Iron Chelation Inhibits NF- B-Mediated Adhesion Molecule Expression by Inhibiting p22 phox Protein Expression and NADPH Oxidase Activity

【摘要】  Objective- Excess iron may increase oxidative stress and play a role in vascular inflammation and atherosclerosis. Here we determined whether the iron chelator, desferrioxamine (DFO), ameliorates oxidative stress and cellular adhesion molecule expression in a murine model of local inflammation.

Methods and Results- Dorsal air pouches were created in C57BL/6J mice by subcutaneous injection of air. DFO (100 mg/kg body weight) was injected into the air pouch once a day for two days followed immediately on the second day by lipopolysaccharide (LPS; 2.5 mg/kg body weight). The animals were euthanized 24 hours later for analysis of oxidative stress markers and adhesion molecules in air pouch tissue. LPS treatment enhanced protein levels of p22 phox, a catalytic subunit of NADPH oxidase, and increased NADPH oxidase activity and levels of superoxide radicals and hydrogen peroxide. Furthermore, LPS activated NF- B and increased expression of adhesion molecules. All of these inflammatory responses were strongly suppressed by DFO, but not iron-loaded DFO.

Conclusions- Our data show that DFO inhibits LPS-induced, NADPH oxidase-mediated oxidative stress and, hence, NF- B activation and adhesion molecule expression in a murine model of local inflammation. Iron chelation may be helpful in treating atherosclerotic vascular diseases by ameliorating oxidative stress and inflammation.

This study shows that the iron chelator, desferrioxamine (DFO), but not iron-loaded DFO, inhibits NF- B activation and cellular adhesion molecule expression in a murine model of local inflammation by inhibiting p22 phox protein expression, NADPH oxidase activity, and oxidant production. Therefore, chelation of excess iron may help attenuate inflammation and atherosclerosis.

【关键词】  adhesion molecules desferrioxamine LPS NADPH oxidase

Introduction

It is now well established that atherosclerosis is a vascular inflammatory disease. 1 Plasma or serum levels of inflammatory mediators such as C-reactive protein, soluble adhesion molecules, and cytokines and chemokines predict an increased risk of cardiovascular events in patients with coronary artery disease or coronary risk factors. 1-3 Recent evidence indicates that some standard therapies known to reduce cardiovascular risk, such as lipid-lowering statins, also exert antiinflammatory effects. 3

LPS is an endotoxin from the outer membrane of Gram-negative bacteria and a potent proinflammatory agonist. LPS has been recently recognized as a potential mediator of inflammatory responses in the setting of atherosclerosis. 4 LPS is known to increase oxidative stress and expression of cellular adhesion molecules, 5-7 two important factors in the development of atherosclerosis. 8-9 Vascular cell adhesion molecule-1 (VCAM-1) appears to play a particularly important role in early atherosclerosis. 10-11 Increased expression of VCAM-1 and other adhesion molecules on the vascular endothelium results in increased recruitment of monocytes to the arterial wall, where they undergo differentiation to resident macrophages that can further enhance vascular inflammation and trigger atherosclerotic lesion formation. 9-11

NADPH oxidase-derived reactive oxygen species (ROS), in particular superoxide radicals (O 2 ·-) and their dismutation product, hydrogen peroxide (H 2 O 2 ), have been implicated in the development of human atherosclerotic lesions. 12 Intracellular ROS may increase transcription of inflammatory genes, including cellular adhesion molecules, via activation of the transcription factor, nuclear factor B (NF- B). 13,14 Animal studies have shown that NADPH oxidase-derived O 2 ·- plays a key role in regulating VCAM-1 expression in the arterial wall. 15-17

Interestingly, current strategies to reduce ROS and oxidative stress by antioxidant supplements do not seem effective in treating patients with coronary artery disease. 18-20 It has been suggested, but remains unproven, that iron plays a role in vascular inflammation and atherosclerosis, 21,22 and that iron chelating agents may help ameliorate inflammation and atherosclerosis by lowering oxidative stress. 14,23 Desferrioxamine (DFO) is a potent iron chelator used clinically to remove both intra- and extracellular iron. 24,25 In the present study, we investigated whether DFO inhibits LPS-induced oxidative stress, NF- B activation, and cellular adhesion molecule expression in a murine model of local inflammation.

Materials and Methods

For detailed Materials and Methods, please see http://atvb.ahajournals.org.

