Results

Slow degradation of the CO-RM

A complete color change of the CO-RM in dimethyl sulfoxide (DMSO) solution, from yellow to colorless, occurred gradually within 60 min in open air at room temperature (Figure 1). We surmised that CO may therefore be released slowly from the base lotion also, and be available for a period after topical application for delivery into the skin layers, perhaps including the dermis, to mediate biological effects. In sealed or closed containers, the color change of the CO-RM/DMSO solution was observed to take much longer (not shown). The effect of the lotion emulsion on the release of CO from the CO-RM was not tested, but the lotion in the applicator syringe barrel, in which air was excluded, remained yellow during the treatments, and converted to white only when discarded into air contact after daily use.

Figure 1.

Time release of CO from the CO-RM. Slow decrease of absorbance over time by carbon monoxide-releasing molecule (CO-RM) in dimethyl sulfoxide, indicative of slow release of CO from the solution.

Full figure and legend (8K)

Lotions containing increasing concentrations of either fresh or aged CO-RM were tested for possible immunomodulatory effects from topical application (Figure 2). Fresh CO-RM lotions were not significantly immunosuppressive. The highest tested concentration of the aged CO-RM lotions, 500 M, did reveal a slight but significant suppression of CHS (p=0.017 by ANOVA), but the Tukey test showed no significant difference; therefore, the biological relevance is uncertain.

Figure 2.

Effect of fresh and aged CO-RM lotions on CHS. Contact hypersensitivity response to oxazolone in groups of five mice treated with increasing concentrations of fresh carbon monoxide-releasing molecule (CO-RM) (no significant differences) and aged CO-RM lotions, which became slightly immunosuppressive at 500 M.

Full figure and legend (24K)

CO-RM protects against SSUVR- or cis-UCA-induced immunosuppression

Irradiation with SSUVR resulted in 32% suppression of CHS (Figure 3). Treatment with 500 M CO-RM lotion did not significantly affect CHS, but markedly reduced the SSUVR-induced immunosuppression to 15% suppression; p=0.003. In mice in which HO enzyme activity was inhibited by injection with SnPP, the SSUVR was similarly immunosuppressive (48% suppression) as without SnPP treatment (p=0.102). Application of 500 M CO-RM again effectively (p<0.0001) reduced the SSUVR suppression to 18%, similar to the protection without SnPP inhibition, indicating that the protective effect was because of the exogenous CO-RM and had no significant contribution from endogenously produced CO. The aged CO-RM lotion appeared to protect slightly from SSUVR suppression of CHS, but this was not statistically significant.

Figure 3.

Effect of CO-RM lotion on SSUVR-supressed CHS in the presence of the endogenous HO inhibitor SnPP. Contact hypersensitivity response to oxazolone in groups of five mice immunosuppressed by solar-simulated ultraviolet radiation (SSUVR) exposure, showing the partial protective effect of 500 M carbon monoxide-releasing molecule (CO-RM) lotion, but not of 500 M aged CO-RM lotion. Inhibition of endogenous heme oxygenase activity by injection of Sn protoporphyrin-IX (SnPP) slightly exacerbated SSUVR immunosuppression, but did not alter the CO-RM protection.

Full figure and legend (13K)

Consistent with the responses to SSUVR, the topical application of cis-UCA resulted in 23% suppression of CHS (Figure 4; p=0.028), and this was abrogated by 500 M CO-RM lotion (p=0.001). In the presence of SnPP the suppression by cis-UCA (31% suppression) was not significantly different, and again the suppression was abrogated by 500 M CO-RM. The protection by the CO-RM against cis-UCA was thus more complete than against SSUVR, suggesting that other contributing mediators of photoimmunosuppression, such as prostaglandins, may be less sensitive to the CO-dependent pathways. This is consistent with the superior protection by UVA radiation against cis-UCA compared with UVB-induced suppression of CHS that we have previously observed (^{Reeve and Tyrrell, 1999}).

Figure 4.

Effect of CO-RM lotion on cis-UCA supressed CHS in the presence of the endogenous HO inhibitor SnPP. Contact hypersensitivity response to oxazolone in groups of five mice immunosuppressed by topical cis-urocanic acid (cis-UCA), showing the complete protective effect of 500 M carbon monoxide-releasing molecule (CO-RM) lotion, but not of 500 M aged CO-RM lotion. Inhibition of endogenous heme oxygenase activity by injection of Sn protoporphyrin-IX did not alter either cis-UCA immunosuppression, or the CO-RM protection.

Full figure and legend (16K)

Thus the results show that the immunosuppressive effects of SSUVR or its immunosuppressive photoproduct, cis-UCA, were strongly inhibited by the presence of the exogenous source of CO, independent of endogenous HO activity, and that the CO-RM treatment mimicked the immunoprotective role of UVA-induced HO activity in the skin.

