• Experimental allergic encephalomyelitis;
  • Heme oxygenase-1;
  • Multiple sclerosis;
  • Oxidative stress;
  • NADPH cytochrome P450 reductase;
  • Heat shock protein


  1. Top of page
  2. Abstract
  4. Induction of EAE
  6. HO-1 expression in EAE
  7. Reductase expression in EAE
  9. Acknowledgements

Abstract: Oxidative stress is implicated in the pathogenesis ofexperimental allergic encephalomyelitis (EAE), a model for multiple sclerosis.Heme oxygenase-1 (HO-1) is a heat shock protein induced by oxidative stress.HO-1 metabolizes the pro-oxidant heme to the antioxidant biliverdin and CO.HO-1 requires electrons, donated by NADPH cytochrome P450 reductase(henceforth, reductase), for catalytic activity. EAE was induced with apeptide of proteolipid protein in SJL mice, and the expression of HO-1 andreductase in the hindbrain was analyzed. HO-1 protein levels weresignificantly increased in EAE animals compared with control mice. HO-1expression was present in ameboid macrophages, reactive microglia, andastrocytes in white matter tracks. Bergmann glia and ameboid macrophages alsowere occasionally stained in the molecular layer of the cerebellum. UnlikeHO-1, reductase protein levels decreased with disease severity. HO-1 andreductase were associated with each other in endoplasmic reticulum micelles,suggesting that the decrease in reductase does not interfere with itsassociation with HO-1. In cells that express HO-1, the association ofreductase with HO-1 should competitively inhibit the interaction of reductasewith cytochrome P450 isozymes and thereby limit free radical production as thelatter two enzymes act cooperatively to produce superoxide. The increase inHO-1 together with the decrease in reductase may be part of a common defensemechanism attempting to minimize tissue damage in several neurologicalconditions.

Multiple sclerosis (MS) is a demyelinating disease of the CNS with an autoimmune component implicated in the pathogenesis (Raine, 1984; Cross et al., 1996). Experimental allergic encephalomyelitis (EAE) is a commonly used animal model of MS (Bourrie et al., 1999). The autoimmune inflammation that exists in MS and EAE is considered to have both T- and B-cell components (Misko et al., 1995; Raine et al., 1999). The active phase of EAE and MS is associated with the production of Th1 cytokines, namely, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin (IL)-1, IL-2 (Reder et al., 1994; Jung et al., 1997; Bourrie et al., 1999), as well as the production of antibodies that react with myelin/oligodendrocyte glycoprotein (Raine et al., 1999). In both diseases, there is loss of blood-brain barrier integrity, infiltration of macrophages and CD4+ T cells into the CNS, activation of microglia, and demyelination (Prineas and McDonald, 1997; Tran et al., 1997). However, a difference between the two diseases is the presence of extravasation of red blood cells in EAE but not in MS (Raine et al., 1980; Hansen and Pender, 1989).

Oxidative stress is implicated in the pathogenesis of EAE and MS (Cross et al., 1996, 1997; Hewett et al., 1996; Forge et al., 1998; Pedchenko and LeVine, 1998). Production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) occurs in these diseases (Cross et al., 1996, 1997; Hewett et al., 1996; Pedchenko and LeVine, 1998). Oxidative stress activates the stress response, an endogenous mechanism including induction of proteins and immune-related molecules that attempts to counteract a disturbance in homeostasis (Massa et al., 1996). Inducible nitric oxide synthase and cyclooxygenase-2 are two protein components of the stress response that are induced in EAE and MS (Cross et al., 1996; Sahrbacher et al., 1998; Deininger and Schluesener, 1999). Heme oxygenase-1 (EC; HO-1), another stress-related protein, is induced by oxidative stress (Ewing and Maines, 1993; Dalton et al., 1995); however, its expression in EAE or MS is unknown.

HO-1 is a 32-kDa member of the heat shock protein family (HSP32) (Ewing and Maines, 1991; Ewing et al., 1992). HO-1 and the constitutive isozyme HO-2 catalyze the breakdown of the pro-oxidant heme into the antioxidant biliverdin (Tenhunen et al., 1968; Maines, 1988). To perform this function, HO-1 needs reducing equivalents supplied by NADPh ferrihemoprotein reductase [EC; NADPH cytochrome P450 reductase (henceforth, reductase)], and thus reductase is found in all tissues that exhibit HO activities (Maines et al., 1986; Maines, 1988). Byproducts of biliverdin production are CO and the release of iron from the porphyrin ring of heme (Maines, 1988). The released iron is a stimulus for down-regulation of the transferrin receptor (Elbirt and Bonkovsky, 1999) and up-regulation of the translation of ferritin, which binds and stores the liberated iron, preventing its participation in redox reactions (Eisenstein et al., 1991; Balla et al., 1992; Vile and Tyrrell, 1993; Vile et al., 1994; Elbirt and Bonkovsky, 1999). Iron can also become sequestered in mitochondria, which may be toxic (Schipper et al., 1999). If liberated iron is not appropriately handled by cellular systems (e.g., up-regulation of ferritin), then inhibition of HO may be protective, as shown by Dwyer et al. (1998), because free iron can catalyze free radical production.

