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Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis
NITRIC OXIDE
Biology and Chemistry
Nitric Oxide 9 (2004) 153–164 www.elsevier.com/locate/yniox
Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis Daniela Dal Secco,a Juliane Alves Paron,a Sandra H.P. de Oliveira,c Sergio Henrique Ferreira,a Jo~ ao Santana Silva,b and Fernando de Queiroz Cunhaa,* a
b
Department of Pharmacology, School of Medicine of Ribeir~ ao Preto, University of S~ ao Paulo, Avenida Bandeirantes, 3900, 14049-900-Ribeir~ ao Preto, S~ ao Paulo, Brazil Department of Biochemistry and Immunology, School of Medicine of Ribeir~ ao Preto, University of S~ ao Paulo, Avenida Bandeirantes, 3900, 14049-900-Ribeir~ ao Preto, S~ ao Paulo, Brazil c Department of Basic Science, School of Dentistry, Aracßatuba State University of S~ ao Paulo, Brazil Received 12 June 2003; received in revised form 23 October 2003
Abstract There is controversy in the literature over whether nitric oxide (NO) released during the inflammatory process has a pro- or inhibitory effect on neutrophil migration. The aim of the present investigation was to clarify this situation. Treatment of rats with non-selective, N G -nitro-L -arginine (nitro), or selective inducible NO synthase (iNOS), aminoguanidine (amino) inhibitors enhanced neutrophil migration 6 h after the administration of low, but not high, doses of carrageenan (Cg) or Escherichia coli endotoxin (LPS). The neutrophil migration induced by N -formyl–methionyl–leucyl–phenylalanine (fMLP) was also enhanced by nitro or amino treatments. The enhancement of Cg-induced neutrophil migration by NOS inhibitor treatments was reversed by co-treatment with L -arginine, suggesting an involvement of the L -arginine/NOS pathway in the process. The administration of Cg in iNOS deficient (iNOS = ) mice also enhanced the neutrophil migration compared with wild type mice. This enhancement was markedly potentiated by treatment of iNOS = mice with nitro. Investigating the mechanisms by which NOS inhibitors enhanced the neutrophil migration, it was observed that they promoted an increase in Cg-induced rolling and adhesion of leukocytes to endothelium and blocked the apoptosis of emigrated neutrophils. Similar results were observed in iNOS = mice, in which these mechanisms were potentiated and reverted by nitro and L -arginine treatments, respectively. In conclusion, these results suggest that during inflammation, NO released by either constitutive NOS (cNOS) or iNOS down-modulates the neutrophil migration. This NO effect seems to be a consequence of decreased rolling and adhesion of the neutrophils on endothelium and also the induction of apoptosis in migrated neutrophils. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Nitric oxide synthase; Inflammation; Apoptosis; Adhesion; Neutrophil migration
Neutrophils are the principal cells involved in host defense against acute bacterial and fungal infections [1]. Although they have a protective effect, the tissue damage observed in diseases such as rheumatoid arthritis, glomerulonephritis, immune vasculitis, ulcerative colitis, and inflammatory bowel disease is, at least, in part, a
* Corresponding author. Fax: +55-856332301. E-mail address: [email protected] (F.Q. Cunha).
1089-8603/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2003.11.001
consequence of neutrophil accumulation [2–5]. The neutrophil migration during an inflammatory response results mainly from the release by resident cells of neutrophil chemotactic factors, which induce the rolling and adhesion of neutrophils on endothelial cells, followed by their transmigration to the extravascular space [6,7].
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Several neutrophil chemoattractant factors have been described in the literature, such as TNF-a,1 PAF, C5a, LTB4 , and a variety of chemokines, including IL-8, GRO-a, and SDF-1 [8–11]. On the other hand, the production of chemoattractant factors is concomitant with the release of anti-inflammatory mediators, including IL-10, IL-4, and lipoxin, which negatively modulate the neutrophil recruitment [12–14]. Recent studies have shown that NO might also counteract the neutrophil migration [15–19]. NO is produced in mammalian cells from L -arginine and oxygen by a family of enzymes known as NO synthases (NOS). Three NOS isoforms are described: neuronal (nNOS or type I), inducible (iNOS or type II), and endothelial (eNOS or type III). The nNOS and eNOS are constitutively expressed, whereas the iNOS can be induced during the immune and inflammatory responses by bacterial lipopolysaccharide and cytokines, such as TNF-a, IL-1, IFN-c, a, and b, and chemokines [20–22]. Although the mechanisms by which NO attenuates neutrophil accumulation are not fully elucidated, evidence suggests that NO, released by either eNOS or iNOS, modulates the leukocyte–endothelial cell interaction. It was observed that selective inhibitors of iNOS and cNOS increase neutrophil adhesion to endothelial cells, while NO donors decrease both adhesion and leukocyte transmigration to inflammatory sites. Moreover, these parameters are also increased in iNOS = mice [17,23–33]. Furthermore, expression of the cell adhesion molecules CD11b/CD18, L-, P-, E-selectin, ICAM-1, and VCAM-1 among others is down-regulated by NO donors and up-regulated by NOS inhibitors [27,30,34–37]. On the other hand, while this evidence suggests that NO reduces the neutrophil migration, there are also studies in the literature that contradict these findings. For example, it was observed that mice treated with selective iNOS inhibitors (L -NIL or amino) or with nonselective NOS inhibitors (L -NMMA or L -NAME), or
1 Abbreviations used: Cg, carrageenan; LPS, lipopolysaccharide; fMLP, N -formyl-methionyl-leucyl-phenylalanine; nitro, N G -nitroG L -arginine; L -NAME, N -nitro-L -arginine methyl ester; L -NMMA, N G -monomethyl-L -arginine; SIN-1, 3-morpholinosyndnonimine N-ethylcarbamide; amino, aminoguanidine; PBS, phosphate buffered saline; AE-ITU, (S-(2-aminoethyl) isothiourea); NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; cNOS, constitutive nitric oxide synthase; eNOS, endothelial nitric oxide synthase; iNOS = , inducible nitric oxide synthase deficient mice; 1400 W, N-(3(aminomethyl)-benzyl) acetamidine; L -NIO, L -N(5)(1-iminoethyl)-ornithine; L -NIL, L -iminoethyl-lysine; TNF-a, tumor necrosis factor-a; PAF, platelet-activating factor; LTB4 , leukotriene B4 ; IL-8, interleukin-8; IL-10, interleukin-10; IL-4, interleukin-4; IL-1, interleukin-1; GRO-a, growth-related oncogene-alpha; SDF-1, stromal cell-derived factor 1; SEB, staphylococcal enterotoxin B; SCW, streptococcal cell wall; ICAM-1, intracellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; IFN-c, interferon-c.
iNOS = mice present a reduction in neutrophil migration to inflammatory sites [19,38–40]. Taking in to account these controversies, the aim of the first part of the present study was to investigate in two experimental species (rats and mice) the effects of a selective iNOS (amino) and non-selective NOS (nitro) inhibitors on neutrophil migration induced by low and high doses of the inflammatory stimuli Cg or LPS, and by a direct chemotactic mediator, fMLP. The neutrophil migration induced by Cg in iNOS = mice was also investigated. It was found that the animals treated with both NOS inhibitors or iNOS = mice exhibited enhanced neutrophil migration to the peritoneal cavity in response to low doses of the inflammatory stimuli. It is accepted that the disappearance of the migrated neutrophils from inflammatory sites is a consequence of the apoptotic process, which results in the recognition and uptake of these cells by macrophages [41–44]. Within the last decade, several studies demonstrated that NO is involved in the induction of apoptosis in several cell types, including neutrophils [45–51]. Thus, the treatment of animals with NOS inhibitors could reduce the apoptotic rate of migrated neutrophils, extending their life and allowing them to linger at the inflammatory site. To investigate whether an enhancement of neutrophil accumulation in the peritoneal cavities of the animals treated with NOS inhibitors or in iNOS = mice was mostly a consequence of the inhibition of apoptosis, and/or due to an increase in the mechanism of neutrophil/endothelium adhesion, we investigated the percentage of apoptosis in the Cg-induced neutrophil migration in animals treated with NOS inhibitors (nitro and amino) and in iNOS = mice. Furthermore, the effect of treatment with these NOS inhibitors on the Cginduced rolling/adhesion of neutrophils to endothelial cells was also investigated, in rats. It was found that the treatment of the animals with either amino or nitro enhances the rolling and adhesion of the neutrophils on endothelium and also blocked the apoptosis process in migrated neutrophils. Similar results were observed in iNOS = mice.
Materials and methods Animals Adult male Wistar rats weighing 180–200 g and adult male C57BL-6 (wild type) or iNOS deficient (iNOS = ) mice weighing 18–20 g were used in this study. The animals were housed in temperature-controlled rooms (22– 25 °C), with access to water and food ad libitum, until use in the Departments of Pharmacology and Immunology of the School of Medicine of Ribeir~ ao Preto, University of S~ao Paulo. The wild type and iNOS = mice were housed in a sterile laminar flow cabinet.