Results

Desferrioxamine Inhibits LPS-Induced p22 phox Protein Expression and NADPH Oxidase Activity

Dorsal air pouches were created in C57BL/6J mice as described in Methods. Twenty-four hours after the injection of LPS (2.5 mg/kg b.w. in 1 mL Hank?s balanced salt solution) into the air pouch, a 3.3-fold increase in leukocyte content in air pouch lavage fluid was observed (from 90±8 leukocytes/µL in control animals [n=8] to 301±58 leukocytes/µL in LPS-treated animals [n=8]). In contrast, no leukocytes were observed in the synovial tissue lining the air pouch, which is comprised mainly of fibroblasts. 5 LPS treatment also led to a 3.5-fold increase in protein levels of p22 phox, a catalytic subunit of NADPH oxidase, in the air pouch synovial tissue ( Figure 1 ). Injection of the iron-chelator, DFO (100 mg/kg b.w.), 24 hours before and a second time immediately before LPS administration into the air pouch significantly inhibited the increase in p22 phox protein levels ( Figure 1 ). In contrast, neither DFO loaded with an equimolar amount of Fe(III) nor apocynin (10 -3 mol/L in 1 mL Hank?s balanced salt solution) inhibited the LPS-induced increase in p22 phox protein levels ( Figure 1 ). Apocynin inhibits the assembly of the active NADPH oxidase complex by preventing translocation of one of its cytosolic subunits, p47 phox, from the cytosol to the plasma membrane. 26 Protein levels of p47 phox also increased 3-fold after LPS treatment, but this increase was not inhibited by DFO or apocynin (data not shown).

Figure 1. Protein levels of p22 phox in mouse air pouch tissue. Animals were treated without or with LPS, desferrioxamine (DFO), apocynin (APO), or Fe(III)-loaded DFO (DFO-FeIII) as described in Methods. Homogenized air pouch tissue was subjected to Western blot analysis. The protein bands of p22 phox and actin (molecular sizes, 22 kDa and 43 kDa, respectively) were scanned by densitometry and the relative protein levels were expressed as the ratio of p22 phox :actin (upper part of the figure). Data shown are the means±SEM of 4 to 6 mice per group; * P <0.05 vs control, # P <0.05 and P 0.05 vs LPS-treated group. The lower part of the figure shows a representative Western blot for two animals per group. "A-10" indicates the A-10 whole cell lysate, which serves as the positive control.

The enzymatic activity of NADPH oxidase increased almost 5-fold in air pouch tissue 24 hours after LPS administration, and DFO completely inhibited this increase ( Figure 2 ). The basal activity of xanthine oxidase, another O 2 ·- -generating enzyme, was more than 6-fold lower than that of NADPH oxidase and was not increased by LPS treatment (supplemental Figure I, available online at http://atvb.ahajournals.org). As expected, apocynin strongly inhibited LPS-stimulated NADPH oxidase activity ( Figure 2 ), while it had no effect on p22 phox protein levels (see above, Figure 1 ). Importantly, Fe(III)-loaded DFO did not inhibit the LPS-induced increase in NADPH oxidase activity ( Figure 2 ). Taken together, these data suggest that DFO inhibits the LPS-induced increase in protein levels of p22 phox -and thus NADPH oxidase activity-by a mechanism involving metal chelation, but not nonspecific antioxidant effects of DFO.

Figure 2. NADPH oxidase activity in mouse air pouch tissue. Animals were treated without or with LPS, desferrioxamine (DFO), apocynin (APO), or Fe(III)-loaded DFO (DFO-FeIII) as described in Methods. NADPH oxidase activity in homogenized air pouch tissue was measured by lucigenin chemiluminescence on the addition of 10 -4 mol/L NADPH. Data shown are the means±SEM of 4 to 8 mice per group; ** P <0.01 vs control, * P 0.05 vs LPS-treated group, ## P <0.01 and # P <0.05 vs LPS-treated group.

Desferrioxamine and Apocynin Inhibit LPS-Induced Production of O 2 ·- and H 2 O 2

In addition to NADPH oxidase activity, which was assessed by measuring O 2 ·- production in air pouch tissue homogenates on addition of NADPH, we also measured endogenous O 2 ·- production in intact tissue without NADPH addition. We found that the rate of endogenous O 2 ·- production was increased nearly 3-fold by LPS, and DFO and apocynin, but not Fe(III)-loaded DFO, significantly inhibited LPS-induced O 2 ·- production (supplemental Figure IIa). These effects of DFO, Fe(III)-loaded DFO, and apocynin were confirmed by in situ staining of LPS-exposed air pouch tissue for O 2 ·- production using dihydroethidium (supplemental Figure IIb).

