Plasminogen activator inhibitor-1 supports IL-8-mediated neutrophil transendothelial migration by inhibition of the constitutive shedding of endothelial IL-8/heparan sulfate/syndecan-1 complexes

The endothelium is the primary barrier to leukocyte recruitment at sites of inflammation. Neutrophil recruitment is directed by transendothelial gradients of IL-8 that, in vivo, are bound to the endothelial cell surface. We have investigated the identity and function of the binding site(s) in an in vitro model of neutrophil transendothelial migration. In endothelial culture supernatants, IL-8 was detected in a trimolecular complex with heparan sulfate and syndecan-1. Constitutive shedding of IL-8 in this form was increased in the presence of a neutralizing Ab to plasminogen activator inhibitor-1 (PAI-1), indicating a role for endothelial plasminogen activator in the shedding of IL-8. Increased shedding of IL-8/heparan sulfate/syndecan-1 complexes was accompanied by inhibition of neutrophil transendothelial migration, and aprotinin, a potent plasmin inhibitor, reversed this inhibition. Platelets, added as an exogenous source of PAI-1, had no effect on shedding of the complexes or neutrophil migration. Our results indicate that IL-8 is immobilized on the endothelial cell surface through binding to syndecan-1 ectodomains, and that plasmin, generated by endothelial plasminogen activator, induces the shedding of this form of IL-8. PAI-1 appears to stabilize the chemoattractant form of IL-8 at the cell surface and may represent a therapeutic target for novel anti-inflammatory strategies.

Efeito anti-inflamatório do heparinóide isolado do camarão Litopenaeus vannamei sobre a peritonite aguda

In recent years the heparin has been the subject of several studies that aim to expand its use as a therapeutic agent, due to its ability to modulate the activity of various proteins that play important roles in the regulation of pathophysiological processes. In several experiments and preclinical trials, heparin has demonstrated an anti-inflammatory role. However, its clinical use is limited, due to its strong anticoagulant activity and hemorrhagic complications. For this reason, considerable efforts have been employed in discovery of heparin analogous (heparinoid) with reduced side effects, that retain the anti-inflammatory properties of heparin. In this context, a heparinoid obtained from the head of Litopenaeus vannamei shrimp, which presents a structural similarity to heparin, showed, in previous studies, anti-inflammatory activity in a model of acute peritonitis with reduced anticoagulant effect in vitro and low hemorrhagic activity. Thus, the present work had as objective to evaluate the effect the heparinoid of the cephalothorax of gray shrimp on the acute inflammatory response in different times (3 or 6 hours after the induction of inflammatory stimulus), using the model of acute peritonitis induced in mice. It was also analyzed the HL effect over the activity of elastase, an enzyme involved in leukocyte recruitment. Furthermore to check if the different doses of heparin and heparinoid change the hemostatic balance in vivo, was assessed the effect of these compounds on the plasma clotting time in animals submitted to inflammation. The results show that in 3 hours, all doses of heparinoid were able to prevent efficiently in the acute inflammatory process without any anticoagulant effects, unlike the extrapolation dose of heparin, which has induced a large hemorrhage due its high anticoagulant activity. However, 6 hours after induction of inflammation, only the dosages of 0.1 and 1.0 μg/Kg of heparin and 1.0 μg/Kg of heparinoid kept anti-migratory effect, without changing of the hemostatic balance. These results indicate that the anti-migratory effect of theses compounds depends on the dosage and time of inflammatory stimulus. The HL and heparin were also able to inhibit the activity of the enzyme elastase. The discovery of this bioactive compound in the cephalothorax of shrimps can arouse great interest in biotechnology, since this compound could be useful as a structural model interesting for the development of new therapeutic agents for peritonitis

O papel da lisofosfatidilcolina na ativação do inflamassoma NLRP3 e sua relação com a formação de células gordurosas

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Molecular, 2016.