The HO system is the most efficient heme-degrading method and the only one that produces biliverdin and CO almost exclusively (Maines, 1988). Biliverdin is reduced to another antioxidant, bilirubin, by biliverdin reductase (Tenhunen et al., 1968; Maines, 1988). In micromolar concentrations in vitro, bilirubin scavenges peroxyl radicals (Stocker et al., 1987), and in vivo it protects against oxidative stress due to CoCl2 administration (Llesuy and Tomaro, 1994). Moreover, bilirubin formed by HO-2 protects cortical and hippocampal cultures from H2O2 (Doré et al., 1999). The CO formed plays a role in second messenger systems through the generation of cyclic GMP (Maines, 1997; Zakhary et al., 1997). In addition to oxidative stress, HO-1 expression can be induced by heme, heat shock, glutathione depletion, and cytokines, such as TNF-α and IL-1α (Maines, 1988; Ewing and Maines, 1991, 1993; Ewing et al., 1992; Terry et al., 1999). Studies using HO-1 knockout mice show that HO-1 is crucial for iron homeostasis, and its up-regulation is an adaptive mechanism to protect cells from oxidative damage during stress (Poss and Tonegawa, 1997a,b). Furthermore, overexpression of HO-1 protects against middle cerebral artery occlusion, suggesting that HO-1 has a protective role during CNS stress in vivo (Panahian et al., 1999).

In addition to shuttling reducing equivalents to HO-1, the ∼78-kDa reductase works in concert with the cytochrome P450 isozymes in the metabolism of xenobiotics and endogenous chemicals (Yasukochi and Masters, 1976; Bergh and Strobel, 1992). A byproduct of the oxidation of chemicals by the cytochrome P450 system is O2•- (Kappus, 1993). In the endoplasmic reticulum, the up-regulation of HO-1 should increase the interaction of reductase with HO-1 while attenuating the interaction of reductase with cytochrome P450 isozymes, resulting in a lower production of O2•-. Thus, the association of reductase with HO-1 should cause a shift toward an antioxidant state.

The purpose of this study was to analyze the expression of HO-1 and reductase during the course of EAE to help elucidate the role of HO-1 and the stress response in this disease.


  1. Top of page
  2. Abstract
  4. Induction of EAE
  6. HO-1 expression in EAE
  7. Reductase expression in EAE
  9. Acknowledgements


All reagents were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.) with the exception of proteolipid peptide (amino acids 139-151; Research Support Facility, University of Kansas, Lawrence, KS, U.S.A.). Mycobacterium tuberculosis H37RA and Freund's incomplete adjuvant (Difco Laboratories, Detroit, MI, U.S.A.), pertussis toxin (List Biological Laboratories, Campbell, CA, U.S.A.), Metofane (Priman-Moore, Mundelein, IL, U.S.A.), protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA, U.S.A.), recombinant rat HSP32, and polyclonal antibodies to HO-1 and reductase (Stressgen, Victoria, BC, Canada).


Female SJL mice, aged 6 weeks and ∼15 g, were obtained from Jackson Laboratories (Bar Harbor, ME, U.S.A.). Original breeder pairs of mice heterozygous for the twitcher mutation were obtained from Jackson Laboratories and subsequently bred in the Laboratory for Animal Resources at the University of Kansas Medical Center. Homozygous offspring for the twitcher mutation have globoid cell leukodystrophy, which leads to progressive trembling starting at day ∼20 and death by day ∼40. All mice had access to food and water ad libitum. All procedures involving animals were in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the institutional animal care and use committee.

Induction of EAE

  1. Top of page
  2. Abstract
  4. Induction of EAE
  6. HO-1 expression in EAE
  7. Reductase expression in EAE
  9. Acknowledgements

Mice were anesthetized with Metofane and given two subcutaneous injections of 75 μg of proteolipid peptide in phosphate-buffered saline (total dose = 150 μg) that had been emulsified in an equal volume of Freund's incomplete adjuvant containing 100 μg of M. tuberculosis H37RA (total dose = 200 μg). One injection was given at the nape and the second was given on the dorsum. Pertussis toxin (100 ng i.v. through the tail vein) was administered on days 0, 3, and 7 following encephalitogen (total dose = 300 ng). Animals were weighed and scored for clinical signs. Scores were based on the following signs: 0 = normal; 1 = flaccid tail, piloerection, and/or weight loss; 2 = hind limb weakness causing righting difficulty; 3 = hind limb weakness causing righting inability; 4 = hind limb paresis, limp walking, incontinence; 5 = partial hind limb paralysis; 6 = total hind limb paralysis plus forelimb weakness; 7 = hind limb paralysis and forelimb paresis or paralysis; 8 = death or moribund requiring killing.

Western blotting

Animals for western blotting analyses were killed according to the following groups: control (no encephalitogen), preclinical (killed before any animal displayed clinical signs), scores 1-3 (animals that displayed a clinical score of 1, 2, or 3), and scores ≥4 (animals that displayed a clinical score of ≥4). Hindbrains (cerebellum, pons, and medulla) were removed, frozen on dry ice, and stored at -80°C. Tissues were homogenized in ice-cold homogenization buffer (10 mM Tris-HCl, pH 7.4, 1.5 mM EDTA, pH 8.0, 1 mMβ-mercaptoethanol, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 30 μM phenylmethylsulfonyl fluoride, and 400 mM NaCl). Microsomal fractions were obtained by centrifuging the homogenate at 20,000 g for 20 min at 4°C and the resultant supernatant at 105,000 g for 1 h at 4°C. Total microsomal protein was determine by the Lowry method (Sigma).

