Ann Thorac Surg 2000;69:228-232
© 2000 The Society of Thoracic Surgeons
Original Articles
Effects of mast cell membrane stabilizing agents in a rat lung ischemia-reperfusion model
Kerem M. Vural, MDa,
Hui Liao, MDb,
Mehmet C. Oz, MDa,
David J. Pinsky, MDc
a Department of Cardiothoracic Surgery, College of Physicians and Surgeons, Columbia University, New York, New York USA
b Department of Physiology, College of Physicians and Surgeons, Columbia University, New York, New York, USA
c Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, New York, USA
Address reprint requests to Dr Vural, N. Tandogan cad. 5/6 Kavaklidere, 06540 Ankara, Turkey
e-mail: kvural{at}tr-net.net.tr
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Abstract
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Background. The aim of this study was to test the hypothesis that agents which stabilize the mast cell membrane may modulate the phenotype of the vascular wall in a lung ischemia-reperfusion model, including altering expression of endothelial and leukocyte adhesion receptors and the inducible nitric oxide synthase (NOS-2).
Methods. Three sets of rats were given either intravenous saline (group A), ketotifen (group B), or cromolyn (group C), respectively. The left pulmonary artery was ligated temporarily and reopened after 2 hours of ischemia. Then, after a 2-hour period of reperfusion, the left lung was excised. ICAM-1 and NOS-2 were measured at the protein level by Western blotting, and cGMP levels were measured by enzyme-linked immunosorbent assay in the lung tissue specimens for each drug group.
Results. ICAM-1 expressions, determined as the intensity of a given band on the Western blot, were 197 ± 59 in group B and 195 ± 83 in group C versus 369 ± 114 in group A (p = 0.002 for analysis of variance). In contrast with ICAM-1, NOS-2 expression was increased by ketotifen or cromolyn treatment (464 ± 82 in group B and 507 ± 93 in group C, compared with 377 ± 44 for group A, p = 0.007). The finding of increased NOS-2 expression in groups B and C is consistent with the observed increase in tissue cGMP levels in the same groups (1.92 ± 0.9 pmol/mL for group A versus 7.8 ± 3.5 pmol/mL for group B, and 12.4 ± 5.8 pmol/mL for group C, p = 0.0004).
Conclusions. These data establish that mast cell stabilizing agents modulate the vascular phenotype in the setting of pulmonary ischemia and reperfusion by decreasing ICAM-1 expression, augmenting expression of NOS-2, and increasing tissue cGMP levels. As decreasing ICAM-1 expression and increasing cGMP levels have proven useful to limit proinflammatory mechanisms of tissue injury, mast cell stabilizing agents may provide a new therapeutic option to improve organ function in the setting of reperfusion.
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Introduction
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Circulating blood cells, especially monocytes and lymphocytes, contain substantial amounts of histamine that may be released upon stimulation [1]. Histamine-induced recruitment of rolling leukocytes is mediated in part by adhesion receptor expression on the endothelial cell [2]. Adhesion molecules expressed by the endothelial cell play a key role in leukocyte-mediated tissue injury. Circulating leukocytes adhere to vessel wall through ligand-receptor interactions involving potent adhesion receptors such as ICAM-1, after which they leave the blood stream and enter the tissue. Having been activated, they release toxic substances which may cause a considerable amount of damage to adjacent tissues [3].
Ketotifen is a second generation histamine H1 receptor antagonist which has long been used in the management of allergic disorders. In addition to histamine receptor antagonism, ketotifen has been recently found to inhibit the release of mast cell and neutrophil-derived proinflammatory mediators. Ketotifen also stimulates nitric oxide synthase (NOS-2) activity by mechanisms other than H1-receptor antagonism [4]. Cromolyn is another mast cell stabilizing agent reported to have similar properties which may possess even greater potency in terms of its antiinflammatory properties [5, 6]. The aim of this study was to determine the effects of these 2 drugs, with respect to attenuation of the inflammatory response, in terms of their ability to modulate the vascular phenotype, including adhesion receptor and NOS-2 expression. Such an effect may be especially beneficial in reperfusion settings such as organ transplantation or other types of heart or lung operation.
