| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J Thorac Cardiovasc Surg 2000;120:558-565
© 2000 The American Association for Thoracic Surgery
Cardiopulmonary Support and Physiology |
From the Department of Surgery, Course of Interventional Medicine (E1), Osaka University Graduate School of Medicine,a Osaka; the Sumitomo Pharmaceuticals Research Center,b Osaka; and the Second Department of Physiology, Tokai University School of Medicine,c Isehara City, Japan.
Address for reprints: Yoshiki Sawa, MD, Department of Surgery, Course of Interventional Medicine (E1), Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan (E-mail: sawa{at}surg1.med.osaka-u.ac.jp ).
 |
Abstract |
|---|
 Top Abstract IntroductionMethods Results Discussion References |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
1,2 Current evidence indicates strongly that neutrophils play a pivotal role in the development of CPB-induced inflammatory response. 3 Generally, one of the neutrophil-mediated inflammatory responses is the interaction of neutrophils with vascular endothelial cells via adhesion molecules; the selectin family (E-, P-, and L-selectin), the immunoglobulin superfamily (intercellular adhesion molecule-1: ICAM-1), and the integrins (CD11/CD18 complex: Mac-1). 4 Inhibition of the adhesion processes seems beneficial in attenuating the inflammatory response, and prior studies have demonstrated the effects of blocking various adhesion molecules in animal inflammation models. 5,6
P-selectin is an adhesion molecule that is constitutively present in the Weibel-Palade bodies in endothelial cells or 
-granules in platelets, and it participates in "rolling," which is considered the first step of neutrophil migration into the postcapillary venules. 7 Stimulation by histamine, thrombin, oxygen-derived free radicals, or inflammatory cytokines, which are CPB-induced chemotactic mediators, leads to the rapid degranulation and translocation of P-selectin onto the cell surface within a few minutes soon after the initiation of inflammatory response. 7 The subsequent up-regulation of other adhesion molecules follows several hours after "expression of P-selectin." 8 Thus, inhibiting the earliest neutrophil-endothelial interactions, which are mediated by P-selectin, appears suitable for clinical application. The role of P-selectin in CPB-induced inflammatory response has not been clarified, although prior study in the fields of cardiovascular surgery has demonstrated that blockade of P-selectin by species-specific monoclonal antibody attenuates myocardial ischemia-reperfusion injury. 9
Nitric oxide (NO) is one of the most potent vasodilators among the inflammation-mediated vasoactive substances, and CPB has been reported to enhance plasma NO production. 10,11 Interleukin 6 (IL-6) and IL-8 are reported to be produced at the early phase of CPB-induced inflammatory response, 12 and various chemotactic mediators subsequently induced are thought to produce inducible NO synthase (iNOS), 11 as well as to regulate some adhesion molecules. 13 The increase in NO production may result in peroxynitrite formation by rapid reaction of superoxide anion with NO, which is a potent oxidant. 14 Although NO and peroxynitrite have opposite cytologic effects on the development of inflammation and their roles remain unclear, 15-18 NO itself is thought to regulate P-selectin expression. 19 To elucidate whether P-selectin participates in CPB-induced inflammatory response in association with NO production followed by reaction of peroxynitrite, we examined the effect of a species-specific P-selectin monoclonal antibody in a rat model of CPB.
|
Methods |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
9 was added into the priming solution of the bypass circuit before CPB (group P, n = 10). In the other group, a 3-mg/kg dose of PNB1.6 (Sumitomo Pharmaceuticals), which is a "nonblocking monoclonal antibody," was added in the same way for the control group (group C, n = 10). All animals had received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). The rats were anesthetized by intraperitoneal administration of sodium pentobarbital (50 mg/kg) and placed in the supine position. The lungs were ventilated with 100% oxygen through an 18-gauge tracheotomy tube with a tidal volume of 10 mL/kg and a respiratory rate of 60 breaths/min. After exposure of the cannulation sites, heparin (300 units/kg) was injected intraperitoneally. Two 16-gauge catheters were inserted via the right jugular vein into the right atrium and directly into the left femoral vein. A 20-gauge catheter was inserted directly into the left femoral artery.
