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Interleukin-10 (IL-10) is a pleiotropic cytokine with a wide spec-trum of biological effects on lymphoid and myeloid cells1,2. Oneof the known functions of IL-10 is its ability to inhibit the pro-duction of pro-inflammatory mediators, including tumor necro-sis factor-α (TNF-α), IL -6, IL-1, granulocyte-macrophage colonystimulating factor, and the generation of nitric oxide (NO) bylipopolysaccharide (LPS)-activated monocytes/macrophages.However, the molecular mechanisms underlying the anti-in-flammatory effects of this cytokine remain unknown. Earlierstudies on IL-10 receptor-signaling pathways have revealed thatthe JAK1/STAT3-dependent pathway is not sufficient for theanti-inflammatory action3–6. Although IL-10 activates PI-3-ki-nase and p70 S6 kinase, these pathways are also not required forthe anti-inflammatory effects of the cytokine7. Another elusivepoint is that the inhibitory activity of IL-10 on LPS-induced in-flammation can be blocked by the protein synthesis-inhibitorcycloheximide, suggesting the involvement of newly synthe-sized protein(s) in its effects8,9.

Heme oxygenase (HO) is the rate-limiting enzyme in heme ca-tabolism, which leads to the generation of biliverdin, free ironand carbon monoxide (CO)10–12. Three mammalian HO isoformshave been identified, one of which, HO-1, is a stress-responsiveprotein induced by various oxidative agents. Over the pastdecade, HO-1 has been implicated in the cytoprotective defenseresponse against oxidative injury12. In addition to the antioxi-dant activities of biliverdin and its metabolite, bilirubin, therehas been increasing interest in the potential effects of endoge-nous CO, which, like NO, activates the cGMP pathway and elic-its neurotransmission in the central nervous system andvasodilation in the vascular system10. Otterbein et al.13 haveshown that CO inhibits the expression of LPS-induced pro-in-flammatory cytokines and increases LPS-induced expression ofIL-10 in macrophages, suggesting that it is involved in the anti-inflammatory action of HO-1. However, more work is required

to delineate the role of HO-1 in inflammation. Here we exploredthe potential interplay between IL-10 and HO-1 in the inhibi-tion of LPS-induced inflammatory responses. We found IL-10 tobe a potent inducer of HO-1 in mouse primary macrophages andthe J774 cell line, with induction of HO-1 occurring as early asthree hours after IL-10 treatment. Cotreatment with a HO in-hibitor or a CO scavenger significantly suppressed the inhibitoryeffects of IL-10 on LPS-induced TNF-α and NO production, aswell as the expression of matrix metalloproteinase-9 (MMP-9) inmacrophages. HO-1 induction was also seen in mice receivingIL-10 administration. Furthermore, IL-10-mediated protectionagainst LPS-induced septic shock in mice was decreased bycotreatment with a HO inhibitor, supporting the role of HO-1 inthe anti-inflammatory action of IL-10 in vivo.

Induction of HO-1 expression by IL-10 in murine macrophagesTo examine whether IL-10 affects HO-1 gene expression, wetreated the murine macrophage cell line, J774, for 24 hours withvarious concentrations of IL-10. Western-blot analysis revealeddose-dependent induction of HO-1 by IL-10 (Fig. 1a).Concentrations of IL-10 as low as 10 ng/ml were capable of in-ducing maximal expression of HO-1 in this cell line. Using 10ng/ml of IL-10, induction was evident as early as 3 hours, andreached a maximum after 24 hours of treatment with IL-10 (Fig.1b). IL-6, which induces STAT-3 phosphorylation of IL-10 (ref.14), did not induce HO-1 expression at concentrations up to 40ng/ml (Fig. 1c). The induction of HO-1 by IL-10 was also demon-strated in primary peritoneal macrophages isolated from BALB/cmice (Fig. 1d).

p38 activation in IL-10-induced HO-1 gene expression Northern-blot analysis showed that IL-10 (10 ng/ml) treatment ofJ774 cells for 12 hours resulted in an increase in HO-1 mRNA,which was completely blocked by the transcription inhibitor,

