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Modulation of IL-1 -Induced Neutrophil Migration by Dexamethasone and Lipocortin 1
Abstract
IL-1 is a pro-inflammatory cytokine which controls many features of the immune and inflammatory response. When injected into a mouse 6-day-old air-pouch, human rIL-1 (1 to 100 ng) induced in a dose-dependent fashion a migration of PMN that could be reliably assessed 4 h after injection. Both IL-1 alpha and IL-1 beta were active in this model. The effect of the cytokine was inhibited by local administration of actinomycin D (1 to 10 micrograms), alpha-melanocyte-stimulating hormone (200 micrograms), and a mAb recognizing IL-1R type I (10 micrograms). Indomethacin (1 mg/kg), an inhibitor of cyclo-oxygenase, and BW4AC (2 mg/kg), a selective lipoxygenase inhibitor, were without effect but moderate inhibition was seen with the platelet-activating factor antagonist WEB2086 (1 to 10 mg/kg). The glucocorticoid dexamethasone (0.015 to 1.5 mg/kg) potently inhibited the elicitation of neutrophils induced by IL-1 when given systemically 2 h before the cytokine. The steroid-induced anti-inflammatory protein lipocortin 1 (LC1) also produced a dose-dependent inhibition of PMN migration into the pouch with an ED50 of approximately 0.15 to 0.21 mg/kg. The denatured protein was without effect. Passive immunization of mice with a polyclonal sheep antiserum or a mAb raised against LC1 abolished the inhibitory action of dexamethasone whereas preimmune serum or control IgG were without significant effect. These findings provide further evidence that LC1 is involved in the anti-inflammatory action of glucocorticosteroids and suggest that this protein may act as an endogenous regulator of IL-1 action.
... Annexin A1 (AnxA1) was originally identified as a glucocorticoid-regulated protein and is an endogenous anti-inflammatory mediator during the resolution phase of inflammation. It is particularly abundant in various cells of the host immune system, including monocytes, macrophages, T lymphocytes and neutrophils (4,5,24,25). AnxA1 can be mobilized from cytoplasm to membrane and is eventually released in abundance from neutrophils after activation or after adherence to endothelial cells (4,24,25). ...
... It is particularly abundant in various cells of the host immune system, including monocytes, macrophages, T lymphocytes and neutrophils (4,5,24,25). AnxA1 can be mobilized from cytoplasm to membrane and is eventually released in abundance from neutrophils after activation or after adherence to endothelial cells (4,24,25). Functionally, AnxA1 has been shown to attenuate leukocyte recruitment in many experimental inflammatory models by inhibiting cell adhesion and transmigration (22,24). The important anti-inflammatory role of AnxA1 in the normal defense mechanisms of the body has been further supported by animal studies showing that AnxA1-null mice are more prone to both acute and chronic inflammatory reactions (6,8,15,17,18,26,36,39). ...
... AnxA1 can be mobilized from cytoplasm to membrane and is eventually released in abundance from neutrophils after activation or after adherence to endothelial cells (4,24,25). Functionally, AnxA1 has been shown to attenuate leukocyte recruitment in many experimental inflammatory models by inhibiting cell adhesion and transmigration (22,24). The important anti-inflammatory role of AnxA1 in the normal defense mechanisms of the body has been further supported by animal studies showing that AnxA1-null mice are more prone to both acute and chronic inflammatory reactions (6,8,15,17,18,26,36,39). ...
Annexin A1 (AnxA1), originally identified as a glucocorticoid-regulated protein, is an impor- tant endogenous anti-inflammatory mediator during the resolution phase of inflammation, and its cir- culating level has been rarely studied in sepsis patients. Glucocorticoid has been extensively used in treating patients with sepsis. However, it is unclear whether endogenous cortisol or exogenous glucocor- ticoid contributes to the regulation of AnxA1 levels in peripheral blood of sepsis patients. The aim of this study was to investigate: [1] serial changes over time in the plasma levels of AnxA1 and cortisol in sepsis patients; and [2] prognostic value of AnxA1 level in the survival of sepsis patients. Fifty-eight adult sepsis patients admitted to an intensive care unit (ICU) were enrolled. The plasma levels of cortisol and AnxA1 were determined by specific enzyme-link immunosorbent assay. Results show that the median daily levels of cortisol at the 1st, 3rd, 5th and 7th day after admission to ICU were signifi- cantly elevated over the cortisol level of the control subjects. However, the AnxA1 level was elevated in only thirty-three patients (56%) over the observation period. There was no significant correlation between cortisol levels and AnxA1 levels. Further analysis indicated that steroid treatment resulted in significant elevation of the cortisol level over time, but did not affect the AnxA1 level. AnxA1 levels were also not statistically different between surviving and non-surviving patients. In conclusions, the circu- lating level of AnxA1 is elevated in a subgroup of sepsis patients, and the AnxA1 level does not correlate with the cortisol level in the peripheral blood of sepsis patients.
... This model can also be used to assess the role of live bacteria and bacterial products in the promotion or inhibition of inflammatory and biological responses (Jones et al., 2020;Koh et al., 2015), as well as screening of potential anti-inflammatory compounds (Li et al., 2021) or therapeutic molecules. Efficacy of therapeutic agents, antagonists or agonists can be assessed by either by local injection in the air pouch to examine direct interactions (Herrera et al., 2015;Koh et al., 2015;Perretti & Flower, 1993), or inoculation intravenously or interperitoneally (Perretti & Flower, 1993) to examine cell recruitment and activity to the air pouch prior to a stimulus of interest. The use of mice with genetic mutant backgrounds also allows investigation of the role of specific host cell pathways in inflammation and immune cell function. ...
... This model can also be used to assess the role of live bacteria and bacterial products in the promotion or inhibition of inflammatory and biological responses (Jones et al., 2020;Koh et al., 2015), as well as screening of potential anti-inflammatory compounds (Li et al., 2021) or therapeutic molecules. Efficacy of therapeutic agents, antagonists or agonists can be assessed by either by local injection in the air pouch to examine direct interactions (Herrera et al., 2015;Koh et al., 2015;Perretti & Flower, 1993), or inoculation intravenously or interperitoneally (Perretti & Flower, 1993) to examine cell recruitment and activity to the air pouch prior to a stimulus of interest. The use of mice with genetic mutant backgrounds also allows investigation of the role of specific host cell pathways in inflammation and immune cell function. ...
Neutrophils are an important part of the innate immune system and among the first cells to respond to infections and inflammation. Responses include chemotaxis towards stimuli, extravasation from the vasculature, and antimicrobial actions such as phagocytosis, granule release, reactive oxygen species (ROS) production, and neutrophil extracellular trap (NET) formation (NETosis). Studying how neutrophils respond to a variety of stimuli, from biomaterial interactions to microbial insults, is therefore an essential undertaking to fully comprehend the immune response. While there are some immortalized cell lines available that recapitulate many neutrophil responses, ex vivo or in vivo studies are required to fully understand the complete range of neutrophil phenotypes. Here we describe two protocols for neutrophil isolation for further ex vivo study: recovery of neutrophils from human peripheral blood, and isolation of neutrophils from the oral cavity. We also discuss an in vivo model of general inflammation with the murine air pouch that can be used to assess numerous parameters of neutrophil and immune activation, including neutrophil recruitment and biological activity. In these protocols, the cells are isolated to allow for a high degree of experimental control. The protocols are relatively straightforward and can be successfully used by labs with no prior primary cell experience. © 2023 Wiley Periodicals LLC.
Basic Protocol 1 : Neutrophil isolation from human blood
Basic Protocol 2 : Neutrophil isolation from the oral cavity
Basic Protocol 3 : Murine air pouch model of general inflammation
... The endogenous cytokine IL-1 which can be generated from a variety of cells in response to a wide range of stimuli may also be generated in the peritoneal cavity. IL-1 is known to be a potent mediator of neutrophil accumulation in vivo in rabbit skin , mouse peritoneal cavity (Sayers et a l , 1988) and mouse air pouch (Perretti & Flower, 1993). The endogenous production of IL-1 has been demonstrated after challenge with casein, turpentine and zymosan (Goto et a l , 1984;Gershenwald et a l , 1990;Perretti et a l , 1992). ...
... possibly other cell types) to be exposed and so possibly affected directly. In contrast, neutrophils would not be affected directly by local steroids and thus the balance of steroid action may tip towards the contribution and importance of the inhibition of mediator generation. Several studies have provided evidence which may support such a theory.Perretti & Flower (1993) using a model of IL-1-induced inflammation in mouse air pouch found that systemic but not local lipocortin inhibited neutrophil accumulation whereas both systemic (via lipocortin induction) and local dexamethasone were inhibitory. These authors concluded that systemic inhibition was likely to be mediated by an effect on the circulating ...
A characteristic feature of acute inflammation is increased microvascular permeability. Mediators that increase microvascular permeability can act in 2 ways; either directly on the endothelial cell eg. bradykinin, or by a mechanism dependent on circulating neutrophils eg. the chemoattractant FMLP. Oedema can be suppressed by both steroid and non-steroid anti-inflammatory drugs. These compounds may be able to act at several levels. This study was designed to investigate mechanisms of oedema formation and the possible sites of action of anti-inflammatory compounds. FMLP-induced oedema formation was not dependent on endogenous histamine release or pro-inflammatory products of the cyclo-oxygenase pathway. Intravenous infusion of zymosan-activated plasma produced transient neutropenia in rabbits which resulted in inhibition of oedema formation induced by FMLP, but not that induced by bradykinin. Ibuprofen, selectively inhibited FMLP-induced oedema formation when administered intravenously. This drug did not induce neutropenia and the effect was independent of cyclo-oxygenase inhibition. Ibuprofen may interfere with the interaction between circulating neutrophils and venular endothelial cells. The microtubule blocking agent colchicine also selectively inhibited FMLP- induced oedema formation even when it was administered at intervals after intradermal FMLP. This suggests that continuing interactions between functionally active neutrophils and endothelial cells are necessary for the protracted plasma protein leakage induced by this chemoattractant. There is evidence that anti-inflammatory steroids may owe some of their anti-inflammatory actions to effects on the target cells of inflammatory mediators eg. microvascular endothelial cells 2md neutrophils. In an attempt to investigate this possibility in vivo the ability of dexamethasone to modulate oedema formation and ¹¹¹In-neutrophil accumulation in rabbit skin was investigated. Dexamethasone was effective when administered in three ways. Firstly, local pretreatment of skin with dexamethasone inhibited oedema responses but not ¹¹¹In-neutrophil accumulation. Secondly, treatment of neutrophil recipient rabbits intravenously with dexamethasone inhibited both oedema responses and ¹¹¹In-neutrophil accumulation in response to exogenous chemoattractants. Thirdly, systemic treatment of neutrophil donor animals with dexamethasone resulted in suppression of ¹¹¹In-neutrophil accumulation in response to intradermal chemoattractants in recipients. Systemic treatment with dexamethasone suppressed neutrophil accumulation oedema formation and the generation of LTB₄ induced by intraperitoneal-injection of zymosan in the rabbit. The generation of TXB₂ and prostacyclin were not inhibited by dexamethasone. Depleting animals of circulating neutrophils inhibited the generation of LTB₄ suggesting that accumulating neutrophils are responsible for the generation of this chemoattractant. This study shows that anti-inflammatory compounds can inhibit oedema formation and neutrophil-accumulation by acting at several different sites. There is evidence for inhibition by effects on the microvascular endothelial cell and the neutrophil.