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Breeding pairs of mice with targeted disruption of the iNOS = gene were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). All experiments were conducted in accordance with the ethical guidelines of the School of Medicine of Ribeir~ ao Preto, University of S~ ao Paulo, S~ ao Paulo, Brazil. Drugs Carrageenan (Cg), Escherichia coli lipopolysaccharide (LPS), N -formyl-methionyl-leucyl-phenylalanine (fMLP), aminoguanidine (amino), N G -nitro-L -arginine (nitro), and L -arginine were purchased from Sigma (USA). In vivo neutrophil migration For the determination of neutrophil migration, the rats and mice were killed at the specified time and the peritoneal cavity cells were harvested by washing the cavity with 10 and 5 mL, respectively, of PBS containing EDTA (37.2 mg/100 mL). The volumes recovered were similar in all experimental groups and equated to approximately 95% of the injected volume. Total counts were performed in a cell counter (COULTER A C T; Coulter, Miami, FL, USA) and differential cell counts (100 cells total) were carried out on cytocentrifuge (Cytospin 3; Shandon Lipshaw, Pittsburgh, Pennsylvania, USA) slides stained with Rosenfeld. The results are presented as the number of neutrophils per cavity. Determination of neutrophil rolling and adhesion to the mesenteric microcirculation by intravital microscopy The leukocyte rolling and adhesion were examined as previously described [52–54]. Briefly, animals were anesthetized with tribromoethanol (250 mg/kg). The mesenteric tissue was exposed for microscopic examination in situ. This was performed through a longitudinal incision of the skin and abdominal muscle on the right side of the body followed by the exposure of the mesentery. Respiratory movements of the animals did not affect the preparation, and the microcirculatory characteristics remained essentially stable throughout the course of the experiment. The animals were maintained on a special board thermostatically controlled at 37 °C, with a transparent platform on which the tissue to be transilluminated was placed. The preparation was kept moist and warm by irrigating the tissue with warmed (37 °C) Ringer LockeÕs solution, pH 7.2–7.4, containing 1% gelatin. A 500-line television camera was mounted onto a triocular Zeiss microscope to facilitate observation of the enlarged image (3400) on a video screen. Images were recorded on a video recorder with a 40 long distance objective and a 0.65 numerical aperture. An image-splitting micrometer was adjusted to the phototube of the microscope as described by Baez [52].
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The image splitter sheared the optical image into two separate images and displaced one with respect to the other. By rotating the image splitter in the phototube, the shearing was maintained in a direction at right angles to the axis of the vessel. The displacement of one image from the other allowed measurement of the vessel diameter. Vessels selected for study were third-order venules, defined according to their branch-order location within the microvascular network. These vessels corresponded to postcapillary venules, with a diameter of 10–18 lm. The interaction of leukocytes with the luminal surface of the venular endothelium was studied in a segment of the vessel. Rolling leukocytes (‘‘rollers’’) were defined as those white blood cells that moved at a velocity less than that of erythrocytes in the same stream. The number of rolling leukocytes was determined in 10-min intervals. These leukocytes moved sufficiently slowly as to be individually visible and were counted as they rolled past a 10-lm length of venule [53]. A leukocyte was considered to be adherent to the venular endothelium if it remained stationary for >30 s [55]. Adherent cells (‘‘stickers’’) were expressed as the number per 10-lm length of venule. Cells were counted in the recorded image using five different fields for each animal to avoid variability due to sampling. Data were then averaged for each animal. Apoptotic index of migrated neutrophils The apoptotic index was determined according to the instructions of a TACS Annexin V-Biotin Apoptosis Detection Kit (R&D Systems, Minneapolis, USA). Briefly, leukocytes harvested from the peritoneal cavity were washed (500g for 5 min at room temperature) with 500 lL cold (2–8 °C) PBS. Then, the cell pellet (1 106 cells) was resuspended in 100 lL of a solution of annexin V, conjugated to biotin, and propidium iodide before being incubated in the dark at room temperature for 15 min. After centrifugation (500g), the pellet was resuspended in 100 lL of Binding Buffer, which contains fluorescent-streptavidin conjugate, and incubated in the dark for 15 min. The cell suspension was centrifuged again and the pellet was resuspended in 400 lL of Binding Buffer, before being subjected to FACSort flow cytometry (Becton Dickinson, San Jose, CA, USA) analysis. The appropriate gate for neutrophils was determined using purified neutrophils and monocytes harvested from the peritoneal cavity after injection of Cg. Experimental protocols 1. Effect of nitro and amino on the neutrophil migration induced by Cg, LPS or fMLP in rats. The rats were treated subcutaneously (s.c.) with phosphate buffered saline (PBS; 0.5 mL) or with the
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NOS inhibitors, nitro (unspecific NOS inhibitor) or amino (selective inhibitor of iNOS), at doses of 25, 50 or 100 mg/kg. Thirty minutes later Cg (30 lg/1.0 mL) was injected intraperitoneally (i.p.) and the neutrophil migration was determined 6 h after, as described above. The animals in the control group (C) were injected i.p. with PBS (1.0 mL). In other experimental groups, the rats were treated s.c. with nitro or amino at a dose of 50 mg/kg and 30 min later they were injected with Cg at doses of 30 or 100 lg/1.0 mL, LPS at doses of 20 or 100 ng/1.0 mL or fMLP at a dose of 10 nmol/1.0 mL. The neutrophil migration was determined 6 and 48 h after the injection of inflammatory stimuli. The animals of the experimental groups in which the neutrophil migration was determined 48 h after the inflammatory stimulus injection also received nitro or amino (50 mg/ kg) twice a day. The doses of 30 and 100 lg of Cg and of 20 and 100 ng of LPS were chosen on the basis of previous unpublished experiments, in which they induced a mild and intense neutrophil migration, respectively, into peritoneal cavities of rats. In another experimental group, the animals were co-treated s.c. with L -arginine (500 mg/kg), plus nitro (50 mg/kg) or amino (50 mg/kg) and 30 min later Cg (30 lg/1.0 mL) was injected i.p., the neutrophil migration was determined 6 h after. 2. Effect of nitro and amino on the neutrophil migration induced by Cg in wild type and iNOS = mice. The effects of NO synthesis inhibitors upon Cg-induced neutrophil migration in mice were also investigated. For this, wild type mice were treated s.c. with PBS (0.5 mL), nitro or amino, at a dose of 50 mg/kg and 30 min later they were injected with Cg at dose a of 500 lg/ 1.0 mL. Six hours after the animals were killed and the number of neutrophils present in the harvested fluid was determined as described above. Cg (500 lg/1.0 mL) was also injected i.p. in iNOS = mice pretreated s.c. with PBS or nitro (50 mg/kg) and the neutrophil migration was determined 6 h after. The dose of Cg injected in mice was five times higher than that used in rats because in preliminary experiments it was observed that mice are much less responsive than rats to inflammatory stimuli. 3. Effect of nitro and amino on Cg-induced neutrophil rolling and adhesion to the mesenteric microcirculation in rats. The animals were treated s.c. with PBS (0.5 mL), nitro or amino, at a dose of 50 mg/kg. Thirty minutes later, Cg at a dose of 30 lg/1.0 mL was injected i.p. and 2 and 4 h after the leukocyte rolling and adhesion to the ileocaecal portion of mesenteric microcirculation were determined in five different fields for each animal by intravital microscopy, respectively. The data were then averaged for each animal, as described above. The rolling and adhesion were determined 2 and 4 h after Cg injection, respectively, because in a preliminary experiment we observed that rolling and adhesion processes peak at these times after inflammatory stimuli injection.
4. Effect of nitro and L -arginine on Cg-induced neutrophil rolling and adhesion to the mesenteric microcirculation in iNOS = mice. The wild type mice were treated s.c. with PBS (0.5 mL), while the iNOS = mice were treated with nitro, at a dose of 50 mg/kg or L -arginine (1 g/kg, s.c.). Thirty minutes later, Cg at a dose of 500 lg/1.0 mL was injected i.p. and the leukocyte rolling and adhesion to the ileocaecal portion of mesenteric microcirculation were determined 2 and 4 h after, respectively, in five different fields for each animal by intravital microscopy, as described above. The wild type mice in the control group (C) were injected i.p. with PBS (1.0 mL). Because the mouse has much more fat deposition on the mesentery, a careful examination of the mesentery microcirculation was done to identify vessel areas without fat deposition. 5. Effect of nitro and amino on the apoptotic index of inflammatory neutrophils in rats, wild type, and iNOS = mice. Rats and wild type mice were treated with PBS s.c., nitro or amino, at doses of 50 mg/kg and, 30 min later, Cg, at doses of 30 lg/1.0 mL (rats), and 500 lg/1.0 mL (mice) was injected i.p. The iNOS = mice were treated with PBS s.c., nitro (50 mg/kg, s.c.) or L -arginine (1 and 2 g/kg, s.c.) and 30 min later Cg (500 lg/1.0 mL) was injected i.p. The cells were harvested and washed 6 h after Cg injection and the apoptotic index was determined, as described above. Statistical analysis The data are reported as means SEM and are representative of two or three different experiments. The means from different treatments in each individual experiment were compared by ANOVA. When significant differences were identified, individual comparisons were subsequently made with BonferroniÕs t test for unpaired values. Statistical significance was set at P < 0:05.
