American Journal of Respiratory and Critical Care Medicine

LPS and selected cytokines upregulate xanthine dehydrogenase/xanthine oxidase (XDH/XO) in cellular systems. However, the effect of these factors on in vivo XDH/XO expression, and their contribution to lung injury, are poorly understood. Rats were exposed to normoxia or hypoxia for 24 h after treatment with LPS (1 mg/kg) and IL-1 β (100 μ g/kg) or sterile saline. Lungs were then harvested for measurement of XDH/XO enzymatic activity and gene expression, and pulmonary edema was assessed by measurement of the wet/dry lung weight ratio (W/D). Although treatment with LPS + IL-1 β or hypoxia independently produced a 2-fold elevation (p < 0.05 versus exposure to normoxia and treatment with saline) in lung XDH/XO activity and mRNA, the combination of LPS + IL-1 β and hypoxia caused a 4- and 3.5-fold increase in these values, respectively. XDH/XO protein expression was increased 2-fold by hypoxia alone and 1.3-fold by treatment with LPS + IL-1 β alone or combination treatment. Compared with normoxic lungs, W/D was significantly increased by exposure to hypoxia, LPS + IL-1 β , or combination treatment. This increase was prevented by treatment of the animals with tungsten, which abrogated lung XDH/XO activity. In conclusion, LPS, IL-1 β , and hypoxia significantly upregulate lung XDH/XO expression in vivo. The present data support a role for this enzyme in the pathogenesis of acute lung injury.

Factors that regulate the metabolism and activity of xanthine dehydrogenase/oxidase (XDH/XO) remain poorly understood despite overwhelming evidence of a role for XDH/XO in disease processes such as ischemia-reperfusion, radiation injury, the adult respiratory distress syndrome, and other inflammatory conditions (1). However, oxygen tension and cytokines appear to be important regulatory factors. Several investigators have now shown that, in cultured pulmonary artery endothelial cells, XDH/XO activity is inversely correlated with the O2 tension to which the cells are exposed (2-5). Furthermore, we have shown that hypoxia upregulates XDH/XO gene expression (5). We have also recently demonstrated an in vivo conversion of XDH to the reactive O2 species-generating enzyme XO in lungs of rats exposed to hypoxia for a total of 5 d (6). Some cytokines such as interferon-γ (IFN-γ), are known to upregulate XDH/XO activity and/or mRNA expression (7). However, with the exception of IFN-γ, the effects of cytokines on XDH/XO have not been tested specifically in lung tissue. Similarly, whereas bacterial lipopolysaccharide (LPS) increases XDH/XO mRNA expression and activity in mouse liver (8), its effect on lung XDH/XO is unclear (9). LPS and cytokines are particularly relevant to lung injury since sepsis is one of the major causes of the acute respiratory distress syndrome (ARDS). Both LPS and IL-1 have been shown to cause pulmonary injury in animal models (10, 11). Furthermore, bronchoalveolar lavages of patients with ARDS display increased expression of certain proinflammatory cytokines (e.g., IL-1 and IL-6) (12-15), some of which correlate with injury and predict poor outcome (12).

The goal of this study was to test the possibility that hypoxia, LPS, and IL-1β might regulate XDH/XO in rat lung, hence contributing to the pathogenesis of lung edema.

All drugs and reagents were obtained from Sigma Chemical (St. Louis, MO) unless specified otherwise.

Exposure of Animals and Drug Administration

Male adult Sprague-Dawley rats weighing approximately 300 to 400 g were exposed to either hypobaric temperature (0.5 atm) or normobaric atmosphere (controls) for 24 h. Half of the animals in each exposure group received, just prior to exposure, an intraperitoneal injection of LPS (from Escherichia coli, Sigma Pharmaceuticals; 1 mg/ kg) and IL-1β (100 μg/kg; Merck Research Laboratories, Rahway, NJ) or the same volume of sterile saline (vehicle). In separate experiments, animals received a tungsten-enriched, molybdenum-deficient diet (0.7 g Na tungstate/kg chow; ICN Biochemicals, Corte Madera, CA) for 3 wk prior to experimentation (a regimen that has been found effective for inhibition XDH/XO activity (16) in order to test the effect of XDH/XO inhibition on the development of pulmonary edema and mortality. After exposure, animals were killed with an overdose of pentobarbital (50 mg/kg) given intraperitoneally, and lungs were harvested en bloc and processed for XDH/XO enzymatic assay, quantitative polymerase chain reaction (PCR), Western blotting, and wet/ dry lung weight ratio as detailed below. All procedures and drug treatments were approved by the Department of Laboratory Animal Medicine at the New England Medical Center, and the animals were killed in accordance with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association.