Increased Endothelial Cell-Leukocyte Interaction in Murine Schistosomiasis: Possible Priming of Endothelial Cells by the Disease

Background and Aims: Schistosomiasis is an intravascular parasitic disease associated with inflammation. Endothelial cells control leukocyte transmigration and vascular permeability being modulated by pro-inflammatory mediators. Recent data have shown that endothelial cells primed in vivo in the course of a disease keep the information in culture. Herein, we evaluated the impact of schistosomiasis on endothelial cell-regulated events in vivo and in vitro. Methodology and Principal Findings: The experimental groups consisted of Schistosoma mansoni-infected and age-matched control mice. In vivo infection caused a marked influx of leukocytes and an increased protein leakage in the peritoneal cavity, characterizing an inflamed vascular and cellular profile. In vitro leukocyte-mesenteric endothelial cell adhesion was higher in cultured cells from infected mice as compared to controls, either in the basal condition or after treatment with the pro-inflammatory cytokine tumor necrosis factor (TNF). Nitric oxide (NO) donation reduced leukocyte adhesion to endothelial cells from control and infected groups; however, in the later group the effect was more pronounced, probably due to a reduced NO production. Inhibition of control endothelial NO synthase (eNOS) increased leukocyte adhesion to a level similar to the one observed in the infected group. Besides, the adhesion of control leukocytes to endothelial cells from infected animals is similar to the result of infected animals, confirming that schistosomiasis alters endothelial cells function. Furthermore, NO production as well as the expression of eNOS were reduced in cultured endothelial cells from infected animals. On the other hand, the expression of its repressor protein, namely caveolin-1, was similar in both control and infected groups. Conclusion/Significance: Schistosomiasis increases vascular permeability and endothelial cell-leukocyte interaction in vivo and in vitro. These effects are partially explained by a reduced eNOS expression. In addition, our data show that the disease primes endothelial cells in vivo, which keep the acquired phenotype in culture.

Amphetamine modulates cellular recruitment and airway reactivity in a rat model of allergic lung inflammation

Asthma is characterized by pulmonary cellular infiltration, vascular exudation and airway hyperresponsiveness. Several drugs that modify central nervous system (CNS) activity can modulate the course of asthma. Amphetamine (AMPH) is a highly abused drug that presents potent stimulating effects on the CNS and has been shown to induce behavioral, biochemical and immunological effects. The purpose of this study was to investigate the effects of AMPH on pulmonary cellular influx, vascular permeability and airway reactivity. AMPH effects on adhesion molecule expression, IL-10 and IL-4 release and mast cell degranulation were also studied. Male Wistar rats were sensitized with ovalbumin (OVA) plus alum via subcutaneous injection. One week later, the rats received another injection of OVA-alum (booster). Two weeks after this booster, the rats were subjected to AMPH treatment 12 h prior to the OVA airway challenge. In rats treated with AMPH, the OVA challenge reduced cell recruitment into the lung, the vascular permeability and the cellular expression of ICAM-1 and Mac-1. Additionally, elevated levels of IL-10 and IL-4 were found in samples of lung explants from allergic rats. AMPH treatment, in comparison, increased IL-10 levels but reduced those of IL-4 in the lung explants. Moreover, the tracheal responsiveness to methacholine (MCh), as well as to an in vitro OVA challenge, was reduced by AMPH treatment, and levels of PCA titers were not modified by the drug. Our findings suggest that single AMPH treatment down-regulates several parameters of lung inflammation, such as cellular migration, vascular permeability and tracheal responsiveness. These results also indicate that AMPH actions on allergic lung inflammation include endothelium-leukocyte interaction mechanisms, cytokine release and mast cell degranulation. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Leukotriene B(4) is essential for selective eosinophil recruitment following allergen challenge of CD4(+) cells in a model of chronic eosinophilic inflammation

Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class It expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4(+) (but not CD4(-)) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4(+) cells ex vivo. MK886 blocked induction of CCL17 Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalburnin-restimulated CD4(+) cells initiate eosinophil recruitment which is strictly dependent on LTB4 production. (C) 2008 Elsevier Inc. All rights reserved.

In vitro infection of human dendritic cells by Aspergillus fumigatus conidia triggers the secretion of chemokines for neutrophil and Th1 lymphocyte recruitment.

