Characterization and Biological Significance of Immunosuppressive Peptide D2702.75– 84(E 3 V) Binding Protein ISOLATION OF HEME OXYGENASE-1*

This is the first report on peptidic inhibitors of heme oxygenase. Such peptides were originally developed from the immunomodulatory peptide 2702.75–84 which corresponds to amino acid residues 75 to 84 of the a 1 - helix of HLA-B2702 (2702.75–84) and has been shown to be immunosuppressive in vitro and in vivo. In vitro , 2702.75–84 inhibited cytotoxic T- and natural killer cell-mediated target cell lysis, and in vivo peptide therapy resulted in prolongation of heart and skin allograft survival in mice. The peptide was also shown to bind to heat shock protein 70. However, D -enantiomers of 2702.75–84 and derivatives thereof, while still being immunosuppressive, did not bind to heat shock protein 70. This study was designed to identify proteins binding to peptide D2702.75–84(E 3 V) (rvnlrialry) consisting of D - amino acids. Compared with 2702.75–84 (RENLRIALRY), glutamic acid residue 76 (E) was replaced with valine (V). Affinity chromatography using immobilized D2702.75–84(E 3 V) and mouse and human cell extracts, resulted in the isolation of heme oxygenase-1 (HO-1). Peptide D2702.75–84 inhibited HO activity in vitro in a dose dependent manner. Similar to what has been observed with Total RNA was prepared from tissues by the method of Chirgwin et al. (20). Poly(A 1 ) RNA was isolated by oligonucleotide(dT) cellulose chromatography, fractionated on a 1.2% agarose gel, and transferred to Nytran (Schleicher & Schuell). Prehybridization, hybridization to the appropriate 32 P-labeled cDNA, and post-hybridization treatment of the blots were preformed as described elsewhere (21). The signals on auto-radiograms were quantitated using an LKB UltroScan densitometer.

Peptides derived from various regions of the HLA class I heavy chain have been shown to exert immunomodulatory effects by influencing T cell responses (1)(2)(3)(4). Clayberger and co-workers (5, 6) studied the less polymorphic region of the HLA class I ␣ 1 -helix (residues 75-84). Peptides corresponding to this region of the HLA-B7 (07.75-84) or HLA-B2702 (2702.75-84) molecule blocked the differentiation of human CTL precursors in vitro in a non-allele-restricted manner (5).
The 2702.75-84 peptide inhibited not only differentiation, but also T and NK 1 cell-mediated lysis of target cells in a nonallele-restricted manner. Woo et al. (7) subsequently demonstrated a more powerful effect when the peptides were used in a dimeric form (2702.84 -75-75-84). These peptides have also been studied in vivo in transplantation models. In rats, 07.75-84 induced permanent acceptance of LEW heart allografts in ACI recipients when combined with subtherapeutic doses of cyclosporine A (6). In congeneic LEW.1W donors and LEW.1A recipients, 07.75-84 induced long term graft survival without additional cyclosporine A treatment (8). In a mouse heart transplant model, where C57Bl/6 mice were used as donors and CBA mice as recipients, treatment with both 2702.75-84 and 2702.84 -75-75-84 prolonged graft survival significantly, with B2702.84 -75-75-84 showing a more powerful effect (7,9,10). Administration of 2702.75-84 has been shown to prolong the survival of skin allografts significantly when the tail skin of C57Bl/6 mice was grafted on CBA mice (9).
Despite the peptide's effectiveness, its mechanism of action is not fully understood. For reasons discussed elsewhere (7,9,10) a direct interaction with T cell receptors or NK cell inhibitory receptors can be excluded. The recent observation that both L-and D-enantiomers of peptide 2702.75-84 prolong heart allograft survival in vivo indicated that the peptides' immunomodulatory activity is probably not based on indirect presentation by major histocompatibility class molecules (10). Similarly, the recent hypothesis that peptide 2702.75-84 modulates immune responses by binding to HSP/HSC70 can be excluded because the D-isomer of 2702.75-84 did not bind such proteins (11,12).