Determination of XDH/XO Activity

XDH/XO whole lung activity was measured using a high-performance liquid chromatography (HPLC) technique as previously reported (6). Briefly, sample preparation and HPLC were obtained as follows.

Sample preparation. Desiccated lung tissue was ground into a fine powder using a mortar and pestle. One-half gram of frozen powder was added to 5 ml of a homogenizing buffer (consisting of 50 mM K+-phosphate buffer containing 10 mM DTT, 1 mM PMSF, and 0.1 mM EDTA at pH 7.0) and centrifuged at 18,000 rpm for 30 min at a temperature of 4° C. One and one-half milliliters of the supernatant were decanted and centrifuged for an additional 15 min, and the sample was then used for measurement of XDH, XO, and uric acid (UA).

Determination of enzymatic activity by HPLC. Activity was measured by monitoring the formation of UA after addition of xanthine (50 μM) for XO measurement, or xanthine (50 μM) + NAD (500 μM) for XO+XDH measurement, to 0.5 ml of tissue supernatant. Identical samples prepared in parallel and treated with allopurinol (1 μM) were used to confirm that the formation of UA detected was from XDH/ XO activity. All samples were incubated at a temperature of 37° C for 30 min. Samples were deproteinized by addition of 20 μl of perchloric acid before centrifugation at high speed for 10 min. The supernatant was used for measurement of UA. Enzymatic activity was expressed in international units where one unit is equal to 1 μM UA/min.

Quantitative PCR for Rat XDH/XO

This was assessed by a quantitative PCR technique we have previously described for determination of XDH/XO mRNA expression in cultured endothelial cells (17). The XDH/XO internal standard, PCR technique, and quantification were obtained as detailed below.

Rat XDH/XO internal standard preparation. The rat insertion mutant internal standard (IS) was prepared from a PCR fragment that was generated by using primers 5′-CGCAGAATACTGGATGAGCGAGGT-3′ (P1) and 5′-GCCGGTGGGTTTCTTCTTCTTGAA-3′ (P2) to amplify 568 bp of rat XDH/XO cDNA from nt 2792-3359 (18). PCR was performed for 30 cycles with 1 min, 94° C denaturation; 2 min, 60° C annealing; and 2 min, 72° C extension. The last cycle extension was for 10 min. This fragment was restricted with PstI and NsiI in two separate reactions to yield fragments with compatible cohesive ends. Because these enzymes cut only once, two subfragments were generated in each reaction. The 50 and 518 bp subfragments from PstI, and the 150 and 418 bp subfragments from NsiI were separated by electrophoresis on 3% NuSieve/1% SeaKem agarose in TRIS/acetic acid/ EDTA buffer at 80 V for 2 h. Subfragments at 518 and 150 bp were excised from the gel and purified on Geneclean (Bio-101, La Jolla, CA) according to the manufacturer's instructions. Equal amounts of the purified subfragments were ligated at ambient temperature for 17 h with T4 DNA ligase to generate the mutant fragment with a 100-bp insertion relative to the wild type cDNA. Serial dilutions of the ligated sample were subjected to PCR with primers P1/P2 as described above. After electrophoresis, the fragment at 668 bp was excised from the gel and purified. It was reamplified with nested primers 5′-AGGTCGCCATAACCTGTGGGCTG-3′ (P3) and 5′-ATTGAGGTCAGCACTGGCAGA- GG-3′ (P4) and purified on Geneclean to generate the 573-bp rat insertion mutant IS.