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed according to Laemmli (1970). Microsomal fractions (20 μg) were mixed with sample loading buffer [62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% (wt/vol) SDS, 640 mMβ-mercaptoethanol, and 0.01% (wt/vol) bromophenol blue] and heated at 95°C for 5 min. The samples were loaded onto a 12% gel in a Bio-Rad Mini-Protean 3 apparatus. Electrophoresis was performed in running buffer (pH 8.3; 25 mM Tris, 192 mM glycine, and 3.5 mM SDS) at 200 V at room temperature until the bromophenol blue dye reached the bottom of the gel.

Gels were allowed to equilibrate in ice-cold transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol) for 10 min. Transfer to polyvinylidene difluoride membrane (Millipore Corp., Bedford, MA, U.S.A.) was performed at 100 V for 1 h in a Bio-Rad Mini Trans-Blot apparatus.

Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS; 20 mM Tris-HCl, pH 7.4, 154 mM NaCl) for 1 h at room temperature. Primary antibody incubation for HO-1 or reductase at a 1:1,000 dilution in TBS was performed overnight at room temperature. Membranes were washed with 0.05% Tween 20-supplemented TBS (TBST). Secondary anti-body incubation was performed for 4 h using a goat anti-mouse IgG conjugated to alkaline phosphatase (Bio-Rad) at a 1:1,000 dilution in TBS at room temperature. Membranes were washed with TBST. The Immun-Star Chemiluminescent Protein Detection System (Bio-Rad) was used to develop the blots, with the signal captured on Hyperfilm ECL film (Amersham Life Science, Buckinghamshire, U.K.). Blots were quantified using a Personal Densitometer SI incorporated with ImageQuaNT version 4.1 software (Molecular Dynamics, Sunnyvale, CA, U.S.A.).


Mice were anesthetized with Metofane and perfused through the left cardiac ventricle with 4% paraformaldehyde in 100 mM potassium phosphate buffer (pH 7.4). Hindbrains were removed and immersion-fixed in the paraformaldehyde fixative for 24 h at room temperature. Hindbrains were embedded in paraffin and sectioned at a thickness of 4 or 8 μm. Immunohistochemical staining for HO-1 (1:1,000) was performed with a Dako Autostainer (Dako, Carpinteria, CA, U.S.A.) following the manufacturer's instructions. Controls included no primary antibody or preadsorbed primary antibody.


Microsomal samples from control or EAE animals were pooled according to treatment group and diluted to 500 μl with TBS (pH 7.5). Reductase antibody incubation was performed at a dilution of 1:100 with agitation for 3 h at room temperature. Protein A-agarose incubation was performed at a dilution of 1:50 with agitation for 2 h at room temperature. The pellets were collected by centrifugation at 1,000 g for 5 min at 4°C and washed four times with TBS (pH 7.5) with 1,000 g centrifugation. The final pellets were resuspended in 25 μl of electrophoresis sample buffer, heated for 5 min at 95°C, chilled on ice, centrifuged, and loaded onto 12% gels. SDS-PAGE and western blotting for HO-1 were performed as described above.

Statistical analysis

Western blots were assessed using one-way ANOVA and Dunnett's post hoc analysis. Orthogonal polynomial linear contrast (two way) was performed across the four groups to evaluate significant trends of protein expression.

HO-1 expression in EAE

  1. Top of page
  2. Abstract
  4. Induction of EAE
  6. HO-1 expression in EAE
  7. Reductase expression in EAE
  9. Acknowledgements

Mice with a clinical score of ≥4 displayed significantly increased levels of HO-1 protein in the hindbrain compared with controls. EAE mice with a score of 1-3 had levels that were between those of control mice and EAE mice with clinical scores of ≥4. Statistical analysis revealed a significant trend for HO-1 levels to increase with disease severity (Fig. 1).


Figure 1. HO-1 is increased in the hindbrain as EAE severity increases. A: Mice with a clinical score of ≥4 displayed significantly increased levels (∼10-fold) of HO-1. *p < 0.05 compared with controls (one-way ANOVA). Orthogonal polynomial linear contrast (two way) across the four conditions revealed a significant trend for HO-1 levels to increase with disease severity (p < 0.002). The numbers in parentheses indicate the number of mice evaluated. B: Representative HO-1 western blot. MW, molecular mass markers; Con, control; Pre, preclinical EAE; 1-3, EAE mice with a clinical score of 1, 2, or 3; ≥4, EAE mice with a clinical score of ≥4; HO-1, HO-1 standard.

Download figure to PowerPoint

HO-1 expression in twitcher mice

Twitcher mice have globoid cell leukodystrophy, a demyelinating disease caused by a mutation leading to defective production of galactosylceramidase (Sakai et al., 1996). This enzymatic defect results in the failure to digest galactosylceramide and psychosine, which leads to demyelination and accumulation of globoid cells in the CNS. Unlike in EAE, the blood-brain barrier remains relatively intact in the twitcher mouse (Kondo et al., 1987; Pedchenko and LeVine, 1999), and thus it was used to evaluate HO-1 expression in a model of demyelination without extravasation of red blood cells.

Western analysis revealed increased HO-1 levels in the hindbrain of twitcher mice compared with littermate controls (Fig. 2).


