The role of heme oxygenase-1 in down regulation of PGE<Subscript>2</Subscript> production by taurine chloramine and taurine bromamine in J774.2 macrophages

Taurine chloramine (TauCl) and taurine bromamine (TauBr), products of myeloperoxidase halide system, exert anti-inflammatory properties. TauCl was demonstrated to inhibit the production of a variety of pro-inflammatory mediators including cyclooxygenase-2 (COX-2) dependent production of prostaglandin E₂ (PGE₂). Recently we have demonstrated that both major leukocyte haloamines, TauCl and TauBr, induced expression of HO-1 in non-activated and LPS-activated J774.2 macrophages. In this study, we have shown that TauCl and TauBr, at non-cytotoxic concentrations, inhibited the production of (PGE₂) without altering the expression of COX-2 protein, in LPS/IFN-γ stimulated J774.2 cells. The inhibitory effect of TauCl and TauBr was reversed by chromium III mesoporhyrin (CrMP), an inhibitor of HO-1 activity. Our data suggest that HO-1 might participate in anti-inflammatory effects of TauCl/TauBr possibly by inhibition of COX-2 activity and decrease of PGE₂ production.     

Taurine bromamine (TauBr) - its role in immunity and new perspectives for clinical use

This review is an attempt to summarize our knowledge about taurine bromamine (TauBr) properties, its role in innate immunity and its therapeutic potential.TauBr and taurine chloramine (TauCl) are major haloamines generated by eosinophils and neutrophils at a site of inflammation. Both haloamines share anti-inflammatory and anti-oxidant properties. TauBr, similarly to TauCl, decreases the production of proinflammatory mediators. Their anti-inflammatory and anti-oxidant activities are enhanced by their ability to induce the expression of heme oxygenase-1 (HO-1). TauCl is more stable than TauBr. On the other hand, only TauBr was found to be highly membrane-permeable showing stronger microbicidal activity than TauCl.In the light of the anti-inflammatory and antimicrobial properties of TauBr we discuss its therapeutic potential in local treatment of inflammation, especially acne vulgaris, the most common inflammatory skin disorder. TauBr, at non-cytotoxic concentrations, is able to kill Propionibacterium acnes, the skin bacteria involved in pathogenesis of acne vulgaris.As topical antibiotics used in the therapy of acne are associated with the emergence of resistant bacteria, topical TauBr seems to be a good candidate for an alternative therapy.Recently, in a double blind trial, the efficacy of TauBr was compared with the efficacy of clindamycin, one of the most common topical antibiotics used in acne therapy. Comparable reduction of acne lesions was observed in the TauBr and clindamycin groups of patients with mild and moderate inflammatory facial acne vulgaris. We conclude that this pilot study supports our concept that TauBr can be used as a topical agent in the treatment of acne vulgaris, especially in patients who have already developed antibiotic resistance. Further studies are necessary to substantiate the more extended use of TauBr as an anti-inflammatory and anti-oxidant agent in human medicine.     

Inhibition of NOS-2 induction in LPS-stimulated J774.2 cells by 1, 5-isoquinolinediol, an inhibitor of PARP.

Activation of both poly (ADP-ribose) polymerase (PARP) and inducible nitric oxide synthase (NOS-2) have been implicated in the pathogenesis of various forms of inflammation, therefore compounds which may simultaneously inhibit both pathways are of potential therapeutic interest. We tested the influence of potent inhibitor of PARP, 1, 5-isoquinolinediol (ISO), on NOS-2 induction in model of mouse macrophages (cell line J774.2) stimulated with lipopolysaccharide (1 microg/ml). Pretreatment with ISO (1-300 microM) resulted in dose-dependent inhibition of accumulation of NOS-2-derived nitrite in culture medium (IC(50) = 9,3 microM) as well as inhibition of NOS-2 protein induction in cultured J774.2 cells; ISO given 10 hours after LPS did not influence activity of NOS-2. Interestingly, another PARP inhibitor, 3-aminobenzamide (3-AB, 10-3000 microM), did not influence 24-hr nitrite accumulation in J774.2 cell culture, either administered 15 minutes prior to LPS or 10 hrs after LPS. Scavenging of reactive oxygen species by use of mixture of SOD and catalase (SOD/Cat, 100/300 - 1000/3000 U/ml) as well as cell permeable SOD-mimetic [Mn(III)TBAP, 1- 100 microM], did not influence NOS-2 induction in J774.2 cells. In summary, we identified 1, 5-isoquinoline as potent inhibitor of induction of NOS-2 in LPS-treated mouse macrophages. The exact mechanism of inhibitory action of this compound on NOS-2 induction requires further investigation.     