In addition, LPS significantly enhanced production of H 2 O 2, which is a dismutation product of O 2 ·-. In agreement with the above data, LPS-induced H 2 O 2 production was significantly inhibited by DFO and apocynin, but not Fe(III)-loaded DFO (supplemental Figure III). These data indicate that NADPH oxidase is the main source of LPS-induced O 2 ·- and H 2 O 2 production in air pouch tissue, and DFO suppresses production of these ROS by inhibiting NADPH oxidase.

Desferrioxamine and Apocynin Inhibit LPS-Induced Nuclear Translocation of NF- B and Expression and Secretion of Cellular Adhesion Molecules

NF- B is a redox-sensitive transcription factor that controls the expression of most inflammatory genes. To monitor activation of NF- B, translocation of its p65 subunit from the cytosol to the nucleus was assessed by immunohistochemistry. As shown in Figure 3 A, in air pouch fibroblasts of unstimulated control mice, p65 (stained green) was located outside the nucleus (dark blue). After LPS treatment, p65 translocated to the nucleus, which now appears light green to turquoise ( Figure 3 A). LPS-induced nuclear translocation of p65 was blocked by DFO and apocynin, but not Fe(III)-loaded DFO ( Figure 3 A). These results were confirmed by quantitation of protein levels of p65 in nuclei isolated from air pouch tissue. As shown in Figure 3 B, LPS treatment caused an 2.5-fold increase in nuclear levels of p65, which was significantly suppressed by DFO and apocynin, but not Fe(III)-loaded DFO.

Figure 3. Nuclear translocation of NF- B in mouse air pouch tissue. Animals were treated without or with LPS, desferrioxamine (DFO), apocynin (APO), or Fe(III)-loaded DFO (DFO-FeIII) as described in Methods. Air pouch tissue sections or nuclear extracts were subjected to immunohistochemical detection of the NF- B subunit, p65 (panel A) or analyzed for p65 protein by ELISA (panel B), respectively. In panel A, positive p65 immunoreactivity and nuclear staining are indicated by green and blue fluorescence, respectively. The tissue sections shown are representative of three separate experiments. The white bar in "Control" indicates 0.001 mm. Data shown in panel B are the means±SEM of 4 mice per group; ** P <0.01 vs control, # P <0.05 vs LPS-treated group, * P 0.05 vs LPS-treated group.

Two inflammatory genes regulated by NF- B are the cellular adhesion molecules, VCAM-1 and intercellular adhesion molecule-1 (ICAM-1). As shown by immunohistochemistry, VCAM-1 protein (stained dark brown) was barely detectable in control air pouch tissue, but clearly visible-localized to fibroblasts-after LPS treatment ( Figure 4 A). Desferrioxamine and apocynin blocked LPS-stimulated VCAM-1 expression, whereas Fe(III)-loaded DFO had no effect ( Figure 4 A). These data were confirmed by Western blot analysis, which showed a significant increase in VCAM-1 protein expression in LPS-treated air pouch tissue and strong inhibitory effects of DFO and apocynin, but not Fe(III)-loaded DFO ( Figure 4 B).

Figure 4. Adhesion molecule expression in mouse air pouch tissue. Animals were treated without or with LPS, desferrioxamine (DFO), apocynin (APO), or Fe(III)-loaded DFO (DFO-FeIII) as described in Methods. Air pouch tissue sections or homogenized air pouch tissue, respectively, were subjected to immunohistochemical analysis (panel A) or Western blot analysis (panel B) of VCAM-1, respectively. In panel a, positive VCAM-1 immunoreactivity is indicated by dark brown staining. The tissue sections shown are representative of four separate experiments. The black bar in "Control" indicates 0.01 mm. In panel b, the protein bands of VCAM-1 and actin (molecular sizes, 110 kDa and 43 kDa, respectively) were scanned by densitometry and the relative protein levels were expressed as the ratio of VCAM-1:actin (upper part of the panel). Data shown are the means±SEM of 4 mice per group; ** P 0.05 vs LPS-treated group, ## P <0.01 vs LPS-treated group. The lower part of the panel shows a representative Western blot for two animals per group. "HUVEC" indicates human umbilical vein endothelial cell lysate, which serves as the positive control.

Concomitantly with increased tissue expression of cellular adhesion molecules, LPS-stimulated release of VCAM-1 and ICAM-1 into air pouch lavage fluid was observed, which was inhibited by DFO and apocynin, but not Fe(III)-loaded DFO (supplemental Figure IVa and IVb). Taken together, these data suggest that NADPH oxidase-derived ROS activate NF- B, which leads to upregulation of VCAM-1 and ICAM-1 gene expression; these inflammatory responses are inhibited by the iron chelator, DFO, most likely because DFO inhibits the LPS-induced increase in protein levels of the catalytic NADPH oxidase subunit, p22 phox.

