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Neutrophil migration in inflammation: Nitric oxide inhibits rolling, adhesion and induces apoptosis
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, NG-nitro-L-arginine (nitro), or selective inducible NO synthase (iNOS), aminoguanidine (amino) inhibitors enhanced neutrophil migration 6h 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.
... Group 5: L-NG-nitro arginine methyl ester (L-NAME, a nonspecific inhibitor of nitric oxide synthase [NOS]; Sigma, Livonia, MI, USA) was injected subcutaneously (100 mg/kg, dissolved in sterile saline) 30 min before the subcutaneous injection of BjV [32]. ...
... Group 6: Aminoguanidine hydrochloride (a selective inducible nitric oxide [iNOS] inhibitor; Sigma, Livonia, MI, USA) was injected intraperitoneally (50 mg/kg, dissolved in sterile saline) 30 min before the subcutaneous injection of BjV [32]. ...
... Research has shown that NO controls leukocyte-endothelium interactions. It is possible to observe more of these cellular events by blocking endothelial nitric oxide synthase (eNOS) and iNOS [32,49]. A study found that nonspecific NOS inhibitors, like L-NAME, do not stop the production of ICAM-1, which helps white blood cells stick firmly to the endothelium when lipopolysaccharide (LPS) or carrageenan is present [50]. ...
In recent years, extensive research has delved into the pathophysiology of local reactions triggered by Bothrops snake venoms. Even though antivenom works well at reducing death and systemic effects, it is still not very effective in treating local reactions because it cannot counteract damage that has already been triggered. This limitation might be attributed to certain molecules that amplify the venom-induced innate response. While evidence suggests endogenous mediators at the venom site play a role in this envenomation, in Brazil, the concurrent use of anti-inflammatory agents or other drugs alongside antivenom remains uncommon. This study evaluated the pharmacological mediation of alterations in leukocyte–endothelium interactions following the experimental envenomation of mice with Bothrops jararaca venom, the main culprit of snake-related accidents in Southeast Brazil. We treated envenomed mice with inhibitors of different pharmacological pathways and observed the cremaster muscle microcirculation with intravital microscopy. We found that eicosanoids related to cyclooxygenase pathways and nitric oxide significantly contributed to B. jararaca venom-induced alterations in leukocyte–endothelium interactions. Conversely, lipoxygenase-mediated eicosanoids, histamine, and serotonin had minimal participation. Notably, dexamethasone and antivenom treatment diminished B. jararaca venom–induced alterations in leukocyte–endothelium interactions. The limited efficacy of the antivenom in managing Bothrops venom-induced local reactions emphasizes the critical need for supplementary treatments to enhance therapeutic outcomes.
... 4) L-NG-nitro arginine methil ester-L-NAME (a nonspecific inhibitor of the enzyme nitric oxide synthase (NOS), Sigma-100 mg/kg, dissolved in sterile saline, s.c.) 30 minutes before the s.c. injection of BjV [31]; 5) Aminoguanidine hydrochloride (a selective enzyme inhibitor inducible nitric oxide (iNOS), Sigma-50 mg/kg dissolved in sterile saline, i.p.) 30 minutes before the s.c. injection of BjV [31]; 6) Promethazine hydrochloride (an H1 receptor inhibitor, Sigma, 10 mg/kg dissolved in sterile saline, i.p.) 30 minutes before the s.c. ...
... injection of BjV [31]; 5) Aminoguanidine hydrochloride (a selective enzyme inhibitor inducible nitric oxide (iNOS), Sigma-50 mg/kg dissolved in sterile saline, i.p.) 30 minutes before the s.c. injection of BjV [31]; 6) Promethazine hydrochloride (an H1 receptor inhibitor, Sigma, 10 mg/kg dissolved in sterile saline, i.p.) 30 minutes before the s.c. injection of BjV [32]; 7) Methysergide maleate salt (a serotonin 5HT receptor antagonist, Sigma-0.8 mg/kg dissolved in sterile saline, i.p.) 30 minutes before the s.c. ...
... A body of research shows that nitric oxide (NO) plays a part in controlling the interaction between white blood cells and endothelium. It is possible to see more of these cellular events by blocking certain forms of the enzymes' endothelial (eNOS) and inducible nitric oxide synthase (iNOS) [31,49]. A study found that NOS inhibitors that do not target specific genes, like L-NAME, do not stop the production of ICAM-1, a molecule that helps white blood cells stick firmly to endothelium when lipopolysaccharide (LPS) or carrageenan is present [49]. ...
In recent years, extensive research has delved into the pathophysiology of local reactions triggered by Bothrops snake venoms. Even though antivenom works well at reducing death and systemic effects, it is still not very effective in treating local reactions because the venom works so quickly, and antivenom cannot fix injuries that have already been triggered. This might be attributed to certain molecules amplifying the venom-induced innate response. While evidence suggests endogenous mediators at the venom site play a role in this envenomation, in Brazil, concurrent use of anti-inflammatories or other drugs associated with the antivenom remains uncommon. The aim of this study was to determine how various inflammatory mediators affected the leukocyte-endothelium interaction (LEI) following a B. jararaca bite. According to our findings, nitric oxide and cyclooxygenase pathway eicosanoids significantly contribute to alteration of LEI induced by B. jararaca venom. Conversely, lipoxygenase-mediated eicosanoids, histamine, or serotonin had minimal participation. Markedly, treatment with dexamethasone alongside antivenom mitigated alterations of LEI induced by the B. jararaca snake venom. The limited efficacy of the antivenom in managing Bothrops venom-induced local reactions emphasizes the critical need for supplementary treatments to enhance therapeutic outcomes.
... NO released during the inflammatory process can downregulate the migration of neutrophils to inflammatory sites due to the decreased rolling and adhesion of neutrophils on the endothelium and also the induction of apoptosis in migrated neutrophils [2,28]. Some studies have observed that inhibitors of NOS increase neutrophil adhesion to endothelial cells [29,30], while NO donors decrease both adhesion and leukocyte transmigration to inflammatory sites [30,31]. ...
... During the inflammatory process, production and release of cytokines, chemokines, prostaglandins and nitric oxide occur, resulting in the down-modulation of leukocyte recruitment to the inflammatory site [3,28]. Therefore, in this study, we verified the influence of the NO pathway on the leukocyte migration induced by carrageenan. ...
... Some studies in the literature have shown that NO is a mediator that contributes to the inflammatory response, stimulating the production of inflammatory cytokines and peroxynitrite [46,47]. Although the beneficial effects of NO during inflammatory insult have also been reported [2,3,28], Iwata et al. [30] showed, in a carrageenan-induced pleurisy model, that administration of an NO donor or NO substrate (Larginine) reduced the migration of inflammatory cells and edema formation, lowered oxidative stress, and normalized antioxidant enzyme activities. Other studies have suggested the importance of more research concerning the role of NO in osteoarthritis, as well as the use of NO-donating drugs to provide a new therapeutic option for the treatment of inflammatory diseases [48,49]. ...
The pyrazole compound LQFM-021 exhibits vasorelaxant, antinociceptive and anti-inflammatory activities. Furthermore, it has low toxicity, indicating that this compound may be considered to be a good prototype for the development of new analgesic/anti-inflammatory drugs. Therefore, the aim of this study was to investigate the potential anti-inflammatory activity of LQFM-021 using a model of carrageenan-induced inflammation as well as the mechanism of action and role of nitric oxide in this effect. Acute treatments with LQFM-021 (30 and 60 mg/kg p.o.) reduced paw edema formation dose-dependently 2 h after carrageenan injection. In the carrageenan-induced pleurisy test, LQFM-021 (30 mg/kg p.o.) reduced the leukocyte (polymorphonuclear) count in the pleural cavity, as well as decreased protein extravasation and myeloperoxidase activity. This dose of LQFM-021 increased the NO (nitrite/nitrate) and IL-4 levels and decreased the TNF-α and IL-1β levels in the pleural cavity. Moreover, pre-treatment with L-NAME reversed the effect of LQFM-021 on NO, leukocyte migration, and the TNF-α and IL-1β levels. Additionally, we observed that LQFM-021 showed weak inhibitory activity on cyclooxygenases, but reduced the PGE2 levels in the pleural cavity. Immunoblot analyses showed that LQFM-021 promoted a decrease in COX-2 levels and increase in iNOS levels. In conclusion, we demonstrated that LQFM-021 has marked anti-inflammatory activity by reducing polymorphonuclear recruitment, which is associated with the inhibition of the production of inflammatory cytokines and eicosanoids. In addition, we found that the synthase/release of nitric oxide promoted by LQFM-021 is essential for the anti-inflammatory effect observed.
... However, NO can also be neuroprotective. It can limit inflammation by decreasing production of cytokines and expression of cell adhesion molecules (15)(16)(17)(18), and inhibit caspase activity (19), suggesting an anti-apoptotic action. In addition, the activation of NOS-2 has been linked with neurogenesis (20,21), and NOS-2 inducing cytokines such as TNF-␣ and IL-1 display neuroprotective properties (22,23). ...
... Contralateral Sensorimotor Deficits Are Reduced in NOS-2 Ϫ/Ϫ Mice after ACI When performing the grid test (Fig. 5), mild and severely injured animals from both genotypes displayed a greater percentage of footslips on their contralateral limbs when compared to sham controls (wild-type Ϫ F (2,15) ϭ 12.97; p Ͻ 0.0001, sample sizes ϭ 5 for all groups; NOS-2 Ϫ/Ϫ Ϫ F (2,15) ϭ 41.38; p Ͻ 0.0001, sample sizes: sham ϭ 6, mild ϭ 5, severe ϭ 4). When comparing genotypes, NOS-2 Ϫ/Ϫ mice showed a significantly decreased percentage of errors on the contralateral side after severe injury (F (1,9) ϭ 7.188; p ϭ 0.0104), but not after mild injury (F (1,9) ϭ 0.068; p ϭ 0.796). ...
... The beam balance (Fig. 6) and Rotarod tests (Fig. 7 genotypes (beam balance: wild-type Ϫ F (2,15) ϭ 33.30; p Ͻ 0.0001, n ϭ 5 for all groups; NOS-2 Ϫ/Ϫ Ϫ F (2,14) ϭ 41.61; p Ͻ 0.0001, sample sizes: sham ϭ 5, mild ϭ 5, severe ϭ 4; Rotarod (sample sizes as for beam balance): wild-type Ϫ F (2,15) ϭ 17.89; p Ͻ 0.0001; NOS-2 Ϫ/Ϫ Ϫ F (2,15) ϭ 5.268; p ϭ 0.0075), and these deficits persisted for 7 days. However, there were no significant differences between genotypes on the beam balance (severe injury: F (1,9) ϭ 0.6272; p ϭ 0.4328) or Rotarod (severe injury: F (1,9) ϭ 0.0009; p ϭ 0.798) after either mild or severe injury. ...
Nitric oxide (NO) synthesized froth the inducible isoform of nitric oxide synthase (NOS-2) has been suggested to play both beneficial and deleterious roles in various neuropathologies. To define the role of nitric oxide in traumatic brain injury, we subjected male mice lacking a functional NOS-2 gene (NOS-2(-/-)) and their wild-type littermates (NOS-2(+/+)) to mild or severe aseptic cryogenic cerebral injury. Expression of NOS-2 mRNA and protein was observed in NOS-2(+/+) animals following injury. Lesion volume (as measured by histology and brain imaging) and neurological outcome (using motor and cognitive behavioral paradigms) were assessed at various times after injury. While magnetic resonance imaging revealed the extent of edema of the 2 genotypes to be similar, histology showed a reduced (32%) lesion volume in severely injured NOS2(-/-) compared with NOS-2(+/+) mice. In addition, NOS-2(-/-) mice showed significant improvements in both contralateral sensorimotor deficits (grid test: p = 0.011) and cognitive function (Morris water maze: p = 0.009) after severe injury compared to their wild-type littermates. This indicates that lesion volume is reduced and neurological recovery is improved after acute traumatic injury in mice lacking a functional NOS-2 gene, and strongly suggests that the post-trauma production of NO from this source contributes to neuropathology.
... Our group has previously demonstrated that NO produced by iNOS reduces neutrophil migration to the site of infection. The failure of neutrophil migration was associated with the down-regulation of CXCR2 on the surface of neutrophils and reduced ICAM-1 expression on ECs (27,42,43). Additionally, HO-1 products were linked to impaired neutrophil rolling and adhesion in mesentery venules and to the internalization of CXCR2 (44). ...
... However, the association among IL-6, M2 macrophages, lower NO levels, and improvement in sepsis survival should be taken carefully. Our laboratory has reported that NO has a dual role in sepsis: it impairs neutrophil migration to the focus of infection and is also essential to control the growth of microorganisms when released inside phagosomes (42,43,86). Benjamin and colleagues observed that low doses of iNOS inhibitors improve sepsis outcome by increasing neutrophil recruitment to the site of infection. ...
Sepsis is a life-threatening organ dysfunction caused by a deregulated host response to infection. This inappropriate response to microorganism invasion is characterized by an overwhelmed systemic inflammatory response and cardiovascular collapse that culminate in high mortality and morbidity in critical care units. The occurrence of sepsis in diabetes mellitus (DM) patients has become more frequent, as the prevalence of DM has increased dramatically worldwide. These two important diseases represent a global public health concern and highlight the importance of increasing our knowledge of the key elements of the immune response related to both conditions. In this context, it is well established that the cells taking part in the innate and adaptive immune responses in diabetic patients have compromised function. These altered responses favor microorganism growth, a process that contributes to sepsis progression. The present review provides an update on the characteristics of the immune system in diabetic and septic subjects. We also explore the beneficial effects of insulin on the immune response in a glycemic control-dependent and independent manner.
... Neovestitol reduces neutrophil migration by a nitric oxide-dependent mechanism in the peritoneal cavity of mice. Nitric oxide plays a crucial role in modulating neutrophil migration in LPS-induced peritonitis in mice 15,16 . This study investigated the activity of neovestitol in neutrophil migration and expression of ICAM-1 against a pretreatment with an inducible nitric oxide synthase (iNOS) inhibitor (aminoguanidine 50 mg/kg). ...
... Studies have shown that nitric oxide plays a crucial role in modulating leukocyte adhesion during the inflammatory process. The action of nitric oxide (iNOS pathway) in LPS-induced inflammation is associated with a reduction in ICAM-1 expression in the mesenteric microcirculation of mice 15,16 . In this study, we found that treatment with an iNOS inhibitor abolished the inhibitory effect of neovestitol on ICAM-1 expression and neutrophil migration. ...
Isoflavonoids have been largely studied due to their distinct biological activities identified thus far.
Herein, we evaluated the activity of neovestitol, an isoflavonoid isolated from Brazilian red propolis,
in acute and chronic inflammation. As for acute inflammation, we found that neovestitol reduced
neutrophil migration, leukocyte rolling and adhesion, as well as expression of ICAM-1 in the mesenteric
microcirculation during lipopolysaccharide-induced acute peritonitis. No changes were observed in the
levels of TNF-α, CXCL1/KC and CXCL2/MIP-2 upon pretreatment with neovestitol. The administration
of an inducible nitric oxide synthase (iNOS) inhibitor abolished the inhibitory effects of neovestitol in
neutrophil migration and ICAM-1 expression. Nitrite levels increased upon treatment with neovestitol.
No effects of neovestitol were observed on the chemotaxis of neutrophils in vitro. As for chronic
inflammation, neovestitol also reduced the clinical score and joint damage in a collagen-induced
arthritis model. There was no change in the frequency of IL-17-producing TCD4+ cells. In addition,
pretreatment with neovestitol reduced the levels of IL-6. These results demonstrate a potential
anti-inflammatory activity of neovestitol, which may be useful for therapeutic purposes and/or as a
nutraceutical.
... www.nature.com/scientificreports/ Inflammatory cytokines have been hypothesised to induce apoptosis 75 . Apoptosis can also be triggered by the binding of ligands (such as inflammatory cytokines) to specific death receptors on the cell membrane, leading to the recruitment of adaptor proteins and activation of cysteine protease cascades that ultimately induce cell death 76 . ...
