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Nitric oxide released from iNOS in polymorphonuclear leukocytes makes them deformable in an autocrine manner
NITRIC OXIDE
Biology and Chemistry
Nitric Oxide 7 (2002) 221–227 www.academicpress.com
Brief communication
Nitric oxide released from iNOS in polymorphonuclear leukocytes makes them deformable in an autocrine manner Hirosuke Kobayashi,a,* Tailin Cui,a Miyuki Ando,a Ryuji Hataishi,a Takao Imasaki,a Hisashi Mitsufuji,a Izumi Hayashi,b and Tomoyuki Tomitaa b
a Department of Medicine, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan Department of Pharmacology, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan
Received 30 November 2001; received in revised form 18 March 2002
Abstract The objective of this study was to determine whether endogenous nitric oxide (NO) derived from reaction catalyzed by the inducible isoform of NO synthase (iNOS: NOS II) in polymorphonuclear leukocytes (PMNs) makes the PMNs deformable. Previous studies have shown that NO increases the deformability of PMNs and decreases the sequestration of PMNs in the lungs. However, there was little information regarding the effect of PMN-derived NO on the cells’ deformability. In the present study PMNs were isolated from the blood of rats 24 h after ip injection of saline (control) or lipopolysaccharide (LPS), and expression of iNOS in the PMNs of the LPS group was confirmed by immunocytochemistry. PMN deformability was evaluated by measuring the pressure generated during their passage through a microfilter at a constant flow rate. The nitrite/nitrate content of the solution in which the isolated PMNs were incubated was measured by the Griess method. In the control group, no iNOS was detectable in the PMNs, and the nitrite/nitrate level in the PMN incubation solution was low. Deformability was unchanged after incubation with specific iNOS inhibitor aminoguanidine, but decreased after incubation with N-formyl-methionyl-leucyl-phenyl-alanine. In the LPS group, PMN deformability was decreased compared to that of the control group. iNOS was detectable in the PMNs, and the deformability further decreased after incubation with aminoguanidine. These results suggest that endogenous NO generated during reactions catalyzed by iNOS in PMNs makes them deformable in an autocrine manner. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: ARDS; Polymorphonuclear leukocyte; Deformability; Inducible nitric oxide synthase; Sequestration
Polymorphonuclear leukocytes (PMNs)1 play an important role in the pathogenesis of acute respiratory distress syndrome (ARDS) [1], and in addition to the expression of adhesion molecules, such as selectins for leukocyte rolling and integrin for leukocyte adhesion [2,3], the initiating event in the development of the injury is PMN sequestration in pulmonary microvessels [4]. Decreased deformability results in PMN sequestration as a result of PMN plugging or their prolonged transit time in the microvascular beds [5], since the diameter of
*
Corresponding author. Fax: +81-42-778-8441. E-mail address: [email protected] (H. Kobayashi). 1 Abbreviations used: PMN, polymorphonuclear leukocyte; ARDS, acute respiratory distress syndrome; fLMP, N-formyl-methionyl-leucyl-phenyl-alanine; ZAP, zymosan-activated plasma; LPS, lipopolysaccharide.
a PMN (7 lm) is smaller than that of pulmonary capillaries (5 lm). Previous studies have shown that stimulation with N-formyl-methionyl-leucyl-phenyl-alanine (fMLP) or zymosan-activated plasma (ZAP) [6–8] decreases PMN deformability, resulting in PMN sequestration in the lungs, and inhaled nitric oxide (NO) has been shown to reduce the change in deformability in rabbits following ZAP infusion [9]. Inhaled nitric oxide or inhibition of endogenous nitric oxide formation is reported to reduce hyperoxic lung injury [10]. In wild-type and iNOS-deficient mice the increased expression levels of the inducible isoform of NO synthase (iNOS: NOS II) in the lung has been shown to protect the lung against the influx of PMNs and PMN-associated injury in both LPS-treated mice [11] and hyperoxia-exposed mice [12].
1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 9 - 8 6 0 3 ( 0 2 ) 0 0 1 0 9 - X
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iNOS is induced by lipopoloysaccharide (LPS) administration in many types of lung cell (including alveolar macrophages and type II cells) [13], and iNOS has been found to be expressed in PMNs themselves in both rats [14–16] and humans [17,18]. In many previous studies on lung injury, however, it has been tacitly assumed that the iNOS is expressed in lung cells, and neutrophil iNOS and its role in PMN deformability has never been evaluated. We suspected that NO increases or maintains the deformability of PMNs irrespective of its origin, including the reaction catalyzed by the iNOS in PMNs themselves, and that autocrine maintenance of deformability may contribute to feedback suppression of PMN stiffening. The objective of this in vitro study was to determine whether endogenous NO generated in reactions catalyzed by NO synthase in PMNs, specifically by iNOS, makes the PMNs deformable.
