Article

Tumour necrosis factor-?? and leukotriene B4 mediate the neutrophil migration in immune inflammation

British Journal of Pharmacology

January 2002
134(8):1619-28

DOI:10.1038/sj.bjp.0704403

Source
PubMed

Authors:

Claudio Canetti

Federal University of Rio de Janeiro

Joao Santana Silva

University of São Paulo

Sergio Henrique Ferreira

University of São Paulo

Fernando Cunha

University of São Paulo

We investigated the mediators responsible for neutrophil migration induced by ovalbumin (OVA) in immunized mice and the mechanisms involved in their release. OVA administration promoted dose- and time-dependent neutrophil migration in immunized, but not in non-immunized mice, which was mediated by leukotriene B4 (LTB4) and tumour necrosis factor (TNF)α, since it was inhibited by LTB4 synthesis inhibitor (MK 886) or by LTB4 receptor antagonist (CP 105,696), by dexamethasone and by antiserum to TNFα (82, 85, 63 and 87%, respectively). Confirming TNFα involvement, OVA challenge in immunized p55 TNF receptor deficient mice (p55−/−) did not promote neutrophil migration (control: 2.90±0.68; p55−/−: 0.92±0.23×106 neutrophils cavity−1). OVA-stimulated peritoneal cells from immunized mice released a neutrophil chemotactic factor which mimicked, in naive mice, neutrophil migration induced by OVA. Supernatant chemotactic activity is due to TNFα and LTB4, since its release was inhibited by MK 886 (93%) and dexamethasone (90%), and significant amounts of these mediators were detected. TNFα and LTB4 released by OVA challenge seem to act through a sequential mechanism, since MK 886 inhibited (88%) neutrophil migration induced by TNFα. Moreover, peritoneal cells stimulated with TNFα released LTB4. CD4+ T cells are responsible for TNFα release, because the depletion of this subset prevented the release of TNFα (control: 400±25; immunized: 670±40; CD4+ depleted: 435±18 pg ml−1). In conclusion, neutrophil migration induced by OVA depends on TNFα released by CD4+ cells, which acts through an LTB4-dependent mechanism. British Journal of Pharmacology (2001) 134, 1619–1628; doi:10.1038/sj.bjp.0704403

IL-18 enhances collagen-induced arthritis by recruiting neutrophils via TNF-alpha and leukotriene B-4

Article

Full-text available

Aug 2003

A crucial role for TNF-?? in mediating neutrophil influx induced by endogenously generated or exogenous chemokines, KC/CXCL1 and LIX/CXCL5

Article

Sep 2009
BRIT J PHARMACOL

Chemokines orchestrate neutrophil recruitment to inflammatory foci. In the present study, we evaluated the participation of three chemokines, KC/CXCL1, MIP-2/CXCL2 and LIX/CXCL5, which are ligands for chemokine receptor 2 (CXCR2), in mediating neutrophil recruitment in immune inflammation induced by antigen in immunized mice. Neutrophil recruitment was assessed in immunized mice challenged with methylated bovine serum albumin, KC/CXCL1, LIX/CXCL5 or tumour necrosis factor (TNF)-alpha. Cytokine and chemokine levels were determined in peritoneal exudates and in supernatants of macrophages and mast cells by elisa. CXCR2 and intercellular adhesion molecule 1 (ICAM-1) expression was determined using immunohistochemistry and confocal microscopy. Antigen challenge induced dose- and time-dependent neutrophil recruitment and production of KC/CXCL1, LIX/CXCL5 and TNF-alpha, but not MIP-2/CXCL2, in peritoneal exudates. Neutrophil recruitment was inhibited by treatment with reparixin (CXCR1/2 antagonist), anti-KC/CXCL1, anti-LIX/CXCL5 or anti-TNF-alpha antibodies and in tumour necrosis factor receptor 1-deficient mice. Intraperitoneal injection of KC/CXCL1 and LIX/CXCL5 induced dose- and time-dependent neutrophil recruitment and TNF-alpha production, which were inhibited by reparixin or anti-TNF-alpha treatment. Macrophages and mast cells expressed CXCR2 receptors. Increased macrophage numbers enhanced, while cromolyn sodium (mast cell stabilizer) diminished, LIX/CXCL5-induced neutrophil recruitment. Macrophages and mast cells from immunized mice produced TNF-alpha upon LIX/CXCL5 stimulation. Methylated bovine serum albumin induced expression of ICAM-1 on mesenteric vascular endothelium, which was inhibited by anti-TNF-alpha or anti-LIX/CXCL5. Following antigen challenge, CXCR2 ligands are produced and act on macrophages and mast cells triggering the production of TNF-alpha, which synergistically contribute to neutrophil recruitment through induction of the expression of ICAM-1.

IL-18 Enhances Collagen-Induced Arthritis by Recruiting Neutrophils Via TNF- and Leukotriene B4

Article

Full-text available

Aug 2003

IL-18 expression and functional activity have been associated with a range of autoimmune diseases. However, the precise mechanism by which IL-18 induces such pathology remains unclear. In this study we provide direct evidence that IL-18 activates neutrophils via TNF-alpha induction, which drives the production of leukotriene B(4) (LTB(4)), which in turn leads to neutrophil accumulation and subsequent local inflammation. rIL-18 administered i.p. resulted in the local synthesis of LTB(4) and a rapid influx of neutrophils into the peritoneal cavity, which could be effectively blocked by the LTB(4) synthesis inhibitor MK-886 (MK) or its receptor antagonist CP-105,696. IL-18-induced neutrophils recruitment and LTB(4) production could also be blocked by a neutralizing anti-TNF-alpha Ab. In addition, IL-18 failed to induce neutrophil accumulation in vivo in TNFRp55(-/-) mice. In an IL-18-dependent murine collagen-induced arthritis model, administration of MK significantly inhibited disease severity and reduced articular inflammation and joint destruction. Furthermore, MK-886-treated mice also displayed suppressed proinflammatory cytokine production in response to type II collagen in vitro. Finally, we showed that IL-18-activated human peripheral blood neutrophils produced significant amounts of LTB(4) that were effectively blocked by the MK. Together, these findings provide a novel mechanism whereby IL-18 can promote inflammatory diseases.

Anti-Inflammatory Benefits of Antibiotics: Tylvalosin Induces Apoptosis of Porcine Neutrophils and Macrophages, Promotes Efferocytosis, and Inhibits Pro-Inflammatory CXCL-8, IL1α, and LTB4 Production, While Inducing the Release of Pro-Resolving Lipoxin A4 and Resolvin D1

Article

Full-text available

Apr 2018

Excessive accumulation of neutrophils and their uncontrolled death by necrosis at the site of inflammation exacerbates inflammatory responses and leads to self-amplifying tissue injury and loss of organ function, as exemplified in a variety of respiratory diseases. In homeostasis, neutrophils are inactivated by apoptosis, and non phlogistically removed by neighboring macrophages in a process known as efferocytosis, which promotes the resolution of inflammation. The present study assessed the potential anti-inflammatory and pro-resolution benefits of tylvalosin, a recently developed broad-spectrum veterinary macrolide derived from tylosin. Recent findings indicate that tylvalosin may modulate inflammation by suppressing NF-κB activation. Neutrophils and monocyte-derived macrophages were isolated from fresh blood samples obtained from 12- to 22-week-old pigs. Leukocytes exposed to vehicle or to tylvalosin (0.1, 1.0, or 10 µg/mL; 0.096–9.6 µM) were assessed at various time points for apoptosis, necrosis, efferocytosis, and changes in the production of cytokines and lipid mediators. The findings indicate that tylvalosin increases porcine neutrophil and macrophage apoptosis in a concentration- and time-dependent manner, without altering levels of necrosis or reactive oxygen species production. Importantly, tylvalosin increased the release of pro-resolving Lipoxin A4 (LXA4) and Resolvin D1 (RvD1) while inhibiting the production of pro-inflammatory Leukotriene B4 (LTB4) in Ca²⁺ ionophore-stimulated porcine neutrophils. Tylvalosin increased neutrophil phospholipase C activity, an enzyme involved in releasing arachidonic acid from membrane stores. Tylvalosin also inhibited pro-inflammatory chemokine (C–X–C motif) ligand 8 (CXCL-8, also known as Interleukin-8) and interleukin-1 alpha (IL-1α) protein secretion in bacterial lipopolysaccharide-stimulated macrophages. Together, these data illustrate that tylvalosin has potent immunomodulatory effects in porcine leukocytes in addition to its antimicrobial properties.

Synthesis of Lipid Mediators during UVB-Induced Inflammatory Hyperalgesia in Rats and Mice

Article

Full-text available

Dec 2013
PLOS ONE

Peripheral sensitization during inflammatory pain is mediated by a variety of endogenous proalgesic mediators including a number of oxidized lipids, some of which serve endogenous modulators of sensory TRP-channels. These lipids are eicosanoids of the arachidonic acid and linoleic acid pathway, as well as lysophophatidic acids (LPAs). However, their regulation pattern during inflammatory pain and their contribution to peripheral sensitization is still unclear. Here, we used the UVB-model for inflammatory pain to investigate alterations of lipid concentrations at the site of inflammation, the dorsal root ganglia (DRGs) as well as the spinal dorsal horn and quantified 21 lipid species from five different lipid families at the peak of inflammation 48 hours post irradiation. We found that known proinflammatory lipids as well as lipids with unknown roles in inflammatory pain to be strongly increased in the skin, whereas surprisingly little changes of lipid levels were seen in DRGs or the dorsal horn. Importantly, although there are profound differences between the number of cytochrome (CYP) genes between mice and rats, CYP-derived lipids were regulated similarly in both species. Since TRPV1 agonists such as LPA 18∶1, 9- and 13-HODE, 5- and 12-HETE were elevated in the skin, they may contribute to thermal hyperalgesia and mechanical allodynia during UVB-induced inflammatory pain. These results may explain why some studies show relatively weak analgesic effects of cyclooxygenase inhibitors in UVB-induced skin inflammation, as they do not inhibit synthesis of other proalgesic lipids such as LPA 18∶1, 9-and 13-HODE and HETEs.

Anti-inflammatory effects of LASSBio-998, a new drug candidate designed to be a p38 MAPK inhibitor, in an experimental model of acute lung inflammation

Article

Jul 2011

We investigated the effects of LASSBio-998 (L-998), a compound designed to be a p38 MAPK (mitogen-activated protein kinase) inhibitor, on lipopolysaccharide (LPS)-induced acute lung inflammation in vivo. BALB/c mice were challenged with aerosolized LPS inhalation (0.5 mg/ml) 4 h after oral administration of L-998. Three hours after LPS inhalation, bronchoalveolar lavage fluid was obtained to measure the levels of the proinflammatory cytokines TNF-α (tumor necrosis factor-α) and IL-1 (interleukin-1) and the chemokines MCP-1 (monocyte chemoattractant protein-1) and KC (keratinocyte chemoattractant). In addition, neutrophil infiltration and p38 MAPK phosphorylation was measured. L-998 inhibited LPS-induced production of TNF-α and IL-1β and did not alter KC and MCP-1 levels. Furthermore, L-998 also significantly decreased neutrophil accumulation in lung tissues. As expected, L-998 diminished p38 MAPK phosphorylation and reduced acute lung inflammation. Inhibition of p38 MAPK phosphorylation by L-998 was also demonstrated in LPS-challenged murine C57BL/6 peritoneal macrophages in vitro, with concentration-dependent effects. L-998 suppressed LPS-induced lung inflammation, most likely by inhibition of the cytokine-p38 MAPK pathway, and we postulate that L-998 could be a clinically relevant anti-inflammatory drug candidate.

Neutrophils in Cancer immunotherapy: friends or foes?

Article

Full-text available

May 2024
MOL CANCER

Neutrophils play a Janus-faced role in the complex landscape of cancer pathogenesis and immunotherapy. As immune defense cells, neutrophils release toxic substances, including reactive oxygen species and matrix metalloproteinase 9, within the tumor microenvironment. They also modulate the expression of tumor necrosis factor-related apoptosis-inducing ligand and Fas ligand, augmenting their capacity to induce tumor cell apoptosis. Their involvement in antitumor immune regulation synergistically activates a network of immune cells, bolstering anticancer effects. Paradoxically, neutrophils can succumb to the influence of tumors, triggering signaling cascades such as JAK/STAT, which deactivate the immune system network, thereby promoting immune evasion by malignant cells. Additionally, neutrophil granular constituents, such as neutrophil elastase and vascular endothelial growth factor, intricately fuel tumor cell proliferation, metastasis, and angiogenesis. Understanding the mechanisms that guide neutrophils to collaborate with other immune cells for comprehensive tumor eradication is crucial to enhancing the efficacy of cancer therapeutics. In this review, we illuminate the underlying mechanisms governing neutrophil-mediated support or inhibition of tumor progression, with a particular focus on elucidating the internal and external factors that influence neutrophil polarization. We provide an overview of recent advances in clinical research regarding the involvement of neutrophils in cancer therapy. Moreover, the future prospects and limitations of neutrophil research are discussed, aiming to provide fresh insights for the development of innovative cancer treatment strategies targeting neutrophils.

Dietary fish oil results in a microenvironment with high anti-inflammatory potential, but does not inhibit mice from mounting an effective immune response to Borrelia burgdorferi. (51.9)

Article

May 2012

Dietary fish oil is suggested to alleviate a variety of inflammatory disorders by altering eicosanoid production. However, the extent of changes resulting from a diet high in omega-3 fatty acids is unknown, and any benefits resulting from these changes are unsubstantiated. Several studies conclude that the lack of phenotypic effects of fish oil result from an inability to consume sufficient quantities for any benefits to be evident. We fed mice either a control diet containing 18% by weight soybean oil or fish oil and infected the mice with Borrelia burgdorferi in order to quantify changes in temporal eicosanoid production throughout the course of disease. Using a lipidomics approach, we detected dramatic temporal and compositional changes in the majority of the 103 eicosanoids monitored. LOX metabolites produced in fish oil-fed mice were primarily generated non-enzymatically, and arachidonic acid metabolism was shunted from PGE2 synthesis to PGD2 synthesis resulting in the production and accumulation of 15d-PGJ2. Despite the fact that the fish oil diet led to a highly anti-inflammatory microenvironment, mice were able to mount an immune response and clear infection as well as mice fed a control diet. This study confirms that a diet high in fish oil results in a microenvironment with high anti-inflammatory potential but does not necessarily affect the ability of the organism to mount an effective immune response to infection with Borrelia burgdorferi.

Microphysiological Systems for Studying Cellular Crosstalk During the Neutrophil Response to Infection

Article

Full-text available

Apr 2021

Neutrophils are the primary responders to infection, rapidly migrating to sites of inflammation and clearing pathogens through a variety of antimicrobial functions. This response is controlled by a complex network of signals produced by vascular cells, tissue resident cells, other immune cells, and the pathogen itself. Despite significant efforts to understand how these signals are integrated into the neutrophil response, we still do not have a complete picture of the mechanisms regulating this process. This is in part due to the inherent disadvantages of the most-used experimental systems: in vitro systems lack the complexity of the tissue microenvironment and animal models do not accurately capture the human immune response. Advanced microfluidic devices incorporating relevant tissue architectures, cell-cell interactions, and live pathogen sources have been developed to overcome these challenges. In this review, we will discuss the in vitro models currently being used to study the neutrophil response to infection, specifically in the context of cell-cell interactions, and provide an overview of their findings. We will also provide recommendations for the future direction of the field and what important aspects of the infectious microenvironment are missing from the current models.

The role of the LTB 4 -BLT1 axis in chemotactic gradient sensing and directed leukocyte migration

Article

Full-text available

Oct 2017

Directed leukocyte migration is a hallmark of inflammatory immune responses. Leukotrienes are derived from arachidonic acid and represent a class of potent lipid mediators of leukocyte migration. In this review, we summarize the essential steps leading to the production of LTB4 in leukocytes. We discuss the recent findings on the exosomal packaging and transport of LTB4 in the context of chemotactic gradients formation and regulation of leukocyte recruitment. We also discuss the dynamic roles of the LTB4 receptors, BLT1 and BLT2, in mediating chemotactic signaling in leukocytes and contrast them to other structurally related leukotrienes that bind to distinct GPCRs. Finally, we highlight the specific roles of the LTB4-BLT1 axis in mediating signal-relay between chemotaxing neutrophils and its potential contribution to a wide variety of inflammatory conditions including tumor progression and metastasis, where LTB4 is emerging as a key signaling component.