CO-RM protection against cis-UCA immunosuppression is concentration dependent

The topical application of 50 M CO-RM contributed no significant protection (42% suppression of CHS) against cis-UCA-induced immunosuppression (49% suppression). A clear significant dose response could be seen, however, with 125, 250, and 500 M CO-RM (Figure 5), as the degree of immunosuppression reduced from 49% to 27%, 24% and 14%, respectively (p=0.019, 0.021, p<0.0001, respectively) and the protective effect by 500 M CO-RM restored the CHS reaction to a level not significantly different from the control level.

Figure 5.

CO-RM concentration dependence for immunoprotection against cis-UCA. Contact hypersensitivity response to oxazolone in groups of five mice immunosuppressed by topical cis-urocanic acid, and the concentration-dependent protective effect of increasing carbon monoxide-releasing molecule lotion concentration.

Full figure and legend (12K)

Effect of inhibitors of guanylyl cyclase and p38 MAPK

Topical application of the selective guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-1)quinoxaline-1-one (ODQ) did not alter the CHS response. The 52% suppression of CHS induced by SSUVR was reduced to only 20% suppression by UVA radiation (Figure 6). In the presence of ODQ, however, this immune protection was significantly inhibited, and the CHS response remained suppressed by 44% (p<0.0001). Assay of the cutaneous guanosine 3',5'-cyclic monophosphate (cGMP) levels following daily CO-RM application for 3 d showed an ODQ-inhibitable increase of 34% (Table I), providing support for the availability of CO from topical CO-RM lotion and its action on guanylyl cyclase. In contrast, topical application of the p38 inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl-5-4-(4-pyridyl) 1H-imidazole (SB203580) was apparently, but not significantly immunopotentiating itself (Figure 7; p=0.13). Under conditions of 54% suppression of CHS by SSUVR, and the reduction of this to 7% suppression by UVA plus SSUVR, the SB203580 treatment was not inhibitory and even slightly (p=0.009) but significantly enhanced the UVA immunoprotection against SSUVR. But this response did not differ from UVA plus SB203580 in the absence of SSUVR (p=0.96); therefore, the small enhancement of UVA immunoprotection may not be biologically significant. Thus, these experiments provide evidence that UVA photoimmunoprotection may act by the stimulation of the guanylyl cyclase system by CO, but do not support the involvement of the p38 MAPK pathway.

Figure 6.

Effect of quanylyl cyclase inhibitor ODQ on UVA photoimmunoprotection. Contact hypersensitivity response to oxazolone in groups of five mice immunosuppressed by solar-simulated ultraviolet radiation exposure, showing the protective effect of ultraviolet A (320–400 nm) (UVA) irradiation, and the inhibition of the UVA protection by the guanylyl cyclase inhibitor 1H-(1,2,4)oxadiazolo-(4,3-1)quinoxaline-1-one.

Full figure and legend (14K)

Figure 7.

Effect of p38 MAPK inhibitor SB 203580 on UVA photo immunoprotection. Contact hypersensitivity response to oxazolone in groups of five mice immunosuppressed by solar-simulated ultraviolet radiation exposure, showing the protective effect of ultraviolet A (320–400 nm) irradiation, and the failure to alter this protection by the p38 mitogen-activated protein kinase inhibitor 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl-5-4-(4-pyridyl) 1H-imidazole.

Full figure and legend (13K)

Table I - CO-RM lotion application daily for 3 d upregulated the production of cutaneous cGMP (measured by ELISA), which was prevented by the soluble guanylyl cyclase inhibitor, ODQ.

Full table

Discussion

The study of the effects of gaseous CO on tissues has called for innovative strategies for the presentation of CO experimentally. For in vitro studies, CO has been bubbled into culture media, and inhalation of controlled amounts of CO gas has been used for rodent studies. But inhaled CO will be delivered to tissues as carboxyhemoglobin, and its availability to cells will be affected by oxygen tension, and biological half-life of CO in the blood of 2–5 h (reviewed by^{Von Burg, 1999}). Recently,^{Motterlini et al (2002)} have described a novel approach with the use of transition metal carbonyls as CO-RM, and have demonstrated that the CO-RM could sustain vasodilatation in precontracted aortic rings, attenuate coronary vasoconstriction in the heart ex vivo, and significantly reduce acute hypertension in vivo (^{Motterlini et al, 2002}). Therefore, we have tested the CO-RM, choosing topical application for the first time, to facilitate the direct presentation of the CO to the target tissue, the skin. More recently, a water-soluble CO-RM has been developed by the Motterlini et al group (^{Clark et al, 2003}) and shown to liberate CO when infused intravenously in mice, and to have no toxic effect on heart function or blood pressure, nor to increase carboxyhemoglobin levels in the blood (^{Guo et al, 2004}).