Materials and methods
Immunocytochemistry of iNOS Expression of iNOS in the PMNs of the LPS group was confirmed by immunocytochemistry. PMNs isolated from the control group and LPS group were cytospun onto slide glasses by placing 0.3 mL PMN solution in each funnel of a Cytospin II (Shandon, Cheshire, UK; 1200 rpm for 4 min). The cytospin preparations were fixed in ethanol, rinsed in dH2O (5 min), and quenched in a humid chamber (3% H2 O2 in absolute methanol 10 min). After rinsing in PBS (5 min 3), they were exposed to normal serum (10 min) and incubated overnight at 4 °C with an anti-iNOS rabbit polyclonal IgG antibody (Biomol Research Laboratories, PA) diluted to 1:100 in PBS. The cells were then exposed to a secondary antibody (biotinylated goat anti-rabbit IgG) and an enzyme complex (Histostain SP kit, Zymed Laboratories, South San Francisco, CA). Reactive sites were visualized with aminoethylcarbazole (producing a red reaction product). The specificity of the iNOS antibody was confirmed by Western blotting (see manufacturer’s data sheet) and by preadsorption experiments [19].
Animals and measurements Nitrite/nitrate assay Wistar rats, 7–10 weeks old and weighing between 250 and 300 g (Clea Japan, Tokyo, Japan), were randomly assigned to one of two groups: (i) an LPS group in which rats were injected ip with 5 mg/kg LPS (Escherichia coli, 0127:B8, phenol extract, L-3129, Sigma, St. Louis, MO); and (ii) a control group in which rats were injected ip with 5 mg/kg saline. Animal care was in accordance with the guidelines of the Animal Care Committee of Kitasato University. At 24 h after the injection, rats were anesthetized with ether, and a specimen of whole blood was collected from a carotid artery into an EDTA tube. PMNs were isolated from the blood with PMN isolation reagents for rats (NIM2, Cardinal Associates, Santa Fe, NM) according to the manufacturer’s instructions. In brief, whole blood anticoagulated with EDTA was layered onto reagents NIM2A and NIM2B (Cardinal Associates) and centrifuged in a swinging bucket at 900 rpm at room temperature for 45 min to yield a distinct PMN fraction and a mononuclear leukocyte (MN) fraction. The plasma layer and MN fraction were discarded, the PMNs were harvested from the PMN fraction and were washed in PMN buffer (1.38 mM NaCl, 27 mM KCl, 8.1 mM Na2 HPO4 7H2 O, 1.5 mM KH2 PO4 , and 5.5 mM glucose, pH 7.4), centrifuging at 400g for 10 min, and discarding the supernatant. After resuspending the cells with 2 mL of erythrocyte lysing buffer (E-Lyse, Cardinal Associates), they were centrifuged at 400g for 4 min, and the supernatant was discarded. The cells were then washed once with 5 mL PMN buffer and resuspended in PMN buffer. The purity of the PMNs was over 95%, with 98% viability as assessed by the trypan blue exclusion test.
Nitrite/nitrate concentrations in the PMN incubation solution (1 106 /aliquot) in both the control and the LPS group were measured under the following two sets of conditions: (i) under the PMNs + saline conditions (seven control animals and seven LPS-treated animals) PMNs were incubated at 37 °C for 30 min, and (ii) under the PMNs + aminoguanidine conditions (seven control animals and seven LPS-treated animals) PMNs were incubated with 100 mM aminoguanidine (Sigma Chemical, St. Louis, MO), a selective inhibitor of NOS type II [20], at 37 °C for 30 min. To measure nitrite/nitrate production at the end of the incubation period the reaction was stopped by placing the reaction tube in icy water at 0 °C. The cells were then separated out by centrifuging at 400g for 10 min at 0 °C, and the supernatant was collected for nitrite/nitrate measurement. The nitrite/nitrate content of the incubation solution was determined by converting the nitrate into nitrite through a cadmium column and measuring it by the Griess method with an autoanalyzer (TCI-NOx 1000 and S-3200, Tokyo Kasei Kogyo, Tokyo, Japan). The test tubes were made of borosilica to avoid nitrite/nitrate contamination by tubes. The nitrite/nitrate level in the incubation solution without PMNs was checked to exclude possible contamination, including by environmental air, and it was found to be negligible. Deformability assay The deformability of the PMNs in both the control and the LPS groups was measured under the following
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three sets of conditions: (i) under the PMNs + saline conditions (15 control animals and 16 LPS-treated animals), PMNs were incubated at 37 °C for 30 min; (ii) under the PMNs + aminoguanidine conditions (12 control animals and 14 LPS-treated animals), PMNs were incubated with 100 mM aminoguanidine at 37 °C for 30 min; and (iii) under the PMNs + fMLP conditions (8 control animals and 8 LPS-treated animals), after incubation at 37 °C for 25 min, PMNs were incubated with 10 nM N-formyl-methionyl-leucyl-phenyl-alanine (Sigma), fMLP, at 37 °C for 5 min (total incubation time, 30 min). The fMLP in the deformability assay was used to confirm the reactivity of the PMNs isolated from the blood as a positive control. PMN deformability was examined by the microfilter technique [4,20], which uses the pressure required to force PMN to pass through a polycarbonate filter having a uniform pore diameter of 5 lm (Nucleopore, Pleasanton, USA) as the index of deformability. After suspending 1 106 PMN in PMN buffer, the suspension was introduced into the filter at a constant flow rate of 1.5 mL/min for 6 min with a constant speed infusion pump (Model 22, Harvard Apparatus, South Natick, MA). Filtration pressure was continuously measured with a pressure transducer (TP-400T,
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Nihonkohden, Tokyo, Japan) connected at a right angle to the main perfusion line upstream of the filter, and the differences in peak filtration pressure were evaluated statistically. Statistical analysis Values are expressed as means SE. The Wilcoxon paired signed-rank test was employed to detect statistically significant differences between untreated and treated PMNs from the same rats, and the Mann– Whitney rank test was employed to detect statistically significant differences between the control and LPStreated PMNs. P values less than 0.05 were considered significant.