The CPB circuits were composed of a roller pump (Perista Bio-Minipump AC-2120; Atto Co, Ltd, Tokyo, Japan), a membrane oxygenator (Senko Medical Co, Ltd, Osaka, Japan), a venous reservoir, and a tubing line. None of the materials in the CPB circuit were heparin coated. The bypass circuit was primed with the following solution without blood components: 12 mL of plasma expander containing hydroxyethyl-amylum (Hespander; Kyorin Pharmaceutical, Tokyo, Japan), 8 mL of lactated Ringer's solution, 2 mL of 7% sodium bicarbonate, 2 mL of mannitol, 100 units of heparin, and 1.5 mg of tobramycin. Perfusion flow rate was maintained at 100 mL · kg–1 · min–1, and perfusate temperature was maintained at 34°C with a heat exchanger. No blood components were transfused throughout the experiment. Routinely, termination of CPB was aided by continuous administration of dobutamine (3 µg · kg–1 · min–1). The remaining priming solution was infused gradually after the termination of CPB.
A 1.5-mL sample of arterial blood and exhaled air was obtained at the following 3 times: (1) before the initiation of CPB, (2) at the termination of CPB, and (3) 3 hours after the termination of CPB. Plasma levels of P-selectin, inflammatory cytokines, and nitrate/nitrite, nitrotyrosine, and exhaled NO concentration were measured to evaluate the effect of ARP2-4. Respiratory index was obtained by measuring arterial and alveolar PO 2.
Measurements
Plasma levels of P-selectin
The blood sample was mixed with citrate to a final concentration of 0.314%. Plasma was separated by centrifugation at 2000g for 15 minutes. The plasma level of P-selectin was measured by enzyme-linked immunosorbent assay with the use of anti-human P-selectin polyclonal antibody (09361A; Pharmingen, San Diego, Calif) and peroxidase-conjugated anti-rabbit immunoglobulin (P448; DAKO, Glostrup, Denmark). 20 Plasma P-selectin measured by this method is considered to be released both from platelets destroyed by centrifugal force and from endothelial cells as a result of CPB-induced endothelial activation.
Inflammatory cytokines
We measured plasma levels of IL-6 and IL-8 as markers of CPB-induced inflammatory response. The plasma IL-6 level was measured by enzyme-linked immunosorbent assay with a commercially available kit (Rat ELISA Kits; Biosource International, Camarillo, Calif). The IL-8 level was measured by enzyme immunoassay with a commercially available kit (Rat IL-8 Kit; Panafarm Laboratory, Tokyo, Japan).
Plasma nitrate/nitrite
The plasma fraction after centrifugation was diluted 1:1 with nitrite- and nitrate-free distilled water. Subsequently, 400 µL of diluted plasma was ultrafiltered at 2000g and its filtrates were analyzed by an automated procedure based on the Griess reaction. 21
Nitrotyrosine (peroxynitrite) formation
Peroxynitrite has a very short half-time in vivo and is usually measured in terms of the formation of 3-nitro-L -tyrosine (nitrotyrosine), which is generated by the nitration of tyrosine residues by peroxynitrite itself 22 and is relatively stable. Thus, we measured nitrotyrosine formation as an indicator of peroxynitrite production using a high-pressure liquid chromatography method previously described. 23 In brief, blood samples were centrifuged at 1200g for 15 minutes, and the filtrates were hydrolyzed for 24 hours. The supernatant fluid was analyzed by high-pressure liquid chromatography with a C-18 reverse-phase column (Jasco, Tokyo, Japan), and the peak concentrations were measured with an ultraviolet detector set at 274 nm (UV-970; Jasco). The peak was identified on the basis of the retention time of authentic 3-nitro-L -tyrosine (nitrotyrosine) or tyrosine. Peroxynitrite formation was measured as the formation of nitrotyrosine from tyrosine and expressed as the percentage ratio of nitrotyrosine to tyrosine (%NTY, expressed as [nitrotyrosine/tyrosine + nitrotyrosine] x 100%). 23
Exhaled NO concentration
Mandatory ventilation consisting of 5 mL of room air was performed 10 times with a 10-mL syringe. The last 5 mL of exhaled air was subjected to NO analysis. Three successive 1-mL samples of exhaled air were measured with a chemiluminescence analyzer (Sievers model 270B; Sievers Instruments, Boulder, Colo) sensitive to NO at concentrations of 5 to 5 x 105 ppb within 30 minutes after the collection of exhaled air. The average value from the 3 successive samples was used for analysis. 24
Respiratory index
Respiratory index is an index of oxygenation function, and its increase reflects the presence of pulmonary shunting in a variety of conditions including atelectasis, pulmonary contusion, and pulmonary thromboembolism. Respiratory index was calculated by arterial blood gas assay as follows: Respiratory index = (Alveolar – arterial oxygen tension gradient)/Arterial oxygen tension.