Figure 2. HO-1 is increased in the hindbrain of twitcher mice. HO-1 western blot displays increased HO-1 expression in the hindbrain of twitcher mice. Twitcher mice are the animal model for globoid cell leukodystrophy, another demyelinating disease where extravasation of blood is not characteristic. Con, control; Twi, twitcher; HO-1, HO-1 standard, a 28-amino acid truncated recombinant version of the native HO-1 of 289 amino acids.

Download figure to PowerPoint

HO-1 immunohistochemistry

HO-1 immunohistochemistry revealed extensive staining in the cerebellar white matter of EAE mice (Fig. 3B-D). Staining in the pons and medulla was in cells often associated with blood vessels Fig. 3E and F). Based on morphology, HO-1 staining is present in activated ameboid macrophages and reactive microglia as well as some astrocytes and Bergmann glia in the cerebellum (Fig. 3G-I). Sections from 10 of 12 mice that displayed clinical signs of EAE revealed HO-1 staining in the hindbrain. Examination of sections from six control and six preclinical mice revealed only background staining (Fig. 3A). Sections incubated without primary antibody or with a preadsorbed antibody had no or residual staining, respectively.


Figure 3. HO-1 immunohistochemistry. A: Cerebellum from control (no EAE) stained for HO-1. No staining is present in white or gray matter structures. B: Cerebellum from an affected EAE mouse. HO-1 staining is present in cells confined mostly to white matter tracks. C and D: Cerebellar white matter from an affected EAE mouse. HO-1 staining is present in round and/or process-bearing cells characteristic of ameboid macrophages. E-H: Pons staining in affected EAE mice. HO-1 staining is visible in cells with the morphology of ameboid macrophages, associated with vessels, and reactive microglia (E), reactive microglia and astrocytes (F), fibrous astrocytes (note the cellular process extends to a blood vessel) (G), and in a cell with a highly branched morphology characteristic of a protoplasmic astrocyte (H). I: Cerebellar cortex from an affected EAE mouse. HO-1 staining is present in Bergmann glia. Bar = 100 μm (A and B), 50 μm (C, D, E, and I), 40 μm (F), and 25 μm (G and H).

Download figure to PowerPoint

Reductase expression in EAE

  1. Top of page
  2. Abstract
  4. Induction of EAE
  6. HO-1 expression in EAE
  7. Reductase expression in EAE
  9. Acknowledgements

Mice with a clinical score of μ4 displayed decreased levels of reductase protein in the hindbrain compared with control mice. Orthogonal polynomial linear contrast (two way) across the four conditions revealed a significant trend for reductase levels to decrease with disease severity (Fig. 4). There was evidence that one data point was an outlier based on a studentized residual value of 3.1 (>2.0 suggests an outlier). This one data point was not included in the analysis.


Figure 4. Reductase levels are decreased in the hindbrain as EAE severity increases. A: Mice with a clinical score of ≥4 displayed decreased levels (∼50%) of reductase compared with controls. Orthogonal polynomial linear contrast (two way) across the four conditions revealed p < 0.03, which indicates a significant trend for reductase levels to decrease with disease severity. The numbers in parentheses indicate the number of mice evaluated. B: Representative reductase western blot. Con, control; Pre, preclinical EAE; 1-3, EAE mice with a clinical score of 1, 2, or 3; ≥4, EAE mice with a clinical score of ≥4; MW, molecular mass markers.

Download figure to PowerPoint

Association of reductase and HO-1

Hindbrain microsomal samples were examined by negative staining electron microscopy (Fig. 5A). Microsomes prepared from control or EAE animals had an average diameter of ∼10 nm. Hindbrain microsomal samples were immunoprecipitated with the reductase antibody and subsequently analyzed by western blotting using the HO-1 antibody. Blotting experiments revealed that HO-1 and reductase are associated with each other in the microsomal samples of EAE mice but not in microsomes of control mice (Fig. 5B).


Figure 5. Reductase associates with HO-1 during EAE. A: Electron micrograph of negatively stained microsomes prepared from an EAE animal is shown. Microsomes from control or EAE animals had an average diameter of ∼10 nm. Bar = 0.1 μm. The arrowheads indicate individual microsomes. B: Microsomes from control or EAE animals were pooled and immunoprecipitated with an antireductase polyclonal antibody. The membrane was stained with an anti-HO-1 polyclonal antibody. A band was present in the EAE sample but not the control sample. HO-1, HO-1 standard; Con, control; EAE, EAE mice with a clinical score of ≥4.

Download figure to PowerPoint


  1. Top of page
  2. Abstract
  4. Induction of EAE
  6. HO-1 expression in EAE
  7. Reductase expression in EAE
  9. Acknowledgements

A significant trend was revealed for HO-1 protein levels to increase in the hindbrain with increased EAE severity. The increased expression of HO-1 is located predominantly in white matter in cells associated with demyelination and inflammation, namely, ameboid macrophages, activated microglia, and some astrocytes. The occasional staining of Bergmann glial cells revealed that HO-1 induction can occur in gray matter structures during this demyelinating disease. Ten of 12 affected EAE mice exhibited HO-1 staining in the hindbrain. The two mice without staining likely had pathology in areas other than the hindbrain as EAE can result in lesions at all levels of the spinal cord and brain.