Heme oxygenase-1 mediates the anti-inflammatory effect ofinterleukin-10 in mice

TZONG-SHYUAN LEE & LEE-YOUNG CHAU

Graduate Institute of Life Sciences, National Defense Medical Center, and Institute of Biomedical Sciences,Academia Sinica, Taipei, Taiwan, Republic of China

Correspondence should be addressed to L.-Y.C.; email: lyc@mail.ibms.sinica.edu.tw

The mechanisms underlying the action of the potent anti-inflammatory interleukin-10 (IL-10) arepoorly understood. Here we show that, in murine macrophages, IL-10 induces expression ofheme oxygenase-1 (HO-1), a stress-inducible protein with potential anti-inflammatory effect, viaa p38 mitogen-activated protein kinase-dependent pathway. Inhibition of HO-1 protein synthesisor activity significantly reversed the inhibitory effect of IL-10 on production of tumor necrosis fac-tor-α induced by lipopolysaccharide (LPS). Additional experiments revealed the involvement ofcarbon monoxide, one of the products of HO-1-mediated heme degradation, in the anti-inflam-matory effect of IL-10 in vitro. Induction of HO-1 by IL-10 was also evident in vivo. IL-10-mediatedprotection against LPS-induced septic shock in mice was significantly attenuated by cotreatmentwith the HO inhibitor, zinc protoporphyrin. The identification of HO-1 as a downstream effectorof IL-10 provides new possibilities for improved therapeutic approaches for treating inflamma-tory diseases.

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actinomycin D (5 µg/ml) (Fig. 2a). This finding sug-gests that the induction of HO-1 resulted primarilyfrom transcriptional activation. Cyclohexamide (2 µg/ml), which completely blocks the expressionof A8 gene induced by the combination of LPS (1 µg/ml) and IL-10 (10 ng/ml)15, did not have signif-icant effects on IL-10-induced HO-1 gene expression(Fig. 2a). This indicates that the induction of theHO-1 gene did not require new protein synthesis.

Recently, studies on HO-1 induction by stressstimuli have shown that pathways involving mito-gen-activated protein kinases (MAPKs)16 are respon-sible for the transduction of signals to initiate geneactivation17–19. To determine whether a similar signalmechanism is responsible for the upregulation ofHO-1 gene expression by IL-10 in macrophages, weexamined the activation states of three MAPK sub-families, ERK, JNK and p38, in J774 cells. Treatmentwith IL-10 (10 ng/ml) resulted in rapid phosphoryla-tion of p38, but not of ERK or JNK (Fig. 2b). p38phosphorylation reached a peak at 5 minutes, thendeclined to baseline within 15 minutes. Similar re-sults were observed in primary macrophages uponIL-10 stimulation (Fig. 2b). To exclude the possibilitythat IL-10 used in our experiments was contami-nated with LPS, which has been shown to activatep38 as well as ERK and JNK (ref. 20), we carried out the same ex-periment using an anti-IL-10 neutralizing antibody. Western-blotanalysis showed that the IL-10-induced p38 phosphorylation at 5min was completely abolished by a 30 min preincubation at 37°Cof IL-10 (10 ng/ml) with anti-IL-10 neutralizing antibody (1µg/ml), but not with control IgG (1 µg/ml) (Fig. 2c). This result in-dicates that p38 activation is specifically induced by the cytokine.The IL-10-mediated increase in HO-1 mRNA level was completely

blocked by SB203580, a specific inhibitor of p38, whereas similarconcentrations of PD98059, a specific inhibitor of ERK, had nosignificant effect (Fig. 2d). The inhibitory effect of SB203580 (10µM) on HO-1 protein induction was also observed early (3 h) afterIL-10 treatment in J774 cells (Fig. 2e). In contrast, SB203580 at thesame concentration did not affect the expression of suppressor ofcytokine signaling-3 (SOCS-3), a known IL-10-inducible gene4, at 4hours after cytokine stimulation (Fig. 2f).

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Fig. 1 IL-10 induces HO-1 protein expression in murine macrophages. a and b, Westernblots of HO-1 and β-actin in J774 cells incubated (a) with the indicated concentrations ofIL-10 and (b) with IL-10 for the indicated times. c, J774 cells were incubated with the indi-cated concentrations of IL-6 or IL-10, and the extent of STAT-3 phosphorylation (20 min)and HO-1 induction (24 h) after cytokine stimulation were determined by Western blotanalysis. d, Protein expression of HO-1 and β-actin in mouse peritoneal macrophages incu-bated with the indicated concentrations of IL-10 or IL-6 for 24 h.

