Suppression of Inflammatory Cytokine Production by Carbon Monoxide Involves the JNK Pathway and AP-1

The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress and modulates pro-inflammatory cytokines. As the sepsis syndrome results from the release of pro-inflammatory mediators, we postulated that heme oxygenase-1 and its enzymatic product CO would protect against lethality in a murine model of sepsis. Mice treated with a lethal dose of lipopolysaccharide (LPS) and subsequently exposed to inhaled CO had significantly better survival and lower serum interleukin (IL)-6 and IL-1β levels than their untreated counterparts. In vitro, mouse macrophages exposed to LPS and CO had significantly attenuated IL-6 production; this effect was concentration-dependent and occurred at a transcriptional level. The same effect was seen with increased endogenous CO production through overexpression of heme oxygenase-1. Mutation within the AP-1-binding site in the IL-6 promoter diminished the effect of CO on promoter activity, and treatment of macrophages with CO decreased AP-1 binding in an electrophoretic mobility shift assay. Electrophoretic mobility supershift assay indicated that the JunB, JunD, and c-Fos components of AP-1 were particularly affected. Upstream of AP-1, CO decreased JNK phosphorylation in murine macrophages and lung endothelial cells. Mice deficient in the JNK pathway had decreased serum levels of IL-6 and IL-1β in response to LPS compared with control mice, and no effect of CO on these cytokine levels was seen in Jnk1 or Jnk2 genedeleted mice. In summary, these results suggest that CO provides protection in a murine model of sepsis through modulation of inflammatory cytokine production. For the first time, the effect of CO is shown to be mediated via the JNK signaling pathway and the transcription factor AP-1. The stress-inducible protein heme oxygenase-1 provides protection against oxidative stress and modulates pro-inflammatory cytokines. As the sepsis syndrome results from the release of pro-inflammatory mediators, we postulated that heme oxygenase-1 and its enzymatic product CO would protect against lethality in a murine model of sepsis. Mice treated with a lethal dose of lipopolysaccharide (LPS) and subsequently exposed to inhaled CO had significantly better survival and lower serum interleukin (IL)-6 and IL-1β levels than their untreated counterparts. In vitro, mouse macrophages exposed to LPS and CO had significantly attenuated IL-6 production; this effect was concentration-dependent and occurred at a transcriptional level. The same effect was seen with increased endogenous CO production through overexpression of heme oxygenase-1. Mutation within the AP-1-binding site in the IL-6 promoter diminished the effect of CO on promoter activity, and treatment of macrophages with CO decreased AP-1 binding in an electrophoretic mobility shift assay. Electrophoretic mobility supershift assay indicated that the JunB, JunD, and c-Fos components of AP-1 were particularly affected. Upstream of AP-1, CO decreased JNK phosphorylation in murine macrophages and lung endothelial cells. Mice deficient in the JNK pathway had decreased serum levels of IL-6 and IL-1β in response to LPS compared with control mice, and no effect of CO on these cytokine levels was seen in Jnk1 or Jnk2 genedeleted mice. In summary, these results suggest that CO provides protection in a murine model of sepsis through modulation of inflammatory cytokine production. For the first time, the effect of CO is shown to be mediated via the JNK signaling pathway and the transcription factor AP-1. Heme oxygenase-1 (HO-1) 1The abbreviations used are: HO-1, heme oxygenase-1; LPS, lipopolysaccharide; IL, interleukin; AP-1, activator protein-1; C/EBP, CAAT/enhancer-binding protein; ELISA, enzyme-linked immunosorbent assay; EMSA, electrophoretic