Discussion

The data presented in this article show that in an experimental murine model of local inflammation the iron chelator, desferrioxamine, prevents the LPS-induced increase in p22 phox protein levels, NADPH oxidase activity, and O 2 ·- and H 2 O 2 production; and inhibits nuclear translocation and, thus, activation of NF- B and gene expression of VCAM-1 and ICAM-1. These data are germane to the pathogenesis of atherosclerosis, in which NADPH oxidase-mediated oxidative stress and inflammatory responses in the vascular wall play crucial roles. 1-3,7,8,16,26,27

Although the exact relationship between inflammation and oxidative stress remains to be fully elucidated, LPS has been suggested to cause vascular inflammation and ROS formation in the setting of atherosclerosis. 4 NADPH oxidase, a major enzymatic source of O 2 ·-, is found in various cells and tissues, including vascular endothelial cells, smooth muscle cells, fibroblasts, and adventitia. 17,27-32 In murine air pouch tissue, which is comprised mainly of fibroblasts and did not contain leukocytes, we found that LPS increased NADPH oxidase activity and production of O 2 ·- and H 2 O 2. These ROS were derived primarily from NADPH oxidase, as their formation was strongly inhibited by the NADPH oxidase inhibitor, apocynin. In contrast, xanthine oxidase activity was much lower than NADPH oxidase activity and was not increased by LPS treatment. These observations are consistent with published findings that NADPH oxidase plays a causal role in LPS-induced oxidative stress and inflammatory responses in lung and brain. 33,34

The mechanism by which LPS activates NADPH oxidase remains incompletely understood. The structure of NADPH oxidase is complex, and at least 6 different subunit complexes have been proposed for the various NADPH oxidase isoforms found in different tissues and cells. 6,35-37 Only the phagocytic NADPH oxidase has been fully characterized and has been shown to consist of 6 subunits: two catalytic subunits, p22 phox and gp91 phox (also called NOX 2 ), which together form the superoxide-producing, membrane-bound cytochrome b 558; and the cytosolic regulatory subunits, Rac, p47 phox, p67 phox, and p40 phox. 35 Although not all NADPH oxidase subunits of nonphagocytic cells have been characterized, 35 all NADPH oxidase isoforms require p22 phox, which serves as a docking protein for the other subunits and stabilizes NOX in the membrane. 38 An in vitro study using human HEK293T cells suggested that LPS activates NADPH oxidase and increases oxidative stress through the direct interaction of toll-like receptor 4 (TLR 4) with the NADPH oxidase subunit, NOX 4, a homologue of NOX 2. 39 Another study reported that LPS stimulated NADPH oxidase activity through the release of tumor necrosis factor (TNF)-. 34 Our data demonstrate that LPS increases protein levels of p22 phox, which is the likely mechanism for the observed LPS-induced increase in NADPH oxidase activity and O 2 ·- and H 2 O 2 production.

It should be noted that the above changes in NADPH oxidase activity were observed 24 hours after LPS challenge, but not after 1 or 3 hours (data not shown). This observation is consistent with previous observations that increased production of O 2 ·- by NADPH oxidase required prolonged exposure to LPS. 6,36 Interestingly, prolonged exposure to LPS also has been shown to alter cellular iron status, which may explain why more than 3 hours are required for LPS to increase NADPH oxidase activity. As explained further below, LPS is known to upregulate the divalent metal transporter 1 (DMT1), 40 leading to increased cellular iron uptake, which in turn increases heme biosynthesis and, hence, the stability of the heme-containing protein, p22 phox. 41,42

An important finding of our study is that LPS treatment caused nuclear translocation of the redox-sensitive transcription factor, NF- B, by a NADPH oxidase-dependent mechanism. The notion that NADPH oxidase is a critical upstream mediator of LPS-induced NF- B activation is supported by our findings that apocynin strongly suppressed both NADPH oxidase activity and NF- B activation and by the previously reported observation that LPS failed to activate NF- B in NADPH oxidase-deficient mice. 33 In parallel with NF- B activation, we observed increased expression of VCAM-1 and ICAM-1, which are critical adhesion molecules in monocyte recruitment to the arterial wall and initiation of atherosclerosis. 9-11 It has been reported that inflammation-induced oxidative stress regulates VCAM-1 and ICAM-1 expression 43 and that LPS failed to induce VCAM-1 expression in NF- B-deficient mice. 44 In addition, LPS was shown to activate NF- B and thus upregulate VCAM-1 expression in microvessels of air pouch tissue. 5 In our study we found that LPS-induced VCAM-1 expression was localized to fibroblasts, the main cell type in the synovial tissue of the air pouch. 5 Furthermore, we observed that NADPH oxidase inhibition by apocynin blocked LPS-induced VCAM-1 expression. Taken together, these findings suggest that LPS upregulates VCAM-1 expression via a cascade of events involving increased p22 phox protein levels, NADPH oxidase activity, ROS production, and NF- B activation.