Fig. 2 Involvement of p38 activation in IL-10-induced HO-1gene expression. a, Northern blot showing HO-1 mRNA fromJ774 cells incubated with or without IL-10 in the absence orpresence of actinomycin D (Act. D). The effect of cycloheximide(CHX) on the expression of HO-1 mRNA induced by IL-10, orA8 mRNA induced by LPS plus IL-10, was also examined. b, Western blots showing the extent of phosphorylation of ERK,JNK, and p38 MAPKs at the indicated times after IL-10 stimula-tion of J774 cells or primary peritoneal macrophages. c, p38phosphorylation in J774 cells incubated with IL-10 alone, orwith IL-10 pretreated with IL-10 neutralizing antibody (IL-10Ab) or control IgG. d, HO-1 mRNA expression in the J744 cellsincubated with IL-10 in the absence or presence of indicatedconcentrations of SB203580 or PD98059, then HO-1 mRNAexpression measured. e, HO-1 protein expression in J774 cellsincubated with IL-10 in the absence or presence of SB203580for the indicated times. f, Effect of SB203580 on SOCS-3 pro-tein expression in J774 cells incubated with IL-10.

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HO-1 mediates suppression of LPS-activated TNF-αPretreatment of J774 cells with IL-10 significantly suppressedLPS-induced TNF-α production (Fig. 3a). To determine whetherHO-1 mediates the inhibitory effect of IL-10, we transfected cellswith antisense oligodeoxynucleotides (ODN) complementary toHO-1 mRNA prior to their sequential treatment with IL-10 andLPS. Western-blot analysis revealed that IL-10-induced HO-1protein expression was substantially reduced by treatment withantisense ODN, but not by sense or scrambled ODN (Fig. 3a). Insitu HO-1 immunostaining on transfected cells revealed that theHO-1 expression in 80% of the cells was completely abolished byantisense ODN treatment (data not shown). In parallel, IL-10-mediated suppression of LPS-induced TNF-α production was alsosignificantly attenuated by antisense ODN treatment (Fig. 3a).

To ensure that the inhibitory effect of HO-1 antisense ODN isnot mediated through the induction of interferon and other cel-lular genes caused by the double-stranded RNA formed21, we per-formed additional experiments with antisense ODN to SOCS-3mRNA and found that SOCS-3 antisense ODN abolished IL-10-induced SOCS-3 expression without affecting the inhibitory ef-fect of IL-10 on LPS-induced TNF-α production (Fig. 3b). Thisresult indicates that the ablation of HO-1 is responsible for thesuppression of IL-10 effect by the HO-1 antisense ODN.

The involvement of HO-1 in the anti-inflammatory action ofIL-10 was also confirmed using a specific HO competitive in-hibitor, zinc protoporphyrin IX (ZnPP), which at 1 µM signifi-cantly blocked IL-10-mediated inhibition of LPS-induced TNF-αproduction (Fig. 3c). In contrast, copper protoporphyrin IX

(CuPP) (1 µM), which does not inhibit HO (ref. 22),was not effective. To determine whether CO or ironreleased from heme degradation by HO-1 was respon-sible for the action of IL-10, we examined the effectsof hemoglobin (a scavenger of CO) and desferrioxam-ine (an iron chelator) on the IL-10-mediated inhibi-tion of LPS-induced TNF-α production. We found

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Fig. 3 HO-1 mediates IL-10-induced suppression of TNF-α production inLPS-activated J774 cells. a, Cells transfected with antisense, sense or scrambledODN were left untreated (�) or treated (�) with IL-10 prior to LPS challenge.HO-1 and β-actin protein expression was examined by western blot (upperpanel) and the amounts of TNF-α released into culture medium assayed byELISA (lower panel). *, P < 0.005 compared with control cells treated with IL-10/LPS. b, Cells transfected without (�) or with (�) SOCS-3 antisense ODNwere treated with IL-10 and LPS. The SOCS-3 protein expression and TNF-αproduction were then determined. c–e, Cells treated with IL-10 (10 ng/ml, 4 h), followed by LPS (1 µg/ml, 2 h) in the absence or presence of ZnPP, CuPP,hemoglobin (Hb) or desferrioxamine (Def.). TNF-α released into culture

medium was determined by ELISA. *, P < 0.005 compared with cells treatedwith IL-10/LPS. f, Cells treated with LPS in the presence or absence of bilirubin.Bilirubin had no effect on LPS-induced TNF-α production.

