| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
University of Colorado Health Sciences Center, Denver, Colorado 80262
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
ACKNOWLEDGEMENTS
FOOTNOTES
REFERENCES
ABSTRACT 
Terada, Lance S., Nancy N. Mahr, and Eugene D. Jacobson.
Nitric oxide decreases lung injury after intestinal ischemia. J. Appl. Physiol. 81(6):
2456-2460, 1996.
After injury to a primary organ, mediators are
released into the circulation and may initiate inflammation of remote
organs. We hypothesized that the local production of nitric oxide (NO)
may act to limit the spread of inflammation to secondarily targeted
organs. In anesthetized rats, 30 min of intestinal ischemia followed by
2 h of reperfusion (I/R) did not increase lung albumin leak. However,
after treatment with NG-nitro-L-arginine methyl ester
(L-NAME), intestinal I/R led to increased lung leak, suggesting a protective effect of endogenous NO.
The site of action of NO appeared to be the lung and not the gut
because 1) after treatment with
L-NAME, local delivery of NO to
the lung by inhalation abolished the increase in intestinal I/R-induced
lung leak; 2)
L-NAME had no effect on
epithelial permeability (51Cr-labeled EDTA clearance) of
reperfused small bowel; and 3) after treatment with L-NAME, local
delivery of NO to the gut by luminal perfusion did not improve
epithelial permeability of reperfused intestines. Furthermore,
L-NAME increased, and inhaled NO
de- creased, the density of lung neutrophils in rats subjected to intestinal I/R, and treatment with the selectin antagonist fucoidan abolished L-NAME-induced lung
leak in rats subjected to intestinal I/R. We conclude that
endogenous lung NO limits secondary lung injury after intestinal I/R by
decreasing pulmonary neutrophil retention.
acute respiratory distress syndrome; neutrophils; xanthine oxidase; multiorgan failure; inflammation
INTRODUCTION DYSFUNCTION OF MULTIPLE ORGANS can accompany the acute
respiratory distress syndrome (ARDS), with catastrophic consequences. The determinants of remote organ involvement after an injury to a
primary organ are poorly understood but may reflect a balance between
activation of pro- and anti-inflammatory pathways. Terada et al. (20)
have previously demonstrated that, after intestinal ischemia-reperfusion (I/R), elevations in circulating levels of the
oxidant enzyme xanthine oxidase promote retention of neutrophils in the
lung. Recently, it has also been shown that nitric oxide (NO) can
oppose the acute inflammatory effects of oxidants. For instance, NO
donors can diminish mesenteric leukocyte adherence caused by exogenous
xanthine oxidase (6). Addition of NO also attenuates the intestinal
permeability defect prompted by acute lung damage (2). Conversely, the
leukocyte adherence that accompanies suppression of endogenous NO can
be reversed with the scavenger superoxide dismutase (11). Therefore, we
hypothesized that lung-derived NO may be locally protective against
secondary pulmonary injury after a primary injury to the intestines. We
tested this hypothesis in a rat model of mesenteric ischemia and found
suggestive evidence that local NO production diminishes lung injury by
decreasing neutrophil sequestration.
MATERIALS AND METHODS Sources of Reagents
Experimental Protocol
Adult male Sprague-Dawley rats (300-400 g) were anesthetized with ketamine (80 mg/kg ip) and xylazine (10 mg/kg ip), placed on a warming pad, and allowed to breathe spontaneously. A laparotomy was performed, and the superior mesenteric artery was occluded with a microaneurysm clamp (Miltex, Lake Success, NY) for 30 min and then released. Reperfusion lasted 2 h and was generally accompanied by gross hyperemia and swelling of the bowel. One group of rats received L-NAME (5 mg/kg iv 10 min before ischemia and another 5 mg/kg at reperfusion). Another group received fucoidan (25 mg/kg iv at ischemia). Fucoidan is a heavily sulfated fucose polymer that inhibits P- and L-selectin-mediated neutrophil adhesive interactions (17) and has been shown to reduce neutrophil rolling in mesenteric venules by >90% (10). The exposed abdominal viscera were covered with plastic wrap to minimize heat and water loss. Reperfusion continued for 2 h, and all measurements were obtained at the end of the reperfusion period. Delivery of inhaled NO. Anesthetized rats were placed in a 47.6-liter polycarbonate chamber with a single egress valve. The chamber was flushed with NO gas (50 ppm) at 