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Material and methods
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Animal surgery
Three sets of Harlan male Sprague-Dawley rats (Harlan, Indianapolis, IN), each consisted of 7 animals, were anesthetized by intraperitoneal injection of xylazine (5 mg/kg) and ketamine (50 mg/kg). After ensuring adequate depth of anesthesia, the animals were fixed in a supine position and connected to a rat ventilator (Harvard Rodent Ventilator, Model 683, Harvard Apparatus, Inc, Holliston, MA) through a tracheostomy stoma created in the neck. Anesthesia was maintained during the experiment with repetitive doses of xylazine and ketamine at 45-minute intervals or on return of somatic reflexes. A bilateral clamshell incision was then made to expose the thoracic cavity. The right, left, and main pulmonary arteries were located and dissected free from the surrounding adjacent tissues. Following dissection, animals received an intravenous injection of either physiological saline (group A, controls), ketotifen fumarate (1 mg/kg, group B, prepared as a 1 mg/mL stock solution in physiological saline; Sigma Chemical Co, St. Louis, MO), or cromolyn sodium (20 mg/kg, group C, prepared as a 5 mg/mL stock solution in physiological saline; Sigma Chemical Co). Fifteen minutes later, heparin was administered (1 mg/kg), and the left pulmonary artery was temporarily ligated. To ensure complete pulmonary ischemia, pulmonary hilus was also ligated with a heavy silk tie, interrupting bronchial circulation. Cromolyn injections were repeated at every 1 hour due to the very short in vivo plasma life of this drug [7]. After 2 hours of ischemia, the left pulmonary artery was reopened. At the end of another 2-hour period of reperfusion, the left lung was excised and snap frozen in liquid nitrogen. Animal use complied with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals" (NIH Publication no. 86-23, revised 1985) and was approved by the Institutional Animal Care and Use Committee.
Western blotting
Tissue samples were homogenized for 30 seconds at 4°C with a Polytron homogenizer (Kinematica, GmbH, Krienz-Luzerne, Switzerland) with ice-cold 20 mmol/L Tris hydrogen chloride (HCl), pH 7.4, containing 100 mmol/L sodium chloride (NaCl), 2 mmol/L phenylmethylsulphonyl fluoride (PMSF), 0.5 mg/L leupeptin, and 0.7 mg/L pepstatin. Homogenates were shaken at 4°C for 3 hours, then centrifuged at 13,000 rpm for 10 minutes at 4°C and the supernatant was collected as the source of sample protein. Samples were run in a 7.5% polyacrylamide gel and then transferred to a nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA). The membrane was blocked for nonspecific binding using 1% bovine serum albumin (BSA) in phosphate buffer saline (PBS) for 12 hours at 4°C. The membrane was then incubated in 0.05% BSA and 0.05% Tween 20 in PBS containing goat-anti-rat ICAM-1 (Santa Cruz Inc, Santa Cruz, CA) antibody at 1:1000 concentration for 1 hour. After subsequent washes, the membrane was incubated with anti-goat IgG-peroxidase conjugate (Sigma Chemical Co) at 1:2000 concentration in PBS, 0.5% BSA and 0.05% Tween 20 for 45 minutes. After the subsequent washes, roentgenogram films were obtained using enhanced chemiluminescence (ECL) detection (Amersham, Piscataway, NJ). The staining intensity of specific bands was quantified by densitometric scanning by a computer software (Molecular Analyst, Bio-Rad Laboratories). Those calculated areas were then statistically compared. The same procedure was repeated for the detection of inducible NOS-2 using a rabbit-derived anti-NOS-2 (Santa Cruz Biotechnology Inc) and an anti-rabbit IgG-peroxidase conjugate (Sigma Chemical Co) at 1:2000 concentration.
Enzyme-linked immunosorbent assay (ELISA)
Tissue homogenates in PBS were added to 5% trichloroacetic acid and kept on ice for 30 minutes. Those homogenates were spun at 4,000 rpm for 15 minutes. Supernatant was collected and washed with ether 3 times. After storing the homogenates at 20°C for 5 minutes, cGMP content was measured by an ELISA kit (R&D Systems Inc, Minneapolis, MN) which is based on the competitive binding technique in which cGMP present in a sample competes with a fixed amount of alkaline phosphatase-labeled cGMP for sites on a rabbit polyclonal antibody. That antibody was bound to a goat-anti-rabbit antibody coated onto the microplate. The absorbance was read at 405 nm using a Biokinetics microplate reader Model EL 340 (Biotek Instruments, Winooski, VT).
Statistical analysis
All statistics were performed using SPSS statistical software (release 8.0, SPSS Inc, Chicago, IL). Means are presented ± standard deviation. One-way analysis of variance (ANOVA) was performed first to search any differences between groups A, B, and C. If the result of the ANOVA is significant (p < 0.05), pairwise comparisons between the groups were made by a post-hoc test (Tukey’s HSD procedure). The significance level was set at p less than 0.05.
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Results
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Cellular adhesion molecule expression
The expression of ICAM-1 in ischemic and reperfused rat lung was examined by Western blotting. ICAM-1 expression, quantified as the intensity of a given band on the Western blot was as follows: 369 ± 114 in group A (control group), 197 ± 59 in group B and 195 ± 83 in group C (Figs 1, 2). After ANOVA revealed significant difference between the groups A, B, and C (p = 0.002), the analysis continued with a post-hoc test (Tukey’s HSD test) to make pairwise comparisons, which revealed significant difference between groups A and B, and groups A and C (p < 0.05). No difference was detected between groups B and C. These data demonstrate that in comparison with group A, ICAM-1 expression was significantly lower in both groups of animals treated with a mast cell stabilizing agent. There were no differences in ICAM-1 expression between the two mast cell stabilizers studied.