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Fig. 4 HO-1 inhibition reverses IL-10-induced suppression ofTNF-α production in LPS-activated primary macrophages. a, Primary peritoneal macrophages were transfected with HO-1 antisense, sense or scrambled ODN, followed by stimulationwithout (�) or with (�) IL-10 prior to LPS challenge. HO-1 andβ-actin protein expression and TNF-α release are shown. *, P <0.005 compared with control cells treated with IL-10/LPS. b, Cells were treated with or without IL-10 (10 ng/ml, 4 h), fol-lowed by LPS (1 µg/ml, 2 h) in the absence or presence ofZnPP (1 µM). *, P < 0.025 compared with cells treated with IL-10/LPS.

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that hemoglobin, at concentrations ranging from 2.5 µM to 10 µM, effectively and in a dose-dependent manner reversedthe effect of IL-10 (Fig. 3d), whereas desferrioxamine (50–200µM) had no significant effect (Fig. 3e). To determine whetherbilirubin, the other degradation product of heme, had anti-in-flammatory activity, we treated cells with LPS in the presence orabsence of bilirubin (2.5–10 µM) and found that bilirubin didnot significantly inhibit LPS-induced TNF-α production (Fig.3f). These results suggest that CO, derived from heme degrada-tion, mediates the inhibitory effect of IL-10 on TNF-α produc-tion. Using HO-1 antisense ODN and ZnPP, we alsodemonstrated that HO-1 is involved in IL-10-mediated suppres-sion of LPS-induced TNF-α production in primary macrophages(Fig. 4a and b).

HO-1 mediates suppression of NO and MMP-9LPS-induced expression of inducible nitric oxide synthase(INOS) and production of NO provide important cytotoxicfunction in macrophages23. IL-10 inhibited the induction ofINOS expression by LPS in J774 cells and primarymacrophages, and ZnPP (1 µM) and hemoglobin (10 µM),which reversed the inhibition of TNF-α production by IL-10,markedly suppressed the IL-10-mediated inhibition of INOSexpression and NO production, whereas desferrioxamine (200µM) had no effect (Fig. 5a and b). Bilirubin (10 µM), however,did not show any significant effect on LPS-induced INOS ex-pression and NO production. These results suggest that HO-1and CO mediate the inhibitory effect of IL-10 on LPS-inducedINOS expression.

We next determined whether HO-1 plays a role in the sup-pressive effect of IL-10 on the expression of MMPs (refs. 24,25),which are important in the degradation and remodeling of theextracellular matrix at sites of inflammation26. We examinedthe constitutive expression of MMPs in J774 cells and foundthat IL-10 inhibited MMP-9 expression in these cells, as shownby both zymography and western blotting (Fig. 5c). This effectwas again significantly attenuated by ZnPP and hemoglobin.The expression of MMP-9 in untreated primary macrophageswas undetected but upregulated by LPS. Similar to that observedin J774 cells, MMP-9 expression as assessed by zymography inLPS-activated primary macrophages was markedly inhibited byIL-10 (Fig. 5d). ZnPP and hemoglobin again significantly re-versed the effect of IL-10, supporting the involvement of HO-1in this process.

Induction of HO-1 by IL-10 in vivoWe injected BALB/c mice intraperitoneally with IL-10 and mea-sured HO-1 expression in various tissues and cells. A time-depen-dent increase was observed in peritoneal macrophages, liver andspleen, whereas no significant change was observed in circulat-ing monocytes, lung or kidney up to 24 hours after IL-10 treat-ment (Fig. 6a and b). To examine whether the unresponsivenessof monocytes resulted from improper stimulation, we treatedthe fresh monocytes isolated from mouse peripheral blood withIL-10 for 24 hours in culture and found that the HO-1 was notinduced under this condition. However, the expression of HO-1was induced by IL-10 in macrophage-like cells derived from theperipheral monocytes and maintained in culture for one dayprior to IL-10 treatment (data not shown). These observationssuggest that macrophages respond more profoundly than mono-cytes to IL-10-induced HO-1 gene expression. When mice re-ceived a LPS challenge 3 hours after IL-10 treatment, the survivalrate was markedly increased as compared with that of mice re-ceiving LPS alone (80% versus 13% survival; P < 0.001) (Fig. 6c).

