol. 277, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
DISCUSSION
tion in Control cells is also expressed in ANA-1 macrophages exposed to LPS and the iNOS inhibitor, L-NAME. Repletion of NO to LPS ⫹ L-NAME cells in the form of the exogenous NO donor, SNAP, resulted in detection of increased levels of MIP-1␥ gene transcription, steady state mRNA levels and secreted protein. In all treatment groups, there was no immunoreactive MIP-1␥ protein noted in the cell lysates. These data suggest that LPS and NO not only increase MIP-1␥ gene transcription, but also induce MIP-1␥ protein translation. In addition, NO is necessary but sufficient to induce MIP-1␥ expression; an LPS-dependent signal transduction pathway is also required. MIP-1␥, also known as CCF-18 and MRP-2, is a member of the C-C family of murine chemokines. The human counterpart is, as yet unknown, but it has been hypothesized that CCL15/HCC2/leukotactin/MIP-1␦ may represent the human homologue (3). Little is known of MIP-1␥ function. However, in dendritic cells, Mohamafzadeh and coworkers found that MIP-1␥ functions to chemotactically recruits T cells, activated, nonactivated, CD4⫹, and CD8⫹ (4). Youn and colleagues also found that MIP-1␥ is a potent suppressor of growth factor stimulated colony formation by bone marrow myeloid progenitors (5). Poltorak et al. compared MIP-1␥ with MIP-1␣ and found that it acts as a pyrogen when administered intraventricularly (6). They both engage the same high affinity receptor on neutrophils and activate calcium release following cell contact. Additional data suggest that both MIP-1␥ and MIP-1␣ utilize a common signalling pathway. Of interest, they found that MIP-1␥ is expressed constitutively in a wide variety of tissues, including macrophages, and circulates at high concentrations, ⬃1 g/ml. The authors postulate that MIP-1␥ might regulate the “set point” for cell activation, diminishing responsiveness to itself and other CC chemokines. In stark contrast to our results, previous work by Mohamadzadeh failed to demonstrate LPS mediated increase in baseline XS52 dendritic cell expression of MIP-1␥ mRNA (4). However, in LPS-injected mice, Poltorak and coworkers found that net induction of MIP-1␥ mRNA expression was exhibited in spleen, heart and lung (6). Circulating macrophages or monocytes were not examined. In ad-
The host response to gram-negative endotoxin is characterized by an influx of inflammatory cells into host tissues, mediated in part by localized production of chemokines. In this study, we demonstrate that ANA-1 murine macrophages produce the chemokine, MIP-1␥, in response to LPS-mediated NO production. Transcription of the MIP-1␥ gene is upregulated in the presence of LPS and NO. Although secreted MIP-1␥ protein is only detected in the presence of LPS stimulation, MIP-1␥ gene transcription and steady state mRNA levels are both found in unstimulated Control macrophages. This steady state level of gene transcrip-
FIG. 4. Immunoblot analysis of secreted and cell lysate OPN protein in LPS stimulated ANA-1 macrophages, as described under Methods. Blot is representative of four experiments.
FIG. 3. Effect of LPS induced NO production on MIP-1␥ gene transcription in ANA-1 macrophages. (A) Nuclear run-on blot of MIP-1␥ gene transcription. Target DNA for MIP-1␥ was amplified by PCR based upon the published sequence. -actin and pT-Adv DNA served as positive and negative controls, respectively. Blot is representative of three experiments. (B) Histogram representation of LPS induced MIP-1␥ gene transcription normalized to -actin. (* P ⬍ 0.01 LPS vs Control, LPS ⫹ L-NAME, and L-NAME; ** P ⬍ 0.05 LPS ⫹ L-NAME ⫹ SNAP vs Control, LPS ⫹ L-NAME, and L-NAME by Students t test.) Values are expressed as mean ⫾ SEM of three experiments.