Given the role played by chemokines in the selective homing of immune cells, we sought to characterize the profile of chemokines produced by human dendritic cells (DC) following in vitro Aspergillus fumigatus infection and their ability to recruit cells involved in the antifungal defense. At the onset of A. fumigatus infection, DC released elevated amounts of CXCL8 that promote the migration of polymorphonuclear cells (PMN). Moreover, soluble factors released from A. fumigatus-infected DC increased also the surface expression of two activation markers, CD11b and CD18, on PMN. A. fumigatus infection resulted also in CCL3, CCL4, CXCL10 and CCL20 productions that induce the migration of effector memory Th1 cells. Moreover, the late expression of CCL19 suggests that A. fumigatus-infected DC could be implicated in the migration of CCR7+ naïve T cells and mature DC in lymph nodes. Together these results suggested the involvement of human DC in the regulation of innate and adaptive immunity against A. fumigatus through the recruitment of cells active in the fungal destruction.

Neutrophil-derived CCL3 is essential for the rapid recruitment of dendritic cells to the site of Leishmania major inoculation in resistant mice.

Neutrophils are rapidly and massively recruited to sites of microbial infection, where they can influence the recruitment of dendritic cells. Here, we have analyzed the role of neutrophil released chemokines in the early recruitment of dendritic cells (DCs) in an experimental model of Leishmania major infection. We show in vitro, as well as during infection, that the parasite induced the expression of CCL3 selectively in neutrophils from L. major resistant mice. Neutrophil-secreted CCL3 was critical in chemotaxis of immature DCs, an effect lost upon CCL3 neutralisation. Depletion of neutrophils prior to infection, as well as pharmacological or genetic inhibition of CCL3, resulted in a significant decrease in DC recruitment at the site of parasite inoculation. Decreased DC recruitment in CCL3(-/-) mice was corrected by the transfer of wild type neutrophils at the time of infection. The early release of CCL3 by neutrophils was further shown to have a transient impact on the development of a protective immune response. Altogether, we identified a novel role for neutrophil-secreted CCL3 in the first wave of DC recruitment to the site of infection with L. major, suggesting that the selective release of neutrophil-secreted chemokines may regulate the development of immune response to pathogens.

Leukocyte-derived microparticles in vascular homeostasis.

Leukocyte-derived microparticles (LMPs) may originate from neutrophils, monocytes/macrophages, and lymphocytes. They express markers from their parental cells and harbor membrane and cytoplasmic proteins as well as bioactive lipids implicated in a variety of mechanisms, maintaining or disrupting vascular homeostasis. When they carry tissue factor or coagulation inhibitors, they participate in hemostasis and pathological thrombosis. Both proinflammatory and anti-inflammatory processes can be affected by LMPs, thus ensuring an appropriate inflammatory response. LMPs also play a dual role in the endothelium by either improving the endothelial function or inducing an endothelial dysfunction. LMPs are implicated in all stages of atherosclerosis. They circulate at a high level in the bloodstream of patients with high atherothrombotic risk, such as smokers, diabetics, and subjects with obstructive sleep apnea, where their prolonged contact with the vessel wall may contribute to its overall deterioration. Numbering microparticles, including LMPs, might be useful in predicting cardiovascular events. LMPs modify the endothelial function and promote the recruitment of inflammatory cells in the vascular wall, necessary processes for the progression of the atherosclerotic lesion. In addition, LMPs favor the neovascularization within the vulnerable plaque and, in the ruptured plaque, they take part in coagulation and platelet activation. Finally, LMPs participate in angiogenesis. They might represent a novel therapeutic tool to reset the angiogenic switch in pathologies with altered angiogenesis. Additional studies are needed to further investigate the role of LMPs in cardiovascular diseases. However, large-scale studies are currently difficult to set up because microparticle measurement still requires elaborate techniques which lack standardization.

