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Lipocortin 1 and Chemokine Modulation of Granulocyte and Monocyte Accumulation in Experimental Inflammation
Abstract
Migration of blood-derived leukocytes to tissue sites of inflammation is a hallmark of the response that the host organizes to counteract an insult or a trauma or an infection. A cascade of events is then activated to allow interaction between the leukocyte and the endothelium of postcapillary venule, and this cascade is finely regulated such that mechanisms of negative control are operating side by side with pathways that promote and sustain the extravasation process. Examples of both these positive and negative regulatory systems are discussed here.
... Previous studies have reported that ANXA1 induced in endotoxemia, peritonitis, and arthritis exerts anti-inflammatory properties by suppression of leukocyte activation and transmigration (9)(10)(11)(12). Such anti-inflammatory properties of ANXA1 are mediated by autocrine and paracrine signaling of a 26-amino-acid ANXA1 N-terminus peptide, Ac2-26, that is released from the fulllength protein through regulated proteolysis (2,(13)(14)(15)(16). ...
... Such important reparative responses are modulated by mucosal mediators, such as anti-inflammatory protein ANXA1 (60)(61)(62)(63). The beneficial effects of ANXA1 are exerted by a unique N-terminal peptide that is released by proteolysis from the full-length protein (2,13,14,64). The importance of the N-terminal domain in mediating anti-inflammatory activity is further supported by studies wherein deletion of the N terminus induced inactivation of the protein, while use of synthetic N terminus peptides exerted pharmacologic activity (65,66). ...
N-formyl peptide receptors (FPRs) are critical regulators of host defense in phagocytes and are also expressed in epithelia. FPR signaling and function have been extensively studied in phagocytes, yet their functional biology in epithelia is poorly understood. We describe a novel intestinal epithelial FPR signaling pathway that is activated by an endogenous FPR ligand, annexin A1 (ANXA1), and its cleavage product Ac2-26, which mediate activation of ROS by an epithelial NADPH oxidase, NOX1. We show that epithelial cell migration was regulated by this signaling cascade through oxidative inactivation of the regulatory phosphatases PTEN and PTP-PEST, with consequent activation of focal adhesion kinase (FAK) and paxillin. In vivo studies using intestinal epithelial specific Nox1-/-IEC and AnxA1-/- mice demonstrated defects in intestinal mucosal wound repair, while systemic administration of ANXA1 promoted wound recovery in a NOX1-dependent fashion. Additionally, increased ANXA1 expression was observed in the intestinal epithelium and infiltrating leukocytes in the mucosa of ulcerative colitis patients compared with normal intestinal mucosa. Our findings delineate a novel epithelial FPR1/NOX1-dependent redox signaling pathway that promotes mucosal wound repair.
... The expression of proteins that control vascular tone, such as nitric oxide synthase (NOS) and leukocyte interaction, such as platelet endothelial cell adhesion molecule 1 (PECAM-1) and intercellular cell adhesion molecule 1 (ICAM-1), can be modulated by hormonal profiles [1,2]. Hormones released according to the circadian rhythm, such as corticosterone and melatonin, control endothelial-mediated immune cell migration [3][4][5]. ...
... Corticosterone impairs leukocyte migration owing to the externalization of lipocortin 1 at the plasma membrane of adherent neutrophils [3] and by the reduced expression of L-selectin by endothelial cells [4]. The melatonin effect is mediated by the inhibition of the nuclear factor kappa B (NF-kB) activation, which impairs the expression of adhesion molecules and enzymes involved in the inflammatory response, such as the inducible nitric oxide synthase (iNOS) [5][6][7][8][9]. ...
The endothelial layer regulates the traffic of cells and substances between the blood and tissues and plays a central role in the mounting of an inflammatory response. We have recently shown that inhibition of the nocturnal melatonin surge during the mounting of an inflammatory response primes endothelial cells to a highly reactive state, increasing the expression of adhesion molecules and inducible nitric oxide synthase (iNOS) as well as the in vitro adherence of leukocytes. Here, we investigated whether physiological variations in the plasma melatonin levels owing to the light/dark environmental cycle could also prime the reactive state of endothelial cells. Cultured endothelial cells (16-20 days) obtained from rats killed during the daytime adhere more neutrophils, expressed more adhesion molecules and iNOS, and had a higher content of the transcription factor nuclear factor kappa B (NF-kB) translocated to the nuclei. We also evaluated the expression of 84 genes (using real-time PCR array) related to the innate inflammatory response and observed a higher expression of 19 genes in cultures obtained during the daytime. In addition, the only gene that was highly expressed in cells obtained from rats killed during nighttime was one that encodes a protein that negatively modulates inflammatory response. In conclusion, the daily rhythm of melatonin also primes the ability of endothelial cells to adhere to neutrophils. This new approach for evaluating the influence of the donor on cells maintained in culture should have applications for the standardization of cell banks.
... A growing body of evidence from studies on inflammation in animal models and humans suggests that annexin I potently inhibits neutrophil extravasation (reviewed in Ref. 35). Annexin I also is strongly induced by glucocorticoids and is considered to mediate some of the well-known antiinflammatory effects of these hormones (36). ...
... The N terminus of annexin I has been shown to play an important modulatory role in the biological effects of the protein. For example, it has been shown by several studies that the N-terminal peptide Ac 2-26 mimics the potent antiinflammatory effects of intact annexin I (35). The amino-terminal domain regulates interactions with membranes (37), and is directly involved in binding to S100 proteins (38,39). ...
We recently showed that a class of novel carboxylated N:-glycans was constitutively expressed on endothelial cells. Activated, but not resting, neutrophils expressed binding sites for the novel glycans. We also showed that a mAb against these novel glycans (mAbGB3.1) inhibited leukocyte extravasation in a murine model of peritoneal inflammation. To identify molecules that mediated these interactions, we isolated binding proteins from bovine lung by their differential affinity for carboxylated or neutralized glycans. Two leukocyte calcium-binding proteins that bound in a carboxylate-dependent manner were identified as S100A8 and annexin I. An intact N terminus of annexin I and heteromeric assembly of S100A8 with S100A9 (another member of the S100 family) appeared necessary for this interaction. A mAb to S100A9 blocked neutrophil binding to immobilized carboxylated glycans. Purified human S100A8/A9 complex and recombinant human annexin I showed carboxylate-dependent binding to immobilized bovine lung carboxylated glycans and recognized a subset of mannose-labeled endothelial glycoproteins immunoprecipitated by mAbGB3.1. Saturable binding of S100A8/A9 complex to endothelial cells was also blocked by mAbGB3.1. These results suggest that the carboxylated glycans play important roles in leukocyte trafficking by interacting with proteins known to modulate extravasation.
... These anti-inflammatory effects are mediated by autocrine and paracrine signaling of the 26-amino acid ANXA1 N-terminal peptide Ac2-26, which is released from the full-length protein through regulated cleavage, making this peptide the pharmacophore region of ANXA1. 8 Strategies for the targeted and specific delivery of ANXA1 and its biomimetic peptides that utilize drug delivery systems are attractive avenues to explore for developing proresolving therapies for both acute and chronic inflammatory conditions. Nanomedicine allows the engineering of polymeric nanoparticle (NP) carriers for controlled and targeted drug delivery. ...
Background
Although in most patients with inflammatory bowel diseases, conservative therapy is successful, a significant proportion of patients still require surgery once in their lifetime. Development of a safe perioperative treatment to dampen colitis activity without disturbance of anastomotic healing is an urgent and unmet medical need. Annexin A1 (ANXA1) has been shown to be effective in reducing colitis activity. Herein, a nanoparticle-based perioperative treatment approach was used for analysis of the effects of ANXA1 on the resolution of inflammation after surgery for colitis.
Methods
Anxa1-knockout mice were used to delineate the effects of ANXA1 on anastomotic healing. A murine model of preoperative dextran sodium sulfate colitis was performed. Collagen-IV-targeted polymeric nanoparticles, loaded with the ANXA1 biomimetic peptide Ac2-26 (Ac2-26-NPs), were synthesized and administered perioperatively during colitis induction. The effects of the Ac2-26-NPs on postoperative recovery and anastomotic healing were evaluated using the disease activity index, histological healing scores, and weight monitoring. Ultimately, whole-genome RNA sequencing of the anastomotic tissue was performed to unravel underlying molecular mechanisms.
Results
Anxa1-knockout exacerbated the inflammatory response in the healing anastomosis. Treatment with Ac2-26-NPs improved preoperative colitis activity (P < 0.045), postoperative healing scores (P < 0.018), and weight recovery (P < 0.015). Whole-genome RNA sequencing revealed that the suppression of proinflammatory cytokine and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling was associated with the treatment effects and a phenotypic switch toward anti-inflammatory M2 macrophages.
Conclusions
Proresolving therapy with Ac2-26-NPs promises to be a potent perioperative therapy because it improves colitis activity and even intestinal anastomotic healing by the suppression of proinflammatory signaling.
... It is mostly found in white blood cells, stromal cells and biological fluids expressed in variety of cells including white blood cells, endothelial cells, fibroblasts and gut epithelial cells [68,69]. It inhibits the formation of lipid mediator by blockade of enzyme phospholipase A 2 , thereby regulating cellular growth and differentiation, neuroendocrine secretion, central nervous system response to cytokines and accumulation of neutrophil to different tissues [70][71][72]. It blocks the release of inflammatory mediators by inhibition of cytosolic phospholipase A 2 enzyme, thus prevents the formation of pro-inflammatory eicosanoid, inducible cyclooxygenase and nitric oxide synthase enzymes [73]. ...
Inflammation is a physiological intrinsic host response to injury meant for removal of noxious stimuli and maintenance of homeostasis. It is a defensive body mechanism that involves immune cells, blood vessels and molecular mediators of inflammation. Glucocorticoids (GCs) are steroidal hormones responsible for regulation of homeostatic and metabolic functions of body. Synthetic GCs are the most useful anti-inflammatory drugs used for the treatment of chronic inflammatory diseases such as asthma, chronic obstructive pulmonary disease (COPD), allergies, multiple sclerosis, tendinitis, lupus, atopic dermatitis, ulcerative colitis, rheumatoid arthritis and osteoarthritis whereas, the long term use of GCs are associated with many side effects. The anti-inflammatory and immunosuppressive (desired) effects of GCs are usually mediated by transrepression mechanism whereas; the metabolic and toxic (undesired) effects are usually manifested by transactivation mechanism. Though GCs are most potent anti-inflammatory and immunosuppressive drugs, the common problem associated with their use is GC resistance. Several research studies are rising to comprehend these mechanisms, which would be helpful in improving the GC resistance in asthma and COPD patients. This review aims to focus on identification of new drug targets in inflammation which will be helpful in the resolution of inflammation. The ample understanding of GC mechanisms of action helps in the development of novel anti-inflammatory drugs for the treatment of inflammatory and autoimmune disease with reduced side effects and minimal toxicity.
... In addition, two other proteins, Annexin A1 and Lamin, not involved in glycolysis, has been identified by our proteomic analysis. Annexin A1 have been implicated in many cellular processes such as inflammation [26][27][28], glucorticoid action [29], secretion and exocytosis [30][31][32], cell growth and differentiation [33][34][35] and is phosphorylated by several protein tyrosine kinase (PTKs) [36,37]. As far as Lamin is concerned, its phosphorylation is mainly dependent on the cell cycle [38][39][40]. ...
Background:
Low Molecular Weight Phosphotyrosine Protein Phosphatase (LMW-PTP) is an enzyme involved not only in tumor onset and progression but also in type 2 diabetes. A recent review shows that LMW-PTP acts on several RTK (receptor tyrosine kinase) such as PDGFR, EGFR, EphA2, Insulin receptor. It is well described also its interaction with cSrc. It is noteworthy that most of these conclusions are based on the use of cell lines expressing low levels of LMW-PTP. The aim of the present study was to discover new LMW-PTP substrates in aggressive human tumors where the over-expression of this phosphatase is a common feature.
Methods:
We investigated, by proteomic analysis, the protein phosphorylation pattern of A375 human melanoma cells silenced for LMW-PTP. Two-dimensional electrophoresis (2-DE) analysis, followed by western blot was performed using anti-phosphotyrosine antibodies, in order to identify differentially phosphorylated proteins.
Results:
Proteomic analysis pointed out that most of the identified proteins belong to the glycolytic metabolism, such as α-enolase, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase and triosephosphate isomerase, suggesting an involvement of LMW-PTP in glucose metabolism. Assessment of lactate production and oxygen consumption demonstrated that LMW-PTP silencing enhances glycolytic flux and slow down the oxidative metabolism. In particular, LMW-PTP expression affects PKM2 tyrosine-phosphorylation and nuclear localization, modulating its activity.
Conclusion:
All these findings propose that tumor cells are subjected to metabolic reprogramming after LMW-PTP silencing, enhancing glycolytic flux, probably to compensate the inhibition of mitochondrial metabolism.
General significance:
Our results highlight the involvement of LMW-PTP in regulating glucose metabolism in A375 melanoma cells.
... Adhesion receptors permit the circulating cell to scan "the endothelium." After the matching, endothelial "signal expression pattern" was found [71], and receptor activation leads to firm adhesion. ...
Mesenchymal stem cells (MSCs) are promising candidates for adult cell therapies in regenerative medicine. To fully exert their potential, efficient homing and migration toward lesion sites play an important role. Local transplantation deposits MSC in spatial proximity to the lesion, but often requires invasive procedures. Systemic administration routes are favored, but require the targeted extravasation of the circulating MSC at the site of injury. Transplanted MSC can indeed leave the blood flow and transmigrate through the endothelial barrier, and reach the lesion site. However, the underlying processes are not completely dissolved yet. Recent in vitro and in vivo research identified some key molecules scattered light on the extravasation mechanism. This review provides a detailed overview over the current knowledge of MSC transendothelial migration. We use the leukocyte extravasation process as a role model to build a comprehensive concept of MSC egress mechanisms from the blood stream and identified relevant similarities as well as important differences between the extravasation mechanisms. Stem Cells 2017;35:1446-1460.