Based on these observations, we looked for additional proteins that may interact with these immunomodulatory peptides. For these studies, we used a 2702.75-84-derived peptide (D2702.75-84(E 3 V)) with two modifications: (i) synthesis with D-amino acids and (ii) substitution of glutamic acid residue at position 76 in 2702.75-84 with valine. Compared with peptide 2702.75-84, this peptide displayed enhanced immunomodulatory activity in vitro and in vivo (12). Affinity purification using this peptide resulted in the isolation of heme oxygenase 1 (HO-1, HSP32). Effects of peptide binding to this enzyme were analyzed in vitro and in vivo.

Animals
Male, 7-8-week-old CBA/J (H-2 k ) and C57BL/6/J (H-2 b ) mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were housed according to the Animal Welfare Guidelines, Department of Health, California.

Peptide Synthesis and Immobilization
Peptides were synthesized using Fmoc chemistry, purified by HPLC and shown to be Ͼ90% homogenous by analytical reverse phase HPLC (SynPep, Dublin, CA). Peptides CSGSGrvnlrialry and CSGSGrvnlrtalry (5 mg) were dissolved in Me 2 SO (60%) and coupled to 2 ml of SulfoLink coupling gel according to the manufacturer's recommendations (Pierce). An additional column was coupled with cysteine-HCl alone. Affinity chromatography using all three gels was performed simultaneously.

Cell Culture and Preparation of Cell Extracts
Human T cell leukemia cell line Jurkat (clone E6-1) and a mouse lymphoma cell line Yac-1 were obtained from ATCC and maintained in RPMI 1640 medium (Sigma) containing 10% fetal bovine serum. For purification of 2702.75-84(E 3 V)-binding proteins, 1 ϫ 10 9 cells were washed in cold PBS and lysed in a buffer containing 50 mM Tris-Cl (pH 7.6), 300 mM NaCl, 1% CHAPS, and 5 g/ml each of leupeptin, aprotinin, pepstatin, chymostatin, antipain and 0.5 mM phenylmethylsulfonyl fluoride. Cells were lysed at 4°C for 30 min. Insoluble material was discarded after centrifugation at 2000 rpm for 10 min, and protein content was estimated by the BCA assay (Pierce).

Affinity Chromatography
SulfoLink gel was washed several times with 10 mM phosphate buffer containing 150 mM NaCl, 0.1% CHAPS, and protease inhibitors (wash buffer) and incubated with cell lysate for 60 min at 4°C with continuous rocking. The gel was packed in a column and unbound cell lysate was recovered. The column was washed with 20 column volumes of wash buffer, followed by wash buffer containing 500 mM NaCl. The latter fraction was termed high salt eluate. Bound proteins were eluted with 100 mM glycine-HCl, pH 3.0, containing 0.1% CHAPS and protease inhibitors in 1-ml fractions that were immediately neutralized to pH 7.0 using 1 M Tris-HCl, pH 8.2. Protein concentration was estimated by the BCA assay (Pierce), and fractions containing protein were pooled, concentrated in a microconcentrator, and analyzed by SDS-polyacrylamide gel electrophoresis and western blotting. All fractions were stored at Ϫ80°C.

Gel Electrophoresis and Western Blotting
Approximately 1 g of pH 3.0 eluate, 5 g of the high salt eluate, and 20 g of cell lysate were boiled in SDS sample buffer containing 2-mercaptoethanol and separated on a 12% SDS-polyacrylamide gel. Proteins were visualized by silver staining (13).