Our experiments have demonstrated that the topically applied CO-RM could reproduce the immunoprotective effect of UVA irradiation against both SSUVR exposure or cis-UCA in the mouse. Because it is established that UVA irradiation induces both HO-1 mRNA and HO enzyme activity in mouse skin (^{Allanson and Reeve, 2004}) and in cultured human skin fibroblasts (^{Applegate et al, 1995}), and in the mouse the capacity of UVA radiation to afford such photoimmunoprotection could be attributed largely to the induced HO activity, we conclude that the product of HO activity, the gaseous molecule CO, is the immunoprotective factor. Inhibition of the endogenous HO enzyme activity by injecting the mice with its inhibitor SnPP eliminated any contribution by a possible CO-RM induction of HO-1 via a hypoxia mechanism, as has been observed by others (^{Murphy et al, 1991; Carraway et al, 2000}). An immunoregulatory role for CO has been found by others in studies in which exogenously applied CO has been shown to prevent pro-inflammatory cytokine production (^{Otterbein et al, 2000}) and to suppress allograft rejection in mouse-to-rat cardiac transplants (^{Sato et al, 2001}). In these studies, the application of CO appears to have stimulated immunosuppressive pathways, whereas our model is evidence for CO protection against immunosuppression. Although this appears to be incongruous, it is reminiscent of the inducibility of HO-1 under the opposite conditions of hypoxia and hyperoxia (^{Clark et al, 2000; Motterlini et al, 2000}), and might indicate the capacity of HO-1-derived CO to restore cellular and tissue immunological homeostasis from either stressor. This response to the CO-RM, [Ru(CO)₃Cl₂]₂ applied directly to the target organ, the skin, constitutes a new in vivo model of the biological effector that to date has been demonstrated mainly in vitro, and in vivo only in the mitigation of acute hypertension induced by L-NAME in the rat (^{Motterlini et al, 2002}). The latter effect was achieved by the intravenous injection in the rat of 20 mol per kg of the CO-RM. Very recently, a newly synthesized water-soluble CO-RM, tricarbonylchloro(glycinato)ruthenium administered intraperitoneally to mice at 40 mg per kg, which we estimate to be approximately equivalent to 60 mol per kg of the non-glycinated [Ru(CO)₃Cl₂]₂ CO-RM, was shown to provide protection from cardiac allograft rejection in mice, thus demonstrating that the transition metal tricarbonyl can provide the CO for an immunoregulatory role (^{Clark et al, 2003}). These concentrations were somewhat higher than in our study, where the topical CO-RM was immunologically protective in the mouse at concentrations of 125–500 M, equivalent to 1.0–4.0 mol per kg. The mechanism of CO liberation from the CO-RM, however, appears to be determined by the relative affinity for the Ru nucleus of the potential substituting ligand in the solution (DMSO in our study). It has been suggested that in vivo, the cellular environment may present other potential ligands such as glutathione, which may facilitate release of the CO (^{Clark et al, 2003}). Therefore, the route of CO-RM administration and tissue localization is likely to play an important role in determining the availability of CO from these donor compounds.

The time course and concentration dependence for the release of CO from the CO-RM have been previously characterized by^{Motterlini et al (2002)}. They detected approximately 0.7 mol of CO liberated from 1 mol [Ru(CO)₃Cl₂]₂ by capturing the CO in a myoglobin solution. Using nuclear magnetic resonance spectroscopy, they also identified a dicarbonyl monomer, Ru(CO)₂(DMSO)Cl₂, when the CO-RM was solubilized in DMSO, demonstrating the capacity of DMSO theoretically to liberate 1 mol of CO from the CO-RM molecule. The time course of CO release reached a plateau at 60 min, a pattern of CO release that mirrors the loss of the yellow color of [Ru(CO)₃Cl₂]₂ solution that we observed, and supports the concept that the colorimetric change indicated the CO-RM degradation and CO release. Our evidence that the degraded colorless product applied at 500 M was slightly but significantly immunosuppressive was unexpected, and the mechanism for such immunosuppression needs to be established. This subtle immunosuppressive effect was absent at the lower concentrations. It may be relevant that cytotoxicity in vitro of the CO-RM/DMSO has been reported with cultured vascular smooth muscle cells at high concentrations of >400 M, although only after prolonged 24 h exposure (^{Motterlini et al, 2002}), during which time it can be assumed that a degradation product like Ru(CO)₂(DMSO)Cl₂ was accumulating in the culture media. The in vitro cytotoxicity was not observed when the cells were treated with the metal ion alone in the form of RuCl₃, nor with DMSO.

It appears, however, that the 500 M aged CO-RM may have retained some CO ligands that could be further liberated because, rather than exacerbating the SSUVR immunosuppression, the aged CO-RM solution displayed a slight, although not statistically significant protection against SSUVR-induced immunosuppression. Because the aged CO-RM did not appear to protect against cis-UCA-induced immunosuppression, we surmise that CO-RM applied topically to the skin remained in the skin until its exposure to SSUVR 1 h later, and that the irradiation facilitated the liberation of further CO from the molecule. Such photodissociation and CO release from other metal carbonyls have been reported (^{Hughey et al, 1975;Wrighton and Ginley, 1975}), and might be advantageous if topical CO-RM were to be used therapeutically to protect from photoimmunosuppression. Further studies should clarify the potential for [Ru(CO)₃Cl₂]₂ to undergo photolysis on the skin, and the waveband of light that might activate this reaction.