Results Expression of iNOS in the PMNs in the LPS group iNOS immunocytochemistry showed that 87% of the PMNs isolated from the blood of the LPS group expressed iNOS and that the PMNs from the control group did not (Fig. 1).
Fig. 1. Immunocytochemistry of iNOS of PMNs. Immunocytochemistry of iNOS showed that 87% of PMNs isolated from the blood of the LPS group expressed iNOS, but the PMNs from the control group did not. Bar, 50 lm.
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incubation with aminoguanidine (Fig. 2). The nitrite/ nitrate level of the incubation solution of the PMNs isolated from LPS-treated rats was significantly higher than the level in the control rats, and it decreased after incubation with aminoguanidine (Fig. 2). Effect of aminoguanidine and fMLP on PMN deformability
Fig. 2. The nitrite/nitrate level of the incubation solution of the polymorphonuclear leukocytes. AG, aminoguanidine. The nitrite/nitrate level of the incubation solution of the PMNs isolated from the control rats was low, but significantly higher than zero, and did not change after incubation with aminoguanidine. The nitrite/nitrate level of the incubation solution of the PMNs isolated from LPS-treated rats was significantly higher than the level in the control rats, and it decreased after incubation with aminoguanidine.
Effect of aminoguanidine on nitrite/nitrate levels The nitrite/nitrate level of the incubation solution of the PMNs isolated from control rats was low, but significantly higher than zero, and it did not change after
Continuous infusion of PMNs across a 5-lm filter resulted in a rapid rise in the differential pressure across the filter followed by a peak or plateau (Figs. 3A and B). In the control group incubation with aminoguanidine did not change the time course or plateau of the pressure. By contrast, when the PMNs were exposed to fMLP, there was a similar increase in pressure, but the plateau was reached at a higher pressure (Fig. 3A). In the LPS group the pressure generated by the PMNs reached a plateau at a higher pressure (Fig. 3B) than the unexposed PMNs in the control group. The time course and plateau of the pressure generated by the PMNs incubated with fMLP were similar to those of the PMNs incubated without fMLP, but the plateau pressure generated by the PMNs treated with aminoguanidine was reached at a higher pressure (Fig. 3B). The peak filtration pressure of PMNs isolated from the control rats was low and did not change after incubation with aminoguanidine, but it increased after incubation with fMLP (Fig. 4). The peak filtration pressure of the PMNs isolated from the LPS-treated rats was higher than in the control rats and increased even
Fig. 3. Time course of filtration pressure of polymorphonuclear leukocytes. PMN, polymorphonuclear leukocyte; AG, aminoguanidine; fMLP, Nformyl-methionyl-leucyl-phenyl-alanine. Continuous infusion of PMNs across a 5-lm filter resulted in a rapid rise in the differential pressure across the filter followed by a peak or plateau (A and B). (A) In the control group incubation with AG did not change the time course or plateau of the pressure. By contrast, when the PMNs were exposed to fMLP, there was a similar increase in pressure, but the plateau was reached at a higher pressure. (B) In the LPS group the pressure generated by the PMNs reached a plateau at a higher pressure than the unexposed PMNs in the control group (A). The time course and plateau of the pressure generated by the PMNs incubated with fMLP were similar to those of the PMNs incubated without fMLP, but the plateau pressure generated by the PMNs treated with AG was reached at a higher pressure.
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Fig. 4. Peak filtration pressure. AG, aminoguanidine; fMLP, N-formyl-methionyl-leucyl-phenyl-alanine. The peak filtration pressure of the PMNs isolated from the control rats was low and did not change after incubation with AG, but it increased after incubation with fMLP. The peak filtration pressure of the PMNs isolated from LPS-treated rats was higher than in the control rats, and it increased even further after incubation with AG, but did not change after incubation with fMLP.
further after incubation with aminoguanidine, but it did not change after incubation with fMLP (Fig. 4).