... Pharmacological intervention of AnxA1 has been shown to decrease neutrophil rolling and adhesion to endothelium while increasing detachment of adherent and inducing neutrophil apoptosis [23]. Perretti and Flower (1993) demonstrated that AnxA1 attenuated IL-1 and IL-8 induced neutrophil migration into the murine air pouch [24]. Additionally, Getting et al. showed that both endogenous and exogenous AnxA1 were able to inhibit the neutrophil and monocyte recruitment in murine peritoneal cavity [25]. ...
... Pharmacological intervention of AnxA1 has been shown to decrease neutrophil rolling and adhesion to endothelium while increasing detachment of adherent and inducing neutrophil apoptosis [23]. Perretti and Flower (1993) demonstrated that AnxA1 attenuated IL-1 and IL-8 induced neutrophil migration into the murine air pouch [24]. Additionally, Getting et al. showed that both endogenous and exogenous AnxA1 were able to inhibit the neutrophil and monocyte recruitment in murine peritoneal cavity [25]. ...
Cardiovascular disease (CVD) continues to be the leading cause of death in the world. Increased inflammation and an enhanced thrombotic milieu represent two major complications of CVD, which can culminate into an ischemic event. Treatment for these life-threatening complications remains reperfusion and restoration of blood flow. However, reperfusion strategies may result in ischemia–reperfusion injury (I/RI) secondary to various cardiovascular pathologies, including myocardial infarction and stroke, by furthering the inflammatory and thrombotic responses and delivering inflammatory mediators to the affected tissue. Annexin A1 (AnxA1) and its mimetic peptides are endogenous anti-inflammatory and pro-resolving mediators, known to have significant effects in resolving inflammation in a variety of disease models. Mounting evidence suggests that AnxA1, which interacts with the formyl peptide receptor (FPR) family, may have a significant role in mitigating I/RI associated complications. In this review article, we focus on how AnxA1 plays a protective role in the I/R based vascular pathologies.
... En favorisant la production de lipocortine1, les glucocorticoïdes inhibent la synthèse de phospholipase A2 et ainsi, la cascade de l'acide arachidonique permettant de réduire la production des leucotriènes pro-inflammatoires [138]. Enfin, la synthèse de la COX-2 [139] et de la NO synthase inductible [140] est aussi inhibée par les corticoïdes (Figure 11). ...
... Le complexe interagit avec l'ADN au niveau de sites accepteurs appelés « Glucocorticoids-Responsive-Elements » ou GRE, et exerce ainsi soit une activation soit une inhibition de la transcription. Lorsque la transcription est activée il se produit une augmentation de production de protéines anti-inflammatoires comme la lipocortine-1 (ou annexine-1), IL 10, protéine IkB [138], [159], [160], cette dernière participe aussi à la transrépression en empêchant la translocation nucléaire de NF-κB et sa fixation sur la séquence NF-κB responsive element [161]. A l'inverse lorsque la transcription est inhibée par régulation négative directe via un site de liaison négatif ou nGRE, la transcription des gènes de l'inflammation comme l'interleukine-1 (IL-1) ou l'interleukine-2 (IL-2) est réprimée par l'interaction directe du RG activé avec des GREs négatifs [162]. ...
Le choc septique est la principale cause de mortalité dans les services de réanimation. Les glucocorticoïdes (GC) et la protéine C activée (APC) sont deux traitements adjuvants recommandés au cours du choc septique. Ce travail a pour objectif d'évaluer l'impact de la combinaison d'APC et des GC sur les paramètres hémodynamiques et la survie. Le sepsis expérimental se caractérise par une hypotension artérielle avec acidose lactique et une hyporéactivité vasculaire. L'administration de Dexa et/ou d'APC permet de diminuer les taux de lactates, d'interleukines et de nitrate/nitrite. Chez les groupes traités, la contraction est améliorée ainsi que la relaxation vasculaire des aortes et des artères mésentériques. L'administration d'APC et de Dexa, seul ou en association, entraine une diminution de l'expression induite d'iNOS et la restauration de la voie Akt. La combinaison APC et Dexa améliore le temps de survie de façon synergique. Nos résultats suggèrent que l'APC et les GC devraient être réévalués en association dans le traitement du choc septique
... LC1 is the first member of the annexin or lipocortin superfamily of phospholipid-and calcium-binding proteins to have been cloned (36), and it is widely accepted that its synthesis and disposition can be modulated by glucocorticoids as well as by the cytokine interleukin-6 (36)(37)(38)(39). Recently, we have demonstrated that administration of LC1 to experimental animals reduced the extravasation of blood-borne neutrophils in simple models of acute inflammation (16,40), apparently by interfering with the process of neutrophil interaction with the activated endothelium (19). Antibodies against LC1 prevented the antimigratory action displayed by DEX (18,20,40). ...
... Recently, we have demonstrated that administration of LC1 to experimental animals reduced the extravasation of blood-borne neutrophils in simple models of acute inflammation (16,40), apparently by interfering with the process of neutrophil interaction with the activated endothelium (19). Antibodies against LC1 prevented the antimigratory action displayed by DEX (18,20,40). Other groups have confirmed these observations (17,21). ...
... In vivo the Ac2-26 peptide has been shown to exert an anti-inflammatory effect in models of myocardial ischaemia reperfusion (I/R) (La, D'Amico et al. 2001), mesentery I/R (Gavins, Yona et al. 2003), glycogen peritonitis (Teixeira, Das et al. 1998) and IL1β airpouch (Perretti, Ahluwalia et al. 1993), where it was reported to significantly reduce the recruitment of neutrophils to the site of injury/inflammation (Perretti and Flower 1993). In other more complex acute models such as zymosan peritonitis apart from a reduction in PMN recruitment at an early time point (4h) a reduction of monocyte recruitment into the peritoneal cavity after 24h was also reported (Getting, Flower et al. 1997). ...
... As described in Chapter 1, Annexin A1 was initially identified as a glucocorticoid inducible protein with the ability to downregulate phospholipase A2 (PLA2) and thus arachidonic acid synthesis (Cirino, Peers et al. 1989). Since then studies conducted by a number of laboratories have shown that this protein, at least in part, exerts its anti-inflammatory effects by regulating cell recruitment to the site of inflammation (Errasfa, Rothhut et al. 1985;Perretti and Flower 1993;Lim, Solito et al. 1998;Hayhoe, Kamal et al. 2006). Subsequent studies, have highlighted that the anti-inflammatory effects of Annexin A1 extend beyond inhibition of cell recruitment to the site of the inflammatory insult. ...
... Administration of nebulized β 2 -agonists (continuous or repetitive), such as salbutamol, causes bronchodilation and is considered a first-line treatment for the management of asthma (NIH, 1997;Wort, 2003). Corticosteroids are the most commonly prescribed therapeutics for controlling nearly all types of inflammatory reactions and exert strong effects on leukocyte recruitment when administered orally or systemically (Harris, 1972;Flower, 1988;Perretti, Flower, 1993). The most striking effect of corticosteroids is their ability to inhibit the expression of multiple inflammatory mediators whose genes are regulated by transcription factors, such as nuclear factor-kB (NF-kB) (Barnes, 1998). ...
... A systematic review conducted by our group showed the anti-eosinophilic effects of medicinal plants and plant-derived substances in eosinophilic models, including models of acute peritonitis induced by either the polysaccharide-rich F1 fraction from Histoplasma capsulatum yeast or by Toxocara canis helminth infection, also known as visceral larva migrans syndrome (VLMS) or toxocariasis, and the classical ovalbumin-induced allergic asthma model (Rogerio, Sá-Nunes, Faccioli, 2010). Eosinophils produce cytokines (e.g., IL-4, IL-5 and IL-13), chemokines (e.g., CCL11) (Perretti, Flower, 1993;Barnes, 1998;Barnes, 2006), lipid mediators (LTB 4 ) and principal cationic proteins (major basic protein, eosinophil-derived neurotoxin, eosinophil cationic protein and eosinophil peroxidase) that can exacerbate airway inflammation and cause tissue damage (Rothenberg, Hogan, 2006;Gleich, Loegering, 1984). Our group was the first to demonstrate that quercetin (10 mg/kg; oral dose) reduces eosinophils in the blood, bronchoalveolar lavage fluid (BALF) and the pulmonary parenchyma in a murine model of ovalbumininduced allergic airways inflammation (Rogerio et al., 2007). ...
Allergic asthma is a complex inflammatory disorder characterized by airway hyperresponsiveness, eosinophilic inflammation and hypersecretion of mucus. Current therapies include β2-agonists, cysteinyl leukotriene receptor 1 antagonists and corticosteroids. Although these drugs demonstrate beneficial effects, their adverse side effects limit their long-term use. Thus, the development of new compounds with similar therapeutic activities and reduced side effects is both desirable and necessary. Natural compounds are used in some current therapies, as plant-derived metabolites can relieve disease symptoms in the same manner as allopathic medicines. Quercetin is a flavonoid that is naturally found in many fruits and vegetables and has been shown to exert multiple biological effects in experimental models, including the reduction of major symptoms of asthma: bronchial hyperactivity, mucus production and airway inflammation. In this review, we discuss results from the literature that illustrate the potential of quercetin to treat asthma and its exacerbations.
... As a calcium-dependent phospholipid-binding protein, AnxA1 is reported to inhibit leukocyte migration to the site of inflammation and aseptic inflammation (8). Ac2-26, which is the pharmacologically active center of AnxA1, was synthesized by Cirino et al (18) as early as 1993 and it has been shown to effectively inhibit the activation of the transcription factor NF-κB and the subsequent production of proinflammatory cytokines, such as TNF-α and IL-1β (19). Although previous studies (20,21) have confirmed that Ac2-26 exhibits anti-inflammatory effects and stability in the blood circulatory system, the inability to quantify it in serum or plasma is a limitation of the present study. ...
Inflammation is one of the most crucial mechanism underlying hepatic ischemia-reperfusion injury (HIRI). Several studies have shown that Ac2-26, the active N-terminal peptide of Annexin A1, could modulate anti-inflammatory processes and protect the organs from ischemia-reperfusion injury (IRI). However the effects of Ac2-26 on an HIRI model have not been reported to date. The purpose of the present study was to determine whether Ac2-26 pretreatment could protect hepatocytes against acute HIRI by inhibiting neutrophil infiltration through regulation of the high mobility group box protein 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. To this end, a total of 72 adult C57BL/6 mice were randomly divided into sham operation (sham), ischemia-reperfusion (I/R), I/R + Ac2-26 and Ac2-26 groups. The HIRI model was established by occluding the branch of the hepatic pedicle to the left and median liver lobes with an atraumatic vascular clamp for 45 min, followed by reperfusion for 24 h. The expression of HMGB1, TLR4, NF-κB, IκBα and lymphocyte antigen 6 complex locus G6D (Ly6G) was detected using reverse transcription-quantitative PCR, western blotting and immunohistochemical staining; serum levels of HMGB1 were evaluated using an enzyme-linked immunosorbent assay. Flow cytometry was used to detect the proportion of neutrophil. The results indicated that Ac2-26 preconditioning rescued hepatocyte dysfunctions induced by HIRI. In addition, HIRI was associated with a significant increase in HMGB1 expression and release, accompanied by increased expression of TLR4, which was significantly inhibited by Ac2-26. Furthermore, the expression of phosphorylated (p)-NF-κB and the ratio of p-NF-κB to NF-κB were markedly increased, while the expression of IκBα was decreased in the I/R group compared with those in the sham group; however, these effects were reversed by Ac2-26 administration. Additionally, Ac2-26 administration significantly inhibited neutrophil infiltration and resulted in low levels of neutrophils and Ly6G as well as reduced myeloperoxidase activity. Taken together, these results indicated that Ac2-26 pretreatment serves a protective role against HIRI by regulating the HMGB1/TLR4/NF-κB signaling pathway and inhibiting neutrophil infiltration.