The increase in HO-1 in EAE mice is not due simply to induction by heme from extravasation of red blood cells. This is evidenced by the increased levels of HO-1 in the hindbrain of twitcher mice, a demyelinating disease that does not exhibit extravasation of blood (Kondo et al., 1987; Pedchenko and LeVine, 1999). A major stimulus for EAE-associated HO-1 induction likely is oxidative stress. Many response elements have been identified in the 5′-untranslated region of HO-1, including the antioxidant response element, which is activated by oxidative stress (Lavrovsky et al., 1994; Elbirt and Bonkovsky, 1999). Furthermore, induction of HO-1 expression also may be stimulated in part by cytokines, for example, IL-1α, IL-1β, IL-11, TNF-α (Fukuda and Sassa, 1993; Strandell et al., 1995; Terry et al., 1999), and/or prostaglandins (Koizumi et al., 1995; Rossi and Santoro, 1995). Both of these classes of molecules are released during EAE-associated inflammation.

A significant trend was revealed for reductase protein levels to decrease in the hindbrain with increased EAE severity. However, reductase was associated with HO-1 in the endoplasmic reticulum. Thus, the decrease in reductase levels may not be occurring in cells that also express HO-1. There are other examples of reductase levels and/or activity decreasing in various experimental paradigms. Acute stress resulted in a 21% decrease in reductase levels in rat Leydig cells (Kostic et al., 1998), and lipopolysaccharide limited the induction of reductase by phenobarbital by ∼20% (Sewerynek et al., 1995). Furthermore, glutathione and glutathione disulfide inhibit reductase activity, which may be a mechanism by which these molecules inhibit NADPH-dependent lipid peroxidation via reductase (Scholtz et al., 1996). Moreover, lower reductase levels result in greater resistance to doxorubicin free radical-induced toxicity (Singh et al., 1990). The present investigation showing lower levels of reductase in EAE mice represents one of the first studies to examine the levels of this enzyme in a pathological state of the brain. Lower reductase levels also were observed in animals with cuprizone-induced demyelination (M. R. Emerson and S. M. LeVine, unpublished observations). A decrease in reductase may be a defense mechanism used to limit cellular damage during disease states.

Association of reductase with HO-1 is required for HO-1 function (Maines, 1988; Elbirt and Bonkovsky, 1999). Both enzymes are located in the endoplasmic reticulum membrane, and this preferential association is present in microsomal samples from affected EAE mice but not in control mice. There is a functional cytochrome P450 system in the brain that also requires reductase for enzymatic function (Ravindranath, 1998). Cytochrome P450 isozymes are present in neurons, astrocytes, microglia, and infiltrating macrophages (Ravindranath, 1998; Nicholson and Renton, 1999; Hodges et al., 2000). Oligodendrocytes contain cytochrome P450 17α-hydroxylase and cytochrome P450 side chain cleavage (Zwain and Yen, 1999; Brown et al., 2000); however, the presence of other P450 isozymes is unknown. Whether the expression of reductase in these cell types responds to various stimuli in the same manner in which its expression is modulated in the liver and other sites remains to be determined.

Another aspect of this microsomal system that could stoichiometrically favor the association of reductase with HO-1 would be a decrease in cytochrome P450 isozyme levels. Down-regulation of cytochrome P450s has been shown in response to the pro-inflammatory cytokines TNF-α, IFN-γ, and IL-1 (Nicholson and Renton, 1999), which are released by Th1 cells in EAE (Reder et al., 1994; Jung et al., 1997; Bourrie et al., 1999). Rats treated with metals display reduced levels of the heme-containing cytochrome P450s in liver as well as an induction of HO-1 (Maines and Kappas, 1977). Furthermore, an increase in HO-1 levels is associated with decreased cytochrome P450s in rat cardiac myocytes treated with antimony (Snawder et al., 1999). HO-1 could also directly attenuate the synthesis of cytochrome P450s by metabolizing heme and thus limiting the amount available for incorporation (Snawder et al., 1999). Lower cytochrome P450 levels together with enhanced HO-1/reductase association would favor an antioxidant state.

HO-1 is a stress-related protein, and altered expression is a marker of stress response activation (Massa et al., 1996; Elbirt and Bonkovsky, 1999). In EAE, the stress response may be an endogenous mechanism attempting to minimize demyelination and limit free radical-mediated damage to oligodendrocytes. Thus, by increasing HO-1 function, a protective pathway may be activated. This could occur by HO-1 together with reductase producing the antioxidant biliverdin and its subsequent reduction to bilirubin by biliverdin reductase. Furthermore, the release of CO in place of O2•- (Elbirt and Bonkovsky, 1999), the latter produced by reductase with cytochrome P450 isozymes, would cause a shift toward activation of a protective pathway and inactivation of a free radical-producing pathway that has the potential to promote pathogenesis (Fig. 6).


Figure 6. Proposed mechanisms of HO-1 and reductase. A: Pro-oxidant pathway. The association of reductase with cytochrome P450 isozymes in the endoplasmic reticulum results in the production of O2•-. The O2•- liberated can react with NO to produce ONOO- or reduce ferric iron to ferrous iron that can react with H2O2 to produce •OH, both highly oxidative species. ROS and RNS are implicated in EAE/MS pathogenesis. B: Protective pathway. The association of reductase with HO-1 in the endoplasmic reticulum results in the production of CO, instead of O2•- through the interaction of reductase with cytochrome P450 isozymes. Furthermore, the pro-oxidant heme is cleaved to form the antioxidant biliverdin. The iron released stimulates translation of ferritin to bind and store liberated iron. Thus, this pathway contributes to a more reduced cellular redox state. HO-1, heme oxygenase-1; Red, NADPH cytochrome P450 reductase; cP450, cytochrome P450 isozymes; X, substrate for cytochrome P450 isozymes.