Western transfer was carried out using the blot module from Novex (San Diego, CA) on nitrocellulose (Hybond-N, Amersham Corp.) in transfer buffer (12 mM Tris, 96 mM glycine, pH 8.3) at 30 V for 45 min. Efficiency of transfer was monitored by including prestained molecular weight markers (Amersham Corp.) on the gel. Nonspecific binding sites were blocked by immersing the membrane in PBS (pH 7.5) containing 1% bovine serum albumin overnight at 4°C. After a short wash in PBS containing 0.1% Tween-20 (PBST), the membrane was incubated in primary antibody at the specified dilution in PBST for 1 h at room temperature with continuous rocking. The primary antibodies tested were rat anti-human HSC/HSP70 (Stressgen, Victoria, BC, Canada) at a 1:10,000 dilution, rabbit anti-rat HSP32 (also called HO-1), rabbit anti-rat heme oxygenase-2 (HO-2), rabbit anti-mouse HSP27 (all from Stressgen), all diluted at 1:5000. Following extensive washing in PBST, the membrane was incubated in the appropriate secondary antibody solution at the specified dilution for 1 h at room temperature with continuous rocking. The secondary antibodies used were rabbit anti-rat at 1:10,000 or donkey anti-rabbit at 1:5000, respectively (Jackson Im-munoResearch Laboratories, West Grove, PA). All secondary antibodies were conjugated to horseradish peroxidase.
After extensive washing, bands were visualized using enhanced chemiluminescence western blotting reagents (Pierce). Exposure to film (Hyperfilm ECL, Amersham Corp.) was performed for 10 -30 s.

Protein Sequencing
Amino acid sequencing of the ϳ30-kDa band was performed by Edman chemistry using an automated ABI 470 A system sequencer with an on-line ABI 128 HPLC and analyzed on ABI 610 system software at the Protein structure laboratory, University of California, Davis, CA. The sequence was compared with known sequences in the University of Wisconsin GCG data base.

Purification of Recombinant HO-1
A first strand HO-1 cDNA was generated with 1 g of testis poly(A) RNA using the cDNA cycle kit (Invitrogen), priming with oligo(dT), and used as a template for polymerase chain reaction using the primers 5Ј-GGGAAGCTTGGAGCGCCCACAGCTCG-3Ј (representing nucleotides 2 to 18 of HO-1 (17) and a HindIII restriction site (italics) and 5Ј-AAGCTTATGATGATGATGATGATGCTTATCGTCATCGTCCAT-GGCATAAATTCCCACTG-3Ј which contains the reverse complements of nucleotides 848 -867, an enterokinase cleavage site (underlined), histidine tag (bold), stop codon (double underline), and HindIII site (italics). The fragment was cloned into the HindIII site of the pBSϩ vector (Stratagene, La Jolla, CA) and the resultant plasmid was transformed into Inv FЈ Escherichia coli cells. Orientation of the insert was determined by restriction analysis and confirmed by sequencing. Fusion proteins were purified from overnight cultures utilizing ProBond TM (Invitrogen) columns in accordance with the manufacturer's instructions. Pooled peak fractions from the ProBond TM column elution were buffer-exchanged into 50 mM Tris-HCl, pH 8.0, containing 10% glycerol, 1 mM CaCl 2 , and 0.1% Tween-20 and concentrated to approximately 1 mg/ml for storage at Ϫ80°C. Preparations were judged to be Ͼ90% homogenous as assessed by SDS-polyacrylamide gel electrophoresis followed by staining with Coomasie Brilliant Blue.

Enzyme Assays
Heme Oxygenase-To determine HO activity in tissues, mouse spleen samples were homogenized on ice in a Tris-HCl lysis buffer (pH 7.4) containing 0.5% Triton X-100 and protease inhibitors. Samples were frozen in small aliquots until use. Spleen microsomes were prepared and used as the source of HO for activity measurements, assessed by bilirubin formation. Spleen homogenate (100 l) was mixed with 0.8 mM NADPH, 0.8 mM glucose 6-phosphate, 1.0 unit of Glu-6-P dehydrogenase, 1 mM MgCl 2 , and 10 l of purified rat liver biliverdin reductase at 4°C. The reaction was initiated by the addition of hemin (final concentration 0.25 mM). The reaction mixture was incubated at 37°C in the dark for 60 min. At the end of the incubation period, any insoluble material was removed by centrifugation, and supernatants were analyzed for bilirubin concentration by a modified procedure of Hillman and Beyer (Sigma Diagnostics, kit no. 552). Controls included spleen samples in the absence of the NADPH-generating system and all components of the reaction mixture in the absence of spleen homogenates. Biliverdin reductase was purified from rat liver by the method described by Kutty and Maines (14).