Histologic analysis
All rats were killed 3 hours after the termination of CPB. Lung specimens were frozen and prepared for immunohistologic examination.
ARP2-4 (Sumitomo Pharmaceuticals; dilution 1/250 in phosphate-buffered saline solution) and peroxidase-conjugated goat anti-mouse immunoglobulin G (Sigma Chemical Company, St Louis, Mo; dilution 1/10,000 in phosphate-buffered saline solution) were used to evaluate the expression of lung vascular P-selectin. 25 Naphthol AS-D chloroacetate esterase was used to evaluate neutrophil adhesion to lung endothelial cells. Accumulated neutrophils and alveoli were counted under a microscope at 100x magnification in 10 fields in all 10 serial sections. The results were corrected by number of alveoli.
Statistical analysis
All data are expressed as mean ± standard deviation. Comparisons between groups were analyzed by 2-way repeated-measures analysis of variance and the unpaired Student t test. The Spearman rank correlation test was used for analysis between exhaled NO concentration and respiratory index. All analyses were performed with the StatView version 5.0 statistical package (Abacus Concepts, Inc, Berkeley, Calif).
|
Results |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
|
).
Plasma IL-8 levels before CPB were not significantly different between the 2 groups, and the levels after the termination of CPB were similar to the pattern of IL-6. Both at the termination and 3 hours after the termination of CPB, plasma IL-8 levels in group P were significantly lower than those in group C (Table I).
Plasma nitrate/nitrite
The plasma level of nitrate/nitrite before CPB was not significantly different between the groups. In both groups, the NO levels after the termination of CPB were significantly higher than those before CPB, and they were significantly higher 3 hours after the termination of CPB than immediately after the termination of CPB. The NO level in group P was significantly lower than that in group C 3 hours after the termination of CPB, although there was no significant difference immediately after the termination of CPB (Table I).
Nitrotyrosine (peroxynitrite) formation
Nitrotyrosine was not detected in the supernatant fluid obtained before CPB in either group. In both groups, it was detected after the termination of CPB, and the %NTY 3 hours after the termination of CPB was significantly higher than that at the termination of CPB. The %NTY in group P was significantly lower than that in group C at the termination of CPB, and the difference between the 2 groups remained at about the same level of significance 3 hours after the termination of CPB (Table I).
Exhaled NO concentration
The 2 groups had similar concentrations of exhaled NO before CPB. Exhaled NO concentration at the termination of CPB was significantly lower than that before CPB in both groups, but it was significantly higher 3 hours after CPB than before CPB. At the termination of CPB, the level in group P was significantly higher than that in group C; however, 3 hours after the termination of CPB, the level in group P was significantly lower than that in group C (Table I).