To further elucidate the role of HO-1, we treated animals withthe HO inhibitor, ZnPP, and assessed its effect on IL-10-medi-ated protection. We noticed that mice receiving ZnPP seemedmore susceptible to LPS-induced septic shock; however, the sur-vival rate was not significantly different from that of littermatesreceiving LPS alone. Nevertheless, the protective effect of IL-10was significantly attenuated and the survival rate reduced to30% (P < 0.005) when the IL-10-treated mice received additionaladministration of ZnPP one hour before LPS challenge (Fig. 6c).

Fig. 5 HO-1 mediates IL-10-induced suppression of NO production andMMP-9 expression. a and b, J774 cells and peritoneal macrophages un-treated or treated with IL-10 (10 ng/ml, 2 h) were incubated with or with-out LPS (1 µg/ml, 18 h). The culture medium was then replaced withmedium without phenol red, and cells incubated for additional 6 h with orwithout ZnPP, hemoglobin (Hb), desferrioxamine (Def.) or bilirubin as indi-cated. HO-1 and INOS protein expression and nitrite accumulation weredetermined. In a, *, P < 0.005; in b, *, P < 0.05 and **, P < 0.025 comparedwith cells treated with IL-10/LPS. c, J774 cells treated with or without IL-10(10 ng/ml, 6 h) were incubated for an additional 6 h in the absence or pres-ence of the indicated agents. The culture medium was subjected toSDS–PAGE zymography to detect the activity of MMPs and a major bandwith a molecular size close to proMMP-9 was detected. Its identity asproMMP-9 was confirmed by western blotting. The HO-1 protein level incell lysates was also determined. d, Peritoneal macrophages untreated ortreated with IL-10 (10 ng/ml, 1 h) were incubated with or without LPS (1µg/ml, 5 h), followed by a 6 h incubation in the presence or absence of theindicated agents. The expression of MMP-9 was detected by zymography.

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To examine whether the inhibitory effect of ZnPP on IL-10-me-diated protection can be countered by CO, we exposed animalsreceiving the treatment of IL-10/ZnPP/LPS to 250 p.p.m. CO for12 hours immediately after LPS challenge and found that thesurvival rate returned to 86% (P < 0.005). To further confirm thespecificity of ZnPP on HO inhibition in vivo, we conducted addi-tional experiments using CuPP. ZnPP treatment significantly re-duced the liver HO activity in mice receiving IL-10 for 12 hours(4.79 ± 2.27 nmol/mg protein/h (n = 8) versus 7.76 ± 2.07nmol/mg protein/h (n = 8), P < 0.02); whereas CuPP treatmentdid not significantly affect the activity (8.31 ± 1.79 nmol/mgprotein/h, n = 8). Likewise, CuPP treatment exhibited no signifi-cant effect on the protective effect of IL-10 (Fig. 6d). The serumTNF-α levels paralleled the severity of LPS-induced shock inthese groups of animals (Fig. 6e). These observations strongly

support the implication of HO-1 in theanti-inflammatory effect of IL-10 in vivo.