... The association of OT and anti-inflammatory potential is also known from previous studies; one of the theories to put mechanism for this effect is that OT may induce elevation in corticosteroid tissue level which will inhibit neutrophil infiltration to the target tissue. [30,31] Reperfusion of the globally ischemic myocardium will be a signal not just for recruiting more neutrophils but also increases the grade of neutrophils activation which will emit more free radicals and reactive oxygen molecules which in turn augment tissue injury after reperfusion. [32] This will be a vicious circle that is the more injury then the more neutrophil infiltration. ...
Cardiac oxytocin (OT) is structurally identical to that found in the hypothalamus, which thereby indicates that cardiac OT is derived from the same gene and is an active form of OT. The abundance of OT and OT receptors in atrial myocytes shows that, directly and/or via the release of the cardiac hormone atrial natriuretic peptide, OT can regulate the force of cardiac contractions. Previous studies have demonstrated the role of OT in the myocardial inflammatory response. The mechanism by which OT elicits protective myocardial effects in the immediate post-transplantation period is not yet clear, and the role of the early phase inflammatory elements in this mechanism has not yet been studied. As a result, in this study, we have investigated the anti-inflammatory effects of OT on myocardial protection in the immediate post-transplantation period.
Methods: Adult male Albino rats were grouped into: Sham, Control, and OT-treated groups. The control and treated groups sustained cervical heterotopic heart transplant. Myocardial injury was assessed by measuring: Plasma cardiac troponin I, myocardial proinflammatory cytokines, and histopathological assessment for score of injury, and degree of apoptosis. Myocardial myeloperoxidase, neutrophil infiltration, and neutrophil chemotactant agents, reactive oxygen species, and reactive nitrogen species formation all were measured in the myocardium after 3 hour of reperfusion to assess the neutrophil-dependant myocardial injury and the mechanism involved.
Results: Oxytocin down-regulates the neutrophil chemotactant agents the KC/CXCL1 and MIP-2/CXCL2 which recruit less neutrophil into myocardium, this decrement in myocardial PMN infiltration is associated with less reactive oxygen species and reactive nitrogen species formation in the myocardium after 3 hours of global ischemia reperfusion. These oxytocin-induced down-regulation inflammatory and oxidative processes will end in less myocardial injury through impedance in the post-myocardial ischemia/reperfusion apoptotic process.
Conclusion: Oxytocin ameliorates myocardial injury in heart transplant through down-regulation the myocardial inflammatory response, reactive oxygen species, and neutrophil-dependant myocardial apoptosis.
... This pathway describes the stages involved in the movement or migration of leukocytes out of the circulatory system to the site of tissue damage or infection during an inflammatory response [32][33][34] . Chemotactic molecules (chemokines) like those secreted by monocytes, macrophages and other immune cells play an important role during this process 35,36 . Function of chemokines is mainly to bring about migration (homing) of leukocytes in the respective sites during homeostasis and inflammatory processes 37 . ...
Post-traumatic stress disorder patients experience chronic systemic inflammation. However, the molecular pathways involved and mechanisms regulating the expression of genes involved in inflammatory pathways in PTSD are reported inadequately. Through RNA sequencing and miRNA microarray, we identified 326 genes and 190 miRNAs that were significantly different in their expression levels in the PBMCs of PTSD patients. Expression pairing of the differentially expressed genes and miRNAs indicated an inverse relationship in their expression. Functional analysis of the differentially expressed genes indicated their involvement in the canonical pathways specific to immune system biology. DNA methylation analysis of differentially expressed genes also showed a gradual trend towards differences between control and PTSD patients, again indicating a possible role of this epigenetic mechanism in PTSD inflammation. Overall, combining data from the three techniques provided a holistic view of several pathways in which the differentially expressed genes were impacted through epigenetic mechanisms, in PTSD. Thus, analysis combining data from RNA-Seq, miRNA array and DNA methylation, can provide key evidence about dysregulated pathways and the controlling mechanism in PTSD. Most importantly, the present study provides further evidence that inflammation in PTSD could be epigenetically regulated.
... AnxA1 is an antiinflammatory mediator and we proposed that it may also have a role in the function of the adipose tissue inflammation. AnxA1, a 37-kDa and Ca2+ -and phospholipidbinding protein, is an endogenous glucocorticoid regulated protein, which mediates Chapter 3 Attenuation of plasma AnxA1 in human obesity __________________________________________________________________________________ systemic anti-inflammatory processes (D'Acquisto et al., 2008b, Pupjalis et al., 2011, Dalli et al., 2011 including reducing eicosanoid synthesis (Sudlow et al., 1996), antipyretic effects (Davidson et al., 1991), an antiendotoxic action (Wu et al., 1995) and reduced neutrophil emigration from the systemic circulation (Perretti, 1998). ...
Introduction: The escalating public health problem represented by obesity has spurred multidisciplinary research into adipose tissue and importantly, the molecular biology of the adipocyte. The concept of adipose tissue as an endocrine organ, in addition to an energy storage compartment, is now pivotal in linking excess adiposity to disease states. Recent studies suggest that obesity related metabolic disorders are characterised by mild chronic inflammation as a result of adipocytokine production from fat tissue leading to dysregulation in the pro/anti?inflammatory systemic balance. Adipokines and pro?inflammatory markers are implicated in insulin insensitivity, blood glucose dysregulation, inflammation and atherosclerosis. There is a considerable amount of research into the characterization of adipokines and pro?inflammatory cytokines, the antiinflammatory adipocytokines warrant further exploration. Research studies and design: This PhD research was set out to investigate potential antiinflammatory molecules that could be used as markers of, and therapeutics for, metabolic syndrome associated maladies. This PhD consists of three studies including Study 1: a characterisation study of pro/anti?inflammatory mediators carried out on 116 men of various BMI and body composition; Study 2: an in vitro study design carried out in human Simpson Golabi Behmel Syndrome (SGBS) adipocyte cell line investigating a glucocorticoid regulated anti?inflammatory protein, annexin A1 (AnxA1) and its role in fat tissue function; and Study 3: a double-blind cross-over randomised trial in 15 borderline metabolic syndrome males investigating the effect of a supplemental antiinflammatory agent, resveratrol (250 mg/day for two weeks, from 500 mg of Polygonum cuspidatum (from root)), on metabolic parameters. Key findings: Study 1: We demonstrated for the first time that AnxA1 is significantly inversely correlated with increasing BMI (R = -0.424**, P < 0.001), increasing body fat % (R = -0.192, P = 0.037) and a larger waist size (R = -0.390**, P < 0.001) in 118 men aged 19 to 61 years, with BMI between 16.8 – 56.4 kg/m2, BF % between 4.3 to 51.8 %. The negative correlation of decreasing plasma AnxA1 was strongest statistically when compared with WHR, rather than total body fat, suggesting that centrally located fat may be more influential at reducing plasma AnxA1 concentrations. Study 2: We have shown that ANXA1 gene is expressed in human SGBS adipocytes and hypoxia reduces the expression of ANXA1 gene showing that AnxA1 may act as a counter regulator of adipose tissue inflammation. We found that CRP expression was significantly down-regulated following 4 (P=0.015), 8 (P=0.035) and 24 (P=0.037) of hypoxia treatment in the cells also treated with Ac2-26 peptide compared to vehicle alone. IL-6 was also found to be significantly down?regulated after 24 hour hypoxia treatment in the Ac2-26 treated cells compared to vehicle (P=0.022). Study 3: The effect of resveratrol on metabolic function had no significant effect on the metabolic markers measured including blood pressure, blood glucose, blood cholesterol and glycated LDL. Conclusion: These data demonstrate that AnxA1 could potentially represent a (fat) depot specific biomarker whose decline with increasing central adiposity may relate to the phenomena of increasing systemic inflammation and associated disease risk. We also demonstrate for the first time that an AnxA1 is expressed in human SGBS preadipocytes and mature adipocytes and AnxA1 mimetic, Ac2-26 peptide, regulates pro?inflammatory markers in human SGBS adipocytes. We showed that it may be difficult to improve the metabolic profile of individuals through supplementation of exogenous anti-inflammatory agent, resveratrol. Whilst anti-inflammatory agents such as AnxA1 may propose novel therapeutics for metabolic syndrome associated diseases, to date regular exercise and weight loss remain the main interventions that significantly.
... The profile of effect was similar to that attained with LC1 (i.e., inhibition of IS and IS/LV, reduction in MPO values, TNF-α and MIP-1α levels, and no effect on the parameters of systemic cell activation). Treatment with DEX is likely to increase LC1 levels in circulating leukocytes (18,38,54), thereby augmenting the inhibitory action of endogenous LC1 on neutrophils adherent to the endothelium (15,55,56). Further investigation is required to study the potential role of endogenous LC1 in a systematic manner. ...
... Annexins consist of a conserved C-terminal domain that confers calcium-dependent phospholipid binding and a variable N-terminal domain that is responsible for the specific properties of each annexin I [1,2] . As a steroid-regulated protein annexin I has been found to participate in the cell differentiation and anti-inflammatory effects [3,4] . It is also a major substrate of EGF receptor, which related to endocytic trafficking and sorting of EGFR in multivesicular endosomes [5] . ...
AIM: To investigate the alteration of the annexin I subcellular localization in esophageal squamous cell carcinoma (ESCC) and the correlation between the translocation and the tumorigenesis of ESCC.
METHODS: The protein localization of annexin I was detected in both human ESCC tissues and cell line via the indirect immunofluorescence strategy.
RESULTS: In the normal esophageal epithelia the annexin I was mainly located on the plasma membrane and formed a consecutive typical trammels net. Annexin I protein also expressed dispersively in cytoplasm and the nuclei without specific localization on the nuclear membrane. In esophageal cancer annexin I decreased very sharply with scattered disappearance on the cellular membrane, however it translocated and highly expressed on the nuclear membrane, which was never found in normal esophageal epithelia. In cultured esophageal cancer cell line annexin I protein was also focused on the nuclear membrane, which was consistent with the result from esophageal cancer tissues.
CONCLUSION: This observation suggests that the translocation of annexin I protein in ESCC may correlate with the tumorigenesis of the esophageal cancer.
... In contrast, expression of ANXA1 in inflammatory cells increased at 3 months and then decreased by 5 months, perhaps as a consequence of immunosuppression during the chronic phase of opisthorchiasis (7,43,44). ANXA1 is an anti-inflammatory mediator capable of down-modulating leukocyte tissue localization (45). Alternatively, decreased expression of ANXA1 at 1 month might be related to a switching from innate immunity during acute infection (35) to an adaptive response in chronic infection. ...
We investigated the cytokine/chemokine secretions and alteration of protein expression from peripheral blood mononuclear cells (PBMCs) co-cultured with adult liver flukes, Opisthorchis viverrini, for 6 h to 24 h. PBMC-derived proteins were identified by two-dimensional electrophoresis and mass spectrometry, and the cytokines/chemokines in the supernatant were assessed using a cytokine array. Exposure to O. viverrini induced increase in secretion of pro-inflammatory cytokines, co-stimulating protein, adhesion molecule, and chemotactic chemokines relative to untreated controls. In contrast, secretion of CD40 ligand, interleukin-16, and macrophage inflammatory protein-1b decreased. Proteomic analysis revealed that expression of 48 proteins was significantly altered in PBMCs stimulated with O. viverrini. Selected for further study, immunoblotting of annexin A1 (ANXA1) showed upregulation in PBMCs after 12 and 24 h co-cultured with liver flukes. In in vivo study, transcription and translation of ANXA1 significantly increased in livers of hamsters infected with O. viverrini at 21 days and from 3 months onwards compared to normal controls. Interestingly, immunohistochemistry revealed that ANXA1 was present not only in the cytoplasm of inflammatory cells but also in the cytoplasm of cholangiocytes, in close contact with the parasite and its excretory/secretory products in the biliary system. Expression of ANXA1 increased with time concomitant with bile duct enlargement, bile duct formation and epithelial cell proliferation. In conclusion, several cytokines/chemokines secreted by PBMCs and upregulation of ANXA1 in PBMCs and biliary epithelial cells might have a role in host-defense against O. viverrini infection and tissue resolution of inflammation.
... The association of OT and anti-inflammatory potential is also known from previous studies; one of the theories to put mechanism for this effect is that OT may induce elevation in corticosteroid tissue level which will inhibit neutrophil infiltration to the target tissue. [30,31] Reperfusion of the globally ischemic myocardium will be a signal not just for recruiting more neutrophils but also increases the grade of neutrophils activation which will emit more free radicals and reactive oxygen molecules which in turn augment tissue injury after reperfusion. [32] This will be a vicious circle that is the more injury then the more neutrophil infiltration. ...
Cardiac oxytocin (OT) is structurally identical to that found in the hypothalamus, indicating that this active form of OT is derived from the same gene. The abundance of OT and its receptors in atrial myocytes suggests that, directly and/or via the release of the cardiac hormone atrial natriuretic peptide, this hormone regulates the force of cardiac contractions. Previous studies have demonstrated a role of OT in myocardial inflammatory responses. We sought to study protective myocardial and anti-inflammatory effects of OT in the immediate post-transplant period.
We grouped adult male Albino rats into sham, control, and OT-treated groups. Control and treated groups underwent heterotopic cervical heart transplantation. Myocardial injury was assessed by measuring plasma cardiac troponin I, and myocardial proinflammatory cytokines as well as by performing histopathologic assessments injury score, and of apoptotic degree. Myocardial myeloperoxidase, neutrophil infiltration, neutrophil chemotactic mediators as well as formation of reactive oxygen and reactive nitrogen species were measured in the myocardium at 3 hours after reperfusion to assess neutrophil-dependent myocardial injury.
OT downregulates neutrophil chemotactic molecules - KC/CXCL1 and MIP-2/CXCL2. The decrement in myocardial PMN infiltration was associated with reduced reactive oxygen and reactive nitrogen species formation in the myocardium at 3 hours after reperfusion following global ischemia. OT reduced postmyocardial ischemia/reperfusion apoptotic processed.
OT ameliorated immediate myocardial injury in heart grafts, through downregulation of the inflammatory response, of reactive oxygen species, and of neutrophil dependent apoptosis.