Type 2 diabetes (T2D) can cause severe cardiac complications at functional, histologic and molecular levels. These pathological complications could be mediated by ATP-releasing channels such as Panx1 and ATP receptors, in particular P2X7. The aim of our study was to investigate the effect of high-intensity interval training (HIIT) on T2D-induced cardiac complications at the functional, histopathological and molecular levels, with a particular focus on ATP-releasing channels. 48 male Wistar rats at the age of 8 weeks were randomly allocated into four groups: control (Con), Diabetes (T2D), Training (TR), and Diabetes + Training (T2D + TR). T2D was induced by a high-fat diet plus a low dose (35 mg/kg) of STZ administration. Rats in the TR and T2D + TR groups underwent an 8-weeks training program involving intervals ranging from 80 to 100% of their maximum running speed (Vmax), with 4–10 intervals per session. Protein expression of Interleukin 1β (IL1β), Interleukin 10 (IL-10), Pannexin 1 (Panx1), P2X7R (purinergic P2X receptor 7), NLRP1 (NLR Family Pyrin Domain Containing 1), BAX, and Bcl2 were measured in the heart tissue. Additionally, we assessed heart function, histopathological changes, as well as insulin resistance using the homeostasis model assessment of insulin resistance (HOMA-IR). In contrast to the T2D group, HIIT led to increased protein expression of Bcl2 and IL-10 in the heart. It also resulted in improvements in systolic and diastolic blood pressures, heart rate, ± dp/dt (maximum and minimum changes in left ventricular pressure), while reducing protein expression of IL-1β, Panx1, P2X7R, NLRP1, and BAX levels in the heart. Furthermore, left ventricular diastolic pressure (LVDP) was reduced (P ≤ 0.05). Moreover, heart lesion scores increased with T2D but decreased with HIIT, along with a reduction in fibrosis percentage (P ≤ 0.05). The results of this study suggest that the cardioprotective effects of HIIT on the diabetic heart may be mediated by the modulation of ATP-releasing channels. This modulation may lead to a reduction in inflammation and apoptosis, improve cardiac function, and attenuate cardiac injury and fibrosis.
... The reduction in O 2 − availability prevents its dismutation to hydrogen peroxide (H 2 O 2 ). This chemical species is responsible for inducing leukocyte adhesion by generating PAF (platelet-activating factor) and increasing the expression of other molecules involved in this process [60,61]. ...
Citation: da Silva, P.R.; Apolinário, N.d.M.; Silva, S.Â.S.d.; Araruna, M.E.C.; Costa, T.B.; e Silva, Y.M.S.d.M.; da Silva, T.G.; de Moura, R.O.; dos Santos, V.L. Anti-Inflammatory Activity of N-(3-(1H-indol-3-yl)benzylidene)-2-cyanoacetohydrazide Derivative via sGC-NO/Cytokine Pathway. Abstract: The N-acylhydrazone function has been reported as a pharmacophore group of molecules with diverse pharmacological activities, including anti-inflammatory effects. Therefore, this study was designed to evaluate the anti-inflammatory potential of the compound N-(3-(1H-indol-3-yl)benzylidene)-2-cyanoacetohydrazide (JR19) in vivo. The study started with the carrageenan-induced peritonitis model, followed by an investigation of leukocyte migration using the subcuta-neous air pouch test and an assessment of the antinociceptive profile using formalin-induced pain. A preliminary molecular docking study focusing on the crystallographic structures of NFκB, iNOS, and sGC was performed to determine the likely mechanism of action. The computational study revealed satisfactory interaction energies with the selected targets, and the same peritonitis model was used to validate the involvement of the nitric oxide pathway and cytokine expression in the peritoneal exudate of mice pretreated with L-NAME or methylene blue. In the peritonitis assay, JR19 (10 and 20 mg/kg) reduced leukocyte migration by 59% and 52%, respectively, compared to the vehicle group, with the 10 mg/kg dose used in subsequent assays. In the subcutaneous air pouch assay, the reduction in cell migration was 66%, and the response to intraplantar formalin was reduced by 39%, particularly during the inflammatory phase, suggesting that the compound lacks central analgesic activity. In addition, a reversal of the anti-inflammatory effect was observed in mice pretreated with L-NAME or methylene blue, indicating the involvement of iNOS and sGC in the anti-inflammatory response of JR19. The compound effectively and significantly decreased the levels of IL-6, TNF-α, IL-17, and IFN-γ, and this effect was reversed in animals pretreated with L-NAME, supporting a NO-dependent anti-inflammatory effect. In contrast, pretreatment with methylene blue only reversed the reduction in TNF-α levels. Therefore, these results demonstrate the pharmacological potential of the novel N-acylhydrazone derivative, which acts through the nitric oxide pathway and cytokine signaling, making it a strong candidate as an anti-inflammatory and immunomodulatory agent.
... During sepsis, increased amount of NO lowers the neutrophil migration by inhibiting the process of rolling and adherence to the endothelium of blood vessels. 78 On the other hand, NO is also reported to increase IL8 amounts, a potent chemoattractant and an activator of neutrophils. 79 These results illustrate that the functions of NOS2 may vary depending on the cellular location, temporal expression and type of inflammatory responses. ...
... The injection of carrageenan into the subcutaneous air pouch induces an inflammatory process with characteristics and development similar to that observed in rheumatoid arthritis, with polymorphonuclear infiltration and the release of pro-inflammatory mediators [23].In the initial phase (2-4 hs) after the carrageenan injection, an intense process of leukocyte rolling and cell adhesion occurs. Six hours after the injection, there is a large influx of neutrophils and the release of chemotactic agents at the inflammatory site [24]. After 24 h cellular influx, exudate formation and the production of inflammatory mediators (i.e., NO, PGs, leukotrienes, cytokines)are augmented [25][26][27][28][29]. ...
Background:
N-octadecanoyl-5-hydroxytryptamide (C18-5HT) is an amide that can be obtained by the coupling of serotonin and octadecanoic acid. This study aims to characterize the in vivo and in vitro anti-inflammatory activity of C18-5HT.
Methods:
A subcutaneous air pouch model (SAP) was used. The exudates were collected from SAP after carrageenan injection to assess cell migration and inflammatory mediators production. RAW 264.7 cells were used for in vitro assays.
Results:
C18-5HT significantly inhibited leukocyte migration into the SAP as well as nitric oxide (NO) and cytokines production and protein extravasation. We also observed an reduction in some cytokines and an increase in IL-10 production. Assays conducted with RAW 264.7 cells indicated that C18-5HT inhibited NO and cytokine produced.
Conclusions:
Taken together, our data suggest that C18-5HT presents a significant effect in different cell types (leukocytes collected from exudate, mainly polumorphonuclear leukocytes and cell culture macrophages) and is a promising compound for further studies for the development of a new anti-inflammatory drug.
... This effect was dose-dependent, and treatment with HO-1 inhibitors showed the opposite effect. Delving into potential mechanisms, the authors identified that CO dependent effects appear to be mediated through soluble guanylate cyclase, while HO-1 activity may mediate the inhibition of neutrophil trafficking observed after treatment with a nitric oxide donor [111,112]. Interestingly, these findings support the hypothesis that HO-1 maintains soluble guanylate cyclase in a reduced state capable of responding to NO [113]. Thus, there appears to be a role for HO-1 in modulating the trafficking of neutrophils to inflammatory sites. ...
Excessive inflammation and tissue damage are pathological hallmarks of chronic pulmonary tuberculosis (TB). Despite decades of research, host regulation of these clinical consequences is poorly understood. A sustained effort has been made to understand the contribution of heme oxygenase-1 (HO-1) to this process. HO-1 is an essential cytoprotective enzyme in the host that controls inflammation and oxidative stress in many pathological conditions. While HO-1 levels are upregulated in animals and patients infected with Mycobacterium tuberculosis (Mtb), how it regulates host responses and disease pathology during TB remains unclear. This lack of clarity is due in part to contradictory studies arguing that HO-1 induction contributes to both host resistance as well as disease progression. In this review, we discuss these conflicting studies and the role of HO-1 in modulating myeloid cell functions during Mtb disease progression. We argue that HO-1 is a promising target for host-directed therapy to improve TB immunopathology.
... Given that one of the reasons for the modulated leukocyte profiles is likely initiated by the direct contact between patient blood and the non-endothelial surfaces of ECMO circuits, components with the capacity to reduce leukocyte activation and adhesion, or to boost pathogenic function, may be beneficial. Nitric oxide is an element capable of enhancing the antiinflammatory capacity of the immune system as well as reducing cellular activation and adhesion (112)(113)(114). An early ex vivo ECMO study has also highlighted the potential importance of delivery methods when using supplemental gas to improve biological and clinical outcomes (e.g. ...
A plethora of leukocyte modulations have been reported in critically ill patients. Critical illnesses such as acute respiratory distress syndrome and cardiogenic shock, which potentially require extracorporeal membrane oxygenation (ECMO) support, are associated with changes in leukocyte numbers, phenotype, and functions. The changes observed in these illnesses could be compounded by exposure of blood to the non-endothelialized surfaces and non-physiological conditions of ECMO. This can result in further leukocyte activation, increased platelet-leukocyte interplay, pro-inflammatory and pro-coagulant state, alongside features of immunosuppression. However, the effects of ECMO on leukocytes, in particular their phenotypic and functional signatures, remain largely overlooked, including whether these changes have attributable mortality and morbidity. The aim of our narrative review is to highlight the importance of studying leukocyte signatures to better understand the development of complications associated with ECMO. Increased knowledge and appreciation of their probable role in ECMO-related adverse events may assist in guiding the design and establishment of targeted preventative actions.
... Cells were counted, and the image was recorded using five different fields for each animal to avoid variability due to sampling. Data were then averaged for each animal (Dal Secco et al., 2003;Freitas et al., 2006). ...
The present study examines the possible effect of the novel hybrid molecule JM-20 (3-ethoxycarbonyl-2-methyl-4-(2-nitrophenyl)-411-dihydro-1H-pyrido[2,3-b] [1,5] benzodiazepine) on pain-related behaviours in a persistent pain model (5% formalin test) and in the neutrophil migration events during the inflammatory process. It further introduces JM-20 in a chronic constriction injury (CCI) model to clarify the possible subjacent mechanisms with its consequent clinical relevance. A single administration of JM-20 (20 or 40 mg/kg, per os [p.o.]) decreased licking/biting exclusively in the tonic phase of the formalin test in a GABA/benzodiazepine (BZD) receptor antagonist flumazenil-sensitive manner. JM-20 reduced in vivo neutrophil migration, rolling and adhesion to the endothelium induced by intraperitoneal administration of carrageenan in mice. In addition, plasma extravasation and tumour necrosis factor alpha production in the peritoneal fluid were decreased. Treatment with JM-20 (20 mg/kg, p.o.) for 7 days after CCI reduced mechanical hypersensitivity in a NG-monomethyl-l-arginine (L-NMMA)/methylene blue/glibenclamide-sensitive manner. Histopathological signs of Wallerian degeneration (WD) of the sciatic nerve were also attenuated, as well as interleukin-1 beta release in the spinal cord. The nitrate/nitrite concentration was increased centrally and did not show differences at the peripheral nerve level. The findings of this study suggest JM-20 can decrease persistent pain. A transient activity of its BDZ portion on nociceptive pathways mediated by GABA/BDZ receptors in association with its anti-inflammatory properties could be at least partially involved in this effect. JM-20 decreased CCI-induced mechanical hypersensitivity via the l-arginine/nitric oxide (NO)/cyclic GMP-sensitive ATP-sensitive potassium channel pathway. Its neuroprotective ability by preventing WD could be implicated in its anti-neuropathic mechanisms.
... In addition, NO may act on the vasculature to limit immune cell infiltration from blood, for instance by influencing vessel integrity and modifying vessel leaking as well as adhesion properties. NO-dependent mechanisms that modulate leukocyte adhesion and extravasation have been already described and therefore likely involved in our model (Biffl et al., 1996;Dal Secco et al., 2006;Secco et al., 2003). ...
Recruitment of immune cells during infection is essential to fueling the immune response but can also trigger immunopathology. A critical question is how the immune system can sense inflammation levels and self-adjust accordingly to limit tissue damage while removing the pathogen. During my Ph.D. I studied the self-resolving cutaneous infection with Leishmania major parasites where tissue damage arises when inflammation is allowed to become excessive. At the site of infection, the immune reaction is driven by recruited monocyte-derived cells that represents the major population of infected cells and are also actively involved in fighting the infection. They secrete pro-inflammatory cytokines but also produce nitric oxide (NO), critical to regulate the outcome of the infection: iNOS KO mice are susceptible and do not control the parasite load, subsequently developing severe tissue damage because of excessive immune cell infiltration. My work demonstrated that monocyte-derived cells at the site of infection are regulated by NO that limits their cellular respiration, lowers their energetic resources and consequently their activity in vivo. This regulation relies on tissue-wide NO diffusion and only exists when a sufficient cell density has been reached, revealing that monocyte-derived cells are endowed with a quorum sensing mechanism that adjusts their population size and activity in time and space to avoid immunopathology.
... MaL could reduce TNF-α and iNOS gene expression and increase IL-10 (anti-inflammatory cytokine) production. TNF-α and NO play crucial roles in the late inflammatory process, stimulating the production of proinflammatory cytokines [55] and modulating leukocyteendothelial cell interaction [56]. ...
Inflammatory response occurs when tissues are injured by pathogens, trauma, toxins, or heat. Lectins are proteins that recognize and bind reversibly to glycans and glycoconjugates and can modulate inflammatory responses in in vitro and in vivo models. As such, this study aimed to evaluate the potential of an anti-inflammatory lectin isolated from Machaerium acutifolium seeds (MaL) in mice and LPS-stimulated macrophage models. The protein was solubilized in sterile saline (0.9 % NaCl) immediately before treatment of mice by intraperitoneal routes at doses of 0.02 mg/kg, 1 mg/kg and 5 mg/kg. MaL significantly decreased inflammation in the formalin test, inhibited cell migration in experimental models of carrageenan-induced peritonitis, and blocked the formation of paw edema induced by carrageenan and dextran. In vitro studies showed that MaL downregulated the proinflammatory cytokine genes inducible nitric oxide synthase (iNOS) and tumor necrosis factor-α (TNF-α), but upregulated the anti-inflammatory IL-10 gene in LPS-stimulated macrophages. Therefore, this study suggests that MaL has an anti-inflammatory effect relative to modulated levels of pro- and anti-inflammatory cytokines, indicating that MaL can be used as a potential therapeutic agent in cellular inflammatory events.
... In addition, increased generation of endothelial nitric oxide (NO) in RIPC-treated mice and humans relative to non-RIPC treated controls has been noted [51][52][53][54]. This is relevant as NO has been shown to dampen neutrophil rolling, adhesion and migration (and hence inflammation) in mice, as well as in isolated human neutrophils [55]. In summary, sterile inflammation leads to neutrophil activation, which can be induced by tissue damage and release of DAMPs. ...
Neutrophil recruitment is not only vital for host defense, but also relevant in pathological inflammatory reactions, such as sepsis. Model systems have been established to examine different steps of the leukocyte recruitment cascade in vivo and in vitro under inflammatory conditions. Recently, tissue-specific recruitment patterns have come into focus, requiring modification of formerly generalized assumptions. Here, we summarize existing models of neutrophil recruitment and highlight recent discoveries in organ-specific recruitment patterns. New techniques show that previously stated assumptions of integrin activation and tissue invasion may need revision. Similarly, neutrophil recruitment to specific organs can rely on different organ properties, adhesion molecules, and chemokines. To advance our understanding of neutrophil recruitment, organ-specific intravital microscopy methods are needed.
... NO also modulates chemotactic migration of neutrophils to inflammatory foci. Secco et al. 35 reported enhanced migration and better survival of migrated neutrophils following administration of iNOS inhibitors. An increase in reactive nitrogen intermediates (RNI) and infiltration of activated neutrophils in synovial fluid have been reported in rheumatoid arthritis. ...
Neutrophils play a key role in innate immune responses against foreign intrusion and influence the subsequent instigation of adaptive immune response. Nitric oxide (NO) synthesized by neutrophil nitric oxide synthase (NOS) profoundly modulates their diverse physiological responsibilities furthermore encompassing pathological implications. Neutrophils are the active participants in diverse inflammatory and cardiovascular disorders but neutrophil nitric oxide synthase (NOS) remains enigmatic on various aspects. This review focuses on inducible NOS (iNOS) and makes an attempt to address its potential impact in neutrophil pathophysiology, their differentiation, functionality, and survival. We described the scenario from its expressional modulation, by pro- and anti-inflammatory cytokines governing the extent and duration of neutrophil immune response, to iNOS catalysis, the intracellular compartmentalization, and protein-protein interactions determining its microenvironment, activity and its contribution as a potential signaling protein apart from its role as signal transducer. Further, the relevance of investigating the unexplored facets of iNOS biology in neutrophils and possible prototypes of iNOS regulation is also exemplified in related cellular systems.
... NO also modulates chemotactic migration of neutrophils to inflammatory foci. Secco et al. 35 reported enhanced migration and better survival of migrated neutrophils following administration of iNOS inhibitors. An increase in reactive nitrogen intermediates (RNI) and infiltration of activated neutrophils in synovial fluid have been reported in rheumatoid arthritis. ...