Discussion The main findings in this study were as follows. (i) iNOS was expressed in PMNs isolated from LPS-treated rats, and the nitrite/nitrate level of the PMNs isolated from LPS-treated rats was significantly higher than in the control rats and decreased after incubation with aminoguanidine. (ii) The peak filtration pressure of the PMNs isolated from LPS-treated rats was higher than that of the PMNs from the control rats and increased even further after incubation with aminoguanidine. The results of this study showed that the PMNs of the control rats were deformable and that they were rapidly stiffened by fMLP within 5 min of the start of incubation. We attempted to use fMLP in the deformability assay as a positive control, since fMLP is known to assemble the filamentous actin (F-actin) in PMNs in an immediate and transient reaction [21], thereby stiffening their cytoskeleton. However, the deformability of the PMNs of the LPS-treated rats in the absence of fMLP was similar to results when incubated with fMLP. Whether this finding is attributable to a saturated Factin assembly or whether the fMLP signal pathway was inhibited in the PMNs of the LPS-treated rats should be investigated. The PMNs of the control rats produced low concentrations of nitrite/nitrate, but they were significantly
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greater than zero, and their production was not suppressed by the iNOS inhibitor aminoguanidine. Since low levels of neuronal NOS (NOS I) have been shown to be constitutively expressed in PMNs [22], the basal NO levels observed in the control rats in our study may have been generated by the neuronal NOS. However, it should be noted that incomplete inhibition of iNOS by aminoguanidine in our study cannot be ruled out. The PMNs of the LPS-treated rats were less deformable than those of the control rats. PMNs newly released from the bone marrow are reported to sequester preferentially in the pulmonary microvessels [23,24]. Although it has been reported that the F-actin content of the newly released PMNs does not differ from that of aged PMNs, since the bone marrow PMNs express higher L -selectin levels [25], it is likely that the PMNs newly released from bone marrow during the 24 h after LPS exposure in vivo had phenotypic differences from the mature PMNs present in the control animals, possibly contributing to the reduction in deformability. The possibility that the isolation procedure in this study caused a reduction in the deformability of the PMNs of the LPS-treated animals and immediately induced iNOS in them cannot be ruled out. The possible impact of this isolation procedure is the general limitation of investigations of PMN biology. However, the objective of our study was to determine whether endogenous NO generated in reactions catalyzed by iNOS in PMNs makes the PMNs deformable, and the results showed that endogenous NO makes them deformable in an autocrine manner. Expression of iNOS in PMNs is induced by several cytokines. There is also evidence that iNOS mRNA expression is mediated through Toll-like receptor 4 [26], through which endotoxin activates intracellular signal transduction, leading to iNOS and cytokine production. Several mechanisms have been suggested to be responsible for the inhibitory effect of NO on neutrophil stiffening. NO activates guanylate cyclase and increases the cGMP concentration [27], which prevents the increase in intracellular Ca2þ by inhibiting Ca2þ influx through Ca2þ channels [28], thereby inhibiting the activation of cellular kinases and phosphatases, which are important in F-actin assembly [29]. NO promotes adenosine diphosphate ribosylation of actin, which also inhibits F-actin assembly [30]. Ras-related low-molecular-mass GTPases, Rho, have been shown to activate a Rho-associated kinase, which in turn phosphorylates the myosin binding subunit of the myosin light-chain phosphatase, leading to an increase in phosphorylation of myosin light chain and of force at constant Ca2þ [31,32]. In mammals, expression of the Rho family GTPase Rac2 is restricted to hematopoietic cells, and it is considered to be an essential regulator of multiple specialized neutrophil functions [33]. Phosphoinositide 3-kinase has also been shown to
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play a central role in cell polarity and chemotaxis [34]. However, the link between NO and these key enzymes has not yet been elucidated. It is also interesting that, in contrast to iNOS-deficient mice, heme oxygenase-1-deficient mice lacking carbon monoxide (CO) production exhibit no defect in PMN migration in response to intrapulmonary LPS [35]. Superoxide is likely to be produced in the PMNs of LPS-treated animals. In contrast to CO, NO combines with superoxide, thereby producing peroxynitrite. Peroxynitrite is known to cause nitration of tyrosine [36]. NO is also a source of nitrite, and neutrophil MPO can convert nitrite into NO2 Cl, resulting in tyrosine nitration [37]. Since nitrotyrosination of a-tubulin is reported to cause microtubule dysfunction [38], tyrosine nitration may cause malfunction of several cellular components, including Rac2, phosphoinositide 3-kinase, and F-actin. It is also possible that NO regulates protein function by S-nitrosylation of critical cysteine residues [39]. These possibilities should further be investigated. It should be noted that the protective effect of NO on the ZAP-induced increase in F-actin formation was reported to be only partial [9], suggesting that unknown mechanisms other than alterations in F-actin assembly are also important in the inhibitory effect of NO on the increased deformability of activated PMNs. The iNOS expression and large amount of NO production in PMNs is expected to make them more deformable in vivo as well. The inability of PMNs to express iNOS and produce NO may be related to PMN sequestration in the lung and development of ARDS. Alteration of PMN function by aging or infection should further be evaluated in relation to the ability of PMNs to express iNOS. It has been reported that NO released by PMNs modulates red blood cell deformability [40], and it is possible that NO generated by iNOS reduces the deformability of not only the PMNs themselves, but of red blood cells as well, thereby maintaining the passage of blood cells through the pulmonary circulation. In summary, the results of this study suggest that endogenous NO generated by iNOS in PMNs makes the PMNs deformable in an autocrine manner.
Acknowledgment The authors thank Ms. Masumi Tanaka for her excellent technical support in this study.