... In contrast to the PLA 2 inhibition by annexin I, a property shared with all other annexins tested so far, the inhibitory effect on neutrophil extravasation has been regarded as specific for this annexin (Goulding et al., 1998;Perretti, 1998). To verify this hypothesis using controlled cell culture conditions, we employed a two-chamber model and examined in vitro the transendothelial passage of neutrophils in the presence or absence of different purified annexins. ...
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.
... 11,12 The mechanism(s) of action of annexin 1 and its mimetic has long remained elusive. 13 A recent study by Walther et al 10 has indicated involvement of the receptor for the tripeptide formyl-Met-Leu-Phe (fMLP), termed formyl-peptide receptor or FPR. FPR is the prototype of a group of G protein-coupled receptors: 3 receptor members have been described in the human system, 14 whereas 6 genes, of which possibly 3 translated into protein, have been reported in the mouse. ...
... Although necrotic neutrophils in the airways release abundant amounts of proteases including neutrophil elastase that can be measured in bronchoalveolar lavage (BAL) fluid (BALF) [10,11] , it is not easy to determine whether BALF elastase is actively released by neutrophils or released from necrotic neutrophils. We previously observed that neutrophil elastase in BALF of CF patients readily cleaved a BALF 36 kDa protein annexin 1 [11] which is also abundant in circulating neutrophils and monocytes [12]. In this study we used neutrophil intracellular annexin 1 as a marker to determine whether neutrophil apoptosis and/or necrosis were associated with intense airway inflammation in lung transplant recipients with CF during episodes of bacterial tracheobronchitis. ...
Background
Neutrophils sequestered in lower respiratory tract secretions in the inflamed lung may undergo apoptosis and/or necrosis and release toxic cellular contents that can injure airways or parenchyma. This study examined the viability of neutrophils retrieved from the proximal airways of lung transplant recipients with bacterial tracheobronchitis.
Methods
Integrity and stability of intracellular proteins in neutrophils from proximal airways and peripheral blood from lung transplant recipients with bacterial tracheobronchitis were analyzed via Western blot analysis and determination of neutrophil viability by morphologic appearance and flow cytometry.
Results
Neutrophils in tracheobronchial secretions from lung transplant recipients with cystic fibrosis who had normal chest radiographic imaging but bronchoscopic evidence of purulent tracheobronchitis post-transplant were necrotic and associated with degradation of intracellular protein annexin 1. The neutrophil influx was compartmentalized to large airways and not detected in peripheral bronchoalveolar airspaces sampled via bronchoalveolar lavage. Peripheral blood neutrophils from healthy subjects cultured in vitro demonstrated that annexin 1 degradation, particularly to a 33 kDa annexin 1 breakdown product (A1-BP), was associated with neutrophil necrosis, but not apoptosis. Although annexin 1 degradation was not specific to neutrophil necrosis, it was a sensitive marker of intracellular protein degradation associated with neutrophil necrosis. Annexin 1 degradation to 33 kDa A1-BP was not observed in peripheral blood neutrophils from healthy subjects, but annexin 1 appeared to be degraded in peripheral blood neutrophils of lung transplant recipients despite a normal morphologic appearance of these cells.
Conclusions
Neutrophils were necrotic from the proximal airways of lung transplant recipients with bacterial tracheobronchitis, and this process may begin when neutrophils are still in the systemic circulation prior to sequestration in inflamed airways. Annexin 1 degradation to 33 kDa A1-BP may be useful as a sensitive marker to detect neutrophil necrosis.
... The liver of infected animals treated with the highest dose of dexamethasone showed that the bacteria were present not only in polymorphoneutrophils and macrophages but also in hepatocytes, again probably due to the increased bacterial load in these animals. Dexamethasone suppresses lipopolysaccharide-induced polymorphoneutrophil and macrophage migration (Perretti, 1998), but does not appear to suppress polymorphoneutrophil migration into lesions in response to S. Typhimurium infection in vivo. The suppression of inflammatory gene expression by glucocorticoids is well known and, indeed, we showed iNOS mRNA suppression by dexamethasone in uninfected animals. ...
Salmonella enterica serovar Typhimurium (S. Typhimurium) infection causes an inflammatory response through activation of Toll-like receptor 4 by lipopolysaccharide. Dexamethasone, a glucocorticoid analogue, suppresses inflammatory responses by many mechanisms including inhibition of the lipopolysaccharide-induced production of proinflammatory mediators. There is little information on the effect of glucocorticoids on murine salmonellosis. In this study, we treated susceptible BALB/c mice by subcutaneous implantation of slow-release dexamethasone pellets before infection with S. Typhimurium. Dexamethasone promotes bacterial growth early in infection and induces a dose-dependent increase in bacterial growth within mouse livers and spleens. The bacterial load in organs from infected placebo-treated mice was lower than that in dexamethasone-treated mice. Glucocorticoids inhibit lipopolysaccharide-induced inflammation partially through the steroid-inducible protein annexin-A1 (ANXA1). Infection of wild-type and ANXA1 knock-out mice with S. Typhimurium led to similar organ bacterial loads. ANXA1 also did not affect the bacterial load in organs from infected dexamethasone-treated mice. This suggests that glucocorticoids, independently of ANXA1, accelerate S. Typhimurium growth in vivo in susceptible BALB/c mice.
... The profile of effect was similar to that attained with LC1 (i.e., inhibition of IS and IS/LV, reduction in MPO values, TNF-α and MIP-1α levels, and no effect on the parameters of systemic cell activation). Treatment with DEX is likely to increase LC1 levels in circulating leukocytes (18,38,54), thereby augmenting the inhibitory action of endogenous LC1 on neutrophils adherent to the endothelium (15,55,56). Further investigation is required to study the potential role of endogenous LC1 in a systematic manner. ...
We assessed here the effect of the glucocorticoid-regulated protein lipocortin 1 (LC1) in a model of rat myocardial ischemia reperfusion. Treatment of animals with human recombinant LC1 at the end of a 25-min ischemic period significantly reduced the extent of infarct size in the area at risk as measured 2 h later, with approximately 50% inhibition at the highest dose tested of 50 microg per rat (equivalent to 5.4 nmol/kg). The protective effect of LC1 was abolished by protein denaturation and not mimicked by the structurally related protein annexin V. A combination of electron and light microscopy techniques demonstrated the occurrence of the myocardial damage at the end of the reperfusion period, with loss of fiber organization. LC1 provided a partial and visible protection. The dose-dependent protection afforded by LC1 was paralleled by lower values of myeloperoxidase activity, tumor necrosis factor a, and macrophage inflammatory protein-1a. The functional link between migrated leukocytes and the myocardial damage was confirmed by electron and light microscopy, and a significantly lower number of extravasated leukocytes was counted in the group of rats treated with LC1 (50 microg). In conclusion, we demonstrate for the first time that LC1 reduces the leukocyte-dependent myocardial damage associated with an ischemia-reperfusion procedure.
... Therefore, this model of acute peritonitis can be added to the list of experimental systems in which neutrophil extravasation is down-regulated by systemic injection of ANX-A1. 41 Human and rodent neutrophils have abundant ANX-A1 9,34,42-44 . A substantial amount of protein externalization and secretion (Ͼ60% of total content) has been observed on adhesion of this cell type to monolayers of endothelial cells in vitro. ...
Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.
... Since anti-ANX-1 antibodies can block some of the anti-inflammatory effects of dexamethasone [14] it has been suggested that ANX-1 mediates some, though by no means all [15], of the anti-inflammatory actions of glucocorticoids. Indeed, exogenous ANX-1 reduces neutrophil infiltration in several animal models and against various inflammatory stimuli including IL-1b and zymosan (for review see [16]). However, the effect of exogenous ANX-1 has yet to be investigated in a rat vascular bed or in a model of inflammation as severe as that produced by LPS. ...
Annexin 1 (ANX-1) can reduce leucocyte migration in response to cytokines and chemokines in some rodent models of inflammation. However, its effectiveness against an inflammatory stimulus as strong as bacterial lipopolysaccharide (LPS) is unknown. Thus, we have examined whether ANX-1 can modulate LPS-induced neutrophil accumulation in the rat, as assessed by intravital microscopy and by myeloperoxidase (MPO) assay. The anti-inflammatory glucocorticoid, dexamethasone (DEX) was also studied for comparison. LPS superfusion induced adhesion of leucocytes to the endothelium and a subsequent increase in emigration from rat post-capillary venules over 2 h as assessed by intravital microscopy. Either ANX-1 or DEX was able to attenuate this adhesion and emigration of leucocytes. MPO activity in the lung, kidney and ileum was elevated after a 6-h exposure to LPS (intraperitoneal), indicating accumulation of neutrophils in these tissues. DEX attenuated the LPS-induced increase in MPO in the ileum but had no effect on MPO in the lungs or kidneys. This would suggest that the underlying mechanism by which neutrophils accumulate in the ileum, and more generally in the gastrointestinal compartment, is different from other vascular beds. ANX-1 had no effect on the LPS-induced increase in MPO activity in any of the tissues studied. Thus, from these data, ANX-1 appears to reduce leucocyte adhesion and emigration induced by a short-term (2 h), but not a longer (6 h) exposure to LPS.
Although medical treatment is sucessful in most cases in patients with inflammatory bowel diseases (IBD), a percentage of patients require surgical resection of diseased bowel segments at least once in their lifetime. Healing success of the intestinal anastomosis is at high risk, especially in presence of acute inflammation. Failure of anastomotic healing is a life-threatening complication and causes high socioeconomic costs. Common anti-inflammatory medications can have detrimental effects on wound healing. Thus, targeted perioperative therapeutics supporting anastomotic healing during colitis are an urgent medical need. Here, we develop a novel basal membrane targeted controlled release, pectin-coated polymeric nanoparticle (NP) encapsulating a highly potent inflammation resolving mediator, the peptide Ac2-26. These NPs can undergo gastric passage and facilitate localized release of the therapeutic peptide in the colon via degradation of their pectin-chitosan coating by microbial pectinases, which subsequently exposes a collagen IV targeted NP surface, allowing for further binding and retention of the NPs at the intestinal wound. To test these NPs, we used a murine surgical model combining the formation of an intestinal anastomosis with the induction of a preoperative colitis by dextran sodium sulfate. In this model, perioperative administration of pectin-chitosan coated NPs containing Ac2-26 (P-C-Col IV-Ac2-26-NP) led to the reduction of colitis activity in the postoperative phase. Macroscopic wound closure was improved by P-C-Col IV-Ac2-26-NP treatment as evaluated by endoscopy and intraabdominal adhesion scoring. Microscopic analysis of the healing process showed an improved semiquantitative healing score in the treatment group. In this proof-of-concept study we demonstrate that novel P-C-Col IV-Ac2-26-NP could be a promising and clinically feasible perioperative treatment strategy for IBD patients.
Although medical treatment is sucessful in most cases in patients with inflammatory bowel diseases (IBD), a percentage of patients require surgical resection of diseased bowel segments at least once in their lifetime. Healing success of the intestinal anastomosis is at high risk, especially in presence of acute inflammation. Failure of anastomotic healing is a life-threatening complication and causes high socioeconomic costs. Common anti-inflammatory medications can have detrimental effects on wound healing. Thus, targeted perioperative therapeutics supporting anastomotic healing during colitis are an urgent medical need. Here, we develop a novel basal membrane targeted controlled release, pectin-coated polymeric nanoparticle (NP) encapsulating a highly potent inflammation resolving mediator, the peptide Ac2-26. These NPs can undergo gastric passage and facilitate localized release of the therapeutic peptide in the colon via degradation of their pectin-chitosan coating by microbial pectinases, which subsequently exposes a collagen IV targeted NP surface, allowing for further binding and retention of the NPs at the intestinal wound. To test these NPs, we used a murine surgical model combining the formation of an intestinal anastomosis with the induction of a preoperative colitis by dextran sodium sulfate. In this model, perioperative administration of pectin-chitosan coated NPs containing Ac2-26 (P-C-Col IV-Ac2-26-NP) led to the reduction of colitis activity in the postoperative phase. Macroscopic wound closure was improved by P-C-Col IV-Ac2-26-NP treatment as evaluated by endoscopy and intraabdominal adhesion scoring. Microscopic analysis of the healing process showed an improved semiquantitative healing score in the treatment group. In this proof-of-concept study we demonstrate that novel P-C-Col IV-Ac2-26-NP could be a promising and clinically feasible perioperative treatment strategy for IBD patients.
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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.
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.
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.
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.
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.
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.
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.
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.
Expression profiles of CXC-and CC-chemokines in various forms of tonsillar disease were studied to evaluate whether certain chemokines play a predominant role in a specific subset of tonsillar disease. Total RNA was isolated from 89 biopsies (21 hyperplastic palatine tonsils, 25 adenoids, 16 chronic inflammatory palatine tonsils and 27 chronic inflammatory palatine tonsils with histological prove of acute inflam-mation), reverse transcribed and subjected to PCR amplifying IL-8, Gro-alpha, eotaxin-1, eotaxin-2, MCP-3, MCP-4 and RANTES. 2% agarose gel electrophoresis revealed a predominance of IL-8 in the chronic inflammatory palatine tonsil group compared to tonsillar hy-perplasia. Furthermore, eotaxin-2 was strongly overexpressed in adenoid samples compared to chronic inflammatory specimens. Our data suggest that the majority of diseases related to adenoid formation are mediated via an eotaxin-2 expression, whereas chronic inflammatory tonsillitis is associated with IL-8 upregulation. These data imply that adenoids are related to a Th-2, and chronic inflammatory tonsillitis to a Th-1 based immune response.
Amoebiasis is a disease that affects 10 % of the world population, and it may have a different behavior when attacks bowels, liver, lungs, brain, etc. Its biological cycle is well known, as well as its symptoms and signs of its penetration into those organs, its diagnosis and treatment, but it is still a controversy on the molecular mechanism of its pathogenesis; to study them it, the experimental hepatic abscess in hamsters has been employed. For years it was considered that the pathogenicity of E. Histolítica was due to its capacity to destroy tissues, but we found that virulent E. Histoliticaperse is unable to produce liver damage in leucopenic hamster; we therefore studied the mechanisms of virulence of the amoeba by functional and molecular comparison between virulent and non virulent E. Histolitica. We found that the parasit virulence cannot be explained only by the activity of citotoxic or proteolytic molecules (adhesines, phospholypases and amebopores, or proteases), and the findings suggest that when amoebas arrives to the hamster liver and find a toxic concentration of oxygen, this sensibilizes them to lysis by complement, hydrogen peroxide and hypoclorose acid. The consequences of those findings may open new perspectives for the design of new therapies for the treatment of this disease.