20 l/min for 5 min before flow was decreased to 2 l/min. Rats were allowed to breathe the gas mixture spontaneously. Delivery of intestinal NO. A 3-cm jejunal segment, ~15 cm distal to the ligament of Treitz, was cannulated at its proximal and distal ends with 15-gauge Luer stub adapters (Intramedic, Becton-Dickinson, Sparks, MD) and secured with ligatures, with care taken to preserve the vascular supply of the segment. Saline was first deoxygenated by bubbling with N2 gas and then was bubbled through a fine glass filter with NO (833 ppm, 100 ml/min) and perfused via a peristaltic pump (Masterflex, Cole Parmer, Chicago, IL) into the lumen of the intestinal segment at ~1 ml/min. Lung albumin leak. Pulmonary transvascular protein leak was measured as previously described (20). 125I-albumin (1 µCi) was injected intravenously 30 min before the termination of the experiment. At the end of the experiment, rats were ventilated through a tracheostomy, heparin (200 U iv) was injected into the right ventricle, and the lungs were perfused blood free with modified Krebs-Henseleit buffer. The lung leak index was calculated as the percentage of radioactivity in both lungs relative to that in 1 ml of blood. EDTA clearance. Gut mucosal integrity was assessed by measuring blood-to-lumen clearance of EDTA as previously described (8). A jejunal segment was isolated and perfused with saline as described in Delivery of intestinal NO, and 40 min before the termination of the experiment the renal pedicles were ligated to reduce EDTA clearance. 51Cr-EDTA (100 µCi) was then injected into the inferior vena cava, the luminal perfusate was collected, and the clearance was calculated (2, 18). For rats receiving luminal NO, the clearance was calculated from the same intestinal segment that received luminal NO. Lung neutrophil quantitation. Pulmonary neutrophil retention was assessed by blinded morphometry. After perfusion with modified Krebs-Henseleit buffer, lungs were airway fixed with 10% buffered Formalin at 20 cmH2O pressure and embedded in paraffin. Sagittal sections (5 µm) of the left lung were cut at a point ~2 mm distal to the entry of the main-stem bronchus into the parenchyma. After incubation with rabbit anti-rat neutrophil primary antisera (1:500), a commercial avidin-biotin (avidin-biotin complex) peroxidase staining kit (Pierce Chemical, Rockford, IL) and 3,3
-diaminobenzidine were used to visualize
neutrophils. Slides were counterstained with Gill's
modified hematoxylin and examined in a blinded fashion. For each lung, neutrophils from 30 random fields were counted. The
average for each rat was considered a single value.
Statistical Analysis
Parametric data were anaylzed by using one-way analysis of variance combined with the Student-Newman-Keuls multiple-comparison test. Significance was accepted at a P value of <0.05.RESULTS
Effect of NO on Intestinal I/R-Induced Lung Leak
Rats pretreated with the NO synthase inhibitor L-NAME and subjected to intestinal I/R had increased lung leak compared with L-NAME-treated sham rats (P < 0.001) or untreated I/R rats (P < 0.001; Fig. 1A). In untreated rats, intestinal I/R did not increase (P > 0.05) lung albumin leak. Inhalation of NO gas abrogated (P < 0.001) lung leak in I/R rats pretreated with L-NAME, compared with air-breathing I/R rats pretreated with L-NAME (Fig. 1B). Neither NO nor L-NAME significantly affected lung leak in sham animals.
Effect of NO on Intestinal I/R-Induced Gut Mucosal Permeability
Intestinal I/R increased gut 51Cr-EDTA clearance in untreated (P < 0.001) and L-NAME-treated (P < 0.001) rats (Fig. 2A). Treatment with L-NAME did not significantly affect 51Cr-EDTA clearance in either sham or intestinal I/R rats (P > 0.05). Luminal perfusion of the intestines with NO did not significantly affect 51Cr-EDTA clearance in any of the four treatment groups (Fig. 2B).
Effect of NO on Lung Neutrophil Retention
Rats pretreated with L-NAME and subjected to intestinal I/R had increased lung neutrophils sequestered in buffer-perfused lungs compared with L-NAME-treated sham rats (P < 0.05) or untreated I/R rats (P < 0.05; Fig. 3). In untreated rats, intestinal I/R did not increase lung neutrophil numbers (P > 0.05). Inhalation of NO gas decreased lung neutrophils in I/R rats pretreated with L-NAME, compared with air-breathing I/R rats treated with L-NAME (P < 0.05). L-NAME with or without inhaled NO had no significant effect on lung neutrophil numbers in sham rats.
