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Experimental Biology and Medicine 228:514-516 (2003)
© 2003 Society for Experimental Biology and Medicine

HEME OXYGENASE

Participation of Heme Oxygenase-1 in a Model of Acute Inflammation

Ana María Vicente, María Isabel Guillín and María Josí Alcaraz1

Department of Pharmacology, University of Valencia, 46100 Burjasot, Valencia, Spain

Abstract

In this study, the role of heme oxygenase-1 (HO-1) in the inflammatory response elicited by zymosan in the mouse air pouch model has been examined. This response is characterized by a time-dependent increase in HO-1 expression in the leukocytes migrating into the exudates. At 24–48 h maximal HO-1 expression was accompanied by reduced cyclooxygenase-2 (COX-2) and nitric oxide synthase-2 (NOS-2) expression as well as low levels of inflammatory mediators. Hemin administration into the air pouch caused an elevation of HO-1 protein and bilirubin levels induced by zymosan with inhibition of COX-2 expression. In mouse peritoneal macrophages from hemin-injected mice, we also observed an increased expression of HO-1 with inhibition of COX-2 expression and prostaglandin E2 (PGE2) levels. Our data suggest an anti-inflammatory role for HO-1 in the response induced by this phagocytic stimulus.

Key Words: adhesion molecule • inflammation • cyclooxygenase

Heme oxygenase-1 (HO-1) is part of the integrated response to oxidative stress. The expression of this protein increases in inflammatory cells upon stimulation with a variety of agents and may be associated with the resolution phase of acute inflammation (1). Activation of phagocytes is a key component of the pathophysiology of inflammation, which leads to the generation of reactive oxygen and nitrogen intermediates, in addition to a number of mediators contributing to tissue injury. However, it is known that monocytes/macrophages participate in the negative regulation of inflammatory responses through the phagocytosis of apoptotic cells and production of several proteins, which could play potentially important roles in the resolution phase. Previous studies demonstrated the expression of HO-1 in the RAW 264.7 cell line of murine macrophages activated by zymosan (2). In the present work, we address the participation of HO-1 in the regulation of the in vivo inflammatory response to zymosan.

Materials and Methods

Mouse Air Pouch.
Air pouch was produced in female Swiss mice (25–30 g) as previously described (3). Six days after the initial air injection, 1 ml of sterile saline or 1 ml of 1% w/v zymosan in saline was injected into the air pouch. Hemin (50 nmol/pouch) was administered 30 min before and 8 and 18 hr after zymosan. At different time points, animals were killed by cervical dislocation and exudates collected. After centrifugation, the supernatants were used to assay bilirubin spectrophotometrically (4), leukotriene B4, and prostaglandin E2 (PGE2) by radioimmunoassay, nitrite (3), and tumor necrosis-{alpha} and interleukin-1ß by fluoroimmunoassay. Cell pellets were used to study protein expression by Western blot analysis with specific polyclonal antibodies (2).

Mouse Peritoneal Macrophages.
After 18 hr of intraperitoneal administration of either saline or hemin (2.6 mg/kg ip) to female Swiss mice, resident peritoneal macrophages were harvested by peritoneal lavage and resuspended at 3 x 106 cells/ml in Dulbecco’s modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, and penicillin/streptomycin. After incubation at 37°C for 2 hr, adherent cells were stimulated with zymosan (0.05 mg/ml) for 6 hr.

Results and Discussion

We were interested to find out whether HO-1 induction participated in the inflammatory response to zymosan in the mouse air pouch as well as its relationship with other inducible enzymes. Figure 1Go shows the time course of HO-1, cyclo-oxygenase-2 (COX-2), and nitric oxide synthase-2 (NOS-2) induction after zymosan administration. None of these enzymes was detected in saline-injected mice. COX-2 expression peaked at 12 hr and disappeared by 48 hr after zymosan administration, whereas HO-1 and NOS-2 showed similar profiles, with initial detection at 12 hr followed by increased levels at 24–48 hr.



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  		Figure 1. Time-course of HO-1, COX-2, and NOS-2 protein expression in the mouse air pouch. Band intensity is represented as arbitrary units and mean ± SE mean of three experiments was calculated.