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Fig 1. Representative immunoblot demonstrating ICAM-1 expression of rat lung tissue from groups A, B, and C. (+) = positive control lane for ICAM-1 molecule.
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Fig 2. ICAM-1 expression of rat lung tissue, quantified as the intensity of a given band on the Western blot in Figure 1, presented as means ± standard deviation. The difference is significant between the groups (ANOVA, p = 0.002), pairwise comparisons between the groups were made by a post-hoc test (Tukey’s HSD test). (*) = significant difference at a confidence level of p < 0.05; NS = not significant.
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Inducible nitric oxide synthase (NOS-2) detection
To examine the effect of mast cell stabilizing agents on another inflammatory mediator gene, the expression of the inducible isoform of NOS-2 in the setting of pulmonary ischemia and reperfusion was also studied by Western blotting. NOS-2 expression, determined as the intensity of a given band on the Western blot, was as follows: 377 ± 44 in group A, 464 ± 82 in group B, and 507 ± 93 in group C (Figs 3, 4). The difference between the groups was significant by ANOVA (p = 0.007); pairwise comparisons revealed that NOS-2 expression was significantly higher in group B and group C than that observed in group A (p < 0.05). Again, as was seen for ICAM-1 expression, there were no differences detected between groups B and C.
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Fig 3. Representative immunoblot demonstrating NOS-2 expression of rat lung tissue from groups A, B, and C. (+) = positive control lane for NOS-2 molecule.
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Fig 4. NOS-2 expression of rat lung tissue, quantified as the intensity of a given band on the Western blot in Figure 3, presented as means ± standard deviation. The difference is significant between the groups (ANOVA, p = 0.007), pairwise comparisons between the groups were made by a post-hoc test (Tukey’s HSD test). (*) = significant difference at a confidence level of p < 0.05; NS = not significant.
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cGMP content
Because nitric oxide stimulates soluble guanylate cyclase to form the cyclic nucleotide cGMP, ELISAs were performed to quantify tissue cGMP levels. Tissue cGMP levels were 1.92 ± 0.9 pmol/mL for group A, 7.8 ± 3.5 pmol/mL for group B, and, 12.4 ± 5.8 pmol/mL for group C (Fig 5). The difference between the groups was significant (p = 0.0004). Pairwise comparisons revealed that tissue cGMP levels in groups B and C were significantly higher when compared to those in group A (p < 0.05). There was no difference between groups B and C.
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Fig 5. Tissue cGMP levels, determined by ELISA, presented as means ± standard deviation. The difference is significant between the groups (ANOVA, p = 0.0004), pairwise comparisons between the groups were made by a post-hoc test (Tukey’s HSD test). (*) = significant difference at a confidence level of p < 0.05; NS = not significant.
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Comment
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Leukocyte adhesion to vascular endothelium is a critical step during the early stages of inflammation, and is mediated by the interaction of adhesion receptors expressed on the surfaces of both endothelial cells and leukocytes. The expression of adhesion receptors, therefore, plays a key role in leukocyte-mediated tissue injury in the setting of reperfusion, as pointed out in detail in previous publishings, such as the comprehensive review of Novick and associates [8]. Intercellular adhesion receptors, expressed on endothelial cells, mediates leukocyte binding to endothelial cells through interactions with their integrin counter receptors on leukocytes, such as CD 11/18 and very late antigen-4 (VLA-4). Proinflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor-alpha (TNF-
) can further amplify the local proinflammator milieu by further inducing the expression of endothelial adhesion receptors [9].
In contrast to TNF- and IL-1, which require de novo protein synthesis for their expression, histamine, also a key inflammatory mediator, is stored in preformed granules. Histamine may therefore be rapidly released, playing an important early role in amplifying the inflammatory cascade, by increasing vascular permeability and expression of endothelial leukocyte adhesion receptors [2]. Histamine induces the expression of ICAM-1 synergistically with TNF- through histamine H1 receptors. Histamine H1 receptor antagonists have been shown to inhibit the expression of ICAM-1 [9].
In the current study, an investigation of mast cell stabilizing agents carried out in an in vivo model of lung ischemia-reperfusion injury, we studied the effects of several prototypical mast cell membrane stabilizing agents in the setting of lung reperfusion injury. These agents (ketotifen and cromolyn) favorably modulated cellular adhesion molecule expression and NOS-2 expression in a rat lung ischemia-reperfusion model. Since the nitric oxide (NO)-related vascular response uses cGMP as second messenger, the increased cGMP levels seen with infusions of these are consistent with the finding of increased NOS-2 induction by these agents. These findings may help to explain a mechanism by which cGMP confers important vascular protective properties [10, 11] and why others have shown that mast cell stabilizing agents can mitigate against ischemia-reperfusion injury [6].