mobility shift assay; JNK, c-Jun NH2-terminal kinase; MAPK, mitogen-activated protein kinase. is a microsomal enzyme responsible for the degradation of heme, generating biliverdin, iron, and carbon monoxide (CO). Induction of HO-1 has been reported in response to a wide variety of oxidant stimuli, and this enzyme is a vital component of cellular adaptation to stress. There is growing interest in the role of CO in the anti-inflammatory and cytoprotective function of HO-1. It has recently been demonstrated that the administration of exogenous CO inhibits lipopolysaccharide (LPS)-induced production of tumor necrosis factor-α while increasing interleukin (IL)-10 production both in vitro and in vivo (1Otterbein L.E. Bach F.H. Alam J. Soares M. Tao Lu H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1851) Google Scholar). This effect is independent of the guanylyl cyclase/cGMP pathway, and at least in the case of tumor necrosis factor-α, the effect is post-transcriptional. More recently, Fujita et al. (2Fujita T. Toda K. Karimova A. Yan S.F. Naka Y. Yet S.F. Pinsky D.J. Nat. Med. 2001; 7: 598-604Crossref PubMed Scopus (374) Google Scholar) demonstrated that inhaled CO can rescue HO-1-deficient mice from lethal ischemic lung injury, further strengthening the paradigm of CO as a direct mediator of cellular protection. The sepsis syndrome is the leading cause of death in intensive care units in the United States, and its incidence continues to rise. Despite an increasing incidence, little impact on associated mortality rates has been made over the past several decades (3Friedman G. Silva E. Vincent J.L. Crit. Care Med. 1998; 26: 2078-2086Crossref PubMed Scopus (500) Google Scholar). It has long been recognized that invading microorganisms induce the release of a large number of humoral and cellular pro-inflammatory mediators, causing a systemic inflammatory response syndrome (4Wenzel R.P. Pinsky M.R. Ulevitch R.J. Young L. Clin. Infect. Dis. 1996; 22: 407-412Crossref PubMed Scopus (123) Google Scholar). Elevated plasma concentrations of a variety of cytokines and chemokines such as tumor necrosis factor-α, IL-1, and IL-6 have been described in septic patients (5Arnalich F. Garcia-Palomero E. Lopez J. Jimenez M. Madero R. Renart J. Vazquez J.J. Montiel C. Infect. Immun. 2000; 68: 1942-1945Crossref PubMed Scopus (228) Google Scholar, 6Van der Poll T. van Deventer S.J. Infect. Dis. Clin. N. Am. 1999; 13: 413-426Abstract Full Text Full Text PDF PubMed Scopus (292) Google Scholar). IL-6 is one of the most reliably elevated markers in sepsis (7Selberg O. Hecker H. Martin M. Klos A. Bautsch W. Kohl J. Crit. Care Med. 2000; 28: 2793-2798Crossref PubMed Scopus (209) Google Scholar) and one of the few serum markers that has a demonstrable correlation with mortality rates in humans (8Spittler A. Razenberger M. Kupper H. Kaul M. Hackl W. Boltz-Nitulescu G. Fugger R. Roth E. Clin. Infect. Dis. 2000; 31: 1338-1342Crossref PubMed Scopus (94) Google Scholar). Knowing the anti-inflammatory effects of CO, we hypothesized that CO would protect against lethality in an animal model of sepsis. We further postulated that modulation of IL-6 production may contribute to the salutary effects of CO. We tested these hypotheses using a model of LPS-induced sepsis syndrome. Animals—Male C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME); male Jnk1 –/– and Jnk2 –/– mice and the corresponding control mice were generated as previously described (9Kuan C.Y. Yang D.D. Samanta D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar). Mice were allowed to acclimate for 1 week with rodent chow and water ad libitum. Cell Culture Experiments—RAW 264.7 mouse peritoneal macrophages and MH-S mouse alveolar macrophages were purchased from American Type Culture Collection (Manassas, VA). Both cell types