The mechanism of action of CO and its precise molecular target(s) remain controversial. Currently two possible pathways have been described, via either the p38 protein of MAPK, or the soluble guanylyl cyclase, which is a hemoprotein and a defined receptor for another gaseous signalling molecule, nitric oxide. The p38 MAPK inhibitor SB203580 was shown to abrogate CO-mediated cytoprotection in rat hepatic ischemia/reperfusion injury, and there is recent evidence that CO protection was mediated by the selective activation of the MKK3/p38 protein MAPK pathway (^{Otterbein et al, 2000; Amersi et al, 2002; Otterbein et al, 2003b}). We did not find that SB203580 altered the UVA protection against SSUVR-suppressed CHS, and thus no evidence for the p38 pathway being activated immunologically by UVA radiation. SB203580 inhibits only selective isoforms of p38, however, and the unaffected p38 may have remained active (^{Otterbein et al, 2003}). On the other hand, other investigators have demonstrated that CO, like nitric oxide, binds to and activates guanylyl cyclase to produce cGMP (^{Kharitonov et al, 1995; Steiner et al, 2001}). This is supported by reports that CO-mediated protection against lethal ischemic lung injury in mice was abrogated in the presence of the guanylyl cyclase inhibitor ODQ. Our studies in mouse skin suggest that the immunoprotective properties of CO act via this guanylyl cyclase pathway, because UVA immunoprotection against SSUVR was antagonized by ODQ, and CO-RM application increased the cutaneous cGMP concentration. Interestingly, a recent study reports that UVB-induced cell death and apoptosis in cultured mouse and human keratinocytes was reduced by an exogenous nitric oxide donor, inhibitable by ODQ, consistent with a protective role for the guanylyl cyclase pathway in the skin (^{Weller et al, 2003}). Future studies may reveal tissue or stressor specificities for CO target pathways. Future studies in the skin might also lead to methods by which the CO-protective pathway might be harnessed to inhibit the development of photoimmunosuppression from chronic solar UVB exposure, and thus reduce the cutaneous cancer risk in humans.

In summary, these studies have identified that the photoimmunoprotective product of UVA-induced cutaneous HO activity is the gaseous molecule CO. Our results demonstrate that the murine cutaneous immune system is modulated by CO in vivo, possibly via the activation of soluble guanylyl cyclase, and evidence for the activity of this mechanism, stimulated by UVA irradiation, should now be sought in human skin.

Materials and Methods

Mice

Female inbred albino Skh:hr-I hairless mice, age 8–12 wk old, were provided from the Veterinary Science breeding colony. They were maintained under conventional animal house conditions in wire-topped plastic cages on compressed paper bedding (Fibrecycle Pty, Mudgeeraba, Queensland, Australia) at an ambient temperature of 25°C, and under gold lighting (GEC F40GO) that does not emit any UVB radiation, on a 12 h on/off cycle. They were fed stock laboratory mouse pellets (Norco Stockfeeds, Lismore, New South Wales, Australia) and tap water ad libitum. All procedures were approved by the University of Sydney Animal Ethics Committee and complied with the State Animal Research Act 1985.

UV radiation

The UVA source and its spectral properties have been described (^{Reeve and Domanski, 2002}), and consisted of a planar bank of seven 120 cm fluorescent UVA tubes (Hitachi 40W F40T10BL, Tokyo, Japan) held in a reflective batten at 19 cm above the irradiation table. The radiation was filtered through a selected sheet of 6 mm window glass to block contaminating wavelengths below 320 nm. Irradiance was measured with a IL 5000 radiometer (International Light Inc. Newburyport, Massachusetts), with UVA and UVB detectors (SE 015/UVA and SEE 240/UVB) calibrated to the spectral irradiances of the sources. This source provided 2.7 10^{- 3} W per cm² UVA and 2.3 10^{- 8} W per cm² UVB. The SSUVR source consisted of one UVB tube (Phillips TL-40W/12 RS, Eindhoven, the Netherlands) flanked by two banks of three UVA tubes, and the radiation was filtered through a sheet of 0.125 mM cellulose acetate filter (Eastman Chemical Products, Kingsport, Tennessee) to block contaminating wavelengths below 290 mm. This source provided 3.43 10^{- 3} W per cm² UVA and 2.14 10^{- 4} W per cm² UVB. The sources were allowed to stabilize for 15 min after switching on, and the temperature in the irradiation area below the lamps was controlled by an electric fan. Groups of up to five mice were placed in a box containing food and water, and exposed to 387 kJ per m² of UVA radiation (4 h exposure; equivalent to the UVA content of approximately 6–8 minimal erythemal dose (MED) of sunlight in humans (^{Reeve and Tyrrell, 1999})), or exposed to SSUVR containing 63.8 kJ per m² UVA radiation and 3.98 kJ per m² UVB radiation (31 min exposure, or approximately 3 MED). The UVA-alone dose was suberythemogenic, and could reasonably be absorbed by a human during a day of sunbathing through a UVB-absorbing sunscreen; the SSUVR dose resulted in a moderate but non-burning edematous erythema at 24 h.