Neutrophils play a key role in innate immune responses against foreign intrusion and influence the subsequent instigation of adaptive immune response. Nitric oxide (NO) synthesized by neutrophil nitric oxide synthase (NOS) profoundly modulates their diverse physiological responsibilities furthermore encompassing pathological implications. Neutrophils are the active participants in diverse inflammatory and cardiovascular disorders but neutrophil nitric oxide synthase (NOS) remains enigmatic on various aspects. This review focuses on inducible NOS (iNOS) and makes an attempt to address its potential impact in neutrophil pathophysiology, their differentiation, functionality, and survival. We described the scenario from its expressional modulation, by pro- and anti-inflammatory cytokines governing the extent and duration of neutrophil immune response, to iNOS catalysis, the intracellular compartmentalization, and protein–protein interactions determining its microenvironment, activity and its contribution as a potential signaling protein apart from its role as signal transducer. Further, the relevance of investigating the unexplored facets of iNOS biology in neutrophils and possible prototypes of iNOS regulation is also exemplified in related cellular systems.
Review on expressional modulation, inducible catalysis, intracellular compartmentalization and protein-protein interactions of neutrophil NOS determining microenvironment, activity and its contribution as a potential signaling protein.
... TRPV4-mediated rise in Ca 2+ induces the activation of nitric oxide synthases (eNOS and iNOS) constitutively expressed in epithelial cells, increasing nitric oxide (NO) production in the luminal compartment, resulting in a bactericidal effect (Figure 3). On the other hand, NO would presumably diffuse to the basal face of the epithelial layer, inducing bronchodilation through its relaxing action on airway smooth muscle [142,143], and decreasing leukocyte adhesion to endothelial cells [144][145][146][147][148] through down-regulation of cell adhesion molecules [149][150][151]. Similar to other TRPV4 agonists, such as heat and GSK1016790A [140], LPS-induced TRPV4 activation increases the CBF, suggesting that TRPV4 could also enhance the clearance of pathogens from the airways. ...
The cellular and systemic effects induced by bacterial lipopolysaccharides (LPS) have been solely attributed to the activation of the Toll-like receptor 4 (TLR4) signalling cascade. However, recent studies have shown that LPS activates several members of the Transient Receptor Potential (TRP) family of cation channels. Indeed, LPS induces activation of the broadly-tuned chemosensor TRPA1 in sensory neurons in a TLR4-independent manner, and genetic ablation of this channel reduced mouse pain and inflammatory responses triggered by LPS and the gustatory-mediated avoidance to LPS in fruit flies. LPS was also shown to activate TRPV4 channels in airway epithelial cells, an effect leading to an immediate production of bactericidal nitric oxide and to an increase in ciliary beat frequency. In this review, we discuss the role of TRP channels as sensors of bacterial endotoxins, and therefore, as crucial players in the timely detection of invading gram-negative bacteria. Key Contribution: Here we review the current knowledge about the role of Transient Receptor Potential cation channels in the detection of bacterial lipopolysaccharides.
... Our data suggest that CCR2-expressing IMs or IM-derived DCs, which express CD11c, could be an important component of the NO-producing cells observed by Davis et al. NO has been shown to inhibit rolling and adhesion of neutrophils and induces the apoptosis of neutrophils (39). Additionally, Ly6C ϩ IMs suppress the activity of neutrophils through the production of the lipid mediator prostaglandin E 2 (PGE 2 ) in response to a broad range of bacterium-derived ligands, including those recognized by Toll-like receptor 2 (TLR2), TLR4, TLR5, and TLR9 (37). ...
Murine Ly6C hi inflammatory monocytes (IMs) require CCR2 to leave the bone marrow and enter mesenteric lymph nodes (MLNs) and other organs in response to Yersinia pseudotuberculosis infection. We are investigating how IMs, which can differentiate into CD11c ⁺ dendritic cells (DCs), contribute to innate and adaptive immunity to Y. pseudotuberculosis . Previously we obtained evidence that IMs are important for a dominant CD8 ⁺ T cell response to the epitope YopE 69-77 and host survival using intravenous infections with attenuated Y. pseudotuberculosis . Here, we challenged CCR2 +/+ or CCR2 -/- mice orally with wild-type Y. pseudotuberculosis to investigate how IMs contribute to immune responses during intestinal infection. Unexpectedly, CCR2 -/- mice did not have reduced survival, but retained body weight better and their MLNs cleared Y. pseudotuberculosis faster with reduced lymphadenopathy as compared to controls. Enhanced bacterial clearance in CCR2 -/- mice correlated with reduced IMs in spleens and increased neutrophils in livers. In situ imaging of MLNs and spleens from CCR2-GFP mice showed that GFP ⁺ IMs accumulate at the periphery of neutrophil-rich Yersinia- containing pyogranulomas. GFP ⁺ IMs co-localized with CD11c ⁺ cells and YopE 69-77 -specific CD8 ⁺ T cells in MLN, suggesting that IM-derived DCs prime adaptive responses in Yersinia pyogranulomas. Consistently, CCR2 -/- mice had reduced numbers of splenic DCs, YopE 69-77 -specific CD8 ⁺ T cells, CD4 ⁺ T cells and B cells in organs and lower levels of serum antibodies to Y. pseudotuberculosis antigens. Our data suggest that IMs differentiate into DCs in MLN pyogranulomas and direct adaptive responses in T cells at the expense of innate immunity during oral Y. pseudotuberculosis infection.
... The last inflammatory parameter reduced by the coadministration of propranolol with carrageenan in the TMJ was the neutrophil migration, a key event related to tissue injury (Bradley et al., 1982;Dal Secco et al., 2003). This finding suggests that the activation of the b-adrenoceptors by sympathomimetic amines released during TMJ inflammation is critical for the neutrophils recruitment to the injured joint. ...
Background:
β-Blockers reduce temporomandibular joint (TMJ) pain. We asked whether they also reduce TMJ inflammation and, if so, whether this anti-inflammatory effect contributes to its analgesic action.
Methods:
We measured many parameters of the inflammatory response after co-administration of the β-blocker propranolol with the inflammatory agent carrageenan in the TMJ of female rats. We also hypothesized that the activation of β-adrenoceptors in the TMJ induces nociception mediated, at least in part, by the inflammatory response. To test this hypothesis, we examined the nociceptive response induced by the activation of the β-adrenoceptors in the TMJ in female rats pretreated with thalidomide and fucoidan.
Results:
We found that the co-administration of propranolol with carrageenan in the TMJ of female rats significantly reduced several parameters of the inflammatory response induced by carrageenan such as plasma extravasation, neutrophil migration and the release of the pro-inflammatory cytokines TNF-α, IL-1β and CINC-1. Furthermore, the injection of the β-adrenergic receptor agonist isoproterenol in the TMJ induced nociception that was significantly reduced by thalidomide, fucoidan and by the co-administration of propranolol but not of the α-adrenergic receptor antagonist phentolamine.
Conclusions:
Propranolol has anti-inflammatory effects that contribute to its antinociceptive action in the TMJ of females.
Significance:
β-Blockers have an anti-inflammatory effect on temporomandibular joint (TMJ) that contributes to its analgesic effect. The results of this work suggest that β-blockers can be used to treat the painful conditions of TMJ, especially when they are associated with an inflammatory process.
... Furthermore, trimucrin significantly blocked the expression of nuclear factor kappaB (NF-κB)-related downstream inducible enzymes, such as inducible nitric oxide synthase (iNOS) and COX-2. NO has vasodilative effects, antiplatelet activity and anti-inflammatory activity, all of which are beneficial for cardiac-related improvement after myocardial I-R injury (Dal Secco et al., 2003;Park et al., 1992). Trimucrin therefore not only exerts anti-inflammatory effects via the inhibition of LPS-TLR4 ligation-mediated release of proinflammatory mediators through blockade of monocyte/macrophage αvβ3 integrins, but also inhibits platelet aggregation through blockade of integrin αIIbβ3 (GPIIb-IIIa) (Hung et al., 2016). ...
Trimucrin, a novel small-mass Arg-Gly-Asp (RGD)-containing disintegrin, has been demonstrated to possess anti-platelet and anti-inflammatory effect through blockade of platelet αIIbβ3 and phagocyte αvβ3 integrin. In this study, we found that the platelet-rich plasma prepared from trimucrin-treated rats platelet aggregation was diminished in response to adenosine diphosphate (ADP). We tried to determine whether trimucrin is cardioprotective in rats subjected to myocardial ischemia-reperfusion (I-R) injury. The left anterior descending coronary artery of anesthetized rats was subjected to 1 h occlusion and 3 h reperfusion. The animals received intravenous trimucrin or saline, and the severities of I-R-induced arrhythmia and infarction were compared. Trimucrin significantly reduced I-R-induced arrhythmias and reduced mortality, as well as infarct volume, troponin-I levels, creatine kinase, and lactate dehydrogenase activity in carotid blood compared with vehicle-treated animals during the same period. Trimucrin also improved cardiac function and survival rates after I-R injury. In addition, trimucrin concentration-dependently inhibited platelet adhesion on collagen- and fibrinogen-coated surfaces without affecting platelet counts. Trimucrin also significantly reduced neutrophil infiltration into heart tissues after I-R compared with controls. Furthermore, trimucrin treatment caused significant downregulation of Bax, Caspase-3 apoptotic proteins and upregulation of anti-apoptotic Bcl-2 protein. These results demonstrate that trimucrin exerts cardioprotective property against myocardial I-R injury mediated through antiplatele, anti-inflammatory, anti-apoptotic mechanism, as well as improvements in cardiac function.
... (3,12,13) Nitric oxide, not only participates in the non-specific defense of an organism, but also plays an important role in the regulation of cell death by apoptosis and necrosis. (18) It was shown that newborns with moderate pneumonia have an increased level of neutrophil apoptosis, which acts as a compensatory mechanism to diminish inflammation. Normally, after performing their protective antibacterial function, neutrophils undergo apoptosis, which preserves the integrity of the membranes and prevents the uncontrolled release of toxic contents of these cells in the environment. ...
Introduction. Nitric oxide (NO) is an important diagnostic marker and mediator in the inflammatory process, which plays a key role in the mechanism of programmed cell death, thus, forming the basis of many pathological diseases. Methods. The study involved 73 newborns with pneumonia (moderate severity in 44 neonates (group 1), severe pneumonia in 29 (group 2)). The intensity of neutrophil apoptosis and necrosis was determined by flow cytometry, whereas nitric oxide metabolites were measured by spectrophotometry. Results. The level of nitric oxide metabolites (NO2+NO3) in newborns with pneumonia was higher than in healthy children (16.93 (15.82; 17.79) μmol/ml) and correlated with disease severity (in group 1 – 22.65 (21.42; 23.40) μmol/ml in group 2 – 26.82 (25.81; 27.91) μmol/ml). The level of NO3 increased moderately, while NO2 generation was more intense, exceeding control indexes in both groups (рc- 1<0.001; рc-2<0.001; р1-2<0.001). The occurrence of intensive neutrophil apoptosis was revealed in newborns with pneumonia of moderate severity (рc- 1<0.001), while necrosis prevailed in severe pneumonia (рc-2<0.001). Inverse correlation (R=-0.63; р<0.05) was found between the level of nitric oxide metabolites and neutrophil apoptosis; and direct correlation (R=0.68; р<0.05) was revealed between NO metabolites and neutrophil necrosis indices. Conclusions. Increased generation of nitric oxide metabolites, that directly correlated with disease severity in newborns with pneumonia, was found. NO2 has multidirectional effects on neutrophil apoptosis and necrosis, leading to toxic accumulation of neutrophils in the organism, thus enhancing the inflammatory and intoxication process that impact disease severity.
... Neutrophils obtained from inflammatory exudate of carrageenin-induced experimental pleurisy in rats, treated with ovine PRL or from rats with hyperprolactinemia (HPRL), produced higher amounts of nitric oxide (NO) and TNF-α [12]. NO down-modulates neutrophils migration as a consequence of decreased rolling and adhesion on endothelium and induction of apoptosis in migrating neutrophils [13]. Monocyte and macrophage migration to sites of inflammation is facilitated by intercellular adhesion molecule 1 (ICAM-1), which is stimulated by PRL [14]. ...
Prolactin, a 23-kDa peptide hormone, is produced by the anterior pituitary gland and extrapituitary sites including the immune cells. Prolactin (PRL) participates in innate and adaptive immune response. PRL stimulates the immune cells by binding to receptor (PRL-R). Binding of PRL to its receptor activates the Janus kinase-signal transducer (JAK-STAT). Activation of these cascades results in endpoints such as immunoestimulator and immunosupressor action. Prolactin belongs to the network of immune-neuroendocrine interaction. Hyperprolactinemia has been found in patients with systemic lupus erythematosus (SLE), and new evidence has confirmed a significant correlation between serum PRL levels and disease activity. PRL participates in activation of SLE during pregnancy and in pathogenesis of lupus nephritis, neuropsychiatric, serosal, hematologic, articular, and cutaneous involvement. Hyperprolactinemia was associated with increase IgG concentrations, anti-DNA antibodies, immune complex, glomerulonephritis, and accelerated mortality in murine lupus. Bromocriptine, a dopamine analog that suppresses PRL secretion, was associated with decreased lupus activity, prolonged lifespan, and restoration of immune competence in experimental model. In clinical trials, bromocriptine and derivative drugs showed beneficial therapeutic effect in treating human lupus, including pregnancy. Taken together, clinical and experimental results leave little doubt that PRL indeed contributes to the pathogenesis and clinical expression of SLE.
... eNOS-derived ROS has been linked to a variety of inflammatory diseases like acute lung injury 64 , diabetes mellitus 65 and Ang II-induced hypertension 66 . NO, a pleotropic signalling molecule has been shown to attenuate neutrophil rolling and adhesion 67 . Dal Secco et al. 68 have performed experiments in LPS-treated iNOS -/mice and found increased neutrophil migration compared with the wild-type mice. ...
Free radicals, including reactive oxygen species (ROS) as well as reactive nitrogen species (RNS), and inflammation increase with advancing age. Evidence suggests that oxidative stress and inflammation both lead to impaired vascular function. There is also evidence to suggest that inflammation may cause an increase in radical production leading to enhanced oxidative/nitrosative stress. In addition, higher concentration of free radicals also modulates inflammation by increasing the expression of inflammatory proteins, including cytokines. Although ROS/RNS are predominantly implicated in causing cell damage, they also play a major physiological role in several aspects of intracellular signalling and regulation. ROS/RNS are known to play a dual role in biological systems since they can be either harmful or beneficial to living systems.
... It has been postulated that, in addition to pro-inflammatory effects, iNOS, and to some extent also other NOS isoforms (nNOS and eNOS), limits immune responses and has anti-inflammatory functions [11]. These include, for example, the eNOS-, iNOS-, or eNOSdependent inhibition of leukocyte adhesion and transendothelial migration [38,39]. NO may also exert antiinflammatory effects by affecting polarization of macrophages due to reduced proinflammatory M1 and increased antiinflammatory M2 phenotypic properties [15]. ...
Nitric oxide (NO) is a gaseous free radical molecule involved in several biological processes related to inflammation, tissue damage, and infections. Based on reports that NO inhibits migration of granulocytes and monocytes, we became interested in the role of inducible NO synthetase (iNOS) in pharmacological mobilization of hematopoietic stem/progenitor cells (HSPCs) from bone marrow (BM) into peripheral blood (PB). To address the role of NO in HSPC trafficking, we upregulated or downregulated iNOS expression in hematopoietic cell lines. Next, we performed mobilization studies in iNOS−/− mice and evaluated engraftment of iNOS−/− HSPCs in wild type (control) animals. Our results indicate that iNOS is a novel negative regulator of hematopoietic cell migration and prevents egress of HSPCs into PB during mobilization. At the molecular level, downregulation of iNOS resulted in downregulation of heme oxygenase 1 (HO-1), and, conversely, upregulation of iNOS enhanced HO-1 activity. Since HO-1 is a negative regulator of cell migration, the inhibitory effects of iNOS identified by us can be at least partially explained by its enhancing the HO-1 level in BM cells.
Electronic supplementary material
The online version of this article (doi:10.1007/s12015-016-9693-1) contains supplementary material, which is available to authorized users.
... Cell death is induced following enhanced levels of NO from inducible nitric oxide synthase (iNOS) during inflammation, ischaemia/reperfusion or by NO donors such as DETA-NO, sodium nitroprusside and S-nitroso-N-acetyl-penicillamine. [8][9][10] Our previous work has demonstrated a dose-dependent pro-and anti-apoptotic effect of NO on promyelocytic cell line HL-60. 11 Two isoforms of NOS-iNOS and nNOS are constitutively expressed in human and mice PMNs 12 but their regulation and interplay in neutrophil apoptosis is still enigmatic. ...