were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 100 μg/ml gentamycin in a humidified atmosphere of 5% CO2 in air with or without additional CO as described below. Cell culture experiments were conducted in triplicate. Stable Cell Transfection—RAW 264.7 cells were stably transfected with a pSFFV/HO-1 plasmid construct as previously described (1Otterbein L.E. Bach F.H. Alam J. Soares M. Tao Lu H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1851) Google Scholar). IL-6 Promoter Constructs—The IL-6 promoter constructs were kindly provided by Drs. Alaina J. Ammit (Faculty of Pharmacy, University of Sydney, Sydney, Australia) and Oliver Eikelberg (Giessen University School of Medicine, Giessen, Germany). The 651-bp fragment of the human IL-6 promoter was subcloned (5′-KpnI, 3′-XhoI) into the pGL3-Basic luciferase reporter gene vector plasmid (Promega, Madison, WI) to give the parental pIL6-luc651 construct. Within pIL6-luc651, consensus transcription factor-binding sites were inactivated by site-directed mutagenesis, including AP-1, C/EBP-β, and NF-κB as previously described (10Ammit A.J. Lazaar A.L. Irani C. O'Neill G.M. Gordon N.D. Amrani Y. Penn R.B. Panettieri Jr., R.A. Am. J. Respir. Cell Mol. Biol. 2002; 26: 465-474Crossref PubMed Scopus (172) Google Scholar, 11Eickelberg O. Pansky A. Mussmann R. Bihl M. Tamm M. Hilbebrand P. Perruchoud A.P. Roth M. J. Biol. Chem. 1999; 274: 12933-12938Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Transient Cell Transfection and Luciferase Assay—Transfection of RAW 264.7 cells was performed with LipofectAMINE 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, cells were plated onto 12-well plates at a density of 2.5 × 105 cells/plate for 18 h and then transfected with 600 ng of IL-6 construct as well as 30 ng of pRL-TK control vector (Promega) to normalize transfection efficiencies. Following transfection, cells were cultured for 24 h and then treated with 1 μg/ml LPS. Cells were harvested after 8 h, and luciferase activity was assessed using the Dual-Luciferase reporter assay system (Promega) according to manufacturer's instructions. Carbon Monoxide Exposures—Mice or macrophages were exposed to compressed air with varying concentrations of CO (0–500 ppm). For cell culture experiments, the atmosphere included 5% CO2 for adjustment of pH. Details of CO administration have been previously described (1Otterbein L.E. Bach F.H. Alam J. Soares M. Tao Lu H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1851) Google Scholar). Animal Experiments—Mice were exposed to 250 ppm CO or room air for 1 h prior to intraperitoneal administration of LPS, Escherichia coli serotype 0127:B8 (Sigma). In lethality experiments, a dose of 40 mg/kg LPS was administered, and animals were returned to the exposure chamber until 48 h had elapsed or until death had ensued; the time of death was recorded. In cytokine experiments, 1 mg/kg LPS was administered by intraperitoneal injection, and blood was collected after 5 h; the animals were killed at this time. Cytokine Analysis—Serum and media samples were analyzed with enzyme-linked immunosorbent assay (ELISA) kits purchased from R&D Systems (Minneapolis, MN) following the manufacturer's instructions. RNA Extraction and Northern Blot Analysis—Total RNA was isolated from RAW 264.7 macrophages using TRIzol lysis buffer (Invitrogen), followed by chloroform extraction. The details of this technique have been previously described (1Otterbein L.E. Bach F.H. Alam J. Soares M. Tao Lu H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1851) Google Scholar). Electrophoretic Mobility Shift Assay (EMSA)—RAW 264.7 cells were exposed to LPS (1 mg/ml) alone or ambient CO (250 ppm) as described above. Nuclei were extracted as previously described (12Camhi S.L. Alam J. Wiegand G.W. Chin B.Y. Choi A.M. Am. J. Respir. Cell Mol. Biol. 1998; 18: 