... Annexin proteins bind to negatively charged phospholipids in a Ca 2+ -dependent manner and have been recognized for their role in membrane repair, in addition to cell migration and adhesion (17)(18)(19)(20)(21)(22). In the setting of muscle membrane repair, a repair cap forms at the site of injury. ...
Membrane instability and disruption underlie myriad acute and chronic disorders. Anxa6 encodes the membrane-associated protein annexin A6 and was identified as a genetic modifier of muscle repair and muscular dystrophy. To evaluate annexin A6's role in membrane repair in vivo, we inserted sequences encoding green fluorescent protein (GFP) into the last coding exon of Anxa6. Heterozygous Anxa6gfp mice expressed a normal pattern of annexin A6 with reduced annexin A6GFP mRNA and protein. High-resolution imaging of wounded muscle fibers showed annexin A6GFP rapidly formed a repair cap at the site of injury. Injured cardiomyocytes and neurons also displayed repair caps after wounding, highlighting annexin A6-mediated repair caps as a feature in multiple cell types. Using surface plasmon resonance, we showed recombinant annexin A6 bound phosphatidylserine-containing lipids in a Ca2+- and dose-dependent fashion with appreciable binding at approximately 50 μM Ca2+. Exogenously added recombinant annexin A6 localized to repair caps and improved muscle membrane repair capacity in a dose-dependent fashion without disrupting endogenous annexin A6 localization, indicating annexin A6 promotes repair from both intracellular and extracellular compartments. Thus, annexin A6 orchestrates repair in multiple cell types, and recombinant annexin A6 may be useful in additional chronic disorders beyond skeletal muscle myopathies.
... This identified two gene sets which were significantly underrepresented (neutrophil-mediated immunity and antibacterial Dual RNA-seq in Experimental Pneumococcal Infection humoral response) and one gene set which was overrepresented (positive regulation of neutrophil extravasation). The fold changes between the pleural and neutrophil samples of the individual genes contained in these different gene sets are shown in Fig. 3. Genes significantly upregulated within the positive regulation of neutrophil extravasation included CD99L, a murine homolog of human CD99 that is a key mediator of the transendothelial migration of neutrophils (27), and the IL-1 type 1 receptor, which mediates neutrophil migration induced by IL-1 (28). In contrast to the upregulation of genes involved in neutrophil migration, genes involved in immunity and bacterial killing were downregulated. ...
The factors that regulate the passage of bacteria between different anatomical compartments are unclear. We have used an experimental model of infection with Streptococcus pneumoniae to examine the host and bacterial factors involved in the passage of bacteria from the lung to the pleural space. The transcriptional profile of host and bacterial cells within the pleural space and lung was analyzed using deep sequencing of the entire transcriptome using the technique of dual RNA-seq. We found significant differences in the host and bacterial RNA profiles in infection, which shed light on the key factors that allow passage of this bacterium into the pleural space.
... Among M3 hub genes, which reflect the expression of the entire module, we found that both the RNA and protein levels of Anxa1 (annexin A1) demonstrated a strong ability to discriminate between injuries of di erent severities. Annexin A1 is a member of the annexin superfamily of calcium dependent phospholipidbinding proteins, and plays a role in mediating anti-inflammatory e ects through inhibition of phospholipase A2 activity (Elderfield et al., 1993;Liu et al., 2007), decreasing leukocyte activation (Perretti and Flower, 1993;D'Acquisto et al., 2007) and reducing expression of pro-inflammatory cytokines (Sudlow et al., 1996;McArthur et al., 2010). Anxa1 is primarily expressed in microglia, where it regulates the selective removal of apoptotic neurons (McArthur et al., 2010). ...
Spinal cord injury (SCI) is a devastating neurological condition for which there are currently no effective treatment options to restore function. A major obstacle to the development of new therapies is our fragmentary understanding of the coordinated pathophysiological processes triggered by damage to the human spinal cord. Here, we describe a systems biology approach to integrate decades of small-scale experiments with unbiased, genome-wide gene expression from the human spinal cord, revealing a gene regulatory network signature of the pathophysiological response to SCI. Our integrative analyses converge on an evolutionarily conserved gene subnetwork enriched for genes associated with the response to SCI by small-scale experiments, and whose expression is upregulated in a severity-dependent manner following injury and downregulated in functional recovery. We validate the severity-dependent upregulation of this subnetwork in rodents in primary transcriptomic and proteomic studies. Our analysis provides a systems-level view of the coordinated molecular processes activated in response to SCI.
... IL-8, which is a ligand for the gene product of CXCR1, and the integrin β-2, are both known to be involved in stimulating neutrophil activation and migration, which in turn stimulates the release of a variety of cytokines and chemokines through an NF-κB pathway in both murine and human models [31][32][33][34]. Cytokines released by neutrophils include IL-1β and IL-1α; these are ligands for the receptor that is associated with the gene product of IL-1RAP, which, similar to IL-8, also serve to both stimulate neutrophil activation and migration [34][35][36]. Therefore, the gene products of our Hg-associated genes play a role in regulation of the inflammatory response through release by NF-κB pathways or the promotion of those pathways, suggesting a common modulation by Hg that may become more apparent in a study with a larger sample size. ...
There is growing evidence of immunotoxicity related to exposure to toxic trace metals, and an examination of gene expression patterns in peripheral blood samples may provide insights into the potential development of these outcomes. This pilot study aimed to correlate the blood levels of three heavy metals (mercury, cadmium, and lead) with differences in gene expression in 24 participants from the Long Island Study of Seafood Consumption. We measured the peripheral blood mRNA expression of 98 genes that are implicated in stress, toxicity, inflammation, and autoimmunity. We fit multiple linear regression models with multiple testing correction to correlate exposure biomarkers with mRNA abundance. The mean blood Hg in this cohort was 16.1 µg/L, which was nearly three times the Environmental Protection Agency (EPA) reference dose (5.8 µg/L). The levels of the other metals were consistent with those in the general population: the mean Pb was 26.8 µg/L, and the mean Cd was 0.43 µg/L. The expression of three genes was associated with mercury, four were associated with cadmium, and five were associated with lead, although none were significant after multiple testing correction. Little evidence was found to associate metal exposure with mRNA abundance for the tested genes that were associated with stress, toxicity, inflammation, or autoimmunity. Future work should provide a more complete picture of physiological reactions to heavy metal exposure.
... En favorisant la production de lipocortine1, les glucocorticoïdes inhibent la synthèse de phospholipase A2 et ainsi, la cascade de l'acide arachidonique permettant de réduire la production des leucotriènes pro-inflammatoires (Perretti and Flower 1993). Enfin, la synthèse de la COX-2 (Bailey, Makheja et al. 1988) et de la NO synthase inductible (Radomski, Palmer et al. 1990) est aussi inhibée par les corticoïdes (Figure 30). ...
L'ischémie/reperfusion (I/R) est un phénomène très fréquent en clinique humaine. Ce phénomène est observé lors de la désobstruction d'une artère digestive, du traitement d'un état de choc, ainsi qu'au cours d'autres pathologies. L'interruption de la perfusion tissulaire (ischémie) et le rétablissement de celle-ci (reperfusion) sont la cause de la mise en place de troubles hémodynamiques et métaboliques. L'I/R est souvent présentée comme étant la principale source de l'hyperlactatémie et le moteur de la réponse inflammatoire lors des états de choc (cardiogénique, hypovolémique, septique). Parallèlement, elle est responsable de l'induction de la production de la libération des espèces réactives de l'oxygène, des cytokines et du monoxyde d'azote. Suite à un choc hémorragique par Ischémie/reperfusion chez le rat, nous avons montré que 1) le NaHS, donneur d'H2S limite la diminution de la pression artérielle moyenne et diminue le lactate plasmatique, témoin de la souffrance tissulaire, 2) cette amélioration hémodynamique est associée à une baisse de l'expression myocardique des ARNm d'iNOS, une diminution de la concentration des dérivés NOx plasmatiques et une diminution des concentrations aortiques et myocardiques de NO et d'anion superoxyde et 3) l'inhibition d'H2S par la DL-propargylglycine aggrave le tableau hémodynamique et les conséquences tissulaires du choc. Dans un autre modèle d'ischémie/reperfusion intestinale, les résultats obtenus, montrent que l'administration de la Protéine C activée (PCa) ou de la dexaméthaosne (Dexa) : 1) améliore la PAM et la réactivité vasculaire, 2) permet d'augmenter le pH et de diminuer la lactatémie, 3) diminue la production des cytokines pro-inflammatoires et 4) inhibe les médiateurs de l'apoptose. Ces résultats sont reliés à une down régulation d'iNOS, une restauration de la voie Akt/eNOS et à une resensibilisation des adrénorécepteurs alpha. Ces résultats ouvrent de nouvelles perspectives cliniques dans les traitements de l'I/R
... Moreover, treatment with DEXA showed leukocyte count and MPO activity reduction in mouse skeletal muscle (Fig. 5), and similar DEXA effect has been reported with B. jararacussu inoculated in the peritoneum (Pereira et al. 2009). Perretti and Flower (1993), although not using venoms in their investigations, described an antimigratory effect of DEXA on mouse leukocytes and correlated this effect with annexin-1 production. Mancuso et al. (1995) showed that DEXA did not alter the rolling and adherence of leukocytes to the vascular wall, but increases their detaching rate from the endothelium, making the white cells return to the bloodstream. ...
Treatment of snakebite around the world is a challenge because of the diversity of snake species and the complexity of their venom components. Most snakebites induce intense local inflammatory response that leads to extensive tissue damage which can result in late important disabilities. Among the pit vipers in the American continent, the snakes of genus Bothrops are very important, due to the great number of snakebites and mainly because their venom induces prompt local injury characterized by hemorrhage, edema, and myonecrosis. The main protocol for the treatment is based on the administration of animal-derived antivenom. However, despite reducing some systemic effects, this therapy is not able to completely neutralize all of the local venom effects. The problem is bigger when the therapy is delayed because of geographical problems or lack of accessibility to health support. The use of the glucocorticoid is proposed to decrease the acute inflammatory response to snakebites, and recent data indicate that dexamethasone is effective in reducing the local inflammation response and myonecrosis caused by Bothrops envenomation. This chapter shows a general view of venom injury caused by snakebites and revises the experimental use of dexamethasone as adjuvant treatment for these accidents, while detailed descriptions of specific venom components and its effects are present in other sections of this book.