Neutrophils play an indispensable role in killing of invading pathogens by enhancing reactive oxygen species (ROS) and NO generation, and subsequently undergoing apoptosis. Unlike ROS/NOX2, role of NO/NOS still remains undefined in the apoptosis of neutrophils (PMNs) and the present study attempts to decipher the importance of NO/NOS in the neutrophil apoptosis. Prolonged treatment of human PMNs or mice bone marrow derived neutrophils (BMDN) with NO led to enhanced ROS generation, caspase-8/caspase-3 cleavage, reduced mitochondrial membrane potential and finally cellular apoptosis. NO-induced ROS generation led to caspase-8 deglutathionylation and activation, which subsequently activated mitochondrial death pathway via BID (Bcl-2 family protein) cleavage. NO-mediated augmentation of caspase-8 and BID cleavage was significantly prevented in BMDN from neutrophil cytosolic factor-1 (NCF-1) knockout (KO) mice, implying the involvement of NOX2 in NO-induced apoptosis of PMNs. Furthermore, ROS, NO generation and inducible nitric oxide synthase (iNOS) expression were enhanced in a time-dependent manner in human PMNs and mice BMDN undergoing spontaneous apoptosis. Pharmacological and genetic ablation of iNOS in human PMNs and mice BMDN significantly reduced the levels of apoptosis. Impaired apoptosis of BMDN from iNOS KO mice was due to reduced caspase-8 activity which subsequently prevented caspase-3 and -9 activation. Altogether, our results suggest a crucial role of NO/iNOS in neutrophil apoptosis via enhanced ROS generation and caspase-8 mediated activation of mitochondrial death pathway.
... Endotoxemia is well associated with the increased release of neutrophils from the bone marrow into the circulation; however, extensive evidence shows that it also inhibits their migration out of the circulation (Wagner and Roth, 1999). The direct effects of LPS as well as the indirect effects through mediators such as NO, discussed previously, result in the inhibition of neutrophil migration (Wagner and Roth, 1999;Dal Secco et al., 2003). These effects may delay the response of neutrophils to infection of the mammary. ...
The dairy industry continues to suffer severe economic losses due to the increased disease incidence cows experience during the transition period. It has long been the classical view that the major contributing factor to the development of these periparturient diseases is the considerable increase in nutritional demands for milk production. This classical view, however, fails to account for the substantial correlation between both metabolic and infectious diseases and the detrimental effects that can occur with the provision of high-energy diets to support these nutritional demands. Currently, increasing evidence implicates bacterial endotoxins in the etiopathology of most periparturient diseases. Bacterial endotoxins are components of the outer cell wall of gram-negative and gram-positive bacteria that are highly immunostimulatory and can trigger proinflammatory immune responses. The ability of endotoxins to translocate from the mucosal tissues, including the gastrointestinal tract, mammary gland, and uterus, into the systemic circulation has been observed. Once they have entered the circulation, endotoxins potentially contribute to disease either directly, through eliciting an inflammatory response, or indirectly through other factors such as the overreaction of the natural protective mechanisms of the host. Although the evidence implicating a role of endotoxins in the pathogenesis of transition diseases continues to grow, our current knowledge of the host response to mucosal endotoxin exposure and pathogenic mechanisms remain largely unknown. Developing our understanding of the connection between endotoxemia and dairy cattle disease holds significant potential for the future development of preventative measures that could benefit the productivity of the dairy industry as well as animal welfare.
... The total leukocytes counts were performed with a Newbauer chamber, and differential cell counts (100 cells total) were carried out on cytocentrifuge slides (Cytospin 3; Shandon Southern Products, Astmoore, UK) stained with panotic solutions (Laborclin, Pinhais, Paraná, Brazil). The results are the number of total leukocytes, neutrophils or mononuclear cells x 10 6 [17,18,24]. ...
Pimaradienoic acid (PA; ent-pimara-8(14),15-dien-19-oic acid) is a pimarane diterpene found in plants such as Vigueira arenaria Baker (Asteraceae) in the Brazilian savannas. Although there is evidence on the analgesic and in vitro inhibition of inflammatory signaling pathways, and paw edema by PA, its anti-inflammatory effect deserves further investigation. Thus, the objective of present study was to investigate the anti-inflammatory effect of PA in carrageenan-induced peritoneal and paw inflammation in mice. Firstly, we assessed the effect of PA in carrageenan-induced leukocyte recruitment in the peritoneal cavity and paw edema and myeloperoxidase activity. Next, we investigated the mechanisms involved in the anti-inflammatory effect of PA. The effect of PA on carrageenan-induced oxidative stress in the paw skin and peritoneal cavity was assessed. We also tested the effect of PA on nitric oxide, superoxide anion, and inflammatory cytokine production in the peritoneal cavity. PA inhibited carrageenan-induced recruitment of total leukocytes and neutrophils to the peritoneal cavity in a dose-dependent manner. PA also inhibited carrageenan-induced paw edema and myeloperoxidase activity in the paw skin. The anti-inflammatory mechanism of PA depended on maintaining paw skin antioxidant activity as observed by the levels of reduced glutathione, ability to scavenge the ABTS cation and reduce iron as well as by the inhibition of superoxide anion and nitric oxide production in the peritoneal cavity. Furthermore, PA inhibited carrageenan-induced peritoneal production of inflammatory cytokines TNF-α and IL-1β. PA presents prominent anti-inflammatory effect in carrageenan-induced inflammation by reducing oxidative stress, nitric oxide, and cytokine production. Therefore, it seems to be a promising anti-inflammatory molecule that merits further investigation.
... The venular area in which the adhesion process was determined varied from 350 to 450 μm 2 , and the results were expressed as the number of adherent leukocyte per 100 μm 2 of venule. The time points selected to determine rolling and adhesion (3 h) processes were based on previous studies, which observed that these processes peak at these times after inflammatory stimuli injection 15,16 . ...
Mikania lindleyana is a plant widely distributed in Brazilian Amazonia, popularly known as “sucuriju” and largely used in folk medicine to treat inflammation, chronic ulcers and pain. In the present study, we identified the secondary metabolites of the aqueous extract of M. lindleyana (AEML) and investigate its effects on several models of inflammation and nociception in rodents. Phytochemical screening of the AEML showed the presence of saponins, proteins, amino acids, phenols, tannins, organic acids and flavonoids. Oral pretreatment of mice with AEML significantly (P < 0.05) reduced the abdominal constrictions evoked by acetic acid injection and the licking time in both first and second phases in the formalin test but has no significant effect on hot plate test. In rats, AEML inhibited the edema formation induced by carrageenan and croton oil while has no significant effects on the edema induced by dextran. AEML inhibited the carrageenaninduced neutrophil migration as well as the rolling and the adhesion of leukocytes. The present study shows for the first time that AEML displays antinociceptive and anti-inflammatory activities which could be attributed respectively to a possible opioid mechanism and to an inhibition of the adhesion molecules by interference on pro-inflammatory cytokines. These results support the widespread use of M. lindleyana in popular medicine to treat inflammation and pain.
... In several works (Hickey, 2001;Spiecker et al., 1998;Dal Secco et al., 2003) it was observed that NO donors decrease leukocytes rolling and adhesion to endothelial cells, as wells as neutrophil transmigration to inflammatory sites. Moretti et al. (2012) verified that NO deficiency results in a prolonged inflammatory reaction in model the mesh implant. ...
Schistosomiasis, an immune disease, remains a major public health problem in endemic area. To determine the influence of Nitric Oxide (NO) on this disease, we tested two compounds (Trans-[Ru(bpy)2(NO)SO3](PF6)-PF6and Na2[Fe(CN)5(NO)]-SNP, which releases NO when activated by biological reducing agents, in BALB/c mice infected subcutaneously by Schistosoma BH strains. The parasitic activity of NO-donors was evaluated in this model by measuring the immune cellular response in liver with: Cytokines levels; histopathological characteristics and the number of the granulomatous lesions; and NO levels. We found that NO-donors treated mice were more resistant to infection, since they exhibited higher survival. Furthermore, we observed in histopathological analysis a decreased influx of inflammatory cells in the hepatic tissue of mice treated with both donors. The parasite counting (estimated as eggs and worms number) was also minor in treated mice. Moreover, decreased levels of IL-10 were detected in the liver of infected mice treated with SNP. The animals treated with PF6showed high plasmatic NO levels at 45 days after infection. Altogether, these data suggest that NO is a pivotal factor of resistance during schistossomiasis by controlling parasites proliferation, influencing cytokine production and consequently modulating the development of inflammatory response.
... NO protects against cellular damage by reactive oxygen species and stimulates regeneration of the skin. [14][15][16][17][18][19][20][21] Medical needling is performed with an automated device that penetrates the skin with fine needles, thereby creating channels for improved absorption of topically applied compounds through the top layer of the skin. This is important as large (>20 kDa) and hydrophilic (>500 Da) molecules penetrate poorly into the hydrophobic stratum corneum. ...
The use of platelet-rich plasma and growth factors is emerging as an anti-ageing regimen for the skin. We tested the safety and efficacy of 3D-MatrixLift®, a new treatment regimen for skin rejuvenation that combines medical needling and the application of a stem cell and growth factor-rich solution with irradiation by LED light. A total of 15 participants were enrolled in a single-centre, prospective pilot study. The elasticity parameters of the skin increased significantly after five rounds of treatment, with no signs of adverse effects. 3D-MatrixLift improves the elasticity of the skin and can be used safely in combination with medical needling for skin rejuvenation.
... CO was also reported to have anti-inflammatory effects during inflammation [15,31]. Evidence showed that CO stimulated soluble guanylate cyclase activity and increases cellular levels of cyclic GMP, which are associated with an inhibition of nociceptor hypersensitivity [32]. Therefore, the intraperitoneal administration of CO-RM-2 as well as the HO-1 inducer CoPP could both increase endogenous CO content, thus, significantly reduce the mechanical allodynia and thermal hypersensitivity. ...
Objective:
Heme oxygenase-1 (HO-1) exerts protective effects against ischemia and inflammation in the central nervous system. However, its role in neuropathic pain is still unclear. This study was undertaken to explore the distribution and possible mechanism of HO-1 in a mouse model of peripheral nerve injury.
Design and methods:
The experiment was conducted using a mouse model of L5 spinal nerve ligation (SNL). Mice received repeated intraperitoneal injection of Carbon monoxide-releasing molecule-2 (CO-RM-2), HO-1 inducer cobalt protoporphyrin IX (CoPP) or single intraspinal injection of lentivirus (LV) over-expressing HO-1. The behavior analyses were conducted. The distribution and expression of HO-1 in the spinal cord were analyzed.
Results:
HO-1 but not HO-2 was upregulated in spinal cord microglia cells after nerve injury, and the repeated intraperitoneal administration of CORM-2 (10 mg/kg/d) or CoPP (5 mg/kg/d) both significantly reduced the mechanical allodynia and thermal hyperalgesia induced by SNL (P < 0.01). Intraspinal injection of LV-HO-1 persistently suppresses SNL-induced neuropathic pain (P < 0.01 or P < 0.05), significantly induced the spinal HO-1 protein content (P < 0.01) and inhibited the microglia activation (P < 0.01) 7 days after SNL.
Conclusion:
HO-1 upregulation could elicit potent analgesic effects against neuropathic pain, which might partly be attributed to inhibition of spinal microglia activation. HO-1 signaling pathway may present a novel strategy for the treatment of neuropathic pain.
Forms of extracorporeal circulation are increasingly used to support critically ill patients. Extracorporeal membrane oxygenation (ECMO) and renal replacement therapy (RRT) are two of the most widely used and are employed as bridges to recovery or as definitive therapy. While both are life-saving, they also have limitations. Of note, ECMO and RRT have been associated with dysregulation of the immune system. Both are known to trigger a pro-inflammatory response; alter leukocyte numbers, phenotype, and function; increase platelet/endothelial-leukocyte interplay; and lead to a pro-coagulant state, alongside some features of immunosuppression. These changes may be exacerbated by blood contacting non-endothelialised surfaces as well as the non-physiological conditions associated with these modalities. However, teasing out the immune effects caused by these modalities from the actual underlying disease burden is complex. This chapter describes and highlights the immune responses related to extracorporeal circulations. Additionally, it outlines the factors contributing to ECMO- and RRT-related immune dysregulation, and the role these changes play in device complications. An increased understanding and acknowledgement of the patient-pump and -oxygenator interphase may assist in guiding the design of targeted preventative actions in the future.
Sepsis-associated acute kidney injury (S-AKI) is a frequent complication in critically ill patients, which is often initiated by glomerular endothelial cell dysfunction. Although transient receptor vanilloid subtype 4 (TRPV4) ion channels are known to be permeable to Ca2+ and widely expressed in kidneys, the role of TRPV4 on glomerular endothelial inflammation in sepsis remains elusive. In the present study, we found that TRPV4 expression in mouse glomerular endothelial cells (MGECs) increased after lipopolysaccharide (LPS) stimulation or cecal ligation and puncture (CLP) challenge, which increased intracellular Ca2+ in MGECs. Furthermore, the inhibition or knockdown of TRPV4 suppressed LPS-induced phosphorylation and translocation of NF-κB and IRF-3 in MGECs. Clamping intracellular Ca2+ mimicked LPS-induced responses observed in the absence of TRPV4. In vivo experiments showed that the pharmacological blockade or knockdown of TRPV4 reduced glomerular endothelial inflammatory responses, increased survival rate and improved renal function in CLP-induced sepsis without altering renal cortical blood perfusion. Taken together, these results suggest that TRPV4 promotes glomerular endothelial inflammation in S-AKI, and its inhibition or knockdown alleviates glomerular endothelial inflammation by reducing Ca2+ overload and NF-κB/IRF-3 activation. These findings provide insights that may aid in the development of novel pharmacological strategies for the treatment of S-AKI.
Le sepsis concerne des millions de patients dans le monde et engendre une mortalité importante. La pénétration de l’agent pathogène est responsable d’un ensemble de réactions de l’hôte, avec une altération de grandes fonctions de l’organisme pouvant conduire à la défaillance d’organe. L’endothélium est un élément majeur du système cardiovasculaire assurant notamment la perfusion tissulaire et la régulation des flux hydrosodés entre les secteurs vasculaire et interstitiel. Au cours du sepsis une altération de cet endothélium peut survenir et induire un défaut de microcirculation et une fuite capillaire par altération de la fonction barrière, conduisant à l’hypoxie cellulaire et au syndrome oedémateux.Ce travail de doctorat se divise en une approche clinique évaluant l’effet du syndrome oedémateux sur le pronostic des patients de réanimation, et une approche expérimentale évaluant les effets de nouvelles thérapies sur cet aspect de la fonction endothéliale.Dans la première partie, nous avons identifié que la balance hydrique positive (entrées – sorties de fluides) était associée à la mortalité de patients critiques admis pour assistance circulatoire par ExtraCorporeal Membrane Oxygenation (ECMO) veino-artérielle dans le cadre d’un choc cardiogénique réfractaire. Ceci confirmait des résultats précédents chez les patients septiques et illustrait que ce phénomène concernait également des patients critiques non septiques.Dans la deuxième partie, nous avons évalué dans un premier temps l’effet d’un anticorps anti-VEGF, le bevacizumab, dans un modèle de sepsis par ponction ligature caecale chez la souris. Le VEGF est impliqué dans les phénomènes de fuite paracellulaire et des auteurs avaient préalablement montré un effet bénéfique du bevacizumab sur la fuite capillaire dans le sepsis. Nous n’avons retrouvé aucune modification de la mortalité, malgré un effectif conséquent (n>50), ne justifiant pas la poursuite des investigations. Nous avons ensuite évalué les effets du lactate de sodium molaire (LSM), fluide hypertonique et énergétique ayant suggéré récemment un effet bénéfique sur la macro et la microcirculation dans un modèle endotoxinique, avec notamment une réduction de la balance hydrique. A cette fin nous avons réalisé un modèle de sepsis chez le rat. L’administration continue de LSM induisait une amélioration de la perfusion tissulaire intestinale (microcirculation) et réduisait la fuite capillaire dans l’intestin, le poumon et le foie en comparaison avec le NaCl 0,9%. De plus le LSM réduisait l’inflammation (IL-1b, TNFa, IL-10) et les taux de VEGF-A, et améliorait la fonction cardiaque (echocardiographie : débit cardiaque, fraction de raccourcissement ; cathétérisme cardiaque gauche : pente maximale systolique et compliance ventriculaire). La perfusion de LSM induisait une augmentation de la lactatémie et de la pyruvatémie sans modification du ratio, témoin de la métabolisation du lactate perfusé, avec augmentation de la glycémie (néoglucogenèse) et de l’hydroxybutyrate, suggérant fortement un effet métabolique de sa perfusion.Ainsi le LSM pourrait être un fluide novateur au cours du sepsis et nécessite d’être évalué au cours d’essais cliniques chez l’Homme
Ethnopharmacological relevance
Sphagneticola trilobata (L.) Pruski is a plant species belonging to the Asteraceae family. Kaurenoid acid (KA) is a diterpene metabolite and one of the active ingredients of Sphagneticola trilobata (L.) Pruski. Extracts containing KA are used in traditional medicine to treat pain, inflammation, and infection.