Cis-UCA treatment

Trans-urocanic acid (Sigma-Aldrich, Castle Hill, New South Wales, Australia) was photoisomerized in DMSO solution to an equilibrium mixture of 60%trans- and 40%cis-isomers as previously described (^{Reeve et al, 1993}). A simple cosmetic oil-in-water lotion (^{Reeve et al, 1993}) containing 0.2% (wt/vol) UV-irradiated urocanic acid, called "cis-urocanic" acid here, 0.01 M phosphate-buffered saline at pH 7.2, and 5% DMSO was prepared and stored in the dark at 4°C. The control base lotion was identical in composition, without added urocanic acid. Aliquots of 0.2 mL (400 g urocanic acid) of either lotion were spread evenly over the mouse dorsum using a 1 mL syringe (no needle) and allowed to be absorbed for 30 min. Three applications were made within 24 h, to produce an immunosuppressive response similar to a single SSUVR exposure.

Treatment with CO

The "CO-RM" tricarbonyldichlororuthenium (II) dimer ([Ru(CO)₃Cl₂]₂) (Sigma-Aldrich), here referred to as CO-RM, was dissolved in DMSO and incorporated into base lotion to provide a lotion containing 500 M CO-RM and 10% (vol/vol) DMSO. The fresh CO-RM/DMSO solution is yellow; however, it becomes colorless as it ages and the CO is released from the molecule and becomes available to the target tissue (^{Motterlini et al, 2002}). The degradation of the CO-RM/DMSO, probably to Ru(CO)₂(DMSO)Cl₂ monomer as determined by nuclear magnetic resonance analysis by^{Motterlini et al (2002)}, was observed spectrophotometrically (SpectraMax 250 plate reader, Molecular Devices Corp., Sunnybank, California) at room temperature at 405 nm in a glass well for 3 h. To account for any effect that the degraded CO-RM might have, a control lotion was prepared using the colorless product of the CO-RM/DMSO that had been aged for 4 wk in the dark at room temperature, instead of the fresh CO-RM/DMSO.

Groups of mice were treated topically on days 1–5 with 0.2 mL freshly prepared CO-RM lotion at concentrations between 50 and 500 M, or with lotion containing the CO-RM vehicle (DMSO) only, or with 500 M aged CO-RM lotion, spread over the dorsum. One hour later, on day 1, groups of mice were irradiated with SSUVR, or received topical applications of cis-UCA lotion (or its control lotion) on the dorsum. To eliminate the contribution of endogenously released CO from the constitutive HO activity, or from the UVA component of SSUVR, some groups of mice were injected subcutaneously on the ventrum with 15 mg per kg body weight (0.38 mg per mouse) of a specific inhibitor of this enzyme, SnPP (Porphyrin Products, Logan, Utah), immediately following SSUVR or cis-UCA treatment, and again 19 h later, as previously described (^{Reeve and Tyrrell, 1999}). Control mice were injected with the NaOH/saline vehicle only.

Induction of CHS

All mice were sensitized with 0.1 mL of 2% oxazolone in ethanol applied to the non-irradiated and non-treated ventral skin on days 8 and 9 following the SSUVR or cis-UCA treatment (^{Reeve and Tyrrell, 1999}). On day 15, ear thickness was measured (pre-challenge) using a spring micrometer (Interapid, Geneva, Switzerland), followed by application of 5 L of 2% oxazolone in ethanol to each surface of both pinnae. The peak ear swelling for the control treatments was determined by measuring ear thickness repeatedly during 18–24 h post-challenge (post-challenge ear thickness- pre-challenge ear thickness), and the group average ear swelling of all treated mice was determined at this time point.

Dose responsiveness of CO-RM effect

Fresh CO-RM lotions of 50, 125, 250, and 500 M were applied daily for 5 consecutive days. Immunosuppression was induced on day 1 by three applications within 24 h of cis-UCA lotion, as above, and CHS to oxazolone was induced and elicited as described above. cis-UCA was chosen as the immunosuppressive agent rather than SSUVR, which contains UVA radiation that could produce endogenous CO from induced HO-1 activity. Control mice received the same concentration of CO-RM without cis-UCA treatment.

Treatment with specific inhibitors

The guanylyl cyclase inhibitor, ODQ (Sigma-Aldrich), and the p38 MAPK inhibitor SB203580 (Calbiochem, Croydon, Victoria, Australia) were each dissolved in DMSO, and then incorporated into the base lotion for topical application. The final lotions contained 10% DMSO and 10 M ODQ or 10 M SB203580, and the control lotion contained only DMSO. Aliquots of 200 L were applied to the dorsal skin immediately following UV irradiation or cis-UCA treatment and prior to the applications of the CO-RM, daily for the next 4 d. The CHS response was induced on days 8 and 9 following the SSUVR.