226-234Crossref PubMed Scopus (126) Google Scholar). A double-stranded oligonucleotide containing the consensus transcription factor-binding site for AP-1 (5′-CGCTTGATGAGTCAGCCGGAA-3′) was purchased from Promega. Labeling and DNA binding were performed as previously described (12Camhi S.L. Alam J. Wiegand G.W. Chin B.Y. Choi A.M. Am. J. Respir. Cell Mol. Biol. 1998; 18: 226-234Crossref PubMed Scopus (126) Google Scholar). For the supershift against c-Jun and JunB, JunD, and c-Fos were with samples after the binding protein and the consensus of JNK kits were purchased from and used following the manufacturer's instructions. as in the and control were assessed with for the of dose was using of and was at CO against in a of the used a murine LPS model to the effect of inhaled dose CO on C57BL/6 mice were treated with a lethal dose of intraperitoneal LPS and subsequently exposed to 250 ppm CO or ambient As shown in the animals had significantly better survival than their untreated counterparts. A dose of CO is to rescue animals from lethality to LPS. CO Cytokine in inflammatory cytokines to a role in the of the sepsis we to the effects of CO on cytokine production in murine It has previously been demonstrated by (1Otterbein L.E. Bach F.H. Alam J. Soares M. Tao Lu H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1851) Google Scholar) that CO LPS-induced production of tumor necrosis factor-α and the production of in the effect of CO to IL-6 and we administered a dose of LPS (1 to mice in the or of inhaled CO (250 ppm). Mice treated with CO had lower serum levels of IL-6 and IL-1β 5 h after LPS than control mice as by HO-1 and CO IL-6 in the effect of CO on IL-6 in vitro, we exposed cell of mouse macrophages 264.7 and to 1 μg/ml LPS in the or of CO and assessed IL-6 production by IL-6 was in the cell medium from untreated macrophages or from macrophages treated with CO Cells exposed to LPS had an elevated of IL-6 in the medium after 5 h, this in IL-6 was significantly attenuated by exposure to CO A and The effect of CO was over a of ppm increased CO production would have the same effect as administered CO, we exposed a cell HO-1 to 1 μg/ml LPS and for IL-6 by The control cells were transfected with the The control cells a in IL-6 levels than the cells that CO production has an effect to that of exogenous administration of CO. CO IL-6 in RAW 264.7 assessed the of IL-6 in RAW 264.7 macrophages exposed to 1 μg/ml LPS in the or of CO. Cells were harvested 5 h after LPS treatment for RNA and by Northern As shown in the untreated cells and treated with CO alone have IL-6 by Northern LPS a large in IL-6 and this was by treatment with CO. This that CO IL-6 production in RAW 264.7 cells at a transcriptional level. CO via of IL-6 gene is by the binding of transcription to consensus within the promoter including binding sites for AP-1, or (10Ammit A.J. Lazaar A.L. Irani C. O'Neill G.M. Gordon N.D. Amrani Y. Penn R.B. Panettieri Jr., R.A. Am. J. Respir. Cell Mol. Biol. 2002; 26: 465-474Crossref PubMed Scopus (172) Google Scholar). be responsible for of IL-6 by CO, RAW 264.7 macrophages were transfected with or constructs of the human IL-6 promoter to a luciferase promoter constructs were with site-directed in the or and a parental construct. Luciferase activity was by LPS treatment of cells transfected with constructs to at least the same as the parental construct. CO treatment decreased luciferase activity to a in constructs the AP-1 construct CO AP-1 in that CO can the of AP-1, were performed using from RAW 264.7 macrophages treated with LPS alone or with LPS and CO (250 ppm). Cells were harvested 30 after LPS As shown in this of CO was of AP-1 binding in the of the AP-1 and to the supershift experiments were by against JunB, JunD, and c-Fos to the can be by a in the mobility of the and a of the AP-1 that the AP-1 