Download figure to PowerPoint

These studies provide insight into the role of HO-1 and reductase in the stress response under demyelinating and inflammatory conditions. A decreased level of reductase and its favored association with HO-1 is a newly described constituent of a potential protective pathway that could have an impact in several neurological disorders.


  1. Top of page
  2. Abstract
  4. Induction of EAE
  6. HO-1 expression in EAE
  7. Reductase expression in EAE
  9. Acknowledgements

We thank Julie Collins and Matt Oswald for help with the HO-1immunohistochemistry studies, Don Warn and Patrick Moonasar for assistancewith the preparation of the figures, and Dr. Dennis Wallace for help withstatistical analyses. This work was supported by NIH (NS 33596) and an NICHDMental Retardation Research Center grant (HD 02528).

  • 1
    Balla G., Jacob H.S., Balla J., Rosenberg M., Nath K., Apple F., Eaton J.W., Vercellotti G.M. (1992) Ferritin: a cytoprotective antioxidant stratagem of endothelium.J. Biol. Chem. 2671814818153.
  • 2
    Bergh A.F. & Strobel H.W. (1992) Reconstitution of the brain mixed function oxidase system: purification of NADPH-cytochrome P450 reductase and partial purification of cytochrome P450 from whole rat brain.J. Neurochem. 59575581.
  • 3
    Bourrie B., Bribes E., Esclangon M., Garcia L., Marchand J., Thomas C., Maffrand J.P., Casellas P. (1999) The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood-brain barrier disruption: implications for multiple sclerosis treatment.Proc. Natl. Acad. Sci. USA 961285512859.
  • 4
    Brown R.C., Cascio C., Papadopoulos V. (2000) Pathways of neurosteroid biosynthesis in cell lines from human brain: regulation of dehydroepiandrosterone formation by oxidative stress and beta-amyloid peptide.J. Neurochem. 74847859.
  • 5
    Cross A.H., Keeling R.M., Goorha S., San M., Rodi C., Wyatt P.S., Manning P.T., Misko T.P. (1996) Inducible nitric oxide synthase gene expression and enzyme activity correlate with disease activity in murine experimental autoimmune encephalomyelitis.J. Neuroimmunol. 71145153.
  • 6
    Cross A.H., Manning P.T., Stern M.K., Misko T.P. (1997) Evidence for the production of peroxynitrite in inflammatory CNS demyelination.J. Neuroimmunol. 80121130.
  • 7
    Dalton T., Pazdernik T.L., Wagner J., Samson F., Andrews G.K. (1995) Temporal spatial patterns of expression of metallothionein-I and -III and other stress related genes in rat brain after kainic acid-induced seizures.Neurochem. Int. 275971.
  • 8
    Deininger M.H. & Schluesener H.J. (1999) Cyclooxygenases-1 and -2 are differentially located to microglia and endothelium in rat EAE and glioma.J. Neuroimmunol. 95202208.
  • 9
    Doré S., Takahashi M., Ferris C.D., Hester L.D., Guastella D., Snyder S.H. (1999) Bilirubin, formed by activation of heme oxygenase-2, protects neurons against oxidative stress injury.Proc. Natl. Acad. Sci. USA 9624452450.
  • 10
    Dwyer B.E., Lu S.Y., Laitinen J.T., Nishimura R.N. (1998) Protective properties of tin- and manganese-centered porphyrins against hydrogen peroxide-mediated injury in rat astroglial cells.J. Neurochem. 7124972504.
  • 11
    Eisenstein R.S., Garcia-Mayol D., Pettingell W., Munro H.N. (1991) Regulation of ferritin and heme oxygenase synthesis in rat fibroblasts by different forms of iron.Proc. Natl. Acad. Sci. USA 88688692.
  • 12
    Elbirt K.K. & Bonkovsky H.L. (1999) Heme oxygenase: recent advances in understanding its regulation and role.Proc. Assoc. Am. Phys. 111438447.
  • 13
    Ewing J.F. & Maines M.D. (1991) Rapid induction of heme oxygenase 1 mRNA and protein by hyperthermia in rat brain: heme oxygenase 2 is not a heat shock protein.Proc. Natl. Acad. Sci. USA 8853645368.
  • 14
    Ewing J.F. & Maines M.D. (1993) Glutathione depletion induces heme oxygenase-1 (HSP32) mRNA and protein in rat brain.J. Neurochem. 6015121519.
  • 15
    Ewing J.F., Haber S.N., Maines M.D. (1992) Normal and heat-induced patterns of expression of heme oxygenase-1 (HSP32) in rat brain: hyperthermia causes rapid induction of mRNA and protein.J. Neurochem. 5811401149.
  • 16
    Forge J.K., Pedchenko T.V., LeVine S.M. (1998) Iron deposits in the central nervous system of SJL mice with experimental allergic encephalomyelitis.Life Sci. 6322712284.
  • 17
    Fukuda Y. & Sassa S. (1993) Effect of interleukin-11 on the levels of mRNAs mRNAs encoding heme oxygenase and haptoglobin in human HepG2 hepatoma cells.Biochem. Biophys. Res. Commun. 193297302.
  • 18