To support that CO released from topical CO-RM was available to activate a relevant biological target in the skin, groups of three mice were treated with topical 500 M CO-RM lotion daily for 3 d, or received 10 M ODQ application (inhibitor of soluble guanylyl cyclase) 30 min prior to the CO-RM. Skin was taken 3 h after the last CO-RM application, frozen, homogenized, freed of protein and fat, and the supernatant was analyzed for cGMP by a commercial ELISA kit (R&D Systems, Minneapolis, Missouri).

Statistics

The significance of the differences between treatment groups was evaluated using ANOVA followed by analysis by the Tukey (HSD) test (XLSTAT program) with a confidence range of 95%.

References

1. Allanson, M, Reeve, VE: Immunoprotective UVA (320–400 nm) irradiation upregulates heme oxygenase-1 in the dermis and epidermis of hairless mouse skin. J Invest Dermatol 2004 122: 1030–1036, 10.1111/j.0022-202X.2004.22421.x | Article | PubMed | ISI | ChemPort |
2. Amersi, F, Shen, XD, Anselmo, D, et al: Ex vivo exposure to carbon monoxide prevents hepatic ischemia/reperfusion injury through p38 MAP kinase pathway. Hepatology 2002 35: 815–823, 10.1053/jhep.2002.32467 | Article | PubMed | ISI | ChemPort |
3. Applegate, LA, Noel, A, Vile, G, Frenk, K, Tyrell, RM: Two genes contribute to different extents to the heme oxygenase enzyme activity measured in cultured human skin fibroblasts and keratinocytes implications for protection against oxidant stress. Photochem Photobiol 1995 61: 285–291,  | PubMed | ISI | ChemPort |
4. Aubin, F, Dall'Acqua, F, Kripke, ML: Local suppression of contact hypersensitivity in mice by a new bifunctional psoralen, 4,4'5'-trimethylazapsoralen, and UVA radiation. J Invest Dermatol 1991 97: 50–54, 10.1111/1523-1747.ep12478010 | Article | PubMed | ISI | ChemPort |
5. Bestak, R, Halliday, GM: Chronic low-dose UVA irradiation induced local suppression of contact hypersensitivity, Langerhans cell depletion and suppressor cell activation in C3H/HeJ mice. Photochem Photobiol 1996 64: 969–974,  | PubMed | ISI | ChemPort |
6. Brouard, S, Otterbein, LE, Anrather, J, Tobiasch, E, Bach, FH, Choi, AM, Soares, MP: Carbon monoxide generated by heme oxygenase 1 suppresses endothelial cell apoptosis. J Exp Med 2000 192: 1015–1026,  | Article | PubMed | ISI | ChemPort |
7. Carraway, MS, Ghio, AJ, Carter, JD, Piantadosi, CA: Expression of heme oxygenase-1 in the lung in chronic hypoxia. Am J Physiol 2000 278: L806–L812,  | ISI | ChemPort |
8. Clark, JE, Foresti, R, Green, CJ, Motterlini, R: Dynamics of haem oxygenase expression and bilirubin production in cellular protection against oxidative stress. Biochem J 2000 348: 615–619,  | Article | PubMed | ISI | ChemPort |
9. Clark, JE, Naughton, P, Shurey, S, et al: Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule. Circ Res 2003 93: e2–e8,  | Article | PubMed | ISI | ChemPort |
10. Damian, DL, Barnetson, RStC, Halliday, GM: Low-dose UVA and UVB have different time courses for suppression of contact hypersensitivity to a recall antigen in humans. J Invest Dermatol 1999 112: 939–944, 10.1046/j.1523-1747.1999.00610.x | Article | PubMed | ISI | ChemPort |
11. DiBello, MG, Berni, L, Gai, P, et al: A regulatory role for carbon monoxide in mast cell function. Inflam Res 1998 47: S7–S8,  | ChemPort |
12. Dumay, O, Karam, A, Vian, L, et al: Ultraviolet A1 exposure of human skin results in Langerhans cell depletion and reduction of epidermal antigen-presenting cell function: partial protection by a broad spectrum sunscreen. Br J Dermatol 2001 144: 1161–1168, 10.1046/j.1365-2133.2001.04225.x | Article | PubMed | ISI | ChemPort |
13. Durante, W: Heme oxygenase-1 in growth control and its clinical application to vascular disease. J Cell Physiol 2003 95: 373–382, 10.1002/jcp.10274
