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Department of Medicine, Division of Newborn Medicine, Children’s Hospital and Department of Pediatrics, Harvard Medical School, Boston, Massachusetts 02115
Abstract
An increasing number of studies implicate heme oxygenase-1 (HO-1) in the regulation of inflammation. Although the mechanisms involved in this cytoprotection are largely unknown, HO-1 and its enzymatic products, carbon monoxide and bilirubin, downregulate the inflammatory response by either attenuating the expression of adhesion molecules and thus inhibiting leukocyte recruitment or by repressing the induction of cytokines and chemokines. In the present study we used genetically engineered mice that express high levels of a human cDNA HO-1 transgene in lung epithelium to assess the effect of HO-1 on lung inflammation. Two separate models of inflammation were studied: hypoxic exposure and lipopolysaccharide (LPS) challenge. We found that both mRNA and protein levels of specific cytokines and chemokines were significantly elevated in response to hypoxia in the lungs of wild-type mice after 2 and 5 days of exposure but significantly suppressed in the hypoxic lungs of transgenic mice, suggesting that inhibition of these cytokines was caused by overexpression of HO-1. However, LPS treatment resulted in a very pronounced increase in mRNA levels of several cytokines in both wild-type and transgenic mice. Despite the high mRNA levels, significantly lower cytokine protein levels were detected in the bronchoalveolar lavage of HO-1 overexpressing mice compared with wild type, indicating that HO-1 leads to repression of cytokines in the airway. These results demonstrate that HO-1 activity operates through distinct molecular mechanisms to confer cytoprotection in the hypoxic and the LPS models of inflammation.
Key Words: sepsis shock • hypoxia • adhesion molecule
Heme oxygenase (HO) catalyzes the rate-limiting step of heme oxidation to biliverdin, carbon monoxide, and iron (1). Biliverdin is rapidly converted to bilirubin, a potent endogenous antioxidant (2). Three isoforms of HO have been reported: the inducible HO-1 and the constitutively expressed HO-2 and HO-3 (3).
An increasing number of studies implicate HO-1 in the regulation of inflammation. It was shown to have protective effects in cardiac xenograft rejection (4), endotoxin challenge (5), as well as in hyperoxic lung injury and ischemia–reperfusion in the liver, two well-known models of inflammation-mediated injury (6, 7). HO-1 deficiency, on the other hand, is associated with a chronically inflamed state and increased leukocyte recruitment as reported in the human (8) and in mice null for the HO-1 gene (9, 10).
Although the mechanisms involved in this cytoprotection are largely unknown, HO-1 activity and its enzymatic products, carbon monoxide and bilirubin, have been shown to downregulate the inflammatory response by either attenuating the expression of adhesion molecules and thus inhibiting leukocyte recruitment (11, 12), or by repressing the induction of cytokines and chemokines (5, 13). In the present study, we used genetically engineered mice that constitutively express HO-1 in the lung. Transgenic mice harboring the human HO-1 gene under the control of human surfactant protein-C promoter shown to express high levels of HO-1 mRNA and protein (13) were used to assess the effect of HO-1 on lung inflammation. Two separate models of inflammation were studied: hypoxic exposure and lipopolysaccharide (LPS) treatment. For the hypoxic treatment, mice were exposed to an 8–10% O2 environment for 3 hr and up to 14 days and lung samples were harvested for RNA extraction. To estimate the expression of cytokine genes, RNase Protection Assay was performed and mRNA levels were quantified using a Phosphorimager (Molecular Dynamics/Amersham, Sunnydale, CA).
As shown in Figure 1
, expression levels of cytokines interleukin (IL)-1ß and IL-6 and chemokines macrophage inflammatory protein (MIP)-1
, MIP-1ß, monocyte chemoattractant protein (MCP)-1, and MIP-2 were significantly elevated in response to hypoxia in the lungs of wildtype mice at 2 and 5 days. These increased levels of cytokine expression peaked at 2 days and were almost totally resolved by 14 days of hypoxia treatment. In contrast, other proinflammatory mediators, such as tumor necrosis factor (TNF)-, interferon (IFN)-
, and IL-1 and anti-inflammatory mediators, such as IL-10 were unaffected. More importantly, induction of IL-1ß, IL-6, MCP-1, and MIP-2 was significantly suppressed in the hypoxic lungs of transgenic mice, suggesting that inhibition of these cytokines was caused by overexpression of HO-1. Along with the RNA analysis, protein levels of MIP-2 were measured by enzyme-linked immunosorbent assay (ELISA) in lung extracts. MIP-2 protein was undetectable at baseline in all normoxic samples but, after 2 and 5 days of hypoxia, increased levels of MIP-2 protein were measured in the hypoxic lung of wild-type mice. However, MIP-2 protein was not detected in the hypoxic lung of transgenic mice (13).