    Hansen L.A. & Pender M.P. (1989) Hypothermia due to an ascending impairment of shivering in hyperacute experimental allergic encephalomyelitis in the Lewis rat.J. Neurol. Sci. 94231240.
  • 19
    Hewett S.J., Misko T.P., Keeling R.M., Behrens M.M., Choi D.W., Cross A.H. (1996) Murine encephalitogenic lymphoid cells induce nitric oxide synthase in primary astrocytes.J. Neuroimmunol. 64201208.
  • 20
    Hodges V.M., Molloy G.Y., Wickramasinghe S.N. (2000) Demonstration of mRNA for five species of cytochrome P450 in human bone marrow, bone marrow-derived macrophages and human haemopoietic cell lines.Br. J. Haematol. 108151156.
  • 21
    Jung S., Donhauser T., Toyka K.V., Hartung H.P. (1997) Propentofylline and iloprost suppress the production of TNF-α by macrophages but fail to ameliorate experimental autoimmune encephalomyelitis in Lewis rats.J. Autoimmun. 10519529.
  • 22
    Kappus H. (1993) Metabolic reactions: role of cytochrome P-450 in the formation of reactive oxygen species, inCytochrome P450 (Schenkman J. B. and Grein H., eds), pp. 145154. Springer-Verlag, Berlin.
  • 23
    Koizumi T., Odani N., Okuyama T., Ichikawa A., Negishi M. (1995) Identification of a cis-regulatory element for ▵12-prostaglandin J2-induced expression of the rat heme oxygenase gene. J. Biol. Chem. 2702177921784.
  • 24
    Kondo A., Nakano T., Suzuki K. (1987) Blood-brain barrier permeability to horseradish peroxidase in twitcher and cuprizone intoxicated mice.Brain Res. 425186190.
  • 25
    Kostic T., Andric S., Maric D., Kovacevic R. (1998) The effect of acute stress and opioid antagonist on the activity of NADPH-P450 reductase in rat Leydig cells.J. Steroid Biochem. Mol. Biol. 665154.
  • 26
    Laemmli U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227680685.
  • 27
    Lavrovsky Y., Schwartzman M.L., Levere R.D., Kappas A., Abraham N.G. (1994) Identification of binding sites for transcription factors NF-χB and AP-2 in the promoter region of the human heme oxygenase 1 gene. Proc. Natl. Acad. Sci. USA 9159875991.
  • 28
    Llesuy S.F. & Tomaro M.L. (1994) Heme oxygenase and oxidative stress. Evidence of involvement of bilirubin as physiological protector against oxidative damage.Biochim. Biophys. Acta 1223914.
  • 29
    Maines M.D. (1988) Heme oxygenase: function, multiplicity, regulatory mechanisms, and clinical applications.FASEB J. 225572568.
  • 30
    Maines M.D. (1997) The heme oxygenase system: a regulator of second messenger gases.Annu. Rev. Pharmacol. Toxicol. 37517554.
  • 31
    Maines M.D. & Kappas A. (1977) Metals as regulators of heme metabolism.Science 19812151221.
  • 32
    Maines M.D., Trakshel G.M., Kutty R.K. (1986) Characterization of two constitutive forms of rat liver microsomal heme oxygenase. Only one molecular species of the enzyme is inducible.J. Biol. Chem. 261411419.
  • 33
    Massa S.M., Swanson R.A., Sharp F.R. (1996) The stress gene response in brain.Cerebrovasc. Brain Metab. Rev. 895158.
  • 34
    Misko T.P., Trotter J.L., Cross A.H. (1995) Mediation of inflammation by encephalitogenic cells: interferon-γ induction of nitric oxide synthase and cyclooxygenase 2.J. Neuroimmunol. 61195204.
  • 35
    Nicholson T.E. & Renton K.W. (1999) Modulation of cytochrome P450 by inflammation in astrocytes.Brain Res. 8271218.
  • 36
    Panahian N., Yoshiura M., Maines M.D. (1999) Overexpression of heme oxygenase-1 is neuroprotective in a model of permanent middle cerebral artery occlusion in transgenic mice.J. Neurochem. 7211871203.
  • 37
    Pedchenko T.V. & LeVine S.M. (1998) Desferrioxamine suppresses MBP induced experimental allergic encephalomyelitis in SJL mice.J. Neuroimmunol. 84188197.
  • 38
    Pedchenko T.V. & LeVine S.M. (1999) IL-6 deficiency causes enhanced pathology in twitcher (globoid cell leukodystrophy) mice.Exp. Neurol. 158459468.
  • 39
    Poss K.D. & Tonegawa S. (1997a) Heme oxygenase I is required for mammalian iron reutilization.Proc. Natl. Acad. Sci. USA 941091910924.
  • 40
    Poss K.D. & Tonegawa S. (1997b) Reduced stress defense in heme oxygenase 1-deficient cells.Proc. Natl. Acad. Sci. USA 941092510930.
  • 41
    Prineas J.W. & McDonald W.I. (1997) Demyelinating diseases, inGreenfield's Neuropathology, Vol. 1 (Graham D. I. and Lantos P. L., eds), pp. 813896. Oxford University Press, New York.
  • 42
    Raine C.S. (1984) Biology of disease. The analysis of autoimmune demyelination: its impact on multiple sclerosis.Lab. Invest. 50608635.
  • 43