Effect of Fucoidan on Lung Leak
Rats pretreated with L-NAME and subjected to intestinal I/R had increased (P < 0.001) lung albumin leak compared with L-NAME-treated sham rats (Fig. 4). Fucoidan decreased (P < 0.001) lung leak to baseline levels in L-NAME-treated I/R rats. Fucoidan had no significant effect on L-NAME-treated sham rats.
DISCUSSION
The spread of inflammation from a primarily injured organ to the lung is characteristic of ARDS but occurs in only a fraction of patients at risk for this syndrome. This sporadic occurrence supports the notion that endogenous protective mechanisms prevent the onset of systemic inflammation in the majority of instances. In our study, evidence suggests that endogenous NO limits lung injury and neutrophil recruitment after intestinal I/R in rats.
We found that 30 min of bowel ischemia followed by 2 h of reperfusion did not cause lung leak or neutrophil sequestration. This finding is consistent with prior studies of Terada et al. (20), which demonstrated an increase in lung leak after 90-120 min, but not <60 min, of intestinal ischemia. However, in the present study, the NO synthase inhibitor L-NAME increased pulmonary albumin permeability after an intestinal ischemic insult otherwise subinjurious to the lung, but L-NAME did not increase lung leak under sham conditions. These observations suggest that even 30 min of intestinal I/R can initiate pulmonary inflammatory processes, which are then suppressed by endogenous NO.
Likely sites of action for the protective effects of NO are either the intestines or the lung. It is known, for instance, that NO decreases intestinal injury after mesenteric I/R (14) or lung damage (2). However, our data suggest that NO is acting at the level of the lung and not the intestines. First, L-NAME did not worsen gut mucosal permeability after I/R, in contrast to its striking effect on lung leak. Second, local delivery of NO to the lung, which results in virtually no systemic delivery (19), abolished the effects of L-NAME on lung leak. Finally, local delivery of NO to the intestines by luminal perfusion, which protects the gut from secondary injury in a model of primary lung injury (2), did not diminish gut mucosal leak in our preparation. It is therefore likely that L-NAME causes lung injury by suppressing pulmonary and not intestinal NO. It is interesting to contrast this result with the situation in which the lung is primarily injured by intratracheal acid instillation (2). In this latter instance, NO was found to protect the secondarily damaged small bowel rather than the lung. The common observation that unites both studies is that, in such models of multiple-organ failure, NO affords protection only to secondarily injured organs. A possible conclusion is that NO is not able to protect either gut or lung against an overwhelming primary injury but can mitigate the secondary inflammation that afflicts other organs.
It is also interesting that the function of NO under basal conditions may differ between the lung and the gut. Endogenous NO appears to be involved in the homeostatic maintenance of intestinal mucosal integrity, because L-NAME by itself will increase gut epithelial permeability and leukocyte adherence (9, 13). Indeed, the mean values for gut 51Cr-EDTA clearance in the sham L-NAME group were consistently higher than those for the sham group in the present study, although this effect did not achieve statistical significance. In contrast, neither lung leak nor lung neutrophil sequestration was affected by L-NAME alone, suggesting that endogenous NO may not play a parallel role in preserving mucosal integrity or diminishing the neutrophil population of the lung under basal conditions.
NO may protect the lung at least in part by decreasing neutrophil retention, because L-NAME increased, and inhaled NO decreased, lung neutrophil retention after intestinal I/R. In the mesenteric circulation, it has been demonstrated that NO decreases leukocyte adherence (9, 12), although its effects on lung neutrophil sequestration are not well studied. In this regard, it is interesting to note that NO appears to protect against a neutrophil-dependent ex vivo model of lung injury (7) but not a neutrophil-independent immune-complex model (16). Although the molecular target of NO is not known, P-selectin is a possible candidate. In the mesenteric circulation, for instance, L-NAME increases P-selectin expression and promotes leukocyte rolling and adherence (5). The modulation of P-selectin function by NO is also consistent with our observation that fucoidan blocked L-NAME-induced lung leak after intestinal I/R. In addition, it is likely that P-selectin participates in our model of lung injury, inasmuch as antibodies directed against P-selectin decrease lung injury in a similar model of intestinal I/R (3). Furthermore, exogenous xanthine oxidase increases neutrophil-endothelial cell interactions by increasing pulmonary endothelial P-selectin expression (21).
It is possible, however, that NO also protects lungs by mechanisms not involving neutrophil adherence pathways. For instance, NO decreases superoxide anion production by neutrophils through its actions on a membrane subunit of NADPH oxidase (4), and this effect may potentially diminish the activation of the neutrophils that already reside within the lung. In addition, NO induces endothelial cell antioxidants such as heme oxygenase (15), which may provide additional protection.