of c-Fos to a and treatment of cells with CO to c-Fos to the and and to a CO JNK after with of AP-1 in the of IL-6 production to the JNK pathway be this RAW 264.7 cells were with LPS (1 and exposed to CO (250 ppm) or ambient air as described above. Cells were collected at from to h for of JNK JNK phosphorylation was increased by with this effect was at and JNK phosphorylation was by CO treatment at time and this effect was by 30 The and time CO had no effect on JNK phosphorylation at these of the Jnk1 or Jnk2 and IL-6 in the JNK pathway may the effect of CO on LPS-induced IL-6 and we used mice deficient in the Jnk1 or Jnk2 gene is of of JNK, through the effect of gene is a in JNK activity T. A.J. J. Davis R.J. J. 1996; PubMed Scopus Google Scholar). The effect of Jnk1 on JNK function has been shown to be than that of Jnk2 C. P. Yang D.D. J. A. Flavell R.A. Davis R.J. 2000; PubMed Scopus Google Scholar). The Jnk1 and Jnk2 –/– mice were with a dose of LPS, and cytokine levels were at time 1 h, and 5 h in the or of CO. IL-1β levels were significantly in the control mice than in the Jnk1 –/– or Jnk2 –/– mice at 1 h, by 5 h, this no the time the mice had significantly lower levels of IL-1β than the control mice previously CO had no effect on levels in the Jnk1 –/– and Jnk2 –/– mice IL-6 is to after LPS compared with was significantly in the Jnk1 –/– and Jnk2 –/– mice than in the control mice at 5 h, and was no effect of CO on the levels of IL-6 in the Jnk1 –/– and Jnk2 –/– mice This that the JNK pathway is in the production of IL-1β and IL-6 in response to LPS, that the of this pathway or the effect of CO. of the Jnk1 the of CO in to a of of the Jnk1 gene survival in response to LPS, these mice were with a lethal dose of LPS and subsequently exposed to room air or CO (250 ppm). 24 h, the of survival was h in both that CO had no effect on the Jnk1 –/– mice had a survival than the mice shown in The sepsis syndrome the leading cause of death in intensive care units in the United States, and to impact mortality have been (3Friedman G. Silva E. Vincent J.L. Crit. Care Med. 1998; 26: 2078-2086Crossref PubMed Scopus (500) Google Scholar). from human and animal that HO-1 is by and this a survival Both increased and increased CO have been in patients with sepsis M. E. R. J. 1999; PubMed Scopus Google Scholar, R. M. R. A. Care Med. 2002; 28: PubMed Scopus Google Scholar). LPS administration to HO-1 K. K. T. T. K. M. Dis. 2000; PubMed Scopus Google and in a rodent model of of HO-1 has been shown to be and of HO-1 F. G. J. C. Am. J. Respir. Crit. Care Med. 2001; PubMed Scopus Google Scholar). in a murine of HO-1 to increased mortality Y. J. Full Text PDF PubMed Scopus Google Scholar). The by HO-1 provides cellular protection a of of the of heme have been including by the release of and CO P. A. H. 1999; PubMed Scopus Google Scholar, A. R. 2000; PubMed Scopus Google Scholar, L.E. Choi Am. J. 2000; PubMed Google Scholar). we have the effect of administered CO in a murine LPS model of septic and have shown that CO There is a well described correlation serum IL-6 levels and in sepsis (8Spittler A. Razenberger M. Kupper H. Kaul M. Hackl W. Boltz-Nitulescu G. Fugger R. Roth E. Clin. Infect. Dis. 2000; 31: 1338-1342Crossref PubMed Scopus (94) Google Scholar, C. G. F. P. E. 2001; PubMed Google Scholar, A. A. G. G. M. M. P. K. G. Crit. Care Med. 2002; PubMed Scopus Google Scholar, M. M. Cytokine 2002; 13: Google Scholar, J. C. M. T. L. R. Care Med. 2001; PubMed Scopus Google and increased IL-6 is an independent of survival in human sepsis M. F. A. E. A. E. J.L. 2001; PubMed Scopus Google Scholar). In mouse serum IL-6 and IL-1β were attenuated by exogenous CO The cytokine levels from of CO that the of in vitro suggest that CO has a direct effect on cytokine production. The of IL-6 production by exogenous CO in vitro