Respiratory index
Respiratory indexes were not significantly different before CPB between the 2 groups. Both groups showed significantly higher respiratory indexes at the termination of CPB, and respiratory index 3 hours after the termination of CPB was higher than that at the termination of CPB. In group P, the respiratory indexes were significantly lower than those in group C both at the termination of CPB and 3 hours after the termination of CPB (Table I).
There was a negative correlation between exhaled NO and respiratory index at the termination of CPB (group P: r = –0.781, P = .0056; group C: r = –0.813, P = .0027) (Fig 1, A ). However, there was no significant correlation 3 hours after the termination of CPB (group P: r = 0.570, P = .0825; group C: r = 0.522, P = .1254) (Fig 1, B ).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
26 and has been reported to increase in septic conditions as a result of endothelial activation and damage. 20 No experimental study demonstrating the change in plasma P-selectin in an animal CPB model has been reported; thus, this is the first experimental study examining the change in plasma P-selectin associated with CPB-induced inflammatory response in a species other than Homo sapiens. Our present results in the rat CPB model indicate that P-selectin plays a crucial and interactive role in CPB-induced inflammatory response. Blockade of P-selectin–mediated interaction inhibited neutrophil accumulation in lung capillaries and thereby attenuated the CPB-induced inflammatory response. However, the mechanism by which CPB-induced inflammatory response develops is complex. These histologic findings alone may not account for the effect on attenuating CPB-induced inflammatory response.
P-selectin participates in the development of inflammatory response in various ways other than neutrophil–endothelial cell interaction; platelet-platelet aggregation, and platelet-neutrophil adhesion. 27 Recently, we 28 demonstrated that ARP2-4 inhibits neutrophil–endothelial cell adhesion via activated platelets, resulting in the attenuation of myocardial reperfusion injury. Soon after the initiation of CPB, blood contact with artificial surfaces rapidly induces the up-regulation of P-selectin on the surface of both platelets and endothelial cells. 29,30 The decrease in plasma level of P-selectin reflects the above platelet-mediated interactions, and the increase reflects endothelial activation. At the termination of CPB, the change in plasma P-selectin appears mainly affected by platelet-mediated interactions. Late after the termination of CPB, plasma P-selectin appears mainly affected by the continuation of CPB-induced endothelial activation, as has been speculated. Our results are consistent with the clinical study by Blume and associates, 30 although no quantitative report about the origin of plasma P-selectin has been presented, and further experimental examination is needed.
Neutrophil–endothelial cell interaction is thought to enhance the release of the chemotactic mediators. Inflammatory cytokines, IL-6 and IL-8, are produced at the early phase of CPB-induced inflammatory response, and the production of these inflammatory cytokines continues late after the termination of CPB, which causes a CPB-induced inflammatory response. 12 IL-8, in particular, is produced mainly from neutrophils. It activates the up-regulation of the integrin onto the neutrophils and thereby plays a role in neutrophil transmigration. 13 ARP2-4 may subsequently attenuate further up-regulation of other adhesion molecules and abnormal release of inflammatory cytokines, which are considered as positive feedback processes of the development of CPB-induced inflammatory response.
The enhancement of plasma NO production during and after CPB is considered dependent on the following 2 pathways. After the initiation of CPB, mechanical stimulation of vascular wall by CPB-induced blood flow and hemodilution by CPB priming volume activate endothelial constitutive NOS. 10 Several hours after CPB, excessive NO is produced by the induction of iNOS from activated blood components and inflammatory cytokines. 11 After the termination of CPB, ARP2-4 reduced significantly the enhanced release of inflammatory cytokines such as IL-6 and IL-8. Although these cytokines themselves have no iNOS-inducing activity, the development of inflammation induces subsequent iNOS expression several hours after CPB. 11 Furthermore, tumor necrosis factor and IL-1ß, which induce iNOS production, are released late after the termination of CPB when inflammation is severe. 31 In this short-term CPB model, ARP2-4 did not attenuate the increase in NO at the termination of CPB. The reason for this lack of effect may be partially that the increase in NO at the termination of CPB mainly results from endothelial-constitutive NOS activation induced by CPB initiation itself. 10
On the other hand, %NTY was significantly reduced both at the termination of CPB and 3 hours after the termination of CPB, whereas plasma NO levels at the termination of CPB did not differ significantly between the groups. Peroxynitrite production is defined by the amounts of NO and superoxide anion. 23,24 One of the possible reasons is that ARP2-4 probably reduced superoxide production as a result of attenuating P-selectin–mediated inflammatory interactions, as well as the release of inflammatory cytokines.