Dermatan sulfate reduces monocyte chemoattractant protein 1 and TGF-β production, as well as macrophage recruitment and myofibroblast accumulation in mice with unilateral ureteral obstruction

Selectins play an essential role in most inflammatory reactions, mediating the initial leukocyte-rolling event on activated endothelium. Heparin and dermatan sulfate (DS) bind and block P- and L-selectin function in vitro. Recently, we reported that subcutaneous administration of DS inhibits colon inflammation in rats by reducing macrophage and T-cell recruitment and macrophage activation. In the present study, we examined the effect of porcine intestinal mucosa DS on renal inflammation and fibrosis in mice after unilateral ureteral obstruction (UUO). Twenty-four adult male Swiss mice weighing 20-25 g were divided into 4 groups: group C (N = 6) was not subjected to any surgical manipulation; group SH (N = 6) was subjected to surgical manipulation but without ureter ligation; group UUO (N = 6) was subjected to unilateral ureteral obstruction and received no treatment; group UUO plus DS (N = 6) was subjected to UUO and received DS (4 mg/kg) subcutaneously daily for 14 days. An immunoblot study was also performed for TGF-β. Collagen (stained area ~3700 µm²), MCP-1 (stained area ~1700 µm²), TGF-β (stained area ~13% of total area), macrophage (number of cells ~40), and myofibroblast (stained area ~1900 µm²) levels were significantly (P < 0.05) higher in the UUO group compared to control. DS treatment significantly (P < 0.05) reduced the content of collagen (stained area ~700 µm²), MCP-1 (stained area ~160 µm²) and TGF-β (stained area ~5% of total area), in addition to myofibroblast (stained area ~190 µm²) and macrophage (number of cells ~32) accumulation in the obstructed kidney. Overall, these results indicate that DS attenuates kidney inflammation by reducing macrophage recruitment, myofibroblast population and fibrosis in mice submitted to UUO.

Differential effects of female sex hormones on cellular recruitment and tracheal reactivity after formaldehyde exposure

Female sex hormones (FSHs) exert profound regulatory effects on the course of lung inflammation due to allergic and non-allergic immune responses. As pollution is one of the pivotal factors to induce lung dysfunction, in this study we investigated the modulatory role of FSHs on lung inflammation after a formaldehyde (FA) exposure. For this purpose, lung and systemic inflammatory responses were evaluated in terms of leukocytes countings in bronchoalveolar lavage (BAL), peripheral blood and bone marrow lavage from 7-day ovariectomized (OVx) and Sham-OVx rats subjected to FA inhalation for 3 consecutive days. The hypothesized link between effects of FSHs on expression of adhesion molecules and mast cells degranulation was also studied. Once exposed to FA, Sham-OVx rats increased the number of total cells recovered in BAL and of leukocytes in peripheral blood, and decreased the counts in bone marrow. By contrast, in OVx rats upon FA exposure there was a reduction of the total cells counts in BAL and of blood leukocytes: lung expressions of ICAM-1 and Mac-1 were depressed, but the number of bone marrow cells did not vary. Estradiol treatment of OVx rats increased the total cells in BAL and decreased the number of blood leukocytes, whereas the number of bone marrow cell remained unaltered. Progesterone treatment, in turn increased the total cells in BAL and blood leukocytes, but decreased the number of bone marrow cells. OVx rats exposed to FA developed tracheal hyperresponsiveness to methacholine (MCh). A similarly altered response was found between the tracheal segments of Sham-OVx rats after FA exposure and that found in tracheae of naive rats. Estradiol treatment prevented FA-induced tracheal hyperresponsiveness to MCh whereas progesterone was ineffective in this regard. In addition, OVx rats upon FA exposure significantly increased both, the ability of mast cell degranulation and serum corticosterone levels. In conclusion, it was found that FSHs act by distinct control mechanisms on FA-induced lung inflammation and tracheal hyperresponsiveness, since at low circulating levels of FSHs (such as those after OVx) there is some resistance to the development of a lung inflammatory response, but the cholinergic tracheal responsiveness is exacerbated. Our data also help to understand the involvement of FSHs on mast cells activity after pollutants exposure and add information regarding the role of FSHs on the mechanisms related to endothelium-leukocyte interactions. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Leukotriene B(4) is essential for selective eosinophil recruitment following allergen challenge of CD4(+) cells in a model of chronic eosinophilic inflammation

Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class It expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4(+) (but not CD4(-)) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4(+) cells ex vivo. MK886 blocked induction of CCL17 Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalburnin-restimulated CD4(+) cells initiate eosinophil recruitment which is strictly dependent on LTB4 production. (C) 2008 Elsevier B.V. All rights reserved.