Results Effect of nitro and amino on the neutrophil migration induced by Cg, LPS or fMLP The i.p. injection of a low dose of Cg (30 lg/1.0 mL) induced a significant neutrophil migration in rats, determined 6 h after. The pretreatment of the animals with nitro (Fig. 1A) or amino (Fig. 1B) enhanced the observed neutrophil migration in a dose-dependent manner. Moreover, treatment with nitro or amino (50 mg/ kg) significantly enhanced the neutrophil migration triggered i.p. injection of Cg (30 lg/1.0 mL) or LPS (20 ng/1.0 mL) and determined either 6 or 48 h later Figs. 2A and 3A, respectively. By contrast, nitro or amino (50 mg/kg) did not enhance the neutrophil migration in
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Fig. 1. The NO synthesis inhibitors, nitro and amino, enhance the neutrophil migration induced by Cg. The rats were treated s.c. with PBS or with indicated doses of nitro (A) or amino (B). After 30 min, Cg was injected at a dose of 30 lg/cavity and the neutrophil migration was determined 6 h after. The first bar in each panel represents the neutrophil migration induced by PBS (C, control group). Data are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to the C group; # P < 0:05 compared to PBS treated Cg-injected group (ANOVA followed by BonferroniÕs t test).
response to high doses of Cg (100 lg/1.0 mL) or LPS (100 ng/1.0 mL) when migration was determined 6 h after the inflammatory stimulus injection (left panels of Figs. 2B and 3B, respectively). On the other hand, the neutrophil migration determined 48 h after the injection of high doses of Cg or LPS was enhanced by nitro and amino treatment (right panels of Figs. 2B and 3B). The neutrophil migration determined 6 and 48 h after injection of a direct chemotactic factor, fMLP (10 nmol/ 1.0 mL), also was enhanced by the pretreatment of the rats with nitro or amino at a dose of 50 mg/kg (Fig. 4). The enhancement of Cg (30 lg/1.0 mL)-induced neutrophil migration by the pretreatment of the rats with nitro (50 mg/kg) or amino (50 mg/kg) was inhibited by
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Fig. 2. Effects of nitro and amino NOS inhibitors upon neutrophil migration induced by different doses of Cg. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with Cg at doses of 30 lg/cavity (A) or 100 lg/cavity (B). The neutrophil migration was evaluated 6 or 48 h after Cg injection. The animals in the experimental group in which the neutrophil migration was determined 48 h after Cg injection also received nitro or amino (same doses) twice a day. The first bar in each panel represents the neutrophil migration induced by PBS injected i.p. (C). The values are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated Cg-injected group (ANOVA followed by BonferroniÕs t test).
the co-administration of L -arginine at a dose of 500 mg/ kg (Fig. 5). Effect of nitro and amino upon leukocyte–endothelium interaction (rolling and adhesion) in rats To clarify the mechanisms by which the NOS inhibitors increase the neutrophil migration to the
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Fig. 4. Effects of nitro and amino upon fMLP-induced neutrophil migration. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with fMLP at a dose of 10 nmol/cavity. The neutrophil migration was evaluated 6 or 48 h later. The animals in the experimental group in which the neutrophil migration was determined 48 h after fMLP injection also received nitro or amino (same doses) twice a day. The white bars (C) represent the neutrophil migration induced by PBS injected i.p. The values are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated fMLP-injected group (ANOVA followed by BonferroniÕs t test).
injection of PBS. The pretreatment of the Cg-injected rats with nitro or amino at a dose of 50 mg/kg significantly enhanced leukocyte rolling and adhesion.
Fig. 3. Effects of nitro and amino upon neutrophil migration induced by different doses of LPS. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with LPS at doses of 20 ng/cavity (A) or 100 ng/cavity (B). The neutrophil migration was evaluated 6 or 48 h after LPS injection. The animals in the experimental group in which the neutrophil migration was determined 48 h after LPS injection also received nitro or amino (same doses) twice a day. The first bar in each panel represents the neutrophil migration induced by PBS injected i.p. (C). The values are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated LPS-injected group (ANOVA followed by BonferroniÕs t test).
inflammatory site, we investigated whether these drugs increased leukocyte–endothelium interaction in vivo. Leukocyte–endothelium interaction (rolling and adhesion) was examined in mesenteric postcapillary venules with diameters ranging from 10 to 18 lm. The i.p. injection of Cg (30 lg/1.0 mL) caused a significant increase in rolling (Fig. 6A) and adhesion (Fig. 6B) of leukocytes on endothelium, compared with the i.p.
Effect of nitro and amino upon the apoptotic index of migrated neutrophils The results described above suggest that NO inhibits the neutrophil migration to the inflammatory site. This inhibitory effect seems to be a consequence of the ability to down-regulate the neutrophil rolling and adhesion to endothelium. Since there is evidence in the literature that NO is also an apoptotic mediator, we investigated whether the increase of neutrophil migration observed in animals treated with NOS inhibitors might be also a consequence of reduced apoptosis in emigrated neutrophils. Annexin V binds preferentially to phosphatidylserine that is exposed on the outer face of the cytoplasmic membrane in apoptotic cells and the percentage of the migrated cells exhibiting annexin V binding can be determined by FACSort and used as an index of apoptosis. It was observed that 6 h after Cg (30 lg/1.0 mL) injection, the rats pretreated with PBS (0.5 mL, s.c.) presented around 70% of neutrophils in apoptosis. Pretreatment of the rats with nitro or amino at a dose of 50 mg/kg, 30 min before the Cg injection, blocked apoptosis in the emigrated neutrophils (Fig. 7).
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Fig. 5. The enhancement of Cg-induced neutrophil migration by nitro and amino treatments was prevented by co-administration of L -arginine. The rats were co-treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before), plus PBS or L -arginine (500 mg/kg, s.c.) and then injected i.p. with Cg at a dose of 30 lg/cavity. The neutrophil migration was evaluated 6 h after Cg injection. The first bar represents the neutrophil migration induced by PBS injected i.p. (C). The values are means SEM and are representative of two separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated Cg-injected group, and r P < 0:05 compared to the group treated with nitro or amino, respectively (ANOVA followed by BonferroniÕs t test).
Adhesion, migration, and apoptotic index of neutrophils in iNOS = mice It is accepted that the iNOS = mouse is a more accurate model in which to establish the role of iNOSderived NO in the inflammatory process, since many iNOS inhibitors (including amino) are not totally selective for the iNOS isoform. Therefore, we used iNOS = mice to investigate whether the NO that downmodulates the neutrophil migration and rolling/adhesion on endothelial cells, and induces apoptosis is derived from iNOS. First, we demonstrated that, similar to the results observed in rats, the neutrophil migration triggered by i.p. injection of Cg and determined 6 h after in wild type mice is enhanced by treatment of the animals with nitro or amino at a dose of 50 mg/kg (Fig. 8A). Furthermore, we also observed that 80% of the neutrophils present in the peritoneal cavity of the mice 6 h after Cg injection were apoptotic and the pretreatment of the animals with nitro or amino blocked the apoptotic process (Fig. 9). The Cg-induced neutrophil migration, rolling, and adhesion were also enhanced in iNOS = mice compared with wild type mice, suggesting that this isoform of NOS is a source of the NO that down-modulates the neutrophil/endothelium adhesion mechanism. Interestingly, these enhancements were
Fig. 6. The nitro and amino treatments enhance leukocyte rolling and adhesion on venular endothelial cells. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected with Cg (30 lg/cavity). The leukocyte rolling (A) and adhesion (B) were evaluated by intravital microscopy in the mesentery 2 and 4 h, respectively, after Cg injection (see Materials and methods). The first bar in panels A and B represents the rolling and adhesion, respectively, in PBS i.p. injected animals (C). The values are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated Cg-injected group (ANOVA followed by BonferroniÕs t test).
potentiated by the treatment of the iNOS = mice with nitro at a dose of 50 mg/kg and inhibited by the treatment of the iNOS = mice with a high dose (1 g/kg) of L arginine (Figs. 8B and C). These results suggest that the down-modulation of the neutrophil rolling and adhesion on venular endothelium and, consequently, the neutrophil migration also involves NO released by cNOS. It was also observed that the neutrophils migrated in response to Cg in iNOS = mice were not apoptotic, and that this was not changed by the pretreatment of the
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Fig. 7. The nitro and amino treatments reduce apoptosis in Cg-induced migrated neutrophils. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with Cg (30 lg/cavity). The neutrophil apoptosis index (% of gated) was measured, 6 h after Cg injection, by the cell-surface expression of annexin V, using an annexin V-biotin/ streptavidin FITC-conjugated monoclonal antibody and determining the annexin V expression by FACSsort flow cytometry. The bars represent the percentage of neutrophils staining positive. The values are means SEM and are representative of two separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to PBS treated Cg-injected group (ANOVA followed by BonferroniÕs t test).
animals with nitro. However, the administration of partially re-established the apoptosis in the emigrated neutrophils (Fig. 9). L -arginine
Discussion In the present study, we demonstrated that the pretreatment of rats with nitro, an inhibitor of cNOS and iNOS [22,56,57], or with amino, a selective iNOS inhibitor [22,57] promoted a dose-dependent (25–100 mg/ kg) enhancement in the number of neutrophils that emigrated to the peritoneal cavity 6 h after i.p. administration of a low dose of Cg (30 lg/cavity). Furthermore, the nitro or amino pretreatment also enhanced the neutrophil migration in response to i.p. injection of low doses of Cg, LPS and fMLP in rats, or Cg in mice. The effects of the NOS inhibitors upon the neutrophil migration appear to be a consequence of the inhibition of the L -arginine/NOS pathway, because the enhancement of neutrophil migration was reversed by the co-treatment of the animals with L -arginine, the NOS substrate [58,59]. The fact that the inhibition of NO production enhances the neutrophil migration, suggests that during the inflammatory process, simultaneous with the release of neutrophil chemotactic factors, NO is produced and
Fig. 8. Effects of nitro and amino treatment upon Cg-induced neutrophil migration (A), rolling (B), and adhesion (C) on venular endothelial cells in wild type (WT) and iNOS = mice. The WT mice were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with Cg. The iNOS = mice were treated with PBS, nitro or L -arginine (1 g/kg, s.c., 30 min before) and then also injected i.p. with Cg. The neutrophil migration, rolling, and adhesion were evaluated 6, 2, and 4 h later, respectively. The first bar in each panel represents the neutrophil migration (A), rolling (B), and adhesion (C) induced by PBS injected i.p. (C). The values are means SEM and are representative of two separate experiments with five mice per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to Cg-injected WT mice pretreated with PBS; r P < 0:05 compared to Cg-injected iNOS = mice pretreated with PBS (ANOVA followed by BonferroniÕs t test).