In summary, pulmonary NO protects the lung from secondary injury after intestinal I/R. This conclusion is consistent with the protective effects of exogenous NO in humans with ARDS (1) and further extends the role of NO as an endogenous protective agent in inflammatory states.
ACKNOWLEDGEMENTS
This work was supported by American Heart Association Grants 95008180 and 96001480 and by National Institutes of Health Grants R01-DK-37050 and R29-HL-52591. L. S. Terada is an Established Investigator of the American Heart Association.
FOOTNOTES
Address for reprint requests: L. S. Terada, Univ. of Colorado Health Sciences Center, Box C322, 4200 E. Ninth Ave., Denver, CO 80262 (E-mail: lance.terada{at}UCHSC.edu).
Received 4 June 1996; accepted in final form 26 July 1996.
REFERENCES
| 1. | Benzing, A., P. Brautigam, K. Geiger, T. Loop, U. Beyer, and E. Moser. Inhaled nitric oxide reduces pulmonary transvascular albumin flux in patients with acute lung injury. Anesthesiology 83: 1153-1161, 1995. |
| 2. | Brooks, E. C., E. D. Jacobson, N. N. Mahr, and L. S. Terada. Nitric oxide attenuates gut epithelial leak following lung damage (Abstract). Gastroenterology 110: A315, 1996. |
| 3. | Carden, D. L., J. A. Young, and D. N. Granger. Pulmonary microvascular injury after intestinal ischemia-reperfusion: role of P-selectin. J. Appl. Physiol. 75: 2529-2534, 1993. |
| 4. | Clancy, R. M., J. Leszczynska-Piziak, and S. B. Abramson. Nitric oxide, an endothelial cell relaxation factor, inhibits neutrophil superoxide anion production via a direct action on the NADPH oxidase. J. Clin. Invest. 90: 1116-1121, 1992. |
| 5. | Davenpeck, K. L., T. W. Gauthier, and A. M. Lefer. Inhibition of endothelial-derived nitric oxide promotes P-selectin expression and actions in the rat microcirculation. Gastroenterology 107: 1050-1058, 1994. |
| 6. | Gaboury, J., R. C. Woodman, D. N. Granger, P. Reinhardt, and P. Kubes. Nitric oxide prevents leukocyte adherence: role of superoxide. Am. J. Physiol. 265 (Heart Circ. Physiol. 34): H862-H867, 1993. |
| 7. | Guidot, D. M., M. J. Repine, B. M. Hybertson, and J. E. Repine. Inhaled nitric oxide prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L2-L5, 1995. |
| 8. | Kubes, P. Ischemia-reperfusion in feline small intestine: a role for nitric oxide. Am. J. Physiol. 264 (Gastrointest. Liver Physiol. 27): G143-G149, 1993. |
| 9. | Kubes, P., and D. N. Granger. Nitric oxide modulates microvascular permeability. Am. J. Physiol. 262 (Heart Circ. Physiol. 31): H611-H615, 1992. |
| 10. | Kubes, P., M. Jutila, and D. Payne. Therapeutic potential of inhibiting leukocyte rolling in ischemia/reperfusion. J. Clin. Invest. 95: 2510-2519, 1995. |
| 11. | Kubes, P., S. Kanwar, X. F. Niu, and J. P. Gaboury. Nitric oxide synthesis inhibition induces leukocyte adhesion via superoxide and mast cells. FASEB J. 7: 1293-1299, 1993. |
| 12. | Kubes, P., M. Suzuki, and D. N. Granger. Nitric oxide: an endogenous modulator of leukocyte adhesion. Proc. Natl. Acad. Sci. USA 88: 4651-4655, 1991. |
| 13. | Kurose, I., P. Kubes, R. Wolf, D. C. Anderson, J. Paulson, M. Miyasaka, and D. N. Granger. Inhibition of nitric oxide production: mechanisms of vascular albumin leakage. Circ. Res. 73: 164-171, 1993. |
| 14. | Kurose, I., R. Wolf, M. B. Grisham, and D. N. Granger. Modulation of ischemia/reperfusion-induced microvascular dysfunction by nitric oxide. Circ. Res. 74: 376-382, 1994. |
| 15. | Motterlini, R., R. Foresti, M. Intaglietta, and R. M. Winslow. NO-mediated activation of heme oxygenase: endogenous cytoprotection against oxidative stress to endothelium. Am. J. Physiol. 270 (Heart Circ. Physiol. 39): H107-H114, 1996. |