Adrenal glucocorticoids (GC) and their synthetic analogues are steroid molecules endowed with powerful anti-inflammatory and immunosuppressive properties. The mode of action of GC is very complex and not yet fully elucidated, but a number of mechanisms have been described. Traditionally, selected anti-inflammatory actions of GC have been largely ascribed to the synthesis of a protein termed lipocortin 1 or annexin 1 (AnxAl), which inhibits phospholi-pase A2 activity with subsequent reduction in arachidonic acid release from the cell membrane and pro-inflammatory eicosanoid production [1, 2]. The immunosuppressive effect, on the other hand, is thought to be related to the inhibition of several immune functions, such as cytotoxicity, phagocytosis, and the synthesis of inflammatory cytokines like TNF-α IL-1, IL-2 and IL-8 [3].
Dysregulated inflammation is a central pathological process in diverse disease states. Traditionally, therapeutic approaches have sought to modulate the pro- or anti-inflammatory limbs of inflammation, with mixed success. However, insight into the pathways by which inflammation is resolved has highlighted novel opportunities to pharmacologically manipulate these processes - a strategy that might represent a complementary (and perhaps even superior) therapeutic approach. This Review discusses the state of the art in the biology of resolution of inflammation, highlighting the opportunities and challenges for translational research in this field.
Urinary tract infection (UTI) may cause inflammation of the renal parenchyma and may lead to impairment in renal function and scar formation. Oxidant injury and reactive oxygen species (ROS) have been found responsible in the pathogenesis of UTI. The neurohypophyseal hormone oxytocin (OT) facilitates wound healing and is involved in the modulation of immune and inflammatory processes. We investigated the possible therapeutic effects of OT against Escherichia coli induced pyelonephritis in rats both in the acute and chronic setting.
Twenty-four Wistar rats were injected 0.1 ml solution containing E. coli ATCC 25922 10(10) colony forming units/ml into left renal medullae. Six rats were designed as sham group and were given 0.1 ml 0.9% NaCl. Pyelonephritic rats were treated with either saline or OT immediately after surgery and at daily intervals. Half of the pyelonephritic rats were decapitated at the 24th hour of E. coli infection, and the rest were followed for 7 days. Renal function tests (urea, creatinine), systemic inflammation markers [lactate dehydrogenase (LDH) and tumor necrosis factor alpha (TNF-alpha)] and renal tissue malondialdehyde (MDA) as an end product of lipid peroxidation, glutathione (GSH) as an antioxidant parameter and myeloperoxidase (MPO) as an indirect index of neutrophil infiltration were studied.
Blood urea, creatinine, and TNF-alpha levels were increased, renal tissue MDA and MPO levels were elevated and GSH levels were decreased in both of the pyelonephritic (acute and chronic) rats. All of these parameters and elevation of LDH at the late phase were all reversed to normal levels by OT treatment.
OT alleviates oxidant renal injury in pyelonephritic rats by its anti-oxidant actions and by preventing free radical damaging cascades that involves excessive infiltration of neutrophils.
This is a personal account of the discovery of synexin (annexin VII) in 1977, along with some selected observations on the
development of the annexin field since that time.
The potential involvement of endogenous lipocortin 1 in the process of cellular apoptosis, particularly in cells of the myelo-monocytic lineage, has been investigated. U937 cells were transfected either with an antisense or a sense DNA for lipocortin 1 and the stable clones 36.4AS clone (20–40% lower lipocortin 1 levels) and 15S (30% higher lipocortin 1 levels) were obtained. Cell apoptosis was induced by incubation with tumor necrosis factor-α: optimal responses were observed within a 24 h incubation period at a 5 concentration. Apoptosis was assessed both morphologically, by annexin V binding and cell cycle analysis with propidium iodide. Whilst no consistent difference was seen between wild type cells and clone 36.4AS, a higher incidence of apoptosis (ranging from +30% to + 60%) was observed in the 15S clone. Release of arachidonic acid from loaded cells was promoted by 24 h incubation with the cytokine, and a higher degree of release was measured in the 15S clone. These data indicate that endogenous intracellular lipocortin 1 is involved in the promotion of apoptosis in cells of the myelo-monocytic derivation.
There is a constant search for biodegradable polymers with biocompatible characteristics. However, the reported materials are rarely tested for their immunostimulatory properties, which is an important issue as immune cells activated by the polymers might cause their rejection and lead to further injury to the host tissues. Therefore, the aim of the present study was to determine if biodegradable polymers are able to activate RAW 264.7 macrophages. Aliphatic polyesters, poly(l-lactide) (PLLA), poly(l-lactide-co-trimethylene carbonate) (PLTMC), poly(glycolide-co-l-lactide) (PGLA), poly(glycolide-co-l-lactide-co-ɛ-caprolactone) (PGLCap) and poly(glycolide-co-ɛ-caprolactone) (PGCap), processed into foils by slip-casting, were characterized in terms of their structure ((1)H-NMR, GPC, DSC) and surface properties (chemical composition, water contact angle, surface free energy, topography and roughness). RAW 264.7 cells were cultured on the materials for 3 or 5 days and their adherence, numbers of apoptotic/necrotic cells, as well as production of several cytokines/chemokines and other inflammation-related molecules (matrix metalloproteinases, nitric oxide) was evaluated. The study demonstrated that PLLA and PGLA did not influence macrophage activation and survival. In contrast, PLTMC, PGLCap and PGCap significantly decreased macrophage adherence, increased ratio of apoptosis and up-regulated synthesis/release of numerous inflammatory mediators. Thus, the latter materials might initiate an undesired inflammatory reaction. The above effects of the polymers were attributed to their high hydrophobicity and low polarity due to the presence of ɛ-caproyl blocks (PGLCap and PGCap), and/or high flexibility and susceptibility to mechanical deformation due to low glass-transition temperature (PLTMC, PGLCap and PGCap). In conclusion, while PLLA and PGLA do not affect macrophage functioning, the other materials (PLTMC, PGLCap, PGCap) up-regulate macrophage activity.
AIM: To investigate the alteration of the annexin I subcellular localization in esophageal squamous cell carcinoma (ESCC) and the correlation between the translocation and the tumorigenesis of ESCC.
METHODS: The protein localization of annexin I was detected in both human ESCC tissues and cell line via the indirect immunofluorescence strategy.
RESULTS: In the normal esophageal epithelia the annexin I was mainly located on the plasma membrane and formed a consecutive typical trammels net. Annexin I protein also expressed dispersively in cytoplasm and the nuclei without specific localization on the nuclear membrane. In esophageal cancer annexin I decreased very sharply with scattered disappearance on the cellular membrane, however it translocated and highly expressed on the nuclear membrane, which was never found in normal esophageal epithelia. In cultured esophageal cancer cell line annexin I protein was also focused on the nuclear membrane, which was consistent with the result from esophageal cancer tissues.
CONCLUSION: This observation suggests that the translocation of annexin I protein in ESCC may correlate with the tumorigenesis of the esophageal cancer.
Annexin-1 (ANXA1) is a mediator of the anti-inflammatory actions of endogenous and exogenous glucocorticoids (GC). The mechanism of ANXA1 effects on cytokine production in macrophages is unknown and is here investigated in vivo and in vitro. In response to LPS administration, ANXA1(-/-) mice exhibited significantly increased serum IL-6 and TNF compared with wild-type (WT) controls. Similarly, LPS-induced IL-6 and TNF were significantly greater in ANXA1(-/-) than in WT peritoneal macrophages in vitro. In addition, deficiency of ANXA1 was associated with impairment of the inhibitory effects of dexamethasone (DEX) on LPS-induced IL-6 and TNF in macrophages. Increased LPS-induced cytokine expression in the absence of ANXA1 was accompanied by significantly increased LPS-induced activation of ERK and JNK MAPK and was abrogated by inhibition of either of these pathways. No differences in GC effects on MAPK or MAPK phosphatase 1 were observed in ANXA1(-/-) cells. In contrast, GC-induced expression of the regulatory protein GILZ was significantly reduced in ANXA1(-/-) cells by silencing of ANXA1 in WT cells and in macrophages of ANXA1(-/-) mice in vivo. GC-induced GILZ expression and GC inhibition of NF-kappaB activation were restored by expression of ANXA1 in ANXA1(-/-) cells, and GILZ overexpression in ANXA1(-/-) macrophages reduced ERK MAPK phosphorylation and restored sensitivity of cytokine expression and NF-kappaB activation to GC. These data confirm ANXA1 as a key inhibitor of macrophage cytokine expression and identify GILZ as a previously unrecognized mechanism of the anti-inflammatory effects of ANXA1.
The S100 protein CP-10 (chemotactic protein, 10 kD), a potent chemotactic factor for murine and human polymorphonuclear cells (PMN) and murine monocytes, has been purified in small amounts from supernatants of activated murine spleen cells (Lackmann et al., 1992). To obtain a more abundant source of the protein, CP-10 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The property of S100 proteins to undergo calcium-dependent conformational changes was used in a novel approach to optimize the release of recombinant (r) CP-10 by thrombin cleavage. Purified rCP-10 was characterized by amino-terminal sequence analysis and bioassays. Optimal chemotactic activity of rCP-10 for murine PMN and WEHI-265 monocytoid cells was 10(-11) M (native protein has optimal chemotactic activity between 10(-11) and 10(-13) M). Immunization of rabbits with the GST/CP-10 fusion protein bound to glutathione-agarose beads resulted in high titer, specific antibodies that neutralized CP-10-initiated chemotaxis and were suitable for immunoblotting. A combination of Western and Northern analyses identified CP-10 in murine peritoneal exudate PMN and macrophages, splenocytes, bone marrow cells, and WEHI-265 cells (all of myeloid origin), but not in thymus, liver, lung, 3T3 fibroblasts, EL4 lymphoma cells, or bEND 3 brain endothelial cells, indicating cell-specific regulation of CP-10 expression.
Bactericidal, proteolytic and signal proteins released by activated neutrophils play a major role in infection fighting and inflammatory processes. These proteins are mainly stored in organelles called granules until induction of their controlled exocytosis. The present work deals with the characterization of the proteins which are secreted upon activation of human neutrophils by ionomycin and calcium. Proteins were separated by two-dimensional gel electrophoresis and identified by peptide mass fingerprinting. Almost all the previously described soluble components of neutrophil granules could be identified. Moreover, several additional proteins were shown to be secreted by activated neutrophils, namely calgranulins, human cartilage glycoprotein of 39 kDa (HC gp-39), chitotriosidase, and annexin XI. Their subcellular localization and possible functions are discussed.
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 glucocorticoid hormones and their synthetic derivatives are potent suppressors of inflammatory and allergic pathologies. Their widespread efficacy is the result of multiple modes of action occurring predominantly at the level of the microcirculation. Indeed the glucocorticoids interfere with the function of all of the cellular components of the microcirculation associated with an inflammatory response. These agents inhibit vasodilatation of the arteriolar and capillary beds. therefore preventing the increase in blood flow that characterizes the initial stages of the inflammatory response. They also prevent increases in vascular permeability in the capillary and post-capillary venule, thereby reducing exudate formation. Finally, the glucocorticoids potently suppress leukocyte emigration across post-capillary venules. However, this promiscuity of the glucocorticoids to act at multiple sites also endows this class of (drugs with major side effects associated with chronic treatment. We propose that one way to progress forward is to understand better the effects of glucocorticoids within the microcirculation. This may aid identification of specific molecular sites of action and therefore the development of novel glucocorticoid molecules with fewer side effects.
Leukocyte trafficking into pulmonary tissue and airspaces is a critical component of the host defense response. Activation and migration of polymorphonuclear leukocytes (PMNs) into lungs also contribute to inflammatory tissue injury and remodeling of tissue architecture. There have been considerable advances in our understanding of the mechanisms that control PMN adhesion and transendothelial migration (TEM). Mechanisms of migration unique to the lungs have been described with regard to the profile of adhesion molecules, cytokines, and chemokines elicited during PMN emigration from blood vessels. This work reviews general mechanisms of TEM of PMNs and discusses the nature of PMN recruitment in several models of airway inflammation that illustrate how various stimuli elicit different responses. Pharmacologic manipulation of adhesive interactions between PMNs and endothelial cells is a current area of research aimed at developing pharmacologic agents to control inflammation during pulmonary and other inflammatory diseases. A summary of some of these agents and their actions is presented.
This study investigated the effects of intra-abdominal blood on the systemic response to peritonitis using a murine model of hemorrhage, peritonitis, and multiple organ dysfunction syndrome.
The model used male ICR mice subjected to hemorrhage and intraperitoneal zymosan. Half of the mice received intraperitoneal blood. Outcome measures included lung myeloperoxidase, lung edema, lung injury score, and plasma and lung tissue chemokine production.
Peritoneal blood (in association with peritoneal inflammation) increased lung neutrophil sequestration (myeloperoxidase) (2.56 +/- 1.42 vs. 1.45 +/- 0.49 U/left lung, p = 0.04) and lung weight (0.11 +/- 0.04 vs. 0.07 +/- 0.02 g/left lung, p = 0.02), and was associated with significantly higher chemokine levels in plasma (KC and MCP-1) and lung tissue (KC, MIP-2, and MCP-1). Both plasma and lung tissue neutrophil chemoattractants KC and MIP-2 were significantly linearly correlated with myeloperoxidase (p < 0.009), and lung tissue KC (a neutrophil chemokine) and MCP-1 and MIP-1alpha (mononuclear cell chemokines) correlated with lung injury score (p < 0.003).
Although blood alone in the peritoneal cavity was well tolerated, in conjunction with inflammation, it was synergistic in amplifying the systemic inflammatory response. The amplified lung injury in this model was associated with significant increases in circulating and lung tissue chemokine concentrations.