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Fig. 9. The apoptotic index of migrated neutrophils induced by Cg in wild type (WT) mice treated with PBS (0.5 mL, s.c.), nitro or amino (50 mg/kg, s.c., 30 min before) or in iNOS = mice treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or L -arginine (1 and 2 g/kg, s.c., 30 min before). The animals were treated with the indicated drugs and 30 min later they were injected i.p. with Cg at a dose of 500 lg/cavity. Neutrophil apoptosis was evaluated 6 h after by cellsurface expression of annexin V using annexin V-biotin/streptavidin FITC-conjugated monoclonal antibody and determining expression with a FACSsort flow cytometer. The bars represent the percentage of neutrophils stained positive (means SEM) and are representative of two separate experiments with five mice per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to PBS treated Cginjected WT mice; # P < 0:05 compared to PBS and nitro treated Cginjected iNOS = mice; r P < 0:05 compared to L-arg (1g/1kg) treated Cg-injected iNOS = mice (ANOVA followed by BonferroniÕs t test).
down-modulates the recruitment of neutrophils to the inflammatory site. The fact that the IC50 of nitro for the inhibition of eNOS is around 10 times lower than that for iNOS inhibition [22] suggests that, at low doses, nitro is inhibiting predominantly the eNOS isoform. This, together with the demonstration that amino, a selective inhibitor of iNOS [22,57] enhanced the neutrophil migration suggests that during the inflammatory process NO, which modulates the neutrophil migration, is synthesized by either eNOS or iNOS. In this context, there is evidence in the literature that during the inflammatory process the expression of cNOS or its activity is enhanced [60] and iNOS is induced [61,62]. We confirmed that the NO involved in the down-modulation of neutrophil migration is synthesized by the iNOS isoform by
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showing that Cg-induced neutrophil migration is enhanced in iNOS = mice. The fact that nitro was able to potentiate the enhancement of Cg-induced neutrophil migration in iNOS = mice, which do not express iNOS, suggests that eNOS also is involved in the suppression of neutrophil migration. Other authors have demonstrated that NO down-modulates the neutrophil migration during the inflammatory process and that it is released by both NOS isoforms. For example, animals treated with selective iNOS inhibitors (amino, 1400 W, L -NIL, and AE-ITU), or animals treated with a selective eNOS inhibitor (L -NIO) present enhanced neutrophil migration to inflammatory sites [17–19,63]. Furthermore, the NO donors, SIN-1, NOC-18, spermine-NO, and sodium nitroprusside, inhibit both adhesion and neutrophil migration induced by LPS, IL-1, Cg or ischemia/reperfusion [17,25,26,33,64]. On the other hand, there are some studies that contradict these reports and our own findings. It was observed that animals treated with NOS inhibitors (L -NAME, L -NIL, L -NMMA, and amino) or iNOS = mice present a reduction in the neutrophil migration induced by staphylococcal enterotoxin B (SEB), zymosan, streptococcal cell wall (SCW) or Cg [19,38–40]. It has been suggested that these apparently conflicting data regarding the suppressor effects of NOS inhibitors upon neutrophil migration could be a consequence of vasoconstriction, leading to a reduction in local blood flow, due to the use of high doses and/or systemic administration of the drugs [17,18]. However, a decrease in blood flow in the microcirculation cannot alone explain the reduction in neutrophil migration induced by zymosan and Cg observed in iNOS = mice [38,39], which do not present changes in blood pressure after the inflammatory stimuli injection. We do not have an explanation for these differences, but in our study the iNOS = mice, similar to wild type mice and rats treated with aminoguanidine or nitro-L -arginine and injected with Cg, presented a massive neutrophil migration. It is important to point out that iNOS = mice or wild type mice and rats treated with aminoguanidine at the used dose did not present a significant increase in blood pressure [17]. We showed that when the animals were stimulated with high doses of Cg or LPS, the nitro and amino NOS inhibitors were unable to enhance the neutrophil migration. A possible explanation for this finding is that the large amount of the inflammatory stimulus caused the generation of a substantial concentration of chemotactic mediators, the activities of which could not be countered by the released NO. This may also explain other studies in the literature showing that NOS inhibitors or NO donors do not affect the neutrophil migration to the inflammatory site [65–68]. The fact that the NOS inhibitors were able to potentiate the neutrophil migration 48 h after administration of high doses of the inflammatory stimuli could be explained by the finding
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that at this time point the enhancement of neutrophil migration by the treatment of the animals with NOS inhibitors is mainly due to a reduction in apoptosis (see below). The fact that the inhibition of NO production was able to enhance the neutrophil migration induced fMLP, which is a direct chemotactic mediator, suggests that the inhibitory effect of NO upon neutrophil migration is not dependent upon endogenous release of chemotactic mediators. This fact, together with the substantial evidences in the literature showing that NO reduces neutrophil adhesion to endothelial cells [23–25,28,29,31,32] and that it is involved in the induction of neutrophil apoptosis [48,49,51,69,70], prompted us to investigate the effects of NOS inhibitors on rolling and adhesion of the neutrophils in the mesenteric microcirculation and also on the apoptosis in the migrated neutrophils. These parameters were also analyzed in iNOS = mice. It was observed that both nitro and amino enhanced the rolling and the adhesion of the neutrophil on the mesenteric venules. Furthermore, the neutrophil rolling and adhesion in Cg-injected iNOS = mice were higher than those observed with wild type mice. Moreover, nitro greatly enhanced and L -arginine inhibited these parameters in iNOS = mice. These results suggest that NO produced by both NOS isoforms during the inflammatory process down-modulates the neutrophil/endothelium adhesion mechanism. In this context, there are in vitro and in vivo studies showing that NOS inhibitors increase neutrophil rolling and, later, adhesion, while NO donors decrease leukocyte adhesion on endothelial cells [23–25,28,29,31–33,71–73]. In addition, NOS inhibitors up-regulate and NO donors down-regulate expression of the cell adhesion molecules CD11b/CD18, L-, P-, E-selectin ICAM-1, and VCAM-1 [27,30,34–37]. Furthermore, we also demonstrated that NO released during the inflammatory process by cNOS and iNOS isoforms mediates the apoptosis of the emigrated neutrophils. It was observed that approximately 75% of migrated neutrophils 6 h after Cg injection in rats or mice are apoptotic, as determined by expression of annexin V. Pretreatment of the Cg-injected rats or wild type mice with nitro or amino almost prevented the annexin V expression. Similarly, the Cg-induced migrated neutrophils in iNOS = mice did not exhibit annexin expression. So, the observed increase in the number of neutrophils accumulated in the inflammatory site, when the NO production is pharmacologically or genetically (use of iNOS = mice) inhibited, which is a consequence of the increase in the rolling/adhesion process is due to an increase of survival rate of the emigrated neutrophils, as a result of the reduction of apoptosis rate. We confirmed that cNOS also might be a source of NO involved in the apoptosis since the apoptosis of migrated neutrophils in iNOS = mice was partially re-established by the administration of L -argi-
nine, a substrate for NOS. On the other hand, there are also reports that selective iNOS (L -NIL) inhibitor or iNOS = mice increase the levels of apoptosis in neutrophils, Kupffer cells, endothelial cells, and hepatocytes from liver of animals treated with LPS or during Salmonella infection [74,75]. We do not have an explanation for this apparent incongruence; however, it is under investigation by our group. In conclusion, the results of this study suggest that during the inflammatory process, NO released by either cNOS or iNOS down-modulates the neutrophil migration to inflammatory sites, due to decreased rolling and adhesion of the neutrophils on the endothelium. In addition, the released NO has a fundamental role on apoptosis process of migrated neutrophils.
Acknowledgments ao This work was supported by CAPES (Fundacß~ Coordenacß~ao de Aperfeicßoamento de Pessoal de Nıvel Superior), FAPESP (Fundacß~ao de Amparo a Pesquisa do Estado de S~ao Paulo), and PRONEX (Programa de N ucleos de Excel^encia). We thank Alexandra Rosa Vieira Dias for FACS analysis and Ana K atia dos Santos, Fabıola Leslie Mestriner, Giuliana Bertozi Francisco, Diva Amabile Montanha de Sousa, Sergio Roberto Rosa, and Ieda Regina dos Santos Schivo for technical assistance. We would also like to thank Prof. Dr. Jamil Assreuy Filho for valuable comments and advice.