Human blood monocytes and lymphocytes contain substantial amounts of histamine which may be released upon stimulation with either substance P, C5a, or the calcium ionophore [1]. Histamine release from activated mast cells is attenuated by exogenous NO and exacerbated by NO synthesis inhibitors. Moreover, the mast cell produces a NO-like factor that can directly feed back to decrease histamine release [12]. NO is an endogenous inhibitor of leukocyte adhesion, activation and chemotaxis. NO activates guanylate cyclase and increases the conversion of GTP to cGMP, which in turn reacts with cGMP-dependent protein kinase and causes a cascade of changes in protein phosphorylation, including dephosphorylation of myosin light chain, leading to cell relaxation. Therefore, a reduction in cGMP may cause endothelial cell contraction and increase the size of interendothelial junctions, resulting in a leaky endothelial barrier. Absence of NO synthesis also leads to superoxide accumulation, which could directly cause an increase in endothelial permeability and the release of various mast cell-derived chemical agents, including PAF and histamine [12]. NO modulates mast cell degranulation by competing with superoxide anion, a potent activator of mast cell activation and degranulation. On the other hand, endothelial free radical generation plays an important role in regulation of intercellular adhesion molecules and leukocyte recruitment [13, 14]. Therefore, the suppression of ICAM-1 expression on the endothelial cells by endogenous NO might contribute to suppressing inflammation in vivo.
Ketotifen, a second generation histamine H1 receptor antagonist, has long been used in the management of allergic disorders. In addition to its properties as a histamine receptor antagonist, it may also inhibit the release of mast cell and neutrophil-derived proinflammatory mediators. In various experimental and clinical conditions, ketotifen was noted to reduce mast cell degranulation and to decrease the release of histamine, mast cell proteases, myeloperoxidase, leukotrienes, platelet activating factor (PAF), and various prostaglandins [15]. Ketotifen also stimulates NO synthase activity by mechanisms other than H1-receptor antagonism, and administration of this drug results in modest declines in blood pressure and reduced vascular resistance [4]. Ketotifen stabilizes the cell membrane and/or alters its properties with respect to calcium permeability. It causes a dose-related decrease in acetyl-CoA acetyltransferase stimulation and antigen-induced PAF release. Ketotifen may also promote vascular homeostasis by its properties to prevent a decline in cAMP levels in stimulated leukocytes [15].
Cromolyn is another mast cell stabilizing agent with similar properties as ketotifen, but which may be more potent in effect. Cromolyn modulates both the early and late phases of immunoallergic response. During the early phase response, cromolyn inhibits histamine release from mast cells through cell membrane stabilization. This inhibition may be related to either the ability of the drug to elevate intracellular cAMP levels or its action on receptor mediated calcium channels. As late phase responses are thought to be primarily neutrophil mediated, cromolyn may inhibit these responses by decreasing neutrophil chemotaxis and inhibiting neutrophil activation and lysozyme secretion. One additional site of action for cromolyn may be at the GTP-binding protein, which is related to phospholipase C activation [5]. Cromolyn blocks the release of calcium from intracellular stores. The alteration in calcium mobilization is indicative of impaired neutrophil signaling. Cromolyn also inhibits the superoxide anion and assembly of the NADPH oxidase, which is an important source for free radical production [6].
Taken together, the data presented in this manuscript show additional mechanisms by which mast cell stabilizing agents may promote vascular homeostasis. These agents reduce ICAM-1 expression and increase NOS-2 expression in the setting of lung ischemia and reperfusion injury. As histamine induces the expression of ICAM-1 on the endothelial cells, which may promote leukocyte-induced tissue damage, it is reasonable to speculate that histamine blockers which suppress ICAM-1 expression might reduce the inflammatory reaction following ischemia by an ICAM-1 dependent mechanism. Similarly, the ability of these agents to increase NOS-2 expression and to augment cGMP levels, which has marked antiinflammatory properties, may contribute to a suppression of inflammation in the setting of ischemia and reperfusion. These properties may prove beneficial in clinical settings in terms of reducing reperfusion injury to the heart or lungs following cardiothoracic surgery or organ transplantation.
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Acknowledgments
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This study was supported in part by the United States Public Health Service (grants HL55397 and HL59488). Doctor Oz is an Irving Assistant Professor of Surgery, and Dr Pinsky is a Clinician Scientist for the American Heart Association. Doctor Vural was supported by a research grant from the Turkish Educational Foundation.
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Accepted for publication June 9, 1999.
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Abstract
Introduction