Macrophage inflammatory protein-1 (MIP) is a recently cloned cytokine that causes neutrophilic infiltration and induces an inflammatory response. We studied the effect of MIP-1 alpha on histamine secretion from basophils and mast cells. Leukocytes from allergic and normal subjects were studied. MIP-1 alpha caused dose-dependent release of histamine from basophils of 14 of 20 allergic donors at concentrations of 10(-9)-10(-7) M, and the mean release was 13.50 +/- 2.9% at the highest concentration. In the same experiments, the mean histamine release by anti-immunoglobulin E and monocyte chemotactic and activating factor (MCAF) (10(-7) M) was 32 +/- 7% and 31 +/- 3%, respectively. The cells from only 2 of 10 normal subjects released histamine in response to MIP-1 alpha. Histamine release by MIP-1 alpha was rapid, and almost complete within the first 3 min. MIP-1 alpha-induced degranulation was a calcium-dependent noncytotoxic process. MIP-1 alpha showed chemotactic activity for purified basophils that was comparable to MCAF. Both MIP-1 alpha and MCAF at 10(-7) M concentration elicited a chemotactic response that was 40% of the maximal response to C5a (1 microgram/ml). Murine MIP-1 alpha induced histamine release from mouse peritoneal mast cells in a dose-dependent manner. Thus, we have established that MIP-1 alpha is a novel activator of basophils and mast cells.
The ability of glucocorticosteroids to inhibit tissue eosinophilia may be an important feature of their anti‐inflammatory action in allergic diseases. Our previous work showed that an effect of dexamethasone on the release of eosinophils from the bone marrow could explain its inhibitory action on eosinophil accumulation in a mouse air‐pouch model. Thus, it was unclear from that study whether dexamethasone could interfere with the process of eosinophil trafficking. In the present study, therefore, we used a newly developed mouse model to evaluate the effects of systemic treatment with dexamethasone on the recruitment of ¹¹¹ In‐labelled blood eosinophils to sites of cutaneous inflammation in the mouse and whether lipocortin‐1 (LC‐1) was involved.
The i.d. injection of ovalbumin (OVA) in sensitized mice induced a dose‐dependent recruitment of ¹¹¹ In‐labelled blood eosinophils which peaked at 4 to 8 h after antigen challenge. Systemic treatment with dexamethasone (50 μg per mouse, 3 h after antigen) effectively inhibited ¹¹¹ In‐eosinophil recruitment in this reaction by 70 to 85%. Similarly, a 1 h pretreatment with dexamethasone significantly suppressed ¹¹¹ In‐eosinophil induced by platelet‐activating factor (PAF), leukotriene B 4 (LTB 4 ) and the chemokine macrophage inflammatory protein‐1α (MIP‐1α) by 40 to 70%.
Two experimental approaches were used to evaluate the role of LC‐1: treatment with LC‐1 fragment Ac2‐26 and use of an anti‐LC‐1 antiserum. LC‐1 fragment Ac2 ‐26 (100 μg per mouse) failed to affect ¹¹¹ In‐eosinophil recruitment. Moreover, pretreatment of animals with an anti‐LC‐1 antiserum failed to reverse the inhibitory effects of dexamethasone on ¹¹¹ In‐eosinophil recruitment induced by MIP‐1α and by antigen in sensitized mice.
In contrast, the LC‐1 fragment significantly inhibited glycogen‐induced neutrophil recruitment into the peritoneal cavity of mice. Furthermore, the anti‐LC‐1 antiserum reversed the inhibitory effects of dexamethasone on the glycogen‐induced neutrophil recruitment.
Thus, our results suggest that dexamethasone can inhibit the recruitment of eosinophils in mouse skin independent of an action on the bone marrow. However, by use of two different approaches, we showed that LC‐1 does not play a role in mediating the inhibitory action of dexamethasone on eosinophil migration into cutaneous inflammatory reactions in the mouse. These data add further support to a LC‐1‐independent action of dexamethasone on eosinophils in vivo .
British Journal of Pharmacology (1998) 123 , 538–544; doi: 10.1038/sj.bjp.0701625
In vivo interactions between neutrophils and endothelial cells (EC) follow a multistep process involving two distinct neutrophil adhesion receptors. L-selectin, constitutively functional on resting neutrophils, mediates an activation-independent primary interaction resulting in rolling along the venular wall. Subsequent activation of rolling neutrophils induces upregulation and functional activation of beta 2-integrins (CD11/CD18) leading to firm attachment. Based on previous findings we hypothesized that, under shear force, rolling may be essential for successful neutrophil-EC recognition. Here we report results of our studies of human neutrophil behavior in interleukin (IL)-1-activated rabbit mesentery venules, an interaction that requires both L-selectin and beta 2-integrins. Rolling of human neutrophils is L-selection mediated; it was strongly reduced by monoclonal antibody inhibition or enzymatic removal of L-selectin. Furthermore, activation induced L-selectin shedding and, in a dose- and time-dependent fashion, rendered neutrophils unable to recognize inflamed EC despite expression of active beta 2-integrins, which promoted adhesion in vitro. Neutrophils activated for 5 min or longer lost most of their ability to roll. However, 1-3 min after activation, rolling was reduced (not abolished), and cells that were still able to roll displayed a significant tendency for a CD18-dependent transition from rolling to sticking. The whole sequence of events, rolling, sticking, and transendothelial migration, could be observed if an extravascular chemotactic stimulus was applied by superfusing mesenteries with leukotriene B4. Under such conditions, sticking and emigration was blocked when rolling was inhibited by enzymatic removal of L-selectin. Our results indicate that primary neutrophil interaction with inflamed EC through the L-selectin is a prerequisite for neutrophil function at physiological shear rates in vivo.
The mechanism and cofactor requirements of exocytotic membrane fusion in neutrophils are unknown. Cytosolic proteins have been implicated in membrane fusion events. We assessed neutrophil cytosol for the presence of fusogenic proteins using a liposome fusion assay (lipid mixing). A fusogenic 36-kD protein containing amino acid sequence homology with human annexin I was purified from the cytosol of human neutrophils. This protein also shared functional characteristics with annexin I: it associated with and promoted lipid mixing of liposomes in a Ca(2+)-dependent manner at micromolar Ca2+ concentrations. The 36-kD protein required diacylglycerol to promote true fusion (contents mixing) at the same Ca2+ concentrations used for lipid mixing. The 36-kD protein exhibited a biphasic dose-response curve, by both promoting and inhibiting Ca(2+)-dependent lipid-mixing between liposomes and a plasma membrane fraction. The 36-kD protein also promoted Ca(2+)-dependent increases in aggregation of a specific granule fraction, as measured by a turbidity increase. Antiannexin I antibodies depleted the 36-kD protein from the cytosol by greater than 70% and diminished its ability to promote lipid mixing. Antiannexin I antibodies also decreased by greater than 75% the ability of neutrophil cytosol to promote Ca(2+)-dependent aggregation of the specific granules. These data suggest that annexin I may be involved in aggregation and fusion events in neutrophils.
The injection of a suspension of a polyacrylamide gel (bio gel) into the dorsal subcutaneous area of mice induced an inflammatory reaction and the migration of neutrophils towards the inflamed site.
The intravenous administration of anti‐inflammatory drugs (dexamethasone, indomethacin and lysine‐acetylsalicylate) to Polyacrylamide gel‐treated mice inhibited the accumulation of neutrophils in the inflamed site.
A similar administration of a 36 K mouse lipocortin, induced a strong dose‐dependent inhibition of neutrophil accumulation in the inflamed site.
Dexamethasone and lipocortin inhibited the production of eicosanoids, prostaglandin E 2 (PGE 2 ) and leukotriene B 4 (LTB 4 ) in the inflamed site of Polyacrylamide gel‐treated mice.
Lipocortin impaired both phospholipase A 2 (PLA 2 ) activity and chemotaxis of isolated inflammatory neutrophils.
The present studies show an in vivo anti‐inflammatory effect of lipocortin similar to that of glucocorticosteroids. In agreement with recent data on the extracellular effects of various lipocortins, these results might implicate lipocortin(s) in the anti‐inflammatory effects of glucocorticosteroids.
The objective of this study was to define the nature, magnitude, and mechanisms of histamine-induced leukocyte-endothelial cell interactions in postcapillary venules of the rat mesentery using intravital microscopic techniques. Superfusion of the mesentery with histamine (10(-7)-10(-5) M) resulted in a dose-related increase in the number of rolling leukocytes, a reduction in rolling velocity, and an increased clearance of FITC-labeled rat albumin from blood to superfusate. The histamine-induced recruitment of rolling leukocytes and increased albumin clearance were prevented by histamine H1 (hydroxyzine, diphenhydramine) but not H2 (cimetidine) receptor antagonists. Because histamine induces expression of the adhesion molecule P-selectin in cultured endothelial cells, a monoclonal antibody directed against rat P-selectin and soluble sialyl-LewisX oligosaccharide (the carbohydrate ligand to P-selectin) were also tested as inhibitors. Both were effective in preventing the histamine-induced recruitment of rolling leukocytes, but neither agent attenuated the increased albumin clearance. These observations suggest that (a) histamine recruits rolling leukocytes and increases albumin leakage in postcapillary venules via H1 receptor activation, (b) histamine-induced recruitment of rolling leukocytes is mediated in part by P-selectin expressed on the endothelial cell surface, and (c) the histamine-induced vascular albumin leakage is unrelated to leukocyte-endothelial cell adhesion. Our results are consistent with the view that histamine may act as a mediator of acute inflammatory reactions.
The S100 protein CP-10 (chemotactic protein, 10 kD), a potent chemotactic factor for murine and human polymorphonuclear cells (PMN) and murine monocytes, has been purified in small amounts from supernatants of activated murine spleen cells (Lackmann et al., 1992). To obtain a more abundant source of the protein, CP-10 was expressed in Escherichia coli as a fusion protein with glutathione S-transferase (GST). The property of S100 proteins to undergo calcium-dependent conformational changes was used in a novel approach to optimize the release of recombinant (r) CP-10 by thrombin cleavage. Purified rCP-10 was characterized by amino-terminal sequence analysis and bioassays. Optimal chemotactic activity of rCP-10 for murine PMN and WEHI-265 monocytoid cells was 10(-11) M (native protein has optimal chemotactic activity between 10(-11) and 10(-13) M). Immunization of rabbits with the GST/CP-10 fusion protein bound to glutathione-agarose beads resulted in high titer, specific antibodies that neutralized CP-10-initiated chemotaxis and were suitable for immunoblotting. A combination of Western and Northern analyses identified CP-10 in murine peritoneal exudate PMN and macrophages, splenocytes, bone marrow cells, and WEHI-265 cells (all of myeloid origin), but not in thymus, liver, lung, 3T3 fibroblasts, EL4 lymphoma cells, or bEND 3 brain endothelial cells, indicating cell-specific regulation of CP-10 expression.
In the 9 years since the last review on leukocyte and endothelial interactions was published in this journal many of the critical structures involved in leukocyte adherence to and migration across endothelium have been elucidated. With the advent of cell and molecular biology approaches, investigations have progressed from the early descriptions by intravital microscopy and histology, to functional and immunologic characterization of adhesion molecules, and now to the development of genetically deficient animals and the first phase I trial of “anti-adhesion” therapy in humans. The molecular cloning and definition of the adhesive functions of the leukocyte integrins, endothelial members of the Ig gene superfamily, and the selectins has already provided sufficient information to construct an operative paradigm of the molecular basis of leukocyte emigration. The regulation of these adhesion molecules by chemoattractants, cytokines, or chemokines, and the interrelationships of adhesion pathways need to be examined in vitro and, particularly, in vivo. Additional studies are required to dissect the contribution of the individual adhesion molecules to leukocyte emigration in various models of inflammation or immune reaction. Certainly, new adhesion structures will be identified, and the current paradigm of leukocyte emigration will be refined. The promise of new insights into the biology and pathology of the inflammatory and immune response, and the potential for new therapies for a wide variety of diseases assures that this will continue to be an exciting area of investigation.
The role played by endogenous lipocortin 1 in the anti-migratory action exerted by dexamethasone (Dex) on monocyte recruitment in an in vivo model of acute inflammation was investigated by use of several neutralizing polyclonal antibodies raised against lipocortin 1 or a lipocortin 1-derived N-terminus peptide (peptide Ac2-26). The efficacy of peptide Ac2-26 in inhibiting monocyte and polymorphonuclear leucocyte (PMN) recruitment was also tested.
Intraperitoneal (i.p.) injection of zymosan A (1 mg) produced a time-dependent cell accumulation into mouse peritoneal cavities which followed a typical profile of acute inflammation: PMN influx was maximal at 4 h post-zymosan (between 15 and 20×106 cells per mouse), and this was followed by an accumulation of monocytes which peaked at the 24 h time-point (between 10 and 15×106 cells per mouse).
Dex administration to mice reduced zymosan-induced 4 h PMN infiltration and 24 h monocyte accumulation with similar efficacy: approximately 50% of inhibition of recruitment of both cell types was achieved at the dose of 30 μg per mouse (∼1 mg kg−1, subcutaneously (s.c.)). Maximal inhibitions of 64% and 67% on PMN and monocyte recruitment, respectively, were measured after a dose of 100 μg per mouse (∼3 mg kg−1, s.c.).
Dex (30 μg s.c.) inhibited monocyte (53%) and PMN (69%) accumulation in response to zymosan application in mice which had been treated with a non-immune sheep serum (50 μl s.c.). In contrast, the steroid was no longer active in reducing cell accumulation in mice which had been passively immunized against full length human recombinant lipocortin 1 (serum LCS3), or against lipocortin 1 N-terminus peptide.
Treatment of mice with vinblastine (1 mg kg−1, intravenously (i.v.)) produced a remarkable leucopenia as assessed 24 h after administration. This was accompanied by a 60% reduction in 4 h-PMN influx, and by a 27% reduction in 24 h-monocyte accumulation, measured after zymosan administration. The inhibitory effect of Dex on monocyte recruitment was not significantly modified in vinblastine-treated mice, with 36% and 57% of inhibition calculated at the dose of 30 μg Dex, and 70% and 60% of inhibition at 100 μg Dex, in vehicle- and vinblastine-treated mice, respectively.