Polymerase chain reaction. A master PCR reagent mixture was prepared such that each 50 μl contains 0.4 μm of each primer P3/P4, and 52 μM dNTPs, in 15 mM TRIS-HCl, 50 mM KCl, 1.5 mM MgCl2, and 0.01% gelatin buffer at pH 8.5. A constant amount of cDNA was added to the master mix of PCR primers and buffer, and 50-μl aliquots were placed in several tubes. Subsequently, variable amounts of IS were added to each 50-μl PCR reagent mix. Samples were overlaid with light mineral oil and held at 80° C for hot-start PCR. Then 1.25 units Taq polymerase in 2 μl of water were added to each sample by underlaying, and PCR was carried out for 35 cycles with 1 min, 94° C denaturation; and 2 min, 68° C combined annealing and extension. The last cycle extension was for 10 min. Primer pairs presumably span introns because genomic DNA is not amplified. Controls prepared from RNA without MMLV-RT were negative.

Quantification. After PCR, 10-μl sample aliquots were electrophoresed on 2% NuSieve/1% SeaKem agarose in TRIS/acetic acid/ EDTA buffer for 2 h at 80 V, stained with ethidium bromide, and photographed. Densitometry was done with a Millipore Densitometer with Visage v4.6p software (Millipore, Bedford, MA). Because the XDH/XO target was smaller than the mutant, target optical density was multiplied by a correction factor of 1.22 to normalize the value before calculating the molar ratio of 573 bp mutant to 473 bp target. This ratio was used for relative quantification. For absolute quantification, a fixed amount of cDNA was added to the master mix prior to aliquoting. Subsequently, the IS was serially diluted and added to each sample. When PCR was performed with a fixed amount of cDNA and serial dilutions of IS per sample, optical densities for XO and IS were plotted versus copies of IS to assess amplifications as a function of IS input. The equivalence point, where target and IS copies are equal, was determined from a plot of IS/XO ratio versus copies of IS. This value was divided by 0.4 to compensate for the less than 100% reverse transcription efficiency, which was presumed to be 40% as determined from the manufacturer's instructions. Also, the value was multiplied by 2 to compensate for differential amplification during the first PCR cycle where the single-stranded cDNA target is rendered double-stranded, whereas the double-stranded IS is amplified geometrically. Corrected values are reported as copies of XDH/XO mRNA normalized to tubulin.

Determination of Lung Injury

Pulmonary edema was estimated by the wet/dry lung weight ratio, a technique commonly used for assessment of experimental lung injury (16, 19). Briefly, after exposure to the desired experimental condition, animals were killed with an overdose of pentobarbital. A sternotomy incision was performed and the heart, lungs, and trachea were removed en bloc. The right lower lobe was isolated and immediately weighed (wet weight) before being dried for 24 h at 90° C and then weighed again (dry weight).

Western Blot Analysis

Sample preparation. Desiccated rat lung tissue was placed in cell lysis buffer containing 50 mM TRIS HCl at pH 7.5, 1 mM sodium orthovandate, 10 mM sodium fluoride, 1 mM sodium molybdate, 10 μg/ml aprotine, 10 μg/ml leupeptin, 10 μg/ml soybean trypsin inhibitor, 0.07 μg/ml pepstatin, 1% NP-40, and 5 mM EDTA for 10 min at 4° C. Insoluble material was removed by centrifugation (14,000 × g for 10 min) and the supernatant used for analysis. Protein measurements of the supernatant (samples) were performed using the method of Lowry and colleagues (20).

Polyacrylamide gel electrophoresis. Samples corresponding to equal amounts of total protein (about 50 μg) were diluted (1:5 by volume of buffer) in electrophoresis sample buffer (0.2 M EDTA, 40 mM dithiothreitol, 6% SDS, and 0.06 mg/ml pyronine at pH 6.8) and boiled for 3 to 5 min to denature protein. Samples were then subjected to sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) on 7% slab gel in a model SE-600 apparatus (Hoefer Scientific, San Francisco, CA).