Participation of heme oxygenase-1 in a model of acute inflammation

In this study, the role of heme oxygenase-1 (HO-1) in the inflammatory response elicited by zymosan in the mouse air pouch model has been examined. This response is characterized by a time-dependent increase in HO-1 expression in the leukocytes migrating into the exudates. At 24-48 h maximal HO-1 expression was accompanied by reduced cyclooxygenase-2 (COX-2) and nitric oxide synthase-2 (NOS-2) expression as well as low levels of inflammatory mediators. Hemin administration into the air pouch caused an elevation of HO-1 protein and bilirubin levels induced by zymosan with inhibition of COX-2 expression. In mouse peritoneal macrophages from hemin-injected mice, we also observed an increased expression of HO-1 with inhibition of COX-2 expression and prostaglandin E(2) (PGE(2)) levels. Our data suggest an anti-inflammatory role for HO-1 in the response induced by this phagocytic stimulus.     

Taurine and inflammatory diseases

Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in humans and plays an important role in several essential biological processes such as bile acid conjugation, maintenance of calcium homeostasis, osmoregulation and membrane stabilization. Moreover, attenuation of apoptosis and its antioxidant activity seem to be crucial for the cytoprotective effects of taurine. Although these properties are not tissue specific, taurine reaches particularly high concentrations in tissues exposed to elevated levels of oxidants (e.g., inflammatory cells). It suggests that taurine may play an important role in inflammation associated with oxidative stress. Indeed, at the site of inflammation, taurine is known to react with and detoxify hypochlorous acid generated by the neutrophil myeloperoxidase (MPO)–halide system. This reaction results in the formation of less toxic taurine chloramine (TauCl). Both haloamines, TauCl and taurine bromamine (TauBr), the product of taurine reaction with hypobromous acid (HOBr), exert antimicrobial and anti-inflammatory properties. In contrast to a well-documented regulatory role of taurine and taurine haloamines (TauCl, TauBr) in acute inflammation, their role in the pathogenesis of inflammatory diseases is not clear. This review summarizes our current knowledge concerning the role of taurine, TauCl and TauBr in the pathogenesis of inflammatory diseases initiated or propagated by MPO-derived oxidants. The aim of this paper is to show links between inflammation, neutrophils, MPO, oxidative stress and taurine. We will discuss the possible contribution of taurine and taurine haloamines to the pathogenesis of inflammatory diseases, especially in the best studied example of rheumatoid arthritis.     

Up-regulation of heme oxygenase-1 expression through the Rac1/NADPH oxidase/ROS/p38 signaling cascade mediates the anti-inflammatory effect of 15-deoxy-delta 12,14-prostaglandin J2 in murine macrophages

We investigated the signaling pathway that leads to the expression of heme oxygenase-1 (HO-1) in murine macrophages in response to 15-deoxy-delta 12,14-prostaglandin J2 (15dPGJ2). 15dPGJ2 caused dose- and time-dependent activation of Rac1, followed by a transient increase in reactive oxygen species (ROS) via NADPH oxidase, which leads to downstream activation of p38 kinase. Inhibition of 15dPGJ2-dependent HO-1 expression significantly attenuated suppression by 15dPGJ2 of LPS-induced iNOS expression and subsequent production of nitric oxide (NO). Our findings strongly suggest that 15dPGJ2 exerts its anti-inflammatory activity through the Rac1-NADPH oxidase-ROS-p38 signaling to the up-regulation of HO-1 in an in vitro inflammation model.     

Cross-regulation of carbon monoxide and the adenosine A2a receptor in macrophages

Adenosine and heme oxygenase-1 (HO-1) exert a wide range of anti-inflammatory and immunomodulatory actions, making them crucial regulatory molecules. Despite the diversity in their modes of action, the similarity of biological effects of adenosine and HO-1 led us to hypothesize a possible interrelationship between them. We assessed a potential role for HO-1 in the ability of adenosine or 5'-N-ethylcarboxamidoadenosine (NECA), a stable adenosine analog, to modify the response of LPS-stimulated macrophages. Adenosine and NECA markedly induced HO-1 and blocked LPS-induced TNF-alpha production via adenosine A2aR-mediated signaling; blocking of HO-1 by RNA interference abrogated the effects of adenosine and NECA on TNF-alpha. HO-1 overexpression or exposure to carbon monoxide (CO), a product of HO-1 enzymatic activity, resulted in augmented A2aR mRNA and protein levels in RAW264.7 cells and primary macrophages. The induction of A2aR expression by HO-1 or CO resulted in an increase in the sensitivity to the anti-inflammatory effects of adenosine and NECA, which was lost in macrophages isolated from A2aR-deficient mice. Moreover, a decrease in cAMP levels upon NECA stimulation of naive macrophages was counterbalanced by CO exposure to up-regulate A2aR levels. This implies adenosine receptor isoform switch as a selective modification in macrophage phenotype. Taken together, these data suggest the existence of a positive feedback loop among adenosine, HO-1, CO, and the A2aR in the chronological resolution of the inflammatory response.     