    Raine C.S., Barnett L.B., Brown A., Behar T., McFarlin D.E. (1980) Neuropathology of experimental allergic encephalomyelitis in inbred strains of mice.Lab. Invest. 43150157.
  • 44
    Raine C.S., Cannella B., Hauser S.L., Genain C.P. (1999) Demyelination in primate autoimmune encephalomyelitis and acute multiple sclerosis lesions: a case for antigen-specific antibody mediation.Ann. Neurol. 46144160.
  • 45
    Ravindranath V. (1998) Metabolism of xenobiotics in the central nervous system: implications and challenges.Biochem. Pharmacol. 56547551.
  • 46
    Reder A.T., Thapar M., Sapugay A.M., Jensen M.A. (1994) Prostaglandins and inhibitors of arachidonate metabolism suppress experimental allergic encephalomyelitis.J. Neuroimmunol. 54117127.
  • 47
    Rossi A. & Santoro M.G. (1995) Induction by prostaglandin A1 of haem oxygenase in myoblastic cells: an effect independent of expression of the 70 kDa heat shock protein.Biochem. J. 308455463.
  • 48
    Sahrbacher U.C., Lechner F., Eugster H.P., Frei K., Lassmann H., Fontana A. (1998) Mice with an inactivation of the inducible nitric oxide synthase gene are susceptible to experimental autoimmune encephalomyelitis.Eur. J. Immunol. 2813321338.
  • 49
    Sakai N., Inui K., Tatsumi N., Fukushima H., Nishigaki T., Taniike M., Nishimoto J., Tsukamoto H., Yanagihara I., Ozono K., Okada S. (1996) Molecular cloning and expression of cDNA for murine galactocerebrosidase and mutation analysis of the twitcher mouse, a model of Krabbe's disease.J. Neurochem. 6611181124.
  • 50
    Schipper H.M., Bernier L., Mehindate K., Frankel D. (1999) Mitochondrial iron sequestration in dopamine-challenged astroglia: role of heme oxygenase-1 and the permeability transition pore.J. Neurochem. 7218021811.
    Scholz R.W., Reddy P.V., Liken A.D., and Reddy C.C. (1996) Inhibition of rat liver microsomal NADPh cytochrome P450 reductase by glutathione and glutathione disulfide.Biochem. Biophys. Res. Commun. 226475480.
  • 52
    Sewerynek E., Abe M., Reiter R.J., Barlow-Walden L.R., Chen L., McCabe T.J., Roman L.J., Diaz-Lopez B. (1995) Melatonin administration prevents lipopolysaccharide-induced oxidative damage in phenobarbital-treated animals.J. Cell. Biochem. 58436444.
  • 53
    Singh S.V., Iqbal J., Krishnan A. (1990) Cytochrome P450 reductase, antioxidant enzymes and cellular resistance to doxorubicin.Biochem. Pharmacol. 40385387.
  • 54
    Snawder J.E., Tirmenstein M.A., Mathias P.I., Toraason M. (1999) Induction of stress proteins in rat cardiac myocytes by antimony.Toxicol. Appl. Pharmacol. 1599197.
  • 55
    Stocker R., Yamamoto Y., McDonagh A.F., Glazer A.N., Ames B.N. (1987) Bilirubin is an antioxidant of possible physiological importance.Science 23510431046.
  • 56
    Strandell E., Buschard K., Saldeen J., Welsh N. (1995) Interleukin-1 beta induces the expression of hsp70, heme oxygenase and Mn-SOD in FACS-purified rat islet beta-cells, but not in alpha-cells.Immunol. Lett. 48145148.
  • 57
    Tenhunen R., Marver H.S., Schmid R. (1968) The enzymatic conversion of heme to bilirubin by microsomal heme oxygenase.Proc. Natl. Acad. Sci. USA 61748755.
  • 58
    Terry C.M., Clikeman J.A., Hoidal J.R., Callahan K.S. (1999) TNF-α and IL-1α induce heme oxygenase-1 via protein kinase C, Ca2+, and phospholipase A2 in endothelial cells.Am. J. Physiol. 276H1493H1501.
  • 59
    Tran E.H., Hardin-Pouzet H., Verge G., Owens T. (1997) Astrocytes and microglia express inducible nitric oxide synthase in mice with experimental allergic encephalomyelitis.J. Neuroimmunol. 74121129.
  • 60
    Vile G.F. & Tyrrell R.M. (1993) Oxidative stress resulting from ultraviolet A irradiation of human skin fibroblasts leads to a heme oxygenase-dependent increase in ferritin.J. Biol. Chem. 2681467814681.
  • 61
    Vile G.F., Basu-Modak S., Waltner C., Tyrrell R.M. (1994) Heme oxygenase-1 mediates an adaptive response to oxidative stress in human skin fibroblasts.Proc. Natl. Acad. Sci. USA 9126072610.
  • 62
    Yasukochi Y. & Masters B.S. (1976) Some properties of a detergent-solubilized NADPH-cytochrome c (cytochrome P-450) reductase purified by biospecific affinity chromatography.J. Biol. Chem. 25153375344.
  • 63
    Zakhary R., Poss K.D., Jaffrey S.R., Ferris C.D., Tonegawa S., Snyder S.H. (1997) Targeted gene deletion of heme oxygenase 2 reveals neural role for carbon monoxide.Proc. Natl. Acad. Sci. USA 941484814853.
  • 64
    Zwain I.H. & Yen S.S. (1999) Neurosteroidogenesis in astrocytes, oligodendrocytes, and neurons of cerebral cortex of rat brain.Endocrinology 14038433852.