DiscussionHere we provide both in vitro and in vivodata in support of a role HO-1 in the anti-inflammatory effects of IL-10. Our dataclearly show that IL-10 is a potent inducerof HO-1 in murine primary macrophagesand a murine macrophage cell line. The in-duction of HO-1 was also observed in humanperipheral monocyte-derived macrophagestreated with IL-10 (data not shown), sug-gesting that the effect is not limited to ro-dents. Treatment with actinomycin Dcompletely blocked the induction of HO-1by IL-10, indicating that the regulation ofHO-1 gene expression occurs at the tran-scriptional level. Although a putative STAT-responsive element is located in thepromoter region of the murine HO-1gene27, IL-6, which uses a similar STAT-3signaling pathway to that used by IL-10 butlacks anti-inflammatory function14, did notinduce HO-1 gene expression in murinemacrophages. This result confirms an ear-lier report in human endothelial cells28,whereas a study on rabbit microvessel en-dothelial cells has yielded contradictory re-sults29. We speculate that the differentialeffects of IL-6 on HO-1 gene expression isdependent on cell type and species.Regardless, our data is in agreement withprevious reports by others showing thatthe STAT-3 dependent pathway is not suffi-cient to mediate the anti-inflammatory ac-tion of IL-10 (refs. 4–6). In fact, exposure ofmacrophages to IL-10 led to early but tran-sient phosphorylation of p38, a MAPK thatis commonly activated by inflammatorycytokines and stress stimuli30. We testedthe role of p38 in HO-1 induction medi-ated by IL-10 and found that inhibition ofp38 resulted in a complete blockade of thiseffect, supporting the role of p38 in thisprocess. However, the detailed mechanisminvolved in the activation of HO-1 gene

transcription by IL-10 in macrophages remains to be clarified.The finding that the p38 signal pathway is involved in the

IL-10-mediated anti-inflammatory effect is unexpected. Earlierstudies on LPS-induced TNF-α biosynthesis have revealed the im-portant role of the p38-dependent pathway in the regulation ofTNF-α translation31–33. Furthermore, a recent report byKontoyiannis et al.34 has demonstrated that the IL-10 suppressionof TNF-α translation in LPS-stimulated macrophages is mainlythrough the inhibition of p38 activation. As IL-10 was coadminis-tered in their study with LPS and the effect on p38 phosphoryla-tion was examined at 15 minutes after challenge, it is unlikely thatHO-1, which is induced by IL-10 at a later time period, has a rolein mediating the inhibition of p38 activation observed under theirexperimental setting. In addition, we found that IL-10-inducedp38 phosphorylation reaches a peak at 5 minutes, which is much

Fig. 6 HO-1 is essential for IL-10-mediated protection against LPS-induced septic shock. a and b,BALB/c mice were injected intraperitoneally with IL-10. Changes in HO-1 protein expression in vari-ous tissues and cells were examined by western blot (a), and quantitative data was obtained by den-sitometry (b). c and d, Mice received different treatments as indicated and the lethality within 72 hwas determined. In c, �, control (n = 15); �, ZnPP (n = 5); �, IL-10 (n = 5); � (with dashed line), LPS(n = 15); ⊗ , IL-10/ZnPP (n = 5); �, IL-10/LPS (n = 15); �, LPS/ZnPP (n = 10); � (with dashed line),IL-10/ZnPP/LPS (n = 20); , IL-10/ZnPP/LPS/CO (n = 15). In d, �, CuPP (n = 5); �, LPS (n = 15); �, IL-10/LPS (n = 15); �, IL-10/CuPP (n = 5); � (with dashed line), CuPP/LPS (n = 10); � (withdashed line), IL-10/CuPP/LPS (n = 10). *, P < 0.005 compared with mice receiving IL-10/LPS; #, P <0.005 compared with mice receiving IL-10/ZnPP/LPS. e, Serum TNF-α levels in all mice were deter-mined at 2 h after challenge with or without LPS. *, P < 0.01 compared with group treated with IL-10/LPS; #, P < 0.01 compared with group treated with IL-10/ZnPP/LPS.

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earlier than the time point of LPS-induced maximal p38 phospho-rylation (15 min)4,34. It will be intriguing to know whether the p38signaling induced by IL-10 feeds back on the p38 activation laterinduced by LPS. Although research on p38 function has revealedthe important role of its activation in inflammatory responses30,and inhibition of p38 is considered a potential therapeutic ap-proach for anti-inflammatory therapy, some recent studies yieldcontradictory results. For example, treatment with a p38 inhibitorin mast cells results in enhanced production of TNF-α (ref. 35). Arecent study by van den Blink et al.36 has also demonstrated thatinhibition of p38 results in reduced cytokine production in mostof the cell types tested, but increases cytokine release in LPS-acti-vated macrophages in vitro and in murine models of endotoxemiaand infection with pneumococcal pneumonia. The role of p38 inmodulating the inflammatory responses therefore appears to de-pend on the cell type and stimulus. The observation that p38 me-diates the induction of HO-1 by IL-10 in macrophages mayexplain part of the mechanism(s) by which p38 exerts anti-in-flammatory effects in certain circumstances.