Treatment of mice with peptide Ac2-26 dose-dependently attenuated PMN influx at 4 h post-zymosan with a significant effect at 100 μg per mouse (45% of inhibition, n=9, P<0.05) and a maximal effect of 61% inhibition at the highest dose tested of 200 μg s.c. (n=14, P<0.05). No effect of peptide Ac2-26 (200 μg s.c.) was seen on zymosan-induced 24 h monocyte recruitment. In contrast, administration of 200 μg peptide Ac2-26 every 6 h was effective in reducing the number of monocytes harvested from the inflamed peritoneal cavities at 24 h post-zymosan: 9.40±0.58×106 monocytes per mouse (n=13) and 5.74±0.34 monocytes per mouse (n=14) in vehicle- and peptide Ac2-26-treated mice, respectively (P<0.05).
Finally, peptide Ac2-26 produced a concentration-dependent inhibition of the rate of phagocytosis of mouse resident peritoneal macrophages as measured by flow cytometry, with a maximal reduction of 34% at the highest concentration tested of 100 μg ml−1 (n=8 experiments performed in duplicate; P<0.05).
In conclusion, this study suggests that in vivo monocyte recruitment during acute inflammation is, at least in part, under the negative modulatory control of endogenous lipocortin 1 (as seen after administration of Dex by using the specific antisera) and exogenous lipocortin 1 mimetics (as observed with peptide Ac2-26). In addition to the neutrophil, we can now propose that the monocyte also can be a target for the in vivo anti-inflammatory action of lipocortin 1.
British Journal of Pharmacology (1997) 120, 1075–1082; doi:10.1038/sj.bjp.0701029
A multi-faceted approach was used to investigate the effect of an anti-inflammatory peptide derived from human lipocortin 1 N-terminus region (amino acid 2–26; termed human Ac2–26) on human neutrophil activation in vitro. When incubated with purified human neutrophils. human Ac2–26 produced a concentration-dependent inhibition of elastase release stimulated by formyl-Met-Leu-Phe (fMLP), platelet-activating factor, or leukotriene B4, with an approximate EC50 of 33 μM (100 μg/ml). At this concentration, human Ac2–26 also inhibited (77%) the release of [3H]-arachidonic acid from neutrophils stimulated with fMLP. The peptide, however, did not inhibit the up-regulation of the β2-integrin CD11b and the concomitant shedding of L-selectin from neutrophil plasma membrane induced by fMLP. In adhesion experiments, human Ac2–26 inhibited neutrophil adhesion to endothelial monolayers when this was stimulated with fMLP, but not when this followed endothelial cell activation with histamine or platelet-activating factor. Again, the effect of the peptide was concentration-dependent, and an approximate EC50 of 33 μM was calculated. When a preparation of 125I-labeled human Ac2–26 was incubated with the neutrophils, the peptide was internalised in an energy-dependent fashion. All together, these observations lead us to propose a model in which this peptide derived from the N-terminus of human lipocortin 1 alters a common cellular mechanism producing a selective inhibition of neutrophil activation.
The purpose of this work was to analyze cDNA encoding human monocyte chemoattractant protein-1 (MCP-1), previously isolated from glioma cell line culture fluid. Screening of a cDNA library from total poly(A) RNA of glioma cell line U-105MG yielded a clone that coded for the entire MCP-1. Nucleotide sequence analysis and comparison with the amino acid sequence of purified MCP-1 showed that the cDNA clone comprises a 53-nucleotide 5′-non-coding region, an open reading frame coding for a 99-residue protein of which the last 76 residues correspond exactly to pure MCP-1, and a 389-nucleotide 3′-untranslated region. The hydrophobicity of the first 23 residues is typical of a signal peptide. Southern blot analysis of human and animal genomic DNA showed that there is a single MCP-1 gene, which is conserved in several primates. MCP-1 mRNA was induced in human peripheral blood mononuclear leukocytes (PBMNLs) by PHA, LPS and IL-1, but not by IL-2, TNF, or IFN-γ. Among proteins with similar sequences, the coding regions of MCP-1 and mouse JE show 68% identity. This suggests that MCP-1 is the human homologue of the mouse competence gene JE.
Putative tissue receptors for leukocyte attractants, including neutrophil attractant/activation protein-1 (interleukin 8, NAP-1/IL-8), have been implicated in the regulation of neutrophil emigration into the tissues. An in-situ binding assay and an ex-vivo autoradiographic approach were used to investigate the binding of radiolabeled NAP-1/IL-8 to human and animal skin. These methods revealed the presence of saturable NAP-1/IL-8-binding sites on the endothelial cells of venules and veins but not arteries or capillaries of the dermis. In addition, the binding of NAP-1/IL-8 to dermal macrophages and perivascular mast cells was observed. We suggest that the NAP-1/IL-8-binding sites described here could be involved in the regulation of NAP-1/IL-8-induced neutrophil emigration.
Accumulation of leukocytes in tissues is essential for effective host defense. To fulfill this role the cell must interact with and penetrate the vessel wall and migrate in the tissue. It is now clear that cell adhesion molecules play a crucial role in orchestrating these processes. A number of families of such adhesion molecules that mediate the interaction of circulating leukocytes and vascular endothelial cells have been identified. These include the leukocyte integrins, the selectins, members of the immunoglobulin family, and certain carbohydrates. Studies in vitro have elucidated which of these adhesion molecules are important in the interaction of different leukocyte classes with the endothelium under both basal and stimulated conditions. With the aid of monoclonal antibodies, the role that these molecules play in the interaction of inflammatory cells in the microvasculature in vivo is being assessed. Studies to date have demonstrated the key role of cell adhesion molecules in inflammation.
In this article Antal Rot calls into question the generally accepted notion that gradients of soluble chemotactic factors are responsible for leukocyte emigration from the circulation into sites of inflammation. He presents an alternative model in which chemoattractants bound to the surface of endothelial cells promote neutrophil adhesion and emigration, while soluble blood-borne attractants inhibit adhesion and emigration.
Rat cytokine-induced neutrophil chemoattractant (CINC) is a member of the IL-8 family and its human counterpart is MGSA/gro. Rat neutrophil responses in vitro to rat CINC, human IL-8, and human MGSA/gro were studied. CINC concentrations as low as 1 nM induced apparent chemotaxis of rat neutrophils, but human IL-8 and MGSA/gro required concentrations one or two orders higher than that of CINC to attract neutrophils. These data indicate that human IL-8 and MGSA/gro cannot sufficiently substitute for rat counterparts such as CINC in rats. Therefore, the effect of rat CINC on rats was studied. Intradermally injected 10(-10)-10(-7) M CINC dose-dependently caused infiltration of neutrophils. Significant migration of neutrophils appeared by 30 min, and maximum infiltration was observed around 1-2 hr after the injection. CINC induced quick and transient neutrophil accumulation without lymphocyte and monocyte migration or edema formation. CINC, a member of the IL-8 family but a counterpart of human MGSA/gro-related proteins, is a specific neutrophil chemoattractant and can be distinguished from IL-8, which is a chemotactic factor for lymphocytes and neutrophils.
An in vivo experimental peritonitis model was investigated in the rabbit using zymosan as the inflammatory stimulus. After an i.p. injection of zymosan, exudate was removed at intervals and tested in the back skin of assay rabbits. Assay rabbits received i.v. injections of 125I-albumin and 111In-neutrophils, and the local accumulation of each label was measured in response to intradermal injections of exudate samples mixed with a potentiating dose of PGE2. When peritoneal exudate samples were tested in the presence of a specific anti-C5a antibody, virtually all the edema-inducing and neutrophil chemoattractant activity was abolished in samples taken up to 2 h after the zymosan injection. Later samples, however, contained increasing levels of a non-C5a component. In C5a-depleted 6-h exudate two peaks of inflammatory activity were separated using cation exchange HPLC. Evidence is presented that C5a itself is unable to stimulate the production of these activities. Both peaks of activity appear related to IL-8/NAP-1 as they inhibited the binding of 125I-IL-8/NAP-1 to human neutrophils.
Neutrophil attractant/activation protein-1 (NAP-1) is a recently described cytokine that attracts neutrophils, but not monocytes or eosinophils. This leukocyte specificity is not absolute, in that NAP-1 attracts basophils and small numbers of lymphocytes. Our purpose was to determine in vivo effects of NAP-1, and to compare them to the reported action of the complement attractant, C5a. Intradermal injection into normal human subjects of 40 microliters of NAP-1, over a concentration range of 4 x 10(-8) M to 10(-6) M, caused no symptoms or signs such as wheal-and-flare, itching, induration, or tenderness. However, biopsies of injection sites showed perivascular neutrophil infiltration as early as 30 min, which increased at 1 and 3 h. The mean number of neutrophils per mm2 of dermis for 15 biopsies taken 3 h after intradermal injection of 2 x 10(-7) M or 10(-6) M NAP-1 was 164 +/- 41; the response to saline or a NAP-1 inactive fragment was 5 or less. Intradermal NAP-1 did not cause basophil or lymphocyte infiltration. Consistent with the absence of a wheal-and-flare, acid toluidine blue-stained sections showed no evidence of mast cell degranulation, in contrast to previously reported results with C5a. Thus, the predominant response by human subjects to intradermal NAP-1 was neutrophil accumulation in proximity to dermal blood vessels.
Migration of neutrophils across epithelial or endothelial barriers in response to chemotactic stimuli occurs in inflammation and host defense. Leukotriene B4 (LTB4) may be synthesized by and certainly induces chemotaxis of neutrophils. To better understand the interaction between LTB4, neutrophils, and endothelium and epithelium, we compared the effects of LTB4 on human peripheral blood neutrophil migration through filters alone and on human umbilical vein endothelial (HUVE) cells and three different epithelial cell types, Madin-Darby canine kidney (MDCK) cells, human colon carcinoma (T84) cells, and rat type II alveolar cells, cultured on these filters. Significant LTB4-stimulated neutrophil migration occurred at the lowest (1 nM) dose and in the shortest period of time (15 min) across endothelial cells vs. all three epithelial cell types, and interestingly, vs. filters alone. Dose-response experiments (1-100 nM) indicated that at equimolar LTB4 concentrations neutrophil migration across endothelium was two- to threefold greater than that observed across filters alone and the three epithelial barriers. At higher LTB4 concentrations (100 nM), the degree of neutrophil migration through the three epithelial barriers was equivalent to that observed for filters alone. Overall, the data indicate that the various cellular barriers play an active role in inflammatory processes by regulating the transmigration of neutrophils in response to certain inflammatory chemotactic stimuli.
Human recombinant lipocortin 1 has been tested for anti-inflammatory activity in a conventional model of acute inflammation. Microgram amounts of the protein, locally administered, inhibited edema of the rat paw when induced by subplantar injections of carrageenin: the ED50 was 10-20 micrograms per paw, and inhibition (maximum of 60-70%) was not dependent upon an intact adrenal cortex. Doses of lipocortin that produced approximately 50% inhibition in the carrageenin test were inactive against edema elicited by bradykinin, serotonin, platelet-activating factor-acether, or dextran, whereas edema caused by Naja mocambique venom phospholipase A2 was strongly inhibited by lipocortin. The protein inhibited edema when rats were pretreated with agents that depleted mast-cell amines, kininogen, or polymorphonuclear leukocytes prior to initiation of the carrageenin edema but had no inhibitory action when rats were pretreated with the dual cyclooxygenase/lipoxygenase inhibitor BW 755C. These results demonstrate that human recombinant lipocortin has potent local anti-inflammatory activity, probably through selectively interfering with eicosanoid generation. Lipocortin is relatively ineffective against edema caused by mast-cell degranulation or kinins, except when degranulation is caused by phospholipase A2.
The movement of mononuclear phagocytes and neutrophils from the circulation into tissues is a process which is not completely understood. Monoclonal antibody 5.5 is specific for an 8/14-kDa molecule known variously as the CF antigen, L1 molecule or MRP8 and 14. We show that this molecule, which will be named p8,14 in this study, is expressed in all circulating monocytes and neutrophils as an intracellular product (as well as some types of epithelium). Tissue staining patterns suggest that when monocytes and neutrophils adhere to vascular endothelium, they release this molecule onto the associated endothelium. This process occurs with single monocytes and when monocytes form part of an inflammatory infiltrate. Monoclonal antibody 5.5 does not react with cultured endothelial cells even when stimulated with phorbol ester, tumor necrosis factor, interferon-gamma or interleukin 1 alpha providing further evidence that myeloid cells are the source of the p8,14 in this interactive process. Monocytes which have moved further into such tissues and tissue macrophages in general are monoclonal antibody 5.5 negative, suggesting that the ability to synthesize this molecule may be lost when monocytes leave the circulation and enter tissues. These results indicate that p8,14 plays a role in the interaction between myeloid cells and the vascular endothelium to which they adhere prior to leaving the circulation.
The anti-inflammatory action of glucocorticoids has been attributed to the induction of a group of phospholipase A2 inhibitory proteins, collectively called lipocortin. These proteins are thought to control the biosynthesis of the potent mediators of inflammation, prostaglandins and leukotrienes, by inhibiting the release of their common precursor, arachidonic acid, a process that requires phospholipase A2 hydrolysis of phospholipids. Lipocortin-like proteins have been isolated from various cell types, including monocytes, neutrophils and renal medullary cell preparations. The predominant active form is a protein with an apparent relative molecular mass (Mr) of 40,000 (40K). These partially purified preparations of lipocortin mimic the effect of steroids, and mediate anti-inflammatory activity in various in vivo model systems. Using amino-acid sequence information obtained from purified rat lipocortin, we have now cloned human lipocortin complementary DNA and expressed the gene in Escherichia coli. Our studies confirm that lipocortin is a potent inhibitor of phospholipase A2 activity.