Interestingly, treatment with the iron-chelator DFO suppressed the LPS-induced increase in p22 phox protein levels, and hence all subsequent events of the cascade leading to increased adhesion molecule expression. These data are in agreement with in vitro findings from our laboratory 45 and other laboratories 14 showing that DFO inhibits TNF- -induced VCAM-1 expression in human endothelial cells by suppressing activation of NF- B and another redox-sensitive transcription factor, Sp-1. Conversely, incubation with iron enhanced adhesion molecule expression in human endothelial cells, and this effect was inhibited by preincubation of the cells with DFO. 46

The mechanism by which iron chelation inhibits the LPS-induced increase in p22 phox protein levels remains to be fully elucidated. As indicated above, NADPH oxidase is a heme protein and, hence, iron is important for its activity. 47 Accordingly, phagocytic NADPH oxidase is impaired in iron-deficient patients with anemia and restored to normal levels after iron supplementation. 48 Furthermore, iron and NADPH oxidase both are elevated in human atherosclerotic plaques, 49,50 and iron chelation by DFO inhibited NADPH oxidase-dependent ROS production in mice. 24 Cytochrome b 558, the catalytic subunit of phagocytic NADPH oxidase, contains two heme groups in its catalytic site. 47,51 It is plausible that DFO directly inactivates NADPH oxidase by chelating the active site heme iron, thus blocking the transfer of electrons from NADPH to oxygen and its reduction to O 2 ·-. This notion is supported by the finding that DFO inhibits membrane lipid peroxidation initiated by H 2 O 2 -activated metmyoglobin, suggesting that iron in this heme protein is chelatable by DFO. 52

On the other hand, chelation of heme iron would not explain why DFO inhibited the LPS-induced increase in p22 phox protein levels, as observed in the present study. It is known that the biosynthesis of p22 phox is sensitive to heme availability and, hence, cellular iron status. 41,42 In the absence of heme, p22 phox protein is rapidly degraded, whereas mRNA levels are not affected. 41 Interestingly, TNF- causes intracellular iron sequestration via upregulation of the transferrin receptor and DMT1, 40,53 and, as mentioned above, LPS also upregulates DMT1. 40 Furthermore, DFO enters cells via endocytosis and remains associated with endosomes, 54 which are known to transport iron to mitochondria for heme biosynthesis. 55 It is tempting to speculate, therefore, that the well-known phenomenon of iron sequestration into cells induced by LPS and other inflammatory stimuli 40 not only serves to withdraw iron from extracellular, pathogenic microorganisms, hence stunting their growth, but also causes an increase in protein levels of p22 phox and, hence, increased NADPH oxidase activity and sustained release of bactericidal ROS.

A limitation of the present study is that we did not measure iron levels in air pouch tissue or assess the animals? iron status. Although the mice were fed a standard iron-sufficient diet containing 180 mg Fe/kg, it has been argued that "standard rodent feeds typically contain roughly an order of magnitude more iron than the minimum required to produce a normal hematocrit value", and that "antioxidant and antiinflammatory processes may operate optimally only in the absence of stored iron." 56 Nevertheless, even if our experimental animals contained excess stored iron, DFO exerted significant antioxidant and antiinflammatory effects.

In summary, the present study demonstrates that the iron chelator, desferrioxamine, inhibits LPS-induced NF- B-mediated adhesion molecule expression by decreasing p22 phox protein expression and NADPH oxidase-derived ROS production in a murine model of local inflammation. These data suggest that iron plays a key role in NADPH oxidase-mediated inflammatory responses. Therefore, restriction of intracellular iron may be useful in the treatment of diseases that have a significant inflammatory component, such as atherosclerotic vascular diseases.

Acknowledgments

Source of Funding

This publication was made possible by Grant Number P01 AT002034 from the National Center for Complementary and Alternative Medicine (NCCAM).

Disclosures

The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of NCCAM or the National Institutes of Health.

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作者单位：Linus Pauling Institute, Oregon State University, Corvallis.