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Biology and Chemistry
Nitric Oxide 9 (2004) 153–164 www.elsevier.com/locate/yniox
Neutrophil migration in inflammation: nitric oxide inhibits rolling, adhesion and induces apoptosis Daniela Dal Secco,a Juliane Alves Paron,a Sandra H.P. de Oliveira,c Sergio Henrique Ferreira,a Jo~ ao Santana Silva,b and Fernando de Queiroz Cunhaa,* a
b
Department of Pharmacology, School of Medicine of Ribeir~ ao Preto, University of S~ ao Paulo, Avenida Bandeirantes, 3900, 14049-900-Ribeir~ ao Preto, S~ ao Paulo, Brazil Department of Biochemistry and Immunology, School of Medicine of Ribeir~ ao Preto, University of S~ ao Paulo, Avenida Bandeirantes, 3900, 14049-900-Ribeir~ ao Preto, S~ ao Paulo, Brazil c Department of Basic Science, School of Dentistry, Aracßatuba State University of S~ ao Paulo, Brazil Received 12 June 2003; received in revised form 23 October 2003
Abstract There is controversy in the literature over whether nitric oxide (NO) released during the inflammatory process has a pro- or inhibitory effect on neutrophil migration. The aim of the present investigation was to clarify this situation. Treatment of rats with non-selective, N G -nitro-L -arginine (nitro), or selective inducible NO synthase (iNOS), aminoguanidine (amino) inhibitors enhanced neutrophil migration 6 h after the administration of low, but not high, doses of carrageenan (Cg) or Escherichia coli endotoxin (LPS). The neutrophil migration induced by N -formyl–methionyl–leucyl–phenylalanine (fMLP) was also enhanced by nitro or amino treatments. The enhancement of Cg-induced neutrophil migration by NOS inhibitor treatments was reversed by co-treatment with L -arginine, suggesting an involvement of the L -arginine/NOS pathway in the process. The administration of Cg in iNOS deficient (iNOS = ) mice also enhanced the neutrophil migration compared with wild type mice. This enhancement was markedly potentiated by treatment of iNOS = mice with nitro. Investigating the mechanisms by which NOS inhibitors enhanced the neutrophil migration, it was observed that they promoted an increase in Cg-induced rolling and adhesion of leukocytes to endothelium and blocked the apoptosis of emigrated neutrophils. Similar results were observed in iNOS = mice, in which these mechanisms were potentiated and reverted by nitro and L -arginine treatments, respectively. In conclusion, these results suggest that during inflammation, NO released by either constitutive NOS (cNOS) or iNOS down-modulates the neutrophil migration. This NO effect seems to be a consequence of decreased rolling and adhesion of the neutrophils on endothelium and also the induction of apoptosis in migrated neutrophils. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Nitric oxide; Nitric oxide synthase; Inflammation; Apoptosis; Adhesion; Neutrophil migration
Neutrophils are the principal cells involved in host defense against acute bacterial and fungal infections [1]. Although they have a protective effect, the tissue damage observed in diseases such as rheumatoid arthritis, glomerulonephritis, immune vasculitis, ulcerative colitis, and inflammatory bowel disease is, at least, in part, a
* Corresponding author. Fax: +55-856332301. E-mail address: [email protected] (F.Q. Cunha).
1089-8603/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.niox.2003.11.001
consequence of neutrophil accumulation [2–5]. The neutrophil migration during an inflammatory response results mainly from the release by resident cells of neutrophil chemotactic factors, which induce the rolling and adhesion of neutrophils on endothelial cells, followed by their transmigration to the extravascular space [6,7].
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Several neutrophil chemoattractant factors have been described in the literature, such as TNF-a,1 PAF, C5a, LTB4 , and a variety of chemokines, including IL-8, GRO-a, and SDF-1 [8–11]. On the other hand, the production of chemoattractant factors is concomitant with the release of anti-inflammatory mediators, including IL-10, IL-4, and lipoxin, which negatively modulate the neutrophil recruitment [12–14]. Recent studies have shown that NO might also counteract the neutrophil migration [15–19]. NO is produced in mammalian cells from L -arginine and oxygen by a family of enzymes known as NO synthases (NOS). Three NOS isoforms are described: neuronal (nNOS or type I), inducible (iNOS or type II), and endothelial (eNOS or type III). The nNOS and eNOS are constitutively expressed, whereas the iNOS can be induced during the immune and inflammatory responses by bacterial lipopolysaccharide and cytokines, such as TNF-a, IL-1, IFN-c, a, and b, and chemokines [20–22]. Although the mechanisms by which NO attenuates neutrophil accumulation are not fully elucidated, evidence suggests that NO, released by either eNOS or iNOS, modulates the leukocyte–endothelial cell interaction. It was observed that selective inhibitors of iNOS and cNOS increase neutrophil adhesion to endothelial cells, while NO donors decrease both adhesion and leukocyte transmigration to inflammatory sites. Moreover, these parameters are also increased in iNOS = mice [17,23–33]. Furthermore, expression of the cell adhesion molecules CD11b/CD18, L-, P-, E-selectin, ICAM-1, and VCAM-1 among others is down-regulated by NO donors and up-regulated by NOS inhibitors [27,30,34–37]. On the other hand, while this evidence suggests that NO reduces the neutrophil migration, there are also studies in the literature that contradict these findings. For example, it was observed that mice treated with selective iNOS inhibitors (L -NIL or amino) or with nonselective NOS inhibitors (L -NMMA or L -NAME), or
1 Abbreviations used: Cg, carrageenan; LPS, lipopolysaccharide; fMLP, N -formyl-methionyl-leucyl-phenylalanine; nitro, N G -nitroG L -arginine; L -NAME, N -nitro-L -arginine methyl ester; L -NMMA, N G -monomethyl-L -arginine; SIN-1, 3-morpholinosyndnonimine N-ethylcarbamide; amino, aminoguanidine; PBS, phosphate buffered saline; AE-ITU, (S-(2-aminoethyl) isothiourea); NO, nitric oxide; NOS, nitric oxide synthase; iNOS, inducible nitric oxide synthase; cNOS, constitutive nitric oxide synthase; eNOS, endothelial nitric oxide synthase; iNOS = , inducible nitric oxide synthase deficient mice; 1400 W, N-(3(aminomethyl)-benzyl) acetamidine; L -NIO, L -N(5)(1-iminoethyl)-ornithine; L -NIL, L -iminoethyl-lysine; TNF-a, tumor necrosis factor-a; PAF, platelet-activating factor; LTB4 , leukotriene B4 ; IL-8, interleukin-8; IL-10, interleukin-10; IL-4, interleukin-4; IL-1, interleukin-1; GRO-a, growth-related oncogene-alpha; SDF-1, stromal cell-derived factor 1; SEB, staphylococcal enterotoxin B; SCW, streptococcal cell wall; ICAM-1, intracellular adhesion molecule-1; VCAM-1, vascular cell adhesion molecule-1; IFN-c, interferon-c.
iNOS = mice present a reduction in neutrophil migration to inflammatory sites [19,38–40]. Taking in to account these controversies, the aim of the first part of the present study was to investigate in two experimental species (rats and mice) the effects of a selective iNOS (amino) and non-selective NOS (nitro) inhibitors on neutrophil migration induced by low and high doses of the inflammatory stimuli Cg or LPS, and by a direct chemotactic mediator, fMLP. The neutrophil migration induced by Cg in iNOS = mice was also investigated. It was found that the animals treated with both NOS inhibitors or iNOS = mice exhibited enhanced neutrophil migration to the peritoneal cavity in response to low doses of the inflammatory stimuli. It is accepted that the disappearance of the migrated neutrophils from inflammatory sites is a consequence of the apoptotic process, which results in the recognition and uptake of these cells by macrophages [41–44]. Within the last decade, several studies demonstrated that NO is involved in the induction of apoptosis in several cell types, including neutrophils [45–51]. Thus, the treatment of animals with NOS inhibitors could reduce the apoptotic rate of migrated neutrophils, extending their life and allowing them to linger at the inflammatory site. To investigate whether an enhancement of neutrophil accumulation in the peritoneal cavities of the animals treated with NOS inhibitors or in iNOS = mice was mostly a consequence of the inhibition of apoptosis, and/or due to an increase in the mechanism of neutrophil/endothelium adhesion, we investigated the percentage of apoptosis in the Cg-induced neutrophil migration in animals treated with NOS inhibitors (nitro and amino) and in iNOS = mice. Furthermore, the effect of treatment with these NOS inhibitors on the Cginduced rolling/adhesion of neutrophils to endothelial cells was also investigated, in rats. It was found that the treatment of the animals with either amino or nitro enhances the rolling and adhesion of the neutrophils on endothelium and also blocked the apoptosis process in migrated neutrophils. Similar results were observed in iNOS = mice.
Materials and methods Animals Adult male Wistar rats weighing 180–200 g and adult male C57BL-6 (wild type) or iNOS deficient (iNOS = ) mice weighing 18–20 g were used in this study. The animals were housed in temperature-controlled rooms (22– 25 °C), with access to water and food ad libitum, until use in the Departments of Pharmacology and Immunology of the School of Medicine of Ribeir~ ao Preto, University of S~ao Paulo. The wild type and iNOS = mice were housed in a sterile laminar flow cabinet.