To determine HO activity in purified preparations of HO-1, the procedure described previously was used and the activity was determined in the presence of purified preparations of biliverdin reductase and NADPH-cytochrome P450 reductase (15).
Biliverdin Reductase-Biliverdin reductase activity was determined by measuring the rate of bilirubin formation (14). To a reaction mixture consisting of 0.1 M Tris-HCl (pH 8.7), 0.1 mM NADPH, 5 l (approximately 200 ng of purified enzyme protein), 5.0 M biliverdin, and peptides at a final concentration of 100 M were added. The control reaction mixture did not contain peptides. The reaction was carried out in the dark at 37°C for 30 min, and the bilirubin formed was measured as described above.
Lactate Dehydrogenase and Horseradish Peroxidase-Lactate dehydrogenase enzyme activity was determined using whole cells (E6-1) as the source as described previously (16). Horseradish peroxidase enzyme activity was measured by addition of o-phenylenediamine. Following incubation at room temperature, the reaction was stopped by the addition of HCl, and the optical density was measured at 490 nm.
Total RNA was prepared from tissues by the method of Chirgwin et al. (20). Poly(A ϩ ) RNA was isolated by oligonucleotide(dT) cellulose chromatography, fractionated on a 1.2% agarose gel, and transferred to Nytran (Schleicher & Schuell). Prehybridization, hybridization to the appropriate 32 P-labeled cDNA, and post-hybridization treatment of the blots were preformed as described elsewhere (21). The signals on autoradiograms were quantitated using an LKB UltroScan densitometer.

In Vivo Effects of Peptide Treatment
Normal CBA mice were treated with a single injection (intraperitoneal) of peptide D2702.75-84(E 3 V) at 80 mg/kg dissolved in PBS ϩ 10% Me 2 SO. Control animals were treated with an equal volume of PBS ϩ 10% Me 2 SO. At 6, 24, and 48 h post-treatment, animals were killed, and liver, spleen, kidney, heart, and blood (serum) were collected and rapidly frozen. Tissue samples were used for northern blotting to detect HO-1 mRNA and western blotting to detect HO-1 protein levels. Microsomes were prepared from spleen samples and used for HO activity measurements and western blotting.

RESULTS
Isolation of Heme Oxygenase-1-Recently, peptide 2702.75-84 (RENLRIALRY) and 2702.75-84(E 3 V) (RVNL-RIALRY) were shown to bind to HSC70 and HSP70 (11,12). In contrast, D-enantiomers of these peptides did not bind to HSC70, although these peptides inhibited cytotoxicity and prolonged murine heart allograft survival to a similar extent as the L-enantiomers. These findings prompted us to explore additional peptide binding proteins that may mediate the peptide's immunosuppressive effects. Whole cell lysates from Jurkat cells (Fig. 1A), a human T cell lymphoma, and YAC-1 cells (Fig. 1B), a mouse lymphoma, were incubated with an affinity matrix consisting of peptides coupled to agarose via an Nterminal cysteine residue. Proteins eluted from the affinity gel at low pH were analyzed by SDS-gel electrophoresis. Using peptide D2702.75-84(E 3 V) (rvnlrialry) the pH 3.0 eluate contained a major band of approximately 30 kDa (Fig. 1, lane  4). This protein was purified from both human Jurkat and murine YAC-1 cells. Control runs using SulfoLink-agarose gel containing cysteine or peptide rvnlrtalry (negative control) did not result in the purification of the 30-kDa protein (data not shown).