Aim
The goal of the present study was to investigate the in vivo effects of KA (1-10 mg/kg, per oral gavage) upon LPS inoculation in mice by intraperitoneal (i.p.) or intraplantar (i.pl.; subcutaneous plantar injection) routes at the dose of 200 ng (200 μL or 25 μL, respectively).
Methods
In LPS paw inflammation, mechanical and thermal hyperalgesia MPO activity and oxidative imbalance (TBARS, GSH, ABTS and FRAP assays) were evaluated. In LPS peritonitis we evaluated leukocyte migration, cytokine production, oxidative stress, and NF-κB activation.
Results
KA inhibited LPS-induced mechanical and thermal hyperalgesia, MPO activity and modulated redox status in the mice paw. Pre- and post-treatment with KA inhibited migration of neutrophils and monocytes in LPS peritonitis. KA inhibited the pro-inflammatory/hyperalgesic cytokine (e.g., TNF-α, IL-1β and IL-33) production while enhanced anti-inflammatory/analgesic cytokine IL-10 in peritoneal cavity. In agreement with the effect of KA over pro-inflammatory cytokines it inhibited oxidative stress (total ROS, superoxide production and superoxide positive cells) and NF-κB activation during peritonitis.
Conclusion
KA efficiently dampens LPS-induced peritonitis and hyperalgesia in vivo, suggesting it as a suitable candidate to control excessive inflammation and pain during gram-negative bacterial infections and bringing mechanistic explanation to the ethnopharmacological application of Sphagneticola trilobata (L.) Pruski in inflammation and infection.
Nitric oxide (NO) regulates various physiological and pathophysiological functions in the lungs. However, there is much less information about the effects of NO in the pleura. The present review aimed to explore the available evidence regarding the role of NO in pleural disease. NO, has a double-edged role in the pleural cavity. It is an essential signaling molecule mediating various physiological cell functions such as lymphatic drainage of the serous cavities, the immune response to intracellular multiplication of pathogens, and downregulation of neutrophil migration, but also induces genocytotoxic and mutagenic effects when present in excess. NO is implicated in the pathogenesis of asbestos-related or exudative pleural disease and mesothelioma. From a clinical point of view, the fraction of exhaled NO has been suggested as a potential non-invasive tool for the diagnosis of benign asbestos-related disorders. Under experimental conditions, NO-mimetics were found to attenuate hypoxia-induced therapy resistance in mesothelioma. Similarly, hybrid agents consisting of an NO donor coupled with a parent anti-inflammatory drug showed an enhancement of the anti-inflammatory activity of anti-inflammatory drugs. However, given the paucity of research work performed over the last years in this area, further research should be undertaken to establish reliable conclusions with respect to the feasibility of determining or targeting the NO signaling pathway for pleural disease diagnosis and therapeutic management.
The new pandemic disease COVID-19 has wreaked havoc worldwide. Its infectious agent, SARS-CoV-2, uses two key human enzymes called angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2) to invade body cells. The first one is encoded by the ACE2 gene and the second by the TMPRSS2 gene. Both have an outstanding expression of RNA and proteins in the small intestine compared with other tissues. This prominent location may be related to the main entry route of SARS-CoV-2 into the organism. In the process of infection, two other genes can play a fundamental role: NOS2, which expresses inducible nitric oxide synthase (iNOS), and AOC1, which encodes diamine oxidase (DAO). Both also highlight in the small intestine and are involved in polyamine metabolism. These biogenic amines are important for viral replication, being enhanced when NOS2 and AOC1 genes are downregulated. In addition, NOS2 shows a negative correlation with ACE2 and TMPRSS2, while nondegraded histamine by DAO can lead to an upregulation of both genes on which the virus depends. Taken together, these data suggest that inhibition or underexpression of NOS2 and AOC1 determines the susceptibility to get sick, increasing the risk of infection. On the other hand, a therapeutic approach to the disease could be made with homeopathic medicines. Experiments show the remedies' ability to stimulate gene and protein expression, but a correlation between the symptoms of each drug and these expressions has not yet been established. Here an analysis of the pathogenesis of Silicea terra and Arsenicum album supported on the scientific literature is done. The objective is to propose a theory about their relationship with key genes whose protein expressed in deficiency can give rise to the chain of events that imbalance the internal environment (homeostasis) and allow the development of symptoms. Silicea seems to be related to NOS2 (gene)/iNOS (protein) and Arsenicum with AOC1 (gene)/DAO (protein), being necessary to carry out studies to corroborate these links. Therefore, the aim of this article is to show the importance of NOS2 and AOC1 genes in the development of COVID-19 and to propose a line of investigation to evaluate if homeopathy can improve their protein expression.
Neutrophils, the phagocytic and short-lived cells, were initially noticed for powerful microbicidal action; however, their specific depletion helped in gaging their unidentified importance in myocardial ischemia-reperfusion injury. Moreover, change in the number of circulating/or migrating neutrophils at the inflammatory site attracted scientists to investigate their significance in various pathologies. Importantly, inhibition of neutrophil recruitment and reactive oxygen species (ROS) generation ability improved cardiac function including cardiac hypertrophy and remodeling in diverse conditions. ROS and protease release from neutrophils have been associated with tissue damages including myocarditis, myocardial infarction, and ischemia-reperfusion injury. Nitric oxide (NO), a pleotropic molecule, modulates various physiological functions including vascular tone and cardiac homeostasis. NO controls most of neutrophil functions including ROS generation that influence release of several inflammatory mediators. Neutrophil ROS that depends on NADPH oxidase (NOX-2) system is regulated by diverse mechanisms including posttranslational modifications, protein interactions, and cofactors. In this chapter, we discuss various regulatory mechanisms involved in NO-mediated modulation of neutrophil reactive oxygen and nitrogen species (RONS) generation, and also NO production by neutrophils, which has impacted our understanding of the inflammatory diseases including cardiovascular disorders.
Wound dressings and the healing enhancement (increasing healing speed and quality) are two components of wound care that lead to a proper healing. Wound care today consists mostly of providing an optimal environment by removing waste and necrotic tissues from a wound, preventing infections, and keeping the wounds adequately moist. This is however often not enough to re-establish the healing process in chronic wounds: with the local disruption of vascularization, the local environment is lacking oxygen, nutrients, and has a modified ionic and molecular concentration which limits the healing process. This disruption may affect cellular ionic pumps, energy production, chemotaxis, etc., and will affect the healing process. Biomaterials for wound healing range from simple absorbents to sophisticated bioactive delivery vehicles. Often placing a material in or on a wound can change multiple parameters such as pH, ionic concentration, osmolarity, and it can be challenging to pinpoint key mechanism of action. This article will review the literature of several inorganic ions and molecules and their potential effects on the different wound healing phases and their use in new wound dressings.
The present investigation was aimed to develop SA loaded nanoemulsion by implementing response surface methodology (RSM) and hydrophilic-lipophilic balance (HLB) approach, in amalgamation. The concentration of oil phase (Lemongrass oil containing SA, corresponding to HLB from 13.43 to 14.88) and the ratio of surfactants were taken as independent factors while particle size and polydispersibility index as dependent variables in RSM. The droplet size and polydispersibility index of the optimized nanoemulsion (SANE 14) were 134.70 ± 4.51 nm and 0.168 ± 0.011 respectively, corresponding to the HLB value of 14.8. The retention of SA from SANE 14 in skin layers was found to be 97.23% after 24 h study period. Moreover, a 2.8 and 4.13 folds amount of SA from SANE 14 was retained in epidermis and dermis, respectively, when compared with the drug solution. Therefore, the SANE 14 was capable of delivering the SA into the deeper skin layers with notable retention which was further verified by the confocal microscopy images. The TPA induced skin inflammation in mice ear was significantly reduced (80.25%) in terms of ear thickness. Significant inhibition of pro-inflammatory cytokines proved the efficacy of SANE 14. Conclusively, the controlled dermal release of salicylic acid for improved anti-inflammatory action was successfully achieved.
Objective
SLE serves as an independent risk factor` for endothelial dysfunction (ED) not explained by Framingham risk factors. We sought to understand the development of SLE-induced ED on a cellular level in order to develop strategies aimed at reversing cellular abnormalities. This study assessed the impact of SLE patient serum on endothelial nitric oxide synthase (eNOS), nitric oxide (NO) production and functional changes in the cell.
Methods
Human umbilical vein endothelial cells (HUVECs) cultured in serum of either SLE (n=25) or healthy patients (n=14) or endothelial basal medium 2 (EBM-2) culture media supplemented with fetal bovine serum with or without L-sepiapterin were used for our studies. We applied the fluorescent probe DAF-FM diacetate for intracellular NO detection using flow cytometry. Total RNA isolates were analysed using reverse transcription PCR for eNOS mRNA expression. Oxygen consumption rate was determined using seahorse analysis. Neutrophil adhesion and migration were determined using a calcein AM microscopy assay.
Results
The mRNA expression of eNOS was increased in SLE cultured HUVECs compared with healthy control (p<0.05). The SLE eNOS mRNA level correlated with SLE patient age (p=0.008); however, this trend was not observed with healthy patients. SLE serum reduced NO production in HUVECs compared with EBM-2 cultured cells (p<0.05). Co-treatment of endothelial cells with L-sepiapterin preserved HUVEC capacity to produce NO in SLE conditions (p<0.01). SLE serum enhanced neutrophil migration (p<0.01) but not neutrophil adhesion compared with healthy controls. The bioenergetic health index was not different.
Conclusions
SLE likely causes disruption of endothelial cell eNOS function and NO modulated pathways.
Lipopolysaccharides (LPS), the major components of the wall of gram-negative bacteria, trigger powerful defensive responses in the airways via mechanisms thought to rely solely on the Toll-like receptor 4 (TLR4) immune pathway. Here we show that airway epithelial cells display an increase in intracellular Ca²⁺ concentration within seconds of LPS application. This response occurs in a TLR4-independent manner, via activation of the transient receptor potential vanilloid 4 cation channel (TRPV4). We found that TRPV4 mediates immediate LPS-induced increases in ciliary beat frequency and the production of bactericidal nitric oxide. Upon LPS challenge TRPV4-deficient mice display exacerbated ventilatory changes and recruitment of polymorphonuclear leukocytes into the airways. We conclude that LPS-induced activation of TRPV4 triggers signaling mechanisms that operate faster and independently from the canonical TLR4 immune pathway, leading to immediate protective responses such as direct antimicrobial action, increase in airway clearance, and the regulation of the inflammatory innate immune reaction.
The role of chitinases from the latex of medicinal shrub Calotropis procera on viability of tumor cell lines and inflammation was investigated. Soluble latex proteins were fractionated in a CM Sepharose Fast-Flow Column and the major peak (LPp1) subjected to ion exchange chromatography using a Mono-Q column coupled to an FPLC system. In a first series of experiments, immortalized macrophages were cultured with LPp1 for 24 h. Then, cytotoxicity of chitinase isoforms (LPp1-P1 to P6) was evaluated against HCT-116 (colon carcinoma), OVCAR-8 (ovarian carcinoma), and SF-295 (glioblastoma) tumor cell lines in 96-well plates. Cytotoxic chitinases had its anti-inflammatory potential assessed through the mouse peritonitis model. We have shown that LPp1 was not toxic to macrophages at dosages lower than 125 μg/mL but induced high messenger RNA expression of IL-6, IL1-β, TNF-α, and iNOs. On the other hand, chitinase isoform LPp1-P4 retained all LPp1 cytotoxic activities against the tumor cell lines with IC50 ranging from 1.2 to 2.9 μg/mL. The intravenous administration of LPp1-P4 to mouse impaired neutrophil infiltration into the peritoneal cavity induced by carrageenan. Although the contents of pro-inflammatory cytokines IL-6, TNF-α, and IL1-β were high in the bloodstreams, such effect was reverted by administration of iNOs inhibitors NG-nitro-L-arginine methyl ester and aminoguanidine. We conclude that chitinase isoform LPp1-P4 was highly cytotoxic to tumor cell lines and capable to reduce inflammation by an iNOs-derived NO mechanism.
To investigate the hepatoprotective effect of Cymbopogon citratus or lemongrass essential oil (LGO), it was used in an animal model of acute liver injury induced by acetaminophen (APAP). Swiss mice were pretreated with LGO (125, 250 and 500[Formula: see text]mg/kg) and SLM (standard drug, 200[Formula: see text]mg/kg) for a duration of seven days, followed by the induction of hepatotoxicity of APAP (single dose, 250[Formula: see text]mg/kg). The liver function markers alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and gamma-glutamyl transferase were determined to evaluate the hepatoprotective effects of the LGO. The livers were used to determine myeloperoxidase (MPO) activity, nitric oxide (NO) production and histological analysis. The effect of LGO on leukocyte migration was evaluated in vitro. Anti-oxidant activity was performed by assessing the free radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) in vitro. LGO pretreatment decreased significantly the levels of ALT, AST and ALP compared with APAP group. MPO activity and NO production were decreased. The histopathological analysis showed an improved of hepatic lesions in mice after LGO pretreatment. LGO inhibited neutrophil migration and exhibited anti-oxidant activity. Our results suggest that LGO has protective activity against liver toxicity induced by paracetamol.
Vestitol is an isoflavonoid isolated from Brazilian red propolis with potential anti-inflammatory activity. This study investigated the mechanism of action of vestitol on the modulation of neutrophil migration in the inflammatory process. Pre-treatment with vestitol at 1, 3, or 10 mg/kg reduced LPS- or mBSA-induced neutrophil migration and the release of CXCL1/KC and CXCL2/MIP-2 in vivo. Likewise, pre-treatment with vestitol at 1, 3, or 10 μM reduced the levels of CXCL1/KC and CXCL2/MIP-2 in macrophage supernatants in vitro. Moreover, the administration of vestitol (10 mg/kg) reduced leukocyte rolling and adherence in the mesenteric microcirculation of mice. The pre-treatment with vestitol (10 mg/kg) in iNOS(-/-) mice did not block its activity concerning neutrophil migration. With regard to the activity of vestitol on neutrophils isolated from the bone marrow of mice, there was a reduction on the chemotaxis of CXCL2/MIP-2 or LTB4-induced neutrophils and on calcium influx after pre-treatment with the compound at 3 or 10 μM. There was no change in CXCR2 expression by neutrophils treated with vestitol at 10 μM. These findings demonstrate that vestitol is a promising novel anti-inflammatory agent.
Depuis le début des années 1970, ľexistence ďune relation étroite entre le statut nutritionnel et les capacités de réponse inflammatoire et immunitaire (RII) à ľagression est admise. Les premiers travaux ont montré qu’une dénutrition protéino-énergétique, altérait significativement la réponse immunitaire, tant innée qu’acquise, augmentant ainsi le risque infectieux et le taux de mortalité des malades les plus dénutris. Plus récemment, il est également apparu que ľobésité interférait avec la RII. Ces relations et les principaux mécanismes qui y conduisent seront brièvement revus dans la première partie de ce chapitre, après un rappel des étapes clés de la RII.
Severe haemorrhage can lead to global ischaemia and haemorrhagic shock (HS), resulting in multiple organ failure (MOF) and death. Restoration of blood flow and reoxygenation is associated with an exacerbation of tissue injury and inflammatory response. The neuronal nitric oxide synthase (nNOS) has been implicated in vascular collapse and systemic inflammation of septic shock; however the role of nNOS in HS is poorly understood. The aim of this study is to evaluate the role of nNOS in the MOF associated with HS. Rats were subjected to HS under anaesthesia. Mean arterial pressure was reduced to 30 mmHg for 90 min, followed by resuscitation with shed blood. Rats were randomly treated with 2 chemically distinct nNOS inhibitors [ARL 17477 (1 mg/kg) and 7-nitroindazol (5 mg/kg)] or vehicle upon resuscitation. Four hours later, parameters of organ injury and dysfunction were assessed. HS was associated with MOF development. Inhibition of nNOS activity at resuscitation protected rats against the MOF and vascular dysfunction. In addition, treatment of HS-rats with nNOS inhibitors attenuated neutrophil infiltration into target organs and decreased the activation of NF-[kappa]B, iNOS expression, NO production and nitrosylation of proteins. Furthermore, nNOS inhibition also reduced the levels of pro-inflammatory cytokines TNF-[alpha] and IL-6 in HS-rats. In conclusion, two distinct inhibitors of nNOS activity reduced the MOF, vascular dysfunction and the systemic inflammation associated with HS. Thus, nNOS inhibitors may be useful as an adjunct therapy before fluids and blood administration in HS patients in order to avoid the MOF associated with reperfusion injury during resuscitation.
Background
Kaurenoic acid (KA), a diterpene extracted from copaíba oil-resin, is known to have potent antioxidant and anti-inflammatory properties. l-Arginine (LA) is an amino acid and a nitrogenous precursor for the synthesis of nitric oxide (NO). NO paper in wound healing has already been well documented. The aim of this study was to investigate the protective effects of LA and KA against ischemia reperfusion injury in a randomized skin flap model in rats.