Immunoblotting. After electrophoresis, gel proteins were electrophoretically transferred to PVDF membrane (Tropifluor; Tropix, Bedford, MA) or Immobilon-P (Millipore) according to Towbin and colleagues (21). After transfer, nonspecific PVDF binding sites were blocked with 5% HiPure liquid gelatin (Norland, New Brunswick, NJ) in 5× PBS and 0.1% Tween-20. Blocking was performed for 1 h at ambient temperature. The membrane was then treated with a 1/1,000 to 1/2,500 dilution of AP-conjugated polyclonal antibody against XDH/ XO (developed in Dr. Parks's laboratory [22]) in blocking buffer for 90 min at ambient temperature with gentle agitation. Nitro-block (Tropix) was used according to the manufacturer's instructions. Subsequent washing, detection with the chemiluminescent substrate (CSPD) (Tropix), and film exposure were then performed.

Statistical Analysis

All values are shown as means ± SD. Experiments were obtained with n of at least 4 for each experimental and control condition. Student's unpaired t test was used whenever indicated. Animal mortality after 24 h of exposure to specific experimental conditions was assessed using the chi-square method. For all studies involving more than two groups, Scheffe's multiple comparison test (SPSS version 6.1; SPSS, Chicago, IL) was used to determine significant changes occurring between and within control cells or animals and counterparts exposed to experimental conditions. Significance in all cases was assumed at p < 0.05.

Additive Upregulation of XDH/XO Enzymatic and mRNA Expression by Hypoxia and LPS + IL-1 β Treatment in Rat Lung

In preliminary experiments on cultured pulmonary microvascular endothelial cells (EC), a combination of LPS and IL-1 was found to synergistically upregulate intracellular XDH/XO activity and mRNA expression and to further enhance the stimulation of this enzyme by hypoxia (23). This was the rationale for testing the effects of combined stimuli on the in vivo regulation of XDH/XO. Sprague-Dawley rats were exposed to normobaric (normoxia) or hypobaric (hypoxia) atmosphere for 24 h immediately after receiving a single intraperitoneal injection of LPS + IL-1β (1 mg/kg and 100 μg/kg, respectively) or the equivalent volume of sterile saline, as detailed in Methods. At the end of exposure, lungs were removed and XDH/ XO activity and gene expression were assessed. Hypoxia or LPS + IL-1β treatment alone caused 2- and 2.7-fold increase, whereas combined therapy caused a 4- and 4.7-fold increase, in XO and XO+XDH activity, respectively, as compared with values from animals exposed to normoxia and treated with saline (Figure 1). Similar results were obtained for XDH/XO mRNA expression. The determination of lung XDH/XO mRNA levels obtained by quantitative PCR from one normoxic saline-treated rat (control) and one hypoxic rat treated with LPS + IL-1β, using an XDH/XO insertion mutant as internal standard is illustrated in Figure 2 (see Methods for details). Averages of XDH/XO mRNA copies (corrected for the number of tubulin copies) for each treatment group (n of at least 4 for each experimental group) are shown in Figure 3. Although hypoxia or LPS + IL-1β treatment alone caused a 2- and 2.5-fold increase in the number of XDH/XO mRNA copies, respectively, combination treatment produced a 3.5-fold increase in this value as compared with that from normoxic animals treated with saline (Figure 3).

Western blot analysis of desiccated lungs obtained from rats that had been exposed for 24 h to normoxia or hypobaric hypoxia, after treatment with LPS + IL-1β or saline injection, was performed using a polyclonal antibody against xanthine oxidase. Bovine milk xanthine oxidase was used as a positive control. Preincubation of the primary antibody with control protein (1:10 mixture) prior to immunoblotting abrogated the 150-kD band, confirming specificity of this band for XDH/XO (not shown). As illustrated in Figure 4, there was increased XDH/XO protein expression in hypoxic lungs, and in lungs from normoxic and hypoxic animals treated with LPS + IL-1β as compared with normoxic animals treated with saline. Densitometric analysis of such blots revealed an average 1.3-fold (for normoxic and hypoxic animals treated with LPS + IL-1β) and a 2-fold (for hypoxic animals treated with saline) increase in this expression as compared with that of normoxic lungs.