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Breeding pairs of mice with targeted disruption of the iNOS = gene were obtained from Jackson Laboratories (Bar Harbor, Maine, USA). All experiments were conducted in accordance with the ethical guidelines of the School of Medicine of Ribeir~ ao Preto, University of S~ ao Paulo, S~ ao Paulo, Brazil. Drugs Carrageenan (Cg), Escherichia coli lipopolysaccharide (LPS), N -formyl-methionyl-leucyl-phenylalanine (fMLP), aminoguanidine (amino), N G -nitro-L -arginine (nitro), and L -arginine were purchased from Sigma (USA). In vivo neutrophil migration For the determination of neutrophil migration, the rats and mice were killed at the specified time and the peritoneal cavity cells were harvested by washing the cavity with 10 and 5 mL, respectively, of PBS containing EDTA (37.2 mg/100 mL). The volumes recovered were similar in all experimental groups and equated to approximately 95% of the injected volume. Total counts were performed in a cell counter (COULTER A C T; Coulter, Miami, FL, USA) and differential cell counts (100 cells total) were carried out on cytocentrifuge (Cytospin 3; Shandon Lipshaw, Pittsburgh, Pennsylvania, USA) slides stained with Rosenfeld. The results are presented as the number of neutrophils per cavity. Determination of neutrophil rolling and adhesion to the mesenteric microcirculation by intravital microscopy The leukocyte rolling and adhesion were examined as previously described [52–54]. Briefly, animals were anesthetized with tribromoethanol (250 mg/kg). The mesenteric tissue was exposed for microscopic examination in situ. This was performed through a longitudinal incision of the skin and abdominal muscle on the right side of the body followed by the exposure of the mesentery. Respiratory movements of the animals did not affect the preparation, and the microcirculatory characteristics remained essentially stable throughout the course of the experiment. The animals were maintained on a special board thermostatically controlled at 37 °C, with a transparent platform on which the tissue to be transilluminated was placed. The preparation was kept moist and warm by irrigating the tissue with warmed (37 °C) Ringer LockeÕs solution, pH 7.2–7.4, containing 1% gelatin. A 500-line television camera was mounted onto a triocular Zeiss microscope to facilitate observation of the enlarged image (3400) on a video screen. Images were recorded on a video recorder with a 40 long distance objective and a 0.65 numerical aperture. An image-splitting micrometer was adjusted to the phototube of the microscope as described by Baez [52].
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The image splitter sheared the optical image into two separate images and displaced one with respect to the other. By rotating the image splitter in the phototube, the shearing was maintained in a direction at right angles to the axis of the vessel. The displacement of one image from the other allowed measurement of the vessel diameter. Vessels selected for study were third-order venules, defined according to their branch-order location within the microvascular network. These vessels corresponded to postcapillary venules, with a diameter of 10–18 lm. The interaction of leukocytes with the luminal surface of the venular endothelium was studied in a segment of the vessel. Rolling leukocytes (‘‘rollers’’) were defined as those white blood cells that moved at a velocity less than that of erythrocytes in the same stream. The number of rolling leukocytes was determined in 10-min intervals. These leukocytes moved sufficiently slowly as to be individually visible and were counted as they rolled past a 10-lm length of venule [53]. A leukocyte was considered to be adherent to the venular endothelium if it remained stationary for >30 s [55]. Adherent cells (‘‘stickers’’) were expressed as the number per 10-lm length of venule. Cells were counted in the recorded image using five different fields for each animal to avoid variability due to sampling. Data were then averaged for each animal. Apoptotic index of migrated neutrophils The apoptotic index was determined according to the instructions of a TACS Annexin V-Biotin Apoptosis Detection Kit (R&D Systems, Minneapolis, USA). Briefly, leukocytes harvested from the peritoneal cavity were washed (500g for 5 min at room temperature) with 500 lL cold (2–8 °C) PBS. Then, the cell pellet (1 106 cells) was resuspended in 100 lL of a solution of annexin V, conjugated to biotin, and propidium iodide before being incubated in the dark at room temperature for 15 min. After centrifugation (500g), the pellet was resuspended in 100 lL of Binding Buffer, which contains fluorescent-streptavidin conjugate, and incubated in the dark for 15 min. The cell suspension was centrifuged again and the pellet was resuspended in 400 lL of Binding Buffer, before being subjected to FACSort flow cytometry (Becton Dickinson, San Jose, CA, USA) analysis. The appropriate gate for neutrophils was determined using purified neutrophils and monocytes harvested from the peritoneal cavity after injection of Cg. Experimental protocols 1. Effect of nitro and amino on the neutrophil migration induced by Cg, LPS or fMLP in rats. The rats were treated subcutaneously (s.c.) with phosphate buffered saline (PBS; 0.5 mL) or with the
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NOS inhibitors, nitro (unspecific NOS inhibitor) or amino (selective inhibitor of iNOS), at doses of 25, 50 or 100 mg/kg. Thirty minutes later Cg (30 lg/1.0 mL) was injected intraperitoneally (i.p.) and the neutrophil migration was determined 6 h after, as described above. The animals in the control group (C) were injected i.p. with PBS (1.0 mL). In other experimental groups, the rats were treated s.c. with nitro or amino at a dose of 50 mg/kg and 30 min later they were injected with Cg at doses of 30 or 100 lg/1.0 mL, LPS at doses of 20 or 100 ng/1.0 mL or fMLP at a dose of 10 nmol/1.0 mL. The neutrophil migration was determined 6 and 48 h after the injection of inflammatory stimuli. The animals of the experimental groups in which the neutrophil migration was determined 48 h after the inflammatory stimulus injection also received nitro or amino (50 mg/ kg) twice a day. The doses of 30 and 100 lg of Cg and of 20 and 100 ng of LPS were chosen on the basis of previous unpublished experiments, in which they induced a mild and intense neutrophil migration, respectively, into peritoneal cavities of rats. In another experimental group, the animals were co-treated s.c. with L -arginine (500 mg/kg), plus nitro (50 mg/kg) or amino (50 mg/kg) and 30 min later Cg (30 lg/1.0 mL) was injected i.p., the neutrophil migration was determined 6 h after. 2. Effect of nitro and amino on the neutrophil migration induced by Cg in wild type and iNOS = mice. The effects of NO synthesis inhibitors upon Cg-induced neutrophil migration in mice were also investigated. For this, wild type mice were treated s.c. with PBS (0.5 mL), nitro or amino, at a dose of 50 mg/kg and 30 min later they were injected with Cg at dose a of 500 lg/ 1.0 mL. Six hours after the animals were killed and the number of neutrophils present in the harvested fluid was determined as described above. Cg (500 lg/1.0 mL) was also injected i.p. in iNOS = mice pretreated s.c. with PBS or nitro (50 mg/kg) and the neutrophil migration was determined 6 h after. The dose of Cg injected in mice was five times higher than that used in rats because in preliminary experiments it was observed that mice are much less responsive than rats to inflammatory stimuli. 3. Effect of nitro and amino on Cg-induced neutrophil rolling and adhesion to the mesenteric microcirculation in rats. The animals were treated s.c. with PBS (0.5 mL), nitro or amino, at a dose of 50 mg/kg. Thirty minutes later, Cg at a dose of 30 lg/1.0 mL was injected i.p. and 2 and 4 h after the leukocyte rolling and adhesion to the ileocaecal portion of mesenteric microcirculation were determined in five different fields for each animal by intravital microscopy, respectively. The data were then averaged for each animal, as described above. The rolling and adhesion were determined 2 and 4 h after Cg injection, respectively, because in a preliminary experiment we observed that rolling and adhesion processes peak at these times after inflammatory stimuli injection.
4. Effect of nitro and L -arginine on Cg-induced neutrophil rolling and adhesion to the mesenteric microcirculation in iNOS = mice. The wild type mice were treated s.c. with PBS (0.5 mL), while the iNOS = mice were treated with nitro, at a dose of 50 mg/kg or L -arginine (1 g/kg, s.c.). Thirty minutes later, Cg at a dose of 500 lg/1.0 mL was injected i.p. and the leukocyte rolling and adhesion to the ileocaecal portion of mesenteric microcirculation were determined 2 and 4 h after, respectively, in five different fields for each animal by intravital microscopy, as described above. The wild type mice in the control group (C) were injected i.p. with PBS (1.0 mL). Because the mouse has much more fat deposition on the mesentery, a careful examination of the mesentery microcirculation was done to identify vessel areas without fat deposition. 5. Effect of nitro and amino on the apoptotic index of inflammatory neutrophils in rats, wild type, and iNOS = mice. Rats and wild type mice were treated with PBS s.c., nitro or amino, at doses of 50 mg/kg and, 30 min later, Cg, at doses of 30 lg/1.0 mL (rats), and 500 lg/1.0 mL (mice) was injected i.p. The iNOS = mice were treated with PBS s.c., nitro (50 mg/kg, s.c.) or L -arginine (1 and 2 g/kg, s.c.) and 30 min later Cg (500 lg/1.0 mL) was injected i.p. The cells were harvested and washed 6 h after Cg injection and the apoptotic index was determined, as described above. Statistical analysis The data are reported as means SEM and are representative of two or three different experiments. The means from different treatments in each individual experiment were compared by ANOVA. When significant differences were identified, individual comparisons were subsequently made with BonferroniÕs t test for unpaired values. Statistical significance was set at P < 0:05.