14. Garssen, J, de Gruijl, F, Mol, D, deKlerk, A, Roholl, P, van Loveren, H: UVA exposure affects UVB and cis-urocanic acid-induced systemic suppression of immune responses in Listeria monocytogenes-infected Balb/c mice. Photochem Photobiol 2001 73: 432–438, 10.1562/0031-8655(2001)073&#60;0432:UEAUAC>2.0.CO;2 | Article | PubMed | ISI | ChemPort |
15. Gensler, HL, Magdaleno, M: Topical vitamin E inhibition of immunosuppression and tumorigenesis induced by ultraviolet irradiation. Nutr Cancer 1991 15: 97–106,  | PubMed | ISI | ChemPort |
16. Guo, Y, Stein, AB, Wu, WJ, Tan, W, Zhu, X, Li, QH, Dawn, B: Administration of a CO-releasing molecule at the time of reperfusion reduces infarct size in vivo. Am J Physiol 2004 286: H1649–H1654,  | ISI | ChemPort |
17. Hughey, JL, Bock, CR, Meyer, TJ: Flash photolysis evidence for metal–metal bond cleavage and loss of CO in the photochemistry of [N⁵-(C₂H₅)Mo(CO)₃]₂. J Am Chem Soc 1975 97: 2065–2072,  | Article | ISI | ChemPort |
18. Iwai, I, Hatao, M, Naganuma, M, Kumano, Y, Ichihashi, M: UVA-induced immune suppression through an oxidative pathway. J Invest Dermatol 1999 112: 12–18,
19. Kharitonov, V, Sharma, VS, Pilz, RB, Magde, D, Kosling, D: Basis of guanylate cyclase activation by carbon monoxide. Proc Natl Acad Sci USA 1995 92: 2568–2571,  | PubMed | ChemPort |
20. Khaskhely, NM, Maruno, M, Takamiyagi, A, et al: Pre-exposure withy low-dose UVA suppresses lesion development and enhances Th1 response in BALB/c mice infected with Leishmania amazonensis. J Dermatol Sci 2001 26: 217–232, 10.1016/S0923-1811(01)00098-6 | Article | PubMed | ISI | ChemPort |
21. Maines, MD: The heme oxygenase system: A regulator of second messenger gases. Ann Rev Pharmacol Toxicol 1997 37: 517–554, 10.1146/annurev.pharmtox.37.1.517 | Article | ISI | ChemPort |
22. Motterlini, R, Clark, JE, Foresti, R, Sarathchandra, P, Mann, BE, Green, CJ: Carbon monoxide-releasing molecules: Characterization of biochemical and vascular activities. Circ Res 2002 90: e17–e24, 10.1161/hh0202.104530 | Article | PubMed | ISI | ChemPort |
23. Motterlini, R, Foresti, R, Bassi, R, Calabrese, V, Clark, JE, Green, CJ: Endothelial heme oxygenase-1 induction by hypoxia: Modulation by inducible nitric oxide synthase (iNOS) and S-nitrosothiols. J Biol Chem 2000 275: 13613–13620, 10.1074/jbc.275.18.13613 | Article | PubMed | ISI | ChemPort |
24. Murphy, BJ, Laderoute, KR, Short, SM, Sutherland, RM: The identification of heme oxygenase as a major hypoxic stress protein in Chinese hamster ovary cells. Br J Cancer 1991 64: 69–73,  | PubMed | ISI | ChemPort |
25. Nakamura, T, Pinnell, SR, Darr, D, Kurimoto, I, Itami, S, Yoshikawa, K, Streilein, JW: Vitamin C abrogates the deleterious effects of UVB radiation on cutaneous immunity by a mechanism that does not depend on TNF-alpha. J Invest Dermatol 1997 109: 20–24, 10.1111/1523-1747.ep12276349 | Article | PubMed | ISI | ChemPort |
26. Ndisang, JF, Gai, P, Berni, L, Mirabella, C, Baronti, R, Mannaioni, PF, Masini, E: Modulation of the immunological response of guinea pig mast cells by carbon monoxide. Immunopharmacology 1999 43: 65–73, 10.1016/S0162-3109(99)00045-4 | Article | PubMed | ISI | ChemPort |
27. Ngheim, DX, Kazimi, N, Clydesdale, G, Ananthaswamy, H, Kripke, ML, Ullrich, SE: Ultraviolet A radiation suppresses an established immune response: Implications for sunscreen design. J Invest Dermatol 2001 117: 1193–1199, 10.1046/j.0022-202x.2001.01503.x | Article | PubMed |
28. Otterbein, LE, Bach, FH, Alam, J, et al: Carbon monoxide has anti-inflammatory effects involving the mitogen activated protein kinase pathway. Nat Med 2000 6: 422–428,  | Article | PubMed | ISI | ChemPort |
29. Otterbein, LE, Otterbein, SL, Ifedigbo, E, et al: MKK3 mitogen-activated protein kinase pathway mediates carbon monoxide-induced protection against oxidant induced lung injury. Am J Path 2003 163: 2555–2563,  | PubMed | ISI | ChemPort |
30. Peyton, KJ, Reyna, SV, Chapma, GB, et al: Heme oxygenase-1-derived carbon monoxide is an autocrine inhibitor of vascular smooth muscle cell growth. Blood 2002 99: 4443–4448,  | Article | PubMed | ISI | ChemPort |