Methods
A modified McFarlane flap model measuring 2.5 wide × 8 cm long was established in 36 anesthetized rats and evaluated within 3 groups: group control, group l-arginine, and group kaurenoic acid. Each group was subdivided into two subgroups (T1 and T2, n = 6 each). Samples were collected 24 h (T1)/48 h (T2) postoperatively for oxidative stress (glutathione), as non-protein thiols, malondialdehyde (MDA), NO2, inflammation [myeloperoxidase (MPO)], and cytokines TNF-α and IL-1β assays.
Results
KA promoted a significant decrease of TNF-α and IL-1 expression and MPO activity at T1/T2 time points. NSGH levels increased significantly in KA-treated rats, while MDA levels decreased significantly in the same rats. Arginine promoted a significant decrease in MDA levels at the T1 time point and a significant increase in non-protein thiols concentrations at T1/T2 time points. NO2 concentration also decreased at the T1 time point.
Conclusions
KA may attenuate the oxidative stress and the inflammation, thereby reducing tissue damage induced by ischemia/reperfusion in rats subjected to dorsal skin flaps.
No Level Assigned
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This review concentrates on advances in nitric oxide synthase (NOS) structure, function and inhibition made in the last seven years, during which time substantial advances have been made in our understanding of this enzyme family. There is now information on the enzyme structure at all levels from primary (amino acid sequence) to quaternary (dimerization, association with other proteins) structure. The crystal structures of the oxygenase domains of inducible NOS (iNOS) and vascular endothelial NOS (eNOS) allow us to interpret other information in the context of this important part of the enzyme, with its binding sites for iron protoporphyrin IX (haem), biopterin, L-arginine, and the many inhibitors which interact with them. The exact nature of the NOS reaction, its mechanism and its products continue to be sources of controversy. The role of the biopterin cofactor is now becoming clearer, with emerging data implicating one-electron redox cycling as well as the multiple allosteric effects on enzyme activity. Regulation of the NOSs has been described at all levels from gene transcription to covalent modification and allosteric regulation of the enzyme itself. A wide range of NOS inhibitors have been discussed, interacting with the enzyme in diverse ways in terms of site and mechanism of inhibition, time-dependence and selectivity for individual isoforms, although there are many pitfalls and misunderstandings of these aspects. Highly selective inhibitors of iNOS versus eNOS and neuronal NOS have been identified and some of these have potential in the treatment of a range of inflammatory and other conditions in which iNOS has been implicated.
We have developed a novel model of allergen-induced eosinophil extravasation into mouse air-pouches following sensitization and challenge with ovalbumin (Ova). This model was used to investigate the mechanism(s) underlying the anti-inflammatory action of the glucocorticoid hormone dexamethasone (Dex).
Injection of 10 μg Ova into 6-day-old dorsal air-pouches of mice sensitized to the same antigen provoked an intense cell accumulation as early as 6 h post-challenge (0.08±0.03 and 4.0±1.0×105 leucocytes in saline and Ova-treated air-pouches, respectively), maximal at 24 h (0.02±0.01 and 6.0±0.8×105 leucocytes in saline and Ova-treated air-pouches, respectively) and persisted up to 48 h. At the 24 h time-point, the cellular infiltrate consisted of 37% eosinophils, 18% neutrophils and 45% mononuclear cells, as assessed by histological examination. The same ratio of eosinophil/neutrophil was obtained by fluorescence-activated cell sorting (FACS) analysis, since 72% of the polymorphonuclear (PMN) population was positive for very-late antigen-4 (VLA-4) expression.
Subcutaneous (s.c.) administration of Dex (50 or 100 μg per mouse, −1 h) inhibited eosinophil accumulation into Ova challenged air-pouches by about 70% (P<0.05) and 75% (P<0.05), respectively, when compared to controls. Cell accumulation measured at 48 h after Ova injection was also significantly reduced (−75%) by Dex administration at the 24 h time-point (n=12, P<0.05).
Eosinophil numbers in the bone marrow and blood were quantitated. We found that the sensitization protocol induced a 3 fold increase in eosinophil numbers in the bone marrow (naive mice: 2.7±0.3×105; sensitized mice: 8.7±1.7×105, P<0.05) and blood (naive mice: 0.5±0.2×105; sensitized mice: 1.5±0.3×105, P<0.01). However, 24 h following Ova challenge, the eosinophil numbers in the bone marrow had dropped (3.7±0.8×105) with no change in the circulating pool, suggesting an equilibrium within the eosinophil pools had been reached.
Dex administration provoked a profound eosinopaenia in the blood of naive (5.2±1.5 to 0.9±0.6×104) and sensitized mice (1.5±0.3 to 0.08±0.02×105) at 4 h. This effect was reversed within 24 h. Dex also inhibited the release of eosinophils from the bone marrow in response to Ova challenge.
We show for the first time that eosinophils express the steroid-inducible protein lipocortin 1 (LC1). FACS analysis of eosinophils emigrated into the Ova challenged air-pouches revealed detectable LC1-like immunoreactivity (373×103). These data were also substantiated by LC1 detection in circulating eosinophils of interleukin-5 transgenic mice (strain: CBA/Ca). However, s.c. injection of Dex (50 μg) did not alter LC1 levels in blood eosinophils, such that 235±21×103 LC1-like molecules per cell were measured after vehicle treatment (n=5), and 224±8×103 LC1-like molecules per cell were associated with this cell type 1 h after steroid treatment (n=5, not significant). Finally, resident eosinophils (in the pleural cavity) were found to have much higher LC1 levels than that found in the blood circulation (2 fold increase, P<0.05).
Passive immunization of mice against LC1 with a validated antiserum (termed LCS3) and protocol failed to modify the anti-migratory activity exerted by Dex towards eosinophil extravasation into Ova-challenged air-pouches. The steroid (50 μg s.c., −1 h) produced a similar degree of inhibition of eosinophil accumulation both in control animals (treated with a non-immune sheep serum) and LCS3-treated mice (−56% and 59%, respectively, n=15–21, not significant).
In conclusion, the air-pouch provides a novel and convenient cavity to study allergen-induced cell recruitment which is sensitive to glucocorticoid hormone treatment. The effect of Dex on eosinophil distribution in these experimental conditions has been studied in detail and we failed to find an important role for endogenous LC1 in these actions of the steroid.
British Journal of Pharmacology (1997) 121, 97–104; doi:10.1038/sj.bjp.0701122
Clinical reports indicate that acute ethanol intoxication in chronic ethanol abusers is associated with neutropenia. We hypothesize that ethanol accelerates the apoptosis of neutrophils thus decreasing the peripheral blood count of neutrophils. We studied the effect of ethanol on neutrophil apoptosis in vivo as well as in vitro. Human neutrophils harvested from healthy subjects after an alcohol drinking binge showed enhanced apoptosis (before, 0.5+/-0.25 vs. after, 26.1+/-2.6% apoptotic neutrophils/field). Peritoneal neutrophils isolated from ethanol-treated rats also showed increased (P < 0.0001) apoptosis when compared with neutrophils isolated from control rats (control, 0.8+/-0.2% vs. ethanol, 11.8+/-0.7% apoptotic neutrophils/field). In in vitro studies, ethanol in concentrations of 50 mM and higher accelerated the apoptosis of human and rat neutrophils. This effect of ethanol on human neutrophils was time dependent. DNA isolated from ethanol-treated human neutrophils displayed integer multiples of 180 base pairs (ladder pattern), further confirming the effect of ethanol on neutrophil apoptosis. N(G)-monomethyl-L-arginine monoacetate and N(G)-nitro-L-arginine methyl ester, inhibitors of nitric oxide (NO) synthase, attenuated the ethanol-induced neutrophil apoptosis. Sodium nitroprusside, a NO donor, also promoted neutrophil apoptosis. Moreover, ethanol enhanced neutrophil expression of inducible NO synthase. In addition, ethanol stimulated neutrophil NO generation. These results suggest that ethanol accelerates neutrophil apoptosis. This effect of ethanol on neutrophil apoptosis seems to be mediated through the generation of NO.
Drug-induced leucopenia renders rats hyporeactive to various inflammatory stimuli. Administration to leucopenic rats of suspensions of lymphocytes, sufficient to apparently correct the induced lymphocytopenia, led to a partial but marked reversal of the inhibited responses. Similar results were observed when lysates of lymphocytes or filtrates of the disintegrated cells were injected. Suspensions of polymorphonuclear granulocytes, on the contrary, were ineffective in producing a reversal of inhibited inflammatory reactions in leucopenic rats. The presence of a proinflammatory factor (LpIF) in lymphocytes, which might be involved in the modulation of acute inflammatory responses is suggested.
Guinea pig alveolar macrophages obtained by bronchoalveolar lavage were isolated by adherence for 2 h and stimulated with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) for different time intervals. The supernatants then were tested for their chemotactic effect on guinea pig peritoneal normodense eosinophils and for release of thromboxane B2, leukotriene B4 (LTB4), and platelet activating factor (PAF). The supernatant from fMLP-stimulated alveolar macrophages induced a significant eosinophil attraction (96.0 +/- 11.9, number of migrating eosinophils [mean +/- SEM], n = 17) as compared to unstimulated macrophages (4.8 +/- 1.4, n = 15). This effect was not accounted for by fMLP carry-over to the macrophages because, in contrast to human eosinophils, fMLP has no chemotactic effect on guinea pig eosinophils. Pretreatment of eosinophils with BN 52021 (100 microM), a specific PAF antagonist, and with indomethacin (10 microM), a cyclooxygenase inhibitor, failed to inhibit migration of eosinophils induced by supernatants from either stimulated or unstimulated alveolar macrophages. In contrast, inhibition of the 5-lipoxygenase enzyme with N-(3-phenoxycinamyl)-acetohydroxamic acid (1 microM) suppressed eosinophil migration by alveolar macrophage supernatants (94.1 +/- 2.6% of inhibition, n = 6). Desensitization of eosinophils by and to LTB4 (10 nM) inhibited migration induced by supernatants from stimulated alveolar macrophages (87.5 +/- 5.4% of desensitization toward LTB4 and 83.1 +/- 5.4% of desensitization toward supernatants, n = 5). Under the present experimental conditions, LTB4 is the only agent implicated in eosinophil migration induced by supernatants from fMLP-stimulated alveolar macrophages.
The objective of this study was to determine whether endogenous nitric oxide (NO) inhibits leukocyte adhesion to vascular endothelium. This was accomplished by superfusing a cat mesenteric preparation with inhibitors of NO production, NG-monomethyl-L-arginine (L-NMMA) or NG-nitro-L-arginine methyl ester (L-NAME), and observing single (30-microns diameter) venules by intravital video microscopy. Thirty minutes into the superfusion period the number of adherent and emigrated leukocytes, the erythrocyte velocity, and the venular diameter were measured; venular blood flow and shear rate were calculated from the measured parameters. The contribution of the leukocyte adhesion glycoprotein CD11/CD18 was determined using the CD18-specific monoclonal antibody IB4. Both inhibitors of NO production increased leukocyte adherence more than 15-fold. Leukocyte emigration was also enhanced, whereas venular shear rate was reduced by nearly half. Antibody IB4 abolished the leukocyte adhesion induced by L-NMMA and L-NAME. Incubation of isolated cat neutrophils with L-NMMA, but not L-NAME, resulted in direct upregulation of CD11/CD18 as assessed by flow cytometry. Decrements in venular shear rate induced by partial occlusion of the superior mesenteric artery in untreated animals revealed that only a minor component of L-NAME-induced leukocyte adhesion was shear rate-dependent. The L-NAME-induced adhesion was inhibited by L-arginine but not D-arginine. These data suggest that endothelium-derived NO may be an important endogenous modulator of leukocyte adherence and that impairment of NO production results in a pattern of leukocyte adhesion and emigration that is characteristic of acute inflammation.
A basic protein having chemotactic activity for neutrophils is secreted by the rat kidney epithelioid cell line NRK-52E in response to interleukin-1 beta (Watanabe, K., Kinoshita, S., and Nakagawa, H. (1989) Biochem. Biophys. Res. Commun. 161, 1093-1099). The protein, which is referred to as cytokine-induced neutrophil chemoattractant (CINC), has been shown to be a dimer of identical subunits; and the complete amino acid sequence of the subunit has been established. Sequence determination has been achieved by automated Edman degradation of reduced and carboxymethylated CINC and of peptides generated by cleavage with cyanogen bromide and lysyl endopeptidase. The CINC subunit consists of 72 amino acid residues. The amino acid sequence of CINC shows striking similarities to the sequences of the proteins encoded by the mouse platelet-derived growth factor-inducible KC gene and human and hamster gro genes, suggesting that CINC is the rat counterpart of the KC/gro protein.
Mechanisms governing the normal resolution processes of inflammation are poorly understood, yet their elucidation may lead to a greater understanding of the pathogenesis of chronic inflammation. The removal of neutrophils and their potentially histotoxic contents is one prerequisite of resolution. Engulfment by macrophages is an important disposal route, and changes in the senescent neutrophil that are associated with their recognition by macrophages are the subject of this investigation. Over 24 h in culture an increasing proportion of human neutrophils from peripheral blood or acutely inflamed joints underwent morphological changes characteristic of programmed cell death or apoptosis. Time-related chromatin cleavage in an internucleosomal pattern indicative of the endogenous endonuclease activation associated with programmed cell death was also demonstrated. A close correlation was observed between the increasing properties of apoptosis in neutrophils and the degree of macrophage recognition of the aging neutrophil population, and a direct relationship between these parameters was confirmed within aged neutrophil populations separated by counterflow centrifugation into fractions with varying proportions of apoptosis. Macrophages from acutely inflamed joints preferentially ingested apoptotic neutrophils and histological evidence was presented for occurrence of the process in situ. Programmed cell death is a phenomenon of widespread biological importance and has not previously been described in a cell of the myeloid line. Because it leads to recognition of intact senescent neutrophils that have not necessarily disgorged their granule contents, these processes may represent a mechanism for the removal of neutrophils during inflammation that also serves to limit the degree of tissue injury.
Although nitric oxide (NO) and antioxidants inhibit adhesion molecule expression, their inhibitory effects on nuclear factor κB (NF-κB) activation may differ. The NO donors, but not 8-bromo-cGMP, decreased tumor necrosis factor α (TNF-α)-induced VCAM-1, ICAM-1, and E-selectin expression by 11-70%. In contrast, NAC completely abolished VCAM-1 and E-selectin expression and decreased ICAM-1 expression by 56%. Gel shift assays demonstrate that NF-κB activation was inhibited by both NO and antioxidants. The activation of NF-κB involves the phosphorylation and degradation of its cytoplasmic inhibitor IκB-α by 26S proteasomes. The 26S proteasome inhibitor MG132 prevented the degradation of phosphorylated IκB-α. NAC inhibited IκB kinase (IKK) activity and prevented IκB-α phosphorylation and degradation. In contrast, NO did not inhibit IKK activity, IκB-α phosphorylation, or IκB-α degradation. However, NO, but not antioxidants, induced IκB-α promoter activity. The inhibitory effects of NO on adhesion molecule expression, therefore, differs from that of antioxidants in terms of the mechanism by which NF-κB is inactivated. J. Leukoc. Biol. 63: 732–739; 1998.
During the past few years, an enormous amount of research has been conducted on the vascular L-arginine/nitric oxide (NO) system. Alterations of NO production and/or bioavailability have been shown to occur both in experimental animal models (Osborne et al., 1989b; Tsao et al., 1990; Gauthier et al., 1995; Scalia et al., 1996) and in humans (Chester et al., 1990; Boger et al., 1997) in diverse settings such as hypertension, hypercholesterolemia, diabetes, ischemia-reperfusion, and heart failure.
Xanthine oxidase-derived oxidants and leukocytes have been implicated in the microvascular injury associated with reperfusion of ischemic intestine. The objective of this study was to determine whether xanthine oxidase-derived oxidants play a role in the leukocyte-microvascular interactions initiated by ischemia-reperfusion. Adherence and extravasation of leukocytes were monitored in cat mesenteric venules subjected to 1 h of ischemia (blood flow reduced to 20% of control) and reperfusion. Leukocyte rolling velocity, vessel diameter, and red cell velocity were also measured in control (untreated) animals and in animals pretreated with either allopurinol or superoxide dismutase. The responses of venular blood flow, wall shear rate, and leukocyte rolling velocity to ischemia and reperfusion did not differ between the three experimental groups. In control animals, 1 h of ischemia was associated with significant adherence and extravasation of leukocytes with reperfusion greatly enhancing these responses. Allopurinol treatment did not alter the responses to ischemia per se, yet it largely prevented the further increment in adherence and extravasation associated with reperfusion. Superoxide dismutase treatment attenuated the leukocyte responses elicited by both ischemia and reperfusion. Our observations that both allopurinol and superoxide dismutase attenuate reperfusion-induced leukocyte adherence and extravasation are consistent with the hypothesis that xanthine oxidase-derived oxidants initiate the leukocyte infiltration induced by reperfusion of ischemic intestine.
Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue1 and for the inhibition of platelet aggregation2 and platelet adhesion3 attributed to endothelium-derived relaxing factor4. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay5, chemiluminescence1 or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.
Several recent studies have suggested that nitric oxide (NO) derived from the inducible isoform of NO synthase (NOS) may act as an endogenous modulator of the inflammatory response by inhibiting adhesion of leukocytes to endothelial cellsin vitro.Few studies have addressed specifically the role of iNOS in regulating leukocyte recruitmentin vivoin a model of acute inflammation. Thus, the objective of this study was to assess the role of iNOS in modulating neutrophil (PMN) extravasation in an oyster glycogen-induced model of acute peritonitis in rats. Data obtained in the present study demonstrates that injection (IP) of oyster glycogen induces massive and selective PMN recruitment into the peritoneal cavity of rats at 6 hrs following OG administration. These extravasated cells were found to contain significant amounts of iNOS protein as assessed by Western blot analysis. Treatment of rats with the selective iNOS inhibitor L-iminoethyl-lysine (L-NIL) dramatically reduced NO levels in lavage fluid as measured by decreases in nitrate and nitrite concentrations without significantly affecting iNOS protein levels. Although L-NIL inhibited NO production by >70%, it did not alter oyster glycogen-induced PMN recruitment when compared to vehicle-treated rats. We conclude that PMN-associated, iNOS-derived NO does not play an important role in modulating extravasation of these leukocytes in this model of acute inflammation.
Neutrophils constitute over 90% of cells found in the synovial fluid of rheumatoid arthritis (RA) patients. Since such fluids also contain immune complexes (IgG-IgG and IgG-IgM rheumatoid factors) and complement split products (C5, C5A, DES, ARG, C3B, etc.), all of the reactants are present for a local Arthus lesion. Moreover, neutrophils from RA patients endocytose these immune complexes and complement components in vivo and in vitro. In consequence, it has been suggested that lysosomal enzymes and other mediators of inflammation released by neutrophils after uptake of immune complexes (in the bulk phase or on the surface) account, at least in part, for rheumatoid inflammation. Secretion of lysosomal hydrolases, especially neutral proteases, which provoke tissue injury and generation of reactive oxygen species (e.g. O2) is part of a stimulus-secretion response to a variety of secretagogues, including immune complexes and complement components. However, the pathways of secretion and O2 generation are stimulus-specific and can be dissected to establish cause and effect relationships by (a) kinetic analysis, (b) varying the stimulus, (c) use of impermeant reagents to block discrete responses. Neutrophils also generate products of 11-cyclo-oxygenase (e.g., PGE2, TXA2) and of the 5- and 15-lipoxygenase (mono-, di-and trihetes, LTB4 and their isomers). However, the cyclo-oxygenase products (except TXA2) do not cause inflammation acting alone; indeed, they inhibit the function of neutrophils, platelets, macrophages and mast cells. The most potent proinflammatory agent yet identified as a product of arachidonate is LTB4. LTB4 is a potent Ca ionophore, a strong chemo-attractant, induces local inflammation, and activates neutrophils.
Recent evidence indicates that chronic hyperhomocysteinemia, which is found in from 9 to 15% of the general population, is an independent risk factor for the development of atherosclerosis. We sought to elucidate the mechanism by which exposure of the vascular wall to high levels of homocysteine initiates this inflammatory reaction. We examined the acute effect of homocysteine on endothelial dysfunction in isolated rat arteries and on microcirculatory leukocyte–endothelium interaction in vivo. Intravital microscopy of rat mesenteric venules was performed by superfusing the mesentery with increasing concentrations of homocysteine (1–5 mmol/l). There was a significant concentration- and time-dependent increase in leukocyte rolling, adherence, and extravasation compared with control rats superfused with Krebs-Henseleit solution (p < 0.01). Moreover, immunohistochemical staining demonstrated significantly increased P-selectin and intercellular adhesion molecule-1 (ICAM-1) expression on intestinal venules after homocysteine superfusion. In contrast, mesenteric superfusion with the nitric oxide donor 4-hydroxymethyl-furazan-3-carboxylic acid oxide (CAS1609, 1 μmol/l) significantly attenuated homocysteine-induced leukocyte rolling, adherence, and transmigration to control levels (p < 0.01). CAS1609 also attenuated both P-selectin and ICAM-1 expression on mesenteric venules and decreased CD18 expression on isolated leukocytes. Superior mesenteric arteries incubated with 5 mmol/l homocysteine developed significant (p < 0.01) endothelial dysfunction (i.e., impaired relaxation to endothelium-dependent dilators). Acute hyperhomocysteinemia induces endothelial dysfunction, characterized by a loss of endothelium-derived nitric oxide, leading to an inflammatory state. This state results in increased leukocyte rolling, adherence, and transmigration by upregulation of cell adhesion molecules. Our data suggest that hyperhomocysteinemia inhibits the important homeostatic role of nitric oxide in preventing endothelial dysfunction.
This study investigated the regulatory effects of the major inflammatory mediator, nitric oxide (NO), on human neutrophil apoptosis in vitro. Co-culture of human neutrophils with the NO donors GEA 3162 (1,2,3,4-oxatriazolium,5-amino-3-(3,4-dichlorophenyl)-chloride) (10-100 microM) and 3-morpholino-sydnonimine (SIN-1) (0.3-3 mM) caused a dramatic and concentration-dependent induction of apoptosis. However, N-formyl-methionyl-leucyl-phenylalanine (FMLP)-induced neutrophil activation (actin reorganization and chemotaxis) was inhibited by GEA 3162 treatment. The pro-apoptotic effects of the NO donors were (i) unaffected by the soluble guanylate cyclase inhibitor LY-83583 (6-anilino-5,8-quinolinedione; 100 microM), (ii) antagonized by superoxide dismutase (6 microg/mL), (iii) mimicked by exogenous peroxynitrite (at concentrations >100 microM), and (iv) inhibited by the caspase inhibitor Z-Val-Ala-DL-Asp-fluoromethylketone (100 microM). The pro-apoptotic effect of the NO donors was not mimicked by the cell-permeable cyclic nucleotide analogue, N6,2-O-dibutyrylguanosine-3',5'-cyclic monophosphate (dibutyryl-cGMP) at concentrations < or =0.2 mM. Indeed, at high concentrations (> or =2 mM), dibutyryl-cGMP caused an inhibition of apoptosis. These results suggest that NO-mediated apoptosis, although caspase-dependent, is mediated by a cGMP-independent mechanism and involves the concurrent generation of oxygen free radicals and, potentially, peroxynitrite. Our data reveal a unique role for NO in inflammatory responses with differential effects upon neutrophil activation and survival, with important implications for the successful resolution of inflammation.
Schistosomulum-released products (SRP) have been shown to enhance both expression of rat and human eosinophil Fc receptors and IgG-dependent cytotoxicity. The present work provides additional evidence of the secretion of eosinophil-enhancing factors by schistosomula and other developmental stages of schistosomes, including adult worms. The heat lability, as well as the strong inhibition of the stimulating activity of SRP by the protease inhibitor Trasylol, suggest that thermolabile proteases secreted by the parasite are involved in this mechanism. The purification of the schistosome proteases by preparative isoelectric focusing and gel filtration demonstrated that neutral proteases able to hydrolyze the collagenase substrates Azocoll and Z-Gly-Pro-Leu-Gly-Pro are able to significantly enhance eosinophil effector functions. Purified Clostridium histolyticum collagenase was also able to mimic the enhancing effect of schistosome proteases, suggesting involvement of a collagenase-like activity of the enzymes in the eosinophil stimulation.
To test the hypothesis that mesothelial cells play a role in regulating inflammatory responses within the pleural space, we examined neutrophil chemotactic activity released by cytokine-stimulated mesothelial cells. Human mesothelial cells were isolated from patients with transudative pleural effusions and cultured. The purity of the cell population was assessed by morphologic, immunocytochemical, and biochemical characteristics. Confluent fourth passage mesothelial cell plates were exposed to varying concentrations of the recombinant human cytokines IL-1 alpha, TNF-alpha, or IFN-gamma, or Escherichia coli endotoxin (LPS). Polymorphonuclear neutrophil (PMN) chemotactic activity in the conditioned media was measured in microchemotaxis chambers. Although none of the cytokines demonstrated inherent chemotactic activity, each stimulated mesothelial cells to produce PMN chemotactic activity in a dose-dependent manner. TNF-alpha stimulated the release of the greatest quantity, whereas stimulation with IFN-gamma and IL-1 alpha resulted in the release of lesser but still significant quantities of PMN chemotactic activity. By contrast, LPS did not increase the basal level of chemotactic activity produced by the cells. The cytokine-induced chemotactic activity was proteinaceous, required de novo synthesis, and had a predominant m.w. of 10,000. Significant quantities of immunoreactive neutrophil-activating peptide-1 (NAP-1)/IL-8 were detected in mesothelial cell supernatants after stimulation with each of the cytokines. The neutrophil chemotactic activity of supernatants from mesothelial cells stimulated with either IL-1 alpha or IFN-gamma was completely neutralized with rabbit anti-human NAP-1/IL-8 polyclonal antiserum. The same antiserum neutralized the majority, but not all, of the neutrophil chemotactic activity in supernatants from TNF-stimulated mesothelial cells. Stimulated mesothelial cells also expressed an inducible mRNA transcript that hybridized with a specific oligonucleotide probe for human NAP-1/IL-8. These observations provide a mechanism whereby mesothelial cells could respond to inflammatory stimuli in the underlying lung and regulate inflammatory responses within the pleural space.
A sensitive enzyme-linked immunosorbent assay (ELISA) for rat interleukin 8/cytokine-induced neutrophil chemoattractant (CINC) has been established by using biotin-conjugated anti-CINC rabbit IgG. The biotin-streptavidin sandwich ELISA detected CINC at concentrations from 3 pg/ml up to 30 ng/ml. The concentration of CINC in the pouch fluid (exudate) of rat carrageenin-induced inflammation was measured by the ELISA. After a time lag of about 2 h, neutrophils steadily accumulated in the carrageenin/air-pouch until 8 h. Similarly, the CINC level of exudate increased after about a 2-h lag, and reached a maximum (134 ng/ml) at 8 h, and thereafter decreased to a negligible concentration at 24 h after carrageenin injection. In association with the rise in CINC level, the concentration of exudate 96-kDa gelatinase corresponding to neutrophil gelatinase/type IV collagenase increased with time. The results suggest that CINC contributes, at least in part, to the neutrophil migration into the inflammatory lesion of the carrageenin-induced inflammation in rats.
The hyperalgesic activities in rats of interleukin‐1β (IL‐1β), IL‐6, IL‐8, tumour necrosis factor α (TNFα) and carrageenin were investigated.
IL‐6 activated the previously delineated IL‐1/prostaglandin hyperalgesic pathway but not the IL‐8/sympathetic mediated hyperalgesic pathway.
TNFα and carrageenin activated both pathways.
Antiserum neutralizing endogenous TNFα abolished the response to carrageenin whereas antisera neutralizing endogenous IL‐1β, IL‐6 and IL‐8 each partially inhibited the response.
The combination of antisera neutralizing endogenous IL‐1β + IL‐8 or IL‐6 + IL‐8 abolished the response to carrageenin.
These results show that TNFα has an early and crucial role in the development of inflammatory hyperaglesia.
The delineation of the roles of TNFα, IL‐1β, IL‐6 and IL‐8 in the development of inflammatory hyperalgesia taken together with the finding that the production of these cytokines is inhibited by steroidal anti‐inflammatory drugs provides a mechanism of action for these drugs in the treatment of inflammatory hyperalgesia.
Using immunohistochemistry and a panel of monoclonal antibodies, we have compared T-lymphocyte, eosinophil, macrophage, and neutrophil infiltration in bronchial biopsies from 10 intrinsic (nonallergic) asthmatics (IA) and seven extrinsic (allergic) asthmatic (EA), with similar degrees of disease severity. The results were compared with 12 normal healthy nonatopic controls (NC). All subjects were nonsmokers and were not taking oral or inhaled corticosteroids. An intense mononuclear cell infiltrate was identified in IA with an increase in the number of CD45+ cells (total leukocytes), CD3+ and CD4+ lymphocytes, and CD68+ macrophages (p < 0.03, p < 0.01, p < 0.03, and p < 0.03, respectively), compared with NC. Increases were also found in CD4+ (p < 0.05) and CD68+ (p < 0.05) cell numbers between IA and EA. IL-2 receptor-bearing cells (CD25+) and the number of total (MBP+) and actively secreting (EG2+) eosinophils, were also increased in IA compared with NC (p < 0.01, p < 0.01, and p < 0.01, respectively). Similar increases in EG2+ eosinophils and CD25+ (IL-2 receptor-positive) cells were observed in EA (p < 0.01 and p < 0.02, respectively). No differences were detected in the three groups for the number of elastase-positive cells (neutrophils). EG2+ numbers in IA correlated with the Aas asthma symptoms score (r = 0.65, p < 0.05), whereas EG2+ cell numbers in all asthmatics (IA + EA) correlated with airway methacholine responsiveness (r = -0.55, p < 0.03) and with the Aas asthma symptom score (r = 0.54, p < 0.03).(ABSTRACT TRUNCATED AT 250 WORDS)
We have examined the hypothesis that cytokines mediate the enhanced responsiveness of eosinophils to PAF in sensitized mouse skin. PAF (10 ng per site) resulted in a considerable degree of eosinophil accumulation in ovalbumin (OA)-sensitized mice but not in non-sensitized mice. Intradermal preadministration of cytokines (IL-5, IL-3 and GM-CSF) also significantly enhanced PAF-induced migration of eosinophils in a dose-dependent manner. The relative potency with which these cytokines primed cell migration was IL-5 greater than IL-3 greater than GM-CSF, however, each cytokine alone showed no direct effect. We conclude that the sensitization or the exogenous application of cytokines is capable of augmenting PAF-induced eosinophil migration in mice in vivo, and the cytokines thus elicited by sensitization may contribute to the extensive recruitment of inflammatory cells in allergic diseases.
Phagocyte recognition of cells that have undergone apoptosis (programmed cell death) is an event of broad biological significance. Characterized by endogenous endonuclease activation, which results in chromatin fragmentation and nuclear condensation, apoptosis leads to swift ingestion of intact but 'senescent' or 'unwanted' cells by phagocytes in processes as diverse as the physiological involution of organs, the remodelling of embryonic tissues, and metamorphosis. The cell-surface mechanisms by which macrophages recognize apoptotic cells as 'senescent-self' have remained obscure. Here we report that macrophage recognition of apoptotic cells (both neutrophils and lymphocytes) is mediated by the vitronectin receptor, a heterodimer belonging to the beta 3 or cytoadhesin family of the integrins. Previously, the functions of the vitronectin receptor were believed to be limited to cell anchorage, but our findings indicate that the receptor has a novel and direct role in self-senescent-self intercellular recognition leading to macrophage phagocytosis of cells undergoing apoptosis.
Intravenous injection of Sephadex G200 particles into rats on days 0, 2 and 5 caused an increase in eosinophil numbers in blood and lung tissues. Peak numbers were obtained on days 3-12 and thereafter declined to approach control values by day 21. The rise in eosinophil numbers was paralleled by an increase in lung cell fragility as measured by a transient reduction in the number of viable cells isolated from parenchymatous tissue following the digestion of lung fragments in vitro. This decrease in lung cell viability was not seen in rats given a single injection of Sephadex. Dexamethasone, dapsone and isoprenaline given before each injection of Sephadex reduced lung and blood eosinophil numbers and prevented lung cell death. Aspirin and indomethacin were without effect. Incubation of normal lung tissues with disrupted peritoneal eosinophils reduced the numbers of viable cells recovered. No such effects were seen using intact eosinophils and disrupted or intact neutrophils and mononuclear cells. This system provides a model of lung cell damage associated with eosinophil infiltration in vivo.
Changes in eosinophil counts after intrathoracic (it) injection of endotoxin (LPS) were investigated in Wistar rats (150-180 g). Increasing doses of endotoxin (62.5-500 ng/cavity) induced a dose-dependent increase in the number of eosinophils recovered from the rat pleural cavity 24 h later. The eosinophilia was apparent within 24 h, peaked within 48 h (from 0.76 +/- 0.12 to 3.68 +/- 0.51 eosinophils x 10(6)/cavity, P less than 0.001) and returned to basal levels 120 h after the it injection of endotoxin (250 ng/cavity). Endotoxin (3 ng-4 micrograms/ml) failed to attract eosinophils in vitro under conditions in which PAF-acether induced a dose-related response. These findings indicate that endotoxin-induced eosinophil migration in vivo is mediated by a secondary mechanism.