Results Effect of nitro and amino on the neutrophil migration induced by Cg, LPS or fMLP The i.p. injection of a low dose of Cg (30 lg/1.0 mL) induced a significant neutrophil migration in rats, determined 6 h after. The pretreatment of the animals with nitro (Fig. 1A) or amino (Fig. 1B) enhanced the observed neutrophil migration in a dose-dependent manner. Moreover, treatment with nitro or amino (50 mg/ kg) significantly enhanced the neutrophil migration triggered i.p. injection of Cg (30 lg/1.0 mL) or LPS (20 ng/1.0 mL) and determined either 6 or 48 h later Figs. 2A and 3A, respectively. By contrast, nitro or amino (50 mg/kg) did not enhance the neutrophil migration in
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Fig. 1. The NO synthesis inhibitors, nitro and amino, enhance the neutrophil migration induced by Cg. The rats were treated s.c. with PBS or with indicated doses of nitro (A) or amino (B). After 30 min, Cg was injected at a dose of 30 lg/cavity and the neutrophil migration was determined 6 h after. The first bar in each panel represents the neutrophil migration induced by PBS (C, control group). Data are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to the C group; # P < 0:05 compared to PBS treated Cg-injected group (ANOVA followed by BonferroniÕs t test).
response to high doses of Cg (100 lg/1.0 mL) or LPS (100 ng/1.0 mL) when migration was determined 6 h after the inflammatory stimulus injection (left panels of Figs. 2B and 3B, respectively). On the other hand, the neutrophil migration determined 48 h after the injection of high doses of Cg or LPS was enhanced by nitro and amino treatment (right panels of Figs. 2B and 3B). The neutrophil migration determined 6 and 48 h after injection of a direct chemotactic factor, fMLP (10 nmol/ 1.0 mL), also was enhanced by the pretreatment of the rats with nitro or amino at a dose of 50 mg/kg (Fig. 4). The enhancement of Cg (30 lg/1.0 mL)-induced neutrophil migration by the pretreatment of the rats with nitro (50 mg/kg) or amino (50 mg/kg) was inhibited by
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Fig. 2. Effects of nitro and amino NOS inhibitors upon neutrophil migration induced by different doses of Cg. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with Cg at doses of 30 lg/cavity (A) or 100 lg/cavity (B). The neutrophil migration was evaluated 6 or 48 h after Cg injection. The animals in the experimental group in which the neutrophil migration was determined 48 h after Cg injection also received nitro or amino (same doses) twice a day. The first bar in each panel represents the neutrophil migration induced by PBS injected i.p. (C). The values are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated Cg-injected group (ANOVA followed by BonferroniÕs t test).
the co-administration of L -arginine at a dose of 500 mg/ kg (Fig. 5). Effect of nitro and amino upon leukocyte–endothelium interaction (rolling and adhesion) in rats To clarify the mechanisms by which the NOS inhibitors increase the neutrophil migration to the
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Fig. 4. Effects of nitro and amino upon fMLP-induced neutrophil migration. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with fMLP at a dose of 10 nmol/cavity. The neutrophil migration was evaluated 6 or 48 h later. The animals in the experimental group in which the neutrophil migration was determined 48 h after fMLP injection also received nitro or amino (same doses) twice a day. The white bars (C) represent the neutrophil migration induced by PBS injected i.p. The values are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated fMLP-injected group (ANOVA followed by BonferroniÕs t test).
injection of PBS. The pretreatment of the Cg-injected rats with nitro or amino at a dose of 50 mg/kg significantly enhanced leukocyte rolling and adhesion.
Fig. 3. Effects of nitro and amino upon neutrophil migration induced by different doses of LPS. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with LPS at doses of 20 ng/cavity (A) or 100 ng/cavity (B). The neutrophil migration was evaluated 6 or 48 h after LPS injection. The animals in the experimental group in which the neutrophil migration was determined 48 h after LPS injection also received nitro or amino (same doses) twice a day. The first bar in each panel represents the neutrophil migration induced by PBS injected i.p. (C). The values are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated LPS-injected group (ANOVA followed by BonferroniÕs t test).
inflammatory site, we investigated whether these drugs increased leukocyte–endothelium interaction in vivo. Leukocyte–endothelium interaction (rolling and adhesion) was examined in mesenteric postcapillary venules with diameters ranging from 10 to 18 lm. The i.p. injection of Cg (30 lg/1.0 mL) caused a significant increase in rolling (Fig. 6A) and adhesion (Fig. 6B) of leukocytes on endothelium, compared with the i.p.
Effect of nitro and amino upon the apoptotic index of migrated neutrophils The results described above suggest that NO inhibits the neutrophil migration to the inflammatory site. This inhibitory effect seems to be a consequence of the ability to down-regulate the neutrophil rolling and adhesion to endothelium. Since there is evidence in the literature that NO is also an apoptotic mediator, we investigated whether the increase of neutrophil migration observed in animals treated with NOS inhibitors might be also a consequence of reduced apoptosis in emigrated neutrophils. Annexin V binds preferentially to phosphatidylserine that is exposed on the outer face of the cytoplasmic membrane in apoptotic cells and the percentage of the migrated cells exhibiting annexin V binding can be determined by FACSort and used as an index of apoptosis. It was observed that 6 h after Cg (30 lg/1.0 mL) injection, the rats pretreated with PBS (0.5 mL, s.c.) presented around 70% of neutrophils in apoptosis. Pretreatment of the rats with nitro or amino at a dose of 50 mg/kg, 30 min before the Cg injection, blocked apoptosis in the emigrated neutrophils (Fig. 7).
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Fig. 5. The enhancement of Cg-induced neutrophil migration by nitro and amino treatments was prevented by co-administration of L -arginine. The rats were co-treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before), plus PBS or L -arginine (500 mg/kg, s.c.) and then injected i.p. with Cg at a dose of 30 lg/cavity. The neutrophil migration was evaluated 6 h after Cg injection. The first bar represents the neutrophil migration induced by PBS injected i.p. (C). The values are means SEM and are representative of two separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated Cg-injected group, and r P < 0:05 compared to the group treated with nitro or amino, respectively (ANOVA followed by BonferroniÕs t test).
Adhesion, migration, and apoptotic index of neutrophils in iNOS = mice It is accepted that the iNOS = mouse is a more accurate model in which to establish the role of iNOSderived NO in the inflammatory process, since many iNOS inhibitors (including amino) are not totally selective for the iNOS isoform. Therefore, we used iNOS = mice to investigate whether the NO that downmodulates the neutrophil migration and rolling/adhesion on endothelial cells, and induces apoptosis is derived from iNOS. First, we demonstrated that, similar to the results observed in rats, the neutrophil migration triggered by i.p. injection of Cg and determined 6 h after in wild type mice is enhanced by treatment of the animals with nitro or amino at a dose of 50 mg/kg (Fig. 8A). Furthermore, we also observed that 80% of the neutrophils present in the peritoneal cavity of the mice 6 h after Cg injection were apoptotic and the pretreatment of the animals with nitro or amino blocked the apoptotic process (Fig. 9). The Cg-induced neutrophil migration, rolling, and adhesion were also enhanced in iNOS = mice compared with wild type mice, suggesting that this isoform of NOS is a source of the NO that down-modulates the neutrophil/endothelium adhesion mechanism. Interestingly, these enhancements were
Fig. 6. The nitro and amino treatments enhance leukocyte rolling and adhesion on venular endothelial cells. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected with Cg (30 lg/cavity). The leukocyte rolling (A) and adhesion (B) were evaluated by intravital microscopy in the mesentery 2 and 4 h, respectively, after Cg injection (see Materials and methods). The first bar in panels A and B represents the rolling and adhesion, respectively, in PBS i.p. injected animals (C). The values are means SEM and are representative of three separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to PBS treated Cg-injected group (ANOVA followed by BonferroniÕs t test).
potentiated by the treatment of the iNOS = mice with nitro at a dose of 50 mg/kg and inhibited by the treatment of the iNOS = mice with a high dose (1 g/kg) of L arginine (Figs. 8B and C). These results suggest that the down-modulation of the neutrophil rolling and adhesion on venular endothelium and, consequently, the neutrophil migration also involves NO released by cNOS. It was also observed that the neutrophils migrated in response to Cg in iNOS = mice were not apoptotic, and that this was not changed by the pretreatment of the
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Fig. 7. The nitro and amino treatments reduce apoptosis in Cg-induced migrated neutrophils. The rats were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with Cg (30 lg/cavity). The neutrophil apoptosis index (% of gated) was measured, 6 h after Cg injection, by the cell-surface expression of annexin V, using an annexin V-biotin/ streptavidin FITC-conjugated monoclonal antibody and determining the annexin V expression by FACSsort flow cytometry. The bars represent the percentage of neutrophils staining positive. The values are means SEM and are representative of two separate experiments with six rats per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to PBS treated Cg-injected group (ANOVA followed by BonferroniÕs t test).
animals with nitro. However, the administration of partially re-established the apoptosis in the emigrated neutrophils (Fig. 9). L -arginine
Discussion In the present study, we demonstrated that the pretreatment of rats with nitro, an inhibitor of cNOS and iNOS [22,56,57], or with amino, a selective iNOS inhibitor [22,57] promoted a dose-dependent (25–100 mg/ kg) enhancement in the number of neutrophils that emigrated to the peritoneal cavity 6 h after i.p. administration of a low dose of Cg (30 lg/cavity). Furthermore, the nitro or amino pretreatment also enhanced the neutrophil migration in response to i.p. injection of low doses of Cg, LPS and fMLP in rats, or Cg in mice. The effects of the NOS inhibitors upon the neutrophil migration appear to be a consequence of the inhibition of the L -arginine/NOS pathway, because the enhancement of neutrophil migration was reversed by the co-treatment of the animals with L -arginine, the NOS substrate [58,59]. The fact that the inhibition of NO production enhances the neutrophil migration, suggests that during the inflammatory process, simultaneous with the release of neutrophil chemotactic factors, NO is produced and
Fig. 8. Effects of nitro and amino treatment upon Cg-induced neutrophil migration (A), rolling (B), and adhesion (C) on venular endothelial cells in wild type (WT) and iNOS = mice. The WT mice were treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or amino (50 mg/kg, s.c., 30 min before) and then injected i.p. with Cg. The iNOS = mice were treated with PBS, nitro or L -arginine (1 g/kg, s.c., 30 min before) and then also injected i.p. with Cg. The neutrophil migration, rolling, and adhesion were evaluated 6, 2, and 4 h later, respectively. The first bar in each panel represents the neutrophil migration (A), rolling (B), and adhesion (C) induced by PBS injected i.p. (C). The values are means SEM and are representative of two separate experiments with five mice per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to C group; # P < 0:05 compared to Cg-injected WT mice pretreated with PBS; r P < 0:05 compared to Cg-injected iNOS = mice pretreated with PBS (ANOVA followed by BonferroniÕs t test).