620
Vol. 277, No. 3, 2000
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
dition, to date, no studies have been performed which examine protein expression in the presence and absence of LPS. Our data indicates that LPS mediated NO production significantly increase secreted MIP-1␥ protein levels by a mechanism of increased gene transcription and translation. The potential interaction between NO and MIP-1␥ metabolism has not been previously examined. However, Muhl and Dinarello examined production of a homologous CC chemokine, MIP-1␣, in human adherent peripheral blood mononuclear cells (7). They determined that the iNOS inhibitor, N-monomethyl-Larginine (L-NMMA), decreased LPS-induced MIP-1␣ protein release and steady state mRNA levels. In addition, IL-1 and TNF-␣ release were inhibited in this setting. In contrast, MCP-1 release was unaffected, while IL-10 release was enhanced. The authors did not examine MIP-1␥ mRNA half-life or MIP-1␥ gene transcription. They conclude that NO acts as a proinflammatory agent by inducing MIP-1␣ release in the setting of LPS stimulation. In our study, using LPSstimulated murine macrophages in culture, we demonstrate that both MIP-1␥ gene transcription and protein expression are NO-dependent. The paradigm of NO dependent gene transcription in the setting of LPS stimulation is not unique to the chemokine, MIP-1␥. Based upon our SSH data from murine macrophages, CC chemokines such as JE/ MCP-1 and C10/MRP-1, also exhibit increased mRNA and protein expression in the presence of LPS mediated NO production (unpublished observations). In a murine model of wound healing, Frank and coworkers recently found that NO is a negative regulator of RANTES chemokine expression (8). However, the effect of NO on RANTES gene transcription was not addressed. Nevertheless, expression of numerous proteins, such as gamma-glutamylcysteine synthetase and osteopontin, have been demonstrated to be influenced by NO and its effects upon gene transcription (9, 10). The underlying mechanisms are varied, but the majority center upon the participation of NO in redox chemistry. As an example, the formation of S-nitrosothiols underlies pathways of NO oxidation which lead to surrogate NO-like bioactivity and can result in allosteric receptor modification, inhibition of sulfhydryl-enzyme activities, and down-regulation of transcriptional activators or repressors (11). Certainly,
in the setting of LPS stimulation, our data suggest that NO is a necessary, but insufficient cofactor for upregulation of MIP-1␥ gene transcription and protein translation. MIP-1␥ chemokine expression requires both NO and LPS dependent signalling pathways. REFERENCES 1. Yang, G. P., Ross, D. T., Kuang, W. W., Brown, P. O., and Weigel, R. J. (1999) Combining SSH and cDNA microarrays for rapid identification of differentially expressed genes. Nuc. Acids Res. 27, 1517–1523. 2. Kuang, W. W., Thompson, D. A., Hoch, R. V., and Weigel, R. J. (1998) Differential screening and suppression subtractive hybridization indentified genes differentially expressed in an estrogen receptor-positive breast carcinoma cell line. Nuc. Acids Res. 26, 1116 –1123. 3. Rossi, D., and Zlotnick, A. (2000) The biology of chemokines and their receptors. Annu. Rev. Immunol. 18, 217–242. 4. Mohamadzadeh, M., Poltorak, A. N., Bergstresser, P. R., Beutler, B., and Takashima, A. (1996) Dendritic cells produce macrophage inflammatory protein-1gamma, a new member of the CC chemokine family. J. Immunol. 156, 3102–3106. 5. Youn, B. S., Jang, I. K., Broxmeyer, H. E., Cooper, S., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., Elick, T. A., Fraser, M. J., and Kwon, B. S. (1995) A novel chemokine, macrophage inflammatory protein-related protein-2, inhibits colony formation of bone marrow myeloid progenitors. J. Immunol. 155, 2661–2667. 6. Poltorak, A. N., Bazzoni, F., Smirnova, I. I., Alejos, E., Thompson, P., Luheshi, G., Rothwell, N., and Bianchi, M. (1995) MIP1gamma: molecular cloning, expression and biological activities of a novel CC chemokine that is constitutively secreted in vivo. J. Inflammation 45, 207–219. 7. Muhl, H., and Dinarello, C. A. (1997) Macrophage inflammatory protein-1alpha production in lipopolysaccharide stimulated human adherent blood mononuclear cells is inhibited by the nitric oxide synthase inhibitor N-monomethyl-L-arginine. J. Immunol. 159, 5063–5069. 8. Frank, S., Kampfer, H., Wetzler, C., Stallmeyer, B., and Pfeilschifter, J. (2000) Large induction of the chemotactic cytokine RANTES during cutaneous wound repair: a regulatory role for nitric oxide in keratinocyte-derived RANTES expression. Biochem. J. 347, 265–273. 9. Kuo, P. C., Abe, K. Y., and Schroeder, R. A. (1996) Interleukin-1 induced nitric oxide production modulates glutathione synthesis in cultured rat hepatocytes. Am. J. Physiol. 271, C851–C862. 10. Takahashi, F., Takahashi, K., Maeda, K., Tominaga, S., and Fukuchi, Y. (2000) Osteopontin is induced by nitric oxide in RAW 264.7 cells. IUBMB Life 49, 217–221. 11. delaTorre, A., Schroeder, R. A., Punzalan, C., and Kuo, P. C. (1999) Endotoxin-mediated S-nitrosylation of p50 alters NFkappa B-dependent gene transcription in ANA-1 murine macrophages. J. Immunol. 162, 4101– 4108.
621