31. Reeve, VE, Bosnic, M, Boehm-Wilcox, C, Nishimura, N, Ley, RD: Ultraviolet A radiation (320–400 nm) protects hairless mice from immunosuppression induced by ultraviolet B radiation (280–320 nm) or cis-urocanic acid. Int Arch Allergy Immunol 1998 115: 316–322, 10.1159/000069463 | Article | PubMed | ISI | ChemPort |
32. Reeve, VE, Bosnic, M, Rozinova, E: Carnosine (beta-alanylhistidine) protects from the suppression of contact hypersensitivity by ultraviolet B (280–320 nm) radiation or by cis urocanic acid. Immunology 1993 78: 99–104,  | PubMed | ISI | ChemPort |
33. Reeve, VE, Domanski, D: Refractoriness of UVA-induced protection from photoimmunosuppression correlates with heme oxygenase response to repeated UVA exposure. Photochem Photobiol 2002 76: 401–405, 10.1562/0031-8655(2002)076&#60;0401:ROUIPF>2.0.CO;2 | Article | PubMed | ISI | ChemPort |
34. Reeve, VE, Tyrrell, RM: Heme oxygenase induction mediates the photoimmunoprotective activity of UVA radiation in the mouse. Proc Natl Acad Sci USA 1999 96: 9317–9321, 10.1073/pnas.96.16.9317 | Article | PubMed | ChemPort |
35. Sato, K, Balla, J, Otterbein, L, et al: Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J Immunol 2001 166: 4185–4194,  | PubMed | ISI | ChemPort |
36. Skov, L, Hansen, H, Barker, JNWN, Simon, JC, Baadsgaard, O: Contrasting effects of ultraviolet-A and ultraviolet-B exposure on induction of contact hypersensitivity in human skin. Clin Exp Immunol 1997 107: 585–588, 10.1046/j.1365-2249.1997.d01-944.x | Article | PubMed | ISI | ChemPort |
37. Skov, L, Villadsen, B, Ersboll, BJ, Simon, JC, Barker, J, Baadsgaard, O: Long-wave UVA offers partial protection against UVB-induced immune suppression in human skin. Acta Pathologica Microbiologica et Immunologica Scandinavica 2000 108: 825–830,  | ChemPort |
38. Song, R, Mahidhara, RS, Liu, F, Ning, W, Otterbein, LE, Choi, AM: Carbon monoxide inhibits airway smooth muscle cell proliferation via mitogen-activated protein kinase pathway. Am J Respir Cell Mol Biol 2002 27: 603–610,  | PubMed | ISI | ChemPort |
39. Steiner, AA, Branco, LG, Cunha, FQ, Ferreira, SH: Role of the haem oxygenase/carbon monoxide pathway in mechanical nociceptor hypersensitivity. Br J Pharmacol 2001 132: 1673–1682, 10.1038/sj.bjp.0704014 | Article | PubMed | ISI | ChemPort |
40. Stocker, R, Glazer, RN, Ames, BN: Antioxidant activity of albumin-bound bilirubin. Proc Natl Acad Sci USA 1987 84: 5918–5922,  | Article | PubMed | ChemPort |
41. Van Uffelen, BE, de Koster, BM, Van Steveninck, J, Elferink, JG: Carbon monoxide enhances human neutrphil migration in a cyclic GMP-dependent way. Biochem Biophys Res Comm 1996 226: 21–26, 10.1006/bbrc.1996.1305 | PubMed | ChemPort |
42. Vile, GF, Tyrrell, RM: Oxidative stress resulting from ultraviolet A irradiation of human skin fibroblasts leads to a heme oxygenase dependent increase in ferritin. J Biol Chem 1993 268: 14678–14681,  | PubMed | ISI | ChemPort |
43. Von Burg, R: Carbon monoxide. J Appl Toxicol 1999 19: 379–386, 10.1002/(SICI)1099-1263(199909/10)19:5&#60;379::AID-JAT563>3.0.CO;2-8 | Article | PubMed | ISI | ChemPort |
44. Weller, R, Schwentker, A, Billiar, TR, Vodovotz, Y: Autologous nitric oxide protects mouse and human keratinocytes from ultraviolet B radiation-induced apoptosis. Am J Physiol 2003 284: C1140–C1148,  | ISI | ChemPort |
45. Wrighton, MS, Ginley, DS: Photochemistry of metal–metal bonded complexes: II The photochemistry of rhenium and manganese carbonyl complexes containing a metal–metal bond. J Am Chem Soc 1975 97: 2065–2072,  | Article | ISI | ChemPort |

Acknowledgments

This study was supported by the National Health and Medical Research Council of Australia, and the University of Sydney Cancer Research Fund. We are grateful to Ms Diane Domanski for excellent research assistance.