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Fig. 9. The apoptotic index of migrated neutrophils induced by Cg in wild type (WT) mice treated with PBS (0.5 mL, s.c.), nitro or amino (50 mg/kg, s.c., 30 min before) or in iNOS = mice treated with PBS (0.5 mL, s.c.), nitro (50 mg/kg, s.c., 30 min before) or L -arginine (1 and 2 g/kg, s.c., 30 min before). The animals were treated with the indicated drugs and 30 min later they were injected i.p. with Cg at a dose of 500 lg/cavity. Neutrophil apoptosis was evaluated 6 h after by cellsurface expression of annexin V using annexin V-biotin/streptavidin FITC-conjugated monoclonal antibody and determining expression with a FACSsort flow cytometer. The bars represent the percentage of neutrophils stained positive (means SEM) and are representative of two separate experiments with five mice per group. The data used for statistical analyses were from the individual experiment and are the same presented in the figure. I P < 0:05 compared to PBS treated Cginjected WT mice; # P < 0:05 compared to PBS and nitro treated Cginjected iNOS = mice; r P < 0:05 compared to L-arg (1g/1kg) treated Cg-injected iNOS = mice (ANOVA followed by BonferroniÕs t test).
down-modulates the recruitment of neutrophils to the inflammatory site. The fact that the IC50 of nitro for the inhibition of eNOS is around 10 times lower than that for iNOS inhibition [22] suggests that, at low doses, nitro is inhibiting predominantly the eNOS isoform. This, together with the demonstration that amino, a selective inhibitor of iNOS [22,57] enhanced the neutrophil migration suggests that during the inflammatory process NO, which modulates the neutrophil migration, is synthesized by either eNOS or iNOS. In this context, there is evidence in the literature that during the inflammatory process the expression of cNOS or its activity is enhanced [60] and iNOS is induced [61,62]. We confirmed that the NO involved in the down-modulation of neutrophil migration is synthesized by the iNOS isoform by
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showing that Cg-induced neutrophil migration is enhanced in iNOS = mice. The fact that nitro was able to potentiate the enhancement of Cg-induced neutrophil migration in iNOS = mice, which do not express iNOS, suggests that eNOS also is involved in the suppression of neutrophil migration. Other authors have demonstrated that NO down-modulates the neutrophil migration during the inflammatory process and that it is released by both NOS isoforms. For example, animals treated with selective iNOS inhibitors (amino, 1400 W, L -NIL, and AE-ITU), or animals treated with a selective eNOS inhibitor (L -NIO) present enhanced neutrophil migration to inflammatory sites [17–19,63]. Furthermore, the NO donors, SIN-1, NOC-18, spermine-NO, and sodium nitroprusside, inhibit both adhesion and neutrophil migration induced by LPS, IL-1, Cg or ischemia/reperfusion [17,25,26,33,64]. On the other hand, there are some studies that contradict these reports and our own findings. It was observed that animals treated with NOS inhibitors (L -NAME, L -NIL, L -NMMA, and amino) or iNOS = mice present a reduction in the neutrophil migration induced by staphylococcal enterotoxin B (SEB), zymosan, streptococcal cell wall (SCW) or Cg [19,38–40]. It has been suggested that these apparently conflicting data regarding the suppressor effects of NOS inhibitors upon neutrophil migration could be a consequence of vasoconstriction, leading to a reduction in local blood flow, due to the use of high doses and/or systemic administration of the drugs [17,18]. However, a decrease in blood flow in the microcirculation cannot alone explain the reduction in neutrophil migration induced by zymosan and Cg observed in iNOS = mice [38,39], which do not present changes in blood pressure after the inflammatory stimuli injection. We do not have an explanation for these differences, but in our study the iNOS = mice, similar to wild type mice and rats treated with aminoguanidine or nitro-L -arginine and injected with Cg, presented a massive neutrophil migration. It is important to point out that iNOS = mice or wild type mice and rats treated with aminoguanidine at the used dose did not present a significant increase in blood pressure [17]. We showed that when the animals were stimulated with high doses of Cg or LPS, the nitro and amino NOS inhibitors were unable to enhance the neutrophil migration. A possible explanation for this finding is that the large amount of the inflammatory stimulus caused the generation of a substantial concentration of chemotactic mediators, the activities of which could not be countered by the released NO. This may also explain other studies in the literature showing that NOS inhibitors or NO donors do not affect the neutrophil migration to the inflammatory site [65–68]. The fact that the NOS inhibitors were able to potentiate the neutrophil migration 48 h after administration of high doses of the inflammatory stimuli could be explained by the finding
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that at this time point the enhancement of neutrophil migration by the treatment of the animals with NOS inhibitors is mainly due to a reduction in apoptosis (see below). The fact that the inhibition of NO production was able to enhance the neutrophil migration induced fMLP, which is a direct chemotactic mediator, suggests that the inhibitory effect of NO upon neutrophil migration is not dependent upon endogenous release of chemotactic mediators. This fact, together with the substantial evidences in the literature showing that NO reduces neutrophil adhesion to endothelial cells [23–25,28,29,31,32] and that it is involved in the induction of neutrophil apoptosis [48,49,51,69,70], prompted us to investigate the effects of NOS inhibitors on rolling and adhesion of the neutrophils in the mesenteric microcirculation and also on the apoptosis in the migrated neutrophils. These parameters were also analyzed in iNOS = mice. It was observed that both nitro and amino enhanced the rolling and the adhesion of the neutrophil on the mesenteric venules. Furthermore, the neutrophil rolling and adhesion in Cg-injected iNOS = mice were higher than those observed with wild type mice. Moreover, nitro greatly enhanced and L -arginine inhibited these parameters in iNOS = mice. These results suggest that NO produced by both NOS isoforms during the inflammatory process down-modulates the neutrophil/endothelium adhesion mechanism. In this context, there are in vitro and in vivo studies showing that NOS inhibitors increase neutrophil rolling and, later, adhesion, while NO donors decrease leukocyte adhesion on endothelial cells [23–25,28,29,31–33,71–73]. In addition, NOS inhibitors up-regulate and NO donors down-regulate expression of the cell adhesion molecules CD11b/CD18, L-, P-, E-selectin ICAM-1, and VCAM-1 [27,30,34–37]. Furthermore, we also demonstrated that NO released during the inflammatory process by cNOS and iNOS isoforms mediates the apoptosis of the emigrated neutrophils. It was observed that approximately 75% of migrated neutrophils 6 h after Cg injection in rats or mice are apoptotic, as determined by expression of annexin V. Pretreatment of the Cg-injected rats or wild type mice with nitro or amino almost prevented the annexin V expression. Similarly, the Cg-induced migrated neutrophils in iNOS = mice did not exhibit annexin expression. So, the observed increase in the number of neutrophils accumulated in the inflammatory site, when the NO production is pharmacologically or genetically (use of iNOS = mice) inhibited, which is a consequence of the increase in the rolling/adhesion process is due to an increase of survival rate of the emigrated neutrophils, as a result of the reduction of apoptosis rate. We confirmed that cNOS also might be a source of NO involved in the apoptosis since the apoptosis of migrated neutrophils in iNOS = mice was partially re-established by the administration of L -argi-
nine, a substrate for NOS. On the other hand, there are also reports that selective iNOS (L -NIL) inhibitor or iNOS = mice increase the levels of apoptosis in neutrophils, Kupffer cells, endothelial cells, and hepatocytes from liver of animals treated with LPS or during Salmonella infection [74,75]. We do not have an explanation for this apparent incongruence; however, it is under investigation by our group. In conclusion, the results of this study suggest that during the inflammatory process, NO released by either cNOS or iNOS down-modulates the neutrophil migration to inflammatory sites, due to decreased rolling and adhesion of the neutrophils on the endothelium. In addition, the released NO has a fundamental role on apoptosis process of migrated neutrophils.
Acknowledgments ao This work was supported by CAPES (Fundacß~ Coordenacß~ao de Aperfeicßoamento de Pessoal de Nıvel Superior), FAPESP (Fundacß~ao de Amparo a Pesquisa do Estado de S~ao Paulo), and PRONEX (Programa de N ucleos de Excel^encia). We thank Alexandra Rosa Vieira Dias for FACS analysis and Ana K atia dos Santos, Fabıola Leslie Mestriner, Giuliana Bertozi Francisco, Diva Amabile Montanha de Sousa, Sergio Roberto Rosa, and Ieda Regina dos Santos Schivo for technical assistance. We would also like to thank Prof. Dr. Jamil Assreuy Filho for valuable comments and advice.
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