... For instance, early studies implicated AnxA1 to control the adhesion and migration of leucocytes, such as neutrophils and granulocytes, during the inflammatory response induced by glucocorticoids. 14,15 Soon thereafter, AnxA2 was reported to affect the migratory behavior of endothelial cells and neoangiogenesis. 16,17 Similarly, recombinant AnxA5 was initially shown to inhibit lung carcinoma cell migration and T cell adhesion. ...
Annexin A6 (AnxA6) belongs to a highly conserved protein family characterized by their calcium (Ca(2+)) -dependent binding to phospholipids. Over the years, immunohistochemistry, subcellular fractionations, and live cell microscopy established that AnxA6 is predominantly found at the plasma membrane and endosomal compartments. In these locations, AnxA6 acts as a multifunctional scaffold protein, recruiting signaling proteins, modulating cholesterol and membrane transport and influencing actin dynamics. These activities enable AnxA6 to contribute to the formation of multifactorial protein complexes and membrane domains relevant in signal transduction, cholesterol homeostasis and endo-/exocytic membrane transport. Hence, AnxA6 has been implicated in many biological processes, including cell proliferation, survival, differentiation, inflammation, but also membrane repair and viral infection. More recently, we and others identified roles for AnxA6 in cancer cell migration and invasion. This review will discuss how the multiple scaffold functions may enable AnxA6 to modulate migratory cell behavior in health and disease.
... Also glucocorticoids inhibit transcription of IL-1,2,3,4,5,6,8, TNF-α , and GM-CSF [12] and inhibit expression of adhesion molecules such as ICAM-1 and EIAM-1 through inhibitory effects on cytokines like IL-1 and TNF-α [14].In addition, glucocorticoids have inhibitory effects in leukocytes migration by stimulating production of lipocortin -1 [1]. Lipocortin -1 in turn decrease eicosanoid level which inhibit migration of leukocytes [13].It is known that the traveling of phagocytic cells depend on amoeboid movement of the cells, chemotactic substances which released by the inflamed tissues, and number of phagocytic cells . So we believed that the Dexamethasone drug which is glucocorticoids derived in nature has inhibited releasing of chemotactic substances or slowed the amoeboid movement of phagocytic cells or the drug might inhibit the leukocytes production and decreased their numbers.We recommended a separate study to recognize the mechanisms by which Dexamethasone exerts its action on the chemotaxis of leukocytes . ...
The aim of this work was to asses whether use of Dexamethasone in treatment of Systemic Lupus Erythematosus(SLE) patients women affects migration of leukocyte. 24 samples of patients attended to Al sadr teaching hospital in city of Al Najaf were used .Study revealed a significant reducing (P<001) in leukocyte migration after treatment .
... Moreover, treatment with DEXA showed leukocyte count and MPO activity reduction in mouse skeletal muscle (Fig. 5), and similar DEXA effect has been reported with B. jararacussu inoculated in the peritoneum (Pereira et al. 2009). Perretti and Flower (1993), although not using venoms in their investigations, described an antimigratory effect of DEXA on mouse leukocytes and correlated this effect with annexin-1 production. Mancuso et al. (1995) showed that DEXA did not alter the rolling and adherence of leukocytes to the vascular wall, but increases their detaching rate from the endothelium, making the white cells return to the bloodstream. ...
Treatment of snakebite around the world is a challenge because of the diversity
of snake species and the complexity of their venom components. Most snakebites
induce intense local inflammatory response that leads to extensive tissue
damage which can result in late important disabilities. Among the pit vipers in
the American continent, the snakes of genus Bothrops are very important, due to
the great number of snakebites and mainly because their venom induces prompt
local injury characterized by hemorrhage, edema, and myonecrosis. The main
protocol for the treatment is based on the administration of animal-derived
antivenom. However, despite reducing some systemic effects, this therapy is
not able to completely neutralize all of the local venom effects. The problem is
bigger when the therapy is delayed because of geographical problems or lack of
accessibility to health support. The use of the glucocorticoid is proposed to
decrease the acute inflammatory response to snakebites, and recent data indicate
that dexamethasone is effective in reducing the local inflammation response and
myonecrosis caused by Bothrops envenomation. This chapter shows a general
view of venom injury caused by snakebites and revises the experimental use of
dexamethasone as adjuvant treatment for these accidents, while detailed descriptions
of specific venom components and its effects are present in other sections
of this book.
... Le MIF s'oppose aux effets inhibiteurs des corticoïdes sur la production de cytokines de l'inflammation par les monocytes humains activés par le LPS. La production des corticoïdes -defensine qui entre en compétition avec En favorisant la production de lipocortine1, les glucocorticoïdes inhibent la synthèse de arachidonique permettant de réduire la production des leucotriènes pro-inflammatoires (Perretti and Flower 1993). ...
Ischemia/reperfusion (I/R) is a very common phenomenon, observed during intestinal artery surgery, shock treatment, as well as in several other diseases. The disruption of tissue perfusion (ischemia) and recovery (reperfusion) induce hemodynamic and metabolic dysfunction. Gut ischemia/reperfusion is often presented as the main source of lactate and the motor of the inflammatory response, such as cardiogenic, hypovolemic and septic shock. In parallel, gut reperfusion produces numerous mediators such as reactive oxygen metabolites, pro-inflammatory cytokines, and high concentrations of nitric oxide.
In a model of ischemia/reperfusion induced by hemorrhagic shock, we found that 1) NaHS an injectable form of H2S, limited the decrease in arterial pressure induced by shock and decreased plasmatic lactate, a witness of tissue suffering, 2) this hemodynamic improvement was associated with a fall in myocardial iNOS mRNA expression, a reduction in the concentration of plasmatic NOx and a reduction of aortic and myocardial concentrations of NO and superoxide anion and 3) the inhibition of H2S with DL-propargylglycine worsened hemodynamics and tissue consequences of shock
An experimental model of intestinal I/R has been developed, we demonstrated that the administration of APC or Dexa : 1) Improves MAP and vascular reactivity, 2) increased pH and decreased lactate, 3) decreased pro-inflammatory cytokines production and 4) inhibited apoptosis mediators expression. These results are related to a down regulation of iNOS, to a restoration of the AKT/eNOS pathway, and to alpha-adrenoreceptor resensitization.
These results open new perspectives in clinical treatment of I/R.
... Reinforcing the involvement of AnxA1 pathway in neutrophil recruitment, AnxA1-null mice demonstrated a higher extent of neutrophil extravasation in animal models of peritonitis [35,74], allergic conjunctivitis [39], and uveitis [37]. [27,48,54,55] Neutrophil/endothelial interaction (in vivo) ↓ PMN rolling, adhesion, and emigration ↑ Detachment of adherent PMN [27,56] Human PMN ↑ L-selectin shedding [57,58] IL-1 inflamed air pouch ↓ PMN migration [26,59] Carrageenan-induced paw edema ↓ edema ↓ leukocyte infiltration [27] SAnxA1 ...
Neutrophils (also named polymorphonuclear leukocytes or PMN) are essential components of the immune system, rapidly recruited to sites of inflammation, providing the first line of defense against invading pathogens. Since neutrophils can also cause tissue damage, their fine-tuned regulation at the inflammatory site is required for proper resolution of inflammation. Annexin A1 (AnxA1), also known as lipocortin-1, is an endogenous glucocorticoid-regulated protein, which is able to counterregulate the inflammatory events restoring homeostasis. AnxA1 and its mimetic peptides inhibit neutrophil tissue accumulation by reducing leukocyte infiltration and activating neutrophil apoptosis. AnxA1 also promotes monocyte recruitment and clearance of apoptotic leukocytes by macrophages. More recently, some evidence has suggested the ability of AnxA1 to induce macrophage reprogramming toward a resolving phenotype, resulting in reduced production of proinflammatory cytokines and increased release of immunosuppressive and proresolving molecules. The combination of these mechanisms results in an effective resolution of inflammation, pointing to AnxA1 as a promising tool for the development of new therapeutic strategies to treat inflammatory diseases.
... 79 This drug also inhibited neurogenic inflammation caused by antidromic stimulation of the rat saphenous nerve but this was partly abolished by 80 passive immunisation with a neutralizing anti-Anx-A1 antiserum. Anx-A1 or bioactive peptide fragments block neutrophil 81 but not eosinophil 83 infiltration into murine models of inflamed skin and passive immunisation of the mice with a neutralizing anti-Anx-A1 antibodies greatly exacerbated skin inflammation caused by the injection of zymosan activated serum. 14 Although there was no evidence for disordered skin histology in the Anx-A1 -/mice, preliminary data suggests that this tissue is more susceptible to inflammation caused by the subcutaneous injection of zymosan (Ahluwalia A, Flower RJ, unpublished). ...
... Eine Liganden-Rezeptor-Interaktion führt zur Dissoziation der HSPs und damit zur Exponierung von DNS-Bindungsstellen für Transkriptionsfaktoren [183].Zusammen mit NF-kB führt der Liganden-Rezeptor-Komplex außerdem zu einer verminderten Synthese zahlreicher proinflammatorischer Zytokine und Chemokine [46,56,138,137] und zu einer gesteigerten Synthese von MIF [49]. Kortisol hemmt außerdem die induzierbare NO-Synthase [162,163], Zyklooxygenase 2 [5], Phospholipase A 2 und den Arachidonsäurestoffwechsel [148]. Zu einer Downregulation der Entzündungsantwort führt auch eine Signaltransduktion über den Adenosin-A2a-Rezeptor [144]. ...
Das angeborene („innate“) Immunsystem bekämpft erfolgreich den größten Teil unserer Infektionen, noch bevor das erworbene Immunsystem aktiviert wird. Genauere Kenntnisse über Wirkmechanismen des angeborenen Immunsystems lassen uns die Pathophysiologie systemischer Infektionen, wie der Sepsis, besser verstehen und dienen der Entwicklung neuer Therapiestrategien. Das angeborene Immunsystem ist für die erste Abwehrreaktion auf eine Infektion verantwortlich. Darüber hinaus aktiviert und interagiert es mit dem erworbenen Immunsystem. Wechselwirkungen werden über Immunzellen, wie Makrophagen und dendritische Zellen, sowie lösliche Faktoren, wie Zytokine, vermittelt. Aufgrund neuer Erkenntnisse aus Tiermodellen, die neben der Inflammation auch ein Fortschreiten der Bakterienabwehr berücksichtigen, mussten bisherige Erkenntnisse überprüft und korrigiert werden. Die Vorstellung von Sepsis als überwiegende „Überreaktion auf eine Entzündung“ wich neuen Theorien. Die Zellen des angeborenen Immunsystems erkennen Eindringlinge mit Hilfe spezieller Rezeptoren. Nach Rezeptorinteraktion führen intrazelluläre Signalkaskaden zur Zellaktivierung, durch die zahlreiche Zytokine und antimikrobielle Substanzen freigesetzt werden. Im Verlauf einer Sepsis kommt es durch verschiedene Regelkreise zu zunehmender Immunsuppression. Mit dem angeborenen Immunsystem verknüpft ist die Migration von Leukozyten in entzündetes Gewebe. Chemokine und Adhäsionsmoleküle übernehmen dabei Schlüsselrollen. Auch die Blutgerinnung ist mit dem angeborenen Immunsystem eng verzahnt. Immunzellen sezernieren „tissue factor“; dies führt über eine Kaskade u. a. zur Bildung von Thrombin. Dieses kann unter den besonderen Bedingungen der Sepsis eine disseminierte intravasale Gerinnung verursachen. Umgekehrt werden durch Thrombin auch Endothelzellen zur Freisetzung von Chemokinen und Adhäsionsmolekülen angeregt; dies stellt einen positiven „Feedback-Mechanismus“ für die angeborene Immunantwort dar. Neue Therapieansätze zur Sepsis versuchen diese Regelkreise zu durchbrechen und durch Blockade von Zytokinen, Rezeptoren oder durch Aktivierung immunstimulierender Systeme eine ausgewogene Reaktion des angeborenen Immunsystems ohne Suppression der antibakteriellen Funktion zu erzielen.