Involvement of HO-1 in the anti-inflammatory response hasbeen shown in a few disease models13,37–39, including endotox-emia. Although biliverdin and bilirubin, produced by hemedegradation, are antioxidants and may play a role in the protec-tive response to tissue injury occurring during inflammation, re-cent studies have demonstrated that CO is the key moleculemediating the protective effect of HO-1 (refs. 13,40,41). Here weconfirmed that CO mediates the inhibitory effects of IL-10 onLPS-induced inflammatory responses. Our data show that scav-enging of CO by hemoglobin significantly reduced the in-hibitory effect of IL-10 on LPS-induced TNF-α and NOproduction and MMP-9 expression in macrophages.Furthermore, in animal experiments we showed that CO expo-sure could fully restore the protective effect of IL-10, which wasinhibited by additional ZnPP treatment in mice following LPS-induced septic shock. Because of the involvement of p38 in bothIL-10-mediated HO-1 induction and the inflammatory responseselicited by LPS stimulation in macrophages20,31–33, the role of p38in CO-mediated effects is difficult to prove in our experimentalsetting. Nevertheless, we suggest that IL-10 and HO-1 activate apositive-feedback circuit to amplify the anti-inflammatory ca-pacity by upregulation of their respective expressions via COand a p38-dependent mechanism.

In summary, the data presented here provide evidence to sup-port the essential role of HO-1 in the anti-inflammatory func-tion of IL-10 both in vitro and in vivo. CO appears to mediatemost if not all of the protective effects of IL-10, although the de-tailed mechanism by which CO induces downregulation of in-flammation-associated genes in macrophages remains to bedetermined. Identifying the cellular signal pathway and the re-sponsive gene that are essential for the anti-inflammatory effectsof IL-10 provides important information for the design of newtherapeutic strategies to treat inflammatory diseases.

MethodsMaterials. LPS from Escherichia coli (serotype 055:B5) was from DIFCO(Detroit, Michigan). Recombinant mouse IL-10, IL-6 and anti-IL-10 neutral-izing antibody were from R&D Systems (Minneapolis, Minnesota). Zincprotoporphyrin IX and copper protoporphyrin IX were from PorphyrinProduct (Logan, Utah). SB203580 and PD98059 were from Calbiochem (LaJolla, California). HO-1 antibody was from StressGen (Victoria, Canada).INOS antibody was from Transduction Laboratories (San Diego, California).MMP-9, STAT-3, phospho-STAT-3 and SOCS-3 antibodies were from SantaCruz Biotechnology(Santa Cruz, California). Phospho-JNK, phospho-p38

and phospho-ERK MAP kinase antibody kits were from New EnglandBioLabs (Beverly, Massachusetts).

Cell culture. Primary peritoneal macrophages were harvested from BALB/cmice and plated on a culture dish with RPMI 1640 medium containing0.1% BSA. After 2 h of incubation in culture, nonadherent cells were re-moved by washing with serum-free medium. Adherent cells were then usedfor experiments. Murine J774 macrophages (ATCC TIB-67) were cultured inRPMI 1640 medium supplemented with 10% FBS. Cells at 60% confluencywere changed to serum-free medium containing 0.1% BSA and subjectedto various treatments.

Transfection with ODN. The antisense phosphorothioated ODN comple-mentary to murine HO-1 translation initiation codon and 6 base pairs on ei-ther side (5′-ACGCTCCATCACCGG-3′) was synthesized. The sense ODN(5′-CCGGTGATGGAGCGT-3′) and a scrambled ODN (5′-CACGTCAC-CTCAGCG-3′) were used as negative controls. The murine SOCS-3 anti-sense ODN (5′-GGTGACCATGGCGCA-3′) was used as an additionalnegative control. Cells (1 × 106/plate) were transfected with 1 µg ODN pre-mixed with 10 µl LipofectAMINE reagent (Gibco BRL, Rockville, Maryland)according to the manufacturer’s instructions. After incubation for 5 h, cellswere washed once with serum-free medium, treated with 10 ng/ml IL-10for 2 h, and followed by LPS (1 µg/ml) treatment for another 2 h prior towestern-blot analysis and determination of TNF-α production.