Short-Term Regulation of Fc γ R-Mediated Phagocytosis by TLRs in Macrophages: Participation of 5-Lipoxygenase Products

Article

Full-text available

Aug 2017
MEDIAT INFLAMM

TLRs recognize a broad spectrum of microorganism molecules, triggering a variety of cellular responses. Among them, phagocytosis is a critical process for host defense. Leukotrienes (LTs), lipid mediators produced from 5-lipoxygenase (5-LO) enzyme, increase Fc γ R-mediated phagocytosis. Here, we evaluated the participation of TLR2, TLR3, TLR4, and TLR9 in Fc γ R-mediated phagocytosis and whether this process is modulated by LTs. Rat alveolar macrophages (AMs), murine bone marrow-derived macrophages (BMDMs), and peritoneal macrophages (PMs) treated with TLR2, TLR3, and TLR4 agonists, but not TLR9, enhanced IgG-opsonized sheep red blood cell (IgG-sRBC) phagocytosis. Pretreatment of AMs or BMDMs with drugs that block LT synthesis impaired the phagocytosis promoted by TLR ligands, and TLR potentiation was also abrogated in PMs and BMDMs from 5-LO −/− mice. LTB 4 production induced by IgG engagement was amplified by TLR ligands, while cys-LTs were amplified by activation of TLR2 and TLR4, but not by TLR3. We also noted higher ERK1/2 phosphorylation in IgG-RBC-challenged cells when preincubated with TLR agonists. Furthermore, ERK1/2 inhibition by PD98059 reduced the phagocytic activity evoked by TLR agonists. Together, these data indicate that TLR2, TLR3, and TLR4 ligands, but not TLR9, amplify IgG-mediated phagocytosis by a mechanism which requires LT production and ERK-1/2 pathway activation.

A critical role of leukotriene B-4 in neutrophil migration to infectious focus in cecal ligaton and puncture sepsis

Article

Jan 2003

Neutrophil migration to an infectious focus is essential for control and resolution of infection. Early studies demonstrated that the failure of such migration is observed in lethal sepsis induced by cecal ligation and puncture (L-CLP), whereas intense neutrophil migration is seen in sublethal CILP (SL-CLP). In this study, we found that inhibition of synthesis of prostaglandins or leukotriene B-4 (LTB4) did not modify the failure of neutrophil migration or the survival rate of L-CLP mice. In addition, pretreatment of L-CLP mice with a platelet activating factor (PAF) receptor antagonist (UK74505), despite not interfering with the failure process, significantly increased (33%) the survival rate of the animals. Inhibitors of prostaglandin synthesis (indomethacin and meloxican) and UK74505 did not modify the neutrophil migration observed in SL-CLP. On the other hand, the blockade of LTB4 Synthesis (MK886, a 5-lipoxygenase-activating protein inhibitor) or of its receptors (CP-105,696) resulted in reduced neutrophil migration to the peritoneal cavity in SL-CLP mice (62% and 60%, respectively), a consequent increase in the number of bacteria in the inflammatory focus, and a reduced survival rate of the animals (43% and 38%, respectively). Both SL-CLP and L-CLIP animals presented significant levels of LTB4 in the peritoneal exudate (3- and 8-fold higher than sham group, respectively) and these were reduced by the pretreatment of mice with LTB4 inhibitors. In conclusion, our results suggest that LTB4, but not prostaglandins or PAF, is an important chemoattractant involved in neutrophil recruitment to infection sites in SL-CLP, a crucial event in confining the invading pathogens to a restricted area. However, in circumstances in which the infection turns to a lethal sepsis, LTB4 is not involved in the observed failure of neutrophil migration to the infectious focus.

Neutrophil Extracellular Traps Induce Organ Damage during Experimental and Clinical Sepsis

Article

Full-text available

Feb 2016
PLOS ONE

Organ dysfunction is a major concern in sepsis pathophysiology and contributes to its high mortality rate. Neutrophil extracellular traps (NETs) have been implicated in endothelial damage and take part in the pathogenesis of organ dysfunction in several conditions. NETs also have an important role in counteracting invading microorganisms during infection. The aim of this study was to evaluate systemic NETs formation, their participation in host bacterial clearance and their contribution to organ dysfunction in sepsis. C57Bl/6 mice were subjected to endotoxic shock or a polymicrobial sepsis model induced by cecal ligation and puncture (CLP). The involvement of cf-DNA/NETs in the physiopathology of sepsis was evaluated through NETs degradation by rhDNase. This treatment was also associated with a broad-spectrum antibiotic treatment (ertapenem) in mice after CLP. CLP or endotoxin administration induced a significant increase in the serum concentrations of NETs. The increase in CLP-induced NETs was sustained over a period of 3 to 24 h after surgery in mice and was not inhibited by the antibiotic treatment. Systemic rhDNase treatment reduced serum NETs and increased the bacterial load in non-antibiotic-treated septic mice. rhDNase plus antibiotics attenuated sepsis-induced organ damage and improved the survival rate. The correlation between the presence of NETs in peripheral blood and organ dysfunction was evaluated in 31 septic patients. Higher cf-DNA concentrations were detected in septic patients in comparison with healthy controls, and levels were correlated with sepsis severity and organ dysfunction. In conclusion, cf-DNA/NETs are formed during sepsis and are associated with sepsis severity. In the experimental setting, the degradation of NETs by rhDNase attenuates organ damage only when combined with antibiotics, confirming that NETs take part in sepsis pathogenesis. Altogether, our results suggest that NETs are important for host bacterial control and are relevant actors in the pathogenesis of sepsis.

In Vivo Availability of Pro-Resolving Lipid Mediators in Oxazolone Induced Dermal Inflammation in the Mouse

Article

Full-text available

Nov 2015
PLOS ONE

The activation and infiltration of polymorphonuclear neutrophils (PMN) are critical key steps in inflammation. PMN-mediated inflammation is limited by anti-inflammatory and pro-resolving mechanisms, including specialized pro-resolving lipid mediators (SPM). We examined the effects of 15-epi-LXA4 on inflammation and the biosynthesis of pro-inflammatory mediators, such as prostaglandins, leukotriene B4 and various hydroxyeicosatetraenoic acids and SPM, in an oxazolone (OXA)-induced hypersensitivity model for dermal inflammation. 15-epi-LXA4 (100 μM, 5 μL subcutaneously injected) significantly (P < 0.05) reduced inflammation in skin, 24 hours after the OXA challenge, as compared to skin treated with vehicle. No significant influence on the biosynthesis of prostaglandins or leukotriene B4 was observed, whereas the level of 15S-hydroxy-eicosatetraenoic acid was significantly (P < 0.05) lower in the skin areas treated with 15-epi-LXA4. In spite of the use of a fully validated analytical procedure, no SPM were detected in the biological samples. To investigate the reason for the lack of analytical signal, we tried to mimic the production of SPM (lipoxins, resolvins, maresin and protectin) by injecting them subcutaneously into the skin of mice and studying the in vivo availability and distribution of the compounds. All analytes showed very little lateral distribution in skin tissue and their levels were markedly decreased (> 95%) 2 hours after injection. However, docosahexaenoic acid derivatives were biologically more stable than SPM derived from arachidonic acid or eicosapentaenoic acid.

Neutrophil activation by MNCF lectin results in gene transcription and secretion of cytokines, even in anti-inflammatory conditions

Article

Full-text available

Dec 2007

Karina alves toledo

Leukocytes are accumulated, in the inflammatory process, because to action of the wide array of stimuli. This event envolved several and coodenated steps whose inhibited by glucocoticoids, such as dexamethasone. Dexamethasone affects human neutrophils in different ways. It shows negative effects (on synthesis and secretion of pro-inflammatory mediators) and positive effects (for example, on annexin I). Among the inducers of leukocyte migration, MNCF, a galactose-binding lectin, has been described as an agonist and chemoattractant for neutrophils, both in vivo and in vitro. MNCF shows as peculiar activity the migration of neutrophils resistant to dexamethasone actions which awakes great interest in undertanding the mechanism of action on polymorphonuclears by this lectin. Our first step was study human MNCF-stimulated neutrophils pre-incubated with dexamethasone. In these conditions, MNCF, in the absence of F-actin polymerization: (a) protects neutrophils from spontaneous apoptosis, (b) induces tyrosine and p38 MAP quinases phosphorylation, (c) induces CD62L shedding, (d) degranulates secretory vesicles and secundary granules, but not azurophilic granules, in the dependent manner of tyrosine and MAP quinases and Src family, (e) translocates the transcription factor NF-kB and, (f) induces transcription and secretion of pro-inflammatory cytokines and chemokines. In parallel, human neutrophils pre-incubated with dexamethasone and stimulated with MNCF did not show CD62L shedding and F-actin polarization, but the in vitro migration was maintained. Besides, we already observe translocation of NF-kB from cytoplasm to nucleous which it activates the genic transcription and secretion of inflammatory mediators, such as CXCL8. The results, showed here, strengthens previous results, demonstranting that MNCF as a agonist to neutrophils, beyond increase the half life them. Although, dexamethasone modifies some effects of MNCF on neutrophis, this glucocorticoid does not inhibit the cellular response to the studied lectin. Thus, the maintenance of inflammatory mediators, dependents to NF-kB, during the inflammatory process, could explain, even parcialy, the break in the resitance to glucocorticoids actions by MNCF in the neutrophil migration.

Citronellol, a natural acyclic monoterpene, attenuates mechanical hyperalgesia response in mice: Evidence of the spinal cord lamina I inhibition

Article

Jun 2015

Immunomodulatory effects of tulathromycin on apoptosis, efferocytosis, and proinflammatory leukotriene B 4 production in leukocytes from Actinobacillus pleuropneumoniae –or zymosan-challenged pigs

Article

Full-text available

Jun 2015
AM J VET RES

Objective: To investigate the anti-inflammatory and immunomodulatory properties of tulathromycin in vitro and in experimental models of Actinobacillus pleuropneumoniae-induced pleuropneumonia and zymosan-induced pulmonary inflammation in pigs. Animals: Blood samples from six 8- to 30-week-old healthy male pigs for the in vitro experiment and sixty-five 3-week-old specific pathogen-free pigs. Procedures: Neutrophils and monocyte-derived macrophages were isolated from blood samples. Isolated cells were exposed to tulathromycin (0.02 to 2.0 mg/mL) for various durations and assessed for markers of apoptosis and efferocytosis. For in vivo experiments, pigs were inoculated intratracheally with A pleuropneumoniae, zymosan, or PBS solution (control group) with or without tulathromycin pretreatment (2.5 mg/kg, IM). Bronchoalveolar lavage fluid was collected 3 and 24 hours after inoculation and analyzed for proinflammatory mediators, leukocyte apoptosis, and efferocytosis. Results: In vitro, tulathromycin induced time- and concentration-dependent apoptosis in neutrophils, which enhanced their subsequent clearance by macrophages. In the lungs of both A pleuropneumoniae- and zymosan-challenged pigs, tulathromycin promoted leukocyte apoptosis and efferocytosis and inhibited proinflammatory leukotriene B4 production, with a concurrent reduction in leukocyte necrosis relative to that of control pigs. Tulathromycin also attenuated the degree of lung damage and lesion progression in A pleuropneumoniae-inoculated pigs. Conclusions and clinical relevance: Tulathromycin had immunomodulatory effects in leukocytes in vitro and anti-inflammatory effects in pigs in experimental models of A pleuropneumoniae infection and nonmicrobial-induced pulmonary inflammation. These data suggested that in addition to its antimicrobial properties, tulathromycin may dampen severe proinflammatory responses and drive resolution of inflammation in pigs with microbial pulmonary infections.

5-Lipoxygenase Inhibitors Attenuate TNF-α-Induced Inflammation in Human Synovial Fibroblasts

Article

Full-text available

Sep 2014
PLOS ONE

The lipoxygenase isoform of 5-lipoxygenase (5-LOX) is reported to be overexpressed in human rheumatoid arthritis synovial tissue and involved in the progress of inflammatory arthritis. However, the detailed mechanism of how 5-lipoxygenase regulates the inflammatory response in arthritis synovial tissue is still unclear. The aim of this study was to investigate the involvement of lipoxygenase pathways in TNF-α-induced production of cytokines and chemokines. Human synovial fibroblasts from rheumatoid patients were used in this study. 5-LOX inhibitors and shRNA were used to examine the involvement of 5-LOX in TNF-α-induced cytokines and chemokines expression. The signaling pathways were examined by Western Blotting or immunofluorescence staining. The effect of 5-LOX inhibitor on TNF-α-induced chemokine expression and paw edema was also explored in vivo in C57BL/6 mice. Treatment with 5-LOX inhibitors significantly decreased TNF-α-induced pro-inflammatory mediators including interleukin-6 (IL-6) and monocyte chemo-attractant protein-1 (MCP-1) in human synovial fibroblasts. Knockdown of 5-LOX using shRNA exerted similar inhibitory effects. The abrogation of NF-κB activation was involved in the antagonizing effects of these inhibitors. Furthermore, 5-LOX inhibitor decreased TNF-α-induced up-regulation of serum MCP-1 level and paw edema in mouse model. Our results provide the evidence that the administration of 5-LOX inhibitors is able to ameliorate TNF-α-induced cytokine/chemokine release and paw edema, indicating that 5-LOX inhibitors may be developed for therapeutic treatment of inflammatory arthritis.

Tulathromycin Exerts Proresolving Effects in Bovine Neutrophils by Inhibiting Phospholipases and Altering Leukotriene B-4, Prostaglandin E-2, and Lipoxin A(4) Production

Article

Full-text available

Jul 2014
ANTIMICROB AGENTS CH

The accumulation of neutrophils and proinflammatory mediators, such as leukotriene B4 (LTB4), is a classic marker of inflammatory disease. The clearance of apoptotic neutrophils, inhibition of proinflammatory signaling, and production of proresolving lipids (including lipoxins, such as lipoxin A4 [LXA4]) are imperative for resolving inflammation. Tulathromycin (TUL), a macrolide used to treat bovine respiratory disease, confers immunomodulatory benefits via mechanisms that remain unclear. We recently reported the anti-inflammatory properties of TUL in bovine phagocytes in vitro and in Mannheimia haemolytica-challenged calves. The findings demonstrated that this system offers a powerful model for investigating novel mechanisms of pharmacological immunomodulation. In the present study, we examined the effects of TUL in a nonbacterial model of pulmonary inflammation in vivo and characterized its effects on lipid signaling. In bronchoalveolar lavage (BAL) fluid samples from calves challenged with zymosan particles (50 mg), treatment with TUL (2.5 mg/kg of body weight) significantly reduced pulmonary levels of LTB4 and prostaglandin E2 (PGE2). In calcium ionophore (A23187)-stimulated bovine neutrophils, TUL inhibited phospholipase D (PLD), cytosolic phospholipase A2 (PLA2) activity, and the release of LTB4. In contrast, TUL promoted the secretion of LXA4 in resting and A23187-stimulated neutrophils, while levels of its precursor, 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], were significantly lower. These findings indicate that TUL directly modulates lipid signaling by inhibiting the production of proinflammatory eicosanoids and promoting the production of proresolving lipoxins.

Immunity to Lutzomyia intermedia Saliva Modulates the Inflammatory Environment Induced by Leishmania braziliensis

Data

Jan 2010

Background: During blood feeding, sand flies inject Leishmania parasites in the presence of saliva. The types and functions of cells present at the first host-parasite contact are critical to the outcome on infection and sand fly saliva has been shown to play an important role in this setting. Herein, we investigated the in vivo chemotactic effects of Lutzomyia intermedia saliva, the vector of Leishmania braziliensis, combined or not with the parasite. Methods and Findings: We tested the initial response induced by Lutzomyia intermedia salivary gland sonicate (SGS) in BALB/c mice employing the air pouch model of inflammation. L. intermedia SGS induced a rapid influx of macrophages and neutrophils. In mice that were pre-sensitized with L. intermedia saliva, injection of SGS was associated with increased neutrophil recruitment and a significant up-regulation of CXCL1, CCL2, CCL4 and TNF-a expression. Surprisingly, in mice that were pre-exposed to SGS, a combination of SGS and L. braziliensis induced a significant migration of neutrophils and an important modulation in cytokine and chemokine expression as shown by decreased CXCL10 expression and increased IL-10 expression. Conclusion: These results confirm that sand fly saliva modulates the initial host response. More importantly, pre-exposure to L. intermedia saliva significantly modifies the host's response to L. braziliensis, in terms of cellular recruitment and expression of cytokines and chemokines. This particular immune modulation may, in turn, favor parasite multiplication. Citation: de Moura TR, Oliveira F, Rodrigues GC, Carneiro MW, Fukutani KF, et al. (2010) Immunity to Lutzomyia intermedia Saliva Modulates the Inflammatory Environment Induced by Leishmania braziliensis. PLoS Negl Trop Dis 4(6): e712. doi:10.1371/journal.pntd.0000712 This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. Funding: This work was supported by grants from FAPESB, PAPES/FIOCRUZ, and CNPq. TRdM was supported by a CNPq fellowship. MB-N, CB, AB, and CIdO are senior investigators from CNPq. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.

Analgesic and anti-inflammatory studies of cyclopeptide alkaloid fraction of leaves of Ziziyphus nummularia

Article

Full-text available

Oct 2013

Ziziyphus nummularia (family: Rhamnaceae) is a thorny small bush, grows in abundance in the grazing lands of the arid areas of Rajasthan, India. It is an important ethnomedicinal plant of the Thar Desert; local inhabitants use every part of the plant as medicine. Kernels are prescribed in pregnancy as soporific, antiemetic and for relieving abdominal pain. The insect gall is powered and given orally with water to cure bone fracture. Crushed root is applied on the paining shoulder of the bullock. The decoction of leaves is used for the treatment of cough and cold; leaves are also regarded as diaphoretic and prescribed in typhoid. Paste of leaves is used for healing of cuts, boils and cutaneous disease. It is widely used in pain and inflammatory conditions. Z. nummularia contains a unique group of alkaloids known as cyclopeptide alkaloids, in continuation of our work carried out on the leaves of Z. nummularia , present study was initiated to explore antiinflammatory and analgesic potential of cyclopeptide alkaloids isolated from the leaves of Z. nummularia (IFZN). Anti-inflammatory activity was tested against rat paw oedema, mouse peritonitis and cotton pellet granuloma. For screening of analgesic activity, acetic acid induced writhing, tail flick and hot plate test were performed. IFZN 30 mg/kg shows the anti-oedematogenic effect against paw oedema induced by carrageenan, dextran, serotonin and histamine; IFZN 20 and 30 mg/kg were found to have highly significant anti-nociceptive effects. Result of pharmacological studies indicated that IFZN is a potent and efficacious analgesic agent. The analgesic activity of IFZN is mediated by the peripheral as well as central pathways.