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Biology and Chemistry
Nitric Oxide 7 (2002) 221–227 www.academicpress.com
Brief communication
Nitric oxide released from iNOS in polymorphonuclear leukocytes makes them deformable in an autocrine manner Hirosuke Kobayashi,a,* Tailin Cui,a Miyuki Ando,a Ryuji Hataishi,a Takao Imasaki,a Hisashi Mitsufuji,a Izumi Hayashi,b and Tomoyuki Tomitaa b
a Department of Medicine, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan Department of Pharmacology, Kitasato University School of Medicine, Kitasato 1-15-1, Sagamihara, Kanagawa 228-8555, Japan
Received 30 November 2001; received in revised form 18 March 2002
Abstract The objective of this study was to determine whether endogenous nitric oxide (NO) derived from reaction catalyzed by the inducible isoform of NO synthase (iNOS: NOS II) in polymorphonuclear leukocytes (PMNs) makes the PMNs deformable. Previous studies have shown that NO increases the deformability of PMNs and decreases the sequestration of PMNs in the lungs. However, there was little information regarding the effect of PMN-derived NO on the cells’ deformability. In the present study PMNs were isolated from the blood of rats 24 h after ip injection of saline (control) or lipopolysaccharide (LPS), and expression of iNOS in the PMNs of the LPS group was confirmed by immunocytochemistry. PMN deformability was evaluated by measuring the pressure generated during their passage through a microfilter at a constant flow rate. The nitrite/nitrate content of the solution in which the isolated PMNs were incubated was measured by the Griess method. In the control group, no iNOS was detectable in the PMNs, and the nitrite/nitrate level in the PMN incubation solution was low. Deformability was unchanged after incubation with specific iNOS inhibitor aminoguanidine, but decreased after incubation with N-formyl-methionyl-leucyl-phenyl-alanine. In the LPS group, PMN deformability was decreased compared to that of the control group. iNOS was detectable in the PMNs, and the deformability further decreased after incubation with aminoguanidine. These results suggest that endogenous NO generated during reactions catalyzed by iNOS in PMNs makes them deformable in an autocrine manner. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: ARDS; Polymorphonuclear leukocyte; Deformability; Inducible nitric oxide synthase; Sequestration
Polymorphonuclear leukocytes (PMNs)1 play an important role in the pathogenesis of acute respiratory distress syndrome (ARDS) [1], and in addition to the expression of adhesion molecules, such as selectins for leukocyte rolling and integrin for leukocyte adhesion [2,3], the initiating event in the development of the injury is PMN sequestration in pulmonary microvessels [4]. Decreased deformability results in PMN sequestration as a result of PMN plugging or their prolonged transit time in the microvascular beds [5], since the diameter of
*
Corresponding author. Fax: +81-42-778-8441. E-mail address: [email protected] (H. Kobayashi). 1 Abbreviations used: PMN, polymorphonuclear leukocyte; ARDS, acute respiratory distress syndrome; fLMP, N-formyl-methionyl-leucyl-phenyl-alanine; ZAP, zymosan-activated plasma; LPS, lipopolysaccharide.
a PMN (7 lm) is smaller than that of pulmonary capillaries (5 lm). Previous studies have shown that stimulation with N-formyl-methionyl-leucyl-phenyl-alanine (fMLP) or zymosan-activated plasma (ZAP) [6–8] decreases PMN deformability, resulting in PMN sequestration in the lungs, and inhaled nitric oxide (NO) has been shown to reduce the change in deformability in rabbits following ZAP infusion [9]. Inhaled nitric oxide or inhibition of endogenous nitric oxide formation is reported to reduce hyperoxic lung injury [10]. In wild-type and iNOS-deficient mice the increased expression levels of the inducible isoform of NO synthase (iNOS: NOS II) in the lung has been shown to protect the lung against the influx of PMNs and PMN-associated injury in both LPS-treated mice [11] and hyperoxia-exposed mice [12].
1089-8603/02/$ - see front matter Ó 2002 Elsevier Science (USA). All rights reserved. PII: S 1 0 8 9 - 8 6 0 3 ( 0 2 ) 0 0 1 0 9 - X
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iNOS is induced by lipopoloysaccharide (LPS) administration in many types of lung cell (including alveolar macrophages and type II cells) [13], and iNOS has been found to be expressed in PMNs themselves in both rats [14–16] and humans [17,18]. In many previous studies on lung injury, however, it has been tacitly assumed that the iNOS is expressed in lung cells, and neutrophil iNOS and its role in PMN deformability has never been evaluated. We suspected that NO increases or maintains the deformability of PMNs irrespective of its origin, including the reaction catalyzed by the iNOS in PMNs themselves, and that autocrine maintenance of deformability may contribute to feedback suppression of PMN stiffening. The objective of this in vitro study was to determine whether endogenous NO generated in reactions catalyzed by NO synthase in PMNs, specifically by iNOS, makes the PMNs deformable.