occurred at concentrations of CO ppm). For this of CO is to the levels that can be in the of ppm) J. H. T. N. J. Med. 2002; PubMed Scopus Google Scholar). Northern we have shown that the in vitro of LPS-induced IL-6 production at a transcriptional level. using IL-6 promoter constructs with at of transcription factor-binding sites AP-1, and that the of CO may be through modulation of AP-1. This be made as of binding site had no effect on the of promoter by LPS, that no transcription factor is for IL-6 in response to LPS. was a decreased IL-6 promoter activity after treatment with CO in the AP-1 that of AP-1 binding may for the effect of CO. of NF-κB binding by CO has previously been reported S.L. F. L.E. Choi A.M. Am. J. Respir. Cell Mol. Biol. 2002; PubMed Scopus Google IL-6 promoter suggest that this is a factor in We have shown that the AP-1 components by CO JunB, JunD, and have shown that inhibits IL-6 transcription in human via of AP-1-binding activity and of c-Fos F. 1996; Google Scholar). The of the results of that to is the that HO-1 the anti-inflammatory effects of in mice Nat. Med. 2002; PubMed Scopus Google Scholar). The JNK pathway is by cellular stress and is with the of AP-1 transcription et al. Flavell R.A. Davis R.J. Mol. Biol. PubMed Scopus Google Scholar) recently reported that the JNK pathway is for the of AP-1 activity by tumor necrosis JNK is of binding the NH2-terminal of AP-1 by its component AP-1 can then into the to transcription of R.J. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The of JNK with AP-1, with the of the JNK pathway in the of cytokines by Y. C. J. 2002; Scopus Google made the JNK signaling an as a for CO. We have shown in a mouse cell CO can JNK at time JNK to and 30 after LPS in in vitro cellular mediated by JNK from an and of this using mice suggest that the effect of CO on the JNK pathway is is over time in The mice we used deficient in the Jnk1 or Jnk2 of both is lethal C. P. Yang D.D. J. A. Flavell R.A. Davis R.J. 2000; PubMed Scopus Google Scholar). JNK activity is in these mice, is and the Jnk1 has been shown to have a effect on JNK activity than the Jnk2 C. P. Yang D.D. J. A. Flavell R.A. Davis R.J. 2000; PubMed Scopus Google Scholar). Both Jnk1 –/– and Jnk2 –/– mice levels of IL-1β in response to LPS at 1 h, the levels to of the control mice after 5 The in IL-1β to levels may be to the that JNK activity is and signaling in these mice. The IL-6 levels were elevated in the control or mice at 1 h, at 5 h, the mice had levels of IL-6 than the Jnk1 –/– or Jnk2 –/– mice. More the Jnk1 –/– and Jnk2 –/– mice to exogenous CO in the same as the the levels of IL-1β and IL-6 were by CO in these mice. The effect of CO in survival was seen in the Jnk1 –/– mice and their of survival at was increased compared with C57BL/6 mice. The results of these experiments with mice suggest that CO the JNK pathway in the of inflammatory cytokine production. There have been no CO to the JNK have demonstrated an in vitro effect of CO on the pathway (1Otterbein L.E. Bach F.H. Alam J. Soares M. Tao Lu H. Wysk M. Davis R.J. Flavell R.A. Choi A.M. Nat. Med. 2000; 6: 422-428Crossref PubMed Scopus (1851) Google Scholar, L.E. J. E. Bach F.H. Choi A.M. Soares J. Med. 2000; PubMed Scopus Google Scholar). these time as as or 30 after cell It is that CO has effects on signaling in and as results the of transcription and signaling the JNK pathway a into the of CO effects in In summary, we have demonstrated for the first time a role for CO in a murine model of sepsis and have shown that this is associated with decreased production of IL-1β and IL-6 in vivo and in We have that the effect of CO the signaling We that decreased of in the of CO to a effect on inflammatory cytokine production and protection against sepsis. We Drs. Oliver Eikelberg and Alaina J. Ammit for the IL-6 promoter