Effect of exogenous galectin-1 on leukocyte migration: modulation of cytokine levels and adhesion molecules

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Pulmonary neutrophil recruitment and bronchial reactivity in formaldehyde-exposed rats are modulated by mast cells and differentially by neuropeptides and nitric oxide

We have used a pharmacological approach to study the mechanisms underlying the rat lung injury and the airway reactivity changes induced by inhalation of formaldehyde (FA) (1% formalin solution, 90 min once a day, 4 days). The reactivity of isolated tracheae and intrapulmonary bronchi were assessed in dose-response curves to methacholine (MCh). Local and systemic inflammatory phenomena were evaluated in terms of leukocyte countings in bronchoalveolar lavage (BAL) fluid, blood, bone marrow lavage and spleen. Whereas the tracheal reactivity to MCh did not change, a significant bronchial hyporesponsiveness (BHR) was found after FA inhalation as compared with naive rats. Also, FA exposure significantly increased the total cell numbers in BAL, in peripheral blood and in the spleen, but did not modify the counts in bone marrow. Capsaicin hindered the increase of leukocyte number recovered in BAL fluid after FA exposure. Both compound 48/80 and indomethacin were able to prevent the lung neutrophil influx after FA, but indomethacin had no effect on that of mononuclear cells. Following FA inhalation, the treatment with sodium cromoglycate (SCG), but not with the nitric oxide (NO) synthase inhibitor L-NAME, significantly reduced the total cell number in BAL. Compound 48/80, L-NAME and SCG significantly prevented BHR to MCh after FA inhalation, whereas capsaicin was inactive in this regard. on the other hand, indomethacin exacerbated BHR. These data suggest that after FA inhalation, the resulting lung leukocyte influx and BHR may involve nitric oxide, airway sensory fibers and mast cell-derived mediators. The effect of NO seemed to be largely restricted to the bronchial tonus, whereas neuropeptides appeared to be linked to the inflammatory response, therefore indicating that the mechanisms responsible for the changes of airway responsiveness caused by FA may be separate from those underlying its inflammatory lung effects. (c) 2005 Elsevier B.V. All rights reserved.

A novel biological activity for galectin-1: Inhibition of leukocyte-endothelial cell interactions in experimental inflammation

Galectin-1 (Gal-1), the prototype of a family of β -galactoside-binding proteins, has been shown to attenuate experimental acute and chronic inflammation. In view of the fact that endothelial cells (ECs), but not human polymorphonuclear leukocytes (PMNs), expressed Gal-1 we tested here the hypothesis that the protein could modulate leukocyte-EC interaction in inflammatory settings. In vitro, human recombinant (hr) Gal-1 inhibited PMN chemotaxis and trans-endothelial migration. These actions were specific as they were absent if Gal-1 was boiled or blocked by neutralizing antiserum. In vivo, hrGal-1 (optimum effect at 0.3 μg equivalent to 20 pmol) inhibited interleukin-1β-induced PMN recruitment into the mouse peritoneal cavity. Intravital microscopy analysis showed that leukocyte flux, but not their rolling velocity, was decreased by an anti-inflammatory dose of hrGal-1. Binding of biotinylated Gal-1 to resting and post-adherent human PMNs occurred at concentrations inhibitory in the chemotaxis and transmigration assays. In addition, the pattern of Gal-1 binding was differentially modulated by PMN or EC activation. In conclusion, these data suggest the existence of a previously unrecognized function of Gal-1, that is inhibition of leukocyte rolling and extravasation in experimental inflammation. It is possible that endogenous Gal-1 may be part of a novel anti-inflammatory loop in which the endothelium is the source of the protein and the migrating PMNs the target for its anti-inflammatory action.