Materials and methods
Immunocytochemistry of iNOS Expression of iNOS in the PMNs of the LPS group was confirmed by immunocytochemistry. PMNs isolated from the control group and LPS group were cytospun onto slide glasses by placing 0.3 mL PMN solution in each funnel of a Cytospin II (Shandon, Cheshire, UK; 1200 rpm for 4 min). The cytospin preparations were fixed in ethanol, rinsed in dH2O (5 min), and quenched in a humid chamber (3% H2 O2 in absolute methanol 10 min). After rinsing in PBS (5 min 3), they were exposed to normal serum (10 min) and incubated overnight at 4 °C with an anti-iNOS rabbit polyclonal IgG antibody (Biomol Research Laboratories, PA) diluted to 1:100 in PBS. The cells were then exposed to a secondary antibody (biotinylated goat anti-rabbit IgG) and an enzyme complex (Histostain SP kit, Zymed Laboratories, South San Francisco, CA). Reactive sites were visualized with aminoethylcarbazole (producing a red reaction product). The specificity of the iNOS antibody was confirmed by Western blotting (see manufacturer’s data sheet) and by preadsorption experiments [19].
Animals and measurements Nitrite/nitrate assay Wistar rats, 7–10 weeks old and weighing between 250 and 300 g (Clea Japan, Tokyo, Japan), were randomly assigned to one of two groups: (i) an LPS group in which rats were injected ip with 5 mg/kg LPS (Escherichia coli, 0127:B8, phenol extract, L-3129, Sigma, St. Louis, MO); and (ii) a control group in which rats were injected ip with 5 mg/kg saline. Animal care was in accordance with the guidelines of the Animal Care Committee of Kitasato University. At 24 h after the injection, rats were anesthetized with ether, and a specimen of whole blood was collected from a carotid artery into an EDTA tube. PMNs were isolated from the blood with PMN isolation reagents for rats (NIM2, Cardinal Associates, Santa Fe, NM) according to the manufacturer’s instructions. In brief, whole blood anticoagulated with EDTA was layered onto reagents NIM2A and NIM2B (Cardinal Associates) and centrifuged in a swinging bucket at 900 rpm at room temperature for 45 min to yield a distinct PMN fraction and a mononuclear leukocyte (MN) fraction. The plasma layer and MN fraction were discarded, the PMNs were harvested from the PMN fraction and were washed in PMN buffer (1.38 mM NaCl, 27 mM KCl, 8.1 mM Na2 HPO4 7H2 O, 1.5 mM KH2 PO4 , and 5.5 mM glucose, pH 7.4), centrifuging at 400g for 10 min, and discarding the supernatant. After resuspending the cells with 2 mL of erythrocyte lysing buffer (E-Lyse, Cardinal Associates), they were centrifuged at 400g for 4 min, and the supernatant was discarded. The cells were then washed once with 5 mL PMN buffer and resuspended in PMN buffer. The purity of the PMNs was over 95%, with 98% viability as assessed by the trypan blue exclusion test.
Nitrite/nitrate concentrations in the PMN incubation solution (1 106 /aliquot) in both the control and the LPS group were measured under the following two sets of conditions: (i) under the PMNs + saline conditions (seven control animals and seven LPS-treated animals) PMNs were incubated at 37 °C for 30 min, and (ii) under the PMNs + aminoguanidine conditions (seven control animals and seven LPS-treated animals) PMNs were incubated with 100 mM aminoguanidine (Sigma Chemical, St. Louis, MO), a selective inhibitor of NOS type II [20], at 37 °C for 30 min. To measure nitrite/nitrate production at the end of the incubation period the reaction was stopped by placing the reaction tube in icy water at 0 °C. The cells were then separated out by centrifuging at 400g for 10 min at 0 °C, and the supernatant was collected for nitrite/nitrate measurement. The nitrite/nitrate content of the incubation solution was determined by converting the nitrate into nitrite through a cadmium column and measuring it by the Griess method with an autoanalyzer (TCI-NOx 1000 and S-3200, Tokyo Kasei Kogyo, Tokyo, Japan). The test tubes were made of borosilica to avoid nitrite/nitrate contamination by tubes. The nitrite/nitrate level in the incubation solution without PMNs was checked to exclude possible contamination, including by environmental air, and it was found to be negligible. Deformability assay The deformability of the PMNs in both the control and the LPS groups was measured under the following
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three sets of conditions: (i) under the PMNs + saline conditions (15 control animals and 16 LPS-treated animals), PMNs were incubated at 37 °C for 30 min; (ii) under the PMNs + aminoguanidine conditions (12 control animals and 14 LPS-treated animals), PMNs were incubated with 100 mM aminoguanidine at 37 °C for 30 min; and (iii) under the PMNs + fMLP conditions (8 control animals and 8 LPS-treated animals), after incubation at 37 °C for 25 min, PMNs were incubated with 10 nM N-formyl-methionyl-leucyl-phenyl-alanine (Sigma), fMLP, at 37 °C for 5 min (total incubation time, 30 min). The fMLP in the deformability assay was used to confirm the reactivity of the PMNs isolated from the blood as a positive control. PMN deformability was examined by the microfilter technique [4,20], which uses the pressure required to force PMN to pass through a polycarbonate filter having a uniform pore diameter of 5 lm (Nucleopore, Pleasanton, USA) as the index of deformability. After suspending 1 106 PMN in PMN buffer, the suspension was introduced into the filter at a constant flow rate of 1.5 mL/min for 6 min with a constant speed infusion pump (Model 22, Harvard Apparatus, South Natick, MA). Filtration pressure was continuously measured with a pressure transducer (TP-400T,
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Nihonkohden, Tokyo, Japan) connected at a right angle to the main perfusion line upstream of the filter, and the differences in peak filtration pressure were evaluated statistically. Statistical analysis Values are expressed as means SE. The Wilcoxon paired signed-rank test was employed to detect statistically significant differences between untreated and treated PMNs from the same rats, and the Mann– Whitney rank test was employed to detect statistically significant differences between the control and LPStreated PMNs. P values less than 0.05 were considered significant.