Pivotal Role of 5-Lipoxygenase Pathway in Lung Injury After Experimental Sepsis

Article

Full-text available

Aug 2013
AM J RESP CELL MOL

Post-sepsis lung injury is a common clinical problem associated with significant morbidity and mortality. Leukotrienes (LTs) are important lipid mediators of infection and inflammation derived from the 5-lipoxygenase (5-LO) metabolism of arachidonate with the potential to contribute to lung damage post sepsis. In order to test the hypothesis that LTs are mediators of lung injury after sepsis, we assessed lung structure, inflammatory mediators, and mechanic changes following cecal ligation and puncture (CLP) surgery in wild type (WT) and 5-LO knockout (5-LO-/-) mice, as well as in WT mice treated with pharmacologic LT synthesis inhibitor (MK886) and LT receptor antagonists (CP105,696 and montelukast). Sixteen h post-surgery, WT animals exhibited severe lung injury (by histological analysis), a substantial mechanical impairment (increase in static lung elastance), an increase in neutrophil infiltration, and high levels of leukotriene B4 (LTB4), cysteinyl-LTs (cys-LTs), prostaglandin E2 (PGE2), interleukin (IL)-1β, IL-6, IL-10, IL-17, KC (CXCL1), and MCP-1 (CCL2) in lung tissue and plasma. 5-LO-/- mice and WT mice treated with a pharmacological 5-LO inhibitor were significantly protected from lung inflammation and injury. Selective antagonists for BLT1 or cys-LT1, the high affinity receptors for LTB4 and cys-LTs, respectively, were insufficient to provide protection when used alone. In conclusion, these results point to an important role for 5-LO products in sepsis-induced lung injury, and suggest that the use of 5-LO inhibitors may be of therapeutic benefit clinically.

Downregulation of Lymphatic Vessel Formation Factors in PGF2α-induced Luteolysis in the Cow

Article

Full-text available

Mar 2013
J REPROD DEVELOP

Prostaglandin F2α (PGF2α) induces luteolysis in cows and causes infiltration of immune cells, which resembles inflammatory immune response. Since the general immune response is mediated by the lymphatic system, we hypothesized that luteolysis is associated with generation of an immune response that involves lymphatic vessels in the bovine corpus luteum (CL). The CL was obtained from Holstein cows at the mid-luteal phase (days 10-12, ovulation = day 0) by ovariectomy at various time points after PGF2α injection. Lymphatic endothelial cell (LyEC) marker, endothelial hyaluronan receptor 1 (LYVE1), levels decreased significantly 12 h after PGF2α injection. Podoplanin, another LyEC marker, decreased from 15 min after PGF2α injection. PGF2α also diminished mRNA expression of lymphangiogenic factors, such as vascular endothelial growth factor (VEGF) C, VEGFD and VEGF receptor 3 (VEGFR3). During PGF2α-induced luteolysis, the levels of mRNA expression of tumor necrosis factor α (TNFα; the major pro-inflammatory cytokine) and chemokine (C-X-C motif) ligand 1 (neutrophil chemokine) were increased. On the other hand, chemokine (C-C motif) ligand 21, which regulates outflow of immune cells from tissues via the lymphatic vessels during an immune response, was decreased. This study demonstrated that the lymphatic network in the CL is disrupted during luteolysis and suggests that during luteolysis, immune cells can induce a local immune response in the CL without using the lymphatic vessels.

Docking, synthesis and pharmacological activity of novel urea-derivatives designed as p38 MAPK inhibitors

Article

May 2012

Endothelin-1 induces neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2 in mice

Article

Full-text available

Feb 2012

Endothelin mediates neutrophil recruitment during innate inflammation. Herein we address whether endothelin-1 (ET-1) is involved in neutrophil recruitment in adaptive inflammation in mice, and its mechanisms. Pharmacological treatments were used to determine the role of endothelin in neutrophil recruitment to the peritoneal cavity of mice challenged with antigen (ovalbumin) or ET-1. Levels of ET-1, tumour necrosis factor α (TNFα), and CXC chemokine ligand 1 (CXCL1) were determined by enzyme-linked immunosorbent assay. Neutrophil migration and flow cytometry analyses were performed 4 h after the intraperitoneal stimulus. ET-1 induced dose-dependent neutrophil recruitment to the peritoneal cavity. Treatment with the non-selective ET(A)/ET(B) receptor antagonist bosentan, and selective ET(A) or ET(B) receptor antagonists BQ-123 or BQ-788, respectively, inhibited ET-1- and ovalbumin-induced neutrophil migration to the peritoneal cavity. In agreement with the above, the antigen challenge significantly increased levels of ET-1 in peritoneal exudates. The ET-1- and ovalbumin-induced neutrophil recruitment were reduced in TNFR1 deficient mice, and by treatments targeting CXCL1 or CXC chemokine receptor 2 (CXCR2); further, treatment with bosentan, BQ-123, or BQ-788 inhibited ET-1- and antigen-induced production of TNFα and CXCL1. Furthermore, ET-1 and ovalbumin challenge induced an increase in the number of cells expressing the Gr1(+) markers in the granulocyte gate, CD11c(+) markers in the monocyte gate, and CD4(+) and CD45(+) (B220) markers in the lymphocyte gate in an ET(A)- and ET(B)-dependent manner, as determined by flow cytometry analysis, suggesting that ET-1 might be involved in the recruitment of neutrophils and other cells in adaptive inflammation. Therefore, the present study demonstrates that ET-1 is an important mediator for neutrophil recruitment in adaptive inflammation via TNFα and CXCL1/CXCR2-dependent mechanism.

Implication of haematophagous arthropod salivary proteins in host-vector interactions

Article

Full-text available

Sep 2011

The saliva of haematophagous arthropods contains an array of anti-haemostatic, anti-inflammatory and immunomodulatory molecules that contribute to the success of the blood meal. The saliva of haematophagous arthropods is also involved in the transmission and the establishment of pathogens in the host and in allergic responses. This survey provides a comprehensive overview of the pharmacological activity and immunogenic properties of the main salivary proteins characterised in various haematophagous arthropod species. The potential biological and epidemiological applications of these immunogenic salivary molecules will be discussed with an emphasis on their use as biomarkers of exposure to haematophagous arthropod bites or vaccine candidates that are liable to improve host protection against vector-borne diseases.

Agglutinin isolated from the red marine alga Hypnea cervicornis J. Agardh reduces inflammatory hypernociception: Involvement of nitric oxide

Article

Oct 2010

Hypnea cervicornis agglutinin (HCA), a lectin isolated from the red marine alga has been previously shown to have an antinociceptive effect. In the present study in rats, mechanisms of action of HCA were addressed regarding mechanical hypernociception induced by carrageenan, ovalbumin (as antigen), and also by prostaglandin E(2) in rats. The lectin administered intravenously inhibited carrageenan- and antigen-induced hypernociception at 1, 3, 5 and 7h. This inhibitory effect was completely prevented when lectin was combined with mucin, demonstrating the role of carbohydrate-binding sites. The inhibition of inflammatory hypernociception by HCA was associated with the prevention of neutrophil recruitment to the plantar tissue of rats but was not associated with the inhibition of the release of pro-hypernociceptive cytokines (TNF-alpha, IL-1 beta and CINC-1). HCA also blocked mechanical hypernociception induced by PGE(2), which was prevented by the administration of nitric oxide synthase inhibitors. These results were corroborated by the increased circulating levels of NO metabolites following HCA treatment. These findings suggest that the anti-hypernociceptive effects of HCA are not associated with the inhibition of pro-inflammatory cytokine production. However, these effects seem to involve the inhibition of neutrophil migration and also the increase in NO production.

Immunity to Lutzomyia intermedia Saliva Modulates the Inflammatory Environment Induced by Leishmania braziliensis

Article

Full-text available

Jun 2010
PLOS NEGLECT TROP D

During blood feeding, sand flies inject Leishmania parasites in the presence of saliva. The types and functions of cells present at the first host-parasite contact are critical to the outcome on infection and sand fly saliva has been shown to play an important role in this setting. Herein, we investigated the in vivo chemotactic effects of Lutzomyia intermedia saliva, the vector of Leishmania braziliensis, combined or not with the parasite. We tested the initial response induced by Lutzomyia intermedia salivary gland sonicate (SGS) in BALB/c mice employing the air pouch model of inflammation. L. intermedia SGS induced a rapid influx of macrophages and neutrophils. In mice that were pre-sensitized with L. intermedia saliva, injection of SGS was associated with increased neutrophil recruitment and a significant up-regulation of CXCL1, CCL2, CCL4 and TNF-alpha expression. Surprisingly, in mice that were pre-exposed to SGS, a combination of SGS and L. braziliensis induced a significant migration of neutrophils and an important modulation in cytokine and chemokine expression as shown by decreased CXCL10 expression and increased IL-10 expression. These results confirm that sand fly saliva modulates the initial host response. More importantly, pre-exposure to L. intermedia saliva significantly modifies the host's response to L. braziliensis, in terms of cellular recruitment and expression of cytokines and chemokines. This particular immune modulation may, in turn, favor parasite multiplication.

Modeling anti-tumor Th1 and Th2 immunity in the rejection of melanoma

Article

May 2010
J THEOR BIOL

Recent experiments indicate that CD4(+) Th2 cells can reject skin tumors in mice, while CD4(+) Th1 cells cannot (Mattes et al., 2003; Zhang et al., 2009). These results are surprising because CD4(+) Th1 cells are typically considered to be capable of tumor rejection. We used mathematical models to investigate this unexpected outcome. We found that neither CD4(+) Th1 nor CD4(+) Th2 cells could eliminate the cancer cells when acting alone, but that tumor elimination could be induced by recruitment of eosinophils by the Th2 cells. These recruited eosinophils had unexpected indirect effects on the decay rate of type 2 cytokines and the rate at which Th2 cells are inactivated through interactions with cancer cells. Strikingly, the presence of eosinophils impacted tumor growth more significantly than the release of tumor-suppressing cytokines such as IFN-gamma and TNF-alpha. Our simulations suggest that novel strategies to enhance eosinophil recruitment into skin tumors may improve cancer immunotherapies.

Mineral trioxide aggregate stimulates macrophages and mast cells to release neutrophil chemotactic factors: role of IL-1beta, MIP-2 and LTB(4)

Article

Mar 2010

In the present study, the role of macrophages and mast cells in mineral trioxide aggregate (MTA)-induced release of neutrophil chemotactic factor was investigated. MTA suspension (50 mg/mL) was plated over inserts on macrophages or mast cells for 90 minutes. Untreated cells served as controls. Cells were washed and cultured for 90 minutes in RPMI without the stimuli. Macrophages and mast cell supernatants were injected intraperitoneally (0.5 mL/cavity), and neutrophil migration was assessed 6 hours later. In some experiments, cells were incubated for 30 minutes with dexamethasone (DEX, 10 muM/well), BWA4C (BW, 100 muM/well) or U75302 (U75, 10 muM/well). The concentration of Leukotriene B(4) (LTB(4)) in the cell-free supernatant from mast cells and macrophage culture was measured by ELISA. Supernatants from MTA-stimulated macrophages and mast cells caused neutrophil migration. The release of neutrophil chemotactic factor by macrophages and mast cells was significantly inhibited by DEX, BW, or U75. Macrophages and mast cells expressed mRNA for interleukin-1 (IL-1)beta and macrophage inflammatory protein-2 (MIP-2) and the pretreatment of macrophages and mast cells with DEX, BW, or U75 significantly altered IL-1beta and MIP-2 mRNA expression. LTB(4) was detected in the MTA-stimulated macrophage supernatant but not mast cells. MTA-induces the release of neutrophil chemotactic factor substances from macrophages and mast cells with participation of IL-1beta, MIP-2, and LTB(4).

Endothelin Receptors and Pain

Article

Feb 2009

Unlabelled: The endogenous endothelin (ET) peptides participate in a remarkable variety of pain-relatedprocesses. Pain that is elevated by inflammation, by skin incision, by cancer, during a Sickle Cell Disease crisis and by treatments that mimic neuropathic and inflammatory pain and are all reduced by local administration of antagonists of endothelin receptors. Many effects of endogenously released endothelin are simulated by acute, local subcutaneous administration of endothelin, which at very high concentrations causes pain and at lower concentrations sensitizes the nocifensive reactions to mechanical, thermal and chemical stimuli. Perspective: In this paper we review the biochemistry, second messenger pathways and hetero-receptor coupling that are activated by ET receptors, the cellular physiological responses to ET receptor activation, and the contribution to pain of such mechanisms occurring in the periphery and the CNS. Our goal is to frame the subject of endothelin and pain for a broad readership, and to present the generally accepted as well as the disputed concepts, including important unanswered questions.

A Review of Candidate Pathways Underlying the Association Between Asthma and Major Depressive Disorder

Article

Full-text available

Feb 2009
PSYCHOSOM MED

To consider the mechanisms that may link asthma and major depressive disorder (MDD). Asthma and MDD co-occur at higher rates than expected, but whether this reflects shared underlying pathophysiological mechanisms is not known. A review of the epidemiological data linking asthma and MDD was conducted and the possible biological mechanisms that could account for the high rate of this comorbidity were reviewed. MDD occurs in almost half of patients with asthma assessed in tertiary care centers. Dysregulation of the hypothalamic pituitary adrenal axis may predispose people to both MDD and asthma, and similar alterations in the immune, autonomic nervous, and other key systems are apparent and may contribute to this increased risk of co-occurrence. High rates of MDD in asthma may result from the stress of chronic illness, the medications used to treat it, or a combination of the two. The high level of co-occurrence may also reflect dysregulation of certain stress-sensitive biological processes that contribute to the pathophysiology of both conditions.

The pharmacological profile of ovalbumin-induced paw oedema in rats

Article

Full-text available

Jun 2002

Rats are commonly used in anaphylaxis models, mainly in intestinal anaphylaxis. Hypersensitivity mechanisms are complex and they are not clearly defined. Ovalbumin (OVA) is commonly used for studies on the hypersensitivity mechanism. However, the potential pro-inflammatory mediators induced by this antigen in the model of paw oedema in immunized rats are still not completely understood. This work examines the pharmacological modulation of several mediators involved in rat hind paw immune oedema induced by OVA. Wistar rats were previously immunized (14-18 days) with OVA (30 microg, intraperitoneally) or sham-sensitized with aluminum hydroxide (control). The paw volumes were measured before the antigenic stimuli and 1, 2, 3 and 4 h after the intraplantar injection of OVA (10 microg/paw). Subcutaneous injection of dexamethasone, diphenhydramine, cyproheptadine, chlorpromazine or methysergide significantly inhibited (p < 0.05) the allergic paw oedema. The dual inhibitor of cyclooxygenase and lipoxygenase (NDGA), the cyclooxygenase inhibitor (indomethacin), the lipoxygenase inhibitor (MK-886), the PAF antagonist (WEB 2086), the mast cell stabilizer (ketotifen), and the anti-histamine (meclizine) did not inhibit the immune oedema. In addition, thalidomide and pentoxifylline (anti-tumour necrosis factor drugs) were ineffective against OVA-induced oedema. The fact that indomethacin, MK-886, NDGA and WEB 2086 are unable to inhibit this allergic oedema indicates that the dexamethasone action seems not to be via phospholipase A2, but possibly due to the synthesis and/or the inhibitory activity of cytokines. The paw oedema inhibition by diphenhydramine, but not by meclizine, may suggest a different mechanism, which is independent of the effect of histamine. These data indicate that allergic oedema is more sensitive to anti-serotonin drugs, mainly anti-5-HT2, suggesting that the principal mediator of this inflammatory response is serotonin.