... 24 Ac2-26 probably interacts with both FPR1 and FPR2 on human neutrophils to exert its effects, whereas full-length annexin A1 binds only to FPR2 (Figure 3). 34 Although Ac2-26 has been shown to have similar efficacy to full-length annexin A1 in some models, 72 which might relate to the ability of this peptide to promote transportation of full-length endogenous annexin A1 from the cytoplasm to the external surface of the cell, 37 the N-terminal peptide is approximately 200 times less potent than the parent protein. 73 Another FPR2 agonist, the 'W-peptide' (Trp-Lys-Tyr-Met-Val-D-Met), has been shown to inhibit the develop ment of allergen-induced inflammation and T H 1cell/T H 17-cell cytokine production, in situ, in a mouse model of non-eosinophilic asthma. ...
Glucocorticoids have broad-ranging and powerful anti-inflammatory and immunomodulatory effects. Unsurprisingly, therefore, glucocorticoids are widely and persistently used to treat a large number of inflammatory diseases, including rheumatoid arthritis (RA), despite the well-described adverse effects of these drugs. Annexin A1 is a glucocorticoid-induced molecule that is known to replicate many of the described anti-inflammatory effects of glucocorticoids. In addition to the well-documented roles of this protein in neutrophil function, emerging evidence suggests that annexin A1 is involved in the modulation of T-cell function and the adaptive immune responses relevant to RA. Interest in annexin A1 was renewed after the delineation of the receptors for this protein. This breakthrough also led to advances in our understanding of anti-inflammatory annexin A1 mimetic peptides and agonistic compounds targeting these receptors, particularly those specific for the receptor N-formyl peptide receptor 2 (FPR2). Herein, we review the current knowledge of the biological activities of annexin A1 and their relevance to RA pathogenesis. We also discuss the potential of annexin A1 mimics and strategies aimed at potentiating annexin A1 signalling to become viable approaches to minimizing glucocorticoid use in RA and other inflammatory disorders.
... Les corticoïdes empêchent la migration des cellules inflammatoires vers les tissus en bloquant la synthèse de chémokines [12] ; ils bloquent la synthèse de la plupart des molécules pro-inflammatoires -IL-1, IL-2, IL-3, IL-6 (interleukines), IFNγ (interféron-gamma), GM-CSF (Granulocyte Macrophage Colony Stimulating Factor) et TNF-α (tumor necrosis factor-α) -et stimulent la production de MIF (macrophage Migration Inhibitory Factor) [13]. De plus, en stimulant la synthèse de lipocortine-1 [14], les corticoïdes inhibent la PLA 2 (phospholipase A 2 ) et donc la cascade de l'acide arachidonique réduisant la production de leucotriènes. Finalement, les corticoïdes inhibent la synthèse de COX-2 (cyclo-oxygenase-2) [15] et d'iNOS (oxyde nitrique synthase inductible) [16], deux enzymes clé de la réaction inflammatoire au sepsis. ...
... On day 6, 40 ng of mouse recombinant TNF-␣ (R&D Systems) in 1 ml of D-PBS solution was injected into the matured air pouch. To enhance leukocyte response to TNF-␣, we injected the dose of TNF-␣ with 0.5% carboxymethylcellulose (CMC), an inert carrier (19). At 2, 4, and 6 h after administration of TNF-␣, the mice were sacrificed by injections of overdose of combined anesthetics (ketamine, xylazine, and acepromazine). ...
To differentiate the unique and overlapping functions of LFA-1 and Mac-1, LFA-1-deficient mice were developed by targeted homologous recombination in embryonic stem cells, and neutrophil function was compared in vitro and in vivo with Mac-1-deficient, CD18-deficient, and wild-type mice. LFA-1-deficient mice exhibit leukocytosis but do not develop spontaneous infections, in contrast to CD18-deficient mice. After zymosan-activated serum stimulation, LFA-1-deficient neutrophils demonstrated activation, evidenced by up-regulation of surface Mac-1, but did not show increased adhesion to purified ICAM-1 or endothelial cells, similar to CD18-deficient neutrophils. Adhesion of Mac-1-deficient neutrophils significantly increased with stimulation, although adhesion was lower than for wild-type neutrophils. Evaluation of the strength of adhesion through LFA-1, Mac-1, and CD18 indicated a marked reduction in firm attachment, with increasing shear stress in LFA-1-deficient neutrophils, similar to CD18-deficient neutrophils, and only a modest reduction in Mac-1-deficient neutrophils. Leukocyte influx in a subcutaneous air pouch in response to TNF-α was reduced by 67% and 59% in LFA-1- and CD18-deficient mice but increased by 198% in Mac-1-deficient mice. Genetic deficiencies demonstrate that both LFA-1 and Mac-1 contribute to adhesion of neutrophils to endothelial cells and ICAM-1, but adhesion through LFA-1 overshadows the contribution from Mac-1. Neutrophil extravasation in response to TNF-α in LFA-1-deficient mice dramatically decreased, whereas neutrophil extravasation in Mac-1-deficient mice markedly increased.
The COVID-19 pandemics has made sparkly evident the importance of acute inflammation and its timely resolution to protect humans from pathogenic viruses while sparing them from collateral damages due to an uncontrolled immune response. It is clear now that resolution of inflammation is an active process regulated by endogenous specialized proresolving lipid mediators (SPM) biosynthesized from essential polyunsaturated fatty acids. Accruing evidence indicates that SPM are produced during viral infections and play key roles in controlling the magnitude and duration of the inflammatory response and in regulating adaptive immunity. Here, we reviewed biosynthesis and bioactions of SPM in virus-mediated human diseases. Harnessing SPM and their proresolutive actions can help in providing new therapeutic approaches to current and future human viral diseases by controlling infection, stimulating host immunity, and protecting from organ damage.
Introduction:
We aimed to use two indices, amniotic fluid interleukin-6 (IL-6) concentration at diagnosis and diagnosis-to-delivery interval, to clarify the frequencies of maternal inflammatory response (MIR) and fetal inflammatory response (FIR) in the placenta of patients with intra-amniotic infection and intra-amniotic inflammation (IAI).
Methods:
This is a single-center retrospective cohort study. From August 2014 to April 2020, participants were diagnosed with IAI with or without microbial invasion of the amniotic cavity (MIAC) using amniocentesis. IAI was defined as concentrations of amniotic IL-6 ≥ 2.6 ng/mL. MIAC was defined as a positive amniotic fluid culture. IAI with MIAC was defined as an intra-amniotic infection. We calculated the cut-off values for IL-6 concentration in the amniotic fluid at diagnosis and the diagnosis-to-delivery interval for MIR-positive cases among those with intra-amniotic infection.
Results:
The amniotic fluid IL-6 concentration at diagnosis and diagnosis-to-delivery interval were 15.8 ng/mL and 12 h, respectively. Among cases with intra-amniotic infection, MIR was 98% (52/53) positive, i.e., when either of the two cut-off values was exceeded. There were no significant differences between the frequencies of MIR and FIR. In cases with IAI but no MIAC, the frequencies of MIR and FIR were significantly lower than those with intra-amniotic infection, except when neither of the two cut-off values was exceeded.
Discussion:
We clarified the MIR- and FIR-positive cases in intra-amniotic infection and cases with IAI but no MIAC according to condition, including the diagnosis-to-delivery interval.
The acute inflammatory response is the body's first system of alarm signals that are directed toward containment and elimination of microbial invaders. Uncontrolled inflammation has emerged as a pathophysiologic basis for many widely occurring diseases in the general population that were not initially known to be linked to the inflammatory response, including cardiovascular disease, asthma, arthritis, and cancer. To better manage treatment, diagnosis, and prevention of these wide-ranging diseases, multidisciplinary research efforts are underway in both academic and industry settings. This book provides an introduction to the cell types, chemical mediators, and general mechanisms of the host's first response to invasion. World-class experts from institutions around the world have written chapters for this introductory text. The text is presented as an introductory springboard for graduate students, medical scientists, and researchers from other disciplines wishing to gain an appreciation and working knowledge of current cellular and molecular mechanisms fundamental to inflammation.
Acute inflammation is a self-limiting process of the immune system, which resolves through the initiation of a program referred to as the resolution of inflammation. It has been argued that uncontrolled inflammation may be the basis of a variety of chronic inflammatory and autoimmune diseases. The resolution of inflammation is an active process coordinated by the production of proresolving mediators. The release of proresolving mediators prevents further migration of granulocytes, and increases leukocyte apoptosis. Moreover, some proresolving molecules are able to promote the infiltration of nonphlogistic macrophages, which are fundamental cells to efferocyte of apoptotic granulocytes. This event, in turn, triggers macrophage reprogramming towards more restorative and resolutive roles, thereby promoting resolution and reestablishment of tissue homeostasis. Here, we summarize the most prominent pro-resolving mediators relevant to the resolution of inflammation.
Glucocorticoids (GCs) are used as immunosuppressive and antiinflammatory agents in organ transplantation and in treating autoimmune diseases and inflammatory disorders. GCs were shown to exert their antiproliferative effects directly through blockade of certain elements of an early membrane-associated signal transduction pathway, modulation of the expression of select adhesion molecules, and by suppression of cytokine synthesis and action. GCs may act indirectly by inducing lipocortin synthesis, which in turn, inhibits arachidonic acid release from membrane-bound stores, and also by inducing transforming growth factor (TGF)-β expression that subsequently blocks cytokine synthesis and T cell activation. Furthermore, by preferentially inhibiting the production of Th1 cytokines, GCs may enhance Th2 cell activity and, hence, precipitate a long-lasting state of tolerance through a preferential promotion of a Th2 cytokine-secreting profile. In exerting their antiproliferative effects, GCs influence both transcriptional and posttranscriptional events by binding their cytosolic receptor (GR), which subsequently binds the promoter region of cytokine genes on select DNA sites compatible with the GCs responsible elements (GRE) motif. In addition to direct DNA binding, GCs may also directly bind to, and hence antagonize, nuclear factors required for efficient gene expression, thereby markedly reducing transcriptional rate. The pleiotrophy of the GCs action, coupled with the diverse experimental conditions employed in assessing the GCs effects, indicate that GCs may utilize more than one mechanism in inhibiting T cell activation, and warrant careful scrutiny in assigning a mechanism by which GCs exert their antiproliferative effects. © 1998 Elsevier Science Inc.
Bereits zwischen dem 10. und 16. Jahrhundert waren chinesische Alchimisten in der Lage, Steroide nahezu bis zur Homogenität aufzureinigen und für medizinische Zwecke einzusetzen.