Proteins in cell lysates and various fractions eluted from the affinity matrix were analyzed with various antibodies following transfer to nitrocellulose. Incubation of western blots with anti-HSC/HSP70 antibody revealed the presence of these proteins in the whole cell lysate and the high salt wash, but not in the low pH eluate of E6-1 (Fig. 2A, lane 4) or YAC-1 cell lysate (Fig.  2B, lane 4). Probing the western blots with anti-HSP25 and anti-HSP27 did not reveal the presence of these proteins in any of the fractions (data not shown). In contrast, analysis of the blots with anti-HSP32 resulted in a positive signal in the protein fraction eluted at pH 3.0. This result was obtained with proteins purified from both E6-1 (Fig. 3A) and YAC-1 (Fig. 3B) cell extracts. The positive band on the western blot corresponded to the single band observed in the SDS-polyacrylamide gel. No detectable signal was seen with whole cell lysate or the high salt fractions of either cell extract.
In an effort to confirm its identity and compare it to other published sequences, the single ϳ30-kDa band obtained in the pH 3 fractions that bound to anti-HSP32 antibody as well as peptide D2702.75-84(E 3 V), was further analyzed for partial amino acid sequence. Complete homology was observed with human and mouse HSP32 and 97% homology with rat HSP32 (Table I), thus confirming that the eluted protein was HSP32 or HO-1.
Inhibition of Heme Oxygenase Activity by D2702.75-84(E 3 V)-Heme oxygenase catalyzes the degradation of heme into biliverdin which is subsequently reduced to bilirubin by biliverdin reductase. When the activity of purified recombinant HO-1 was assessed in the presence of peptide D2702.75-84(E 3 V), a significant dose dependent reduction of enzyme activity was observed (Fig. 4): half-maximal inhibition of HO-1 activity occurred at about 20 M. The inhibition of bilirubin formation was not due to the inhibition of biliverdin reductase by the peptide because addition of peptide to purified biliverdin reductase had no effect (data not shown). The specificity of the effect was also evaluated using lactate dehydrogenase and horse radish peroxidase. Addition of D2702.75-84(E 3 V) to these enzymes had no effect on their activity (data not shown).
The specificity of peptide-mediated inhibition of HO was studied using various different peptides. Due to the availability of limited amounts of purified recombinant HO-1, the effect of peptides on mouse and rat HO activity was assessed using spleen cell extracts (Table II). HO-1 is by far the most prevalent form of the HO system in the spleen. All of the tested peptides had been evaluated in vitro and in vivo for immunomodulatory with the identical amino acid sequence as peptide D2702.75-84(E 3 V) but consisting of L-instead of D-amino acids inhibited splenic mouse and rat HO activity to a similar extent as peptide D2702.75-84(E 3 V). Similarly, the rationally designed peptide RDP1258 which prolonged heart and kidney allograft survival in both mouse and rats displayed an IC 50 of 20 M with both enzymes. Peptide 2702.75-84 inhibited mouse HO but not rat HO. Half-maximal inhibition of mouse HO was observed at about 1 mM. At this peptide concentration (the highest concentration tested), rat HO activity was not affected at all. Compared with D2702.75-84 and RDP1258, this corresponded to a 50 times lower inhibitory activity with mouse HO. Similarly, this peptide has been shown to be less potent in prolonging mouse heart allograft survival and to be ineffective in prolonging rat allograft survival (12). 2 Peptide 07.75-84 has been shown to prolong rat heart allograft survival (6,8), but not mouse heart allograft survival (7, 9 10, 12). 2 Interestingly, peptide 07.75-84 displayed inhibitory activity, although moderately, on rat splenic HO but had no effect on mouse HO activity (Table II).