Role of XO in the Development of Acute Lung Injury

The effects of hypoxia and treatment with LPS + IL-1β on the development of pulmonary edema (assessed by the wet/dry lung weight ratio) and mortality (estimated by the chi-square method) were examined 24 h after initiation of therapy. In separate experiments, the effect of a tungsten-rich, molybdenum-free diet initiated 3 wk prior to exposure to hypoxia and treatment with LPS + IL-1β, was assessed on the two variables, pulmonary edema and mortality. Control animals received a regular chow. The tungsten-rich diet resulted in undetectable lung XO activity levels in animals treated with saline, hypoxia, LPS + IL-1β, or the combination of hypoxia and LPS + IL-1β (Table 1). When compared with control animals exposed to normoxia and treated with saline injection, hypoxia or LPS + IL-1β therapy alone produced an 18%, whereas combined hypoxia and LPS + IL-1β therapy produced a 25%, increase in wet/dry lung weight ratio (Figure 5). This increase was completely prevented by pretreatment of the animals with tungsten for all three treatment groups (Figure 5). There were no deaths at 24 h in normoxic rats treated with saline (controls, n = 30) or LPS + IL-1β alone (n = 11) and in rats exposed to hypoxia alone (n = 20). However, combined treatment with hypoxia and LPS + IL-1β resulted in significant mortality (13 out of 29 animals, p < 0.05 versus control animals), which was not prevented by pretreatment of the animals with a tungsten-rich diet (three deaths out of 9 animals treated; p > 0.05 versus animals treated with the combination of hypoxia and LPS + IL-1β).


Rats (n)O2 ExposureTreatmentDietXO Activity*(μU/g tissue)
8 (Control)NormoxiaSalineRegular 73 ± 8
8NormoxiaLPS + IL-1Regular131 ± 35
7HypoxiaSalineRegular135 ± 33
8HypoxiaLPS + IL-1Regular260 ± 25
4NormoxiaLPS + IL-1TungstenND
4HypoxiaLPS + IL-1TungstenND

*  Results are expressed as mean ± SD.

  p < 0.05 compared with control.

  p < 0.05 compared with single treatment (hypoxia or cytokine alone).

There is growing evidence that the enzyme XDH/XO might participate in the pathogenesis of pulmonary diseases, presumably because of the ability of XO to produce reactive O2 species that may alter normal cellular functions. At least three separate groups of investigators (16, 19, 24) have causally implicated XO in the development of lung injury after ischemia of the gut or liver, organs that are rich sources of XO. In other animal models of lung injury, XO has been shown to be greatly increased in the bronchalveolar lavage (∼ 400-fold) and serum of mice infected with the influenza virus (25). In humans, XO and its substrates hypoxanthine and xanthine were found to be elevated in the serum of patients with the acute respiratory distress syndrome (ARDS) (26, 27) as compared with normal controls or critical care patients with other organ diseases or those undergoing pulmonary resection. In one of these studies, nonsurvivors of ARDS displayed higher levels of oxidative stress and damage than did survivors (27). Increased hypoxanthine and XO levels have also been demonstrated in the epithelial lining fluid of premature neonates with bronchopulmonary dysplasia (28), implicating this oxyradical-generating system in the early pulmonary inflammatory response in these infants. More recently, XO was found to be significantly elevated in patients with sepsis syndrome and secondary organ dysfunction (29).