Western-blot analysis. Cells lysates were prepared and 50 µg of lysate pro-teins were electrophoresed on 8% or 10% SDS–polyacrylamide gel. Westernblotting was carried out as described42 except that antigens were detectedusing enhanced chemiluminescence system (Pierce, Rockford, Illinois).

Northern-blot analysis. Human HO-1 cDNA was obtained as previouslydescribed42. Murine A8 cDNA was prepared by RT-PCR using RNA isolatedfrom J774 cells treated with LPS and IL-10 (ref.15). Northern blotting wasperformed as described42.

Determination of TNF-α concentration. The TNF-α concentrations in cul-ture media and mouse sera were determined using an IEA kit (AssayDesigns, Ann Arbor, Michigan).

Determination of nitrite production. Accumulated nitrite, a stable break-down product of NO, in culture medium was determined using the Griessreagent43.

SDS–PAGE zymography. Culture media (50 µl/sample) were elec-trophoresed at 4 °C in 8% SDS–PAGE gel containing 0.1% gelatin under anon-reducing conditions. The proteins in the gel were renatured by incuba-tion with 2.5% Triton X-100 at room temperature for 1 h. The gelatinolyticactivity was examined as described44.

Animal experiments. To study the induction of HO-1 in vivo, BALB/c malemice (8-wk-old) received intraperitoneal administration of 1 µg IL-10/mouse. At the indicated times, the animals were killed and various cellsand tissues collected, and HO-1 protein levels were determined by westernblot. To assess the effect of IL-10 on endotoxin-induced shock, BALB/c micereceived intraperitoneal injections of sterile PBS, ZnPP (25 mg/kg bodyweight), IL-10 (1µg/mouse), a lethal dose of LPS (20 mg/kg body weight),ZnPP for 1 h followed by LPS (ZnPP/LPS), IL-10 for 2 h followed by ZnPP (IL-10/ZnPP), IL-10 for 3 h followed by LPS (IL-10/LPS) or IL-10 for 2 h followedby ZnPP for 1 h and then followed by LPS (IL-10/ZnPP/LPS). In a separateexperiment, ZnPP was replaced by CuPP (25 mg/kg body weight) for thetreatment of animals. For CO exposure, animals were placed in a chamber(1.385 cubic ft) containing 250 p.p.m. CO, which was equilibrated by aflow of 0.5% CO (5,000 p.p.m.) mixed with compressed air, for 12 h. Theconcentration of CO in the chamber was continuously monitored by a gasmonitor (Crowcon, Oxfordshire, UK). Serum TNF-α levels were determined2 h after LPS challenge. The handling of animals was in accordance with theguidelines of the Institute of Biomedical Sciences, Academia Sinica.

HO activity assay. To determine hepatic HO activity, liver was homoge-nized in 4 volumes of ice-cold 0.1 M potassium phosphate buffer pH 7.4,

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246 NATURE MEDICINE • VOLUME 8 • NUMBER 3 • MARCH 2002

ARTICLES

followed by centrifugation at 13,000g for 15 min at 4 °C. The supernatant(200 µg) was then incubated with 50 µM hemin, 1 mM NADPH, 2 mM glu-cose-6-phosphate and 1 unit glucose-6-phosphate dehydrogenase in 0.1 Mpotassium phosphate (pH 7.4) at 37 °C for 30 min in the dark. The bilirubingenerated was estimated spectrophotometrically45.

Statistical analysis. Each experiment was performed at least 3 times. Theresults are expressed as means ± s.d. Data on TNF-α and nitrite concentra-tions were analyzed using Student’s t-test. Survival rate data were analyzedusing Fisher’s exact test. A value of P < 0.05 was considered statistically sig-nificant.

AcknowledgmentsThis work was supported by grants from the National Science Council ofTaiwan (NSC-90-2320-B-001-039) and the Institute of Biomedical Sciences,Academia Sinica, Taiwan, ROC.

RECEIVED 14 AUGUST 2001; ACCEPTED 18 JANUARY 2002

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