| 16. | Mulligan, M. S., J. S. Warren, C. W. Smith, D. C. Anderson, C. G. Yeh, A. R. Rudolph, and P. A. Ward. Lung injury after deposition of IgA immune complexes: requirements for CD18 and L-arginine. J. Immunol. 148: 3086-3092, 1992. |
| 17. | Nelson, R. M., S. Dolich, A. Aruffo, O. Cecconi, and M. P. Bevilacqua. Higher-affinity oligosaccharide ligands for E-selectin. J. Clin. Invest. 91: 1157-1166, 1993. |
| 18. | Nylander, O., P. Kvietys, and D. N. Granger. Effects of hydrochloric acid on duodenal and jejunal mucosal permeability in the rat. Am. J. Physiol. 257 (Gastrointest. Liver Physiol. 20): G653-G660, 1989. |
| 19. | Shah, N. S., D. K. Nakayama, T. D. Jacob, I. Nishio, T. Imai, T. R. Billiar, R. Exler, S. A. Yousem, E. K. Motoyama, and A. B. Peitzman. Efficacy of inhaled nitric oxide in a porcine model of adult respiratory distress syndrome. Arch. Surg. 129: 158-164, 1994. |
| 20. | Terada, L. S., J. J. Dormish, P. F. Shanley, J. A. Leff, B. O. Anderson, and J. E. Repine. Circulating xanthine oxidase increases following intestinal ischemia-reperfusion and mediates lung neutrophil sequestration. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L394-L401, 1992. |
| 21. | Terada, L. S., B. M. Hybertson, K. G. Connely, D. Weill, D. Piermattei, and J. E. Repine. Xanthine oxidase increases neutrophil adherence to endothelial cells by a dual ICAM-1 and P-selectin-mediated mechanism. J. Appl. Physiol. In press. |
This article has been cited by other articles:
|
|
 |
|
  C. Glynos, A. Kotanidou, S. E. Orfanos, Z. Zhou, D. C. M. Simoes, C. Magkou, C. Roussos, and A. Papapetropoulos Soluble guanylyl cyclase expression is reduced in LPS-induced lung injury Am J Physiol Regulatory Integrative Comp Physiol, April 1, 2007; 292(4): R1448 - R1455. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Waldow, K. Alexiou, W. Witt, F. M. Wagner, V. Gulielmos, K. Matschke, and M. Knaut Attenuation of Reperfusion-Induced Systemic Inflammation by Preconditioning With Nitric Oxide in an In Situ Porcine Model of Normothermic Lung Ischemia Chest, June 1, 2004; 125(6): 2253 - 2259. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Schreiber, K. Gin-Mestan, J. D. Marks, D. Huo, G. Lee, and P. Srisuparp Inhaled Nitric Oxide in Premature Infants with the Respiratory Distress Syndrome N. Engl. J. Med., November 27, 2003; 349(22): 2099 - 2107. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. B. O'Donnell and B. A. Freeman Interactions Between Nitric Oxide and Lipid Oxidation Pathways : Implications for Vascular Disease Circ. Res., January 19, 2001; 88(1): 12 - 21. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. LEEMAN, V. Z. de BEYL, D. BIARENT, M. MAGGIORINI, C. MELOT, and R. NAEIJE Inhibition of Cyclooxygenase and Nitric Oxide Synthase in Hypoxic Vasoconstriction and Oleic Acid-Induced Lung Injury Am. J. Respir. Crit. Care Med., May 1, 1999; 159(5): 1383 - 1390. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. K. Wright, L. T. Kim, T. E. Rogers, H. Nguyen, and R. H. Turnage Nitric Oxide and Thromboxane A2-Mediated Pulmonary Microvascular Dysfunction Arch Surg, March 1, 1999; 134(3): 293 - 298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. A. Skinner, C. R. White, R. Patel, S. Tan, S. Barnes, M. Kirk, V. Darley-Usmar, and D. A. Parks Nitrosation of Uric Acid by Peroxynitrite. FORMATION OF A VASOACTIVE NITRIC OXIDE DONOR J. Biol. Chem., September 18, 1998; 273(38): 24491 - 24497. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. S. Terada, J. E. Repine, D. Piermattei, and B. M. Hybertson Endogenous nitric oxide decreases xanthine oxidase-mediated neutrophil adherence: role of P-selectin J Appl Physiol, March 1, 1997; 82(3): 913 - 917. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