A Critical Role of Leukotriene B4 in Neutrophil Migration to Infectious Focus in Cecal Ligation and Puncture Sepsis

Article

Feb 2003

Neutrophil migration to an infectious focus is essential for control and resolution of infection. Early studies demonstrated that the failure of such migration is observed in lethal sepsis induced by cecal ligation and puncture (L-CLP), whereas intense neutrophil migration is seen in sublethal CLP (SL-CLP). In this study, we found that inhibition of synthesis of prostaglandins or leukotriene B4 (LTB4) did not modify the failure of neutrophil migration or the survival rate of L-CLP mice. In addition, pretreatment of L-CLP mice with a platelet activating factor (PAF) receptor antagonist (UK74505), despite not interfering with the failure process, significantly increased (33%) the survival rate of the animals. Inhibitors of prostaglandin synthesis (indomethacin and meloxican) and UK74505 did not modify the neutrophil migration observed in SL-CLP. On the other hand, the blockade of LTB4 synthesis (MK886, a 5-lipoxygenase-activating protein inhibitor) or of its receptors (CP-105,696) resulted in reduced neutrophil migration to the peritoneal cavity in SL-CLP mice (62% and 60%, respectively), a consequent increase in the number of bacteria in the inflammatory focus, and a reduced survival rate of the animals (43% and 38%, respectively). Both SL-CLP and L-CLP animals presented significant levels of LTB4 in the peritoneal exudate (3- and 8-fold higher than sham group, respectively) and these were reduced by the pretreatment of mice with LTB4 inhibitors. In conclusion, our results suggest that LTB4, but not prostaglandins or PAF, is an important chemoattractant involved in neutrophil recruitment to infection sites in SL-CLP, a crucial event in confining the invading pathogens to a restricted area. However, in circumstances in which the infection turns to a lethal sepsis, LTB4 is not involved in the observed failure of neutrophil migration to the infectious focus.

PPAR-α and -γ but not -δ agonists inhibit airway inflammation in a murine model of asthma: In vitro evidence for an NF-κB-independent effect

Article

Jun 2003

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that have been proposed to regulate inflammation by antagonising the nuclear factor-κB (NF-κB) signalling pathway. We investigated the role of PPARs using synthetic agonists in murine models of airway inflammation, and addressed the possible effect on NF-κB signalling in vitro using a human epithelial cell line, A549. Sensitised BALB/c mice exposed to an aerosol solution of ovalbumin had an increased number of airway eosinophils, neutrophils and lymphocytes. When given intranasally an hour before the aerosol challenge, a PPAR-α (GW 9578) and PPAR-γ (GI 262570) selective agonist as well as a dual PPAR-α/γ (GW 2331) agonist selectively inhibited allergen-induced bronchoalveolar lavage eosinophil and lymphocyte but not neutrophil influx. In contrast, a PPAR-δ agonist (GW 501516) was inactive. When given intranasally an hour before challenge, PPAR-α and PPAR-γ selective agonists as well as a dual PPAR-α/γ agonist did not inhibit lipopolysaccharide-induced bronchoalveolar lavage neutrophil influx or tumour necrosis factor-α (TNF-α) and KC production. In A549 cells, selective agonists for PPAR-α, -γ and -δ did not inhibit intracellular adhesion molecule-1 expression following stimulation with proinflammatory cytokines. In addition, IL-8 release and the activation of an NF-κB-responsive reporter gene construct were inhibited only at micromolar concentrations, suggesting that these effects were not PPAR-mediated. Our in vivo data show that agonists of PPAR-α and -γ, but not -δ, inhibit allergen-induced bronchoalveolar lavage eosinophil and lymphocyte influx. In vitro data suggest that this effect might not be mediated by antagonism of the NF-κB pathway. British Journal of Pharmacology (2003) 139, 163–171. doi:10.1038/sj.bjp.0705232

Tripterine enhances regulatory effect of TNF- α on Caspase-3 and induces apoptosis of lung cancer cell

Article

Aug 2023

Lung cancer is a common malignancy of the respiratory system with a high morbidity and mortality. We intended to identify the apoptosis-promoting role of tripterine in lung cancer. Lung cancer SPC-A-1 cells were exposed to low, moderate, and high dosage of tripterine (2, 5 and 10 μ mol/L) with the model group not being intervened. After treatment, the role of tripterine in SPC-A-1 cell apoptosis was observed. In addition, lung cancer cells were transfected with TNF- α mimic (pc-TNF- α group) and TNF- α inhibitor (si-TNF- α group). Tripterine +pc-TNF- α group was set up to determine the interaction between tripterine and TNF- α . The cell survival rate, TNF- α and Caspase-3 expression levels then were detected by MTT and flow cytometry. Tripteryglide treatment dose-dependently decreased lung cancer cell viability and induced cell apoptosis, resulting in an increase of TNF- α expression. However, when TNF- α expression was inhibited upon transfection, SPC-A-1 cell apoptosis was suppressed. TNF- α mimics activated apoptosis and up-regulated Caspase-3 expression. Combination of tripteryglide and TNF- α mimics more significantly elevated apoptotic rate of lung cancer cells when elevating the content of Caspase-3. SPC-A-1 cells are highly sensitive to TNF- α and TNF- α significantly increases the activity and expression of Caspase-3. Tripteryglide can up-regulate TNF- α expression to facilitate lung cancer cell apoptosis and increase Caspase-3 expression.

Glucosensing in rainbow trout

Book

Jan 2013

Rainbow trout has been considered for many years as a model of glucose intolerant species. The different hypothesis raised by many researchers to explain such phenomenon has been tested thoroughly in recent years without arriving at a clear explanation. One of the processes that could be involved in its inability to deal with increased levels of glucose could be the absence of glucosensing mechanisms similar to those found in mammals. However, several recent studies in rainbow trout have demonstrated the existence of glucosensor systems in hypothalamus, hindbrain and Brockmann bodies. The fact that this system has been characterized in a species whose natural diet contains less than 1% carbohydrate intake makes rainbow trout an attractive model for glucosensing studies, as so far it is the only vertebrate carnivorous species in which this system has been explored.The glucosensing system has been shown to be activated when glucose levels increase at the same time that food intake decreases while conversely, when glucose levels decrease, glucosensors are inactivated and food intake increases. The mechanisms through which these glucosensor systems operate are similar to those described in mammalian brain regions, though quite different in pancreatic cells. Information has also been reported regarding the molecular characterization of these systems, their specific location in the brain and their endocrine regulation.The present review will provide a general overview of the research carried out in this area in recent years and provide perspectives for future research in this field.

Anti-inflammatory effect of dikaempferol rhamnopyranoside, a diflavonoid from Eugenia jambolana Lam. Leaves

Article

Dec 2016

Traditionally, the Indian Blackberry or locally called Jamun, Eugenia jambolana Lam. (Syn.: Syzygium cumini), is well known for its pharmacological potential, particularly anti-inflammatory. Here, we studied kaempferol-7-O-α-Lrhamnopyranoside]-4'-O-4'- [kaempferol-7-O-α-L-rhamnopyranoside (EJ-01) isolated from the E. jambolana leaves for possible anti-inflammatory activity. EJ-01 (3, 10 and 30 mg/kg, p.o.) was assessed for anti-inflammatory activity using carrageenan-induced paw edema model in mice by determining edema volume, myeloperoxidase (MPO), nitrite plus nitrate (NOx) and cytokine levels in paw edema tissue. EJ-01 significantly attenuated the edema, MPO levels, tumor necrosis factor-alpha (TNF-α) and interleukin-1beta (IL-1β) levels in the edema of paw at the 5th hour after carrageenan injection at all doses. EJ-01 (30 mg/kg) decreased the nitric oxide (NO) levels of the edema of paw at the 5th hour after carrageenan injection. The anti-inflammatory mechanisms of EJ-01 might be related to the decrease in the level of edema paw by reduced activities of NO and MPO. It probably exerts anti-inflammatory effects through the suppression of TNF-α and IL-1β. Therefore, we conclude that EJ-01 could be positively exploited for itspotential benefits against inflammatory diseases and support the pharmacological basis of E. jambolana as traditional herbal medicine for the treatment of inflammatory diseases.

CsTNF1, a teleost tumor necrosis factor that promotes antibacterial and antiviral immune defense in a manner that depends on the conserved receptor binding site

Article

Oct 2015

Tumor necrosis factor (TNF) is one of the most important cytokines involved in inflammation, apoptosis, cell proliferation, and stimulation of the immune system. The TNF gene has been cloned in teleost fish; however, the in vivo function of fish TNF is essentially unknown. In this study, we report the identification of a TNF homologue, CsTNF1, from tongue sole (Cynoglossus semilaevis) and analysis of its expression and biological effect. CsTNF1 is composed of 242 amino acid residues and possesses a TNF domain and conserved receptor binding sites. Expression of CsTNF1 was detected in a wide range of tissues and up-regulated in a time-dependent manner by experimental challenge with bacterial and viral pathogens. Bacterial infection of peripheral blood leukocytes (PBL) caused extracellular secretion of CsTNF1. Purified recombinant CsTNF1 (rCsTNF1) was able to bind to PBL and stimulate the respiratory burst activity of PBL. In contrast, rCsTNF1M1 and rCsTNF1M2, the mutant CsTNF1 bearing substitutions at the receptor binding site, failed to activate PBL. Fish administered with rCsTNF1, but not with rCsTNF1M1 and rCsTNF1M2, exhibited enhanced expression of IL-1, IL-6, IL-8, IL-27, TLR9 and G3BP in a time-dependent manner and augmented resistance against bacterial and viral infection. These results provide the first evidence that the receptor binding sites are essential to a fish TNF, and that CsTNF1 is involved in the innate immune defense of fish against microbial pathogens.

Anti-inflammatory effects of retinoids and carotenoid derivatives on caspase-3-dependent apoptosis and efferocytosis of bovine neutrophils

Article

Full-text available

Dec 2014
AM J VET RES

Objective: To evaluate immunomodulatory properties of all-trans retinoic acid and a fully oxidized β-carotene dietary product in calves with Mannheimia haemolytica-induced pneumonia. Animals: Twenty-five 6- to 10-week-old male Holstein calves for experimental inoculations and three 8- to 30-week-old Angus heifers for blood donations. Procedures: In vitro, neutrophils and monocyte-derived macrophages isolated from blood of healthy Angus heifers were treated with all-trans retinoic acid (1 μM) or fully oxidized β-carotene (8.3 μg/mL) for various times and assessed for markers of cellular death, antimicrobial function, and production of proinflammatory leukotriene B4. Following 28 days of dietary supplementation with fully oxidized β-carotene, Holstein calves were experimentally inoculated with M haemolytica. Bronchoalveolar lavage fluid was collected at 3 and 24 hours after challenge inoculation and analyzed for markers of apoptosis. Results: In vitro, all-trans retinoic acid and fully oxidized β-carotene induced cell-selective, caspase-3-dependent apoptosis in neutrophils, which subsequently enhanced efferocytosis in macrophages. Conversely, neither treatment altered phorbol 12-myristate 13-acetate-induced oxidative burst, phagocytosis of nonopsonized zymosan (complement or antibody independent), or M haemolytica-induced leukotriene B4 production in bovine neutrophils. In vivo, fully oxidized β-carotene enhanced leukocyte apoptosis in bronchoalveolar lavage fluid as well as subsequent efferocytosis by macrophages without altering numbers of circulating leukocytes. Conclusions and clinical relevance: Neutrophil apoptosis and subsequent efferocytosis by macrophages are key mechanisms in the resolution of inflammation. Findings for the present study indicated that all-trans retinoic acid and fully oxidized β-carotene could be novel nutraceutical strategies that may confer anti-inflammatory benefits for cattle with respiratory tract disease.

Targeting endothelin ETA and ETB receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Article

Full-text available

Mar 2009

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ETA and ETB receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ETB receptor antagonism, but rather by endothelin ETA receptor antagonism. Furthermore, endothelin ETA, but not ETB, receptor antagonism inhibited antigen-induced PGE2 production, whereas either selective or combined blockade of endothelin ETA and/or ETB receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.

Leukotriene B4 Mediates Neutrophil Migration Induced by Heme

Article

Full-text available

Jun 2011
J IMMUNOL

High concentrations of free heme found during hemolytic events or cell damage leads to inflammation, characterized by neutrophil recruitment and production of reactive oxygen species, through mechanisms not yet elucidated. In this study, we provide evidence that heme-induced neutrophilic inflammation depends on endogenous activity of the macrophage-derived lipid mediator leukotriene B(4) (LTB(4)). In vivo, heme-induced neutrophil recruitment into the peritoneal cavity of mice was attenuated by pretreatment with 5-lipoxygenase (5-LO) inhibitors and leukotriene B(4) receptor 1 (BLT1) receptor antagonists as well as in 5-LO knockout (5-LO(-/-)) mice. Heme administration in vivo increased peritoneal levels of LTB(4) prior to and during neutrophil recruitment. Evidence that LTB(4) was synthesized by resident macrophages, but not mast cells, included the following: 1) immuno-localization of heme-induced LTB(4) was compartmentalized exclusively within lipid bodies of resident macrophages; 2) an increase in the macrophage population enhanced heme-induced neutrophil migration; 3) depletion of resident mast cells did not affect heme-induced LTB(4) production or neutrophil influx; 4) increased levels of LTB(4) were found in heme-stimulated peritoneal cavities displaying increased macrophage numbers; and 5) in vitro, heme was able to activate directly macrophages to synthesize LTB(4). Our findings uncover a crucial role of LTB(4) in neutrophil migration induced by heme and suggest that beneficial therapeutic outcomes could be achieved by targeting the 5-LO pathway in the treatment of inflammation associated with hemolytic processes.

Anti-Inflammatory Benefits of Antibiotic-Induced Neutrophil Apoptosis: Tulathromycin Induces Caspase-3-Dependent Neutrophil Programmed Cell Death and Inhibits NF- B Signaling and CXCL8 Transcription

Article

Full-text available

Dec 2011
ANTIMICROB AGENTS CH

Clearance of apoptotic neutrophils is a central feature of the resolution of inflammation. Findings indicate that immuno-modulation and induction of neutrophil apoptosis by macrolide antibiotics generate anti-inflammatory benefits via mechanisms that remain obscure. Tulathromycin (TUL), a new antimicrobial agent for bovine respiratory disease, offers superior clinical efficacy for reasons not fully understood. The aim of this study was to identify the immuno-modulating effects of tulathromycin and, in this process, to establish tulathromycin as a new model for characterizing the novel anti-inflammatory properties of antibiotics. Bronchoalveolar lavage specimens were collected from Holstein calves 3 and 24 h postinfection, challenged intratracheally with live Mannheimia haemolytica (2 × 10(7) CFU), and treated with vehicle or tulathromycin (2.5 mg/kg body weight). Terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) staining and enzyme-linked immunosorbent assay (ELISA) revealed that tulathromycin treatment significantly increased leukocyte apoptosis and reduced levels of proinflammatory leukotriene B(4) in M. haemolytica-challenged calves. In vitro, tulathromycin concentration dependently induced apoptosis in freshly isolated bovine neutrophils from healthy steers in a capase-3-dependent manner but failed to induce apoptosis in bovine fibroblasts, epithelial cells, and endothelial cells, as well as freshly isolated bovine blood monocytes and monocyte-derived macrophages. The proapoptotic effects of TUL were also, in part, drug specific; equimolar concentrations of penicillin G, oxytetracycline, and ceftiofur failed to cause apoptosis in bovine neutrophils. In addition, tulathromycin significantly reduced levels of phosphorylated IκBα, nuclear translocation of NF-κB p65, and mRNA levels of proinflammatory interleukin-8 in lipopolysaccharide (LPS)-stimulated bovine neutrophils. The findings illustrate novel mechanisms through which tulathromycin confers anti-inflammatory benefits.

Leukotriene B⁴ and platelet-activating factor [microforme] : assessment of biological significance in neutrophil trafficking /

Article

Full-text available

Hanan Elimam

"Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de Maître ès sciences (M.Sc.) en sciences pharmaceutiques option pharmacologie" Thèse (M. Sc.)--Université de Montréal, 2004. Microfilm du manuscrit.

Leukotriene B⁴ and platelet-activating factor : assessment of biological significance in neutrophil trafficking /

Article

Full-text available

Hanan Elimam

"Mémoire présenté à la Faculté des études supérieures en vue de l'obtention du grade de Maître ès sciences (M. Sc.) en sciences pharmaceutiques option pharmacologie." Thèse (M. Sc.)--Université de Montréal, 2004.

Role of sensory innervation in the rat pulmonary neutrophil recruitment induced by staphylococcal enterotoxins type A and B

Article

May 2009

Rat airways exposure to Staphylococcal enterotoxin A (SEA) and B (SEB) induces marked neutrophil influx. Since sensory neuropeptides play important roles in cell infiltration, in this study we have investigated its contribution in triggering SEA- and SEB-induced pulmonary neutrophil infiltration. Male Wistar rats were exposed intratracheally with SEA (3 ng/trachea) or SEB (250 ng/trachea). Animals received different in vivo pretreatments, after which the neutrophil counts and levels of substance P and IL-1 in bronchoalveolar lavage fluid were evaluated. Alveolar macrophages and peritoneal mast cells were incubated with SEA and SEB to determine the IL-1 and TNF-alpha levels. Capsaicin pretreatment significantly reduced SEA- and SEB-induced neutrophil influx in bronchoalveolar lavage fluid, but this treatment was more effective to reduce SEA responses. Treatments with SR140333 (tachykinin NK(1) receptor antagonist) and SR48968 (tachykinin NK(2) receptor antagonist) decreased SEA-induced neutrophil influx, whereas SEB-induced responses were inhibited by SR140333 only. Cyproheptadine (histamine/5-hydroxytriptamine receptor antagonist) and MD 7222 (5-HT(3) receptor antagonist) reduced SEA- and SEB-induced neutrophil influx. The substance P and IL-1 levels in bronchoalveolar lavage fluid of SEA-exposed rats were significantly higher than SEB. In addition, SEA (but not SEB) significantly released mast cell TNF-alpha. Increased production of TNF-alpha and IL-1 in alveolar macrophages was observed in response to SEA and SEB. In conclusion, sensory neuropeptides contribute significantly to SEA- and SEB-induced pulmonary neutrophil recruitment, but SEA requires in a higher extent the airways sensory innervation, and participation of mast cells and alveolar macrophage products.