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, elevated levels of the proinflammatory cytokines TNF-, IL-1, and IL-1ß were detected in the lungs of both wild-type and transgenic mice after 1 hr of treatment. In addition to the above mediators, a number of cytokines and chemokines were also induced. Extremely robust levels of IL-1 receptor antagonist (IL-1Ra), cytokine IL-6, and chemokines MIP-1, MIP-1ß, MIP-2, IP-10, and MCP-1 were detected in the LPS-treated lungs of both transgenic and wild- type mice. Quantification of relative induction showed that there was no difference in the mRNA level of these inflammatory mediators between the wild-type and transgenic animals.
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B known to be a major regulator of chemokine expression (14–18). Understanding the effect of HO-1 on nuclear factor-B could elucidate the mechanisms of cytoprotection conferred in hypoxia. Exploring the LPS-induced inflammation model, we were unable to detect any effect of HO-1 overexpression on the rapid and robust induction of cytokine mRNA levels in the lung. This could indicate that the target of the HO-1 cytoprotective action is located upstream of the point where the LPS and the hypoxic signaling pathways converge into proinflammatory transcriptional activity in the nucleus. Although this would be the simplest mechanistic explanation, a number of alternative interpretations are possible given the drastically different inflammation models studied here. Prime among the models under active consideration is one incorporating the assumption that, whereas LPS can elicit an acute and widespread inflammatory response in the lung, hypoxia can activate inflammatory pathways that involve distinct cell populations, primarily in areas coinciding with or abutting the Type II alveolar cells. Because HO-1 is specifically overexpressed in the alveoli of transgenic mice (13), the diffusible enzymatic byproducts, CO and biliverdin/bilirubin, could downregulate proinflammatory responses in the immediate vicinity of these cells. This would be easily detected even at the total lung mRNA level of analysis. A more generalized inflammatory response to LPS stimulation would induce high levels of cytokine mRNA from nonalveolar regions. In a total lung RNA analysis this abundance could mask the absence of inflammatory response from the perialveolar areas.
An intriguing observation in the LPS inflammation model is the significant decrease of cytokine levels in the alveolar lavage of transgenic mice, as assessed by MIP-2 immunoreactivity, demonstrating that even in this acute model, HO-1 overexpression can confer anti-inflammatory protection. The crucial role of MIP-2 in lung inflammation, as assessed by neutrophil infiltration, has been demonstrated in studies using neutralizing antibodies to MIP-2 whereby neutrophil accumulation was significantly reduced after LPS (19) or IgG immune complex-induced lung injury (20). The downregulation of MIP-2 protein by HO-1, therefore, could be critical in reducing neutrophil recruitment and thus limiting lung injury from LPS. Because there is no effect on the overall lung cytokine mRNA, one is tempted to speculate that HO-1 overexpression can confer anti-inflammatory protection by decreasing cytokine expression at a post-transcriptional level. Significantly, a similar, post-transcriptional protective action of exogenous CO, the HO-1 enzymatic product, has also been observed in the LPS model as assessed by the decrease of serum cytokine (TNF-) immunoreactivity. In that study, the p38 MAPK pathway was shown to play a critical role in mediating the anti-inflammatory action of CO (5). The unequivocal demonstration of HO-1 activity inhibiting cytokine gene downstream of transcription would further underlie the critical nature of this versatile enzyme in anti-inflammatory processes. Identifying the role of the signaling pathways involved in the inflammatory response and the effects of HO-1 on their activation could provide new insights and point to new directions for the development of anti-inflammatory strategies and therapeutic approaches.
Footnotes
1 To whom requests for reprints should be addressed at Division of Newborn Medicine, Children’s Hospital, Enders 9, 300 Longwood Ave., Boston, MA 02115. E-mail: stella.kourembanas{at}TCH.Harvard.edu 
References
B and IB proteins: New discoveries and insights. Annu Rev Immunol 14:649–681, 1996.[Medline]
B—A pivotal transcription factor in chronic inflammation diseases. N Engl J Med 336:1066–1071, 1997.B and human disease. J Clin Invest 107:3–6, 2001.[Medline]
B signaling pathways in mammalian and insect innate immunity. Genes Dev 15:2321–2342, 2001.B puzzle. Cell 109:S81–S96, 2002.
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