Despite the overwhelming body of evidence linking XDH/ XO to organ injury, little is known about the factors that regulate this enzyme in general, more particularly when it relates to the lung. We have previously demonstrated in vivo upregulation of XDH/XO activity and mRNA expression in hypoxic as compared with normoxic pulmonary artery endothelial cells (5), and have recently extended these studies to show in vivo upregulation of XO activity in the lung of rats exposed to hypobaric atmosphere (hypoxia) for a period of 5 d as compared with counterparts maintained in normobaric atmosphere (normoxia) (6). Regarding regulation of XDH/XO by cytokines, interferon-γ (IFN-γ) has been demonstrated by other investigators to be a potent inducer of XDH/XO activity and mRNA expression in rat pulmonary artery EC (7), whereas tumor necrosis factor, IFN-γ and interleukins 1 and 6 have been shown to upregulate XDH/XO activity and mRNA expression in bovine renal epithelial cells (30). In addition, IFN-α and its inducers poly(I).poly(C) and bacterial LPS increase XDH/XO mRNA expression and activity in mouse liver (8). However, aside from IFN-γ, the effects of other cytokines had not been tested in lung tissue. A study by Kurosaki and colleagues (9) demonstrated a modest upregulation of lung XO and XO+XDH activity (1.5- and 1.7-fold increase, respectively) but without change in XDH mRNA transcript levels (despite significant increases in these levels in other tissues such as the kidney and intestine) 16 h after injection of mice with LPS. The present study was therefore designed to test the possibility that hypoxia and inflammatory factors such as LPS and IL-1 might upregulate lung XDH/XO, thus contributing to the development of acute lung injury.

Preliminary experiments on cultured pulmonary microvascular EC, which demonstrated a synergistic upregulation of XDH/XO by IL-1β and its inducer LPS with maximal effect when cells were concomitantly exposed to hypoxia (23), provided the rationale for testing the combination of these stimuli in vivo. Exposure of rats to hypoxia or a single intraperitoneal injection of LPS + IL-1β produced a significant increase in XDH/XO activity and gene expression, with a further increase when the two stimuli were combined. Some discrepancies related to the effect of hypoxia on rat lung XDH/XO activity between the present study and our prior work, in which rats were exposed for 5 d to hypobaric hypoxia (6), are worth noting. In the latter study, continuous exposure to hypoxia resulted in significant increases in XO activity and the XO to XDH ratio, but without increase in XDH activity, whereas in the present study hypoxia caused a significant elevation in both XO and XDH activity but without causing a change in the XO to XDH ratio. These discrepancies, while not readily explained, could partly be related to the length of exposure to hypoxia, the most notable difference in experimental design between the two studies.

The present study does not allow the identification of a specific pulmonary source of XDH/XO. However, it is reasonable to assume that EC would constitute the main cellular source of this enzyme in this system considering the extensive vascular network of the lung. Other vascular cells such as smooth muscle cells that display significant XDH/XO activity when exposed to hypoxia (5) may have also contributed to enhanced expression of this enzyme in lungs of hypoxic animals. Differences between the present study and that by Kurosaki and colleagues (9) in which only modest increases in lung XDH/XO activity without changes in mRNA transcripts after injection of mice with LPS alone could be explained by differences in species (mouse versus rat) or more likely by a potentiation effect, in the present study, related to cotreatment of the animals with IL-1β and its inducer LPS. An additional finding in the present study is the additive effect of LPS/IL-1β and hypoxia on XDH/XO upregulation in lung tissue.

Because increases in lung XDH/XO protein by hypoxia, LPS + IL-1β, or the combination of these factors were not as dramatic as those obtained with XDH/XO mRNA expression and activity, it is possible that post-translational events might have occurred, in addition to the transcriptional effect caused by hypoxia as noted previously in cellular systems (5). Such post-transcriptional modification of XDH/XO (i.e., inhibition of activity) has previously been demonstrated with reactive oxygen species (31-33). Because general production of reactive oxygen species by EC has been shown to be reduced in hypoxia (34), a subsequent increase in XDH/XO activity can be postulated on that basis (33), at least when it relates to hypoxia. It should be noted that, in a comparable study, treatment of mice with LPS resulted in no increase in lung XDH/XO protein despite increases in lung XDH/XO activity and upregulation of the protein in other organs (e.g., intestines, liver, and spleen) (9).