Generally, exhaled NO production is attributed to neural NOS of lung epithelial cells in the pre-inflammatory state. 32 As inflammation develops, iNOS expression is induced and activated macrophages produce a great deal of exhaled NO. 33 Thus, exhaled NO is considered an indicator of lung injury in respiratory diseases and septic conditions. 34,35 However, the change in exhaled NO after CPB remains unclear, and its association with CPB-induced inflammatory response has not been clarified. 36 The present study first suggested the association between the change in exhaled NO and CPB-induced inflammatory response in an experimental CPB model even though the detailed mechanism, such as the roles of NOSs in exhaled NO production, was not well characterized.
As one of the means of attenuating neutrophil-mediated interaction, leukocyte-depleted extracorporeal perfusion, which Johnson and coworkers 3 demonstrated in a dog CPB model, seems beneficial in attenuating CPB-induced inflammatory response. However, the technique cannot be easily applied clinically, because continuous use of the removal filter may reduce perfusion flow rate and impair its ability to remove leukocytes. Moreover, complete depletion of leukocytes appears to increase the risk of infection. Our experimental results suggest that blockade of P-selectin with species-specific monoclonal antibody may be useful in attenuating neutrophil-mediated CPB-induced inflammatory response in view of suitable clinical application.
In summary, although the present study does not completely simulate the clinical situation because the pulmonary circulation is preserved in this rat CPB model, it is the first to demonstrate the beneficial effect of species-specific P-selectin monoclonal antibody on CPB-induced inflammatory response. P-selectin, which has been already shown to attenuate myocardial ischemia-reperfusion injury, may help to augment CPB-induced inflammatory response in association with NO and peroxynitrite production. Thus, its monoclonal antibody against P-selectin seems to be a promising treatment for CPB-induced inflammatory response, although additional studies in the clinical setting are needed.
|
References |
|---|
|
TopAbstractIntroductionMethods Results Discussion References |
|---|
This article has been cited by other articles:
|
|
 |
|
  H. Komai, Y. Naito, and Y. Okamura Dextran sulfate as a leukocyte-endothelium adhesion molecule inhibitor of lung injury in pediatric open-heart surgery Perfusion, March 1, 2005; 20(2): 77 - 82. [Abstract] [PDF]
|
|
|
|
 |
|
 Y. Hayashi, Y. Sawa, M. Nishimura, N. Fukuyama, H. Ichikawa, S. Ohtake, H. Nakazawa, and H. Matsuda Peroxynitrite, a product between nitric oxide and superoxide anion, plays a cytotoxic role in the development of post-bypass systemic inflammatory response Eur. J. Cardiothorac. Surg., August 1, 2004; 26(2): 276 - 280. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Hayashi, Y. Sawa, N. Fukuyama, H. Nakazawa, and H. Matsuda Inducible nitric oxide production is an adaptation to cardiopulmonary bypass-induced inflammatory response Ann. Thorac. Surg., July 1, 2001; 72(1): 149 - 155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Medhora, M. Bousamra II, D. Zhu, L. Somberg, and E. R. Jacobs Upregulation of collagens detected by gene array in a model of flow-induced pulmonary vascular remodeling Am J Physiol Heart Circ Physiol, February 1, 2002; 282(2): H414 - H422. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| ANN THORAC SURG | ASIAN CARDIOVASC THORAC ANN | EUR J CARDIOTHORAC SURG |
| J THORAC CARDIOVASC SURG | ICVTS | ALL CTSNet JOURNALS |