The guinea-pig perfused isolated lung, used in conjunction with the cascade superfusion system to measure the release of thromboxane A2(TXA2), is a simple and convenient model for assessing the inhibition by glucocorticoids of eicosanoid formation. Dexamethasone inhibits the release of TXA2 from the lung when it is stimulated by agents such as RCS-RF2 of leukotrienes, but not when bradykinin or arachidonic acid are used. Using this model we have shown that the glucocorticoids suppress eicosanoid generation by cells through the induction of a family of phospholipase A2-inhibitory proteins now termed the 'lipocortins'. Recently the primary structure of one form of lipocortin has been elucidated and the human gene cloned. Lipocortin 1 is a polar monomeric protein with anti-phospholipase properties in vitro and we now report that when infused into guinea-pig lung preparations this protein has the same inhibitory profile as the glucocorticoids but with a more rapid onset of action. This is the first demonstration that eicosanoid formation can be inhibited by a recombinant phospholipase inhibitory protein applied extracellularly.
The glucocorticoids inhibit our 'defence reactions' at many levels. One way in which they achieve this is by inhibiting the synthesis of chemicals involved in the promotion of the inflammatory response. The production of many mediators involved in the response to infection, injury, haemorrhage or metabolic disturbances are under glucocorticoid control such that elevated levels of hormone in the blood suppresses their formation. In many cases the action of these mediators is blocked as well. It might be thought that the glucocorticoids act simply by decreasing the synthesis rate of these protein regulators of inflammation such as the lymphokines, or of the enzymes which make prostaglandins. Whilst this undoubtedly does occur, another mechanism is also employed: that is, the glucocorticoid-induced synthesis of inhitibory proteins. Lipocortin (and possibly other related proteins) then is a sort of 'second messenger' of the glucocorticoids. It is only one of many such regulatory proteins but it is an important one, controlling as it does the mediators which promote development of the symptoms of the inflammatory response. It is undoubtedly the significant component of the inbuilt mechanism for terminating the inflammatory response which the physician exploits, for when he gives his patients relatively large doses of steroids to control an inflammatory response, he is in reality increasing the synthesis of these 'second mesenger' proteins such as lipocortin to a near maximum. All the early studies on lipocortin were performed in vitro, that is under conditions in which steroids were not normally present. Under these circumstances the generation and appearance of lipocortin seemed absolutely dependent upon the presence of glucocorticoids in the perfusing medium. These findings have led some to the erroneous notion that lipocortin was only present following treatment with exogenous steroids. Of course, all healthy mammals have circulating glucocorticoids and thus it is more rational to expect that lipocortin is a normal constituent of plasma and tissues (as indeed it appears to be), although the amount present in the cells can be increased by raising the concentration of endogenous or exogenous steroids. There has been a corresponding change in our appreciation of the function of lipocortin. Originally, it was regarded mainly as an 'anti-inflammatory protein' but today it seems more likely that this protein is present in most cells, and that its function is to control phospholipase A2 activity and to allow lipid hydrolysis only under strictly defined circumstances. This reversible inhibitory function of lipocortin could well be controlled by the phosphorylation and dephosphorylation cycle described above. Naturally, during inflammation, phospholipase is substantially activated and thus there is a requirement for a greater than normal supply of the inhibitory protein, hence the relationship between the rate of synthesis and the release of steroid hormones. The discovery, characterisation, islation, sequencing and cloning of lipocortin has opened up an entirely new and exciting chapter in cell biology and also holds out a strong promise for the future of anti-inflammatory therapy. In addition to their beneficial clinical effects, steroids produce a wide spectrum of side effects which preclude the use of these drugs for long periods of time except in the very seriously ill. These side effects are caused by changes in the transcription of specific genes in the same way as the anti-inflammatory effects. It has long been an article of a faith of scientists working in this are that if we could identify and isolate the 'second messengers' of steroid action that are responsible for the anti-inflammatory effects, it should be possible to produce drugs which possess many of the beneficial action of steroids without incurring the heavy penalty of side effects. The real value of this work is that it enables us to take our first stpes in that direction.
LPS stimulated human blood mononuclear leukocytes to produce a chemotactic factor for human neutrophils. The effect of LPS was dose-dependent; 10 micrograms/ml was optimal for production of chemotactic factor. Chemotactic activity was detected 3 hr after LPS stimulation, and reached its peak at 12 hr. No activity was detected in culture supernatants of unstimulated cells, provided LPS-free media were selected. Isoelectric point of the factor, determined by chromatofocusing, was approximately 8 to 8.5. Molecular weight was approximately 10 kilodaltons by Sephacryl S-200 gel filtration or by HPLC gel filtration on TSK-2000 and -3000 columns in succession. The gel filtration fractions were also assayed for IL 1 activity. The elution position of IL 1 activity corresponded to a m.w. of 18. There was no chemotactic activity in the IL 1 activity peak. Furthermore, highly purified natural Il 1 alpha and -beta and recombinant Il 1 alpha and -beta did not exhibit chemotactic activity for neutrophils in our assay. Among mononuclear leukocytes, the monocyte was the principal producer of neutrophil chemotactic factor. These results suggest that a chemotactic factor for neutrophils, different from IL 1, is produced by LPS-stimulated blood monocytes.
Topical glucocorticoid treatment (betamethasone-17-valerate (0.018 mg/cm2, 3 h pretreatment) significantly inhibited neurogenic oedema formation induced by electrical antidromic stimulation (2 Hz, 15 V, 0.1 ms for 5 min) of the rat saphenous nerve; a response mediated by neuropeptides released from activated capsaicin-sensitive sensory C-fibres. Oedema formation was estimated by measurement of extravasation of i.v. injected 125I-albumin into skin. The inhibitory effect of the topical glucocorticoid was reversed by passive immunisation of rats with polyclonal antibody to the glucocorticoid-inducible anti-inflammatory protein lipocortin 1 (1 ml/kg, s.c., 24 h pretreatment) whilst a non-immune serum was without effect. Similarly the glucocorticoid receptor antagonist RU38486 (20 mg/kg, 2 and 20 h pretreatment) abrogated the response indicating specific binding to glucocorticoid receptors. Topical glucocorticoid treatment also inhibited the oedema produced by intradermal substance P (0.1 nmol) in the dorsal skin of rats. Topical glucocorticoid inhibited neurogenic oedema formation partly through a mechanism dependent upon lipocortin 1. This inhibition may be partly due to a post-junctional effect upon substance P activity/binding however a pre-junctional component cannot be excluded.
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The objective of this study was to systematically assess the molecular mechanisms and kinetics of histamine-induced leukocyte rolling in rat mesenteric venules using intravital microscopy. A complicating factor in these studies is surgical preparation-induced leukocyte rolling (spontaneous rolling), which leads to a lack of effect of histamine on this parameter. Therefore, we identified the source of the surgery-induced leukocyte rolling (partial mast cell degranulation) and established that pretreatment of animals with sodium cromoglycate (connective tissue mast cell stabilizer) inhibited spontaneous leukocyte rolling. Superfusion of the mast cell-stabilized rat mesentery with histamine caused a profound increase in leukocyte rolling which persisted for the entire hour of experimentation. Diphenhydramine (H1-receptor antagonist) but not cimetidine (H2-receptor antagonist) prevented the rise in histamine-induced leukocyte rolling. An anti-P-selectin Ab but not an anti-CD18 Ab reversed the histamine-induced leukocyte rolling in a dose-dependent fashion. In this model of low base line rolling, exposure of the mesentery to the chemotactic agent platelet-activating factor did not induce leukocyte rolling or adhesion. However, co-administration of histamine with platelet-activating factor did indeed promote leukocyte adhesion suggesting that the presence of at least one effector of P-selectin is a minimal requirement for chemotactically-stimulated leukocytes to adhere to postcapillary venules. This study demonstrates for the first time that histamine induces leukocyte rolling via a P-selectin-dependent mechanism in vivo. This is a prolonged, H1 receptor-mediated event that may contribute significantly to the early phase of inflammation.
When injected into a 6‐day‐old mouse air‐pouch, human recombinant interleukin‐8 (IL‐8; 0.03–3 μg) induced, in a dose‐dependent fashion, an accumulation of neutrophils which could be reliably assessed 4 h after the injection. No protein extravasation was measured above the values obtained with the vehicle alone (carboxymethylcellulose, CMC, 0.5% w/v in phosphate‐buffered solution, PBS).
The IL‐8 effect (routinely evaluated at 1 μg dose) was inhibited neither by local administration of actinomycin D (1 μg) nor by systemic treatment with indomethacin (1 mg kg ⁻¹ , i.v.), BWA4C (5 mg kg ⁻¹ , p.o.), methysergide (6 mg kg ⁻¹ , i.p.) and RP67580 (2 mg kg ⁻¹ , i.p.).
Treatment of mice with the H 1 antagonist, mepyramine (1–10 mg kg ⁻¹ , i.p.) resulted in a dose‐dependent inhibition of the cell accumulation elicited by the chemokine, with a maximal reduction of approximately 50–60%. The mepyramine effect was not due to a non specific reduction of neutrophil function, since treatment with this drug (6 mg kg ⁻¹ , i.p.) did not modify the cell infiltration measured in response to a challenge with interleukin‐1β (20 ng) or with the vehicle CMC to any extent. Moreover, treatment of mice with mepyramine did not modify cell counts in a peripheral blood film with respect to controls. Two other H 1 antagonists, chemically unrelated to mepyramine, diphenhydramine (9 mg kg ⁻¹ , i.p.) and triprolidine (0.5 mg kg ⁻¹ , i.p.), inhibited IL‐8‐induced migration to a similar extent (∼ 50–60%), whereas the H 2 antagonist, ranitidine (5 mg kg ⁻¹ , i.p.) was without effect.
The concept that endogenous histamine could be involved in the IL‐8 effect was strengthened in two ways: (i) addition of histamine (0.2–2 μg) to a small dose of IL‐8 (0.3 μg) potentiated the cell elicitation induced by the chemokine without having any effect on its own; (ii) IL‐8‐induced neutrophil accumulation was greatly impaired in animals depleted of mast cell amines by sub‐chronic (5 day) treatment with compound 48/80 according to an established protocol.
The glucocorticoid dexamethasone (Dex; 1–50 μg per mouse, i.v., corresponding approximately to 0.03–1.5 mg kg ⁻¹ , given i.v. 2 h prior to challenge with IL‐8) potently inhibited neutrophil infiltration with an approximate ED 50 of 5 μg per mouse (∼ 0.3 mg kg ⁻¹ , i.v.). Passive immunisation of mice with a polyclonal sheep serum raised against the steroid‐inducible anti‐inflammatory protein lipocortin 1 (LC1) abolished the inhibitory action of Dex whereas a control serum was without effect.
Local administration of Dex at a dose which was ineffective when given systemically (1 μg) also reduced neutrophil migration induced by IL‐8, either alone or in combination with histamine. This local inhibition (∼ 50%), also seen with hydrocortisone (30 μg), was prevented by the concomitant administration of the steroid antagonist RU38486 (10 μg) indicating the involvement of glucocorticoid receptor in the response.
These findings characterize further the mechanisms underlying PMN recruitment induced by IL‐8 in vivo , and point to a role for histamine. The anti‐inflammatory action of the glucocorticoids, as in some other models, appears to be LC1‐dependent when these drugs are given systemically and LC1‐independent when the steroids are given locally.
We have determined that several chemokines induce mast cell migration in vitro. This directed migration is dependent on the presence of particular extracellular matrix proteins and the activation status of the cells. Mast cell haptotactic responses were observed in response to various chemokines on vitronectin-, laminin-, and fibronectin-coated filters. Unstimulated mast cells were chemoattracted only by monocyte chemotactic protein-1 and RANTES on vitronectin-coated and, to a lesser extent, laminin-coated filters, whereas IgE-activated mast cells migrated in response to monocyte chemotactic protein-1, regulated on activation normal T expressed and secreted, platelet factor-4, and macrophage inflammatory protein-1 alpha on all three matrix proteins. No significant migration was observed on collagen type IV-coated or uncoated filters. Mast cell migration in response to chemokines on extracellular matrices and its enhancement by IgE-dependent activation provide a mechanism by which cells may be drawn to sites of inflammation. Chemokine-induced mast cell recruitment may be particularly relevant in host defense responses to parasitic infections, allergic reactions, Jones-Mote reactions, and in wound healing.
Cryptococcus neoformans is acquired via the respiratory tract and is the leading cause of fatal mycosis in AIDS. Development of a T cell-mediated pulmonary inflammatory response is critical for clearance of this pathogen; however, the chemotactic factors that mediate inflammatory cell recruitment into the lungs have not been identified. In the present study, the bronchoalveolar lavage (BAL) fluid levels of the C-C chemokine monocyte chemotactic protein-1 (MCP-1) and the recruitment of inflammatory cells both increased following pulmonary infection with C. neoformans. The kinetics of MCP-1 production in the lungs correlated most closely with the recruitment of CD4+ T cells and monocytes/macrophages. Administration of neutralizing anti-MCP-1 Abs in vivo reduced the BAL fluid levels of MCP-1, decreased the recruitment of both macrophages (> 95%) and CD4+ T cells (76 +/- 9%), and inhibited cryptococcal clearance. Although no in vitro neutrophil or B cell chemotactic activity has been reported for MCP-1, recruitment of these leukocytes was also decreased in anti-MCP-1-treated mice (most likely an indirect effect of reducing the number of CD4+ T cells and macrophages). Neutralization of MCP-1 also resulted in decreased BAL fluid levels of TNF-alpha and IL-6. This is the first demonstration of a role for MCP-1 in clearance of an infection, and provides direct evidence that MCP-1 plays a critical role in the T cell-dependent immune response to C. neoformans.