Induction of HO-1 Expression in Vivo-Expression of HO-1 in vivo can be induced by various forms of stress, as well as enzyme inhibitors. For example, administration of cobalt-protoporphyrin, a well characterized inhibitor of HO, was shown to induce a long lasting up-regulation of HO gene transcription and activity in rats (25,27,31). 3 Similarly, northern blot analysis of HO-1 mRNA levels in kidneys from mice treated with a single dose of D2702.75-84(E 3 V) (Fig. 5) showed a significant increase in HO-1 mRNA levels. A pronounced increase (Ͼ10 times) was detected at 6 h following peptide administration. At 24 h, levels were about 2 times higher in treated tissues than controls, while at 48 h, levels had declined to control values. A more subdued but reproducible increase (about 1.6 times) of HO-1 mRNA levels was detected in liver 6 h after treatment (data not shown). HO-2 mRNA levels were not increased in either tissue (data not shown). The increased production of HO-1 mRNA was reflected by increased amounts of HO-1 protein as detected by western blotting (Fig. 6A). Changes in HO-1 levels in spleen were monitored also by measuring enzyme activity. Increased HO-1 protein levels persisted longer than mRNA reflecting the relative half-lives of the transcript and the protein (23). The observed increase in HO-1 protein was also apparent when HO activity was determined (Fig. 6B). Splenic microsomes prepared from peptide treated animals showed an increase in HO activity of 26 and 31%, at 24 and 48 h post-treatment, respectively, when compared with PBS/Me 2 SO-treated control animals. DISCUSSION A single amino acid substitution in peptide 2702.75-84, replacement of glutamic acid residue 75 with valine, and peptide synthesis using D-amino acids, resulted in a peptide (D2702.75-84 (E 3 V)) with increased immunomodulatory activity as analyzed by inhibition of cytotoxicity and prolongation of heart allograft survival. This peptide did not bind to HSC70, indicating that peptide-mediated immunomodulation is independent of binding to this heat shock protein (12). Here we demonstrated that D2702.75-84(E 3 V) bound to HSP32 (HO-1) and inhibited enzyme activity in vitro in a dose-dependent manner. All known inhibitors of HO induce up-regulation of HO-1 transcription in cells or animals (23,24). Similarly, in vivo administration of D2702.75-84(E 3 V) into mice resulted in rapid up-regulation of HO-1 mRNA and HO-1 protein levels with a corresponding increase in HO activity in liver, spleen, and kidneys.   4. Effect of peptide on in vitro HO activity. The activity of purified HO-1 was measured in the presence of increasing amounts of peptide D2702.75-84(E 3 V). Enzyme activity is expressed as nanomoles of bilirubin formed/mg of protein/min.
In an effort to study the specificity of peptide-mediated inhibition, various modifications of the peptide sequence were analyzed for in vitro enzyme inhibitory activity. Immunomodulatory activity correlated with HO-inhibitory activity; peptides without any immunomodulatory activity did not inhibit mouse or rat HO in vitro. In contrast, all tested peptides that prolonged allograft survival in mice or rats inhibited HO activity in vitro. Peptide 2702.75-84 was shown to prolong allograft survival in mice but not in rats (6 -10, 12). This peptide inhibited mouse HO but not rat HO. In contrast, peptide 07.75-84, which prolonged allograft survival in rats but not in mice, was also a more effective inhibitor of rat HO compared with mouse HO. Based on various physicochemical and structural characteristics of peptides that demonstrated in vivo immunosuppressive activity, an "in silico screening" method was used to design a set of peptides. 2 One of these rationally designed peptides, RDP1258, was shown to prolong allograft survival in both mice and rats 2 and was able to inhibit both rat and mouse HO activity. Thus, our results demonstrate a strong correlation between in vitro peptide mediated inhibition of HO activity and immunomodulation.
HO-1 catalyzes the metabolic degradation of heme into biliverdin which is subsequently reduced to the potent anti-oxidant bilirubin by biliverdin reductase (25,26). HO-1, also known as HSP32, is inducible by various forms of stress (heat, radiation, starvation, hypoxia, hyperoxia, ischemia, GSH-depletion) (26) and has been shown to be up-regulated during an inflammatory response and acute allograft rejection (27,28). Up-regulation of HO activity has been shown to protect cells from oxidative injury (29). Apart from stress, compounds such as heme, metalloporphyrins, and heavy metals have been shown to induce expression of HO-1 in vivo (24,26,30,31). Several of the investigated metalloporphyrins are known inhibitors of HO. The mechanism of feedback regulation of heme oxygenase transcription are unknown. However, one may speculate that a decrease in cellular heme oxygenase results in accumulation of heme, which subsequently induces up-regulation of HO expression.