 
In this model of inflammation, cells present in exudates were mainly neutrophils with maximal migration at 12 hr (Table IGo). The concentration of cells in exudates at 48 hr recovered basal levels according to the phase of resolution. The highest concentration of arachidonic acid metabolites was observed at 4 hr (leukotriene B4) and 12 hr (PGE2), which correlated with COX-2 expression. Nitrite concentration in exudates increased with time after NOS-2 upregulation. High levels of cytokines (tumor necrosis factor-{alpha} and interleukin-1ß) were present in the early phase of this response (4 hr), decreasing at later times. It is interesting to note that maximal HO-1 expression coincided with the return to basal levels of inflammatory mediators and COX-2 downregulation.


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  		Table I. Mediators in Exudates of the Zymosan-Treated Mouse Air Pouch
 
We also determined the effect of hemin on the modulation of experimental inflammation after zymosan administration. In Figure 2AGo, we show that HO-1 was induced by hemin at 12 and 24 hr without affecting COX-2 or NOS-2. This induction was accompanied by a transient bilirubin production (4 hr; Fig. 2BGo). Hemin was also able to increase HO-1 expression and bilirubin production induced by zymosan. This treatment reduced COX-2 protein expression at 12 and 24 hr without modifying PGE2 levels (data not shown), whereas NOS-2 expression did not change.



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  		Figure 2. Effect of hemin on HO-1, COX-2, and NOS-2 expression and bilirubin levels in exudates of the mouse air pouch. (A) Figures are representative of three similar experiments. (B) Data are the mean ± SE mean of 12 animals. *P < 0.05, **P < 0.01.

 
We have shown that hemin potentiated the induction of HO-1 by zymosan and reduced COX-2 expression in the mouse air pouch. Because the time of HO-1 appearance coincided with an increase in the presence of monocytes/macrophages in exudates, we have confirmed these results in another experimental model (mouse peritoneal macrophages). Figure 3Go shows that hemin treatment increased HO-1 expression in macrophages either in basal conditions or after zymosan stimulation, whereas COX-2 expression and PGE2 levels decreased. In this model, hemin acts as inducer of HO-1 but it is not present during incubation, thus avoiding effects as co-factor of COX-2 activity that are likely in the mouse air pouch. These results support a negative regulation of COX-2 by HO-1. Recently, it has been demonstrated that the heme-HO system regulates COX enzyme expression in vascular endothelial cells (5).



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  		Figure 3. Effect of hemin administration on HO-1 and COX-2 expression and PGE2 production in mouse peritoneal macrophages. (A) Band intensity is represented as arbitrary units and mean ± SE mean of three experiments was calculated. (B) PGE2 levels were determined in supernatants. Data are the mean ± SE mean of three experiments. *P < 0.05, **P < 0.01. Basal: cells not stimulated with zymosan.

 
We have shown in vivo the participation of HO-1 in zymosan-induced inflammatory responses and the effect of its up-regulation on COX-2 expression, suggesting a role for HO-1 in the control of the inflammatory response.

Acknowledgments

A.M. Vicente thanks Generalitat Valenciana for a fellowship.

Footnotes

This work was supported by grant SAF2001-2919.

1 To whom requests for reprints should be addressed at Department of Pharmacology, University of Valencia, 46100 Burjasot, Valencia, Spain. E-mail: maria.j.alcaraz{at}uv.es Back

References

  1. Willoughby DA, Moore AR, Colville-Nash PR, Gilroy D. Resolution of inflammation. Int J Immunopharmacol 22:1131–1135, 2000.[Medline]
  2. Vicente AM, Guillen MI, Alcaraz MJ. Modulation of haem oxygenase-1 expression by nitric oxide and leukotrienes in zymosan-activated macrophages. Br J Pharmacol 33:920–926, 2001.
  3. Posadas I, Terencio MC, Guillén I, Ferrándiz ML, Coloma J, Payá M, Alcaraz MJ. Co-regulation between cyclo-oxygenase-2 and inducible nitric oxide synthase expression in the time-course of murine inflammation. Naunyn-Schmiedeberg’s Arch Pharmacol 361:98–106, 2000.[Medline]
  4. Turcanu V, Dhouib M, Poindron P. Determination of heme oxygenase activity in murine macrophages for studying oxidative stress inhibitors. Anal Biochem 263:251–253, 1998.[Medline]
  5. Haider A, Olszanecki R, Gryglewski R, Schwartzman ML, Lianos E, Kappas A, Nasjletti A, Abraham NG. Regulation of cyclooxygenase by the heme-heme oxygenase system in microvessel endothelial cells. J Pharmacol Exp Ther 300:188–194, 2002.[Abstract/Free Full Text]



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