Since the 1950s glucocorticoids have been used extensively in the treatment of inflammatory diseases and despite several undesirable side-effects they still remain as one of the most important drugs to control inflammation. Little was known about the mechanism of action of glucocorticoids until the 1970s when a detailed study of their molecular actions was first began. Since that time our knowledge of the cellular mechanisms that mediate the effects of glucocorticoids has grown considerably. Briefly, following an interaction with specific intracellular receptors, and in concert with other transcription factors, the glucocorticoid receptor complex binds to specific DNA response elements within the promoter sequences of key target genes. As a result of this interaction the expression of many pro-inflammatory mediators is suppressed. However, it is now clear that this process also up-regulates the expression of other mediators that have potent anti-inflammatory properties. This chapter reviews the evidence of one such anti-inflammatory mediator of glucocorticoid action - annexin I.
1Lipocortin-1 and its N-terminal derivatives exert potent inhibitory actions in various models of acute inflammation. The present study examined the ability of lipocortin (LC)-1 to suppress the release of the acute pro-inflammatory mediators, tumour necrosis factor (TNFα) and prostaglandin E2 (PGE2) from human peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or recombinant human interleukin-1β (rhIL-1β).2LPS (10 μg ml−1)-stimulated release of TNFα and PGE2 from PBMC was significantly inhibited by (4 h) co-incubation of the cells with 10−6m dexamethasone (Dex), but not with 10−9m to 10−7m of a N-terminal fragment (amino acids 1–188) of recombinant human LC-1 (LC-1 fragment). However, Dex suppression of LPS-stimulated TNFα and PGE2 secretion from PBMC was reversed when polyclonal antibody to LC-1 fragment (1:10,000 dilution) was included in the medium. rhIL-1β (5times10−8m)-stimulated release of TNFα and PGE2 from PBMC (after 18 h) was abolished by co-incubation of the cells with 10−7m LC-1 fragment.3After incubation with Dex (4 h), cellular proteins from PBMC were immunoblotted using anti-LC-1 fragment antibody (which showed no cross-reactivity with human annexins 2 to 6). Dex caused no increase in immunoreactive (ir)LC-1 content of PBMC, although there was a three fold increase in the amount of a lower mass species with LC-1-like immunoreactivity. This was accompanied by the appearance of irLC-1 in the extracellular medium.4The results of the present study implicate endogenous LC-1 in glucocorticoid suppression of TNFα and PGE2 release from human PBMC and suggest an extracellular site of action for LC-1. LC-1 may also inhibit rhIL-1β-stimulated TNFα and PGE2 secretion from PBMC.
There is a growing appreciation of the important role of resolution mediators in the successful termination of the inflammatory response. Here, we discuss the potential importance of the lipid and peptide proresolving mediators, in particular the resolvins and chemerin-derived peptides, which mediate their effects through specific G protein-coupled receptors (GPCRs).
Many clinicians are frequently confronted with an adolescent who comes to the first
aid department in the middle of the night, complaining of breathlessness and chest
tightness. While he was in a smoky environment he became wheezy and felt out of
breath. After taking some bronchodilator puffs his complaints did not improve but got
even worse. Others are more familiar with the picture of the infant, out of breath
sitting on the bench during gymnastics whereas other kids are busy doing their
exercises. All clinicians will immediately recognize the clinical symptoms of an asthma
patient. Bul what exactly is going on wilhin the airways?
Asthma is one of the most common disorders, affecting approximately 10% of the
population in the Western countries. Asthma, was used to describe several disorders characterized by
breathlessness or pain in the chest. Sir John Floyer wrote in his "treatise of the
asthma" in 1698: "I have assigned the immediate cause of asthma to the straitness,
compression, or constriction of the bronchi". Laennec in the eighteenth century
attributed asthma to a spasm of the smooth muscle fibers of the bronchi. In spite of
the fact that our knowledge of the disease has increased since then and asthma is
now considered as a chronic inflammatory disease, we still do not know the fundamental
cause of asthma and all the factors that induce airway inflammation.
Airway inflammation in asthma is characterized by redness and swelling of the
mucosa. These classical signs of inflammation are easily visible at bronchoscopic
examination. Bronchial biopsies not only show activated mast cells, eosinophils
and lymphocytes, but also epithelial shedding and fragility. Structural changes
include hypertrophy and hyperplasia of airway smooth muscle, and thickening of the
basement mem-brane due to the deposition of collagen in the lamina reticularisb.
The glucocorticoid-regulated protein annexin I (lipocortin I) has been shown to mediate antiinflammatory activities of glucocorticoids, but the molecular basis of its action has remained elusive. Here we show that annexin I acts through the formyl peptide receptor (FPR) on human neutrophils. Peptides derived from the unique N-terminal domain of annexin I serve as FPR ligands and trigger different signaling pathways in a dose-dependent manner. Lower peptide concentrations possibly found in inflammatory situations elicit Ca2+ transients without fully activating the MAP kinase pathway. This causes a specific inhibition of the transendothelial migration of neutrophils and a desensitization of neutrophils toward a chemoattractant challenge. These findings identify annexin I peptides as novel, endogenous FPR ligands and establish a mechanistic basis of annexin I–mediated antiinflammatory effects.
The pro-inflammatory cytokines IL-1 and TNF-α are primary mediators of the acute phase response, the complex reaction of the mammalian organism to infection and injury. Among the genes activated by TNF-α and IL-1 in a variety of cells is TNF-stimulated gene 6 (TSG-6t). The TSG-6† cDNA encodes a secreted 35 kDa glycoprotein which is abundant in synovial fluids of patients with various forms of arthritis and detectable in serum of patients with different inflammatory or autoimmune disorders. TSG-6 protein consists of two structural domains: a hyaluronan-binding link module, the characteristic domain of the hyaladherin family of proteins, and a C-terminal CUB domain, present in a variety of diverse proteins. TSG-6 forms a stable complex with components of the plasma protein inter-α-inhibitor (IαI), a Kunitz-type serine protease inhibitor. TSG-6 and IαI synergize to inhibit plasmin, a serine protease involved in the activation of matrix metalloproteinases which are part of the proteolytic cascade associated with inflammation. Recombinant human TSG-6 protein exerts a potent anti-inflammatory effect in a murine model of acute inflammation. Modulation of the proteolytic network associated with inflammatory processes may be a mechanism whereby TSG-6, in cooperation with IαI, inhibits inflammation. Activation of the TSG-6 gene by pro-inflammatory cytokines, presence of TSG-6 protein in inflammatory lesions and its anti-inflammatory effect suggest a role for TSG-6 in a negative feed-back control of the inflammatory response.
Most anti-inflammatory agents used in the treatment of joint diseases exert inhibitory effects on leukocyte infiltration. Methotrexate, a disease-modifying drug, and corticosteroids also inhibit leukocyte accumulation during inflammation. However, the mechanisms of action of these different compounds on leukocytes vary and in the case of non-steroidal anti-inflammatory drugs (NSAIDs) the mechanism(s) may be indirect. No current drug for inflammatory or degenerative joint disease has been proposed to act specifically by an inhibitory action on neutrophilic leukocytes. Oxaceprol is an amino acid derivative that has been used for several years for the treatment of osteoarthritis and rheumatoid arthritis, ameliorating pain and stiffness and showing good gastrointestinal safety, particularly in comparison with NSAIDs. Recent experimental studies have shown that oxaceprol does not inhibit the synthesis of prostaglandins in vitro, but markedly inhibits neutrophil infiltration into the joints of rats with adjuvant arthritis. These results support earlier screening data showing inhibition by oxaceprol of leukocyte infiltration into sites of acute inflammation. In studies on surgical ischemia reperfusion in hamsters in vivo, oxaceprol was an effective inhibitor of leukocyte adhesion and extravasation. It is proposed that oxaceprol represents a therapeutic agent for degenerative and inflammatory joint diseases, which acts predominantly by inhibiting leukocyte adhesion and migration.
: Lipocortin-1 (LC-1), a Ca++-dependent phospholipid binding protein, is believed to be involved in anti-inflammatory actions of glucocorticoids. to prove the hypothesis that steroid-resistant glomerulonephritis would show increased expression of LC-1, we evaluated the expression of LC-1 in various types of glomerulonephritis. Frozen samples of seven normal kidneys and 30 kidney biopsy tissues were stained with indirect immunofluorescent method. In the normal tissues, minimal change disease (n=9), lupus nephritis (n=5) and IgA nephropathy (n=6), glomeruli did not stain for LC-1. Positive reactions for LC-1 were observed along the peripheral capillary walls in all five patients with membranous nephropathy with out hepatitis B surface antigen (HBsAg). In the patients with membranous nephropathy (MN) who also had chronic liver disease and HBsAg (n=3), only weak reactions for LC-1 were found along the capillary walls and mesangial area in 1 patient. Patients with membranoproliferative glomerulonephritis (n=2) showed positive reactions for LC-1 along the capillary walls. Fourteen patients with minimal change disease or lupus nephritis were treated with prednisolone. Ten patients showed substantial reduction of proteinuria, but four patients did not; however, staining for LC-1 was not negative in the kidney tissues of both steroid-responsive and steroid-resistant patients. These findings suggest that LC-1 does not mediate the action of glucocorticoids in human glomerulonephritis.
Cardiac surgical procedures, with or without cardiopulmonary bypass, elicit a systemic inflammatory response in patients that induces the elaboration of multiple cytokines, chemokines, adhesion molecules, and destructive enzymes. This inflammatory reaction involves multiple interdependent and redundant cell types and humoral cascades, which allows for amplification and positive feedback at numerous steps. This systemic inflammatory response ultimately results in a broad spectrum of clinical manifestations, with multiple organ failure being the most severe form. Investigative efforts have focused on understanding the mechanism of this systemic inflammatory response syndrome in order to develop potential therapeutic targets to inhibit it, thereby possibly decreasing postoperative morbidity and mortality. Multiple therapeutic methods have been investigated, including pharmacologic inhibitors and modifications of surgical technique and the cardiopulmonary bypass circuit. Although studies have demonstrated that the use of these therapies in experimental and clinical settings has attenuated the systemic inflammatory response, they have failed to conclusively show clinical benefit from these therapies. These therapies may be too specific to minimize the deleterious effects of a systemic inflammatory response that results from the activation of multiple, interdependent, and redundant inflammatory cascades and cell types. Hence, further studies that investigate the molecular and cellular events underlying the systemic inflammatory response syndrome and the resultant effects of anti-inflammatory therapies are warranted to ultimately achieve improvements in clinical outcome after cardiac surgical procedures.
Lipocortin-1 (annexin-1) is an endogenous peptide with antiinflammatory properties. We have previously demonstrated lipocortin immunoreactivity in certain glial cells and neurons in the rat brain (Strijbos, P.J.L.M., F.J.H. Tilders, F. Carey, R. Forder, and N.J. Rothwell. 1990. Brain Res. In press.), and have shown that an NH2-terminal fragment (1-188) of lipocortin-1 inhibits the central and peripheral actions of cytokines on fever and thermogenesis in the rat in vivo (Carey, F., R. Forder, M.D. Edge, A.R. Greene, M.A. Horan, P.J.L.M. Strijbos, and N.J. Rothwell. 1990. Am. J. Physiol. 259:R266; and Strijbos, P.J.L.M., J.L. Browning, M. Ward, R. Forder, F. Carey, M.A. Horan, and N.J. Rothwell. 1991. Br. J. Pharmacol. In press.). We now report that intracerebroventricular administration of lipocortin-1 fragment causes marked inhibition of infarct size (60%) and cerebral edema (46%) measured 2 h after cerebral ischemia (middle cerebral artery occlusion) in the rat in vivo. The lipocortin-1 fragment was effective when administered 10 min after induction of ischemia. Ischemia caused increased expression of lipocortin-1 around the area of infarction as demonstrated by immunocytochemistry. Intracerebroventricular injection of neutralizing antilipocortin-1 fragment antiserum increased the size of infarct (53%) and the development of edema (29%). These findings indicate that lipocortin-1 is an endogenous inhibitor of cerebral ischemia with considerable therapeutic potential.