The potentiation of the effect of LPS + IL-1β on XDH/XO mRNA expression by hypoxia remains unexplained. However, it is noteworthy that the 5′-flanking region of the human XDH gene contains several consensus sequences, within 2 kb of the transcription site, for possible binding of specific regulatory factors, including a potential IL-1 responsive element (35). A variant of a hypoxia-responsive element may also be present in the 5′-flanking region of the rat XDH/XO gene (Roger Chalkley, personal communication). Whether transcription factors induced by hypoxia and IL-1 might act in concert on the XDH/XO promoter to upregulate the expression of this enzyme remains to be determined. Such a potential synergistic effect between hypoxia and cytokines would not be without precedent since IL-6 and hypoxia, or a combination of IL-1α, TNF-α, IL-6, and hypoxia have been shown to stimulate the expression of the erythropoietin gene above and beyond the effect of hypoxia or cytokines alone (36). Therefore, only studies addressing the direct effects of IL-1 and hypoxia on specific regions of the XDH/XO promoter might help to shed some light on the mechanisms of action involving these stimuli.

Rats treated with hypobaric hypoxia alone, a single injection of LPS + IL-1β alone, or a combination of these treatments for 24 h showed evidence of increased lung water as compared with rats kept in normobaric normoxia. Treatment with LPS and IL-1 has previously been shown to cause lung injury. Studies of intratracheal insufflation of IL-1 in a rat model suggest a neutrophil-dependent mechanism of this cytokine, also requiring TNF activity to trigger the neutrophil respiratory burst and oxidant production, in the pathogenesis of the lung edema (10). A role for XO was not suggested in that model, but it was implicated in the case of LPS in studies performed by Faggioni and colleagues (11). In the case of hypoxia alone, increases in lung permeability have been described in some (37, 38), but not all (39), experimental models. Hypobaric and normobaric hypoxia exposures for 24 h have both been shown to cause increases in lung transvascular protein escape in male Sprague-Dawley rats (37). In the latter study, this effect was not related to restoration of normoxia after hypoxic exposure (i.e., hypoxia-reoxygenation injury) since increased lung leakage was also noted when measurements were done under continuous hypoxic exposure. In the present study, there was a trend for a further increase in wet/dry lung weight ratios in animals treated with the combination of hypoxia and LPS + IL-1β as compared with animals treated with either stimulus alone. However, this difference was not statistically significant, perhaps because of a relative lack of sensitivity of this particular measurement of lung injury.

Although no direct link between upregulation of XDH/XO by hypoxia/LPS + IL-1β treatment and increased lung water can be established in the present study, complete abrogation of XO activity and prevention of pulmonary edema by tungsten support a role for this enzyme in the pathogenesis of pulmonary edema in this model. Nevertheless, tungsten therapy did not affect the 24 h mortality observed with combined hypoxia and LPS + IL-1β treatment despite prevention of pulmonary edema. A small sample size or possible alterations in systemic hemodynamics caused by LPS + IL-1β treatment may have explained the lack of apparent effect of tungsten therapy on mortality. Because tungsten is known to inhibit other molybdenum-containing proteins such as aldehyde oxidase and sulfite oxidase, one cannot exclude a role for these enzymes in our system. Aldehyde oxidase, in particular, shares a number of similarities with XDH/XO (40). Both enzymes are homodimeric molybdoflavoproteins with similar molecular weight (∼ 150 kD for the monomeric subunit). They each contain two Fe-S centers and have overlap substrate specificity (40). However, aldehyde oxidase is not known to be upregulated by hypoxia or cytokines and, to our knowledge, has not been implicated in lung injury.

In summary, this study has demonstrated an upregulation of XDH/XO gene expression in rat lung by hypoxia, LPS, and IL-1 treatment and suggests a role for this enzyme in the pathogenesis of acute lung injury such as in the acute respiratory distress syndrome or sepsis syndrome.

The writers wish to thank Deborah LaPerche for her assistance in preparing the manuscript.

Supported by Grants R29 HL-49411, 5T32 HL-07053, 5K14-HL-03368, and Program Project Grant HL-48676 from the National Heart, Lung, and Blood Institute.

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Correspondence and requests for reprints should be addressed to Paul M. Hassoun, M.D., New England Medical Center, Pulmonary and Critical Care Division, 750 Washington Street, NEMC 128, Boston, MA 02111. E-mail:


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