Role of anti-Nak(a) antibody, monocytes and platelets in the development of transfusion-related acute lung injury

Article

Dec 2008
VOX SANG

Transfusion-related acute lung injury (TRALI) is one of the most serious side-effects of transfusion. We report here the first two cases of TRALI caused by anti-Nak(a) (anti-CD36) antibody from a single blood donor. The aim of this study was to clarify the role of the anti-Nak(a) antibody in TRALI development. Human lung microvascular endothelial cells were co-cultured with Nak(a)-positive monocytes and Nak(a)-positive platelets together with serum prepared from blood products of a TRALI-caused anti-Nak(a) antibody-carrying donor. Expressions of leukotriene B(4) (LTB(4)) and tumour necrosis factor alpha (TNF-alpha) in the co-culture supernatants were determined. The expressions of LTB(4) and TNF-alpha were found to be markedly increased, particularly in the presence of both Nak(a)-positive monocytes and platelets. The expressions of these mediators were almost completed within 4 h after the initiation of co-culture. Monocyte contribution seemed to be stronger than that of platelets. In the absence of human lung microvascular endothelial, no significant LTB(4) or TNF-alpha release was observed. Anti-Nak(a) antibody may be strongly implicated in lung microvascular endothelial dysfunctions that lead to TRALI in a monocyte- and platelet-dependent manner.

Tumor Necrosis Factor-?? and Interleukin-1?? Mediate the Production of Nitric Oxide Involved in the Pathogenesis of Ifosfamide Induced Hemorrhagic Cystitis in Mice

Article

Jun 2002

We investigated the participation of nitric oxide in ifosfamide induced hemorrhagic cystitis in mice, and the involvement of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta in the induction of nitric oxide production in this model. Hemorrhagic cystitis was induced in mice by 100 to 400 mg./kg. ifosfamide and evaluated 6, 12, 24 or 48 hours thereafter by certain parameters, including vesical edema measurements, microscopic analysis and immunohistochemical testing for inducible nitric oxide synthase. Ifosfamide injected mice were pretreated with 10 to 40 mg./kg. of the nitric oxide synthesis inhibitor L-NG-nitroarginine methyl ester, 80 mg./kg. of mesna, a chemical antagonist of acrolein and the urotoxic metabolite of ifosfamide, 50 microl. antiserum against TNF-alpha and IL-1beta per mouse, 45 mg./kg. of the selective TNF-alpha synthesis inhibitor thalidomide or 200 mg./kg. of the TNF-alpha and IL-1beta synthesis inhibitor pentoxifylline. Ifosfamide induced vesical edema, which peaked 12 hours after ifosfamide injection. Microscopic analysis revealed vascular congestion, edema, hemorrhage, fibrin deposition, neutrophil infiltration and epithelial denudation. Inducible nitric oxide synthase immunolocalization demonstrated intense reactivity to inducible nitric oxide synthase in the cytoplasm of bladder epithelial cells, which showed diffuse necrosis. Pretreatment with mesna reduced the increases in vesical edema, while treatment with L-NG-nitroarginine methyl ester, antiserum to TNF-alpha or IL-1beta, thalidomide or pentoxifylline inhibited vesical edema and microscopic alterations. Antiserum treatments also inhibited the expression of inducible nitric oxide synthase in the urothelium. Nitric oxide produced by inducible nitric oxide synthase is involved in urothelial damage and in the inflammatory events leading to hemorrhagic cystitis after ifosfamide administration in mice. The induction of inducible nitric oxide synthase in the urothelium appears to depend on the synergistic effect of IL-1beta and TNF-alpha.

Involvement of NO in the failure of neutrophil migration in sepsis induced by Staphylococcus aureus

Article

Aug 2002

Sepsis induced by S. aureus was used to investigate whether neutrophil migration failure to infectious focus correlates with lethality in Gram-positive bacteria-induced sepsis in mice. By contrast with the sub-lethal (SL-group), the lethal (L-group) intraperitoneal inoculum of S. aureus caused failure of neutrophil migration (92% reduction), high CFU in the exudate, bacteremia and impairment of in vitro neutrophil chemotactic activity. Pre-treatments of L-group with adequate doses of aminoguanidine prevented the neutrophil migration failure and improved the survival of the animals (pre-treated: 43%; untreated: 0% survival). Thus, the impairment of neutrophil migration in the L-group appears to be mediated by nitric oxide (NO). The injection of S. aureus SL-inoculum in iNOS deficient (−/−) or aminoguanidine-treated wild-type mice (pre- and post-treatment), which did not present neutrophil migration failure, paradoxically caused severe peritonitis and high mortality. This fact is explainable by the lack of NO dependent microbicidal activity in migrated neutrophils. In conclusion, although the NO microbicidal mechanism is active in neutrophils, the failure of their migration to the infectious focus may be responsible for the severity and outcome of sepsis. British Journal of Pharmacology (2002) 136, 645–658; doi:10.1038/sj.bjp.0704734

Functional characterisation of the recombinant tumor necrosis factor in rainbow trout (Oncorhynchus mykiss)

Article

Oct 2003
DEV COMP IMMUNOL

Tumor necrosis factor (TNF) is a key mediator in regulating the inflammatory response. Previously two TNF genes have been cloned and sequenced from rainbow trout, Oncorhynchus mykiss. In this study, the mature peptides of the two TNF molecules were produced in bacteria, purified under native conditions and their bioactivities evaluated in vitro. Both trout rTNF1 and rTNF2 induced gene expression of a number of proinflammatory factors including IL1beta, TNF1, TNF2, IL8 and COX2 in freshly isolated head kidney leucocytes and the macrophage cell line RTS11. The stimulatory doses of both rTNFs were >or=10 ng/ml. Moreover, leucocyte migration and phagocytic activity were enhanced in vitro by the rTNFs in a dose dependent manner. Western blot analysis revealed the presence of multiple forms of rTNF structures including monomeric, dimeric and trimeric forms, suggesting that formation of a homotrimeric structure may be essential for the TNF bioactivities.

Bronchoalveolar lavage of allergic asthmatic patients following allergen provocation

Article

Full-text available

May 1986

In these studies, we describe the use of bronchoalveolar lavage (BAL) to study local changes following aerosol bronchoprovocation (BPC) and environmental exposure to antigen in mildly symptomatic asthmatic patients. The BAL was performed in 12 atopic subjects "out of season," and in five normal subjects at baseline, less than or equal to 4, or 24 hours following BPC. Five asthmatic patients were also lavaged during seasonal exposure to allergen. The BAL cells were examined with light and transmission electron microscopy. Bronchoprovocation, by itself, resulted in an average maximal decrease in FEV1 of 13 percent just prior to BAL. There was no significant decrease in FEV1 as a result of BAL. Within four hours after BPC, the number of neutrophils was significantly greater in BAL compared to baseline (1.5 +/- 0.6 X 10(5) vs 3.4 +/- 1.7 X 10(5) cells; p less than 0.01), and the number of eosinophils was significantly greater within four hours and at 24 hours when compared to baseline values (0.4 +/- 0.3 X 10(5) vs 1.9 +/- 0.7 X 10(5) vs 1.2 +/- 0.4 X 10(5) cells; p less than 0.02). Transmission electron micrographs of BAL from lungs of asthmatic patients revealed degranulation of mast cells and loss of core material from eosinophil granules following challenge with aerosolized allergen or with spontaneous environmental exposure. These studies show that in carefully selected, mildly symptomatic asthmatic subjects, BPC and BAL may be useful to evaluate pathogenetic mechanisms in allergic bronchial asthma.

Interleukin-10 inhibits antigen-induced cellular recruitment into the airways of sensitized mice

Article

Full-text available

Jul 1995

This report examines the effect of recombinant murine (rm) IL-10 on antigen-induced cellular recruitment into the airways of sensitized Balb/c mice. The intranasal instillation of 10 micrograms ovalbumin induced an early (6-24 h) increase in the number of neutrophils, and a late rise (24-96 h) in that of eosinophils in the bronchoalveolar lavage (BAL) fluid and bronchial tissue. A single intranasal instillation of 0.01-0.1 microgram of rmIL-10, administered concurrently with ovalbumin, but not 1 or 3 h thereafter, dose-dependently inhibited both airway neutrophilia and eosinophilia. This phenomenon was suppressed by treating the sensitized mice with 1 mg/mouse of a neutralizing anti-IL-10 mAb, which increased significantly ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These results suggest that antigen stimulation may trigger the in vivo generation of IL-10, which, in turn, participates in the leukocyte infiltration into the airways. rmIL-10 also reduced TNF-alpha release in the BAL fluid observed 1 and 3 h after antigen challenge. Furthermore, the intranasal instillation of an anti-TNF-alpha antiserum to sensitized mice markedly reduced ovalbumin-induced neutrophil and eosinophil accumulation in the BAL fluid. These findings indicate that leukocyte infiltration into the airways of antigen-challenged mice is regulated by IL-10. Furthermore, inhibition of TNF-alpha production by rmIL-10 suggests that allergic airway inflammation and TNF-alpha formation are parallel events in this model.

Treatment of Paracoccidioides brasiliensis -Infected Mice with a Nitric Oxide Inhibitor Prevents the Failure of Cell-Mediated Immune Response

Article

Full-text available

Oct 1998

The activation of the nitric oxide (NO) production system and its involvement in the control of the lung fungal burden and in immunosuppression mechanisms were studied during the course of Paracoccidioides brasiliensis-infected mice. Mice that had been infected with the fungus were treated daily with a specific inhibitor of NO synthesis, N omega-nitro-L-arginine, or with buffered saline (control); NO production was assessed on the basis of spontaneous NO2- production by bronchoalveolar and peritoneal macrophages (Mphi) and of serum NO3- levels. The infection coursed with an elevation of NO3- levels. The Mphi produced NO2- and released TNF-alpha only after stimulation with LPS. In addition, the immunoproliferative responses of spleen cells that had been stimulated with the fungus Ag or with Con A were depressed. An examination of the lungs of infected animals showed a progressive increase in the size of the lesions. Treatment of the animals, which resulted in an inhibition of NO2- production by Mphi and a reduction of serum NO3- levels, caused the spontaneous release of TNF-alpha from infected animals and prevented the failure of the lymphoproliferative capacity of spleen cells. Furthermore, the treatment resulted in less pulmonary damage despite the fact that the lung fungal burden increased. It was also demonstrated that the NO donors S-nitroso-acetyl penicillamine and 3-morpholino-sydnonimine-hydrochloride were able to inhibit the growth of P. brasiliensis in vitro. These results suggest that although NO is important for the killing of the fungi, the activation of NO production in P. brasiliensis infection contributes to the occurrence of the immunosuppression observed during the course of the infection.

Role of Resident Peritoneal Macrophages and Mast Cells in Chemokine Production and Neutrophil Migration in Acute Inflammation: Evidence for an Inhibitory Loop Involving Endogenous IL-101

Article

Full-text available

Mar 1999

The roles played by resident macrophages (Mphi) and mast cells (MCs) in polymorphonuclear leukocyte (PMN) accumulation and chemokine production within the mouse peritoneal cavity in response to administration of zymosan (0.2 and 1 mg), LPS (1 mg/kg), and thioglycolate (0.5 ml of a 3% suspension) were investigated. A marked reduction (>95%) in intact MC numbers was obtained by pretreatment with the MC activator compound 48/80, whereas resident Mphi were greatly diminished (>85%) by a 3-day treatment with liposomes encapsulating the cytotoxic drug dichloromethylene-bisphosphonate. No modulation of thioglycolate-induced inflammation was seen with either pretreatment. Removal of either MCs or Mphi attenuated LPS-induced PMN extravasation without affecting the levels of the chemokines murine monocyte chemoattractant protein-1 and KC measured in the lavage fluids. In contrast, MC depletion inhibited PMN accumulation and murine monocyte chemoattractant protein-1 and KC production in the zymosan peritonitis model. Removal of Mphi augmented the accumulation of PMN elicited by the latter stimulus. This was due to an inhibitory action of Mphi-derived IL-10 because there was 1) a time-dependent release of IL-10 in the zymosan exudates; 2) a reduction in IL-10 levels following Mphi, but not MC, depletion; and 3) an increased PMN influx and chemokine production in IL-10 knockout mice. In conclusion, we propose a stimulus-dependent role of resident MCs in chemokine production and the existence of a regulatory loop between endogenous IL-10 and the chemokine-mediated cellular component of acute inflammation.

Rapid Up-Regulation of CXC Chemokines in the Airways after Ag-Specific CD4+ T Cell Activation

Article

Full-text available

Jan 2001

Ag-specific activation of CD4(+) T cells is known to be causative for the cytokine production associated with lung allergy. Chemokine-induced leukocyte recruitment potentially represents a critical early event in Ag-induced lung inflammation. Whether Ag-specific, lung CD4(+) T cell activation is important in lung chemokine production is currently not clear. Using alphabeta-TCR transgenic BALB/c DO11.10 mice, we investigated the ability of Ag-specific CD4(+) T cell activation to induce lung chemokine production and leukocyte recruitment. Within 1 h of exposure of DO11. 10 mice to OVA aerosol, lung mRNA and protein for the neutrophil chemokines KC and macrophage inflammatory protein (MIP)-2 were greatly increased. Accordingly, neutrophils in the airways increased by >50-fold, and KC and MIP-2 proved to be functional because their neutralization significantly reduced airway neutrophilia. CD4(+) T cell activation was critical because CD4(+) but not CD8(+) T cell depletion reduced KC production, which correlated well with the previously observed inhibition of neutrophil influx after CD4(+) T cell depletion. In vitro studies confirmed that OVA-induced KC and MIP-2 production was conditional upon the interaction of CD4(+) T cells with APCs. A likely secondary mediator was TNF-alpha, and a probable source of these chemokines in the lung was alveolar macrophages. Thus, Ag-specific CD4(+) T cell activation in the lung leads to rapid up-regulation of neutrophil chemokines and the recruitment of neutrophils to the site of Ag exposure. This may be a key early event in the pathogenesis of Ag-induced lung inflammation.

Cytokines in inflammatory bowel disease

Article

Full-text available

Feb 1997

Over the past decade, much has been learned regarding the role of various cytokines in the pathogenesis of inflammatory bowel disease. Several cytokine 'knockout' models in mice have been shown to develop colitis, while alterations in the production of various cytokines has been documented in human Crohn's disease and ulcerative colitis. In recent years, attempts have been made to treat these diseases through modulation of cytokine production or action. This review focuses on the cytokines that have been implicated in the pathogenesis of inflammatory bowel disease. The evidence for and against a role for particular cytokines in intestinal inflammation is reviewed, as is the experimental and clinical data suggesting that cytokines are rational targets for the development of new therapies.

Critical protective role of mast cells in a model of acute septic peritonitis

Article

Jan 1997
NATURE

Rheumatoid arthritis

Article

Jun 1984

Neutrophils constitute over 90% of cells found in the synovial fluid of rheumatoid arthritis (RA) patients. Since such fluids also contain immune complexes (IgG-IgG and IgG-IgM rheumatoid factors) and complement split products (C5, C5A, DES, ARG, C3B, etc.), all of the reactants are present for a local Arthus lesion. Moreover, neutrophils from RA patients endocytose these immune complexes and complement components in vivo and in vitro. In consequence, it has been suggested that lysosomal enzymes and other mediators of inflammation released by neutrophils after uptake of immune complexes (in the bulk phase or on the surface) account, at least in part, for rheumatoid inflammation. Secretion of lysosomal hydrolases, especially neutral proteases, which provoke tissue injury and generation of reactive oxygen species (e.g. O2) is part of a stimulus-secretion response to a variety of secretagogues, including immune complexes and complement components. However, the pathways of secretion and O2 generation are stimulus-specific and can be dissected to establish cause and effect relationships by (a) kinetic analysis, (b) varying the stimulus, (c) use of impermeant reagents to block discrete responses. Neutrophils also generate products of 11-cyclo-oxygenase (e.g., PGE2, TXA2) and of the 5- and 15-lipoxygenase (mono-, di-and trihetes, LTB4 and their isomers). However, the cyclo-oxygenase products (except TXA2) do not cause inflammation acting alone; indeed, they inhibit the function of neutrophils, platelets, macrophages and mast cells. The most potent proinflammatory agent yet identified as a product of arachidonate is LTB4. LTB4 is a potent Ca ionophore, a strong chemo-attractant, induces local inflammation, and activates neutrophils.