The binding of neutrophils (polymorphonuclear leukocytes [PMNs]) to endothelial cells (ECs) presents special requirements in the regulation of intercellular adhesion. ECs that are stimulated by certain agonists, including thrombin and cytokines (tumor necrosis factor alpha, interleukin-1), generate molecular signals that induce the adhesion of PMNs (endothelial cell-dependent neutrophil adhesion). Our experiments demonstrate that the mechanism of binding induced by thrombin is distinct from that induced by the cytokines based on the time courses, the requirement for protein synthesis, and differential binding of HL60 promyelocytic leukemia cells to ECs activated by the two classes of agonists. The rapid EC-dependent PMN adhesion (initiated in minutes) that occurs when the ECs are stimulated by thrombin is temporally coupled with the accumulation of platelet-activating factor, a biologically active phosphoglyceride that remains associated with ECs and that activates PMNs by binding to a cell surface receptor. A portion of the newly synthesized platelet-activating factor (PAF) is on the EC surface, as demonstrated by experiments in which the rate of hydrolysis of PAF synthesized by activated ECs was accelerated by extracellular PAF acetylhydrolase. When ECs were treated with exogenous PAF they became adhesive for PMNs; the PMN binding was prevented by incubating the ECs with PAF acetylhydrolase or by treating the PMNs with competitive PAF receptor antagonists. Thus PAF associated with the EC plasma membrane induces PMN binding, an observation supported by experiments in which PAF in model membranes (liposomes) stimulated rapid PMN adhesion to ECs and to cell-free surfaces. In addition, competitive antagonists of the PAF receptor inhibited the binding of PMNs to ECs activated by thrombin and other rapidly acting agonists, but not to ECs activated by tumor necrosis factor alpha, indicating that PAF that is endogenously synthesized by ECs can mediate neutrophil adhesion. These experiments demonstrate a novel mechanism by which a cell-associated phospholipid, PAF, can serve as a signal for an intercellular adhesive event.
Glucocorticoids inhibit superoxide (O2-) generation by phagocytes through a mechanism that remains unclear. We investigated this effect by using dexamethasone on guinea pig alveolar macrophages. O2- generation was induced either by the calcium ionophore A23187, a potent stimulus of phospholipase A2, or by the protein kinase C activator, phorbol myristate acetate (PMA). Dexamethasone inhibited O2- generation initiated by A23187 by 50-55%. This inhibition required: (a) more than 45 min incubation and was maximal after 2 h; (b) glucocorticoid receptor occupancy; and (c) protein synthesis. The inhibitory effect of dexamethasone could not be explained by an interaction with the respiratory burst enzyme NADPH oxidase since O2- generation was only weakly affected upon PMA stimulation. Lipocortin I, a glucocorticoid inducible and phospholipase A2 inhibitory protein, inhibited O2- generation initiated by A23187 but failed to modulate the respiratory burst activated by PMA. These results were obtained with lipocortin I purified from mouse lungs, human blood mononuclear cells, and with human recombinant lipocortin I. We propose that lipocortin I is capable of inhibiting the activation of NADPH oxidase only when membrane signal transduction involves phospholipase A2. By mimicking the effect of dexamethasone, lipocortin I may extend its potential anti-inflammatory action to the partial control of the formation of oxygen reactive species by phagocytes.
In an attempt to understand the regulatory mechanisms governing passage of neutrophils from the vascular bed to the interstitial tissue, we analyzed the effect of the pleiotropic monokines interleukin 1 (IL-1) and tumor necrosis factor (TNF) on transendothelial neutrophil traffic. Short-time preincubation of human umbilical vein endothelial cell (HUVE) monolayers with IL-1 and TNF led to an impressive time- and dose-dependent increase of endothelial cell-associated neutrophils when working in a full plasma system on petri dishes. Electron microscopic analysis revealed junctional penetration of monolayers by neutrophils. More quantitatively, when using a monolayer-on-filter-system, priming led to a severalfold increase in complete layer passage occurring in the absence of an external chemotactic gradient. Direct comparison with an upside-down modification of the system together with data demonstrating the vectorial behavior of such migration revealed that IL-1-stimulated transendothelial neutrophil traffic is polarized. The described enhancement of neutrophil transendothelial passage was found to be a unique feature of IL-1/TNF-activated HUVE since HUVE-dependent transmigration potentiation was not observed as a consequence of mere neutrophil attachment to endothelial cells (e.g., induced by Fc-mediated adherence of PMN to HUVE). IL-1 acts selectively on endothelial cells as demonstrated by total inhibition of its effect by actinomycin D. Moreover, IL-1 does not induce HUVE monolayers to secrete a chemotaxin, and the neutrophil passage guiding principle is removable from the HUVE surface by short trypsin exposure. Congruent results were obtained with human adult arterial as well as saphenous vein endothelial cells. As shown by blockade of neutrophil migration with pertussis toxin, IL-1- and TNF-inducible transendothelial migration can be dissected into an initial anchoring step, which is succeeded by active neutrophil migration, possibly along a putative endothelial membrane-bound gradient.
Mechanisms involved in neutrophil accumulation induced by intradermal injection of interleukin‐1 (IL‐1) in the rabbit were investigated using intravenously‐injected ¹¹¹ In‐labelled neutrophils. C5a des Arg, N‐formyl‐methionyl‐leucyl‐phenylalanine (FMLP) and leukotriene B 4 (LTB 4 ) were included for comparison.
Local inhibition of protein biosynthesis in the skin using actinomycin‐D or cycloheximide blocked ¹¹¹ In‐neutrophil accumulation induced by IL‐1, but not that induced by the other mediators.
Actinomycin‐D and cycloheximide had no effect on local plasma protein leakage induced by intradermally‐injected C5a des Arg, or that induced by zymosan. ¹¹¹ In‐neutrophil accumulation induced by zymosan was, however, partially suppressed.
A monoclonal antibody, MoAb 60.3, recognising neutrophil surface CD18 antigen, was preincubated with ¹¹¹ In‐neutrophils before intravenous injection. This pretreatment did not affect circulating numbers of radiolabelled cells, but it inhibited their accumulation in response to IL‐1, C5a des Arg and the other mediators.
The results suggest that neutrophil accumulation induced by IL‐1, but not the other mediators, requires local protein biosynthesis, probably in the microvascular endothelium. Neutrophil accumulation to IL‐1 and the other mediators appears to require neutrophil surface antigen, CD18. The inflammatory response to zymosan may be mediated by both endogenous C5a des Arg and IL‐1.
The synthetic glucocorticoid dexamethasone (1 microM to 1 pM) strongly (maximum greater than 80%) inhibits proliferation of the A549 human lung adenocarcinoma line (EC50 greater than 1 nM) and leads to the appearance, or a further increase (approximately 3-fold) in the expression on the cell surface, of the calcium and phospholipid binding protein lipocortin (annexin) 1. Both these effects, which are shared by hydrocortisone (1 microM) but not by progesterone or aldosterone (1 microM), are inhibited by the antiglucocorticoids RU38486 and RU43044 (1 microM). The nonsteroidal antiinflammatory drugs indomethacin (1 microM) and naproxen (10 microM) and human recombinant lipocortin 1 (0.05-5.0 micrograms/ml) also produce growth arrest in this cell line. During proliferation A549 cells spontaneously release prostaglandin E2 [10-20 ng (28-57 pmol) per ml per 5-day period] into the growth medium. In concentrations that cause growth-arrest, dexamethasone, indomethacin, and lipocortin 1 abolish the generation of this eicosanoid by A549 cells. Prostaglandin E2 itself (0.01-1 pM) stimulates cell growth and partially reverses (approximately 50%) the inhibition of growth caused by dexamethasone and indomethacin. Addition of the neutralizing anti-lipocortin 1 monoclonal antibody 1A (5 micrograms/ml), but not the nonneutralizing anti-lipocortin monoclonal antibody 1B, substantially reversed (greater than 80%) the inhibitory activity of dexamethasone on both growth and prostaglandin E2 synthesis. The generation of prostaglandin E2 by A549 cells seems to be an important regulator of cell proliferation in vitro and the dexamethasone-induced suppression of proliferation in this model is attributable to eicosanoid inhibition caused by lipocortin 1.
Contractions elicited by CaCl2 on isolated rat stomach strip preparations have been reported to be potentiated by interleukin-1 beta (IL-1 beta). We have investigated whether this effect can be reduced by the putative IL-1 beta antagonist, alpha-melanocyte-stimulating hormone (alpha MSH). Additionally, the effects of alpha MSH on the specific binding of IL-1 beta to B- and T-cells have been investigated to further clarify its inhibitory activities. Both alpha MSH and its carboxyl terminal tripeptide concentration dependently reduced the potentiation of CaCl2-induced contractions caused by IL-1 beta but not those caused by leukotriene D4, the parent molecule being approximately 250 times more active. Additionally, both peptides potently and selectively reduced 125I-IL-1 beta binding to the T-cell sub-clone EL4-6.1 but not to the B-cell sub-clone 1H7. The results indicate that IL-1 beta effects on rat stomach may be mediated through a type-I (80 kDa) IL-1 beta receptor.
Adherence of neutrophils to endothelium is a key event in the sequence of inflammatory leukocyte responses. Double-color FACS analysis was used to determine the extent and kinetics of neutrophil adherence to rIL-1 beta-pretreated endothelial cells (EC). Neutrophils bound very avidly when the EC were prestimulated for 4 to 6 h with rIL-1 beta. Anti-ELAM-1 F(ab)2 fragments inhibited this adherence for more than 80%. On the other hand, anti-CD18 F(ab)2 fragments also inhibited the neutrophil adherence (40 to 50%). Combined use of anti-ELAM-1 and anti-CD18 F(ab)2 fragments completely prevented adherence. Neutrophils became activated as soon as they made contact with the rIL-1 beta-pretreated EC. First, neutrophils depleted of intracellular ATP showed a clearly decreased adherence completely dependent on ELAM-1-mediated binding, i.e., without additional effects of CD18 adhesion proteins. Thus, CD18 is activated during neutrophil adherence and then participates in the binding process. Secondly, the neutrophils responded with a transient rise in [Ca2+]i upon binding to rIL-1 beta-pretreated EC, which was demonstrated to be caused by endothelial cell-associated platelet-activating factor (PAF). However, the extent of neutrophil adherence to rIL-1 beta-pretreated EC was not affected by the use of the PAF-receptor antagonist WEB 2086, or removal of the EC-bound PAF. The only effect was a complete dependency of the neutrophil adherence on ELAM-1-mediated binding, although anti-CD18 mAb still induced 40 to 50% inhibition under these conditions. We therefore conclude that ELAM-1-mediated binding is the major mechanism for CD18 activation during neutrophil adherence to rIL-1 beta-pretreated EC.