This study investigates the effect of dexamethasone on leukocyte extravasation in the post-capillary venules of the hamster cheek pouch, using an intravital microscopy technique, and seeks to clarify the potential involvement of the steroid-inducible protein lipocortin 1. Topical application of FMLP (10 nmol), or substance P (10 nmol), to the superfused cheek pouch induced at the level of the post-capillary venules the three characteristic phenomena of leukocyte rolling, adhesion, and transmigration. Pretreatment of hamsters with an anti-inflammatory dose of dexamethasone (1 mg/kg) increased lipocortin 1 levels in circulating leukocytes as assessed by flow cytometry, but did not modify either leukocyte rolling or the number of adherent cells; however approximately 65% of the adherent leukocytes subsequently detached and returned to the blood stream, whereas those that entered into the diapedesis process exhibited a long latency (approximately three- to fourfold longer than in control animals) before transmigration. In hamsters passively immunized with a polyclonal anti-lipocortin 1 serum, leukocyte diapedesis started at similar times in both control and dexamethasone-treated animals, whereas a significant prolongation was observed in those animals treated with a non-immune sheep serum. These observations indicate that 1) lipocortin 1 is elevated in circulating leukocytes following dexamethasone treatment; 2) the step of leukocyte extravasation affected by dexamethasone in the actual transmigration process, and 3) this specific effect upon leukocyte diapedesis is mediated by endogenous lipocortin 1.
We have tested the histamine releasing properties and priming abilities of a wide range of recombinant or purified cytokines and growth factors on the basophils of 20 subjects (10 atopic and 10 nonatopic). We found that monocyte chemotactic and activating factor/monocyte chemoattractant protein-1 (MCAF/MCP-1), RANTES, human macrophage inflammatory protein-1 alpha and human inflammatory protein-1 beta, Connective tissue activating peptide III and Neutrophil Activating Peptide-2 (NAP-2) cause histamine release from basophils and are all members of the intercrine/chemokine family. MCAF/MCP-1 was as potent as anti-IgE or C5a and it is clearly the major contributor to histamine releasing factor activity. RANTES was the second major histamine releasing factor among the positive cytokines. Both MCAF/MCP-1 and RANTES are present in conditioned mononuclear cell media and can be separated using Mono Q anion exchange chromatography. We also demonstrated that RANTES has unusual chromatographic properties in spite of its isoelectric point of > 9.0 because it is largely found in peak-2 of the Mono Q column rather than peak-1 in which intercrines such as MCAF/MCP-1, IL-8, and connective tissue activating peptide III are found. All other cytokines and growth factors tested were negative, with the exception of IL-3, which caused histamine release in a subpopulation of subjects, and also primed basophils for release by anti-IgE. Other basophil primers for anti-IgE-dependent histamine release were IL-5, mast cell growth factor (c-kit ligand), and insulin-like growth factor II. Using specific neutralizing antibodies we have shown that MCAF/MCP-1, RANTES, and IL-3 contribute significantly to the activity found in mononuclear cell culture supernatants. Granulocyte-macrophage-CSF, IP-10, I-309, IL-7, IL-8, IL-9, IL-10, IL-11, IgE-binding factor, TNF-alpha, TGF-beta 1, fibroblast growth factor, epidermal growth factor, and endothelial cell growth factor were negative for direct histamine release and as primers of basophils. Our results indicate that cytokines belonging to the intercrine/chemokine family are major constituents of the activity known as "histamine releasing factor" found in MNC supernatants.
"Classical" chemoattractants, such as FMLP, C5a, or leukotriene B4, not only elicit directed motility but also activate neutrophils (degranulation, release of active oxygen species). Signal transduction after ligation of receptors for these classical chemoattractants is mediated by pertussis toxin (PT)-sensitive, heterotrimeric G proteins and the early production of lipid messengers via phospholipases. In contrast, we have previously shown that substance P (SP) and transforming growth factor-beta 1 (TGF-beta 1) are "pure" chemoattractants in that they elicit chemotaxis without activating neutrophils. Paradoxically, pure chemoattractants also activate G proteins (plasmalemmal GTPase activity) without eliciting increments in cytosolic calcium ([Ca]i) and thus inositol trisphosphate. We therefore determined lipid remodeling and signal transduction in response to pure chemoattractants. Increments in plasmalemmal GTPase activated by SP (0.1 microM) and TGF-beta 1 (40 fM), like that after FMLP, were PT-sensitive (SP = 6.6 +/- 2 pm/mg/min vs SP + PT = 1.1 +/- 0.9 over basal activity; TGF-beta 1 = 4.3 +/- 1.6 vs TGF-beta 1 + PT = 2.3 +/- 0.9). In parallel, treatment of PMN with PT (1 microgram/ml, 30 min) inhibited chemotaxis (under agarose) after FMLP (2175 +/- 176 (SEM) microns vs 726 +/- 267) and SP (411 +/- 99 microns vs 103 +/- 62 microns) and TGF-beta 1 (40 fM, 375 +/- 53 microns vs 83 +/- 47). However, G proteins coupled to receptors for SP and TGF-beta 1, unlike FMLP, did not appear to be linked to phospholipases in that neither increments in diacylglycerol were detected after receptor ligation (FMLP = 152 +/- 22% resting levels; SP = 101 +/- 5%; TGF-beta 1 = 105 +/- 4%) nor was alkylacylglycerol increased by exposure to SP or TGF-beta 1 (SP = 92 +/- 4%; TGF-beta 1 = 101 +/- 8%; FMLP = 226 +/- 40%). Moreover, polymorphonuclear leukocytes failed to generate phosphatidates (PA) of either species after SP (DA-PA = 79 +/- 9% resting at 60 s; EA-PA = 103 +/- 4%) or TGF-beta 1 (DA-PA = 101 +/- 5%; EA-PA = 98 +/- 9%) in contrast to FMLP (DA-PA = 155 +/- 22%; EA-PA = 149 +/- 16%). The data clearly contravene the current dogma that all chemoattractants use inositol trisphosphate and diglycerides as intracellular signals and suggest the presence of a unique subset of PT-sensitive G proteins, not coupled to "classical" phospholipases, transduce chemoattraction.
The relatively recent appreciation of a new class of cytokines, the chemokines, has done much to enhance our understanding of the extracellular signals involved in the movement of various populations of white blood cells. Investigation of the molecular underpinnings of chemokine function and their involvement in inflammatory processes of all kinds is beginning to yield information about the mechanisms of pathogenesis of a number of conditions, as well as providing hope for new therapeutic insights.
Leukocytes come into intimate contact with the venular endothelium as they extravasate from blood to the interstitium during inflammation. In exteriorized tissues, the number of leukocytes rolling along the vessel wall was markedly increased in adrenalectomized and metyrapone-treated animals, relative to sham-operated and normal animals. During the development of an acute, local inflammatory response, rollers were numerically decreased and a stronger adhesion of the cells to the endothelium, with a concomitant migration into tissues, was observed. Adhesion and migration were much more marked in adrenalectomized and metyrapone-treated animals than in controls, suggesting that secreted glucocorticoids exert a suppressive effect on leukocyte-endothelial interactions. The increased number of rolling leukocytes in adrenalectomized and metyrapone-treated animals apparently resulted in more cells available to migrate into inflamed tissues. The effect appears to involve receptor occupancy and induction of gene expression because normal animals receiving the steroid antagonist RU 38 486, actinomycin D, or cycloheximide behaved as adrenalectomized or metyrapone-treated animals. Administration to adrenalectomized animals of a monoclonal antibody to the membrane glycoprotein complex CD18 did not affect the number of rolling cells, but dramatically reduced the number of adherent or migrated leukocytes. It is suggested that secreted glucocorticoids, in addition to an effect on rolling behavior of circulating leukocytes, might also modulate the activity of the glycoprotein complex CD18 on white blood cells. The ultimate consequence is a restrictive effect on cell emigration in inflammation.
Eotaxin was recently identified as the major eosinophil chemoattractant in bronchoalveolar lavage fluid obtained 3h after allergen challenge of sensitised guinea-pigs. We now report the cDNA cloning of this C-C chemokine. The 777 base-pair clone, pEo3122, consists of a 40 base 5' untranslated region, an open reading frame of 288 bases predicting a 73 amino acid mature protein plus a 23 amino acid signal peptide, and a 3' untranslated region of 449 bases containing a poly A tail. Northern blot analysis showed eotaxin mRNA in the lungs of naive and sensitised guinea-pigs, which was considerably increased after allergen challenge. Eotaxin may be an important mediator of eosinophil accumulation and activation in allergic reactions. As eotaxin stimulates human eosinophils, this chemokine and related molecules may be involved in human diseases such as asthma where eosinophil accumulation is a prominent feature.
The accumulation of leukocytes in inflamed tissue results from adhesive interactions between leukocytes and endothelial cells within the microcirculation. These adhesive interactions and the excessive filtration of fluid and protein that accompanies an inflammatory response are largely confined to one region of the microvasculature: postcapillary venules. The nature and magnitude of the leukocyte-endothelial cell adhesive interactions that take place within postcapillary venules are determined by a variety of factors, including expression of adhesion molecules on leukocytes and/or endothelial cells, products of leukocyte (superoxide) and endothelial cell (nitric oxide) activation, and the physical forces generated by the movement of blood along the vessel wall. The contribution of different adhesion molecules to leukocyte rolling, adherence, and emigration in venules is discussed. Emerging views on potential endogenous antiadhesion molecules produced by endothelial cells as well as the influence of alterations in shear rate on leukocyte adhesion are addressed. Finally, the pathophysiological significance of the microvascular responses to inflammation are discussed in terms of adhesion-directed strategies for the treatment of different cardiovascular diseases and circulatory disorders.
Lipocortin-1, a 37 kDa member of the annexin superfamily of proteins, originally evoked interest as one of the 'second messengers' of the anti-inflammatory actions of the glucocorticoids. Subsequent research has shown that the protein plays a major regulatory role in systems as diverse as cell-growth regulation and differentiation, neutrophil migration, CNS responses to cytokines, neuroendocrine secretion and neurodegeneration. The role of lipocortin-1 in mediating glucocorticoid-induced effects in these systems has been demonstrated using immunoneutralization strategies and by mimicking steroid actions with highly purified or recombinant lipocortin-1 or its biologically active peptide fragments. Originally the mode of action of lipocortin-1 seemed to be largely through inhibition of prostaglandin formation, but it is now clear that it can modify other aspects of cell function, perhaps pointing to a more fundamental mechanism than was originally envisaged. In this article Rod Flower and Nancy Rothwell review the nature, possible mechanisms and clinical relevance of these diverse actions of lipocortin-1.
We have previously reported that monocyte chemoattractant protein-1 (MCP-1) is the most potent histamine-releasing factor (HRF) for basophils. Macrophage inflammatory protein-1 alpha (MIP-1 alpha) has modest histamine-releasing activity. The objective of this study was to investigate whether MCP-1 and MIP-1 alpha would activate mast cells in vivo and induce a cutaneous inflammatory reaction in mice. To this goal, mouse hind footpads were separately injected with 20 microliters of human recombinant MCP-1 or MIP-1 alpha (10(-7) M). Diluent was used as a control in the second footpad. The footpad-swelling response was measured at 30 min, 1 h, and then hourly for 6 h. Both MCP-1 (2.72 +/- 0.2 vs 2.1 +/- 0.03 mm for diluent, n = 8, p < 0.02) and MIP-1 alpha (3.0 +/- 0.1 vs 2.1 +/- 0.03 mm for diluent, n = 8, p < 0.02) induced an immediate swelling reaction. The immediate reaction was followed by a sustained late reaction that peaked within 1 h and lasted for more than 6 h. Histologic examination of the footpads, obtained at hour 2, revealed that MCP-1 caused mild mononuclear cell infiltrates, moderate degranulation of mast cells, and soft tissue swelling. In contrast, MIP-1 alpha induced a severe inflammatory reaction that consisted of neutrophils, mononuclear cells, and degranulated mast cells. Electron microscope examination of the tissue revealed features of extensive mast cell degranulation by MIP-1 alpha and to a lesser extent by MCP-1. Thus, we conclude that mast cells are activated on injection of MCP-1, whereas degranulation of mast cells and recruitment of leukocytes contribute to the footpad reaction induced with MIP-1 alpha.
The activity of the steroid-inducible protein lipocortin-1 (LC1; with a primary sequence of 346 amino acids; also called annexin 1), a fragment corresponding to amino acids 1-188 and a short peptide from the N-terminus (amino acid 2-26) were tested for anti-inflammatory actions in three models of acute inflammation in the mouse in comparison with a mAb anti-CD11b (αCD11b). In the mouse air-pouch model LC1, fragment 1-188 and peptide Ac2- 26 exhibited powerful inhibitory effects (ED50 ≃ 5.2, 38 and 88 μg/mouse, respectively) on leukocyte migration elicited by IL-1. LC1 was approximately 200 times more potent than Ac2-26 on a molar basis although both gave maximal inhibitions, in contrast fragment 1-188 only produced a partial dose-response curve. LC1 was approximately 20 times more potent on a molar basis in this assay than the αCD11b mAb. Peptide Ac2-26 and the mAb αCD11b also blocked cell migration into the air-pouch induced by IL-8 with approximately the same potency. In the mouse skin edema and zymosan peritonitis assays Ac2-26 was inhibitory (ED50 of 200 μg/mouse) but less so than the αCD11b antibody (ED50 ≃ 0.5 mg/mouse). Both LC1 (10 μg) and Ac2-26 (200 μg) completely blocked FMLP-induced neutropenia in the mouse. Studies using an inactivated LC1 preparation, which binds to the same high affinity binding sites as the biologically active material, indicated that the short peptide acts on the same sites as the native LC1. This study confirms the activity of LC1 in another model of experimental inflammation and suggests that it acts partly through inhibition of leukocyte activation with an overall effect qualitatively comparable to the blocking of CD11b portion of a β2-integrin complex. It also shows that peptides derived from the N-terminal domain of LC1 may mimic the activity of the full length molecule and points the way for a new family of anti-inflammatory substances that inhibit leukocyte trafficking.
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.
A local pre‐injection of 1 μg dexamethasone sodium phosphate strongly inhibited (> 60% inhibition at 3 h; P < 0.001 at all time points) the development of carrageenin‐induced paw oedema in the rat induced by a subplantar injection of 0.1 ml, 2% carrageenin.
Coinjection of a polyclonal rabbit antiserum raised against human 1–188 recombinant lipocortin 1, which also recognised the rat protein, reversed the inhibitory action of dexamethasone ( P < 0.05 at 4 h and 5 h). At the highest volume used (40 μl) control antisera were without any effect.
These data further support the concept that lipocortin 1 is involved in the anti‐inflammatory mechanism of action of the glucocorticoids.