Molecular mechanism for adiponectin-dependent M2 macrophage polarization: link between the metabolic and innate immune activity of full-length adiponectin

The anti-inflammatory effects of globular adiponectin (gAcrp) are mediated by IL-10/heme oxygenase 1 (HO-1)-dependent pathways. Although full-length (flAcrp) adiponectin also suppresses LPS-induced pro-inflammatory signaling, its signaling mechanisms are not yet understood. The aim of this study was to examine the differential mechanisms by which gAcrp and flAcrp suppress pro-inflammatory signaling in macrophages. Chronic ethanol feeding increased LPS-stimulated TNF-α expression by Kupffer cells, associated with a shift to an M1 macrophage polarization. Both gAcrp and flAcrp suppressed TNF-α expression in Kupffer cells; however, only the effect of gAcrp was dependent on IL-10. Similarly, inhibition of HO-1 activity or siRNA knockdown of HO-1 in RAW264.7 macrophages only partially attenuated the suppressive effects of flAcrp on MyD88-dependent and -independent cytokine signatures. Instead, flAcrp, acting via the adiponectin R2 receptor, potently shifted the polarization of Kupffer cells and RAW264.7 macrophages to an M2 phenotype. gAcrp, acting via the adiponectin R1 receptor, was much less effective at eliciting an M2 pattern of gene expression. M2 polarization was also partially dependent on AMP-activated kinase. flAcrp polarized RAW264.7 macrophages to an M2 phenotype in an IL-4/STAT6-dependent mechanism. flAcrp also increased the expression of genes involved in oxidative phosphorylation in RAW264.7 macrophages, similar to the effect of flAcrp on hepatocytes. In summary, these data demonstrate that gAcrp and flAcrp utilize differential signaling strategies to decrease the sensitivity of macrophages to activation by TLR4 ligands, with flAcrp utilizing an IL-4/STAT6-dependent mechanism to shift macrophage polarization to the M2/anti-inflammatory phenotype.     

The effect of taurine on polymorphonuclear leukocyte functions in endotoxemia

Summary. The aim of the present study was to measure MPO activity in PMN leukocytes after endotoxin administration, and to compare the levels of NO₂ ⁻ competing with taurine for reaction with HOCl. Furthermore we aimed to determine TauCl levels, a product of MPO–H₂O₂–Halide system, and to evaluate anti-inflammatory properties of PMN in endotoxemia. In addition, our second objective was to investigate the effect of taurine, an antioxidant amino acid, on anti-bactericidal and anti-inflammatory functions of PMN after administration of endotoxin together with taurine. All experiments were performed with four groups (control, taurine, endotoxemia, and taurine plus endotoxin) of ten guinea pigs. After endotoxin administration (4 mg/kg), MPO activities increased and taurine levels decreased. Therefore levels of TauCl, NO₂ ^•− increased. We observed the effects of taurine as conflicting. When taurine was administrated alone (300 mg/kg), all of these parameters decreased. Consequently, we suggested that taurine is influential in infected subjects but not on healthy ones as an antioxidative amino acid. In addition, we believe that in vivo effects of taurine may differ from those in vitro depending on its dosage.     

Induction of heme oxygenase-1 expression in murine macrophages is essential for the anti-inflammatory effect of low dose 15-deoxy-Delta 12,14-prostaglandin J2

15-Deoxy-Delta 12,14-prostaglandin J2 (15d-PGJ2), a cyclopentenone prostaglandin, displays a potent anti-inflammatory effect at micromolar concentrations (>2 microM) through direct inhibition of nuclear factor (NF)-kappa B activation. Here we show that at submicromolar concentrations (0.1-0.5 microM) 15d-PGJ2 retains the ability to suppress the production of tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in lipopolysaccharide (LPS)-activated murine J774 macrophages under the conditions of a prolonged incubation (>12 h). Western blot analysis revealed that the expression of the cytoprotective enzyme, heme oxygenase-1 (HO-1), was induced and coincident with the anti-inflammatory action of 15d-PGJ2. Inhibition of HO-1 activity or scavenging carbon monoxide (CO), a byproduct derived from heme degradation, significantly attenuated the suppressive activity of 15d-PGJ2. Furthermore, LPS-induced NF-kappa B activation assessed by the inhibitory protein of NF-kappa B(I kappa B) degradation and p50 nuclear translocation was diminished in cells subjected to prolonged treatment with the low concentration of 15d-PGJ2. Treatment of cells with the protein synthesis inhibitor, cycloheximide, or the specific p38 MAP kinase inhibitor, SB203580, blocked the induction of HO-1 and suppression of LPS-induced I kappa B degradation mediated by 15d-PGJ2. Likewise, HO inhibitor and CO scavenger were effective in abolishing the inhibitory effects of 15d-PGJ2 on NF-kappa B activation induced by LPS. The functional role of CO was further demonstrated by the use of a CO releasing molecule, tricarbonyldichlororuthenium(II) dimer, which significantly suppressed LPS-induced nuclear translocation of p50 as assessed by confocal immunofluorescence. Collectively, these data suggest that even at submicromolar concentrations 15d-PGJ2 can exert an anti-inflammatory effect in macrophages through a mechanism that involves the action of HO/CO.     