Immunomodulatory effects similar to those mediated by D2702.75-84(E 3 V) were also observed following cobalt-protoporphyrin (CoPP)-induced up-regulation of HO-1. 3 Both D2702.75-84(E 3 V) and CoPP inhibited HO activity in vitro. In contrast, injection of D2702.75-84 (E 3 V) or CoPP into mice resulted in up-regulation of HO-1 gene transcription and HO activity. HO-1 up-regulation following administration of both compounds was associated with inhibition of lymphocyte proliferation and prolongation of heart allograft survival. In summary, these observations suggest that the immunomodulatory activity of CoPP and 2702.75-84-derived peptides is due to their capability to up-regulate HO in vivo. Formal proof of this hypothesis, however, has to await the availability of genetically engineered animals.
Although the mechanism of immunosuppression by HO overexpression remains unclear, several possible explanations can be envisioned. Degradation of heme by HO results in the production of biliverdin, bilirubin, and carbon monoxide. Biliverdin, the intermediate compound of heme degradation, was shown to inhibit human complement in vitro (33). In this context, it is interesting to note that complement deposition has been observed in unsensitized rat allograft recipients (34). Based on this observation, one may speculate that increased production of biliverdin following overexpression of HO may protect a transplanted organ from complement mediated cell injury and prolong graft survival.
Bilirubin, one of the endproducts of heme degradation, has been shown to inhibit responses of human lymphocytes including phytohemagglutinin-induced proliferation, interleukin-2 production, and antibody-dependent and -independent cell-mediated cytotoxicity (35)(36)(37)(38). Even though peptide therapy had no effect on serum bilirubin levels, one cannot exclude the  possibility that overexpression of HO-1 resulted in substantially higher intracellular bilirubin levels. Additional studies evaluating the intracellular concentration of bilirubin in spleen cells of peptide-treated mice by flow cytometry will be necessary to clarify this issue (39).
The gaseous product of heme degradation, CO, like NO, has been shown to stimulate the production of cGMP via activation of guanylate cyclase (40,41). The secondary messenger, cGMP, has been implicated in cell growth arrest and the release of tumor necrosis factor-␣ by activated macrophages (42). In addition, cGMP is involved in the regulation of various protein kinases, phosphodiesterases and ion channels (22,(43)(44)(45). Thus, one may speculate that the increased cGMP levels may modulate several immune effector functions. Besides stimulation of guanylate cyclase, CO may also modulate immune responses via inhibition of inducible NO synthase. Regulation of HO-activity and NO production are intimately linked and an increased production of CO causes decreased NO production and vice versa (32). Whether peptide treatment affects NO production is yet to be investigated.
It is interesting to discover peptides that inhibit HO-1. The mechanism of inhibition could involve factors such as the interaction of peptide with substrate and/or reductase binding sites, thus preventing binding/activation of the substrate; alternatively, the peptide could interact with HO-1 protein, changing its tertiary structure. The possibility that these peptides may bind to the substrate heme, causing inhibition of heme degradation cannot be dismissed. The latter possibility, however, is unlikely since availability of substrate during the in vitro enzyme assay is not limited and heme is concentrated in much higher proportion than the peptide. Whichever may be the case, the interaction of the peptide with HO-1 must share similarities with HO-2, in that the peptide D2702.75-84(E 3 V) inhibits not only HO-1 activity but also that of HO-2, which was recently observed. 4 In conclusion, our data demonstrated binding of peptide D2702.75-84(E 3 V) to HO-1. The peptides and derivatives thereof inhibit HO activity in vitro. Similar to effects observed following administration of CoPP, 3 injection of D2702.75-84(E 3 V) resulted in up-regulation of HO activity in liver, spleen, and kidneys. The mechanism of immunomodulation by HO-1 is not resolved. Our current knowledge of the biological effects of heme degradation products (biliverdin, bilirubin, and carbon monoxide) suggest that elevated HO activity may affect multiple pathways of immune responses. Identification of compounds that specifically modulate HO activity without causing toxic side effects could lead to the development of novel immunotherapeutics.