The number of leukocytes rolling along the venular endothelium of the vascular network of the internal spermatic fascia was determined in nondiabetic control rats and diabetic rats with television microscopy. A marked decrease in the number of rolling cells was observed in animals rendered diabetic by the injection of alloxan 10, 30, or 180 days before relative to matching controls. Blood leukocyte counts, however, were equivalent in both control and diabetic rats. Under the influence of a local inflammatory stimulus, cells emerged into the perivascular tissue in control animals, and this was accompanied by a reduction in the number of rolling leukocytes. In diabetic rats, the number of rolling leukocytes remained unaltered, and only a few cells accumulated in the connective tissue adjacent to the vessel. Reversal of the defective leukocyte-endothelial interaction was attained by treatment of diabetic animals with insulin. Inhibitors of arachidonic acid metabolism were ineffective to improve leukocyte-endothelial interactions in diabetic animals. Control rats injected intravenously with lyophilized plasma constituents, obtained after dialysis of diabetic rat plasma with 12,000-Mr retention dialysis tubing, behaved as diabetic animals in that they exhibited a reduced number of leukocytes rolling along the venular endothelium. In contrast, material obtained from control rat plasma produced no effect. Heating of active samples for 1 h at 56 degrees C resulted in the complete loss of the inhibitory effect. We conclude that a substance or substances present in diabetic plasma induce a defective leukocyte-endothelial interaction that further impairs resistance to infection.
EOSINOPHILS, like neutrophils and basophils, are a type of granulocyte derived from bone marrow, distinguished by their morphologic features, constituents, products, and associations with specific diseases. The original denominating property of eosinophils was the cardinal affinity of their cytoplasmic granules for acid aniline dyes, such as eosin. Eosinophils contain several eosinophil-specific proteins in their cytoplasmic granules, yet to date no cell-surface proteins unique to eosinophils have been recognized. Thus, tinctorial properties remain the routine basis for identifying and enumerating these leukocytes in blood and tissues. Eosinophilia, characterized by both heightened production of eosinophils in bone marrow and the accumulation of . . .
The injection of platelet-activating factor (PAF) into the rat pleural cavity resulted in a marked increase in the number of eosinophils, neutrophils and mononuclear cells recovered from pleural washings after 6 hr. Within 24 hr, neutrophils and mononuclear cell counts returned to control values, whereas the eosinophilia peaked and persisted up to 96 hr. Treatment with the PAF antagonist BN 52021 [3-(1,1-dimethylethyl)hexahydro-1,4,7b-trihydroxy-8-alpha-methyl-9H-1,7- alpha-(epoxymethanol)1H,6aH-cyclopenta (c) furo (2,3-5) (3',2':3,4) cyclopenta (1,2-d) furan-5,9,12(4H)trione)] (5-20 micrograms/cavity) or with dexamethasone (1-50 micrograms/cavity) inhibited the early (6 hr) and late (24 hr) pleural eosinophil accumulation induced by PAF. Dexamethasone, but not BN 52021, was still effective in inhibiting the late eosinophilia if administered 5 hr after the lipid, suggesting that the delayed eosinophilia may involve a secondary mechanism, still responsive to the glucocorticoid, but independent of PAF itself. The coinjection of the protein synthesis inhibitor cycloheximide or of the inhibitor of mRNA synthesis DNA-dependent actinomycin D selectively suppressed the eosinophil mobilization at 24 hr. Transfer of the cell-free 6-hr PAF pleural washing from donor to normal recipient rats led to a selective and delayed pleural eosinophilia. This activity was destroyed by heating (+100 degrees C) or freezing (-20 degrees C) the material recovered from the donor pleural fluid. Treatment of donors, but not recipients, with either BN 25021, dexamethasone, actinomycin D or cycloheximide inhibited the late eosinophil accumulation triggered by transferred PAF pleural washing, indicating that the generation of this eosinophilotactic activity, but not its effects, is suppressed by those agents.(ABSTRACT TRUNCATED AT 250 WORDS)
Eosinophils (EOS) may play an important role in the pathophysiology of bronchial asthma because they can release oxygen free radicals and several basic proteins which are cytotoxic to bronchial epithelium. We have studied the response of EOS isolated from the blood of atopic subjects with symptoms of asthma (AS, n = 7) or rhinitis (AR, n = 4) or without symptoms (AA, n = 5) and of subjects with the hypereosinophilic syndrome (HES, n = 5). EOS were separated using metrizamide density gradients and activated in vitro with platelet-activating factor (PAF, 100 nM) or phorbol 12-myristate-13-acetate (PMA, 100 nM). Oxygen free radical generation was assessed by a lucigenin-enhanced chemiluminescence (CL) assay. EOS purity was 83 +/- 1.7% (mean +/- SEM) with greater than 95% viability. Background CL responses of EOS from HES were significantly higher than those from AA (p less than 0.01) and AR (p less than 0.05). Normodense EOS from AS (PAF-induced CL = 90 +/- 27 mV) were more responsive to PAF than were those from AR (17 +/- 13 mV, p less than 0.01) and from AA (23 +/- 14 mV, p less than 0.01). Similar results were obtained with PMA. Hypodense EOS from HES subjects were as responsive as normodense EOS from AS to PMA and PFA. Thus, EOS from AS have an enhanced potential for activation, particularly by PAF; this may represent an important mechanism for the perpetuation of the inflammatory response in asthma, since EOS can also release PAF.
Many cell types are known to synthesize nitric oxide (NO.) from L-arginine. There appear to be at least two forms of NO. synthase: an inducible, tetrahydrobiopterin- and flavin-dependent activity exemplified by the macrophage enzyme and a constitutive, Ca+(+)-dependent activity exemplified by the endothelial cell enzyme. L-NG-methylarginine inhibits NO. synthesis by both cell types. We now report that L-NG-aminoarginine and L-NG-nitroarginine are about 100-fold more potent than NG-methylarginine in blocking endothelial cell NO. synthesis. In contrast, NG-aminoarginine and NG-methylarginine are about equipotent with macrophages whereas NG-nitroarginine is much less potent. Since macrophage and endothelial cell NO. synthesis are differentially sensitive to the inhibitors, the panel of inhibitors can be used in complex biological systems to determine if macrophage-like or endothelial-like cells are the predominant source of NO.. Indeed, all three inhibitors elicit a strong pressor response in the anesthetized guinea pig, a result consistent with the view that endothelial cells continually produce vasodilatory NO(.).
The intravenous injection of Sephadex particles (G200) into rats produced a specific increase in numbers of blood eosinophils peaking 7 days later. A second injection, given on day 14 when the numbers of blood eosinophils had fallen to control levels, produced a dose-dependent increase in numbers, greater than the first, and peaking 5 days later. At this time there was a dose-dependent increase in numbers of eosinophils, but not of other leucocytes, in broncho-alveolar lavage fluids and lung tissue, together with an increase in sensitivity of the rats to the respiratory effect produced by the intravenous injection of 5-hydroxytryptamine.
The present study examined the potential role of IL-1 and TNF in granuloma formation. Mice were given i.v. injections of Schistosoma mansoni eggs or Sephadex beads to induce synchronous immune T cell-mediated (hypersensitivity type) or nonimmune (foreign-body type) granulomas, respectively. Granuloma macrophages isolated at 2, 4, 8, 16, and 32 days of granuloma formation were evaluated for their capacity to produce IL-1 and TNF in response to 1 microgram/ml LPS. This was related to circulating levels of the acute phase protein, serum amyloid P (SAP) and expression of Ia Ag by monocytes and macrophages. Macrophages from nonimmune bead lesions were generally weak producers of IL-1 and TNF. In contrast, those from T cell-mediated egg lesions produced significant levels of both monokines. Moreover, there was a clear pattern of sequential monokine production such that IL-1 was produced in greatest amounts early (2 to 4 days), whereas TNF was produced later (8 to 16 days). Levels of SAP showed an initial sharp rise following particle embolization, then decreased rapidly in bead injected animals. However, mice with immune granulomas showed a prolonged elevation in SAP levels that corresponded to the period of maximal IL-1 production (2 to 4 days). Macrophage/monocyte Ia Ag expression was greatest at 8 to 16 days, corresponding to the period of TNF production. Bead injected animals showed low levels of Ia expression over the study period. These findings suggest that IL-1 may be important in the early recruitment stages of granuloma formation while TNF may take part in later maintenance or effector functions. The extent of production of both is likely influenced by the local or systemic milieu of lymphokines.
HUMAN neutrophilic polymorphonuclear leukocytes (neutrophils) provide an effective host defense against bacterial and fungal infection, but they are also important in the pathogenesis of tissue damage in certain noninfectious diseases. Some important events in neutrophil function that will be discussed in this review are shown in Figure 1. Mild to moderate abnormalities of neutrophil function have been reported in many acquired and congenital diseases.1 2 3 4 5 In most of these disorders, the biochemical or morphologic basis of the defects is unknown and the relevance of the neutrophil defect to the manifestations of the disease is unclear. In contrast, persons with marked neutropenia6 or . . .
A variety of lung disorders are associated with the accumulation of eosinophils in the alveolar structures. To help understand the role of eosinophils in these disorders, an animal model of eosinophilic lung disease was developed. Administration of an aerosol of polymyxin B to guinea pigs (3 times per wk for 4 wk) produced diffuse interstitial lung disease with alveolar wall thickening and an alveolitis characterized by marked increases in eosinophils and alveolar macrophages. Bronchoalveolar lavage confirmed the presence of significantly increased numbers of eosinophils and alveolar macrophages in polymyxin-B-treated animals compared with those in control animals. Using density gradient centrifugation, approximately 10(7) eosinophils could be purified from the lungs of a single polymyxin-B-treated animal. Importantly, eosinophils purified from the lungs from polymyxin B-treated animals exhibited significant spontaneous cellular cytotoxicity for human fetal lung fibroblasts. In contrast, neither eosinophils from control animals nor alveolar macrophages from either group of animals were cytotoxic. These findings demonstrate that eosinophils possess effector processes capable of injuring the lung parenchyma and suggest that eosinophils can contribute to the pathogenesis of the interstitial lung disease.
The effect of rat antimacrophage serum (rAMS) was tested on the influence of normal or thioglycollate-stimulated macrophage populations of the rat peritoneal cavity on the migration of polymorphonuclear neutrophils (PMN) induced by carrageenin, heterologous serum (rabbit) and sheep red blood cells. The rAMS used did not cross-react with PMN or lymphocytes nor did it affect circulating white cells, complement levels or lysed PMN present in the inflammatory exudate. It did, however, give a positive immunofluorescence reaction with resident and stimulated macrophages. The rAMS inhibited macrophage function as tested by sheep red blood cell phagocytosis in vivo and release of a PMN chemotactic factor(s) in vitro. Thioglycollate-stimulated peritoneal cavities showed an increased macrophage population and responded with increased PMN migration when challenged with heterologous serum or carrageenin, as compared with control rats. The presence of rat antimacrophage antibodies inhibited PMN migration induced by heterologous serum, sheep red blood cells and carrageenin. It is concluded that resident macrophages participate in the control of PMN migration to the site of an acute inflammation by acting as 'alarm cells' and triggering several defence mechanisms which ultimately protect the host from injurious stimuli.
Chemotactic, phagocytic, and oxidative metabolic activity of exudative leukocytes was measured in patients with Crohn's disease (n = 20) and with ulcerative colitis (n = 20). Unstimulated and casein-stimulated migration in Boyden chambers did not differ from that of healthy controls (n = 21). Patients with Crohn's disease had reduced serum-independent phagocytosis compared with healthy controls (p less than 0.01) and patients with ulcerative colitis (p less than 0.01). Serum-dependent phagocytosis by leukocytes from patients with Crohn's disease did not differ from that in controls but was slightly increased in patients with ulcerative colitis (p less than 0.02). Unstimulated leukocytes showed increased oxidative metabolic activity in both patient groups compared with controls (p less than 0.01), which was negatively correlated with the disease activity in Crohn's disease (p less than 0.02). The study shows that mobilized leukocytes from patients with Crohn's disease differ from those mobilized in ulcerative colitis and supports the concept of an abnormal inflammatory reaction in Crohn's disease.
Intravenous injection of dextran in the form of beads regularly induces eosinophil leukocytosis in rats. The response has characteristics of an immunological reaction.
The purpose of this investigation was to measure the radius and wall thickness of small blood vessels in mesentery and striated (cremaster) muscle in the anesthestized rat. Using the method of image-splitting, the vessel images were sheared at magnifications of 3,OOOx and 6,500x on the video screen.
In the resting state, the mean lumen values for the thinnest portion of endothelial capillary were 4.1 ± 1.2µ (SD) in cremaster and 5.6 ± 1.3µ (SD) in mesentery. Lumen and total diameter were also greater at several levels of precapillary arterioles in mesentery than similar vessels in cremaster, suggesting that smaller vessel size in cremaster might be characteristic of this tissue. Vasoconstriction of up to 50% from resting state is associated with an increase in wall thickness and a decrease in lumen to wall ratio due to a disproportional decrease in radius (inner greater than outer radius). Similarly, an increase in vessel radius in a proportion 4 to 1 (inner greater than outer) was sustained up to about 60% of vasodilatation.
In 13 out of 14 vessels, little or no change in wall cross-sectional area occurred in the face of marked changes in lumen cross-sectional area (–95%,+205%). This strongly suggests that other modifications such as changes in length, swelling, or shrinkage would be relatively unimportant during dynamic changes in wall thickness.
Neutrophils constitute over 90% of cells found in the synovial fluid of rheumatoid arthritis (RA) patients. Since such fluids also contain immune complexes (IgG-IgG and IgG-IgM rheumatoid factors) and complement split products (C5, C5A, DES, ARG, C3B, etc.), all of the reactants are present for a local Arthus lesion. Moreover, neutrophils from RA patients endocytose these immune complexes and complement components in vivo and in vitro. In consequence, it has been suggested that lysosomal enzymes and other mediators of inflammation released by neutrophils after uptake of immune complexes (in the bulk phase or on the surface) account, at least in part, for rheumatoid inflammation. Secretion of lysosomal hydrolases, especially neutral proteases, which provoke tissue injury and generation of reactive oxygen species (e.g. O2) is part of a stimulus-secretion response to a variety of secretagogues, including immune complexes and complement components. However, the pathways of secretion and O2 generation are stimulus-specific and can be dissected to establish cause and effect relationships by (a) kinetic analysis, (b) varying the stimulus, (c) use of impermeant reagents to block discrete responses. Neutrophils also generate products of 11-cyclo-oxygenase (e.g., PGE2, TXA2) and of the 5- and 15-lipoxygenase (mono-, di and tri-hetes, LTB4 and their isomers). However, the cyclo-oxygenase products (except TXA2) do not cause inflammation acting alone; indeed, they inhibit the function of neutrophils, platelets, macrophages and mast cells. The most potent proinflammatory agent yet identified as a product of arachidonate is LTB4. LTB4 is a potent Ca ionophore, a strong chemo-attractant, induces local inflammation, and activates neutrophils.
The effects of steroids on the development of injury in two models of experimental glomerulonephritis (GN), (one mediated by neutrophils, the other by macrophages) were compared. The neutrophil-associated lesion [initiated by heterologous antiglomerular basement membrane (GBM) antibody] was characterized by the development of an exudative endocapillary GN with heavy neutrophil accumulation [mean, 6.9 neutrophils/glomerular cross section (N/GCS) +/- 2.9 SD], minor macrophage infiltration [7.9 macrophages/glomerulus (M/G) +/- 2.2 SD] and heavy proteinuria (1905 mg/24 hr +/- 520 SD). Steroid-treated (methylprednisolone, 2 mg/kg/12 hr i.v.) rabbits developed a marked monocytopenia, mild neutrophilia, and significant reduction in glomerular macrophage accumulation (0.3 M/G 0.02 SD). However, neutrophil accumulation (6.1 N/CGS +/- 2.5 SD), histological appearances, and proteinuria (1820 mg/hr +/- 490 SD) were unaffected. The macrophage-associated model of GN was induced by passive autologous rabbit anti-sheep IgG 15 hr after the injection of a subnephritogenic dose of the same anti-GBM antibody. The glomerular lesion was characterized by a diffuse endocapillary proliferative GN with heavy macrophage infiltration (54 M/G +/- 8 SD), insignificant neutrophil accumulation (0.8 N/GCS 0.02 SD), and the regular development of proteinuria (420 mg/24 hr +/- 80 SD). Steroid-treated rabbits developed a mild neutrophilia and a significant monocytopenia associated with abrogation of glomerular macrophage accumulation (2.3 M/G +/- 0.8 SD). This was associated with the prevention of the development of GN and proteinuria (22 +/- 9.5 SD). Thus, steroids produce monocytopenia and prevent glomerular macrophage accumulation and associated injury whereas neutrophil accumulation and injury is unaffected. These data suggest steroids may have widely varying effects on the outcome of leukocyte-associated experimental GN depending on the nature of the infiltrating cells.