Genipin up-regulates heme oxygenase-1 via PI3-kinase-JNK1/2-Nrf2 signaling pathway to enhance the anti-inflammatory capacity in RAW264.7 macrophages

Genipin, an aglycon of geniposide, has been reported to exhibit diverse pharmacological functions such as antitumor and anti-inflammatory effects. This study aimed to elucidate the anti-inflammatory mechanism of genipin, focusing particularly on the role of heme oxygenase-1 (HO-1), a potent anti-inflammatory enzyme. In RAW264.7 cells, genipin increased HO-1 expression and its enzyme activity via a NF-E2-related factor 2 (Nrf2)–antioxidant response element (ARE) pathway. These effects were significantly inhibited by exposure to the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, LY294002, or by expression of a dominant negative mutant of PI 3-kinase. Additional experiments showed that the activation of c-Jun NH₂-terminal kinase 1/2 (JNK1/2) is required for genipin-induced phosphorylation and nuclear translocation of Nrf2 and antioxidant response element (ARE)-driven induction of HO-1, and acts as a downstream effector of PI 3-kinase. Furthermore, functional significance of HO-1 induction was revealed by genipin-mediated inhibition of lipopolysaccharide-stimulated inducible nitric oxide synthase expression or cyclooxygenase-2 promoter activity, the response was reversed by the blocking of HO-1 protein synthesis or HO-1 enzyme activity. Therefore, identification of PI 3-kinase-JNK1/2-Nrf2-linked signaling cascade in genipin-mediated HO-1 expression defines the signaling event that could participate in genipin-mediated anti-inflammatory response.     

Effect of taurine chloramine,the product of activated neutrophils,on the development of collagen-induced arthritis in DBA 1/J mice

Summary.  Taurine chloramine (TauCl), a product of neutrophil myeloperoxidase – halide system, formed by a reaction of taurine with HOCl, is known as an anti-microbial and anti-inflammatory long-lived oxidant. We previously reported that TauCl inhibits in vitro the production of proinflammatory cytokines (IL-6, IL-8) by RA synoviocytes. Therefore we performed this study to investigate the effect of TauCl treatment on the development of collagen-induced arthritis (CIA) in DBA1/J mice. Early administration of TauCl (after primary immunization) resulted in the delay of the onset of CIA, but had no effect on severity of arthritis. TauCl, given daily for 21 days after booster immunization, did not reduce the symptoms of arthritis in those mice, which already developed CIA, but significantly diminished incidence of the disease (55% vs. 90% of placebo mice). The mechanism of this effect is unknown. This is the first in vivo study suggesting that TauCl may be used for immune intervention in chronic inflammatory diseases. Received November 30, 2001 Accepted January 25, 2002 Published online August 20, 2002 Acknowledgments We express our gratitude to K. Szewczyk for skillful technical assistance. This work was supported by grants from the Committee of Scientific Research (Warsaw, Poland) Grant No. 4PO5B01018, and partially by Grant No.: PO 5A 10419. Authors' address: Janusz Marcinkiewicz, Ph.D., M.D., Department of Immunology, Jagiellonian University Medical College, 18 Czysta St., 31-121 Kraków, Poland, E-mail: mmmarcin@cyf-kr.edu.pl     

Tanshinone IIA from Salvia miltiorrhiza induces heme oxygenase-1 expression and inhibits lipopolysaccharide-induced nitric oxide expression in RAW 264.7 cells

Tanshinone IIA exerts anti-inflammatory effects and influences electron transfer reaction in mitochondria. In the present study, we demonstrated that tanshinone IIA increased intracellular production of reactive oxygen species (ROS), which in turn induces heme oxygenase-1 (HO-1) expression in RAW 264.7 macrophages. Tanshinone IIA inhibited COX-2 and iNOS expression in lipopolysaccharide-activated RAW 264.7 macrophages. Inhibition of HO-1 or scavenging of CO significantly reversed the inhibition of LPS-stimulated nitrite accumulation by tanshinone IIA, suggesting a novel role of HO-1 in the anti-inflammatory effect of tanshinone IIA.