In order to clarify the roles of platelet‐activating factor (PAF) in histamine‐ and thrombin‐induced neutrophil adhesion to vascular endothelial cells, the effects of several PAF antagonists were examined. The effects of the glucocorticoid dexamethasone were also examined in order to gain further insight into the anti‐inflammatory actions of glucocorticoids.
In culture, histamine and thrombin stimulated the adherence of rat peritoneal neutrophils to human endothelial cells from the umbilical vein. They did not stimulate neutrophil adherence in the absence of endothelial cells, suggesting that the target cells for the histamine‐ and thrombin‐induced adherence of neutrophils were endothelial cells, not neutrophils.
Several PAF antagonists, such as CV‐3988, L‐652,731 and Y‐24,180 inhibited the histamine‐ and thrombin‐induced neutrophil adherence in a concentration‐dependent manner. Indomethacin failed to inhibit it.
Dexamethasone, a steroidal anti‐inflammatory drug, did not inhibit the histamine‐ and thrombin‐induced adherence of neutrophils to endothelial cells when the drug was present only during the 20 min incubation period for the adherence assay. When the endothelial cells were preincubated for 3 h with dexamethasone, the adherence of neutrophils to endothelial cells induced by histamine or thrombin was not inhibited.
When the neutrophils were preincubated for 3 h with dexamethasone, the histamine‐ and thrombin‐induced adherence of neutrophils to endothelial cells was inhibited.
Our studies indicate that: (a) adherence of neutrophils to endothelial cells induced by histamine and thrombin is mediated by PAF production since PAF antagonists inhibited the adherence of neutrophils; and (b) neutrophils, not endothelial cells, are the target cells through which dexamethasone acts to inhibit adherence.
The human squamous cell carcinoma SqCC/Y1 undergoes spontaneous terminal differentiation in the confluent state. The degree of maturation was markedly increased by glucocorticoids and by both human recombinant and placental lipocortin I. Western analyses demonstrated cellular secretion of lipocortin into the medium. Glucocorticoid-induced maturation was antagonized by a lipocortin I-specific monoclonal antibody, by phospholipase A2 (PLA2), and by arachidonic acid. Induction of the differentiation of SqCC/Y1 cells by lipocortin I was prevented by arachidonic acid. The PLA2 inhibitor, dibromoacetophenone, caused an increase in envelope-competent cells indicating that inhibition of PLA2 results in induction of differentiation. Epidermal growth factor prevented the induction of differentiation by both lipocortin I and by glucocorticoids. The nonsteroidal lipoxygenase/cyclo-oxygenase inhibitor, phenidone, also increased SqCC/Y1 differentiation, suggesting that leukotrienes, thromboxanes, and/or prostaglandins may be involved in lipocortin-mediated regulation of SqCC/Y1 maturation. The findings support a role for lipocortin I in mediating the effects of glucocorticoids on epidermal cell differentiation.
This study was designed to examine the effects of i.p.-injected alpha-melanocyte stimulating hormone (MSH) on murine neutrophil migration into subcutaneously implanted sponges in response to IL-1-alpha, TNF-alpha, and C5a. The results show that as little as 0.1 ml of 5 x 10(-7) M MSH injected i.p. significantly blocked the accumulation of neutrophils in sponges in response to IL-1. This action of MSH was dose dependent, reversible, and was maximally effective if MSH was given at the same time as the injection of IL-1. This effect of MSH was not restricted to IL-1-induced neutrophil emigration, because MSH also antagonized the accumulation of neutrophils in response to both TNF and C5a. The proopiomelanocortin-derived peptide ACTH which contains the MSH sequence also significantly reduced neutrophil accumulation in response to IL-1, although less effectively than MSH. Similar studies with beta-endorphin showed that it had no effect on neutrophil accumulation in this system. The direct injection of MSH, beta-endorphin and ACTH into sponges or i.p. did not stimulate a neutrophil emigration and eliminated the possibility that MSH or ACTH suppressed the neutrophil influx in response to IL-1, TNF, or C5a by competing for circulating neutrophils. The action of MSH on IL-1, TNF, and C5a-induced neutrophil emigration suggests that this peptide may be an important regulator of the inflammatory response.
Glucocorticoids exert their actions through a time-dependent, receptor-mediated, protein synthesis- and RNA synthesis-dependent mechanism. We have assessed the effects of 24-h culture of human neutrophils with dexamethasone on degranulation, chemotaxis, binding to vascular endothelium and formation of leukotriene B4. Purified neutrophils contained an average of 2896 [3H]dexamethasone binding sites per cell with a Kd of 4.1 X 10(-9) M for [3H]dexamethasone binding. Cells exposed to dexamethasone (10(-6) M) released equal or greater quantities of the lysosomal enzymes, lysozyme and beta-glucuronidase in response to formylmethionyl-leucyl-phenylalanine, serum activated zymosan, and the tumor promoting phorbol diester 12-O-tetradecanoylphorbol-13-acetate compared to controls. Culture with dexamethasone also did not inhibit neutrophil chemotaxis in response to a range of concentrations of formylmethionyl-leucyl-phenylalanine, or did it inhibit binding of neutrophils to cultured endothelial cells stimulated by either leukocyte activators (formylmethionyl-leucyl-phenylalanine and platelet-activating factor) or endothelial activators (interleukin-1, lipopolysaccharide or 12-O-tetradecanoylphorbol-13-acetate). Spontaneous adherence of neutrophils to endothelial cells was inhibited (82.9 +/- 6.8% of control, P less than .025, n = 18). Neither in vitro or in vivo glucocorticoids inhibited neutrophil leukotriene B4 formation induced by either the calcium ionophore A23187 or serum activated zymosan. We conclude that human neutrophils are not functionally inactivated by glucocorticoids and suggest that the mechanism by which glucocorticoids inhibit neutrophil accumulation at inflammatory sites may be by inhibition of the production of chemoattractants and endothelial activators rather than inhibition of their actions.
The chemically novel acetohydroxamic acids, BW A4C, BW A137C and BW A797C, are potent inhibitors of the synthesis of leukotriene B 4 (LTB 4 ) from arachidonic acid by human leucocyte homogenates: the concentrations required for 50% inhibition (IC 50 ) were 0.1 μ m , 0.8 μ m and 0.5μ m respectively. Inhibition was less at higher concentrations of arachidonic add.
These compounds also inhibited the synthesis of [ ¹⁴ C]‐5‐HETE from [ ¹⁴ C]‐arachidonic acid and the calcium‐dependent synthesis of LTB 4 from 5‐HPETE. This, therefore, suggests that they inhibit 5‐lipoxygenase and LTA 4 synthase.
Concentrations of acetohydroxamic acids required to inhibit metabolism of arachidonic acid by cyclo‐oxygenase, 12‐lipoxygenase and 15‐lipoxygenase were 10 to 100 times higher than those required to inhibit 5‐lipoxygenase.
The compounds were potent inhibitors of LTB 4 synthesis induced by the ionophore, A23187, in human intact leucocytes. This inhibition was reversed by washing the cells. They were also potent, selective inhibitors of LTB 4 synthesis induced by A23187 in whole rat blood: binding to rat plasma proteins did not greatly reduce the effectiveness of the compounds.
The effects of the acetohydroxamic acids, administered either intravenously or orally to rats, on the synthesis of LTB 4 , and thromboxane B 2 (TXB 2 ) in A23187‐stimulated blood ex vivo was studied. The three compounds caused dose‐dependent inhibition of the synthesis of LTB 4 but not TXB 2 . Inhibition of LTB 4 synthesis persisted for up to 6 h after a single oral dose of 50 mg kg ⁻¹ .
The plasma concentrations of unchanged compound determined by h.p.l.c. correlated with the inhibition of LTB 4 synthesis ex vivo .
Neutrophil accumulation and activation are early events in the inflammatory response in vivo. Using human recombinant forms of the putative inflammatory mediators interleukin-1 (IL-1) and tumour necrosis factor (TNF alpha) we were unable to detect direct effects on human neutrophil locomotion or intracellular free calcium concentration ([Ca2+]i) in vitro. Human recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF) was able to stimulate significant locomotion, but was unable to elevate neutrophil [Ca2+]i. In contrast, supernatant from cultured human synovial cells that had been treated with human recombinant IL-1 alpha (28 pM) released a factor that stimulated both neutrophil locomotion and elevated neutrophil [Ca2+]i. Our studies demonstrate that the production of this factor is time-dependent, requiring exposure of the synovial cells to IL-1 for more than 4 hr, is not influenced by cyclo-oxygenase or lipo-oxygenase inhibition, but can be abolished by dexamethasone (100 nM) or actinomycin D (0.8 microM). The factor has a molecular weight above 10,000 and does not cross-react with anti-C5a antisera. IL-1 beta and TNF alpha were also able to stimulate its production. Our findings suggest that the neutrophil accumulation that is known to occur in response to IL-1 in vivo may be a consequence of the local production of such a factor.
WEB 2086, a thieno-triazolodiazepine, is a potent and specific antagonist of platelet activating factor (PAF) in vitro and in vivo. This compound inhibits PAF-induced human platelet and neutrophil aggregation in vitro (IC50 = 0.17 and 0.36 microM, respectively) but has little or no effect on the action of other platelet aggregating agents. In comparison with kadsurenone, ketotifen or thiazinamium chloride, WEB 2086 was 26 to 200 times more potent in the PAF-induced platelet aggregation. In anesthetized guinea pigs, pretreatment with 0.1 to 2.0 mg/kg p.o. or 0.01 to 0.5 mg/kg i.v. of WEB 2086 inhibits dose-dependently the accumulation and aggregation of 111Indium labeled platelets, bronchoconstriction, systemic hypotension and also the lethal effect due to an i.v. PAF infusion [30 ng/(kg X min)] or intratracheal instillation of PAF (300 micrograms/kg). Under the same experimental conditions in guinea pigs, WEB 2086 given by inhalation achieved a similar anti-PAF activity. In anesthetized rats, the hypotension induced by an i.v. PAF infusion was also reversed (ED50 = 0.052 mg/kg i.v.). The increase in cutaneous vascular permeability due to intradermal PAF (25 ng/site) was inhibited dose-dependently by WEB 2086 (0.025-2 micrograms/site) in rats. Because of its structural relationship to triazolodiazepines, WEB 2086 was examined for anticonvulsant and sedative action. Up to doses of 300 and 800 mg/kg p.o., respectively, no effects were found. In conclusion, WEB 2086 is a potent and specific PAF antagonist with triazolodiazepine structure but without sedative activity.
Hydrocortisone and the glucocorticoid-induced anti-phospholipase protein macrocortin, were tested as inhibitors of PAF generation. The steroid produced a dose-dependent inhibition of the release of the PAF precursor 2-lyso-PAF, and this effect was mimicked by affinity-purified macrocortin. Neither agent had any effect on the acetylation of lyso-PAF to PAF. Of other drugs tested only phospholipase inhibitors blocked lyso-PAF release and sulphydryl reagents blocked the acetylation step. It is concluded that glucocorticoids inhibit the generation of PAF and this could be an important component of their anti-anaphylactic and anti-inflammatory action.
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