Dual regulation of anti-bacterial resistance and inflammatory neutrophil and macrophage accumulation by L3T4+ and Lyt 2+ Listeria-immune T cells

Article

Feb 1987

Adoptive transfer of anti-Listeria resistance by Listeria-immune spleen T cells was markedly reduced by pretreatment of the cells with monoclonal anti-Lyt 2.2 and complement (Lyt 2+C); pretreatment of cells with monoclonal anti-L3T4 and complement (L3T4+C) had a lesser effect on their ability to transfer resistance. Lyt 2+C-treated and L3T4+C-treated Listeria-immune T cells were undiminished in their immediate ability to transfer enhanced accumulation of inflammatory peritoneal neutrophils and macrophages in response to Listeria antigens. When L3T4+C- and Lyt 2+C-treated Listeria-immune spleen cells were cultured in vitro before transfer, however, it became apparent that the L3T4+ subset was particularly important for mediating in vivo accumulation of inflammatory phagocytes. Listeria-immune spleen T cells produced soluble factors during in vitro culture that, when injected i.p., were able to recruit inflammatory neutrophils and macrophages to the peritoneal cavities of recipient mice. Pretreatment of Listeria -immune spleen cells with L3T4+C before culture markedly diminished their ability to produce soluble factors that were capable of attracting neutrophils and macrophages in vivo. The results of this study indicate substantial roles for both Lyt 2+ and L3T4+ T-cell subsets in the dual regulation of inflammation and anti-bacterial resistance; Lyt 2+ T cells appear to be the principal mediator of anti-bacterial resistance, whereas L3T4+ T cells augment the recruitment of inflammatory phagocytes in vivo.

Effects of Drugs on the Acute Inflammation Following Intraperitoneal Injection of Antigen into Actively Sensitised Rats

Article

Feb 1979

When actively sensitised rats were injected intraperitoneally with antigen, the local reaction that ensued can be divided into two phases: an immediate reaction characterised by histamine and SRS-A release with an associated extravasation of plasma proteins, and a later reaction involving infiltration of neutrophilic polymorphonuclear leucocytes. When the immediate reaction was modified by BRL 10833 (which inhibits histamine release from rat mast cells and reduces extravasation of plasma proteins), there was no reduction in neutrophil infiltration. FPL 55712, an SRS-A antagonist, also failed to inhibit neutrophil infiltration. The beta-adrenoreceptor stimulants isoprenaline and salbutamol reduced neutrophil infiltration. Isoprenaline inhibited the extravasation of plasma proteins when given before antigen, but even when administered to rats after antigen, when extravasation was complete, it still inhibited neutrophil infiltration. Propranolol reversed isoprenaline-induced inhibition of neutrophil infiltration.

CD4+ T cells are required for Ag-specific recruitment of neutrophils by BCG-immune spleen cells

Article

Apr 1992

R Appelberg

Mycobacterium bovis bacillus Calmette-Guérin (BCG)-immune spleen cells co-inoculated into the peritoneal cavity of normal mice with BCG sonicate protein as antigen could induce an antigen-specific recruitment of neutrophils, dependent on the antigen dose and cell number. This response was significantly reduced by anti-T lymphocyte and anti-CD4 treatment of the immune spleen cells prior to the inoculation. Removal of adherent or phagocytic cells or lysis of B cells, had no significant effect. Killing of dividing cells in the splenic population induced a slight reduction in the ability of spleen cells to recruit neutrophils. M. avium sonicate protein was also able to induce BCG-immune spleen cells to mobilize neutrophils but bovine serum albumin, Listeria monocytogenes cytosolic protein and 65,000 MW heat shock protein were not. These results show that CD4+ T cells are able to induce neutrophil recruitment in an antigen-specific way during a mycobacterial infection.

Neutrophil Recruitment by Tumor Necrosis Factor from Mast Cells in Immune Complex Peritonitis

Article

Jan 1993

During generalized immune complex-induced inflammation of the peritoneal cavity, two peaks of tumor necrosis factor (TNF) were observed in the peritoneal exudate of normal mice. In mast cell-deficient mice, the first peak was undetected, and the second peak of TNF and neutrophil influx were significantly reduced. Antibody to TNF significantly inhibited neutrophil infiltration in normal but not in mast cell-deficient mice. Mast cell repletion of the latter normalized TNF, neutrophil mobilization, and the effect of the antibody to TNF. Thus, in vivo, mast cells produce the TNF that augments neutrophil emigration.

Phenotypic characterization of murine tumor-infiltrating T lymphocytes

Article

Apr 1991

Tumor infiltrating lymphocytes (TIL) can be isolated from solid tumors and selectively expanded in long term culture with IL-2 and autologous irradiated tumor. Such long term cultured cells express anti-tumor activity in vitro, mediate the regression of established tumor in murine models of cancer, and have been used for the treatment of cancer in humans. We have characterized freshly isolated mouse Thy-1+ TIL populations, as well as long term TIL cultures, from several different C57BL/6 (B6) tumors. Freshly isolated Thy-1+ TIL include both CD4+ and CD8+ cells, as well as cells bearing NK markers. These cells are predominantly TCR αβ+, with a smaller population of TCR γδ+ cells. The TCR αδ+ cells expressed a broad distribution of Vβ phenotypes that was statistically different from that expressed in normal B6 splenic Thy-1+ cells or CD8+ cells, presumably reflecting in vivo selection in the host anti-tumor response. NK cells are present in these tumors at a greater frequency than noted in splenic T cells. Cultured TIL populations rapidly became exclusively Thy-1+/CD8+/CD4- and TCR αβ+/ γδ-. Individual long term TIL populations initially expressed multiple Vβ products, but rapidly restricted their Vβ expression, frequently expressing a single dominant Vβ. The identity of this dominant Vβ varied among different TIL lines, but the overall representation of Vβ phenotypes in these cultures was statistically different from that seen in Thy-1+ or CD8+ splenocytes. No statistical difference was noted between lines derived from antigenically distinct tumors. The selection of tumor specific T cells in vitro is therefore not reflected in any simple predominance of Vβ usage. The complexity of TCR usage in the anti-tumor response may result from the involvement of multiple α- and β-chain regions in the response to a single antigenic determinant, or may reflect multiple antigenic determinants expressed on a single syngeneic tumor.

Assay of Pyrogens by Interleukin‐6 Release from Monocytic Cell Lines

Article

Sep 1991

A novel in-vitro system has been developed for the detection and quantification of pyrogen in pharmaceutical products. The measured variable was evoked secretion of the pyrogenic cytokine interleukin-6 from MONO MAC 6 monocytic cells incubated with the product. The interleukin-6 was detected using a specific and sensitive ELISA developed for this purpose. The test system detected pyrogenic contamination in 3 batches of therapeutic human serum albumin which had caused adverse reactions in recipients. The contamination was not detected in conventional tests: the rabbit pyrogen test and the limulus amoebocyte lysate test.

Pro-inflammatory activity of enterolobin: A haemolytic protein purified from seeds of the Brazilian tree Enterolobium contortisiliquum

Article

Feb 1991
TOXICON

The pro-inflammatory activity of enterolobin, a haemolytic protein from Enterolobium contortisiliquum seeds, was investigated. In doses ranging from 1 to 20 micrograms/site, enterolobin induced a dose-dependent paw oedema and pleurisy in rats. The effect was apparent after 15 min, peaked at 6 hr and decreased 24 hr after enterolobin was administered. One hour after the intrathoracic injection of enterolobin, the total leukocyte content of the pleural cavity increased significantly, mainly due to mononuclear and neutrophil accumulation. At 24 hr, although the number of mononuclear and neutrophil cells tended to decrease, a great rise in eosinophil counts was noted. Intraperitoneal treatment with the dual lipoxygenase and cyclooxygenase blockers, BW 755c (25 mg/kg) and NDGA (50 mg/kg) or the corticosteroid dexamethasone (0.1 mg/kg) inhibited enterolobin-induced paw oedema by 35, 38 and 47% respectively, whereas indomethacin (2 mg/kg) was inactive. The H1 antagonist, meclizine (25 mg/kg), was also effective against enterolobin oedema while the PAF-antagonists WEB 2086 and PCA 4248 (20 mg/kg) did not modify the reaction. It was concluded that enterolobin is a potent inducer of pleural exudation, cellular infiltration and paw oedema. Furthermore, enterolobin-induced oedema is partially dependent on lipoxygenase metabolites and histamine, while PAF and prostaglandins did not seem to be important in this reaction.

PAF antagonists do not modify IgE antibody production in mice

Article

Feb 1991
BRAZ J MED BIOL RES

The effect of selective PAF antagonists on the in vivo production of IgE antibodies was investigated. The anti-ovalbumin IgE antibody content was estimated by passive cutaneous anaphylactic reaction (PCA) in the plasma of Balb/c mice 10 days after immunization with ovalbumin and alum. The PAF antagonists, BN 52021 (5 mg/kg, ip), BN 50730 (20 mg/kg, po), WEB 2086 (2 mg/kg, ip) and WEB 2170 (5 mg/kg, ip) were administered 1 h before immunization and twice a day for 8 days thereafter. The effect of the antagonists on the PAF-induced vasopermeability was also assayed. In the immunized mice the level of antiovalbumin IgE antibody, estimated by PCA titer, was 1/640. The treatment with the PAF antagonists did not change this level. At the concentrations employed, the antagonists BN 50730, WEB 2086 and WEB 2170 significantly reduced the PAF-induced vascular permeability. These results suggest that PAF does not seem to have a relevant effect on the production of IgE antibodies in vivo in the system used in the present study.

Triggered human mucosal T cells release tumour necrosis factor-alpha and interferon-gamma which kill human colonic epithelial cells

Article

Feb 1991

T cell activation can lead to local tissue injury in organ culture studies of human fetal jejunum, either directly through cytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cytotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions are required for killing of human colonic epithelial cells. Cytokine-containing supernatants were induced by incubating unseparated lamina propria lymphocytes (LPL) or mucosal T cell subpopulations (separated by indirect panning) with anti-CD3 and/or K562 target cells for 18 h at 37 degrees C. Cytokines were measured by cytotoxicity assays using L929 (murine fibroblast) and HT-29 (human colonic tumour) lines as target cells in combination with blocking anti-cytokine antibodies. Supernatants derived from unseparated, CD4+ (greater than 95% pure) and CD8+ (greater than 90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/ml tumour necrosis factor-alpha, respectively). All or nearly all of the cytotoxicity was due to the presence of tumour necrosis factor-alpha (little or no tumour necrosis factor-beta was detected). These same supernatants were cytotoxic (up to 32% lysis at 1/4 dilution) to HT-29 targets in a 48-h 111In release assay. Recombinant tumour necrosis factor-alpha and interferon-gamma alone produced minimal killing of HT-29, but together killed the HT-29 target cells. Anti-tumour necrosis factor-alpha or anti-interferon-gamma alone blocked killing of HT-29 target cells by LPL-derived supernatants, although anti-tumour necrosis factor-beta had no effect upon killing of HT-29. These results demonstrate that human LPL T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosal damage in chronic inflammatory states such as inflammatory bowel disease.

Macrophages cultured in vitro release LTB4 and neutrophil attractant/activation protein (interleukin-8) sequentially in response to stimulation with lipopolysaccharide and zymosan

Article

Dec 1990

The capacity of lipopolysaccharide (LPS), zymosan, and calcium ionophore A23187 to induce neutrophil chemotactic activity (NCA), leukotriene B4 (LTB4), and neutrophil attractant/activation protein (NAP-1) release from human alveolar macrophages (AM) retrieved from normal nonsmokers was evaluated. LPS induced a dose-dependent release of LTB4 that began by 1 h, 4.0 +/- 3.2 ng/10(6) viable AM; peaked at 3 h, 24.7 +/- 13.5 ng/10(6) viable AM; and decreased by 24 h, 1.2 +/- 1.0 ng/10(6) viable AM (n = 8). Quantities of LTB4 in cell-free supernatants of AM stimulated with LPS were determined by reverse-phase high-performance liquid chromatography and corresponded well with results obtained by radioimmunoassay. By contrast, NAP-1 release began approximately 3-5 h after stimulation of AM with LPS, 197 +/- 192 ng/ml, and peaked at 24 h, 790 +/- 124 ng/ml. Release of NAP-1 was stimulus specific because A23187 evoked the release of LTB4 but not NAP-1, whereas LPS and zymosan induced the release of both LTB4 and NAP-1. The appearance of neutrophil chemotactic activity in supernatants of AM challenged with LPS for 3 h could be explained completely by the quantities of LTB4 present. After stimulation with LPS or zymosan for 24 h, AM had metabolized almost all generated LTB4. Preincubation of AM with nordihydroguiaretic acid (10(-4) M) completely abolished the appearance of NCA, LTB4, and NAP-1 in supernatants of AM challenged with LPS. Therefore, LPS and zymosan particles were potent stimuli of the sequential release of LTB4 and NAP-1 from AM.

Identification and isolation of membrane protein necessary for leukotriene production

Article

Feb 1990

Several inflammatory diseases, including asthma, arthritis and psoriasis are associated with the production of leukotrienes by neutrophils, mast cells and macrophages. The initial enzymatic step in the formation of leukotrienes is the oxidation of arachidonic acid by 5-lipoxygenase (5-LO) to leukotriene A4. Osteosarcoma cells transfected with 5-LO express active enzyme in broken cell preparations, but no leukotriene metabolites are produced by these cells when stimulated with the calcium ionophore A23187, indicating that an additional component is necessary for cellular 5-LO activity. A new class of indole leukotriene inhibitor has been described that inhibits the formation of cellular leukotrienes but has no direct inhibitory effect on soluble 5-LO activity. We have now used these potent agents to identify and isolate a novel membrane protein of relative molecular mass 18,000 which is necessary for cellular leukotriene synthesis.

The effects of drugs on leucocyte changes following the injection of Ag into the peritoneal cavities of actively sensitised rats

Article

Apr 1986
Agents Actions

The injection of antigen into the peritoneal cavities of actively sensitised rats produced an immediate reaction characterised by an increase in concentrations in the peritoneal fluids, collected 5 min later, of extravasated dye labelled plasma proteins, histamine and slow reacting substance of anaphylaxis (SRS-A). Changes were also produced in the numbers of leucocytes in the blood and peritoneal cavity. 5 min after antigen challenge there was a reduction in the number of cells that could be washed from the peritoneal cavity. 4 h after antigen there was an increase in numbers of neutrophils both in the blood and peritoneal washings and these fell to the levels in control rats at 24 h. 24 h after antigen, and continuing for 72 h, there was an increase in numbers of eosinophils and mononuclear cells in the peritoneal washings. The rats were injected intravenously with sephadex particles to produce a blood eosinophilia at the time of antigen challenge, this increased the numbers of eosinophils migrating into the peritoneal cavity but had no effect on antibody levels, the numbers of other leucocytes or on the immediate reaction. An inhibitor of lipoxygenase and cyclo-oxygenase metabolism of arachidonic acid, phenidone, at 100 mg/kg p.o., inhibited SRS-A release to control levels, in the immediate reaction, but had no effect on the leucocyte changes. The glucocorticosteroid, dexamethasone, at doses of 0.1 and 1 mg/kg p.o., produced little inhibition of SRS-A release but significantly inhibited neutrophil, eosinophil and mononuclear cell infiltration into the peritoneal cavity.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential secretion of tumor necrosis factor and granulocyte/ macrophage colony stimulating factors but not interferon-γ from CD4+ compared to CD8+ human T cell clones

Article

Jan 1989

Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic lymphokine which may have important regulatory effects on immune responses. It is shown here that eight alloreactive CD4+ T cell clones (TCC) secreted significant amounts of TNF-alpha after stimulation with either specific alloantigen or 12-O-tetradecanoylphorbol 13-acetate together with the calcium ionophore ionomycin (up to 50 ng/ml/24 h/10(6) cells) whereas CD8+ TCC failed to do so (max. 2 ng/ml/24 h/10(6) cells). The CD8+ TCC also secreted markedly less granulocyte/macrophage colony-stimulating factor than the CD4+ cells. However, this was not indicative of a general decrease of lymphokine production by CD8+ cells because CD4+ and CD8+ TCC both secreted similar amounts of interferon-gamma. These results show that regulatory CD4+ lymphocytes can produce large amounts of TNF-alpha, whereas CD8+ effector cells cannot do so.

The combination of interleukin 1 plus tumor necrosis factor causes greater generation of LTB4, thromboxanes and aggregation on human macrophages than these compounds alone

Article

Feb 1989
Progr Clin Biol Res

Current concepts: Immunology - Neutrophils in human diseases

Article

Oct 1987

HUMAN neutrophilic polymorphonuclear leukocytes (neutrophils) provide an effective host defense against bacterial and fungal infection, but they are also important in the pathogenesis of tissue damage in certain noninfectious diseases. Some important events in neutrophil function that will be discussed in this review are shown in Figure 1. Mild to moderate abnormalities of neutrophil function have been reported in many acquired and congenital diseases.1 2 3 4 5 In most of these disorders, the biochemical or morphologic basis of the defects is unknown and the relevance of the neutrophil defect to the manifestations of the disease is unclear. In contrast, persons with marked neutropenia6 or . . .

Effect of Drugs on the Increase in Cell Numbers in the Peritoneal Cavity of the Actively Sensitised Mouse after Intraperitoneal Challenge with Antigen

Article

Feb 1986

The injection of antigen into the peritoneal cavity of actively sensitised mice produced an increase in the number of neutrophils in peritoneal washings collected 4 h later but after 1 day the numbers had returned to control levels. The increase in numbers of mononuclear cells and eosinophils in the peritoneal washings peaked at 2 days and persisted for at least 5 days. Dosing the mice with phenidone, a dual inhibitor of the cyclo-oxygenase and lipoxygenase pathways of arachidonic acid metabolism, potentiated the neutrophil infiltration at 4 h but had no significant effect upon the subsequent mononuclear cell and eosinophil infiltration. In contrast, treatment with the corticosteroid, dexamethasone, reduced the infiltration by all three types of cells, providing further evidence that the corticosteroids can inhibit immune-induced cellular infiltrations by mechanisms other than the inhibition of arachidonic acid metabolism. Isoprenaline, given to the mice before antigen challenge, had no effect on the subsequent neutrophil infiltration, but repeated doses did inhibit the mononuclear cell and eosinophil infiltration measured 4 days later. Aminophylline, disodium cromoglycate and cyproheptadine had no effect upon the cellular changes.