Results Expression of iNOS in the PMNs in the LPS group iNOS immunocytochemistry showed that 87% of the PMNs isolated from the blood of the LPS group expressed iNOS and that the PMNs from the control group did not (Fig. 1).
Fig. 1. Immunocytochemistry of iNOS of PMNs. Immunocytochemistry of iNOS showed that 87% of PMNs isolated from the blood of the LPS group expressed iNOS, but the PMNs from the control group did not. Bar, 50 lm.
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incubation with aminoguanidine (Fig. 2). The nitrite/ nitrate level of the incubation solution of the PMNs isolated from LPS-treated rats was significantly higher than the level in the control rats, and it decreased after incubation with aminoguanidine (Fig. 2). Effect of aminoguanidine and fMLP on PMN deformability
Fig. 2. The nitrite/nitrate level of the incubation solution of the polymorphonuclear leukocytes. AG, aminoguanidine. The nitrite/nitrate level of the incubation solution of the PMNs isolated from the control rats was low, but significantly higher than zero, and did not change after incubation with aminoguanidine. The nitrite/nitrate level of the incubation solution of the PMNs isolated from LPS-treated rats was significantly higher than the level in the control rats, and it decreased after incubation with aminoguanidine.
Effect of aminoguanidine on nitrite/nitrate levels The nitrite/nitrate level of the incubation solution of the PMNs isolated from control rats was low, but significantly higher than zero, and it did not change after
Continuous infusion of PMNs across a 5-lm filter resulted in a rapid rise in the differential pressure across the filter followed by a peak or plateau (Figs. 3A and B). In the control group incubation with aminoguanidine did not change the time course or plateau of the pressure. By contrast, when the PMNs were exposed to fMLP, there was a similar increase in pressure, but the plateau was reached at a higher pressure (Fig. 3A). In the LPS group the pressure generated by the PMNs reached a plateau at a higher pressure (Fig. 3B) than the unexposed PMNs in the control group. The time course and plateau of the pressure generated by the PMNs incubated with fMLP were similar to those of the PMNs incubated without fMLP, but the plateau pressure generated by the PMNs treated with aminoguanidine was reached at a higher pressure (Fig. 3B). The peak filtration pressure of PMNs isolated from the control rats was low and did not change after incubation with aminoguanidine, but it increased after incubation with fMLP (Fig. 4). The peak filtration pressure of the PMNs isolated from the LPS-treated rats was higher than in the control rats and increased even
Fig. 3. Time course of filtration pressure of polymorphonuclear leukocytes. PMN, polymorphonuclear leukocyte; AG, aminoguanidine; fMLP, Nformyl-methionyl-leucyl-phenyl-alanine. Continuous infusion of PMNs across a 5-lm filter resulted in a rapid rise in the differential pressure across the filter followed by a peak or plateau (A and B). (A) In the control group incubation with AG did not change the time course or plateau of the pressure. By contrast, when the PMNs were exposed to fMLP, there was a similar increase in pressure, but the plateau was reached at a higher pressure. (B) In the LPS group the pressure generated by the PMNs reached a plateau at a higher pressure than the unexposed PMNs in the control group (A). The time course and plateau of the pressure generated by the PMNs incubated with fMLP were similar to those of the PMNs incubated without fMLP, but the plateau pressure generated by the PMNs treated with AG was reached at a higher pressure.
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Fig. 4. Peak filtration pressure. AG, aminoguanidine; fMLP, N-formyl-methionyl-leucyl-phenyl-alanine. The peak filtration pressure of the PMNs isolated from the control rats was low and did not change after incubation with AG, but it increased after incubation with fMLP. The peak filtration pressure of the PMNs isolated from LPS-treated rats was higher than in the control rats, and it increased even further after incubation with AG, but did not change after incubation with fMLP.
further after incubation with aminoguanidine, but it did not change after incubation with fMLP (Fig. 4).