Function of Exudative Neutrophilic Granulocytes in Patients with Crohn's Disease or Ulcerative Colitis

Article

Dec 1985

Johan H Wandall

Chemotactic, phagocytic, and oxidative metabolic activity of exudative leukocytes was measured in patients with Crohn's disease (n = 20) and with ulcerative colitis (n = 20). Unstimulated and casein-stimulated migration in Boyden chambers did not differ from that of healthy controls (n = 21). Patients with Crohn's disease had reduced serum-independent phagocytosis compared with healthy controls (p less than 0.01) and patients with ulcerative colitis (p less than 0.01). Serum-dependent phagocytosis by leukocytes from patients with Crohn's disease did not differ from that in controls but was slightly increased in patients with ulcerative colitis (p less than 0.02). Unstimulated leukocytes showed increased oxidative metabolic activity in both patient groups compared with controls (p less than 0.01), which was negatively correlated with the disease activity in Crohn's disease (p less than 0.02). The study shows that mobilized leukocytes from patients with Crohn's disease differ from those mobilized in ulcerative colitis and supports the concept of an abnormal inflammatory reaction in Crohn's disease.

Rheumatoid arthritis. The role of neutrophil activation

Article

Jul 1984

Neutrophils constitute over 90% of cells found in the synovial fluid of rheumatoid arthritis (RA) patients. Since such fluids also contain immune complexes (IgG-IgG and IgG-IgM rheumatoid factors) and complement split products (C5, C5A, DES, ARG, C3B, etc.), all of the reactants are present for a local Arthus lesion. Moreover, neutrophils from RA patients endocytose these immune complexes and complement components in vivo and in vitro. In consequence, it has been suggested that lysosomal enzymes and other mediators of inflammation released by neutrophils after uptake of immune complexes (in the bulk phase or on the surface) account, at least in part, for rheumatoid inflammation. Secretion of lysosomal hydrolases, especially neutral proteases, which provoke tissue injury and generation of reactive oxygen species (e.g. O2) is part of a stimulus-secretion response to a variety of secretagogues, including immune complexes and complement components. However, the pathways of secretion and O2 generation are stimulus-specific and can be dissected to establish cause and effect relationships by (a) kinetic analysis, (b) varying the stimulus, (c) use of impermeant reagents to block discrete responses. Neutrophils also generate products of 11-cyclo-oxygenase (e.g., PGE2, TXA2) and of the 5- and 15-lipoxygenase (mono-, di and tri-hetes, LTB4 and their isomers). However, the cyclo-oxygenase products (except TXA2) do not cause inflammation acting alone; indeed, they inhibit the function of neutrophils, platelets, macrophages and mast cells. The most potent proinflammatory agent yet identified as a product of arachidonate is LTB4. LTB4 is a potent Ca ionophore, a strong chemo-attractant, induces local inflammation, and activates neutrophils.

Leukotriene B4, a mediator of inflammation present in synovial fluid in rheumatoid arthritis

Article

Jan 1984

Leukotriene B4 (LTB4), generated from arachidonic acid following lipoxygenase activity by a variety of inflammatory leucocytes, has been shown to be present in synovial fluid from patients with active rheumatoid arthritis. It does not persist as such, being converted to less active metabolites. The role of LTB4 as one of the natural mediators of inflammation is discussed.

Stenson WE Enhanced synthesis of leukotrien B4 by colonic mucosa in inflammatory bowel disease

Article

Apr 1984
GASTROENTEROLOGY

Leukotriene B4, an arachidonic acid metabolite, is a potent chemotactic agent, and is thought to be an important mediator of inflammation. To investigate the role of this compound as a mediator of inflammation in inflammatory bowel disease, arachidonic acid was incubated with ionophore and colonic mucosa from patients with inflammatory bowel disease and from normal subjects. Mucosa from patients with inflammatory bowel disease converted 2.17% of exogenous arachidonate to leukotriene B4; mucosa from normal subjects converted 0.37%. The production of leukotriene was blocked by sulfasalazine. To determine if inflammatory bowel mucosa contained endogenous leukotriene B4, lipid extracts were analyzed by high pressure liquid chromatography. Mucosa from patients with inflammatory bowel disease contained 254 ng of leukotriene B4 per gram and mucosa from normal subjects contained less than 5 ng of leukotriene B4 per gram. The presence of significant amounts of leukotriene B4 in colonic mucosa in patients with inflammatory bowel disease, combined with the known biologic effects of leukotriene B4, suggests that it may be an important mediator of inflammation in inflammatory bowel disease.

Differential effects of steroids on leukocyte-mediated glomerulonephritis in the rabbit

Article

Sep 1984

The effects of steroids on the development of injury in two models of experimental glomerulonephritis (GN), (one mediated by neutrophils, the other by macrophages) were compared. The neutrophil-associated lesion [initiated by heterologous antiglomerular basement membrane (GBM) antibody] was characterized by the development of an exudative endocapillary GN with heavy neutrophil accumulation [mean, 6.9 neutrophils/glomerular cross section (N/GCS) +/- 2.9 SD], minor macrophage infiltration [7.9 macrophages/glomerulus (M/G) +/- 2.2 SD] and heavy proteinuria (1905 mg/24 hr +/- 520 SD). Steroid-treated (methylprednisolone, 2 mg/kg/12 hr i.v.) rabbits developed a marked monocytopenia, mild neutrophilia, and significant reduction in glomerular macrophage accumulation (0.3 M/G 0.02 SD). However, neutrophil accumulation (6.1 N/CGS +/- 2.5 SD), histological appearances, and proteinuria (1820 mg/hr +/- 490 SD) were unaffected. The macrophage-associated model of GN was induced by passive autologous rabbit anti-sheep IgG 15 hr after the injection of a subnephritogenic dose of the same anti-GBM antibody. The glomerular lesion was characterized by a diffuse endocapillary proliferative GN with heavy macrophage infiltration (54 M/G +/- 8 SD), insignificant neutrophil accumulation (0.8 N/GCS 0.02 SD), and the regular development of proteinuria (420 mg/24 hr +/- 80 SD). Steroid-treated rabbits developed a mild neutrophilia and a significant monocytopenia associated with abrogation of glomerular macrophage accumulation (2.3 M/G +/- 0.8 SD). This was associated with the prevention of the development of GN and proteinuria (22 +/- 9.5 SD). Thus, steroids produce monocytopenia and prevent glomerular macrophage accumulation and associated injury whereas neutrophil accumulation and injury is unaffected. These data suggest steroids may have widely varying effects on the outcome of leukocyte-associated experimental GN depending on the nature of the infiltrating cells.

The role of lymphocytes in the neutrophil migration induced by ovalbumin in immunised rats

Article

May 1995

In the present study, we investigated the role of resident cells in the neutrophil migration induced by ovalbumin (OVA) in immunized rats. OVA administration induced dose-dependent neutrophil migration, which was inhibited by pretreating the animals with dexamethasone, but not with indomethacin or BW 70C. Lymphocytes, but not macrophages or mast cells, obtained from sensitized animals and stimulated in vitro with OVA released a factor that induced neutrophil migration in vivo and in vitro. Both the release of this factor in vitro and the neutrophil migration induced in vivo were inhibited by dexamethasone, thus explaining the inhibitory effect of glucocorticoids on the neutrophil migration induced by OVA in immunized animals. Neither indomethacin nor BW 70C had any such effect. The fact that actinomycin D also inhibited the release of the factor from OVA-stimulated lymphocytes suggests that this substance is of a proteinaceous nature. The importance of lymphocytes for neutrophil recruitment in OVA-immunized rats was supported by the fact that homologous lymphocyte transfer into air pouches rendered these cavities responsive to OVA. Lymphocytes obtained from naive rats and stimulated with the lectins concanavalin A (Con A) or phytohaemagglutinin (PHA) were also able to release a factor that induced neutrophil migration in vivo. In vitro incubation of the supernatant from OVA-stimulated lymphocytes with antisera to interleukin-1 beta (IL-1 beta), IL-8 and tumour necrosis factor-alpha (TNF-alpha) did not inhibit the neutrophil chemotactic activity. These data suggest that IL-1 beta, IL-8 and TNF-alpha are not involved in the neutrophil chemotactic activity of the supernatant. Overall, these results indicate the importance of lymphocyte participation in neutrophil recruitment during inflammatory immune reaction, through the release of a neutrophil chemotactic factor different from IL-1 beta, IL-8 and TNF-alpha.

Alveolar macrophage TNFA release and BAL cell phenotypes in sarcoidosis

Article

Sep 1995

The aim of this study was to investigate the relationship between release of tumor necrosis factor-alpha (TNF-alpha) by alveolar macrophages (AM) and the phenotypic characteristics of bronchoalveolar lavage (BAL) cells in sarcoidosis. We studied the spontaneous release of TNF-alpha by AM in vitro and the phenotypic characteristics of freshly recovered BAL T-cells and AM in 31 individuals (13 with active sarcoidosis, nine with inactive sarcoidosis, and nine normal controls). TNF-alpha was measured by enzyme-linked immunosorbent assay (ELISA) in supernatants from unstimulated AM after 24 h culture. Phenotypic markers of BAL cells were determined by an immunocytochemical assay. AM of patients with active sarcoidosis released more TNF-alpha (1,355 +/- 133 pg/ml/ 10(6) AM/24 h) than those of the inactive group (651 +/- 142 pg/ml/10(6) AM/24 h) or the normal controls (425 +/- 121 pg/ml/10(6) AM/24 h), with p < 0.001 for both comparisons. The amount of TNF-alpha released correlated positively with the percentage expression of CD4 (r = 0.72) and CD25 (r = 0.70) by lymphocytes, and of CD14 (r = 0.63), VLA-4 (r = 0.59), FRD1 (r = 0.67) and 27E10 (r = 0.67) by AM, with p < 0.001 for all correlations. In conclusion, this relationship suggests that these antigens may be considered as cellular activation markers, and that some of these AM antigens may indirectly characterize the AM phenotype that is capable of producing TNF-alpha.

Stimulation of IL-2 Production in Murine Lymphocytes by Substance P and Related Tachykinins

Article

Oct 1993

Substance P (SP), a tachykinin neuropeptide, has been previously reported to stimulate IL-2 production in murine T cell lines activated with phorbol esters. Here we extend these observations by establishing the stimulatory effect of SP and related tachykinins on IL-2 production by normal murine lymphocytes and on purified CD4+ T cells. SP proved to be the most efficient IL-2 inducer, exerting its maximal effect at concentrations that were 4 to 5 orders of magnitude lower than the optimal stimulatory concentrations of physalaemin, NKA, or NKB. SP stimulated IL-2 production in a dose-dependent manner, with an optimal concentration range of 10(-10) to 10(-14) M, comparable with physiologic concentrations of SP found in blood and other organs. The effect of SP was carried by the carboxyl-terminal part of the molecule (SP4-11). The specificity of SP activity was confirmed by the inhibitory effect of spantide, a tachykinin antagonist, and of CP-96,345, a nonpeptide antagonist specific for NK-1-type receptors. In unfractionated spleen cell cultures SP induced de novo IL-2 protein synthesis. SP could induce IL-2 production either directly, or in combination with Con A or anti-CD3 antibody treatments. The effect of SP in conjunction with Con A was synergistic, whereas the effect in conjunction with anti-CD3 antibodies was additive, suggesting different molecular mechanisms for these stimulatory factors. In the absence of additional costimuli the effect of SP in unfractionated spleen cell cultures was partially mediated through the induction of IL-1, and both SP and IL-1 were required for IL-2 induction in purified CD4+ T cells. In contrast to its stimulatory effect on the generation of IL-2, SP did not induce IFN-gamma production in murine spleen cells. The stimulatory effect of SP on IL-2 production suggests that some of the already described immunostimulatory activities of SP could be mediated through the up-regulation of IL-2 production in normal lymphocytes.

Characterization and pharmacological modulation of antigen-induced peritonitis in actively sensitised mice

Article

Nov 1993

The intraperitoneal (i.p.) injection of 1 or 10 μg ovalbumin to sensitized Balb/c mice led to an acute histamine release, firstly evidenced 1 min after the challenge and returning to basal levels 30 min thereafter. This phenomenon was unaccompanied by protein extravasation. A dose‐dependent increase in the amounts of immunoreactive leukotriene (LT) C 4 and LTB 4 was observed in the peritoneal washing from sensitized mice 6 h after 1 or 10 μg ovalbumin administration. In separate experiments, the i.p. administration of 1 mg activated zymosan to non‐immunized mice was followed by a marked protein extravasation, and by immunoreactive LTC 4 and LTB 4 , but not histamine, release in mouse peritoneum 1 h after its injection. Mediator release in the mice peritoneal cavity was concomitant with a transient neutrophil infiltration, which peaked at 6 h and returned to basal levels thereafter. An intense eosinophil accumulation starting at 24 h, peaking at 48 h and returning to basal values at 164 h, was also observed. Ovalbumin (1 μg)‐induced eosinophilia, observed at 24 h, was reduced by the pretreatment of the animals with dexamethasone (1 mg kg ⁻¹ , s.c.) or with the 5‐lipoxygenase inhibitor, BWA4C (20 mg kg ⁻¹ , s.c.), whereas indomethacin (2 mg kg ⁻¹ , s.c.) and the platelet‐activating factor (PAF)‐antagonist SR 27417 (10 mg kg ⁻¹ , s.c.) were ineffective. These results indicate that metabolites of arachidonic acid of lipoxygenase pathway, but not cyclo‐oxygenase derivatives or PAF, mediate antigen‐induced eosinophil accumulation in the mouse peritoneum. The histamine H 1 receptor antagonist drug, cetirizine (15–30 mg kg ⁻¹ , s.c.) markedly reduced ovalbumin‐induced eosinophil accumulation under conditions where terfenadine was ineffective, suggesting that the effect of cetirizine was not related to the inhibition of the H 1 receptor effects of histamine. The immunosuppressive agent, FK‐506 (1–2 mg kg ⁻¹ , s.c.) and the protein synthesis inhibitor, cylcoheximide, when administered either in situ (0.06 ng/cavity) or systemically (5 mg kg ⁻¹ , s.c.), prevented antigen‐induced eosinophil accumulation in the mouse peritoneum, contributing to the concept that substances (probably cytokines) originating from lymphocytes may be involved in the modulation of the eosinophilotactic response in this model. The results of the present study indicate that the i.p. administration of ovalbumin to actively sensitized mice induced late eosinophil accumulation in the peritoneal cavity. This phenomenon, which may be in part mediated by the release of lipoxygenase metabolites and/or by newly generated factors, such as T‐lymphocytes‐derived eosinophilotactic cytokines, offers an interesting tool to investigate the mechanism of action of anti‐allergic and anti‐inflammatory drugs.

The in vitro and in vivo pharmacologic activity of the potent and selective leukotriene B4 receptor antagonist CP-105696

Article

May 1995

CP-105696, (+)-1-(3S,4R)-[3-(4-phenyl-benzyl)-4-hydroxy-chroman-7-yl] cyclopentane carboxylic acid, is a structurally novel, selective and potent leukotriene B4 (LTB4) receptor antagonist. In vitro, CP-105696 inhibited [3H]LTB4 (0.3 nM) binding to high-affinity LTB4 receptors on human neutrophils with an IC50 value of 8.42 +/- 0.26 nM. Scatchard analyses of [3H]LTB4 binding to these high-affinity receptors indicated that CP-105696 acted as a noncompetitive antagonist. CP-105696 inhibited human neutrophil chemotaxis mediated by LTB4 (5 nM) in a noncompetitive manner with an IC50 value of 5.0 +/- 2.0 nM. Scatchard analyses of [3H]LTB4 binding to low-affinity receptors on neutrophils indicated that CP-105696 acted as a competitive antagonist at this receptor, and inhibition of LTB4-mediated CD11b upregulation on human neutrophils was competitively inhibited by CP-105696 (pA2 = 8.03 +/- 0.19). CP-105696 at 10 microM did not inhibit either human neutrophil chemotaxis or CD11b upregulation mediated through alternate (i.e., C5a, IL-8, PAF) G-protein coupled chemotactic factor receptors. In isolated human monocytes, LTB4 (5 nM)-mediated Ca++ mobilization was inhibited by CP-105696 with an IC50 value of 940 +/- 70 nM. In vivo, after oral administration, CP-105696 blocked neutrophil and eosinophil infiltration in cavine dermis mediated by either LTB4 or arachidonic acid with ED50 values of 0.3 +/- 0.1 mg/kg. 12(R)-Hydroxyeicosatetraenoic acid-mediated neutrophil infiltration was blocked by 76.4 +/- 14.8% at 3 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ZCR-2060 on allergic airway inflammation and cell activation in guinea-pigs

Article

Dec 1994

The effects of 2-(2-(4-(diphenylmethyl)-1-piperadinyl) ethoxy) benzoic acid malate (ZCR-2060) on allergic airway inflammation and inflammatory cell activation in guinea-pigs were studied. Allergic airway inflammation was induced by inhalation of antigen into actively-sensitized animals and the increase in inflammatory cells into bronchoalveolar lavage fluid (BALF) was measured. Aeroantigen-induced infiltration of inflammatory cells, especially eosinophils and neutrophils, in BALF gradually increased, and reached a peak at 6 or 9 h after the challenge. ZCR-2060 (1 mg kg-1 p.o.) clearly inhibited the increase of eosinophil numbers in BALF. Moreover, the effect of ZCR-2060 on inflammatory cell activation in terms of chemotaxis and superoxide generation in-vitro was studied. ZCR-2060 (10(-6)-10(-4) M) inhibited the platelet-activating factor (PAF)-induced chemotaxis of eosinophils and neutrophils, but did not inhibit the leukotriene B4-induced chemotaxis of eosinophils and the formyl-Met-Leu-Phe-induced chemotaxis of neutrophils. PAF-induced superoxide anion generation by eosinophils, neutrophils and alveolar macrophages was inhibited by ZCR-2060 (10(-6)-10(-4) M). However, ZCR-2060 did not affect phorbol myristate acetate-induced superoxide anion generation by eosinophils, neutrophils and alveolar macrophages. These results indicate that ZCR-2060 inhibits allergic airway inflammation, and PAF-induced inflammatory cell activation in guinea-pigs. ZCR-2060 may prove useful for the treatment of allergic airway inflammation or allergic disorders, especially inflammatory cell infiltration and activation.