Discussion The main findings in this study were as follows. (i) iNOS was expressed in PMNs isolated from LPS-treated rats, and the nitrite/nitrate level of the PMNs isolated from LPS-treated rats was significantly higher than in the control rats and decreased after incubation with aminoguanidine. (ii) The peak filtration pressure of the PMNs isolated from LPS-treated rats was higher than that of the PMNs from the control rats and increased even further after incubation with aminoguanidine. The results of this study showed that the PMNs of the control rats were deformable and that they were rapidly stiffened by fMLP within 5 min of the start of incubation. We attempted to use fMLP in the deformability assay as a positive control, since fMLP is known to assemble the filamentous actin (F-actin) in PMNs in an immediate and transient reaction [21], thereby stiffening their cytoskeleton. However, the deformability of the PMNs of the LPS-treated rats in the absence of fMLP was similar to results when incubated with fMLP. Whether this finding is attributable to a saturated Factin assembly or whether the fMLP signal pathway was inhibited in the PMNs of the LPS-treated rats should be investigated. The PMNs of the control rats produced low concentrations of nitrite/nitrate, but they were significantly
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greater than zero, and their production was not suppressed by the iNOS inhibitor aminoguanidine. Since low levels of neuronal NOS (NOS I) have been shown to be constitutively expressed in PMNs [22], the basal NO levels observed in the control rats in our study may have been generated by the neuronal NOS. However, it should be noted that incomplete inhibition of iNOS by aminoguanidine in our study cannot be ruled out. The PMNs of the LPS-treated rats were less deformable than those of the control rats. PMNs newly released from the bone marrow are reported to sequester preferentially in the pulmonary microvessels [23,24]. Although it has been reported that the F-actin content of the newly released PMNs does not differ from that of aged PMNs, since the bone marrow PMNs express higher L -selectin levels [25], it is likely that the PMNs newly released from bone marrow during the 24 h after LPS exposure in vivo had phenotypic differences from the mature PMNs present in the control animals, possibly contributing to the reduction in deformability. The possibility that the isolation procedure in this study caused a reduction in the deformability of the PMNs of the LPS-treated animals and immediately induced iNOS in them cannot be ruled out. The possible impact of this isolation procedure is the general limitation of investigations of PMN biology. However, the objective of our study was to determine whether endogenous NO generated in reactions catalyzed by iNOS in PMNs makes the PMNs deformable, and the results showed that endogenous NO makes them deformable in an autocrine manner. Expression of iNOS in PMNs is induced by several cytokines. There is also evidence that iNOS mRNA expression is mediated through Toll-like receptor 4 [26], through which endotoxin activates intracellular signal transduction, leading to iNOS and cytokine production. Several mechanisms have been suggested to be responsible for the inhibitory effect of NO on neutrophil stiffening. NO activates guanylate cyclase and increases the cGMP concentration [27], which prevents the increase in intracellular Ca2þ by inhibiting Ca2þ influx through Ca2þ channels [28], thereby inhibiting the activation of cellular kinases and phosphatases, which are important in F-actin assembly [29]. NO promotes adenosine diphosphate ribosylation of actin, which also inhibits F-actin assembly [30]. Ras-related low-molecular-mass GTPases, Rho, have been shown to activate a Rho-associated kinase, which in turn phosphorylates the myosin binding subunit of the myosin light-chain phosphatase, leading to an increase in phosphorylation of myosin light chain and of force at constant Ca2þ [31,32]. In mammals, expression of the Rho family GTPase Rac2 is restricted to hematopoietic cells, and it is considered to be an essential regulator of multiple specialized neutrophil functions [33]. Phosphoinositide 3-kinase has also been shown to
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play a central role in cell polarity and chemotaxis [34]. However, the link between NO and these key enzymes has not yet been elucidated. It is also interesting that, in contrast to iNOS-deficient mice, heme oxygenase-1-deficient mice lacking carbon monoxide (CO) production exhibit no defect in PMN migration in response to intrapulmonary LPS [35]. Superoxide is likely to be produced in the PMNs of LPS-treated animals. In contrast to CO, NO combines with superoxide, thereby producing peroxynitrite. Peroxynitrite is known to cause nitration of tyrosine [36]. NO is also a source of nitrite, and neutrophil MPO can convert nitrite into NO2 Cl, resulting in tyrosine nitration [37]. Since nitrotyrosination of a-tubulin is reported to cause microtubule dysfunction [38], tyrosine nitration may cause malfunction of several cellular components, including Rac2, phosphoinositide 3-kinase, and F-actin. It is also possible that NO regulates protein function by S-nitrosylation of critical cysteine residues [39]. These possibilities should further be investigated. It should be noted that the protective effect of NO on the ZAP-induced increase in F-actin formation was reported to be only partial [9], suggesting that unknown mechanisms other than alterations in F-actin assembly are also important in the inhibitory effect of NO on the increased deformability of activated PMNs. The iNOS expression and large amount of NO production in PMNs is expected to make them more deformable in vivo as well. The inability of PMNs to express iNOS and produce NO may be related to PMN sequestration in the lung and development of ARDS. Alteration of PMN function by aging or infection should further be evaluated in relation to the ability of PMNs to express iNOS. It has been reported that NO released by PMNs modulates red blood cell deformability [40], and it is possible that NO generated by iNOS reduces the deformability of not only the PMNs themselves, but of red blood cells as well, thereby maintaining the passage of blood cells through the pulmonary circulation. In summary, the results of this study suggest that endogenous NO generated by iNOS in PMNs makes the PMNs deformable in an autocrine manner.
Acknowledgment The authors thank Ms. Masumi Tanaka for her excellent technical support in this study.
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