(+)-1-(3S,4R)-[3-(4-Phenylbenzyl)-4- hydroxychroman-7-yl]cyclopentane Carboxylic Acid, a Highly Potent, Selective Leukotriene B4 Antagonist with Oral Activity in the Murine Collagen-Induced Arthritis Model

Article

Oct 1994

Role of Resident Peritoneal Cells in Eosinophil Migration Induced by Saline

Article

Sep 1994

In the present study, we investigated the role of resident peritoneal cells as well as the mediators involved in the eosinophil migration induced by large volumes of physiological saline. Two consecutive intraperitoneal injections of saline given 48 h apart, induced a selective recruitment of eosinophils into the cavity. This response was not observed with phosphate-buffered saline (PBS). Saline-induced eosinophil migration may be mediated at least in part by LTB4, since the lipoxygenase inhibitors BW A4C and MK 886 prevented the response. In the presence of saline, but not of PBS, mast cells and macrophages incubated in vitro released a factor which induced eosinophil migration when injected into the peritoneal cavity of rats. This release was inhibited by BW A4C and MK 886. These results indicate the importance of mast cells and macrophages in the eosinophil migration induced by saline and suggest the participation of LTB4 in this phenomenon. An abrupt reduction in the extracellular potassium concentration at the membrane of resident cells may be responsible for the saline effects since addition of potassium ions to saline abolished the eosinophil chemotactic activity of the same as well as its ability to stimulate the release of eosinophil chemotactic factor in vitro. Dexamethasone blocked both the saline-induced eosinophil migration and the release of eosinophil chemotactic factor by mast cells and macrophages. Pretreatment of the animals with dexamethasone inhibited the eosinophil migration induced either by the supernatants of saline-stimulated mast cells and macrophages or by LTB4. These results indicate that the release of additional mediators is necessary in order to account for the final eosinophil migration.(ABSTRACT TRUNCATED AT 250 WORDS)

Isotypic Residues in the Membrane Proximal Domain of MHC Class II β-Chains Control Activation of CD4+ T Cells

Article

Jul 1993

Andrea Sant

To investigate the role that isotypic residues play during interactions between CD4+ T cells and MHC class II molecules, interisotypic MHC class II β-chains have been generated in which the β1 domain is derived from I-A and the β2, transmembrane, and intracytoplasmic regions are derived from I-E. Interisotypic or wild-type Aβ genes have been transfected into L cells with the genes encoding the wild-type Aα-chain. Transfectants bearing the recombinant β-chain thus express an MHC class II dimer in which the amino-terminal domains that control TCR and peptide-binding interactions are wild-type Aα and Aβ, whereas the membrane proximal domains are derived from Aα and Eβ. L cells expressing this recombinant class II molecule or wild-type AαAβ have been compared functionally for their ability to stimulate Ag-specific T cell hybridomas and normal T cell clones and to activate primary alloreactive and superantigen-specific T cells. Ag-specific T cell hybridomas vary in their ability to be activated by the recombinant class II molecule, and sensitivity to the isotypic form of the membrane proximal domain correlates with expression of the CD4 molecule. CD4+ T cells distinguish between the wild-type and recombinant dinners, whereas CD4- T cells react equivalently with both. The recombinant class II molecules are defective in activation of normal T cell clones and are totally deficient in activation of primary alloreactive and superantigen-reactive T cells, but stimulate CD4- alloreactive T cell hybridomas equivalently. Together, the results from these experiments suggest that the interisotypic dimers possess normal TCR and peptide interactions, but altered CD4-dependent accessory interactions necessary for activation of normal T cells. These findings indicate that isotypic residues in the membrane proximal domains of MHC class II control CD4-linked accessory function and that this accessory function is most critical for freshly isolated CD4 lymphocytes.

Pfeffer K, Matsuyama T, Kundig TM, Wakeham A, Kishihara K, Shahinian A, Wiegmann K, Ohashi PS, Kronke M & Mak TW.Mice deficient for the 55 kd tumor necrosis factor receptor are resistant to endotoxic shock yet succumb to L. monocytogenes infection. Cell 73: 457−467

Article

Jun 1993
CELL

The multiple biological activities of tumor necrosis factor (TNF) are mediated by two distinct cell surface receptors of 55 kd (TNFRp55) and 75 kd (TNFRp75). Using gene targeting, we generated a TNFRp55-deficient mouse strain. Cells from TNFRp55-/-mutant mice lack expression of TNFRp55 but display normal numbers of high affinity TNFRp75 molecules. Thymocyte development and lymphocyte populations are unaltered, and clonal deletion of potentially self-reactive T cells is not impaired. However, TNF signaling is largely abolished, as judged by the failure of TNF to induce NF-kappa B in T lymphocytes from TNFRp55-deficient mice. The loss of TNFRp55 function renders mice resistant to lethal dosages of either lipopolysaccharides or S. aureus enterotoxin B. In contrast, TNFRp55-deficient mice are severely impaired to clear L. monocytogenes and readily succumb to infection. Thus, the 55 kd TNFR plays a decisive role in the host's defense against microorganisms and their pathogenic factors.

Mast cell modulation of neutrophil influx and bacterial clearance at sites of infection through TNF-alpha

Article

Jun 1996

Although mast cells have been implicated in a variety of inflammatory conditions including immediate hypersensitivity and interstitial cystitis, their physiological role in the body is unknown. We investigated the role of mast cells in host defence against bacterial infections using a well characterized mast-cell-deficiency mouse model. We report here that mast cells, which are selectively located at portals of bacterial entry, are important to host defence. Mast-cell-deficient WBB6F1-W/Wv mice (W/Wv) were up to 20-fold less efficient in clearing enterobacteria than control WBB6F1 +/+ (+/+) mice or mast-cell-reconstituted W/Wv (W/Wv+MC) mice. With higher bacteria inocula, only W/Wv mice died (80%). The limited bacterial clearance in W/Wv mice directly correlated with impaired neutrophil influx. The mast-cell chemoattractant TNF-alpha was implicated in the neutrophil response because TNF-alpha was locally released only in +/+ and W/Wv+MC mice, TNF-alpha-specific antibodies blocked over 70% of the neutrophil influx, and purified mast cells released TNF-alpha upon incubation with bacteria. Additionally, the type-1 fimbrial subunit, FimH, was the necessary enterobacterial component for mast-cell activation and neutrophil influx because an isogenic FimH- mutant evoked a limited neutrophil response in +/+ mice compared to wild-type bacteria.

Echtenacher B, Mannel DN, Hultner LCritical protective role of mast cells in a model of acute septic peritonitis. Nature 381:75-77

Article

Jun 1996

Mast cells play a detrimental role in IgE-dependent allergic reactions. In contrast, a protective function for mast cells has been proposed on the basis of some worm infection models. No reports exist on the in vivo significance of these cells in bacterial infections. Here we use congenitally mast-cell-deficient W/Wv mice and normal +/+ littermates to analyse the role of mast cells in a model of acute septic peritonitis (caecum ligation and puncture (CLP)). Following CLP, W/Wv mice showed a significantly increased mortality compared to +/+ mice. The selective reconstitution of W/Wv mice with cultured +/+ mast cells substantially protected them from the lethal effects of CLP, whereas an anti-tumor-necrosis-factor (TNF) antibody injected immediately after CLP completely suppressed this protection. Our results reveal a previously unrecognized protective role of mast cells and mast-cell-derived TNF in acute bacterial peritonitis.

Seeing the wood for the trees: The forgotten role of neutrophils in rheumatoid arthritis

Article

Aug 1997
Immunol Today

Neutrophils infiltrate diseased joints in rheumatoid arthritis in large numbers and possess considerable potential to inflict the tissue damage that is characteristic of this disease. However, these cells are commonly overlooked by immunologists seeking new ways to treat disease progression. In this article, Steven Edwards and Maurice Hallett evaluate the role of neutrophils in disease pathology with the aim of re-awakening interest in this important cell.

Modulation by lipid mediators of immune complex-induced lung inflammation in mice

Article

Dec 1998
EUR J PHARMACOL

The present study characterized a murine model of immune complex-induced pneumonitis and investigated the role of platelet-activating factor (PAF) and eicosanoids as mediators of lung neutrophil infiltration and hemorrhagic lesions. Rabbit antibodies to bovine serum albumin were injected into the airways and bovine serum albumin was injected intravenously into C3H/HePas and BALB/c mice. After 24 h, a significant increase in neutrophil infiltration and hemoglobin concentration in the bronchoalveolar lavage fluid and lung parenchyma was observed in both strains despite the C3H/HePas strain being 10 times more sensitive to PAF. Neutrophil influx and vascular lesions were not affected by pre-treatment of the mice with the PAF receptor antagonist, WEB 2170 (5-(2-chlorphenyl)carbonyl)-3,4-dihydro- 10-methyl-3-((4-morpholinyl)-2H,7H-cyclopenta(4,5)thieno(3,2-f)(1,2,4)-t riazolo-(4,3-a)(1,4)-diazepine). In contrast, neutrophil influx and vascular lesions were increased by the cyclo-oxygenase inhibitor, indomethacin, and reduced by the inhibitor of leukotriene synthesis, MK 886 (3-[1-(4-chlorobenzyl-3-t-butyl-thio-t-isopropyl-indol-2y-1]-2-2-+ ++dimethylpropanoic acid) and by the leukotriene B4 receptor antagonist, RO 0254094 (2-[(5-carboxypentyl)-6-[6-[3,4-dihidro-4-oxo-8-propyl-2H-1-benzop yran-7-yl)hexyl] benzenepropanoic acid). Increased levels of leukotriene B4, leukotriene C4/D4, thromboxane B2 were found in bronchoalveolar lavage fluid 4 h after induction of the reaction. There is also a tendency to increased prostaglandins E2 levels. Neutrophil infiltration and vascular lesions in immune complex-induced pneumonitis in mice are mediated by leukotriene B4.

Contrasting roles for RANTES and macrophage inflammatory protein‐1α (MIP‐1α) in a murine model of allergic peritonitis

Article

Sep 1999

Cell accumulation and CC chemokine production were assessed in the peritoneal cavity of ovalbumin (OVA)-sensitized mice following antigen challenge. Intraperitoneal challenge with OVA induced a significant eosinophil influx from 6 h post-challenge with increased numbers persisting at 24 h. At 6 h there was also a marked presence of neutrophils. Messenger RNA expression and protein levels for the chemokines RANTES and MIP-1 alpha were measured in the cell pellets and supernatants, respectively, from peritoneal washes following OVA challenge. RANTES mRNA was detected from 2 h to 4 h following OVA injection, whereas mRNA for MIP-1 alpha was only detectable at 4 h. RANTES protein was first detected from 4 h after OVA injection and by 24 h the protein levels had increased further. Basal levels of MIP-1 alpha were detected in peritoneal washes. These levels peaked at 2 h after OVA challenge and rapidly declined to basal levels by 6 h. A functional role for the chemokines was assessed using neutralizing polyclonal antibodies. Co-injection of OVA with anti-RANTES antibodies resulted in a significant inhibition of eosinophil infiltration into the cavity at 6 h and 24 h (63% and 52% inhibition, respectively) without significantly influencing the number of neutrophils present. In contrast, injection of anti-MIP-1 alpha antibodies only inhibited neutrophil migration at the 6 h time point by 44% without significantly affecting the accumulation of eosinophils. These results demonstrate an important role for RANTES in mediating eosinophil influx in allergic inflammation and a contrasting role for MIP-1 alpha in mediating neutrophil recruitment.

Pathogenesis of rheumatoid arthritis - The role of T cells and other beasts

Article

Jun 2001
RHEUM DIS CLIN N AM

The evidence coming from the different experimental approaches reviewed in this article strongly supports the hypothesis that RA is T-cell driven at all stages of the disease. Although the effector phases responsible for the events that lead to joint destruction involve several different cell types, cytokines, and other mediators, T cells still direct operations behind the scenes. Direct experimental proof of this proposition in patients is still lacking, but the development of nondepleting modulating CD4 monoclonal antibodies may provide new tools to test this hypothesis. In this respect, it is encouraging that using one such reagent, we have recently shown that not only did the activity of the disease improve but, more importantly, the inflammatory indices and production of non-T-cell cytokines were reduced. This is not to dissimilar from the results of experiments described in animals, where by blocking synovial T cells, the production of IL-1 beta and TNF alpha could be decreased by more than 90%. From this perspective, it may be predicted that by modulating T cells in the joint, it is possible to achieve our ultimate goal of permanently switching off the disease.

Vasculitis: Phatogenic Mechanisms of Vessel Damage In Inflammation: Basic Principles and Clinical Correlates

B F Haynes
B F Haynes

The role of lymphocytes in the neutrophil migration induced by ovalbumin in immunised rats Rapid up-regulation of CXC chemo-kines in the airways after Ag-speci®c CD4 + T-cell activation

A Klein
F Q Cunha
S H Ferreira
P G Knott
P R Gater
P J Dunford
M E Fuentes
C P Bertrand
K Koch
L S Melvin
Reiter Jr
L A Biggers
M S Showell
H J Pettipher
E R Cheng
J B Milici
A J Breslow

KLEIN, A., CUNHA, F.Q. & FERREIRA, S.H. (1995). The role of lymphocytes in the neutrophil migration induced by ovalbumin in immunised rats. Immunol., 84, 577 ± 584. KNOTT, P.G., GATER, P.R., DUNFORD, P.J., FUENTES, M.E. & BERTRAND, C.P. (2001). Rapid up-regulation of CXC chemo-kines in the airways after Ag-speci®c CD4 + T-cell activation. J. Immunol., 166, 1233 ± 1240. KOCH, K., MELVIN, L.S. Jr, REITER, L.A., BIGGERS, M.S., SHOWELL, H.J., PETTIPHER, E.R., CHENG, J.B., MILICI, A.J., BRESLOW, R., et al (1994). (+)-1-(3S,4R)-[3-(4-phenylbenzyl)-4-hydroxychro-man-7-yl]cyclopentane carboxylic acid, a highly potent, selective leukotriene B 4 antagonist with oral activity in the murine collagen-induced arthritis model. J. Med. Chem., 37, 3197 ± 3199.

Eects of drugs on the acute in¯ammation following i.p. injection of Ag into actively sensitised rats

Jan 1979
216-221

T J Smith

SHARPE, T.J. & SMITH, H. (1979). Eects of drugs on the acute in¯ammation following i.p. injection of Ag into actively sensitised rats. Int. Archs. Allergy Appl. Immunol., 60, 216 ± 221.

Role of resident peritoneal cells in eosinophil migration induced by saline Pathogenesis of rheumatoid arthritis. The role of T cells and other beasts

Jan 1994
317-334

S H P Faccioli
L H Cunha
F Q Ferreira
S H Panayi
G S Corrigall
V M Pitzalis

OLIVEIRA, S.H.P., FACCIOLI, L.H., CUNHA, F.Q. & FERREIRA, S.H. (1994). Role of resident peritoneal cells in eosinophil migration induced by saline. Int. Arch. Allergy Immunol., 104, 323 ± 331. PANAYI, G.S., CORRIGALL, V.M. & PITZALIS, C. (2001). Pathogenesis of rheumatoid arthritis. The role of T cells and other beasts. Rheum. Dis. Clin. North. Am., 27, 317 ± 334.

Contrasting roles for RANTES and macrophage inflammatory protein-1 alpha (MIP-1 alpha) in a murine model of allergic peritonitis

A M Ajuebur
M N Flower
R J Perreti
M Mccoll

Tumour necrosis factor-?? and leukotriene B4 mediate the neutrophil migration in immune inflammation

Abstract

No full-text available

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